Diss. ETH No.8150 MICROBIAL DEATH, LVSIS AND •CRYPTIC• GROWTH: FUNDAMENTAL AND APPLIED ASPECTS A dissertation submitted to the SWISS FEDERAL INSTITUTE OF TECHNOLOGY ZURICH for the degree of Doctor of Natural Sciences presented by COLIN ANTHONY MASON BSc (Hons), University of Bath, England Born on 12 October 1959 in London, England accepted on the recommendation of Prof. Dr. G. Hamer, examiner Prof. Dr. P. Peringer, co-examiner Zurich 1986 ADAG Administration & Druck AG
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Diss. ETH No.8150
MICROBIAL DEATH, LVSIS AND •CRYPTIC• GROWTH: FUNDAMENTAL AND APPLIED ASPECTS
A dissertation submitted to the
SWISS FEDERAL INSTITUTE OF TECHNOLOGY ZURICH for the degree of
Doctor of Natural Sciences
presented by COLIN ANTHONY MASON
BSc (Hons), University of Bath, England Born on 12 October 1959 in London, England
accepted on the recommendation of Prof. Dr. G. Hamer, examiner
Prof. Dr. P. Peringer, co-examiner
Zurich 1986
ADAG Administration & Druck AG
The following sections of this thesis have either been published or
Chapter 13: Proceedings Pro Aqua Pro Vita, 10th International
Exhibition and Technical Meeting for Engineering in
Environmental Protection and Ecology, Basel, Switzerland,
108, 7.1-7.28, 1986.
The articles in chapters 4,6,9,10 and 12 have been sublllitted for
publication and are currently under review.
TO MY PARENTS
Its a very odd thing -
As odd as can be -
That whatever Miss T. eats
Turns into Miss T.
Porridge and apples,
Mince, muffins and mutton,
Jam, junket, jumbles -
Not a rap, not a button
It matters; the moment
They're out of her plate,
Though shared by Miss Butcher
And sour Mr Bate,
Tiny and cheerful
And neat as can be,
Whatever Hiss T. eats
Turns into Miss T.
Walter De La Mare.
I am especially ·;Jrateful to Professor Geoffrey Hamer for his invaluable
advice, support and encouragement throughout this study.
I would also like to express my thanks to:
- Professor Paul Peringer for acting as co-examiner.
- Dr. James Bryers for his advice and cooperation during the early part
of this work.
- Dr. Thomas Egli for his expert help on many issues.
Thomas Fleischmann and Candid Lang for their expertise in much of the
technical aspects of this work.
- Ruth Meisser for much of the typing of this manuscript.
- Dr. Mario Snozzi for translating the summary.
- Dr. Willi Gujer for assistance in computing.
- Dr. Ernst Wehrli for the electron microscopy.
- Ors Baier for bacteriological identification.
- Heidi Bolliger for the technical drawing.
- Paul Schlup for preparing slides and photographs.
- All the staff and students in the technical biology department for
their encouragement, criticism, laughter and for putting up with me.
- The Director and staff at EAWAG for contributing financial assistance,
materials and help.
- And to the Swiss National Programme 7D for financially supporting this
work.
TABLE OF CONTENTS
Olapter Page
l General Introduction l Background l Microbial death, lysis and "cryptic" growth - Fundamental aspects 3 Microbial death, lysis and "cryptic" growth - Applied aspects 5
2 'l'he death and lysis of microorganisms in environmental processes S Introduction S Physiological classification of a microbial culture S Starvation Autolysis Microbial death Mathematical modelling of death and lysis Concluding remarks References
13 23 24 26 29 29
3 Activity,death and lysis during microbial growth in a chemostat 37 Introduction 37 Model development Experimental Results Discussion Conclusions References
2. Gray, T.R.G. and Postgate, J.R. (1976) Editors preface. In: The
Survival of Vegetative Microbes, Proc. Symp. Soc. Gen. Microbiol.,
26, pp ix-x, Cambridge University Press, Cambridge.
3. Gray, T.R.G. and Postgate, J.R. (1976) (Eds.) The Survival of
vegetative Microbes. Proc. Symp. Soc. Gen. Microbial., 26, Cambridge
University Press, Cambridge.
-8-
CHAPTER 2
FEMS Mit:ruhiology Ri:vicws J9 ( 198(,) 37)-401 Puhfo.hi:J hy Els,wii:r
FER tl0044
373
The death and lysis of microorganisms in environmental processes (Death; lysis; survival; starvation: maintenance: 'cryptic' growth: environment)
C.A. Mason, G. Hamer and J.D. Bryers •
ln.~1itu1e of Aquutit' Sciencl!.f, S•·iu f"'etlt:rol butimre of TechnolliXY Ziirtch, Utberlmul~lm.~e 133, ('fl.IJt,00 Dlibem/,,,.f. S••it:trltmJ. t1nd " Depu11men1 cf Cit•il und Ent•lnmmemul Ettgineerintf. Dul.·v U11irer:rl1_r, DurJ1,1m. NC 177fJ6, U.S.A.
Rec~lved 24 March l 986 Accepted 5 May 1986
l. INTRODUCTION
One of the major philosophical stumbling blocks in microbiology relates to the question of ageing. For most macrobes. the processes of birth. growth and death are tangible. observable events. The same cannot be said for the majority of microbes. Exceptions such as some rilamentous microbes (e.g .. Sphaerotilus spp.). some budding bacteria such as llyphomicrobium spp. and yeasts which show effects of ageing by bud scars are well known. However, the question of the ultimate destiny of a particular microbe present at any instantaneous moment in time has as yet to be answered.
Very few papers exist that deal directly with microbial death, ahhough a large amount or cir· cumstantial information is available. Understand· ing more about the processes of ageing. death and lysis in microbes has relevance in the following areas: (I) Public health sector. Erricient testing for the presence or and mechanisms for the destruc-tion of pathogenic organisms in food/ feed and water for human and animal consumption. (2) Biological industries sector. Maximising the per-cemage of active strains with respect to total numbers of microbes. manipulating culture condi-tions to affoct desired optimal system stoichiome· try while maintaining activity and preventing pro-
cess inhibition. (3) Medical sector. Efficacy of antimicrobial agents and qualitative disease as-sessment. In order to deal with such problems, an understanding of the intrinsic physiological mech-anisms involved in (a) induced lysis, (b) autolysis (non-induced lysis). (c) death, (d) resistance. (e) dormancy and (f) survival is necessary.
This review will look at certain aspects of the quantification or microbial death and lysis in terms of both the methods available to investigate the phenomena and the physiological means of defer. ring them. and look at changes which may occur as a result of death or lysis within a population. that may enhance the survival pauern of the re· maining microbes.
2. PHYSIOLOGICAL CLASSIFICATION OF A MICROBIAL CULTURE
2.1. Deft11itio11s
A culture of microbes, either in the laboratory or in their natural environment. is composed of various morphological. biochemical and physio-logical groups. TI1e existence of monocultures is very rare in natural environments, even under conditions where the environment requires special-ised forms such as in some thermophilic and/or
0168·6445/K6/S10.U ~-, l9H6 Fcdi:tution of Eu~1pe.-n Micmhiologicat S;,"-'i~ti~~
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374
acidogenic environments. Biochemical variance arises from the efficient interaction of different microbes in either a food or an energy network, such as in the reduction or various chemical species depending on the redox potential of the environ-ment [lj. Microorganisms have been classified on a physiological basis as: (a) dead microbes; (b) non-viable, active microbes; (c) dormant micro-bes; (d) viable, active microbes.
2.1.I. Dead microbes Strictly defined, these are organisms totally de-
void of metabolic activity, but still possessing a cell wall [2J. Everything failing to meet this description must not and cannot be dead. Defini-tions based on the inability to reproduce [3! are false and lead to embarrassing and dangerous misinterpretations of data. Experimentally, it is almost impossible to determine quantitatively the existence of such cells. Consequently, their pres-ence has to be inferred retrospectively from esti· mates of the total cell numbers and numbets of cells railing into the other categories listed above [ 4) for which more precise methods of quantifica-tion have been developed.
This problem has been compounded by the wide range of different methods available for quantifying microbes. This issue will be addressed in a subsequent section in this review. Dead cells have been treated as inert solids in the considera-tion of particulate degradation in continuous cul-ture systems (5), although they are effectively bio-degradable particulates. As such, dead cells may constitute a very large fraction or the total bio-mass present in trickling filters and activated sludge wastewater treatment systems, in cell re-cycle processes, (i.e., ethanol production) and in semi-continuous (fill and draw) fermentations where a significant portion or the biomass is re-tained as inoculum for each new process cycle.
2.1.2. Non-uiab/e microbes These are organisms which have lost the ability
to reproduce. These result from genetic defects such as absence of a critical enzyme necessary for replication, or lethal breaks in the DNA of the microbe. However, such microbes can carry out substrate transformations when they possess ap-
propriate enzymes. For example. at superoptimal growth temperatures it has been shown that sub-strate energy dissipation can be mediated by non-viable cells (6]. The use of non-viable microbial enzymatic conversion is the basis for immobilised whole cell biocarnlysts [7].
2.1.J. Dormant microbes These can be subdivided into two categories:
(1) Spores; (2) temporarily inactive or resting microbes. These two groups diner in that spores are morphologically differentiated structures. Dorma111 microbes are found in a wide range of environments and may represent the largest class of naturally occurring physiological phenotypes: However, they are frequently confused with non-viable cells, due to the difficulty of promoting their growth using artificial laboratory stimuli. These two types of dormant cells also differ from one another functionally, spores serve distribution and survival functions. whilst resting microbes are only intermediates leading either to active micro-bes or to death or lysis [8).
2.1.4. Active microbes These are cells which can actively assimilate
substrate, increase in mass and replicate. Active microbes are most commonly found in laboratory environments, although they are also frequently the major physiological class found in industrial and technical microbial processes. More informa-tion is available on this category than on any other.
2.2. Analytical met/rods for physiological differentia-tion
The ability to grow and multiply is very often the only criterion used in differentiating between the groups mentioned above. Most working defini-tions for the various types of microbes are restricted by the lack of accurate methods with a sound theoretical basis, together with a fanatical adherence to historically proven inaccurate meth-ods. Use of arcane analytical methods has been perpetuated by public health authorities. despite a wealth of evidence as to the limitations and dangers or such techniques. Some of the current
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methods for assessing cell numbers and activities are discussed in the following section. For more deluils on melhods, the reader should consult spe· cialiscd reviews (9-1 JJ.
!.l. I. Cell c11/rioalio11 mc1hods The use of agar as a solidifying agenl in culture
media was first proposed in 1881 [14]. Sin~'e then, its use has escalated, and today it represents the most universal microbiological technique for the cultivation of microbes (15]. Theoretically the numbers of microbes in a sample can be derived from the numbers of colonies growing on the surface of an agar solidified medium in a Petri dish (16), although it was originally intended that the use of such media should be for the isolation and growth characterisation of microbes. Despite the repeated and often vehement criticism which has appeared in the literature [ 15-19), agar colony counts continue to be used extensively for the enumeration of microbes for estimation of survival (20-23], in stress studies [24-26], and perhaps most surprisingly in public health (27-30], even though alternative accurate techniques are availa-ble (31).
It is now universally accepted that in most natural microbial environments only a very small fraction of the microbes present can be enu· merated using the agar plate technique [18,32-34]. Estimates of this error in the literature vary de-pending on the environment. In soil for example, it has been claimed that only 1-10% of the true number of viable organisms are enumerated [35), whilst in seawater values below 0.1 % are con-sidered normal (33.36]. In the testing of water supplies, as many as 90% of the coliforms, the bacteria used to indicate the presence of patho-gens, may not be enumerated [37).
Some of the problems associated with agar plate counting or microbes include the lack of a single universal medium which will allow growth of all organisms [38), since most organisms are sensitive to the type or medium used [17,39-43]. In the literature 'improved' media for the growth of microorganisms are frequently reported, sug-
. gesting that existing compositions are inadequate. This process or new medium formulation is never-ending, and although improvements are
375
continually being made, an acceptable status will never be reached. The technique has to be rede· fined for use only under those circumstances for which it is suitable. Despite these criticisms it should also be noted that if the method is used, interpretation must embrace all or the limitations of the method, if the results obtained in routine screening work are lo be valid.
Failure to grow on an agar surface has been ascribed to environmental fastidiousness, dorman· cy [21), inhibition by neighbouring cells (16). physicochemical differences between the labora· tory and natural environment [38]. clumping (9) and to the fact that stressed and injured cells may have some difficulty in reproduction (44) since sublethal damage and inactivity are not dis· tinguishable (21,45]. Further comment on the widespread misuse of agar-based cultivation media is superfluous, and the reader may consult the reviews of Buck (17) and Fry (18] for more de-tailed discussion.
1.2.1. Aclioity measurements Metabolic activity, as measured by the rate of
oxygen consumption, has been widely used in microbial ecology for the assessment of microbial activities in different environments [46). Electron transport system (ETS) activity measurements are usually used as a guide to metabolic activity. due to their relative simplicity. The ETS is mediated by the action of several dehydrogenase enzymes such as succinate dehydrogenase, and direct mea-surement of dehydrogenase enzyme activity is as-sumed to be a reliable indication of ETS activity in a specific environment (47,48). A large propor-tion of the total metabolic activity has been shown to be linked to ETS activity (49]. The most fre-quently encountered method for ETS activity as· sessment involves the reduction of the tetrazolium salts 2,3,5-triphenyltetrnrolium chloride (TIC), 2,2' -di·p·nitrophenyl-5,5-diphenyl-3,3' -dimethoxy-4,4' -diphenylene (NBT) or 2-(p-iodophenyl)-3· ( p-nitrophenyl)-5-phenyltetrazolium chloride (INT) to insoluble formazan compounds [50,.51], a technique pioneered by Lenhard (52] for the as-sessment of bacteria in soil. The tetrazolium salts compete with oxygen for electrons [50,53]. Since ETS activity is common to virtually all microbes
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376
(48,49] the technique has provoked increasing in· terest, and since it is applicable under both aerobic and anaerobic conditions [46,49,50) it can thus be used for samples from most environments.
Most of the methods described involve disrup· tion of the cells after incubation with a tetra· zolium salt [54-56] followed by solubilisation of the formazan using. typically, Triton X·lOO (57,58) followed by measuring the absorption at 490 nm {46,47). Direct microscopic e.xamination has also been used to give a more precise estimation of the number of active cells based on the assumption that only living cells will contain the formazan crystal (59-61). However, it has been suggested that not all bacteria present in natural environ· ments are actively metabolising. and may be in a state of dormancy (60,62,63); that not all bacteria are capable of tetrazolium salt reduction [48); and that tetrazolium salts may suppress ETS activity (48). Addition of substrate has also been shown to affect the results, although some disagreement still exists as to the necessity of substrate addition. Tabor and Neihof (49) found no increase in counts of microbes from water samples when succinate was included in the INT·incubation tubes. How· ever, Trevors ·(48) found an increase of 100% at 4°C and 327% at l0°C in water samples when substrate was included, and Bright and Fletcher (64) obtained increased counts when the growth substrate (leucine) was included in their bacterial counts in a series of attachment studies. Work in this laboratory (unpublished results) has also clearly shown an enhancement of active cell num· bers after inclusion of an ETS-activating sub-strate.
Various authors have commented on the prob-lems of detection of small microbes which may be present in natural environments (65) and have suggested modifications to the basic technique [19,49,66) while other authors have chosen differ· ent compounds (67). Much attention has also been given to the other methods for assessment of metabolic activities in natural environments, in-cluding the measurement of uptake rates of ra· diolabelled substrates (681 and measurement of production of metabolic products. A technique involving direct examination of cells whose nucleic acid synthesis has been repressed without ap-
parently affecting their growth has also been sue· cessfully used (36]. The measurement of in situ metabolic activity was the subject of a recent review by Findlay and White (69].
2.2.J. Direct counts Information concerning the physiological state
or a microbe is usually expressed with respect to the total numbers of microbes present (18). De-spite the persistence of some agar based counting procedures. most total counts are presently carried out by microscopy. The use or bright-field mi· croscopy is rare and has limited application [70,71), as has phase contrast microscopy (72). However, epifluorescence (incident light fluorescence) mi-croscopy has been successfully used for enumera-tion purposes [73). Cells are counted on a mem-brane rilter after filtration of a known sample volume. This technique was first developed by Strugger [74) and has since been modified and improved by the introduction or different filters [75,76) and microscope lamp/filter arrangements [77). Various stains have been employed, including fluorescein isothiocyanate (FITC) for soils [12.78) and acridine orange for aquatic samples [21. FITC reacts specifically with proteins and fluoresces green (79). Acridine orange stains nucleic acids, and the technique is very good when used for enumerating total cell numbers, but does not dis-tinguish between live and dead cells (71,80). It has been shown, for example, that acridine orange is still taken up by autoclaved cells [81]. By control-ling the pH it has been suggested that dirrerentia· lion between live and dead cells is possible [82) although experimental verification has yet to be reported. A further restriction or this technique is that acridine orange does not distinguish between dormant and growing cells [2). The accuracy of the method has been examined by comparing counts using epifluorescence and electron microscopy [83J. Using the scanning electron microscope, the counts were found to agree, but epifluorescence gives higher counts than can be obtained with transmis-sion electron microscopy ( 64!.
Other fluorescent compounds have also been used and some claims have been attached to their ability to accurately determine numbers of ac-tively metabolising microbes. Rhodamine 123, for
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example, relies on the existence of a proton motive force for uptake (85] although its use so far has been shown to be restricted to Gram-positive cells. Similarly, fluorescein diacetate (FDA) has been used for enumerating freshwater microbes, where this non-fluorescent compound is attacked within the cells by non-specific esterases [86) with the resultant release of fluorescent products (87]. However, FDA also has dirficulty in penetrating the Gram-negative cell wall [81 l and its effective use is most probably restricted to mammalian cells [88J, yeasts [89) and some cyanobacteria (12j. The stain nuorescamine has also been used success-fully to differentiate between microbial cells and detritus particles in marine samples, due to the high affinity or the stain for amino groups [90].
7~ 6 ·~-·---·-----· 5 4
3
S! 2 en 2
8 e 7
"' 6 ..... ...J 5 w u 4
3 2
I
:::--. .......... ·---·
A
~ ····-· B
0 4 8 12 16 DAYS
Fig. l. Detection of E. <t>li hl0407 i110C11la1ed into (A) aged estuarine water where the pH and salinity were adjusted lO simulate environmental condhiont; and (B) in situ experiments in ~mitropical water using membrane chambers. 0. Acrid inc orange direct coont; C acridine orange direct count control; • fluorescent antibody counts~ •· standard plate count; o, eosin methylene blue agar count; •. dire¢t viable count (nalidixic acid). Redrawn from Jl4) with permission.
7
5
2 ....... ~.---·-· ~4
e -'1'> ...J ...J w u 2
2 4 6 DAYS
371
8
Fig. 2. Detection of V. cholera• CA40t exposed to Pa1uxen1 River water in microcosms. 0, Acridine orange direct count~ "· noorescent antibody count; II. direct viable count (nalidix.ic acid); A, thiosulpate citrate bile $UCCOSC agar coun1~ •. tryptic Soy agar count Redrawn from [34) with permission.
Epifluorescence microscopy using acridine orange has also been combined with autoradiogra· phy for the detection of metabolising cells f91 ). Another approach in total cell enumeration is that of fluorescent antibody labelling. The staining of cells by fluorescent antibodies allows the observa-tion of particular strains in a mixed culture (92,93) thus allowing a direct count of individual species. However, the technique does not provide any in-formation as to the physiological state of the cells. The use of fluorescent antibody technique for public h~alth testing for the presence or patho-genic organisms is currently being investigated. A recent paper clearly shows the advantages of this technique over the presently used standard meth-ods [34]. Their paper reports results from water samples from Bangladesh, tested for the presence
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378
7
4 8 12 16 20 DAYS
Fig. J, ~lection of Shi~/a 1tJ1tnti 530 inoculated into aged l!s.tuarine water where the pH and salinity were adjusted to simulate environmental condittoos. 0. Acrkline orange direct count: •· fluorescent antibody count; a direct viable count; •. tryplic soy agar count; A. MacConkey agar count Redrawn from {341 with permission.
Table I
Evaluation or microbial enumeration methods
of Vibrio dwlcme. Using conventional techniques. 7 or the 52 sample.' tested proved positive. fly the fluorescent antibody technique. 51 out of the 52 were positive. Similar comparisons arc made be-tween acridine orange direcl counts and plalc counts (Figs. 1-3). Clearly. these direct counl methods are beuer suited for the enumeration of organisms, especially where potential public health hazards exist.
A summary of the various conditions for and enumeration possibilities of rhe different methods available is given in Table l.
3. STARVATION
Under laboratory conditions, microbes are gen-erally grown in artificially rich environments the like of which are rarely found in nature. Unlike laboratory culture environments, organisms in natural environments are subjected to variations in substrate availability, (i.e., nitrogen, phos-phorus), in temperature, oxygen, toxic chemicals as well as to spatial variations, and if the cells are attached, differences in substratum composi1ion may exist. Despite these condi1ions, indigenous microbes readily survive in their natural environ-
Act. Non-Rep. and Dorm refer lo the physiologically differentiated cell types discussed in section 2. L Parentheses imply that only .tt part of this population was enumera1ed.
Method Time required Environment Requirement Cell types Differentiation At.wracy for test of test for ccH tnumerated between cell types
PhueCount 24-48 h Modified Yes (Act), (Dor) Yes Low MPN 24-48 h Modified No Act. Non-Rep. (Yes) Low
(Dorm) Activity
ETSONn I h Originol/ No Act. Non·Rep. No High modified (Dorm)
ATP 2h (Cell extract) No Act, Non·Rep No Unknown Oire<:t
AODC/FITC JO min Original/ No All No High modified
FDA 2h Original/ No Act. Non.Rep, Ycs/noo1 Unknown modified (Dorm)
stide culture 24-48 h modified Yes Act. Non .. Rep, Yes/no Low (Chum)
lmmunoOuorescc:nc~ 2h Origimd No All Yes Very high
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ments. Therefore, mechanisms obviously exist conferring the necessary properties to compete and survive. In this section those mechanisms by which microbes compete and survive under varia· ble nutrient conditions (i.e., feast/ famine or starvation) will be discussed with emphasis placed on the modes by which cells are able to defer the death process.
In order lo grow, a microbial cell requires a carbon source, an energy source, and nutrients for biomass synthesis and metabolic regulation. How· ever. it is known that bacteria are able to survive, sometimes for very long periods, in the absence of any or all of these requirements. As pointed out by Morita [65] many publications deal with short time survival (days to weeks) or with survival under specific stress conditions. Stevenson [63] suggested that in most aquatic environments a significant proportion of the bacterial community can be described as being physiologically dormant. Similarly, in soil there is evidence to suggest the dormant bacteria outnumber the active ones [94).
For a cell to survive during starvation, only a very small part of its metabolic potential needs to be expressed. These have been collectively referred to as maintenance functions, and include mainte-nance of osmotic potential, turnover of essential cell materials, and maintenance of the membrane potential. If energy for these processes is not provided, it is said that the cell will irreversibly cease to function. Microbial cells are biochem· ically sophisticated, and many of the. inter· mediates of the chemical reactions have higher free energies than their original substrates. Energy must be supplied to counteract a natural tendency towards disorder and the energy required to main-tain the basic requirements of cellular activity is termed maintenance energy [95). Maintenance en-ergy in the absence of an exogenous energy source has to be derived from the oxidation of either endogenous cellular constituents or storage prod· ucts. This degradation is known as endogenous metabolism and can be defined as the summation of all metabolic reactions which occur when a cell is deprived of either compounds or elements which may serve specifically as exogenous substrates [96}.
The theories resulting in the development of the concept of maintenance are complex. Beauchop
379
and Elsden (97) introduced the concept Y,/ATP relating the mass of cells produced per mol ATP obtained from the energy source in the medium. Theoretical calculations of Y,/ATP can be made under anaerobic growth conditions, since the catabolic pathways for anaerobic breakdown of substrates are known [98]. Despite original theo· fies tO the Contrary f,/ATP is now known not IO be a constant for different microorganisms [99]. Fur· thermore, experimental Y,/ATP values are nearly always much lower than those based on theoreti· cal calculations {100). Much of the early work was carried out in batch culture in a dynamic environ-ment such that yield values were affected by physico-chemical changes {101]. In chemostatic culture, yield values can be obtained under condi· tions without such variations. One method is to use the ratio between the specific growth rate and the specific rate of substrate consumption, i.e.,
r,1,-p./q, (l) Where Y,;. is the microbial biomass yield coef·
ficient (mass of cells produced per mass of sub-strate utilized), JI. is the specific growth rate con· slant (1- 1) and q, the specific substrate consump· tion rate (mass substrate consumed per mass of biomass per unit time). When this is carried out over a range of different growth rates a plot of µ against q, will give a straight line, which when extrapolated fails to go through the origin. The implied assumption is that as the growth rate decreases to zero the value for the specific sub· strate uptake rate tends towards a positive value. Pirt [102) explained this with his theory of mainte· nance energy, and deduced that the consumption of substrates was partly for growth dependent processes and partly for growth independent processes. Stouthamer and Bettenhausen [103) ex· pressed this mathematically as: qATP - µ/f,/ATP
or
qATP - µ/ r~:,.. + m.
(2)
(3)
where qATP is the specific rate of ATP production (mo! ATP· g- 1 dry wt.· r 1), Y,;ATP is the molar growth yield for ATP, and Y,~:,.. is the growth yield per mol ATP corrected for the energy of
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380
maintenance (m,). ·Values for the maintenance energy requirements have been published by Stouthamer (98]. However, Neijssel and Tempest [I 04) question the basic assumption made by Pi rt (102], namely that the maintenance rate does not vary with growth rate. Instead, these authors pro-pose that the maintenance rate does vary with growth rate. This led Pirt (105) to propose a modified model, whereby a growth rate-dependent maintenance term is included in Eqn. I for specific substrate utilisation, i.e.,
q, - µ./ Y,1, + m1 + m'(l - kµ.) (4)
where m 1 is the constant maintenance energy coefficient m', the growth rate dependent mainte-nance energy coefficient when µ. - 0 (mass sub-strate per mass cells· t- 1) and k is a constant. Thus the expression m'(l-kµ.) is the growth rate-dependent maintenance energy, and m' its value when µ. • O {105). This treatment of the mainte· nance energy concept was similar to an approach suggested by Neijssel and Tempest [104). They later clearly expressed their interpretation or maintenance energy terms by stating that the maintenance energy rate and maximum growth yield value derived from linear regression analysis of either yield or metabolic rate versus growth rate are essentially mathematical constants and not biological constants. In a strict physiological sense, both may vary with growth rate (106].
One of the major problems in experimental determinations of maintenance energies is that when growing the cells at a very slow growth rate it is almost impossible to attain steady-state con-ditions, due to non-steady-state medium addition and spent medium removal rates, etc., such that experimental verification that the q,/µ. diagram does indeed tend naturally towards zero at lower growth rate exists and thus the conclusions de· rived cannot be satisfactorily resolved (107).
Microbes possess two mechanisms by which energy (ATP) can be produced Crom the oxidation of an energy substrate. The first of these is sub-strate level phosphorylation where the production of ATP is catalysed by soluble enzyme systems within the cell cytoplasm. The second mechanism is oxidative phosphorylation whereby ATP synthesis is coupled to electron transport reactions
which are driven, in most cases, by the oxidation of either organic compounds or of inorganic ions of negative redox potential, with concomitant re-duction of electron acceptors with higher redox potentials (108). The mechanism by which ATP is produced in oxidative phosphorylation is almost universally agreed to be based upon the chemi-osmotic theory or Mitchell [109). Synthesis of ATP is catalysed by anF0 F1 A TPase enzyme located in microorganisms in the cytoplasmic membrane {108), although it is generally considered that large portions of the membrane itself can be considered to be 'energy transducers' (110). The theory in its simplest form states that these energy transducing systems act as electrogenic proton pumps and translocate protons across the cytoplasmic mem-brane. Since the membrane is cCfectively imper· meable to OH - and H + ions the result or their translocation is the generation of a proton gradi-ent (A pH) and since they are ions, of an electrical gradient (AY,) between the cytoplasm of the cell and its immediate environment. Consequently the cell interior becomes alkaline and electrically negative with respect to the exterior. Since both gradients exert an inwardly directed force on the protons, this force can be expressed by the sum of these two components, namely:
Ailw • M - ZApH (5) where djlw is the proton motive force (mV) and is a measure of the combined chemical and electri· cal forces acting on the protons, 4Y, is the electri-cal potential difference across the membrane, A pH is the pH difference across the membrane and Z - 2.3 RT/ F, where R is the gas constant, T, the absolute temperature and F, the Faraday con-stant. The factor Z converts the pH gradient into m V. The Ailw generated by electron transfer is used to drive an A TP-hyrolysing proton pump in reverse, i.e., in the direction of ATP ·synthesis. Thus, the energy transducing membrane contains two proton pumps, one driven by electron transfer and one driven by ATP hydrolysis.
The energetic activation of the membrane is supposedly a major regulatory mechanism in the physiology of the cell. Its possible influence in the action of autolysing enzymes will be discussed in section 4, but it has also been implicated as a
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regulatory control for other physiological func· tions (111]. The proton motive force supplies the energy for flagella movement [U2), solute trans-port, [113), ppGpp breakdown {114], nitrogen fixa· tion {115], DNA transport (116), and pH homeostasis (117]. The processes driven by ATP are different from those driven by the proton motive force. Since ATP is a general intermediate in biosynthetic processes, supplying energy for the transport of some solutes, it can also be used to generate a proton motive force (118]. The interde-pendence between Ll,P and ATP is one mechanism for metabolic control. In the presence of a high NADH concentration resulting from a high sub· strate flux, Llilw will be enhanced and ATP will be produced. When the ATP content is high. and a low proton motive force is operative, the latter can be regenerated by ATP hydrolysis, thus di· verting ATP away from biosynthesis.
Tempest and Neijssel [106], in a critique of maintenance energy suggest that most of the en· ergy required is necessary for the maintenance of an ionic gradient. This suggestion is based on experiments on the K + ion concentration gradient in Klebsiella aerogenes. It was demonstrated that the transmembrane K + gradient increases with increasing growth rate (since the K + requirement of the cells also increased) which was accompa· nied by concommitant increases in either the specific respiration rate or the oxygen consump-tion rate of the culture (119]. Calculating the amount of 0 2 required to maintain the ionic gradient from the specific respiration rate at each of the different growth rates, the authors demon-strated that a plot of q0 , versus growth rate constant would then indeed go through the origin. Tempest and Neijssel thus conclude that more than 90% or the maintenance energy requirement for glucose-limited cultures of Klebsiella aerogenes is necessary for the maintenance of membrane ionic potential. Since the extracellular K + con-centration diminishes as the growth rate and in-tracellular K + concentration increase, then the maintenance energy must vary with growth rate even in carbon substrate limited cultures with a 10-fold excess or K + [106].
Maintenance energy has also been associated with solute uptake. However, this function can
381
also be related to membrane function, and there-fore, to the chemiosmotic theory. Accordingly, there are several mechanisms by which solutes can be 1ranslocated across the cytoplasmic membrane. Most solutes are transported by the so called secondary transport systems which can be either passive, i.e., without the interaction of specific membrane proteins, or active, whereby such medi· ation occurs [111]. As such a process requires energy, a 'driving force' is derived from the proton motive force of the cells which in general can be described by the expression:
([A]in)
Z log (A)..,, + (n + m)Ll,P- nZLlpH (mV)
(6) where m is the charge of .the solute A and n is the number of protons translocated in the symport.
More recently, Michels et al. (120) have de-scribed an 'energy recycling model' which in es-sence is the reverse process of secondary transport of solutes, whereby the energy of an electrochem-ical product gradient is converted into the energy of an electrochemical proton gradient. Such sys-tems have been detected in the homolactic fermentative Streptococcus cremoris and in Escherichia coli when the microbes are growing fermentatively and therefore have no means for proton extrusion by functional electron transport systems [1 21 ].
Another so called 'maintenance function' is microbial motility, but the motor for flagella mo-tion is not an A TPase as has been demonstrated by the continued motility of microbes even after their ATP pool has been significantly reduced (115), the energy for both flagella motion [122) and the signal mechanism in chemotaxis (123) being derived from the proton motive fo~ (pm!). This has been shown to be the case by using a model system of ghost cell envelopes on which flagella rotation could be initiated by imposing a pH gradient across the envelope membrane (112]. There is also the suggestion that there is a tight stoichiometric coupling between proton transfer and flagella rotation [ 113), although there appears to be a threshold pmf below which llagellar mo-tion is impossible (124).
-17-
)32
Three questions concerning survival need to be answered: (i) How can a microbe in a non-hostile, neutral environment without nutrient source sur-vive for extended periods? (ii) ls energy lost via the non-growth associated membrane functions in microbes with a nutrient nux sufficient to meet its growth requirements? (iii) What effect do such energy losses have on the growth/ survival be· haviour of microbes in which they are occurring? Obviously two different points of reference are indicated. In the first question, natural environ-ments are envisaged, whilst the second and third questions relate predominantly to microbes in technical processes and laboratory systems.
Under starvation conditions, the microbe no longer receives an adequate nutrient flux and is compelled to divert its energy towards specific survival functions. Cellular replication is not pos-sible due to the high energy requirement for synthesis of new cell biopolymers. Energy has to be diverted to the so-called maintenance func-tions. In order to achieve this, the cell has at its disposal several possible mechanisms. Initially if the cell has been under a feast regime, it will contain an excess of polymers such as RNA which are now superfluous for metabolic sustenance. Oxidation of these compounds together with the mobilisation and oxidation of any reserve poly-mers constitutes the observed effect known as endogenous metabolism. However, this step is only the start of the process of survival under starva-tion conditions.
A theory has recently been proposed explaining the relationship between substrate consumption and biomass production at low growth rates {125-127). In order to prevent the 'apparatus ef-fect' whereby substrate addition to slow-growing cultures becomes pulsed as opposed to continuous thereby changing substrate uptake characteristics [107), a recycle reactor was used to grow cells with very long residence times. Il was found that the microbe used ( E. coli) went through various 'phases' before activating the 'stringent response', as the residence time (reciprocal dilution rate or reciprocal growth rate constant) was extended. The stringent response is a series of biochemical adjustments resulting from the limitation of amino-acyl-tRNA. As a result the cell makes a
number of major readjustments of activity in· eluding (I) a reduction in RN A synthesis and accumulation; (2) increased protein turnover; (3) reduced membrane transport; (4) reduced endog-enous synthesis or nucleotides; glycolytic inter-mediates, carbohydrates, lipids, fatty acids, poly-amines and peptidoglycans; (5) increased control ('kinetic proof reading') over protein translation and (6) cAMP accumulation (I 28]. This reaction results from the accumulation of guanosine 5'di-phosphate 3'diphosphate (ppGpp) due to an idling reaction of the ribosomes and uncharged tRNA via a protein known as stringent factor [129). A threshold concentration of ppGpp must be ex-ceeded for the response to manifest. Stouthamer (127] suggests that as the growth rate of the organisms slows down three phases can be dis· tinguished, the first with sufficient tRNA, fol-lowed by a phase in which aminoacyl tRNA be-comes limiting and ppGpp formation begins, and finally a phase where the stringent response is manifested, i.e., where cellular metabolism is put in check at the expense of some limited energy expenditure (from cAMP and ppGpp formation and from the proof-reading of proteins). Whether this energy expenditure is equivalent to the main-tenance energy is not yet clear. This theory is one possible mechanism for survival in nutrient poor environments, whereby the accumulated cAMP may be used by the microbes to activate a range of enzymes when suitable substrates become availa-ble.
During endogenous metabolism and long-term survival experiments, it has as yet been impossible to attribute death to the loss of a specific cellular function (96). However, several theories have been proposed associated with the concept of mainte-nance energy. Correlations between the proton motive force and the metabolic state of the cell have been proposed as well as theories relating the death of the microbe to energy exhaustion (130). Konings and Veldkamp [1201 have also suggested that the gradual decrease in the proton motive force found during starvation experiments could be attributed to the gradual accumulation of •non-viable' cells in the culture.
Zilberstein et al. [l I 7) in a study on the effects of external pH on the proton motive force ob-
served that £. coli shifted its membrane potential
and pH in a homeostatic mechanism to maintain
the internal pH al a constant value. Variations in
the external pH were compensated for by varia-
tions in the membrane potential such that the
Ailw also remained effectively constant. How-
ever, when they subjected a mutant, defective in
its Na• /H • anliporler activity, to a pH change
they found that growth ceased when the A pH
collapsed, although a high membrane potential
was still maintained. The actual death of the mi-
crobes (as measured by lack of colony formation)
did not occur for at least 12 h after the change in
ApH. Recently, 0110 el al. (131} reported that in the
homolactive fermentative S. cremoris the ini-
tiation of lactose starvation caused the membrane
potential lo collapse almost immediately, with an
accompanying collapse in internal ATP concentra-
tion. However, at any time up to 24 h, both can be
restored by addition of lactose, thus allowing the
microbes to survive for short periods in the ab-
sence of a measurable pmf. Unfortunately, no
data were provided as to what happened during
extended periods of starva lion.
In acidophilic bacteria, the pH of the cell cyto-
plasm is maintained al values close to neutrality
despite the extremely low pH values of their growth
:i \6
i 1.2 ~ ~ 0.6 .. ..
0.4 <I A
100 200 300 400 500 TIME (Hrs)
Fig. 4. Effects of starvation on various parameters in T.
acidopltilws. The organism wa.s grown hcterotrophicatly at pH
3.0 in a mineral salts-glucose medium. The cells were harvested
by centrifugation and resuspended at time • 0 in deionized
water at pH 3.0 at 29°C in an incubator shaker. 0, ApH: 0.
~'1-; A, ~~H"; •. cellular ATP level; •. cellular poly-/1-hy-
droxybutyric acid level. The scale for ApH and AP.H• is in
negative mV, that for ~ .. in posilive mV. Redrawn from [1301
with permission.
-18-
)8)
environments. In a starvation study with Thioba-
cillus acidopliilus growing in an environment at pH
3, some decline in ApH cou[d be detected, to-
gether with some minor changes in A ./I during a
starvation period of 530 h (130}. or particular
interest is that as soon as all the internally oxidiz-
able substrates had been exhausted, notably poly-p
hydroxybutyrate, A pH declined, and as soon as
all the available ATP had been used up, Ai/I
collapsed (Fig. 4). The authors were able to show
the dependence of colony-forming abi!ity on the
presence of a measurable Ailw· When the dilw
approached zero, no 'viable cells' could be de-
tected. Ten Brink and Konings (132} found that in
batch cultures with S. cremoris with no pH con-
trol, the proton motive force ( dil H •) collapsed lo
zero as soon as the logarithmic growth phase
ceased (Fig. 5). These researchers concluded that
energy was required lo maintain the pH gradient,
as a result of the changing pH in the external
medium due to lac late production in this organism.
Padan el al. (133} have also shown that in aerobi-
cally grown £. coli, inhibition of respiration by
either anaerobiosis or KCN leads to a collapse in
the ApH. Zychlinslci and Matin [130} showed with
lljiH' 7.0 200
160.! 6.5 ~
~ ~
c
~ 120 z .. s.o: w
I-0
80"'
" j w w u
40 .. : .L
Fig. 5. The electrochemical proton gradient in S. cremoris
during growth in batch culture on a complex medium with
lactose (2 gl- 1) as sole energy source. A, ApH; 0 1 A~; •.
Aji.H•· O, pH; (· - - - - ·), time course of protein synthesis.
Redrawn from (132) with permission.
-19-
384
azide-treated cells of the •!cidophilic T. acidopltilus that the pH gradient could be maintained purely pas.~ively.
A major criticism of much of th.e published material regarding the starvation of microbes is that the effects of death and lysis during the experiments are rarely considered. However, these factors can have serious implications in the inter-pretation of the biochemical changes in the cul· lures under study. A typical example can be seen in a recent study on the survival of the organism Brevibacterium linens during starvation (134J. The authors carried out a very broad range of tests during the starvation period including viability by slide culture, dry weight, oxygen consumption, total sugars, intracellular protein, intracellular amino acids, DNA, RNA, and ATP. They ob· tained what appear at first sight to be interesting changes in the concentration of these parameters with time (Figs. 6-8). ATP was only measured during the first 10 h of the experiment, and showed a rapid decline. However, since the viability, which was unfortunately based on a cell propagation method, decreased to 70% or its initial value, and the total cell mass also decreased together with an increase in extracellular NHZ. lysis processes can· not be ignored. The authors suggest that 'cryptic'
"' z: z
"" :It w
"'
DAYS Fig. 6, Changes in •. turbidity; O. viability; and ;., dry weigh1 in a suspension of BMJibacltrilUtt lintns during 30 days of nutrient 5tarvation. Viability and turbidity AR expressed as Ii of initial values. The ceH1 were grown aerobically at 2J°C in a O.JSI baoto-tryptone, 0.2SI yeast e•t-~ 0.12S'I. glucose medium with mineral .. iu at pH 7.0. The cells were resus-pended in a O.S M Tris-HCI huller solulion (pH 8.0) on day 0 and iPCUbated at 21°C. Redrawn lrom (134) with permission.
0AY5
.. :x: z
Fig, 7. Changes ln the contents or a intrai;cllular protein; A. Cree amino--acids; nnd O. 1he quantity o( ammonium Ions in the exuacellular medium in a suspension of 8. linetU during 30 days of nutrient starvation. The ccUs were grown acrobic:ally at 21°C in a 0.3Sf. baeto-lryptone, 0.2SI yeas1 e•1rac1 and 0.12" glucose medium with mineral salls at pH 7.0. The cells were resuspended in a O.S M Tris-He! buffer solution (pH 8.0) on day 0 and incubated at 21°C. Cells initially contained :no p.g protein and 110 ll& of free amino acids·mg-• dry weight. Redrawn from {1J4J whh permission.
growth was not occurring. based on their failure to observe microscopically either cell wall or cell membrane fragments. The observation of such cell debris. even under conditions where lysis is un-
C) z z "" :It w
"' ... 0 10 20 30
DAY5 Fig. 8. Changes in the intracellular levels of o. DNA; ;., RNA; and D, extracellular material absorbing 11 260 nm. The mean initial quanlity of DNA .., .. 27 1'8 · mg-1 dry wei&ht and tha1 of RNA w .. 70 l'&·mg-1 dry weight The bacterium B. //~ens wti grown aerobically at 21°C in a O.lSi bacto-tryp· tone. 0.251 yeast «traet and O. l2SI glucose medium with mineral ,.ll• at pH 7.0. The cells were resuspended in a O.S M Tris-HCI buffer solution (pH 8.0) on day 0 and incubated al 21°C. Redrawn from [ll4] wilh permi5Sion.
-20-
doubtedly occurring, requires the use of either electron microscopy or of highly specialised mi· croscopic methods such as immunonuorescence, so that this evidence for the lack of lysis is incon· elusive. This aside, if one looks at the data, these are presented in terms or mass of the parameter per unit mass dry weight. However, if death and lysis occur in the culture as suggested by the NHt accumulation and the accumulation of 260 nm absorbing compounds, then the biophysical com· position of what actually constitutes the reference quantity, i.e., unit mass dry weight, must be ques-tioned. H this dry weight were composed entirely of living, active cells, then the data could be accepted as presented. However, after 30 days it is most likely to be predominantly composed of inert cell particulates and dead biodegradable biomass, so that the data have to be recalculated and ex-pressed on a more meaningful basis such as the mass of a particular component per cell or, better still, mass per living/ surviving cell.
The authors measured a reduction in protein content from 310 µg/mg dry weight to 60% of its original value, i.e., 190 µg/mg dry weight after 30 days starvation. However. if the lauer solids were composed of only 60% intact cells then the amount of protein per surviving intact cell is unchanged. This result is rather surprising, since a definitive reduction certainly occurs within the population and intuitively one would expect a cell under starvation to degrade some of its proteins to supply any energy that may be necessary for the survival process. Exactly how such results are best inter· preted is now a problem.
Breuil and Patel (135) have looked at the deple· tion of cellular contents in the anaerobic microbe Methanospirillum hungatei GPl during enforced starvation. Their results were effectively similar to those for the aerobic organism B. linens discussed above, namely that DNA appeared to increase in concentration whilst RNA was degraded, initially rapidly and then somewhat more slowly. ATP was also found to rapidly decline in the system. How-ever, all the results were also expressed on a per unit biomass basis and did not take into account the change in the physiological matrix of the sus-pended solids. A detailed analysis of cell numbers and particulate composition is required before.
JSS
during and after experiments where changes in the intracellular pools of starved cells are being in-vestigated. The foregoing description of the ap-proach to starvation experiments is common. Most of the reported results are expressed in terms of the amount of cell component per ml cell suspen-sion. Unfortunately the use of cell propagation methods is also extremely common in s1arvation experiments, thus reducing the confidence level in the interpretation of most published results.
In a subsequent section, it will be shown 1hat microorganisms possess enzymes capable of de-grading their own cell material. This will be limited to the class of enzymes known as autolysins, which function specifically on the cell walls. However, it has been recognised for some time that other enzymes are active within the cell, modifying in· ternal structures and ensuring substrate availabili-ty during times of exogenous nutrient deprivation. However. these enzymes can also function in catabolism of exogenous nutrients resulting from cellular lysis. For example, Parquet et al. (136) reported on the possible physiological function of the enzyme N·acetylmuramoyl-L-alanine amidase in £. coli K 12. This enzyme was found to be either loosely bound to peptidoglycan or en· trapped in the outer membrane-peptidoglycan complex, and to specifically cleave the bond be-tween N-acetylmuramic acid and alanine in the bacterial peptidoglycan. Paradoxically, such an enzymatic cleavage was found to be extremely rare in normal growing cultures. Hence, it was sug· gested that since the enzyme was active under autolytic conditions; it functioned as a hydrolaSt for growth on peptidoglycan fractions. Parquet et al. (136) .were able to demonstrate growth of £. coli on MurNAc-Lala fractions alone as well as on a MurNAc-lala·Xaa fraction suggesting, there-fore, the presence of other hydrolases. A vast array of enzymes specific for turnover of endog· enous macromolecules are present in microbial cells and under normal growth conditions they function as control mechanisms of the growth and metabolic processes. However, under conditions where exogenous substrate becomes limited. they serve lo provide the cell with .a continued sub· strate supply for energy generation in the absence of resynthesis.
-21-
Growth under a nutrient limitation other than carbon orten leads to the accumulation or an endogenous substrate such as glycogen (for review see Preiss [1371), or poly·P·hydroxy acid polymers. In addition, various intracellular compounds have been identified as substrates ror endogenous metabolism including free amino acids, RNA (138,1391 and proteins [140). The release of these compounds as a result of cell lysis provides the possibility for their uptake by other members of the same population as either carbon energy sub· strate or other nutrient sources and may allow the growth of auxotrophic organisms within mixed populations. This process is known as 'cryptic' growth. The term was first used by Ryan [141 J in a study of the turnover of cells during stationary phase batch growth. Koch [142) also recognised the possibility for cellular turnover during the growth process involving recycling of cellular components in a population. Postgate and Hunter [143) demonstrated 'cryptic' growth in a study on starvation of Aerobar:ter aerogenes ( Klebliella pneumoniae ). After prolonged incubation the capacity for cryptic growth was apparent (Fig. 9) although the authors used plate counts for the
e <fl
"' 0 > ;;: "' :::> "' g 4 "' g
30 20 40 60 80 100 120 140 HOURS
Fig. 9. Survival curve or A. 0,~rugeMJ (K. pMumMi•) showing •cryp1ie' growth. The microbes hanested from continuous cul· turc on glycerol at D • 0.2S h- 1 were washed twice by centri· rugadon. resuspended in distilled water. added to 'saline-Tris' bufler at 20 l'I dry weight organisnu/ml and allowed to starve at 40°C at pH 6.95±0.0S, under lorced aeration. Points up to 24 h by slid< cullure, including plate counlS at 0.7 and 24 h. subsequent points by plate counts only. Redrawn from (143] with permission.
~ 80 ~ ~ "'60 w o5 ~ 40 ..... z: ~ 20 "' &
0 50 INCUBATION TIME AT 37°C
Fig, 10. EUect of nutritiomd status on viabilities of ageing stational)' phase populations of A. o4'togenes (K. pntumoniPt). flasks shaken at J7°C in air in defined minimal media ror~ mulatcd to give b. glycerol~Umited; O. NH; .. \imited; and•. Mg2 ~limited scationary populations. Viability determination by slide cuhure, Redrawn from (144) with permission.
enumeration of starved bacteria. Recycling of potentially limiting nutrients such as Mg2 • or NH.;- (Fig. 10) is thus possible under starvation conditions [144).
During starvation conditions, cells have been shown to respond in a variety of ways including lhe use of their own storage products. Physiologi-cally, it is unreasonable for a cell lo undergo depletion of internal structure if the substrate flux for growth is sufficient. This point will be consid· ered further in the section on the mathematical descriptions of microbial growth and substrate utilisation.
Experiments designed to specifically investigate 'cryptic' growth phenomena are rare. Nioh and Furusaka (145) looked at the growth of bacteria on heat-killed suspensions of the same bacteria. They found that the ability to grow 'cryptically' was not universal and produced figures for cell yields based on the number of cells in the heat killed suspension required to produce unit •new' cells. Unfortunately the nature of the experiment was sufficiently damaging to prevent interpreta· lion of the results under natural conditions. Dur-ing the heat killing process where lysis occurred the heat was probably sufficient to break the
-22-
polymeric particulates up into easily assimilable monomers. However, the heat killing process does not necessarily lead to cell lysis so that most of the soluble material which was potentially available for incorporation into new cells was unavailable, being entrapped in heat-stabilised 'dead cells'. Once again, the enumeration method used was the bacterial plate count technique, and therefore, lit· tic reliance can be placed on the numbers ob· tained. Nioh and Furusaka [145) also found that addition of ammonium chloride led to a consider-able increase in the numbers of cells growing in the heat killed suspensions, suggesting that their suspensions were nitrogen limited, and therefore, the full potential of the 'cryptic' growth process could not be realised.
The growth of bacteria in their heat-killed sus· pensions results in lower biomass yield coeffi· cients than are to be expected from theoretical calculations. Hamer and Bryers [146) used the relationship between maximum biomass yield coefficient and the heat of combustion proposed by Linton and Stevenson [147] to predict a theo-retical maximum yield value of 0.66 g bacterial biomass per g substrate bacterial biomass. assum· ing complete conversion of the substrate carbon to either new cells or CO,. although these authors suggest deoptimization to a yield coefficient of about 0.33 would probably be encountered in real 'cryptic' growth situations. Mason et al. (148] have shown this estimate to be valid with reported yield coefficients around 0.4 (g cell carbon/g substrate carbon) in pure cultures of K p11eumaniae.
In this same study. Mason et al. [1481 grew K pneumoniae in continuous culture in a defined medium with glucose as growth limiting substrate. A volume of the culture was removed and used as the growth medium for the same cells after sonica-tion. centrifugation and sterile filtration. The re-sulting solution, comprised of soluble organics, was used as growth medium for the same cells. The cells for inoculation were derived from the same culture after steady slate conditions were reestablished. With this method there was no ap-parent lag phase before exponential growth oc-curred, suggesting that the microbes were already fully equipped with the necessary battery of en-zymes for growth on the complex nutrient source
0.06 0.05 o.o• 0.03
0.02
387
0.010~-~-~-~-~.-...... -~-...... -~
TIME JMuoJ Fig. 11. •Cryptic· growth curve ot K. pneumoniat NCIB418 growing on a mixture of carbon sourcts derived from cell sonicate preparation. K. pr1eumoniatt was grown in a chemostat at D • O.:S h- 1• The •cryptic' growth medium was prepared by sonicadon follo~d by sterile filtration ol the chcmostat cul§ ture. The inocuium (10 µl in ~ ml) was taken Crom the original culture a(ter steady state conditions had been rccstab,. tished. (From lt48J.)
(fig. 11). Deoptimisation of the maximum theo· retical yield coefficient to values of approx. 0.4 nevertheless represents highly efficient conversion, despite the very large range of potential carbon sources of varying complexity in the sonicated suspension.
15 jg A 14~1
BIOLOGICAL SOLIDS " . 400 13 ~} ~
12 ~ e 300
"' 3 200
100 coo
D m • D B m • MINUTES HOURS
Fig. 12. Metabolism of soluble cell components (prepared by subjecting sludge to sonication) by a.tended aeration activated sludge. The lell·hand graph shows the trend during the first 30 min. whilst lhe extended scale -on the right shows 1hc long~term results. Redrawn from (149) with permission.
-23-
388
A similar approach was adopted by Gaudy et al. (149) who used sonicated sludge as a nutrient source for 'cryptic' growth experiments. By follow-ing the COD reduction arter reinoculating with sludge organisms, it was found that approx. 50% or the COD was removed in the first 15 min, and 90% by the end or the experiment (Fig. 12). and is indicative or the greater potential ror 'cryptic' growth to occur under mixed culture conditions than in pure culture. Obviously a tremendous amount or work still needs to be carried out to assess correctly the impact of 'cryptic' growth in processes such as wastewater and waste sludge treatment and in other industrial processes.
4. AUTOL YSIS
The growth or bacteria requires the interaction of biosynthesis and degradation of various struct-ural polymers, notably cell wall polymers such as peptidoglycan, to allow volumetric expansion and cell division. The class of enzymes responsible for cell wall polymer hydrolysis have been termed autolysins and their action can be broadly divided into (a) constructive or voluntary and (b) destruc· tive or involuntary. Constructive properties are exhibited while the control mechanisms of the autolysins are still effective and include daughter cell separation {150, 15 I j. turnover and expansion of cell wall biopolymers (152-156), and morpho-logical differentiation (151,157). Should this tight control of autolysin activity break down, then the destructive roles of the autolysins are manifested by the rupture of cell walls, leading to loss of cell activity and eventually to total dissolution of cel· lular integrity. While several reviews have been published on the constructive (voluntary) roles of autolysins (158-160J, less auention has been focused on the destructive potential of this class of enzymes and the quantitative realisation of this potential. Some suggestion has been made that the destructive aspect of autolysins may be continu· ously manifested at low levels in populations grown under what are assumed to be ideal condi-tions (146,161,162).
The dual potential or the autolysins demands a highly sensitive and efficient control mechanism.
possibly one of the most demanding in the whole cell, since a single error can result in irreversible damage. A close relationship has been demon-strated between protein synthesis and autolysis (153,163-165) and inhibitors of autolysin activity [164) or activating substances (166-169] have been implicated in the control or autolytic enzymes. Evidence suggesting that cell wall turnover and autolysin activity are controlled by different genes was presented by Vitcovic [ 170) who found no correlation between the two processes.
The location of the autolysins is also unknown. Pooley (171.172) and Glaser and Lindsay [154} have suggested that wall turnover occurs on the outer cell surface, the autolytic enzymes being transported through the cell wall and activated in the region of the older peptidoglycan. New pepti· doglycan is laid down in the inner wall area and protected from degradation by its structural and spatial configuration [156). The matrix in which peptidoglycan is embedded is a highly organised three-dimensional structure and as such should precisely tune the activity of the autolysins. The presence of specific teichoic acids in this matrix have been shown to be necessary for the activity of some of these enzymes in Gram-positive micro-bes (150,173-175]. An alternative explanation was proposed by Joliffe et al. [166) who suggested that the surface chemistry of the cell membrane as determined by the magnitude of the proton motive force, has an inhibitory effect on the activity of the autolytic enzymes. As these move further away from the sphere of influence of the membrane their activity increases. They derived this hypothe-sis from the fact that the addition of agents which were capable of dissipating either electrical or pH gradients resulted in both inhibition of growth and the rapid lysis of exponentially growing cells. They also found that bacteria subjected to starva-tion conditions, such as those suspended in buffer solutions in the absence of an oxidisable carbon substrate, rapidly lyse, but lysis was immediately inhibited by re-energising the membrane by the addition of electron-donating agents.
Membrane de-energisation was also implicated as a possible cause of cell lysis following addition of medium chain fatty acids to cultures or Bacillus subtilis (176). Anomalies are also known where the
-24-
control mechanism over autolysin activity breaks down. Cultures of Myxococcus ccralloides D ex-hibit such behaviour whereby all the cells in a batch culture spontaneously lyse at the end of the exponential growth phase [177}. This behaviour appears to be due to the synthesis of an autolysin activating substance which builds up in the medium and on reaching a certain concentration activates the autolytic enzymes and triggers lysis [167).
Autolytic enzyme activity is not just limited to the bacteria. In fungi, autolysis of mycelia has also been investigated and the same precise mecha· nisms of control are apparent. In Botrytis cinerea, the activity of several different autolytic enzymes varied independently during the autolytic phase in the growth of the mycelium [178). Autolytic activ-ity has also been demonstrated in archaebacteria [179} and in yeasts [180). Microbial autolysis was the subject of a recent symposium [181 ]. It should not be forgotten that anomalies also exist espe-cially in the case of the Mycoplasmas. Since these organisms completely lack a cell wall, they do not succumb to autolysis in the same sense as cell-wall-bound microbes. However, lysis by cell mem-brane rupture in such organisms will lead to Joss of function.
5. MICROBIAL DEATH
Microbes can be killed by various means, al-though in order for a microbe to die it must either lose cellular integrity or incur irreversible damage to its genome. Agents by which one or both of these conditions arise include; chemical damage (including antibiotics), refrigeration, freezing, heating, and irradiation. Very often these results in only temporary damage 10 the microbe, if cellu· lar integrity is not lost or if irreversible damage to 1he genome does not occur. However, if sub-le1hal damage has occurred, it does not necessarily fol-low that the microbe will recover. II has been clearly shown that a damaged microbe is more susceptible to further injury than a healthy mi-crobe {182,183).
Unfortunately, in some instances, damage or injury have become synonymous with failure to
389
illicit growth on an agar surface [45,184). The accuracy and meaning of this interpretation is open to doubt. In some instances the test may indeed be indicative of some form of damage. However. the type and extent of damage varies considerably. Some forms or injury tend to make the microbes more susceptible to the stresses dur-ing either surface or submerged cultivation than others. An example of this was reported by Hurst [184) citing results from (,urran and Evans [185) where one medium used for enumerating the mi-crobes as a test for injury resulted in one set of results. but changing the medium composition re-sulted in a totally different picture. Similarly, in-jury has been described as being the inability of microorganisms to form colonies on a defined minimal medium, while retaining the colony for· ming capability when complex nutrients are pre-sent in the medium or vice versa (45). In experi-ments designed to inves1igate the effects of either physical or chemical maltreatment of microbes, very often no differentiation is made between injured and dead microbes.
There are various possibilities for inflicting in-jury on a microbial cell. The outer layer of a microbe ignoring the capsular material and cell appendages, is the cell wall/membrane complex. Gram-positive microbes possess a simpler wall structure than Gram-negative microbes. In the latter, a three-layered structure exists consisting of an innermost layer of peptidoglycan covalently linked lo lipoprotein molecules. The outermost layer is the outer membrane and is covalently linked to the peptidoglycan middle layer and con· sists of lipopolysaecharides (LPS), phospholipids, and proteins [186). The Gram-positive wall, in contrast, is much simpler and consists of pepli· doglycan chains embedded in a teichoic acid ma-trix. The cell wall serves as a barrier between the internal and external cell environments and con-fers s1ability on the cell. Due to the higher osmotic pressure inside the cell, any damage to the wall/ membrane structure can lead to rupture of this structure and loss of cellular integrity. Strictly speaking the cell does not die, but loss of microbes from a population results from either the disin· tegration or lysis of living individuals.
The cytoplasmic membrane is a metabolically
-25-
390
active, mobile, semipermeable structure which mediates transport between the cell and its en-vironment. Since the elucidation of the chemi-osmotic theory of energy generation, the impor-tance of the membrane and membrane potential have become apparent. Many of the enzyme-me<;li· ated reactions in a cell occur attached to the membrane, and the electron transport chain is an integrated part of the membrane structure. Either rupture or damage to the cell membrane will, therefore. result in injury (if the cell can recover) or death by eventual cessation of metabolic func-tions or by lysis.
Potential sites for damage within the cytoplasm also exist. The DNA, for instance, which encodes the information necessary for the correct func-tioning of a cell and the genetic information for replication is particularly vulnerable. The presence of lesions in !he DNA will lead to the production of nonsense proteins and enzymes and, thereby, to the loss of cellular function or to the inability to reproduce. The ribonucleic acids are also potential sites of damage. They are found as structural components in ribosomes, as well as functioning as messengers between the DNA and the ribo· somes and for transporting amino acids during protein synthesis. Therefore, damage to this com-ponent will also result in non-faithful reproduc-tion of enzymes and other proteins and lead to loss of cellular function.
In the following section a cursory glance will be directed towards the question of methods of in-ducing death in microbes.
Subjecting microbes to sub-lethal concenlra· tions of chemical inhibitors can result in the loss of their ability to either reproduce or grow under certain environmental conditions. Specific agar-based enumeration media used routinely to de-termine the extent of bactericidal activity are thus inappropriate, and may result in unacceptable health risks when used for testing for food- and water-borne palhogens. Chemical agents for kill-ing microbes arc used in water treatment, phar· maceutical therapy, disinfection and food in· duslries. Microbes arc susceptible to attack by chemical agents. In contrast to most physical stresses, chemical agents often interact directly with either the metabolism or the physical struc·
ture of the microbe, and as such, chemical damage occurs at the molecular level. The effects of many chemical agents have still to be elucidated.
TI1e cell wall is the target of many antibiotics, but compounds like EDT A, lysozyme, phenol, and chlorine are also known lo act at the outer boundary of the cell (187) together with various surfactants such as Triton X-100 [188J. Similarly, 1he cell membrane succumbs easily to attack by agents capable of disorganizing the structure, or changing specific activities towards ions, or by inhibiting the membrane-bound proteins involved in transport processes such as by the action of some antibiotics !189), or at superop1imal temper-atures or with strong oxidising agents. The mem-brane is also susceptable to attack by agents capa-ble of uncoupling oxidative phosphorylation reac-tions. Organic solvents such as butanol [190), ethanol !l 91), alkali metal ionophores [192) and detergents [193), have their site of action at the cell membrane.
Cytoplasmic targets for chemicals include cyto· plasmic eniymes, nucleic acids and the ribosomes. Coagulation of the cytoplasm can occur at high drug concentrations [194). A range of different antibiotics affect the biosynthesis and functioning of the nucleic acids. The ribosomes tend to be susceptible to agents which cause instability through removal of the Mg i + necessary for the association of the 30S and SOS subunits (19S,196J.
Often ignored under the heading of chemical injury is the effect of aerobiosis and anaerobiosis in obligate aerobes and obligate anaerobes. The sensitivity of anaerobes exposed to oxygen varies with different strains [197). The time required to deactivate a cell appears 10 be relatively long. Shoesmith and Warsly (198) in a review on the exposure of anaerobes to oxygen, summarised the oxygen tolerance data derived from other authors using various strains and found that despite little agreement in assessment methods, most of the strains were still alive 1 h after exposure.
During refrigeration death occurs as a result of a number of different effects not least of which is the rate of cooling and subsequent thawing processes. Thus, experiments designed to investi· gate the effects of refrigeration on microbes must be very carefully controlled (199). Two types of
-26-
refrigeration are common, namely chilling and freezing. These practices arc commonly used in the preservation of microbial cultures in technical processes, but are equally likely to occur under the innuence of seasonal changes in natural environ· men ts. The effects of refrigeration are also im· portant in the food industry where this practice constitutes a major form of preservation, whereby the destruction and inhibition of microbial growth and activity are desired.
Death often results as a consequence of the loss of control of cell permeability-a phenomenon known as cold shock [199j. One theory for the ensuing death after chilling is the loss of activity of the enzyme DNA ligase as a resull of the depletion of magnesium ions from the cell (200]. At temperatures below 0°C cell damage can occur as a result of either extracellular or intracellular ice crystal formation, depending on the cooling rate [201] and from changes in the ice-crystal structure under different physical (i.e., pressure or temperature) conditions [203]. Death during ex-tended frozen storage occurs at a fast initial rate but slows down with time until a stage is reached where constant numbers remain 'viable' (204). Death is thought to occur as a result of exposure to the raised solute concentration as a conse· quencc of ice crystal formation, as well as from physical damage.
Without knowing the physiological reasons for its effectiveness, heat treatment has been used for over 100 years as a means of either destroying or limiting the growth of microbes. Temperature af-fects different microbes in different ways. The growth of a particular microbe normally takes place within a narrow range of temperatures. It has been suggested that heat principally affects the DNA by inducing the action of exo- and endonucleases resulting in multi-strand breaks in the DNA [204,205). In E. coli, the effect of heat has been suggested to result in the denaturation of the cell wall leaving the peptidoglycan layer weakened at one or two points after which the plasma membrane ruptures [206), Some associa-tion of proteins with the nucleoids also occurs as a result of mild heat treatment (207].
Radiation results in a range of alterations in the DNA, including phosphodiester strand breaks,
)91
nucleic acid-protein cross links, pyrimidine dimer formation. etc. [208).
For detailed information on the mechanisms of destruction of microbes. the reader is recom-mended to consult specialised texts (209-211 ).
6. MATHEMATICAL MODELLING OF DEATH AND LYSIS
Models in general can be physical structures, verbal descriptions, or sets of mathematical equa· tions that aid the user in comprehending complex phenomena. In some instances, a model serves as a hypothesis, other models attempt to predict be. haviour and still others aid in understanding. No model will reflect all aspects of •reality'. Neverthe-less, all models should force the investigator to think in a concise manner and to consolidate what information is known plus what new information is required.
Mathematical modelling or microbial systems has been used to help understand the dynamics of microbial behaviour in laboratory, natural, in· dustrial and man-made environments. Application of such models to scale-up laboratory operations into industrial processes and for extrapolation of laboratory behaviour to natural environments has resulted from the construction of such system modelling. Mathematical models also serve to di-rect and to optimise the research experimentation and to allow prediction of microbial behaviour. Biological systems are extremely complex and the actual system has frequently to be replaced by an imaginary model system which is mathematically tractable [212). As a result, a very large number of models can be proposed for any single system each depending on the assumptions made for sim· plifying the real biological case. Many theoretical dynamic studies have been based on models for-mulated from steady state or equilibrium kinetics and thus inadequately describe dynamic be· haviour [213).
The modelling of death and lysis processes in living cultures requires a broad understanding of the physiology of the organisms concerned. The traditional black box approach, whilst describing the behaviour of a population under a given set or
392
constant conditions, might be totally inadequate when those conditions change. Calibration or the mathematical model with carerul experimentation is also necessary.
One or the earliest mathematical considerations or bacterial growth was presented by Buchanan (214), who presented equations describing the lag, logarithmic, stationary, accelerated death and logarithmic death phases during the batch cycle. Buchanan suggested that cell death begins during the late exponential phase and increases to a level in the stationary phase where it exactly balances the rate or growth or the bacteria, and finally exceeds the rate or growth such that a net decrease in the number or live bacteria present results. He described each of the individual phases using mathematical equations.
The first application of continuous culture growth expressions appeared in 1950 (215] and these were latter assessed by using quantitative experimentation (216]. By 1958 the phenomenon of a decreasing yield coefficient at low growth rates was recognised and this biomass-reducing effect was termed 'endogenous metabolism' (217). Herbert [217) described the constant loss of bio-mass at all growth rates using Eqn. 7.
dx/dt - (,1.1-k.)x (7)
where k, is the endogenous metabolism constant. Powell [218) derived an expression for the steady
state biomass concentration using this notion of endogenous decay
"'"'( s0 D K,D ] x • y•/• D + k - -;;--::::o e rrnax
(8)
A large amount of work during the 1950s and 1960s was concerned with the destruction or mi-crobes, particularly in hostile environments as en-countered in sterilisation and pasteurisation processes. This work arose primarily due to strin-gent hygiene requirements for food for public consumption. A distinct paucity of information exists concerning death and lysis of micro-organisms under conditions where microbes are not exposed suddenly to hostile environments. Without any apparent stress, death must occur by processes such as those of involuntary autolysin activity, as described earlier. In continuous culture
-27-
systems at low growth rates it has long been recognised that a large proportion of tbe microbes is •non-viable' or 'dead' [143,129]. Tempest et al. [219) found that the viability or A. t1eroge11es ( K. pneumoniae) was as low as 40% at a dilution rate or 0.004 h - '. but increased to 90% above 0.5 h - '. Although this data represented evidence or death within a continuous culture, note that the cell enumeration method used was the slide cultivation technique or Postgate et al. [220]. The possibility that such slow-growing organisms (at D • 0.004 h-'. the doubling time= 173 h or 7.2 days) fail to adapt to the agar environment and form colonies does exist and limits extrapolation or these results.
Sinclair and Topiwala [221] presented one or the first models for growth in continuous culture which considers the viability concept and uses the results or Tempest et al. (219) and Postgate and Hunter (143] to verify their model. Two mecha-nisms were assumed to act on the living biomass, x: (1) cell death, which was assumed proportional to the living biomass; and (2) endogenous metabolism, also considered proportional to living biomass. Thus, Sinclair and Topiwala present the mass balance equation for biomass as: dx/dt ~ µ(s)x - k. - yx - Dx (9)
Where y is the specific cell death rate constant (1- 1) and D is the dilution rate (r'). Similarly the mass balance for s appears as:
µ(s)x ds/dt • D(s0 - s) - Y"'"" (10)
•/• where s0 is the influent substrate concentration, s the residual substrate concentration and Y,;:" the yield that would be attained in the absence of decay and cell death. Thus, the steady-state concentration of dead cells (xd) can be written as xd - ( y/D)x (11) and for steady-state soluble substrate, (s),
_ K,(D+K+y) s• l'in-(D+K+y) (12)
The viability can be calculated using Eqn. l3
. b'I' ( ) x x D Vla 11ty P =---=-=--(x+id) x, D+y
(13)
-28-
The specific death rate constant can be de-termined, rearranging Eqn. 13,
l/v=l+y/D (14)
and plotting the inverse of viability versus inverse dilution rate, the slope would equal y.
Using these equations, Sinclair and Topiwala [221 J satisfactorily predicted a decrease in biomass at low dilution rates with concomitant low viabil· ity.
Weddle and Jenkins [222] analysed pilot· and full-scale activated sludge systems for cell viability and activity using various techniques including ATP, dissolved oxygen uptake, cell counts (using enrichment on agar} and electron transport system activity using the compound 2,3,5-triphenyltetra-zolium chloride. They suggested the existence of non-viable organisms and of non-degradable inert biomass fractions. Grady and Roper [223] derived a model for the activated sludge process which predicted cell viability as a function of the mean residence time by incorporating the processes of death, maintenance (endogenous decay) and loss of viability in the absence of death. These authors
393
recognised the existence of those cells which while not dead, were incapable of replication. They fur-ther suggested that these cells could have func· tional enzyme systems and were therefore suscep· table to death and decay by endogenous metabo· lism and lysis. They also commented on the in· fluence of 'cryptic' growth on the system but chose not to model it due a lack of experimental capabilities required for verification. The contri· bution of non-viable cells to the metabolic activity or the system was discussed by Jones (224), who divided biochemical activity in a heterogenous plug now industrial process into the following categories, each group having its own appropriate growth kinetics: (i) Bacteria which grow within the system (Monod kinetics); (ii) bacteria which do not divide within the system but are viable (main-tenance kinetics); (iii) Non-viable cells which still retain some metabolic (biochemical} activity (Mi-chaelis-Menten kinetics). The growth or group (i) microbes maintains suitable concentrations of groups (ii) and (iii) in the system, such that sub· strate removal approaches first order kinetics [223).
The effect of non-viable, metabolically active
-29-
394
cells on the yield coerricient was also shown by van Uden and Madeira-Lopes [6) in !heir model of yeast growth in chemostats at superoptimal tern· peratures.
7. CONCLUDING REMARKS
Despite more than a century of microbiological research, emphasis still tends to be placed on the growth of microbes, and little, if any, attention is focused on their demise. In natural and man-made (including laboratory) environments, microbial 'death' or decline is a process which occurs hand· in-hand with microbial growth. Microbes are neither immortal. nor are they any less accident-prone than other creatures. Since they are not the highly efficient machines they are frequently made out to be, it is time that 1he effect of their frailness be properly considered in process design and evaluation, in public health investigations and in eco·physiological research. In order to salisfacto· rily achieve this, it requires a beuer understanding of the methods available, their limitations and the extent to which deductions can be made from the results they generate.
ACKNOWLEDGEMENTS
C.A.M. was supported by grants from the Swiss Federal Institute for Water Resources and Water Pollution Control (EAWAG) and from the Swiss National Programme 7D.
Within the last 10 years, more accurate meth· ods have evolved to determine the various types or microbe that can exist in a culture. Mason et al. [162) depicted the processes of microbial growth and death in continuous culture, as shown in Fig. 13. They reccgnised the possibility for the pres-ence of each or the various categories or cells and their interrelationship and a11emptted to experi· mentally differentiate between them. Differentia-tion between metabolically active and non-active (dead) cells was possible based on ETS activity. Their results suggested that a very low fraction, if any, of the cells present were indeed dead which appears to disagree with much of the earlier work suggesting the contrary. However, it should be noted, that much or that earlier data were derived using plate count methods which were totally in· adequate for quantitative assessment. The model proposed by Mason et al. [162} suggests that the reduction in biomass yield results from a low level or lysis in the culture and in the model this lysis process and the subsequent growth of the intact organisms on their own lysis products ('cryptic' growth) are represented. Consequently this model may be suited to describe more accurately the behaviour of organisms in natural environments where cell decline may result from cell lysis and actual death occurs rarely, if at all. Hamer and Bryers [146) and Hamer [225} using the same principles, incorporated lysis and 'cryptic' growth in models which satisfactorily describe the effects of these processes in wastewater and waste sludge treatment processes.
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(198) Shoesmith, J.O. and Warsley, B. (1984) Anaerobes and exposure to oxygen, in The Revival of Injured Microbes (Andrew, M.H.E. and Russell, A.D .. Eds.) Soc. Appl. Bacttriol. Symp. Ser. 12, pp. 127-146. Academic Press, London.
{199) Macl.eod. R.A. and Calcou, P.H. (1976) Cold shock and freezing damage to microbes, in The survival of Vegeta· tive Microbes (Gray, T.R.G. and Postgate, J.R .. Eds.) Proc. Symp. Soc. Gen. Microbiol. 26. pp. 81-109. Cam· bridge Unive,.ity Press. Cambridge.
(200) Sato, M. and Takahashi, H. (1970) Cold shock or bacteria, 1\1. Involvement or DNA·ligasc reaction in recovery of EsdterU:hla <oli from cold shock. J. Gen. Appl. Micro· biol. u. 217-229.
(201) Edebo. L. and Magnusson, K.·E. (lll7J) Disintegration of cells and protein recovery. Puro Appl. Chem. 36, J2S-338.
(202) Maxur, P. (1966) Physical and chemical basis for injury ln single celled microorganisms subjected to frec:zing and thawing. in Cryobiology (Meryman, H.T., Ed.) pp. 213-JIS. Academic Pres .. London.
(2031 Mackey, P. (1984) Lethal and &ublethal effects or ,.. frigeration. free:r.ing and frcez.c drying on micr<r organisms. in The Revival of lnjured Microbes {Andrew, M.H.E. and Russell, A.O., Eds.) Soc. Appl. Bacteriol. Symp. Ser. 12. pp. 45- 75. Academic Pras. London.
1204) Sedgwick, S.O. and Bridges, B.A. (1912) Evidence for
indirect production of DNA strand scissions during mild heating of Escherichia coli. J. G~n. Microbiol. 71. 191-193.
(20S) Pellon, J.R .. Ulmer. K.M. and Gomez. R.F. (1980) Heat damage to the folded chromosome o( Escherichia coli Kil Appl. Environ. Microbial. 40, 3S8-364.
(2061 Scheie, P. and Ehrensp<ck, S. (1973) Larsc surface blebs on Esc:heridtia c:oll heated to inactivating temperatures. J. Bac1criol. 114, 814-818.
(207] Pellon, J.R. and Gomez. R.F. (1911) Repair of thermal damage to the Eschen'chia toli nucleoid. J. Bacteriol. 145, 1456-1458.
(208] Moseley. B.E.B. (1984) Radiation damage and its repair in non~sporu1ating bacteria. in The Revival of Injured Microbes (Andrew, M.H.E.. and Russell. A.O .. Eds.) Soc. Appl. Bacterial. Symp. Ser. 12, pp. 147-174. Academic Press. London.
f209l Hugo. W.B. (Ed.) (1971) Inhibition and destruction of the microbial cell. Academic Pre5s, London.
1210) Gray, T.R.G. and Postgate, J.R. (Eds.) (1976) The Sutvivat of Vegetative Microbes. Proc. Symp. Soc. Gen Microbiol. 26. Cambridge University Prw. Cambridge.
121!] Andrew. M.H.E. and Rusacll, A.O. (EdJ.) (1984) The Revival of 1'1iurcd Microbe•. Soc. Appl. Bacterial. Symp. Ser. 12. Academic Press, London.
1212] Topiwala. H.H. (1973) Mathematical models in microbi-ology. in Methods In Microbiolol)I (Norris, J.R. and Ribbon., D.W .. (Eds.) Vol. 8, pp. 35-59. Academic Press, London.
(213] Harrison, D.E.F. and Topiwala. H.H. (1974) Transient and oscillatory states or continuous culture. Adv. Bio.. chem. Eng. 3. 167-219.
12141 Buchanan. R.E. (1918) Lile phases in a bacterial culture. J. Infect. Dis. 23, 109-125.
(21Sf Monod, J. (1950) la technique de culture continue. thCorie ct applications. Ann. Inst. Pasteur 79. 390-410.
(216] Herbert. 0., Elsworth, R. and Telling. R.C. (1956) The
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401
rontinuous culture of bac1cri•. a theoretical and expcri .. mental study. J, Gen. Microbial. 14, 601-622.
1217] Herbert. 0. (1958) Some principles of continuous cul· ture, in Recent Progress in Mic:robio1ogy (Tuncvall. G., Ed.) 7th Int. Congr. Microbial., pp. 381-396. Blackwell, Oxford.
[2181 Powell, E.O. (1967) The growth rate of micJ'OO!!lnisms IS a function or substrate concentration, in Microbial Physiolog)I and Continuous Culture (Powell, E.O., Evans, C.G.T., Strange, R.E. and Tempest, D.W., Eds.) Proc. 3rd Int. Symp., pp. 34-56. HMSO, London.
12191 Tempest. D.W .. Herbert, D. and Phipps, PJ. (1967) Studies on the growth of Atr0b«1er OefoSeMl •• low dilution rates in a chemostat, in Microbial Physiology and Continuous Culture (Powell, E..O., Evans, C.G.T., Strange, R.E. and Tempe5~ D.W., Eds.) Proc. 3rd Int. Symp. pp. 240-2S3. HMSO, London.
[2201 Postgate, J.R., Crumpton, J.E. and Hunter. J.R. (1961) The measurement of bacterial cultures by slide culture. J. Gen. Microbial. 24, I S-24.
12211 Sinclair. C.G. and Topiwala, ff.ff. (1970) Model for continuous culture which considcn the viability concept. Biotechnol. Bioeng. 12, 1069-1079.
1222] Weddle, C.l. and Jenkins, D. (1971) Th• 10ability and activity of activated &Judge. Water Res. S, 621-640.
(223] Grady Jr_ C.P.l. and Roper Jr., R.E. (1974) A model for the biooxidation process which incorporates the viability concept. Water Res. 8, 471-483.
(224) Jones. Q.L. (1973) Bacterial growth kinetics; mca&ure· mcnt and significance in the activated stvdp process. Water Res. 7. 1475-1492.
f225J Hamer. G. (1985) lysis and 'cl')lptic' growth in waste· water and sludge treatment processes. Acta Biotechnot 5, 117-127.
12261 Oujer, W. (1980) The effect of particulate organic llijlterial on activated sludge yield and oxygen require· ment. Proc. Water Technol. 12. 79-95.
(Received April 12, 1985;;,, fi11a/ form June 17, 19115)
The significance of death and lysis processes during microbial growth in chemostat cultures is discussed. A structured model is developed to describe such processes and 1he model is partially verified for carbon limited chemostat eul1ures of Klcbsiella p11eumo11iac. It seems probable that death and lysis arc coincident, when non-viable cells are not considered 10 he dead. KEYWORDS Growth Death Lysis Chemoslal Microbe
INTRODUCTION
Viability is a microbiological term which is rarely correctly interpreted. The main reason is that any definition is system specific and subject to the limitations of the methods used. Such operational definitions frequently lead to misinterpretation once they are extrapolated outside of the regime of their applicability. Recently, however, the consequences of inadequate quantification of viable and dead microorganisms have been realised in terms of public health hazards and in the inability to optimize processes orientated towards production of microbial cells and their products. Thus, there is a need to establish functional definitions and accurate methods to describe the physiological status of microorganisms and thereby contribute more meaningful information towards design criteria for industrial processes.
Nearly a quarter of a century ago Humphrey and Nickerson (1961) remarked that in spite of almost sixty years of intensive research, little understanding of the exact nature of bacterial death had been gained. This comment was in reference to sterilization operations' where the work published by Humphrey and his co-workers during the 1960's did much to elucidate the kinetics and mechanisms of death at elevated temperatures. However, the nature of bacterial death at temperatures either coincident with or close to the optimum for growth still requires resolution and one objective of this communication is to contribute to such resolution.
t Present address: Dept. of Civil und Environmental Engineering, Duke University, Durham, NC 27706, USA
163 -
-38-
164 C.A. MASON, J.D. BRYERS AND G. HAMER Hinshelwood (1951) discussed bacterial death, viability and lysis in terms of
their physiological basis in the cell. He proposed a simple model in which he derived equations similar to Volterra's, based on fluctuations in lysis and synthesis within the cell. To test these equations a vast range of methods for viability assessment have been developed and are well documented (Postgate 1967, Costerton & Colwell, 1979).
Several studies have shown the presence of non-viable microbes (e.g. Tempest et al., 1967, van Uden and Maderia-Lopes, 1976), and several existing models have incorporated the concepts of viability and death into traditional continuous culture theory. For example, Sinclair and Topiwala (1970) modelled the natural death of bacteria in steady state continuous culture systems, using the ratio of viable cell mass to total bacterial cell mass as an index of viability.
Their model considered two mechanisms by which viable cell mass could be lost; namely death and endogenous metabolism. Grady and Roper (1974) presented a model of the bio-oxidation process which incorporates a third physiological type of cell, in addition to the viable and dead, which they designated as cells which had lost the ability to divide. Of the processes resulting in the loss of various cell types from their system, endogenous metabolism, autolysis, lysis by other organisms and predation, only the first two processes were considered relevant to pure culture studies and these were grouped into a single term, decay.
Since lysis and endogenous metabolism effectively represent growth on the cells own constituents these can be separated from the lumped parameters of decay and considered as individual processes.
MODEL DEVELOPMENT
Bacteria, growing in continuous culture, are assumed for purposes of this study, to participate in the processes depicted in Figure 1. Based on this rationale, biomass dry weight, the common indicator of bacterial concentration (and activity), is replaced by a more "structured" description which necessitates the following groupings of cellular material within a culture:
Viable bacteria (Xv) are aerobic cells that consume dissolved organic compounds as carbon and energy substrates. Viable cells respire and replicate at a growth rate, µ, which is a function of dissolved carbon substrate concentration.
Non-viable, respiring bacteria (X • .,) are those cells that do not replicate but still moderate (perhaps enzymatically) substrate oxidation and are thus actively respiring. x ... cells arise only by inactivation of viable cells, X, ..
Dead bacteria (Xd) are those cells that are no longer able to respire and replicate. )(1 cells arise from both the "death" of viable and non-viable, respiring cells.
Biopolymeric particulates (P) are colloidal fragments of cellular origin .that arise due to the lysis of viable, non-viable and intact dead cells. Combined X,,, Xm,, X,1 and P would analytically comprise the total biomass concentration, X, in a
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MICROBIAL GROWTH 165
soluble and particulate debris FIGURE 1 Fundamental processes considered to oceur in a chemostat.
culture. A more precise distinction between these biological particulates, based upon specific analytical methods, is detailed subsequently.
Mathematical Derivation
Accumulation of compounds in the bulk liquid of a completely mixed reactor can be described by mass balances of the general form:
net rate of = net rate of + net rate of accumulation transport transformation (1)
Material balances are organized here in matrix notation as described by Roels (1980).
The seven components of interest in this study are primary substrate con-centration, S., dissolved organic carbon compounds released during lysis, S2 ,
oxygen, 0 2 , viable cells, x;,, non-viable respiring cells, X.,.,. dead cells, Xd, and biopolymer particulates, P. The component vector, a, is defined as:
S1 S2 02
a= x., (2) xntl X,i p
Thus the left hand side of Eq. (I) is daldt.
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166 C.A. MASON, J.D. BRYERS AND G. HAMER Jn a chemostat, receiving a sterile feed of substrate SV and being aerated by
sparging with air, the transport vector a• becomes:
a*=
D(S~- S1)
-DS2 K1.,..(0i - 02) - D02
-DXv -DXnu -DXd -DP
(3)
where D is the dilution rate, K1• the overall liquid side oxygen transfer coefficient and a, the specific gas-liquid interfacial area.
Processes in Figure 1 can be translated into the irreversible reactions, written in the unit mass based stoichiometry outlined by Irvine and Bryers (1985) as shown in Table I. Each reaction is normalized with respect to the mass of the individual
TABLE I
Stoichiometric reactions for growth, death, and lysis processes in a chemostat
Y;,1 the stoichiometric cocflicicnt for the jth component in the ith reaction and is a unit mass stoichiometric value having been normalized by the limiting reactant. Units of Yi.; arc (mass of component j per mass of limiting reactant in reaction I). Consequently, Yi.1 values for the limiting reactant arc (-1). Note: rcactnnts arc assigned negative coefficients and products are positive.
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MICROBIAL GROWTH 167
reactions limiting reactant. Reaction rates, r;, where i = I- I I, arc also in terms of the limiting reactant of the specific reaction. Stoichiometric coellicients in Tahle I are ratios between the various products formed or reactants consumed and the reactions rate limiting reactant. Thus these ratios are fundamentally characteristic for the experimental system selected. Observed "yields", which represent ratios between product and total reactant consumed, do not have the fundamental significance of the intrinsic stoichiometry and the two should not be confused. Intrinsic stoichiometry is inherent to the processes involved, whilst observed yields are artifacts of the reactor system and its operating conditions.
Reaction rate expressions for the eleven reactions in Table I are listed in Table II. Growth of viable cells, X.,. is presumed to be the sum of two Monod-type rate expressions; one based upon primary substrate, St> and the other based upon dissolved organics produced via lysis and hydrolysis, S2 • For lack of information and simplicity, all other rate expressions are presumed to be first order in their limiting reactant.
For simplicity, at this stage of model development oxygen consumption coefficients for the death and inactivation processes (Yu, Y... 3 and }".,, 3 ) (Eqns. 6-8) are assumed to be zero such that }".,, 5, Y4•6 and l';,6 are thus equivalent to unity, although in reality this is unlikely to be the case.
Combining stoichiometry and kinetics in a transformation vector, a*, results in: -r1 -r10
Rate expressions for processes illustrated in Figure l
De.scription
Growth of viable cells on S1 Growth of viable cells on S2 Inactivation of viable cells Death of viable cells Death of non-viable cells Lysis of non-viable cells Lysis of dead cells Lysis of viable cells Hydrolysis of particulates Enzymatic conversion of S 1 by X.,., Enzymatic oxidation of S2 by X.,,,
µ: .. o is the appropriate maximum specific growth rate constant, K,w., the appropriate saturation constant. K .. m1,, the appropriate first.order rate constant~ V :uh• the appropriate maximum reaction rate and KM., .. the appropriate Michaelis-Mcntcn constant.
Parameter
s, s, o, x.. X.., xd p
TABLE !II
Material balances for a chemostat incorporating concepts of growth, death, lysis, and paniculate hydrolysis
Complete material balances for the seven components are listed in Table III. Unsteady state solution of the material balances required numerical integration.
EXPERIMENT AL
Organism Klebsiella pneumoniae NCIB 418, maintained by monthly subculture on plate count agar slopes.
Growth conditions Bacteria were grown in a 2.51 fermenter (MBR Bioreactor AG, Wetzikon, CH) on a simple salts medium (Evans et al., 1970) modified by substituting EDT A (100mg1- 1) for citrate as chelating agent. Polypropylene glycol (20mg1- 1) was also added as an antifoaming agent. Glucose (4.8g1- 1) was the sole carbon energy substrate and limited growth. Pure oxygen was mixed with influent air to maintain constant dissolved oxygen tension over the entire range of residence times investigated. The pH of the culture was automatically maintained at 6.80 by controlled addition of a mixture of 1.5 M NaOH and 1.5 M KOH. The temperature was maintained constant at 35"C and the inlet nutrient flow rate was measured by weight displacement.
Analyses
Total Biomass concentration was measured as dry weight (DW) by a filtration/ gravimetric procedure using tared 0.4 µm Nuclepore filters. Filters were dried at 105"C for 1 h.
Glucose was assayed enzymatically using the GOD-PeridR (Boehringer Mann-heim GmbH, D) according to the procedure described by Egli et al. (1983).
Dissolved Organic Carbon (S2) was measured in cell filtrates using a Tocor 2 (Maihak GmbH, Hamburg, D) dissolved organic carbon analyser.
Cell activity Experimental differentiation between living and dead cells was carried out using the INT-reduction assay of Zimmermann et al. (1978). Cells with active dehydrogenase enzymes (i.e., actively respiring cells) reduce the compound 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyltetrazolium chloride (INT) to an insoluble opaque formazan crystal, which is internally deposited in the cell. Cells were diluted using 0.02 M·phosphate-0.11 M saline buffer (pH 6.80). INT was added to the final dilution tube together with glucose (0.25g1- 1,
in 1 ml Evans mineral solution), which served as an electron source. Total cell counts were determined using an acridine orange stain, by counting green and orange fluorescing cells with a Zeiss SM-lux epifluoroescence microscope. Transmitted light was used for enumerating INT-formazan containing cells.
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170 C.A. MASON, J.D. BRYERS AND G. HAMER
Non-respiring cells are determined by difference between the total epi- and INT-cell counts.
RESULTS
Figures 2 to 5 show the results of both the computer predictions and the experiments. The predictions are represented by the Jines and the experimental data as points. The kinetic constants used for the model predictions are listed in Table IV. It should be noted that total biomass comprises viable, dead and non-viable cells and biopolymeric particulates released from cell lysis. The predicted relative proportions of each to the total biomass is shown in Figure 3. The prediction for active cells (Fig. 4) is calculated from the proportion of viable and non-viable respiring cell mass to the total cell biomass. Total biomass agrees well with experimental results as does cell activity. However, no experimental data were available for comparison of particulate lysis prOduct concentrations. Residual glucose concentrations were below the detection limit (2mg1-1
) of the method used. The curves were not extended to include washout because of the interplay of additional factors, i.e., wall growth (Bryers, 1984) at such dilution rates.
FIGURE 2 Steady state total biomass conccn1ra1ioo as a function of dilution rate.
3.0
2.5
{:' 2.0
"' 1.5
ll) <.()
< :::E 1.0 0 OJ
0.5
-45-
MICROBIAL GROWTH
/ /
I I
I I i i I
0.2
..... //
-·-·--·- -·-·-
VIABLE CELLS NON-VIABLE RESPIRING CELLS DEAD CELLS POLYMERIC PARTICULATES TOTAL BIOMASS
0.4 0.6 0.8 DILUTION RATE (h"1J
1.0
171
1.2
FIGURE 3 Predicted concentrations of viable cells, non-viable respiring cells, dead cells, polymeric particulates and total biomas.' as a function of dilution rate.
100 oo 0 -0.- 0 0 0 0
80 0 EXPERIMENT
-- PREDICTION
>- 60 I-> I-u < 40 I-z: w (.) a: w 20 a.
0 0 0.2 0.4 0.6 0.8 1.0 1.2
DILUTION RATE (h"') FIGURE 4 Percent active Klebsiella pneum,miae in steady state continuous culture as a function of dilution rate.
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172 C.A. MASON, J.D. BRYERS AND G. HAMER
JOO-.------------------. u 0: ~-250 '.:::i-0!... a. "" gj .!:. 200 z 0 CD 0:: 6 150
0
PREDICTED POLYMERIC ------- PARTICULATES
--- PREDICTED } DISSOLVED o EXPERIMENTAL g~~~~~
0
0.2 0.4 0.6 0.8 1.0 DILUTION RATE W1J
1.2
FIGURE 5 Steady state concentrations of dissolved organic carbon (S,) and polymeric particulate producrn (P) as a function o( dilution rate.
DISCUSSION
Endogenous metabolism has been considered to be a significant factor affecting the microbial biomass concentration and hence, the overall biomass yield coefficient, particularly in continuous culture systems operating at low growth rates. Traditionally, microbiologists have explained endogenous metabolism on the basis of the maintenance functions of microbes, i.e., turnover of cell materials, osmotic work to maintain concentration gradients between the cell and its exterior and cell mobility. Strictly, endogenous metabolism embraces several additional phenomena including death, lysis and "cryptic growth" (Hamer, 1985).
Very few microbiological studies have sought to differentiate between tradi-tional maintenance energy requirements and energy dissipation by the other phenomena. All of the contributory components of endogenous metabolism impact on the biomass yield coefficient. Biomass yield coefficients depend on the nature of the limiting substrate and on environmental conditions and hence, are not constant. The maximum theoretical yield coefficient for aerobic growth on glucose lies between 0.69 and 0.73 (Stouthamer, 1979) depending on the number of phosphorylation sites. Relatively few experimental studies with K. pneumoniae indicate that such a biomass yield coefficient is obtainable. Paca and Gregr (1977) report achieving biomass yield coefficients of 0.65 at low growth rates, but most
-47-
MICROBIAL GROWTH 173
TABLE IV
Kinetic constants used for computer prediction
Parameter Value Units
Y,,. 0.597 Y,,4 0.25 µj l.22 (h-') K,, 8 <msr'> µ~ 0.05 (h' ) K,, 200 (mgl-1) k, O.OJ (h-') k. 0.04 (h-') k, 0.04 (h-') ko 0.05 (h-') k7 0.8 (h-') k. 0.05 (h-') k. 0.1 (h-') vro 0.J (h-') KM, 8 (mgl- 1)
vr1 0.05 (h-') K.,, 200 (mg 1- 1)
Y10.s 0.1 Y11.s 0.25 Y•.z 0.65 Y •. 1 0.35 Y,,, 0.65 y7,7 0.35 Y,,, 0.65 Y.,, 0.35 Y•.2 I S'i 4800 mg1-•
of the authors report values between 0.43 and 0.54 (Sinclair and Topiwala, 1970, Topiwala and Sinclair, 1971, Harrison and Loveless, 1971, Neijssel and Tempest, 1975). Many reponed experimental biomass yield coefficients fail to consider endogenous metabolism which results in significant reductions in observed, but not necessarily true yield.
Because of endogenous activities, the observed biomass yield coefficients will vary with dilution rate. In view of the fact that the endogenous metabolism concept incorporates other physiological processes, it is proposed that a major contribution results from death and lysis and it is hypothesized that the true maintenance requirement, i.e., utilization of cellular material for energy, is small relative to cell breakdown and death.
Hinshelwood's (1951) theory of cell ageing and death includes the hypothesis that growth involves balanced lysis and synthesis of major structural polymers which results in a net removal from the cell. Sometimes, lysis of the whole cell results when the control over this process is lost. Either way, particulate and/or soluble organic matter is released by the cell into the culture medium.
Whether the products of intracellular lysis remain in the cell to be further hydrolysed and reused (yielding energy or being reincorporated) or whether they
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174 C.A. MASON, J.D. BRYERS AND G. HAMER
are extruded from the cell is not known. However, since new cell wall material is laid down on its inner surface after transport through the cell membrane, it is hypothesized that the older material is lost entirely from the cell.
Whole cell lysis releases products including hydrolyzing enzymes. Conse-quently, much of the polymeric fraction is further broken down into low molecular weight substances which are more readily utilised by the cell. In monocultures this will be a minor activity, but takes on major importance in mixed cultures (Hamer, 1985). The model presented here differentiates between living viable, living non-reproducing, and dead cells as was suggested by van Uden and Madeira-Lopes (1976). It is not known whether death precedes lysis or whether lysis of viable cells (and non-reproducing cells) is possible before death occurs. This work suggests that dead cells do not remain intact for very long, a result supporting an assumption by Drozd et al. (1978) in their consideration of lysis and "cryptic" growth. Physiologically, this makes sense, since a dead cell must be a cell devoid of all metabolic activity. Such cells are difficult to imagine and a more likely scenario is that cell lysis and cell death are synonymous such that the cell dies as a result of lysis. Thus, the concept of dead cells may well be false.
In many cases where viability is considered, anything less than 100 percent viability suggests the presence of cells with genetic defects that are irreversibly non-viable. Under normal conditions (e.g., in water samples) the probability of this is low and any large deviation from 100 percent viability suggests the presence of reversibly non-viable cells which can further propagate given favourable environments.
The biomass of truly non-viable respiring cells in some systems may be small relative to the total biomass. The mass balance assumes that these cells can increase in mass by growth linked enzymatic oxidation. However, after a certain time period they will lyse. Microscopic examination of K. pneumoniae cultures revealed the occasional presence of elongated cells which may be representative of this category. Experimentally, the non-viable and viable respiring cells were not distinguishable. However, on the assumption that non-viable respiring cells only account for a very minor fraction in this system it is considered that measuring cell activity provides a far more accurate a'nd meaningful parameter than using propagation methods. The question must also be raised as to the applicability of using agar propagation methods especially in systems with very long residence times.
Extension of the model to very long residence times suggests that the proportion of active cells still remains >85 percent at dilution rates as low as 0.002 h- 1• This contradicts the findings of Tempest et al. (1967) who report low viabilities (40 percent) at dilution rates of 0.004 h- 1 (residence time of 250 h). Such a result was obtained using a slide culture technique and doubt must be expressed concerning the possibility for individual very slow growing, effectively nutrient starved cells to replicate on solid medium in the short time span over which the test was carried out. More work is obviously needed to confirm this prediction.
The kinetic constants used to evaluate the model were selected, where possible,
-49-
MICROBIAL GROWTH 175
from the literature. Experimental verification of the assumed constants and rate expression (first order, unless growth is involved, when Monod kinetics were used) must follow and will probably account for the discrepancies between prediction and experimental results.
CONCLUSIONS
The structured type of model that has been developed to describe simultaneous growth, death and lysis in chemostat cultures provides an effective framework for the development of a more detailed understanding of the relative importance of cellular death phenomena. Although the experimental data generated so far are insufficient for any major conclusions of a general nature to be drawn, they do support for the concepts proposed by Drozd et al. (1978). Clearly, a great deal still remains to be investigated with respect to microbial death and lysis in non-hostile environments.
ACKNOWLEDGEMENTS
We wish to express our thanks to Dr. W. Gujer for his help in computerizing the model and to the Swiss National Programme 7D for financial support.
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Stouthamcr. A.H .. The Search for Correlation between Theoretical and Exp<'rimental Growth Yields, International Review of Biochemistry, Microbial Biochemistry, Vol. 21 J.R. Quale (ed). University Park Press, Baltimore (1979).
Tempest, D. W., Herbert, D. and Phipps, P.J., Studies on the Growth of Aerobacter Aerogenes at Low Dilution Rates in a Chemostal. In: Microbial Physiology and Continuous Culture, E.O. Powell, C.G.T. Evans, R.E. Strange, D.W. Tempest (eds). H.M.S.O., London (1967).
Topiwala, H. and Sinclair, C.G., Temperature Relationships in Continuous Culture", Biotechnol. Bioeng. 13, 795-813 (1971).
van Udcn, N. and Madeira-Lopes, A .. "Yield and Maintenance Relations of Yeast Growth in the Chemostat at Superoptimal Temperatures", Biotech110/. Bioeng. 18, 719-804 (1976).
Zimmermann, R., lturriaga, R. and Becker-Birck, J., "Simultaneous Determination of the Total Number of Aquatic Bacteria and the Number thereof Involved in Respiration", Appl. Environ. Microbial. 36, 926-935 (1978).
-51-
CHAPTER 4
"CRYPTIC" GROWTH IN KLEBSIELLA PNEUMONIAE
Summary
The ability of Klebsiella pneumoniae to grow on its own soluble lysis products is shown in a series of batch growth experiments. Maximum specific growth rate coefficients ranging from 0.69 to 1.46 h-l were obtained with experi-mental "cryptic" yield coefficients ranging from 0. 42 to 0.52 (mg-cell-C/mg-substrate-C). These kinetic data are used to calibrate a model which demonstrates that the depression of theoreti.cal maximum yield coefficents to experimentally obtained values can be explained by "cryptic" growth pheno-mena without the need to resort to the use of physiolo-gically undefined, mathematical constants. Growth of K. pneumoniae on sonicated cells derived from steady-state chemostat cultures was followed in batch culture and obser-ved to occur with no lag phase. Batch growth curves did not indicate either diauxic or polyauxic growth, suggesting simultaneous utilization of the complex organic substrate mixture. These data suggest that "cryptic" growth is probably a real event occurring in growing chemostat cultures under ideal growth conditions and most probably also under starvation conditions.
-52-
Introduction
Increasing awareness of the importance of lysis and death in technical
and environmental processes such as in wastewater treatment and in
routine environmental monitoring programmes (Mason !!_ al., 1986a) has
stimulated interest in "cryptic" growth phenomena. The term "cryptic"
growth was introduced by Ryan (1959) and describes the re-utilization of
lysis and leakage products by intact cells of the same population as
carbon ene.rgy substrates and nutrient sources. The phenomenon was also
recognised in cultures of Escherichia coli by Koch (1959). I\ recent
model describing growth of Klebsiella pneumoniae in continuous culture
(Mason !!_ al., 1986b) shows that the processes of lysis and "cryptic"
growth could account for the observed depression of maximum biomass
yield coefficients normally attributed to maintenance energy
requirements (Pirt, 1965).
Whilst "cryptic" growth in monospecies cultures can be economically
important in many biotechnological production processes, the efficiency
and relevance can be much greater in mixed culture systems such as in
wastewater treatment processes (Hamer,19851 Hamer et!'!.!_, 1985) and other
processes where biomass concentration subjects the organisms to extended
periods of starvation within a recycle loop. Furthermore, in activated
sludge processes, recycle frequently involves a change from aerobic to
anaerobic conditions, during which lysis is induced in part of the
population enabling growth and metabolism by the remainder of the
population on the released products. Similarly, during the extended
operation of immobilised cell systems, an initial gradual loss followed
by a precipitous drop in performance occurs; the latter probably as a
result of death/lysis phenomena.
Previous investigations of "cryptic" growth have tended to focus on the
ability of cells to grow on heat killed suspensions of the same strain
(Nioh and Furusaka, 1968). This approach is so unlike the situation
likely to prevail in either growing or in starving cultures that the
conclusions derived from it cannot be generally applied. Therefore, it
is necessary to examine the process of "cryptic" growth using more
strictly defined conditions, closer akin to the processes occurring in
-53-
real culture environments. The purpose of this communication is to
de1110nstrate some of the fundamental principles of "cryptic" growth and
provide some kinetic data.
Theoretical
The processes involved in the growth of a microbial culture can be
described by the scheme shown in Fig. 1. A soluble carbon energy
substrate is used for the production of microbial biomass, soluble
products and C02 • Processes such as maintenance energy detract from
product formation via intra-cellular carbon recycling. However, energy
dissipation can also occur via an external cycle involving lysis of the
microbes followed by "cryptic" growth on the soluble lytic products,
thereby reducing the potential for both product and microbe formation.
This can be described in mathematical terms by establishing equations
for each of the various components present in the culture. For reasons
discussed by Mason !.!:_ al. (1986b), dead cells, Le., microbes totally
devoid of enzymic activity, will be assumed to be absent in continuous
cultures. The rate of accumulation of a component in a continuous flow
culture can be described by the generalised mass balance expression:
net rate of net rate of net rate of + (1)
accumulation transport transformation
Subsequent material balances are expressed in matrix notation as applied
by Roels (1980) for microbial processes.
The vector containing the components of interest, namely S, soluble
carbon energy substrate in feed, SL soluble organic lysis product, X,
the bacterial biomass, P, biopolymeric particulates from lysis and o2 ,
oxygen is defined as:
-54-
s
a • (2)
The transport vector will take the form
D(S -S) 0
-DSL a* -DK (3)
-DP
Ke(02*+02)-002
where D is the dilution rate (equivalent to the flow rate per unit
reactor volume), S0
the soluble carbon energy substrate concentration in
the feed, KL the overall liquid side oxygen transfer coefficient, ex the
specific gas-liquid interfacial area per unit
equilibrium saturation concentration of oxygen.
The· most concise means for representation
volume and
of the
0 * 2 the
reactions
(transformations) occurring in the culture is by means of a
stoichiometric matrix:
Component
Growth
cryptic
Growth
Lysis
Hydrolysis
s
-1
y
SL x
+l
-1 +l
Y*
f L -1
+l
-55-
l? 02
Reaction rate
-1-Y )lX y
-1-Y* p*X Y*
fl? K1X
-1 KhP
where Y is the theoretical maximum biomass growth yield on s, Y*, the
theoretical maximum biomass growth yield on SL, fL and f p' the fraction of dissolved and particulate lysis product, p, the specific
growth rate constant for growth on s, p*, the specific growth rate
constant for growth on SL, K and K * the saturation constants s s corresponding to S and SL, respectively, Kh, the hydrolysis rate
constant and K1 , the lysis rate constant.
Combining rate expressions and stoichiometry gives the transformation x vector a :
:1!!. y
-p*X + fLKlX + ~p Y*
It px + p*X - K1X (4) a
fpK1X-~P
·..:::: (JlX) - .};::!_* (p*X) y Y*
-56-
Thus, equation 1 can be rewritten:
da
a* + a"' (5)
dt
Complete material balances are given in table 1.
Methods
Organism. Klebsiella pneumoniae NClB 418 was maintained by monthly
sub-culture on plate count agar slopes incubated at 35°c and stored at
4°c.
Cell cultivation. K. pneumoniae was grown in a 2.5 1 bioreactor (MBR
Bioreactor AG, Wetzikon, CH) in a mineral salts medium modified from the
recipe of Evans .!!,! al. (1974), such that it contained EDTA (100 mg 1-l)
in place of citrate and used only 50% of the concentration of trace
elements. It was also necessary to add polypropylene glycol (20 mg 1-l)
as an antifoaming agent. Glucose (4.8 g 1-1) was the sole carbon energy
source limiting growth. The medium was added by means of a peristaltic
pump and the flow rate monitored by weight displacement from the
substrate resevoir bottle. Spent broth was removed, and the volume
maintained by an overflow weir. The cultivation temperature was
maintained constant at 35.o0 c and the pH at 6.80 by controlled addition
of a mixture of 1.5 M KOH and 1.5 M NaOH. At least five retention times
were allowed for attainment of a steady state when process conditions
were altered. Dissolved oxygen concentration was measured using an
oxygen electrode (Ingold AG, Urdorf, CHJ and was maintained at> 30%
saturation by varying the oxygen/air ratio in a constant volume of gas
sparged to the bioreactor. Verification of monoculture operation was
carried out by periodic microscopic observation and by cell cultivation
on plate count agar followed by identification of selected single
colonies using the API 20E test kit (API System S.A., Montalieu-Vercieu,
F).
-57-
Preparation of "cryptic" growth medium: Approximately 200 ml of a steady
state culture was withdrawn from the bioreactor into pre-cooled flasks
immersed in ice. The cells were lysed/broken by sonicating 100 ml
volumes in an ice bath for 10 minutes at an amplitude of 28 microns
using a MSE Soniprep 150 Ultrasonicator (MSE, Crawley, GB). The soluble
lysis products were separated by centrifugation at 4°c at 30,000 x g for
10 minutes and pre-filtered under pressure through 0. 2 )Im cellulose
Drozd JW, Linton JD, Downs J, Stephenson RJ (1978) An in situ assessment of the specific lysis rate in continuous cultures-ofMethylococcus sp. (NCIB 11083) grown on methane. FEMS Microbiol Letters 4: 311-314
Egli Th, Lindley ND, Quayle JR (1983) Regulation of enzyme synthesis and variation of residual methanol concentration during carbon-limited growth of Kloeckera sp. 2201 on mixtures of methanol and glucose. J Gen Microbiol 129:1269-1281
Evans CGT, Herbert D, Tempest DW (1974) The continuous cultivation of Micro-organisms. 2: construction of a chemostat. In: Norris JR, Ribbons DW (eds) Methods in Microbiology, vol. 2, Academic Press, London, pp 277-327
Gaudy AF, Yang PY, Obayashi AW (1971) Studies on the total oxidation of activated sludge with and without hydrolytic pretreatment. J Water Poll Contr Fed 43: 40-54
Hamer G (1985) Lysis and "cryptic" growth in wastewater and sludge treatment processes. Acta Biotechnol 2: 117-127
Hamer G, Egli Th, Mechsner Kl (1985) Biological treatment of industrial wastewater: a microbiological basis for process performance. J Appl Bacteriol Syrop Supp 127S-140S
Koch AL (1959) Death of bacteria in growing culture. J Bacteriol 77: 623-627
Linton JD, Stephenson RJ (1978) A preliminary study on growth yields in relation to the carbon and energy content of various organic growth substrates. FEMS Microbiol Letters 3: 95-98
Martin EJ, Washington DR (1964) Kinetics of the steady-state bacterial culture. 1. Mathematical model. Proc. Purdue Industrial Waste Conference, Lafayette, Indiana, part 2, 724-737.
Mason CA, Hamer G, Bryers JD (1986a) The death and lysis of microorganisms in environmental processes. FEMS Microbiology Reviews 39:373-401
Mason CA, Bryers JD, Hamer G (1986b) Activity, death and lysis during microbial growth in a chemostat. Chem Engng Commun 45:163-176
Nioh I, Furusaka c (1968) Growth of bacteria in the heat-killed suspensions of the same bacteria. J Gen Appl Microbiol 14: 373-385
Obayashi AW, Gaudy AF (1973) Aerobic digestion of extracellular microbial polysaccharides. J Water Poll Contr Fed 45: 1584-1594
-65-
Pirt SJ (1965) The maintenance energy of bacteria in growing cultures. Proc Roy Soc Lon Ser B 163: 224-231.
Postgate JR, Hunter JR (1962) The survival of starved bacteria. J Gen Microbiol 29: 233-263
Postgate JR, Hunter JR (1963) The survival of starved bacteria. J Appl Bact 26: 295-306
Postgate JR (1976) Death in macrobes and microbes. In: Gray TRG, Postgate JR (eds) The survival of vegetative microbes. Proc Symp Soc Gen Microbiol vol 26, Cambridge University Press, Cambridge, pp 1-18.
Roels JA (1980) Simple model for the energetics of growth on substrates with different degrees of reduction. Biotechnol Bioeng 22: 33-53
Ryan FJ (1959) Bacterial mutation in a stationary phase and the question of cell turnover. J Gen Microbiol 21 530-549
Schlegel HG (1981) Allgemeine Mikrobi.ologie (5th Edn) George Thieme-Verlag, Stuttgart.
Stouthamer AH (1979) The search for correlation between theoretical and experimental growth yield. Int Rev Biochem 21: 1-45
Van Verseveld HW, Chesbro WR, Braster M, Stouthamer AH (1984) Eubacteria have 3 growth modes keyed to nutrient flow - consequences for the concept of maintenance and maximal growth yield. Arch Microbiol 137: 176-184
-66-
Table 1. Material balances for microbial growth in a chemostat
rate constant (pm) in batch simulated "cryptic" growth study
0.69
0.77
0.85 1.10
1. 46
1. 22
( 6)
( 7)
(8)
( 9)
( 10}
-67-
Table 3. Total organic carbon released by sonication of !· pneumoniae and residual total and dissolved concentrations after batch "cryptic" growth
Cells derived Total organic Total organic Dissolved organic Dissolved organic fran culture carbon relea- carbon in the carbon in the carbon at the end with dilution sed by the culture after culture after of the experiment rate (growth cells after batch growth batch growth as percent of initi-rate) sonication al concentration
Figure 3 Computer prediction from the model for bacterial growth in continuous culture. SL, soluble lytic product concentration, never exceeded 3.7 mg 1-1 • Other symbols: X - biomass, P - particulate biomass and S -
Microbially mediated biotechnological processes are dependent on the activities of either growing microbes, in the case of biomass and growth associated product production, or non-growing microbes, which are potentially superior for many transformations. In both cases endogenous activity will detract from overall process yields and productivities. In processes involving growth, cell production will be reduced, whilst in processes employing non-growing cells an initial gradual decline followed by a precipitous decline in productivity occurs with extended operation.
The requirements for effective process cultures for commercial exploitation are:
(1) Maximum yield coefficients for the product or products; (2) Rapid growth rates and/or product production rates; (3) High affinity for carbon energy substrates; (4) High stability, robustness and lack of fastidiousness.
Many natural, mutant, and engineered strains fail to meet these criteria.
-72-
It has long been postulated that microbes require energy for both growth
and so called maintenance requirements. The latter is generally
expressed as a growth rate independent factor which depresses yield
coefficients and productivities.
Production Culture Physiology.
In all microbial production processes characterisation of the process
culture requires strict definition of its physiological status. The
consequences of inadequate characterisation are an inability to optimise
production processes for either biomass or products. Heterotrophic
processes can be represented by the scheme shown in Fig. 1., where
traditional maintenance requirements such as turnover of intra-cellular
materials, osmotic work to maintain transmembrane concentration
gradients and motility involving internal carbon energy fluxes are
differentiated from processes such as death/lysis/"cryptic" growth that
involve external carbon energy fluxes.
The death/lysis/"cryptic" growth cycle is an undesirable feature in all
commercial microbial processes and is a function of culture robustness.
Furthermore, unlike maintenance requirements which are traditionally
modelled as process rate independent, the death/lysis/"cryptic" growth
cycle is process rate dependent. In the case of those cultures with
broad spectrum capacity for complex substrate utilization, the effects
of the cycle are most marked at low growth rates, but where substrate
specificity is restricted, as with methanotrophs and obligate
methylotrophs, manifestation is as growth inhibtion.
"Cryptic• Growth in Klebsiella pneumoniae
The growth potential of Klebsiella pneumoniae NCIB 418 on its own lytic
products was investigated by sonicating cells from a steady-state
chemostat culture and using sterile filtrate as growth substrate for the
batch growth of cells taken from the same chemostat culture after
-73-
steady-state operation had been re-established. "Cryptic" growth was
shown to occur (Fig. 2), without lag and with a high specific growth
rate constant, 1.04 h-1 , which approaches the maximum value of 1.22 h-l
when glucose was used as sole carbon energy substrate. The amount and
type of dissolved organic carbon released from ~· pneumoniae, grown at
different dilution rates, following sonication is shown in Fig. 3.
Discussion
Reduction of the biomass yield coefficient in microbial growth
processes, concentration of active microbes in non-growing (immobilized)
systems and of product production in all microbial processes can be
largely accounted for using the death/lysis/"cryptic" growth concept
(1). This concept involves extra-cellular processes in contrast to the
traditional maintenance requirement concept which involves only
intra-cellular processes. In view of the fact that the former concept is
generally ignored, over-emphasis has frequently been given to the latter
concept, such that it has largely degenerated into a convenient
mathematical factor with little real physiological meaning, when, in
fact, both processes occur. Under optimized process conditions the
contribution of the extra-cellular process will probably markedly exceed
that of the intra-cellular process. The adoption of two different,
simultaneous process mechanisms offers a more plausible explanation of
the process physiology in all types of microbial production processes.
1) Mason,C.A., Bryers,J.D. and Hamer,G. (1986) Activity, death and lysis
during microbial growth in a chemostat. Chem. Engng. Comm. 45, 163-176.
•cryptic• growth of !!· eneumoniae on it• own lysis products. natch growth was carried out using cells derived from a continuous cult\Jre (D • O.S h-ll at pH 6.80 at
Js0 c.
1.0.--...--..-----~--------0.8 0.6 0.5 0.4 0.3
0.2 Ms
540nm 0.1
0.08 0.06 0.05 0.04 0.03
0.02
o.oi o;--:--.,1;--*°3 --....4 --:---:---+--!a TIME lh .. ra)
Figure L Carbon and enerqy tluxee
...
... , ..
. ..
for beterotrophic qrowth .and product prOduction from eoluble carbon en~uqy aub•tratea.
.1 ·" .• .• OikJlion Rate (If')
~ ---le - r:e.leued fr<n !!· I!!!!!!!!!!!!!! aa a functian of dilutian rate. 'Die lar<JI! particulate fr-Ian !oell ...Uls etc.) - ,,.,,.._, bf contrifugatian. 'Die ol<lwly (or non-) biodagradl!lble fr-=tion ia
- bf the li9'ter aree end the - area ._.....,,. tile ooc .n!eh could be dogr-.S "1thin 24 h in "cryptic" 9f<lWtl\
""""""'""" ..
-75-
CHAPTER 6
SURVIVAL AND ACTIVITY OF KLEBSIELLA PNEUMONIAE
AT SUPER-OPTIMAL TEMPERATURES
Abstract
The effect of temperature on a population of Klebsiella pneumoniae was examined together with the imposition of mild starvation conditions at 35°, 41°, 49°, 55° and 6o0 c. Results for changes in biomass, protein and metabolic activity are presented in terms of gross population changes and show that these parameters decline with increasing temperature. Increases in the amount of dissolved organic carbon together with a decrease in the number of cells present with increasing temperature suggest that death and lysis processes are occurring. Regrowth in the bioreactor occurred after returning the temperature to 35°c and starting a flow of carbon and other nutrients. This was
probably due to reinoculation from head space wall growth drainage rather than reversion of heat-stressed microbes. The consequences of this for thermophilic sludge treatment processes are discussed. The concept of endogenous metabolism is questioned with respect to its being a realistic description of the survival process.
-76-
Introduction
The use of heat for rendering a population of microbes inert is commonly
practised. Sterilization processes in the public health sector, and
pasteurization processes in the food and beverage industries have been
used with considerable effect to achieve either reduction or total
inhibition of microbes and their activity. However, the use of heat
treatment has limited application in the wastewater and waste sludge
treatment \,industries. Introduction of pasteurization processes in the
treatment of sewage sludge have proved unsuccesful where inefficient or
insufficient biodegradation of the organic material in the sludge allows
the possibility of post- pasteurization reinfection either from
organisms in aerosols or from other sources in the vicinity of the
treated sludge.
At present, several novel treatment technologies are being assesssed for
their ability to achieve adequate removal of pathogenic organisms from
waste sludge during treatment, but at the same time achieve high levels
of biodegradation and stabilization during exposure to high
temperatures. Such processes operate in the thermophilic temperature
range (55-7o0 c) under either aerobic or anaerobic conditions. The target
of such treatment processes with respect to hygienisation aspects of
sludge are the bacteria, viruses, worm eggs and protozoa which can
potentially result in serious diseases in humans and animals.
Bacteria of enteric origin such as Escherichia coli and Klebsiella
pneumoniae have been used as indicators of more serious pathogens. Thus,
E.coli and .!5_.pneumoniae, which are both themselves potentially
pathogenic, are monitored together with other coliforms to indicate the
possible presence of more seriously pathogenic microbes such as
Salmonella spp. and ~ spp. . Bacteria are found that can grow or
survive over a wide range of temperatures. Although most are found only
at temperatures below 45°c, others can survive and even grow at
temperatures in excess of loo0 c (l). The pathogenic microbes are of the
-77-
former group and are "killed" or inactivated by temperatures above so0 c,
thus, the potential for heat treatment technologies in the treatment of
waste sludges.
Mow heat treatment affects microbes has been the subject of considerable
attention. In !'.·~· physical changes in the cell surface have been
described whereby a weakening of the structure of the peptidoglycan
results in extrusion of cell membrane-bound "blebs" following transfer
to temperatures in excess of so0 c (2). Pellon~ al. (3) and Pellon and
Gomez (4) found extensive damage to the DNA of !!.coli following a shift
of temperature from 37°c to so0 c, with concommitant loss of viability.
They showed single and double strand breaks occurred in the DNA together
with substantial physical association of protein to the DNA molecule,
the DNA scissions resulting from the action of endo- and exo-nuclease
activity (SJ. Returning the microbes to 37°c resulted in regrowth of the
population following a 40 min lag period during which time the DNA was
repaired (6).
The ability to survive heat shock has been shown to be dependent on the
growth rate (7,8), the nutritional status of the culture (9,10) and the
water activity in the bulk liquid (ll). l'iu and Klein (9) found that a
mixed bacterial culture isolated from water showed a decreasing
sensitivity to mild warming stress in relation to increasing time of
nutrient starvation, whilst the reverse was true of E.coli.
The production of !!.! ~ enzymes and other proteins as a result of heat
stress has also been described in considerable detail for a wide range
of organisms (12,13,14), although the function of these proteins has yet
to be elucidated.
In the aerobic thermophilic sludge biodegradation process, hygienisation
requires the destruction of the pathogenic microbes. It is known that
microbial solids removal can occur at a high rate using this process
(15,16,17) but the fate of pathogenic microbes in such environments is
largely unknown, especially when one examines only those effects caused
-79-
of single colonies using the API 20E test kit (API System S.A.,
Montalieu-vercieu, F).
Heat Treatment: The effect of temperature on !· pneumoniae was invest-
igated by continuosly feeding cells from the cultivation bioreactor into
a second, similar bioreactor maintained at various temperatures. The
hydraulic residence time in the second bioreactor was maintained
constant at 32 h (D = O.Ollh -l). The pH was maintained constant. The
second bioreactor received no nutrient flow. Before sampling, at least
five residence times were allowed so as to establish steady state
conditions. The reactor was aerated by sparging with air.
Analyses
Total Biomass Concentration: was measured as dry weight by a
filtration/gravimetric procedure using tared O. 4 pm Nuclepore filters.
The filters were dried at lOS0 c for lh before reweighing.
Dissolved Organic carbon was determined in the filtrate obtained from
dry weight determination after acidifying using concentrated HCl and removing the inorganic carbon by sparging with N2 for 12 min, in a TOCOR
2, total organic carbon analyser (Maihak AG, Hamburg, D).
~ in the cells and in the extracellular medium was measured after
centrifuging at 30,000 x g using the Biuret method with bovine serum
albumin as the standard as described by Herbert.!!:!!_. (20).
Metabolic Activity was measured using 2-(p-iodophenyll-3-(-
p-nitrophenyl)-5-phenyl tetrazolium chloride (INT). One ml INT (2.0g 1-l
aqueous solution) was added to a 10 ml sample immediately after removal
from the bioreactor after suitable dilution in a O.llM NaCl, 0.02M
phosphate buffer (pH 6.80). One hundred pl glucose in Evans mineral salts medium (glucose concentration 2 .Sgl -l) was also added to provide
an electron source. After 20 min incubation, 100 pl 37\ formaldehyde was
added to stop the reaction. The solution was centrifuged at 30,000 x g
-ao-
for 25 min and the solids resuspended in 10 ml tetrachloroethylene (40
v%) / acetone (60 v%) solution. After 30 min incubation at room
temperature in the dark, the solution was centrifuged at 30,000 x g for 10 min and the absorption of the extracted INT-formazan measured at
490nm.
Total Cell Number was assayed by means of the acridine orange direct
count method. Suitable dilutions of the microbes were made using O.llM
NaCl, 0.02~ phospahte buffer (pH 6.80). Five ml were filtered through pre-stained (1:15000 sudan black in 50% absolute ethanol) 0.2 pm
Nuclepore filters. One ml acridine orange solution (1:5000 in 6.6 mM
phosphate buffer, pH 6.80) was added to the filter for 2 min, and then
removed by filtration. Total cell counts were made by counting green and orange fluorescing cells with a Leitz SM-lux epifluorescence microscope
(Leitz GmbH, Wetzlar, D).
Chemicals: All chemicals were of analytical grade and supplied by either
Fluka (Buchs/SG, CH) or Merck (Darmstadt, D).
The effects of heat on a population of !· pneumoniae were examined
together with the effect of mild starvtion conditions by feeding a
steady state culture of the bacterium from a continuously fed bioreactor into a second bioreactor which received no additional nutrient flow, but
was maintained at pH 6.80 and aerated. The second bioreactor was maintained at each of a series of temperatures; 35°c (equivalent to the
growth temperature in the first bioreactor), 41°, 49°, 55° and 6o0 c.
Samples were removed from the second bioreactor once steady state
conditions had been established, and examined with respect to dry weight, dissolved organic carbon concentration, metabolic activity,
protein content, cell number and the accumulation of 260nm absorbing compounds in the culture medium. The results of these analyses are shown
in Figures. 1-3.
-81-
As can be seen from Figure 1., the dry weight decreases as a result of
only the starvation conditions, between 1.09 gl-l in the first
bioreactor and 0.99 gl -l in the bioreactor maintained at the same
temperature as the first (35°cJ but with no nutrient flow. Noticeable
also is the further decrease in dry weight as a function of increasing
temperature such that at 60°c only 72.4% of the concentration of
bacteria being fed into the bioreactor, on a dry weight basis, remained
in the bioreactor at its outflow.
Simultaneous with the decrease in dry weight, there was a decrease in
the number of cells present as determined by the acridine orange direct
count. This reduction from the number present in the first bioreactor
and the feed to the second bioreactor, occurred under all conditions of
starvation and heating. The extent of reduction was a function of
temperature.
Attempts to enumerate stressed microbes have tended to use techniques
requiring cell replication on an agar surface. This technique
drastically underestimates the true number of microbes present since
failure to reproduce can occur purely through the physico-chemical
characterisics of the agar medium, which differ drastically from
conditions in submerged continuous cultures. Moreover, a time limit is
usually enforced on such tests, thus, further negating the application
of this technique to stress studies where a period of adaptation is to
be expected before growth of those microbes capable of repairing damage
caused by heat is likely to occur. As a consequence, a technique was
used which measures metabolic activity of the culture. This technique is
based on the ability of the compound INT to be reduced by dehydrogenase
enzymes to a water insoluble salt, INT-formazan, which is deposited
internally in the cell. Several dehydrogenase enzymes are active in the
electron transport system where INT competes with oxygen for electrons.
Previous studies have shown that a large proportion of the total
metabolic activity can be linked to electron transport system activity
(21). Using this technique, whereby, the INT-formazan salt is extracted
from the cells and its concentration measured by absorption at 490 nm,
-83-
flow (with a dilution rate of 0.08 h-ll containing glucose (2.5 g 1-ll
in Evans mineral salts medium to the previously heat stressed cells in
the bioreactor after cooling the culture to 35°c. In order to ensure all
bacteria in the bioreactor were subjected to the heat stress the
procedure followed was to stop the flow of bacteria from the growth
reactor, and only four hours later to reduce the temperature and start
the nutrient flow. The ensueing washout and regrowth was monitored by
absorption at 546 nm (see Fig 4). This experiment was carried out after
treatment at 49°, 55° and 60°c and in each case regrowth occurred. After
treatment at 49°c, there was a long lag phase lasting approximately 7 h
whilst for the bacteria treated at 55° and 60°c, the lag phase was much
shorter. The identity and purity of the regrown culture was checked
using the API 20E identification system and !· pneumoniae was identified
as the only microbe present in all regrowth experiments.
Discussion
The results with respect to changes in various parameters as a result of
heat shock, shown in Figs. 1-3 are presented in terms of mass per unit
volume. In studies such as this, where the physiological matrix of the
reference quantity is drastically different under different conditions,
the use of this basis is questionable. Every indication suggests that
death/lysis events are occurring in the culture, and therefore, release
of cell contents into the extracellular medium is occurring. The
acridine orange direct count also shows a clear decrease in the number
of cells per ml and thus expression of the amount of protein found on a
mg per ml basis does not take into account that successively fewer cells
are present per ml with increasing temperature. Thus, as examples, the
results for dry weight and cellular protein have been recalculated and
expressed on a mg per cell basis (Fig. 5). The trend here is the reverse
of that described earlier. Whilst in terms of total cellular protein per
-84-
ml, a reduction was measured, the amount per cell is higher under
starvation conditions with a 32 h bioreactor residence time and during
starvation/temperature treatment. Similarly, whilst the dry weight of l
ml culture fluid decreases with increasing temperature, an increase in
the dry weight of individual cells could be measured when calculated on
a different basis.
Historically, the decrease in biomass found as a result of starvation
has been ,,scribed to endogenous metabolism. Endogenous metabolism is
defined as 'the summation of all metabolic reactions which occur when a
cell is deprived of either compounds or elements which may serve spec-
ifically as exogenous substrates (23). Comparing the data for cells
grown at 35°c with nutrient flow, and those maintained under starvation
(32 hour residence time) in the second bioreactor at 35°c would suggest
that metabolism of endogenous substrates has brought about a reduction
in the biomass. That a reduction at 41° and 49°c occurs can be justified
to some extent using this same hypothesis based on the fact that a
significant level of metabolic activity can still be measured. However,
the extremely low level of metabolic activity found at 55° and 6o0c,
resulting from bacteria which had been in the bioreactor for an
extremely short time, is insufficient to justify the biomass reduction
based on the endogenous metabolism hypothesis. The foregoing discussion
taken together with the fact that protein in individual cells is not
declining as suggested by Fig. 3, but accumulating, leads to increasing
reluctance to ascribe the observed effects to endogenous activity.
Certainly, the production of heat shock proteins has been well
documented and may account for some of the increased values for protein
per cell (24). However, as in the example of endogenous degradation of
protein and other cellular components, the low level of metabolic
activity in the culture at 55° and 6o0 c prohibits the extrapolation of
this concept to the higher temperature conditions.
-as-
Evidence for lysis of the cultrue as a consequence of both starvation
conditions and from temperature effects are provided by the increase in
UV absorbing material (protein, nucleic acids) and from the reduction in
cell number. Both of these variables are unaffected by the difficulties
described above such that more reliance can be placed on these data.
Thus the process of "cryptic" growth is expected to be occurring in
those cultures where the temperature does not inhibit the specific
matabolic pathways involved. "Cryptic" growth has been described in
detail by Mason and Hamer (25) for the microbe !:· pneumoniae and may
well be the reason for the apparent absence of accumulation of
extracellular protein at 35°, 41° and 49°c. At 55° and 60°c it is also
assumed that "cryptic" growth is not occurring.
A more likely explanation of the results reported here is that the
permeability of the cell changes as a result of both the imposition of
starvation conditions and from the effects of temperature. As a result,
the contents of the cell become concentrated due to water efflux, thus
resulting in the higher concentrations of protein per cell. An effect of
temperature on the microstructure of the cell contents was suggested
many years ago by Heden and Wyckoff (26) who showed that heating of ~·
coli to temperatures between 50° and 60° C resulted in granulation of
the cytoplasm. This effect began when cells were heated to 5o0 c and was
extensive in cells heated to 60°c. But even in cells heated to 40°c
(from a growth temperature of 37°c), some loss of protoplasmic
homogeneity had already occurred. This change in the protoplasmic matrix
was found to be irreversible above 5o0 c, and partially reversible at 45°
and 50°c when the cells were returned to their growth optimum
temperature. A similar effect was observed more recently where under
starvation conditions only, a marked decrease in the size of the
protoplasm could be discerned with increasing time of starvation (27).
This change in permeability of the membrane may well be accompanied by a
change in the ash content, thus accounting for an increase in the dry
weight of ~· pneumoniae on a per cell basis. At temperatures above 55°c
either the cell membrane or the cell wall may become heat stabilized and
-86-
prevent additional loss of internal contents and this may be the reason
why the amount of DOC released and the amount of 260 nm absorbing
material decreases slightly between 55° and 60°c.
Given these negative aspects with respect to the survival of K.
pneumoniae at high temperatures, the data shown in Fig. 4 still need to
be explained. The most likely reason for the regrowth of ~· pneumoniae
is that microbes were able to reinoculate the cooled culture from head
space splash. Wall growth in the head space is commonly encountered in
continuous culture vessels. Hamer (28) suggested that in nonfoaming
cultures some degree of microbial entrainment by bubble flotation
followed by entrainment of microorganism containing droplets from bubble
bursting at the liquid surface and collisions between the droplets and
the vessel walls can lead to head space wall growth. Such microbes will
be in an atypical environment compared to that in the bulk liquid and
may, therefore, be exposed to much lower temperatures than prevalent in
the bulk liquid. Hamer (28) calculated that in a fermenter operating at
35°c with a sparged air flow of l volume per volume of medium per minute
the volume of head-space drops formed as a result of bubbles bursting on
the surface would be equal to 1.64 litres per litre of medium each hour.
A certain fraction of the droplets produced will collide with the
fermenter walls and can result in a layer (often a thick layer) of cells
in the head space wall area.
The consequences of this finding is that in any incompletely filled
bioreactor, the head space will contain a source for reinoculation, by
head space drainage, even under conditions where careful control is
carried out. In this series of experiments, foaming was avoided by
careful operation, including gradual changes in bioreactor temperature
for enhanced temperature treatment. Moreover, the control of a small 2.5
litre fermenter is better than that of a 10 m3 industrial bioreactor.
Thus in the treatment of waste sludge, reinfection and regrowth may
arise in "treated" sludge as a result of head space reinoculation in the
bioreactor, particularly where only partial biodegradable organic matter
-87-
stabilization has ocurred. The low level of metabolic activity found at
temperatures of ss0 and 60°c also suggests possibilities for regrowth in
partially treated sludge when organisms short-circuit the thermophilic
bioreactor in continuously fed processes, a feature that tends to be
enhanced as system heterogeneity increases.
Conclusions
The use of temperature to kill pathogenic organisms in processes such as
waste sewage sludge treatment requires effective stabilisation and
sensible bioreactor design to minimise the danger of reinfection. In
stress studies on microbes, the use of specific defined parameters to
describe effects on individual microbes as opposed to overall parameters
reveals different trends and casts doubts on the concept of endogenous
metabolism as a realistic explanation of the survival process mechanism.
Such processes can be better described in terms of death/lysis and
"cryptic" growth phenomenon, together with changes in the physical
properties of the cells.
-88-
References
1. Sonnleitner, B. 1 Fiechter, A.: Advantages of using thermophiles in biotechnological processes: expectations and reality. Trends in Biotechnol. 1 (1983) 74-80.
2. Scheie, P; Ehrenspeck, S.: Large surface blebs on Escherichia coli heated to inactivating temperatures. J. Bacteriol. 114 (197'3) 814-818.
3. Pellon, J.R.; Ulmer, K.M. and Gomez, R.F.: Heat damage to the folded chromosome of Escherichia ~ K-12. Appl. Environ. Microbial. 40 (1980) 358-364.
I 4. Pellon, J.R.; Gomez, R.F.: Repair of thermal damage to the
Escherichia coli nucleoid. J. Bacterial. 45 (1981) 1456-1458.
5. Sedgwick, S.G.; Bridges, B.A.: Evidence for indicrect production of DNA strand scissions during mild heating of Escherichia coli. J. Gen. Microbial. 71 (1972) 191-193.
6. Pellon, J.R.: A note on the repair of the Escherichia coli nucleoid structure after heat shock. J. Appl. Bacterial. 54 (1983) 437-439.
7. George, T.K.; Gaudy, A.F.Jr.: Transient response of continuously cultured heterogenous population to changes in temperature. Appl. Microbial. 26 (1973) 796-803.
8. NG, H.: Effects of growth conditions on heat resistance of Arizona bacteria grown in a chemostat. Appl. Environ. Microbial. 43 (1982) 1016-1019.
9. Wu, S-Y.; Klein, D.A.: Starvation effects on Escherichia coli and aquatic bacterial responses to nutrient addition and secondary warming stresses. Appl. Environ. Microbial. 31 (1976) 216-220.
10. Paris, S.; Pringle, J.R.; Saccharomyces cerevisiae: heat and gluculase sensitivities of starved cells. Ann. Microbial. (Inst. Pasteur). 1348 (1983) 379-385.
11. Verrips, C.T.; Glas, R.; Kwast, R.H.: Heat resistance of Klebsiella pneumoniae in media with various sucrose concentrations. Eur. J. Appl. Microbial. Biotechnol. 8 (1979) 299-308.
19. Evans, C.G.T.; Herbert, D.; Tempest, D.W.; The continuous cultivation of micro-organisms. 2 Construction of a chemostat. In: Methods in Microbiology 2 (J.R. Norris, o.w. Ribbons, eds.) pp 277-327. Academic Press, London (1970).
20. Herbert, D.; Phipps, P.J.; Strange, R.E.: Chemical analysis of microbial cells. In: Methods in Microbiology 7B (J .R. Norris and D.W. Ribbons, eds.) pp. 209-344. Academic Press, London (1971).
21. Tabor.. P.S.; Neihof, R.A.: Improved method for determination of respiring individual microorganisms in natural waters. Appl. Environ. Microbiol. 43 (1982) 1249-1255.
G.: INT-dehydrogenase test for Biotechnol. Bioeng. 28 (1986)
23. Dawes, E.A.: Endogenous metabolism and the survival of starved prokaryotes. In: The Survival of Vegetative Microbes (T.R.G. Gray and J.R. Postgate, eds). Proc. Symp. Soc. Gen. Microbiol. 26 (1976) 19-53. Cambridge University Press, Cambridge.
24. Groat, R.G.; Matin, A.: Synthesis of unique proteins at the onset of carbon starvation in Escherichia coli. J. Ind. Microbiol. 1 (1986) 69-73.
26. Heden, c. -G.; Wyckoff, R. W .G.: The electronmicroscopy of heated bacteria. J. Bacteriol. 58 (1949) 153-160.
27. Reeve, C.A.; Amy, P.S.; Matin, A.: Role of protein synthesis in the survival of carbon-starved Escherichia coli K-12. J. Bacteriol. 160 (1984) 1041-1046.
28. Hamer, G.: Discussion on entrained droplets in fermenters used for the cultivation of single-celled microorganisms. Biotechnol. Bioen9. 14 (1972) 1-12.
2.0
>-0
~
-0 c :. 1.0 e g >< :o.5 ... .Q
E "' z
=iii {.) 2.0
Fi2ure
100
80 .,,, ..§. c 60 0 .Q ::a
{.)
u ·;: "' ~ 40
0 -0 ... .. 0 "' "' 20 i5
0
-90-
t:.._ ......
0.8
.:;;..... - ... A... --
0
..I. I
Growing Culture
35
1. Changes and cell
I
40
in dry number
a culture of K.
0
"'
..I. Growing 35 40 Culture
---- ... _ "t>...._
I
45 Temp(°C)
.... _ .......... _6... -- .... _
-a.
weight ( 0) I metabolic activity ([J
(A)' as a result of heat treating
pneumoniae at various temperatures.
/'""- .... 3
/ --/ /
/ 2.5 /,e_._
/:I' /
/ 2 E // c:
/ 0 / <D
/ N
/ 15 .2 Q. 15 .,, .Q <t
05
45 50 55 60 0 Temp(°C)
Fi2ure 2. Production of dissolved organic carbon (o) and
260 nm absorbing material (A) in steady-state
heat treated cultures of K. 2neumoniae.
-! c ·;;; 0 a: ~ :i u
3.5
3.2
2.9
2.6
2.3
2.0
0
A
.L Growing Culture
-91-
1::1:------~-_..
35 40
---- ,,."' __ a
. 45 50 Temp(°C)
0.4
55 60 0
Figure 3. Changes in the cellular (o) and extracellular (A) protein concentrations in cultures of K. pneumoniae exposed to super-optimal temperatures.
5 4
E 3 c:
<D 2 -:t tn c: 0 ·.;:; 0.. 1 .... 0 0.9 .,,
D QS <( 0.7
Q6 Q5
4 E 3 c:
<D -:t tn 2 c: .2 ..... 0.. .... 0
1 .,, D 0.9 <( (18
0.7 0.6 0.5
4
E 3 c:
<.D 2 -4" I()
c: g 0.. 1 .... 0 0.9 .,,
D 0.8 <( 0.7
0.6 0.5
-4
A
8
c
·2
-92-
0 2 4 6 Time(li)
8 10 12
Figure 4; Regrowth of heat treated cultures of K. pneumoniae. Continuous feed of microbes was halted four hours before returning the temperature to 35°c and starting nutrient flow. A, 49°c; B, ss0 c1 c, 60°c. The expected washout curves are also shown (---) •
0.8
\... al v ~ 0.6
0
~ ~
~ 0.4 ~ -0
0.2
A
0 .!. Growing Culture
-93-
35 40 45 50 55 60 Temp(0 C)
3
2.7 ~ a; v en E
2.4:;: Q
c a;
2.1 0 ct
1.5
Figure 5. Expression of protein and dry weight data in units of per cell.
-94-
CHAPTER 7
THE LIMITS OF EFFECTIVE PERFORMANCE OF
THERMOPHILIC AEROBIC SLUDGE TREATMENT.
Abstract
Increasing application of effective sewage and wastewater treatment technology results in an increasing production of sludge for ultimate disposal. In addition, the continued disposal of sludge on agricultural land requires the introduction of improved sludge stabilization processes. Aerobic thermophilic digestion is one such process, and here, it is examined in the light of established microbial process information.
-95-
Introduction
The widespread application of sewage and wastewater treatment processes
to satisfy increasingly stringent legislation concerning aquaeous
discharges into surface waters has resulted in increased sludge
production and has exacerbated problems of sludge disposal. In
conventional treatment processes, sludge is produced from the
preliminary, primary and secondary process stages. It comprises a dilute
putrefactive suspension of biodegradable and non-biodegradable matter
containing pathogenic organisms and potentially toxic chemicals.
Traditional methods of sludge disposal include; (1) dumping at sea, an
option that is unacceptable to land locked countries; (2) incineration
which frequently requires supplementary fuel and results in air
pollution and, ultimately, extensive land and surface water pollutioni
(3) burial with urban garbage, which can result in leachate problems,
(4) composting with either solid biodegradable residues, where the
balance of the materials processed is critial with respect to compost
quality; (5) disposal by spreading on agricultural land after
stabilization. The last approach involves digestion of the sludge to
reduce the fraction of rapidly biodegradable matter present prior to
spreading, and is usually the preferred solution for sludge disposal in
land locked regions. However, increasing concern, on the grounds of both
public/veterinary health aspects and nuisance problems arising from land
application, is becoming evident and more effective and reliable sludge
digestion processes are urgently required if the practice of land
disposal is to continue as an acceptable option.
The most widely used sludge stabilization process presently in use is
mesophilic anaerobic digestion. Alternative processes, with potentially
superior performance and operating economics are the following:
thermophilic anaerobic digestion, mesophilic aerobic digestion, and
thermophilic aerobic digestion. In this paper, factors affecting the
effective performance of the thermophilic aerobic digestion process will
be evaluated.
-96-
'l'herinophilic Microbes
Microbes are a universally available natural resource, but their
effective application in industrial processes depends on a comprehensive
understanding of their physiology and biochemistry. Temperature is a
very important factor affecting microbial kinetics, physiology and
biochemistry, and any species or strain can be broadly classified
according to its optimum temperature for growth. The psychrophiles have
temperature optima for growth of <:10°c, the mesophiles, of between 15° and 40°c, and the thermophiles, of >4s0 c. Generally, the thermophiles
are further sub-divided into; thermotolerant microbes that grow
optimally between 40° and so0 c, a group that comprizes predominantly
facultative thermophiles; moderately thermophilic microbes with growth
temperature optima between so0 and 65°c and exhibit a minimum
temperature for growth of ca. 45°c; extremely thermophilic or
caldoactive microbes that have growth optima )65°c. Caldoactive microbes
are relatively fastidious in their growth requirements (ll, and hence,
are poorly suited for practical sludge digestion processes, whilst
thermotolerant microbes can be considered to offer only minor advantages
over mesophilic microbes. Therefore, as far as improved sludge digestion
processes are concerned, it is moderate thermophiles that offer genuine
practical process potential, and will be discussed here.
Moderate thermophiles are optimally adapted for growth at elevated
temperatures and do not struggle to survive at such temperatures (2). By
analogy with chemical reactions, where increasing temperature usually
increases the reaction rate, it is generally assumed that increasing
temperature will increase the rate of microbially mediated reactions.
Inspection of the relationship between the maximum specific growth rate
constant and temperature for any particular microbial strain shows an
exponential dependency of the maximum specific growth rate constant on
temperature, over a range of ca. 15°c until a maximum value is reached.
This is maintained for a few degrees and then, within a further 4° to
s0 c, a precipitous drop in the maximum specific growth rate constant
occurs.
-97-
In sludge digestion processes, the primary substrates are particular in
nature, and hence, the environment in which the microbes function is
markedly heterogenous, and this is of obvious significance in any
analysis of temperature effects particularly when the rate controlling
step concerns a mass transfer resistance rather than one of a sequence
of biochemical reactions.
Clearly identifiable effects of temperature on microbes include those
related to; (1) the maximum specific growth rate constant; (2) the
affinity of the microbes for their growth limiting substrate; (3)
endogenous metabolism or maintenance requirements; (4) the specific
death and lysis rate constants. In addition, temperature can also be a
selective pressure with respect to metabolic pathways employed, Hence,
the response of either a microbial strain or an association of strains
to temperature will be both complex and varied. However, as far as
effective sludge mass reduction and heat production are concerned, it is
the factors that affect overall yield coefficient that are most
important, and as far as a hygienic final product is concerned, it is
the factors that control thermal death and deactivation processes that
are critical.
Yield Coefficients and Beat Production
The yield coefficient for microbial biomass formation from any substrate
or nutrient is defined as the weight of biomass produced per unit weight
of substrate or nutrient utilized. In the case of substrates that are
insoluble, immiscible and/or partially non biodegradable, a conversion
coefficient, defined as the weight of biomass produced per unit weight
of substrate supplied, is frequently more appropriate. Digestion
processes seek to minimise the fraction of biodegradable matter present
in the sludge by eith<?r utilization of the biodegradable matter under
conditions where the biomass yield coefficient is minimised or by a
sequence of utilization, lysis and reutilization steps where the overall
biomass yield coefficient is minimised.
-98-
The heat produced in any well-defined, microbial bio-oxidation process
can be calculated by application of thermodynamics to the particular
process (3). However, such an approach is much more difficult when
ill-defined, complex substrates, such as waste sludge, are considered.
Microbial biomass constitutes a significant fraction of sewage and
wastewater treatment process sludge and the average heat of combustion
of microbial biomass has been reported to be 5.32 Kcal g-l on an ash and
moisture free basis (4). For the aerobic growth on and utilisation of
any carbon energy substate, Linton and Stephenson (5) have demonstrated
that the heat of combustion per g substrate carbon is related to the
maximum biomass yield coefficient based on unit weight of carbon in the
substrate. Assuming microbial biomass has a carbon content of 47\, the
heat of combustion on a unit weight of carbon basis will be 11.32 Kcal
g -l C. The maximum biomass yield coefficient for a "cryptic" growth
process can then be estimated from the relationship proposed by Linton
and Stephenson (5). This is 0.66g dry biomass per g dry substrate
biomass, assuming complete incorporation of substrate carbon into new
cells and carbon dioxide. De-optimization of the biomass yield
coefficient for "cryptic" growth will probably result in operating yield
coefficients as low as ca. 0.33.
several empirical formulae for microbial biomass, on an ash and moisture
free basis have been proposed, including CH1. 8o0 • 43N0 . 23 (6). For a
carbon content of 47%, this gives corresponding nitrogen and ash
contents of 12.6\ and 6.4%, respectively. Using this empirical formula
and assuming no nitrification, theoretical equations that describe
"cryptic" growth processes, operating at different yield coefficients
can be devised and the respective oxygen demands calculated.
For aerobic growth processes, Cooney .!!.!: al. (7) obtained a linear
correlation between the rates of heat production and of oxygen
consumption. This correlation can be applied to aerobic "cryptic" growth
processes, operating at various productivities for biomass formation on
the basis of oxygen demands calculated from the theoretical equations,
for various produced biomass yield coefficients.
relationships are shown in Figure 1.
Some predicted
-99-
Death, Lysis and •eryptic• Growth
In all digestion processes, one group of bacteria utilizes other
bacteria as their carbon energy substrate. For such processes to occur,
the bacteria that act as the substrate loose viability and subsequently
lyse. The mechanisms involved in such a process are undoubtedly complex.
In thermophilic aerobic digestion processes the substrate bacteria are
first subjected to mild temperature shock that either deactivates or
even kills them and then to the effects of either autolysis or to the
action of lytic enzymes produced by the thermophilic process culture.
Only after lysis is it possible for the process culture to utilize the
lytic products for growth, as direct ingestion of one bacterium by
another cannot be postulated as a utilization mechanism.
For the mixed, continuous flow digestor, operating without recylce,
shown diagramatically in Figure 2, material balance equations can be
established for the lysis of the substrate bacteria, lytic product
formation and growth of the product bacteria on the lytic products.
Substrate bacteria balance
V.dx s (1)
where V is the digestor liquid volume, F, the inlet and outlet liquid
flow, xso and xs' the concentration of substrate bacteria in the inlet liquid and the digestor, respectively, k
1 , the specific lysis rate
constant and, t, time. Rearrangement of equation 1 gives
dx s
dt
where D is the dilution rate.
(2)
-100-
Product bacteria balance
V.dx V.p.x.dt - F.x.dt (3)
where x is the concentration of product bacteria in the digestor.
Rearrangement of equation 3 gives
dx (p-D) x (4)
dt
Lytic product balance
V.dp -F.p.dt - dt + (5)
where p is the lytic product concentration in the digestor, Yx/p' the
yield coefficient for product bacteria formation from lytic product and
Yp/s' the yield coefficient for lytic product formation from substrate
bacteria, which in a first approximation can be assumed to be 1.
Rearrangement of equation 5
dp p.x -Dp - + (6)
dt
For steady state conditions, dxs/dt, dx/dt and dp/dt will be zero, and
hence equations 2,4 and 6 can be rewritten, for steady state conditions
be equating to zero and substituting x5
, i and p for xs, x and p,
respectively. If it is assumed that the Monod relationship applies to
product bacteria growth on lytic products under steady state conditions,
then
(K + p) p
-101-
where Pm is the maximum specific growth rate constant and KP,
saturation constant.
From the steady state version of equation 2
x s
D.x so
(7)
the
(8)
Substituting for p in the steady state version of equation 4 in equation
7 gives
K .D p
Substituting for p, J'• and
gives
kl.xso x Yx/p
(D + K1 )
x
(9)
in the steady state version of equation 6 s
K .D p (10)
(J.lm - Dl
Typical values of the several constants can be inserted in eguations 8,
9, and 10 and the effect of D on x8
, x, and p can be evaluated.
Obviously, the value of k1 is critical as far as digestor performance is
concerned. Specific lysis rate constants for mesophilic bacte~ia in the
thermophilic temperature range under aerobic conditions are unavailable.
However, specific lysis rate constants, during the growth of a
methanotrophic bacterium in continuous culture at 42°c of between O.Ol
and 0.07 h-1 , depending on the dilution rate, have been reported (8).
Therefore, it can be assumed that the k1 values encountered in aerobic
-102-
thermophilic digestors for the bacteria undergoing destruction will
either be similar to or exceed those quoted. Predictions based on k1 values of 0.04 and 0.14 h-l are shown in Figures 3 and 4, respectively.
concluding Remarks
In order to develop competitive aerobic thermophilic sludge
stabilization processes, it is essential to produce sufficient heat to
permit autothermal operation, and simultaneously, achieve effective
destruction of putrefactive matter, particularly pathogenic organisms
present in either an active or a resting state in the sludge undergoing
stabilization. Effective oxygen conversion and short residence times in
the digester are also desirable. The approach presented in this
communication clearly indicates that established microbial process
information can be directly applied to aerobic thermophilic sludge
digestion in order to identify some of the process performance criteria
that will have to be met by such processes.
References
1. B. Sonnleitner, A. Fiechter, Trends Biotechnol., !• 74, 1983.
2. T.D. Brock, Proc. 19th Symp. Soc. Gen. Microbial., London, 15, 1969.
3. P.L. McCarty, Int. J. Air Wat. Poll., ~. 621, 1965.
The effect of dilution rate (0) on steady state substrate
bacteria (i ) , s for a specific
-1 I' • 0.17 h • m
product bacteria (i) and lytic product (f>) -1
lysis rate constant (kl) of 0.14 h when
\;p • 0.33, kp • 0.2 gl·l and x50
• 40 gl•l
-105-
CHAPTER 8
AEROBIC THBRMOPHILIC BIODEGRADATION OF MICROBIAL CELLS
Abstract
Aerobic thermophilic treatment can be used either as a pretreatment step prior to anaerobic mesophilic treatment or
as a total treatment process for waste sewage sludge. In this contribution a laboratory-scale process research study
that has been undertaken to investigate the effects of some process variables on microbial solids solubilization is described. The operating parameters investigated are bioreactor residence time and pH at 60° c and high dissolved oxygen concentrations.
-106-
Introduction
The major by-product of conventional aerobic biological wastewater
treatment processes is waste sludge, a putrefactive, aqueous suspension
of biodegradable, partially biodegradable and essentially
non-biodegradable solid, dissolved and sorbed matter. Waste sludge
presents a serious disposal problem, particularly in regions remote from
the sea, where frequently the policy for ultimate disposal involves
spreading treated sludge on agricultural land so as to cause minimum
offer the greatest potential for effective waste sludge treatment.
However, bioprocess data concerning aerobic thermophilic treatment are strictly limited. In order to rectify this, data for microbial solids
destruction are reported.
Materials and Methods
A laboratory-scale bioreactor (Chemap AG, Volketswil, CH) with an
operating volume of 6 litres was used for the experiments. The
bioreactor was operated in a continuous flow mode with a feed stream
consisting of bakers yeast, as the sole biodegradable carbon and nitrogen source, dispersed in a solution of K2HP04 (5.7 g/l) and KH2Po4 (8 g/ll. No other nutrients were provided. Air or air/oxygen mixtures
were sparged into the bioreactor and the temperature of the bioreactor
was maintained constant at 6o0 c.
Samples of the bioreactor liquor and feed were centrifuged at 30,000 x g
and the supernatants decanted. The solids were transferred into a
crucible and analysed for total suspended solids (TSS) by overnight
drying at ios0 c, and for volatile suspended solids (VSS) by heating at 0 + - -600 c. NH4 -N, N02 -N, and N03 -N were determined in the supernatant
using an automated nitrogen analyser (Skalar, lli:"eda, NL). carboxylic
acids wei:"e analysed by gas chi:"omatogi:"aphy.
-108-
Results and Discussion
In the experiments, the effects of pH and residence time on the
operation of the aerobic thermophilic biodegradaton process were
investigated. Biodegradation of particulate organic matter proceeds
initially by its solubilization thereby enabling utilization of the
lytic products by the aerobic thermophilic bacteria. Particulate
biodegradation is a complex process which has received little attention
when compared to the utilization of soluble substrates in wastewater
treatment processes. Clearly, the rate limiting step in the overall
process is this initial solubilization and, therefore, the various
factors regulating this process must be understood for optimisation of
any sludge treatment technology.
Hamer et al. (1984) and Hamer & Bryers (1985) discussed the limitations
in the performance of the process with respect to the thermophilic
process bacteria and the effects of thermophilic operating temperatures
on mesophilic (substrate) microbes.
To complete their approach a discussion of enzyme production/action by
thermophilic bacteria and of the pattern of biodegradation is required.
Particulate biodegradation can proceed by two mechanisms;
a) physical contact between process bacteria and bioparticulates,
b) exoenzyme production by the process bacteria, and destruction
without direct contact.
In the former case, digestion is more likely to occur by entrapment in
floes rather than by incidental contact within the bioreactor. The
enzymes responsible for biodegradation will be either bound to the cell
surface or released within the floe such that enhanced concentration
will occur within the microenvironments in the trapped
particulates/floe.
-109-
More likely in a well mixed system, is the second mechanism whereby
exoenzymes (e.g. protease, lipase etc.) specific for the polymeric
components of the bioparticulate material are produced. Since
microorganisms account for the major fraction of the solid material in
sludge, their biodegradation is essential. The cell wall forms a
physical barrier between a pool of readily degradable soluble material
and the process bacteria. Thus initial attack by the enzmes produced by
the thermophilic process bacteria, on the cell wall biopolymers enables
the release and utilisation of the intracellularly available or9anic
compounds as carbon and/or ener<JY sources for growth and for autothermal
heat production.
The enzymes attacking and digesting the yeast cells will function
optimally at a particular pH. However, if more than one enzyme type is
acting, it is conceivable that there will be a range of pH values over
which biodegradation can occur - although with varying rates and
efficiencies. From the data in Fig. l where the percentage total
suspended solids degraded are shown, it is clear that most effective
biodegradation occurs around pH 6. 5. At pH 7. 5 the amount of total
solids removed is not as great, whilst at pH 5.7 biodegradation occurs
to an even lesser degree. These results represent steady state values
durin9 continuous flow operation of the process. The foregoin9 comments
are generalised for the two residence times investi9ated ( 3 and 1. 5
days). At the longer residence time biodegradation proceeds to a greater
de9ree. However, if the total solids removal rates are compared (Fi9. 2)
it is obvious that the shorter residence time is more efficient and
operation at pH 6.5 achieved the best results.
The pH also has a stron9 influence on the ash content of the biomass in
the bioreactor (Fi9. 3). At pH 7.5 the total suspended solids are
composed of a lar9er amount of ash than present in the feed, whilst at
pH 6. 5 and 5. 7 the ash content is reduced. Volatile suspended solids
(VSS) are also degraded optimally at pH 6.5 (Fi9. 4) and despite the
varyin9 ash residue contents, the rate of vss biodegradation, in
-110-
agreement with that for total suspended solids, is optimum at 1.5 day
residence times (Fig. 5). Under all conditions examined, insignificant
amounts of carboxylic acids were found, the highest concentration being
45 mg/l acetic acid at pH 5. 7/1.5 day. This is not surprising since
oxygen was always in excess during these experiments.
In the yeast cells, significant amounts of proteinacious material is
present, the biodegradation of which will release the nitrogen necessary + for the thermophilic bacteria. Accumulation of NH 4 -N was found at
extended residence times (3 days) but not at 1.5 days (Fig. 6).The
dependence of pH on the extent of accumulation, and therefore of
degradation, is clearly demonstrated in these data. No2 -N and N03 -N
concentrations were below detection limits (1 mg/l).Some loss of NH 3 in
the exhaust gasses is to be expected and therefore the presented data
probably underestimates the true amount released.
The results presented here are for a feed sludge composed entirely of
yeast cells in the absence of other soluble carbon energy sauces,
bioparticulates of other microbial origin and of inert particulates.
Naturally, real sludges are likely to behave somewhat differently from
the model study conducted here. Nevertheless, the majority of the
secondary sludge solids fraction, and some of the primary sludge solids,
is microbial in nature and, therefore, extrapolation to operating
conditions with real sludges is probably valid. The effect of residence
time is merely prolongation of contact between enzyme and substrate and
it is questionable as to whether the increased level of biodegradation,
which can be as much as 64% higher at the same pH, under this set of
operating conditions, warrant the increase in operating costs, part-
icularly when the aerobic thermophilic treatment step is intended as a
precursor to anaerobic rnesophilic digestion so that pretreatment of
to be more rapid. In the data presented here, aerobic treatment is shown
to be both pH and residence time dependent, reflecting the probable pH
optima of the enzymes involved in cell lysis and subsequent growth on
lytic product.
-lll-
References
Giqer, w., P.H.Brunner and C.Schaffner, Science 225, 623 (1984)
Hamer, G., J.D.Bryers, J.Berqer and C.A.Mason, Proc. 4th C.E.C., Grado,
I. 705 (1984).
Hamer, G. and J.D.Bryers, Conservation and Recyclinq ~. 267 (1985)
Husmann, w. and F.Malz, GWF-Wasser/Abwasser 100, 189 (1959).
-112-
.. .. 1.5
, . ..
••
Figure 1. Total suspended solids biodegraded as a percentage
of the influent total solids concentration at
various values of pH and for residence times of
i 1! •
J ft ' .., :g 1. l a·
3 and 1. 5 days.
1.5
Figure 2. Total suspended solid removal rate as a function
of pH and residence time in the aerobic thermophilic
biodegradation process.
i s'
J '
Figure 3.
..
Figure 4.
-113-
!.5
Ash content in the feed (left hand bar) and in the bioreactor (right hand bar) during aerobic thermophilic digestion of yeast cells.
3 1.5 ..
.. ••
pH
Volatile suspended solids biodegraded as a percentage of the input volatile suspended solids concentration at various residence times of 3 and 1. 5 days •
i • "
j· .. l
I Figure 5.
l •
~ % I . " :i: .. %
Figure 6.
-114-
3 1.5
Volatile suspended solids removal rate as a function of pH and residence time in the aerobic thermophilic biodegradation process.
3 1.5 ...
..
pH
NH 4-N in the feed (left hand bar) and in the bioreactor (right hand bar) during aerobic thermophilic digestion of yeast cells,
-115-
CHAPTER 9
AEROBIC THERMOPHILIC BIODEGRADATION
OF MICROBIAL CELLS:
1. SOME EFFECTS OF DISSOLVED OXYGEN CONCENTRATION
Results are presented comparing the extent of solubilization/biodegradation of yeast cells by aerobic thermophilic bacteria under conditions of oxygen excess and oxygen limitation. The process was most effective at low dissolved oxygen concentration as judged by solids removal data and by the production of often considerable quantities of carboxylic acids.
-116-
Introduction
Effective legislation by the governments of most West European countries
with respect to pollution of the aquatic environment has resulted in the
widespread installation of both municipal sewage and industrial
wastewater treatment during the past 10-15 years (Hamer, 1985).
Conventional treatment technology comprizes the mechanical and physical
separation of pollutants during preliminary and primary treatment and
the bio-oxidation of pollutants in secondary aerobic biotreatment
processes, most commonly of the activated sludge type. In some cases, an
additional tertiary treatment stage is also employed. The principal
by-product of treatment is waste sludge, comprizing a complex mixture of
suspended biodegradable, partially biodegradable and non-biodegradable
solid matter and associated sorbed and dissolved pollutants. A
significant fraction of waste sewage sludge comprizes microbial biomass,
including some potentially pathogenic microbes present in the process
feed and large quantities of process microbes from secondary treatment.
The conventional procedure used for waste sewage sludge treatment prior
to ultimate disposal, frequently by spreading on agricultural land, is
anaerobic mesophilic digestion/stabilization. Increases in the
quantities of sewage undergoing treatment has resulted in corresponding
increases in waste sewage sludge production and attendant difficulties
in finding disposal sites, particularly as some authorities responsible
for public and veterinary health have expressed reservations with
respect to the efficiency of conventional sludge treatment processes for
the destruction of both pathogenic organisms and removal of toxic
chemicals. To solve the problem of sewage sludge hygienization, it has
been suggested that anaerobic treatment be carried out at thermophilic
temperatures, ss-6o0c, in order to ensure the destruction of pathogenic
organisms. However, pre-stressed concrete anaerobic digestors, designed
for mesophilic operation, 30-37°c, and installed at many treatment
plants, cannot be operated in the thermophilic temperature range. Hence,
-117-
in Switzerland, West Germany and Austria, retrospective installation of
relatively short residence time, aerobic thermophilic pre-treatment/-
hygienization p.~ocesses prior to anaerobic mesophilic digestion/
stabilization processes is occurring (Hamer and Zwiefelhofer, 1986).
Such processes have a multiple role1 the destruction (deactivation) of
pathogenic organisms, the solubilization of particulate biodegradable
matter, particularly microbial biomass and the bio-oxidation of sorbed
pollutants, removed with solids during primary treatment, that are
essentially recalcitrant to anaerobic biodegradation (McEvoy and Giger,
1986) •
In this contribution, the question of the effect of dissolved oxygen
concentration on the solubilization and biodegradation of microbial
cells during aerobic thermophilic treatment will be addressed.
Solubilization of the biodegradable particulate fraction of waste sewage
sludges is a prerequisite for complete treatment. Eastman and Ferguson
(1981) recognized that the hydrolysis of particulate matter to soluble
substrates was the real rate limiting step in anaerobic digestion
processes and recent kinetic models for anaerobic digestion (Gujer and
Zehnder, 1983, Bryers ~al., 1985) incorporate this concept. Because of
the variability of real waste sewage sludges, it is necessary to select
a source of microbial biomass that is of standardized and reproducable
quality for experimental process studies. Here, pressed baker's yeast
was used.
llaterials and llethods
Bioreactor and Operating Conditions: Two laboratory scale bioreactors
with operating volumes of 1.3 1 and 9 1 (Bioengineering AG, Wald, CH)
each with full measurement instrumention, i.e., pH, dissolved oxygen
concentration, temperature and impellar speed monitoring were used for
these experiments. The bioreactors were operated in the continuous flow
mode and were continuously sparged with air at 32 1 h -l for oxygen
-118-
sufficient conditions in the smaller bioreactor and 300 1 h-l in the
larger. Oxygen limited conditions were achieved by reducing the air flow
to 6 and 30 1 h-l respectively. The temperature was maintained constant
at 60° c.
Aerobic Thermophilic Culture and Feed: Aerobic thermophilic bacteria
were obtained from an operating municipal waste sewage sludge aerobic
tioning do indeed run under oxygen limited conditions, as suggested by
high oxygen conversion data, process designers still seek to achieve
oxygen excess conditions in aerobic thermophilic bioreactors (e.g. Match
and Drnevich, 1977) thereby ignoring the process microbiology of such
systems and its effect on efficient operation.
-125-
References
Bryers JD, Berger J and Hamer G (1985) Interpretation of thermophilic anaerobic digestion experiments using a dynamic structured model. Proc IWTUS-3 Conserv and Recyc 8:251-266.
Burns RG (1983) Extracellular enzyme-substrate interactions in Slater JH, Whittenbury R, and fiimpenny JWT (eds) Microbes natural environments. Proc 34 t symp SOC Gen Microbial. University Press, Cambridge, p 249.
soil. In: in their
Cambridge
Eastman JA and Furguson JF (1981) Solubilization of particulate organic carbon during the acid phase of anaerobic digestion. J Wat Poll Cont Fed 53:352-366.
Hamer G (1985) The impact of government legislation on industrial effluent treatment. Proc IWTUS-3 Conserv and Recyc 8:25-43.
Hamer G and Zwiefelhofer HP (1986) Aerobic thermophilic hygienization -a supplement to anaerobic mesophilic waste sludge digestion. IChemE symposium series 96: 163-180.
Grueninger H, Sonnleitner B and Fiechter A (1984) Bacterial diversity in thermophilic aerobic sewage sludge. III A source of organisms producing heat-stable industrial useful enzymes, e.g., Ot-amylases. Appl Microbial Biotechnol 19: 414-421.
Gujer W and Zehnder AJB (1983) Conversion processes in anaerobic digestion, Wat Sci Tech 15:127-167.
Matsch LC and Drnevich RF (1977) Autothermal aerobic digestion. J Wat Poll Cont Fed 49:296-310.
McEvoy J and Giger W (1986) Determin<1.tion of line<1.r alkylbenzenesulfonates in sewage sludge by high-resolution gas chromatography/mass spectrometry. Environ Sci Technol 20:376-383.
Norris JR, Berkely RCW, Logan NA and O'Donnell AG (1981) The Genera Bacillus and Sporolactobacillus. In: Starr MP, Stolp H, TrUper HG, Balonos A and Schlegel HG. The Prokaryotes - a h<1.ndbook on habitats, isolation and identification of b<1.cteria. Springer, Berlin, Heidelberg, New York p 1711.
Sonnleitner B and Fiechter A (1983) Bacterial diversity in thermophilic aerobic sewage sludge - II. Types of organism and their capacities. Appl Microbial Biotechnol 18: 174-180.
Wilkinson TG and Hamer G (1979) The microbial oxidation of mixtures of methanol, phenol, acetone and isopropanol with reference to effluent purification. J Chem Tech Biotechnol 29:56-67.
-126-
Table l Change in ash concentration of the total suspended solids after aerobic thermophilic treatment as a percentage of the amount present in the feed yeast cell suspension.
oxygen sufficient
oxygen limited
Time
(d)
1.2 1.5
0.6 1.0 1.5 2.1 5
in ash content after aerobic thermophllic biotreatment
+ 6.83 10.00
- 33. 07
11.48 7. 22 1.88 9.64
21.22
Table 2 Change in dissolved organic carbon concentration after aerobic thermophilic treatment as a percentage of the amount present in the original feed yeast cell suspension.
oxygen sufficient
oxygen limited
Figure 1.
-127-
Scanning electron micrograph showing the process
microbes and the feed yeast cells during
aerobic-thermophilic biotreatment. Several lysed
(ghost) yeast cells are present composed only of
cell wall debris.
'°1 "'
' :§'
~JO 0
"' " .. I! ; 20
Jl ~ ......
a
-128-
' Si
:l!! ~
..
••
b
Residence Time (d)
Figure 2.. Total suspended solids concentration in feed (left hand bar of
each pair) and in the bioreactor (right hand bar) as a function
of residence time under ·a) oxygen sufficient conditions and
b) reduced oxygen concentrations.
a b .. .. "'
.,, ~
.. 'O
:~ f .. "8 iii ., i '° ~
~AO ,:;: i 'O .. "li .. ? .. ~ ~ JI Jl
~ ao .. ~ JO
~ e ll' !! g $0 ii ..
~ l
Figure J. Total suspended solids biodegraded as a percentage of the
influent total solids concentration for various residence times.,
under a) Oxygen sufficient conditions and bl low oxygen concentrations.
i :g. 5 <
-129-
a b 20 20
i " i :g. .! 15 ::.
j .... l
L .
Piqure 4.
a
•
Figure 5.
Residence Time (d)
Total suspended solids removal rate as a function of residence
time and a) oxygen sufficient conditions and b) reduced oxygen
concentrations.
b
~ ~ •
Residence Time (di
Ash content in feed yeast cell suspension (left hand bar of each
pair) and in the bioreactor (right hand bar) as a function of
residence time under a) oxygen sufficient conditions and
b) low oxygen concentrations.
i eo a
I 0 .. iii ~
31 •• "ll ...
'" "' .!! j 20 !$ .. "' .\!! ti •• li "-
F:j.gure 6.
a
i §
8 " 3 " 'i ~ ... ~
j
' Figure 7.
-130-
i •• b ,, i ,, 0 .. iii
j ~o a: 1 lh• ill .!! "' j It) .. :ii' ! .. ;f
Residence Time (d) Resldenee Time (d)
Volatile suspended solids biode9raded as a percentage of the
influent(feedl volatile suspended solids concentration for
various residence times under a) oxygen sufficient conditions
and b) low oxygen concentrations.
b
L. s
i 0
I I
• " Residence nme (d) Residence Tl ... (d)
Dissolved organic carbon in feed yeast cell suspension
(left hand bar of each pair) and in the bioreactor (right hand
bar) as a function of residence time under a) high dissolved
oxygen concentrations and b) low dissolved oxygen concentrations.
3
-I I s
.5
0
Piqure 8.
a ...
..
Figure 9.
-131-
Residence Time (d) Carboxylic acids measure in the bioreactor as a function of
residence time under reduced oxygen conditions. No carboxylic
acids could be detected u.nder oxygen sufficient conditions.
. .. b
~ ... i .
..
Rt•ldenct Time (d)
+ NH4 -N present in the feed yeast cell suspension (left hand bar
of each pair) and in the bioreactor (right hand bar) as a
function of residence time under a) oxygen sufficient conditions
and b) low oxygen concentrations.
-132-
CHAPTER 10
AEROBIC THERMOPHILIC BIODEGRADATION
OF MICROBIAL CELLS.:
2. SOME EFFECTS OF TEMPERATURE
Summary
The effect of temperature on solubilization and biodeg-radation in a laboratory scale bioreactor under aerobic thermophilic operating conditions was assessed using a substrate consisting of whole yeast cells and inorganic nutrients. Under oxygen limited conditions, consistently better results were obtained for operation at 65°c, reflecting the true thermophilic nature of the process microbes. An operating temperature of 70°c probably exceeded the optimum for effective functioning of the thermophilic microbes and resulted in a less efficient process, whilst an operating temperature of 6o0 c was intermediate with respect to its effectiveness.
-133-
Introduction
i During recent years, the process of aerobic thermophitl.ic waste sludge
treatment has been introduced at several municipal sewaf e and industrial
wastewater treatment plants in Austria, Swi t.:erland nd West Germany
prior to conventional anaerobic treatment. Such pre-tr atment processes
offer both hygienization, i.e., the destruction of pat ogenic organisms
pres.ent in the feed, and solubilization, i.e., partiall degradation of
some of the organic particulate material in the s1u9ge. In addition,
some pollutants sorbed on primary sludge which are essentially
recalcitrant under anaerobic treatment conditons are b1' degraded.
Thermophilic operation can be economically achieved from heat-energy
generation by the process microbes during aerobic bi egradation. Only
minor external heat input is required provided that possibilities for
effective heat exchange between processed and Jaw sludges are
incorporated and heat loss to the surroundings is min~mized (Hamer and
Zwiefelhofer, 1986).
Whilst some aerobic thermophilic treatment plants have been reported to
operate between 44°c and 55°c (Gould and Drnevich, !1978; Jewell and
Kabrick, 1980; Wolinski, 1985) temperatures between ~o0c and 10°c are i
also used (Zwiefelhofer, 1985). At these higher teml;>eratures, truely
thermophilic process microbes, i.e., microbes with ph~siological optima
at elevated temperatures, as opposed to the thermot,lerant mesophiles
which are physiologically adapted for survival only al temperatures up
to 55°c, above which they are unlikely to be found, arj able to develop
and assert their potential.
The object of this investigation was to determine the temperature
optimum for the solubilization and degradation of whol+ microbial cells,
as a basis for rational design of microbial solit destruction in
4, 0.5 mg 1-l MgC1 2 , 0.2 g 1-1 . The bioreactor feed was stored at
4°c for no longer than 5 days.
Analytical.
Dry weight: 5 ml samples were centrifuged at 30,000 x g and the
supernatant removed. The solids were resuspended in water, poured into
tared crucibles and heated overnight at 105°c. They were re-weighed
after cooling in a desiccator.
Ash/volatile suspended solids: After drying overnight, the crucibles
used to determine dry weight were placed in an oven at 60o0 c for ca. 5
h, allowed to cool in a desiccator and then re-weighed.
Dissolved Organic carbon: Appropriate dilutions of the supernatant were
made and the DOC assayed directly with a TOCOR 2 total organic carbon
analyser (Maihak AG, Hamburg, D) • Inorganic carbon was removed by
-135-
acidification using concentrated HCl, and the excess co2 removed by
sparging with N2 for 12 minutes.
+ NH4 ~: This was measured in the supernatants using an automated
nitrogen analyser (Skalar, Breda, NL).
Carboxylic Acids: Low molecular weight carboxylic acids were analysed by
acidifying the supernatants with formic acid and injecting into a
Shimadzu GC-RlA gas chromatograph (Shimadzu, Kyoto, J).
Chemicals: All chemicals were of analytical grade and supplied by either
Fluka (Buchs/SG, CH) or Merck (Darmstadt, D).
Results and Discussion
Three operating temperatures, 60°c, 65°c and 7o0 c, were selected for
examination of the solubilization and biodegradation of whole microbial
cells. The reason for this selection was that autothermal operation
could be used to achieve such temperatures during technical scale
operation and that such temperatures would allow rapid and effective
hygienization. Bioreactor residence times were varied between 1.5 and 5
days at each temperature with the exception of 70°c, where only 1.5 and
3.0 day residence times were examined. The combined results are shown in
Figs. l to 6, where the various measured parameters, total suspended
solids (Fig. + 1). DOC (Fig. 2) and NH4 -N (Fig. 3) are shown in bar
charts together with the percentage solids removal (Figs. 4 and 5) and
the soldis removal rate (Fig. 6) obtained by calculation.
Consistently better results were achieved for operation at 65°c compared
with the other temperatures used. A residence time of 1.5 days at 70°c
resulted in very poor solubilization/biodegration of the microbial
cells, whilst extreme solubilization/biodegradation occurred with a 5
-136-
day residence time at 60°c. However, the solids removal rate under the
latter set of conditions was low when compared to removal rates at
shorter residence times. The maximum substrate removal rate (5.4 g 1-l
d-l) was achieved using 65°c and a 1.5 day residence time, a slightly
lower substrate removal rate resulted at the same residence time at 60°c
(4.9 g 1-l d- 1). Operation at 3 days and 65°c also resulted in a high
rate of substrate removal (4.0 g 1-l d-l).
+ NH4 -N accumulated under all conditions, although at short residence
times this was hardly detectable. Ammonia production is indicative of
deamination of the particulate nitrogen containing fraction of the + biomass and thus an increase in NH
4 -N content should reflect the extent
of solubilization. This process probably occurs slower than the overall
solubilization of the organic matter, hence the low level of + + accumulation of NH4 -N at short residence times. Some NH4 -N will also
be lost by stripping in the exhaust gases as a result of non-optimal
condenser operation and thus the measured concentrations can only be
considered indicative of effective solubilization.
An increase in the level of dissolved organic carbon was found under all
process operating conditions. This is the primary parameter indicative
of the extent of solubilization. Accumulation of dissolved organic
carbon occurs as a consequence of carboxylic acid production from the
oxygen limited metabolism of the process microbes. Table 1 shows the
amounts of the various acids produced. Acetate was consistently found in
the highest concentrations. The very low production of carboxylic acids
for 60°c and 3 day residence time indicates that during this set of
operating conditions oxygen was probably not limiting growth.
The solubilization/biodegradation of whole yeast cells has been shown to
be optimal under conditions of low dissolved oxygen (Mason et ~· 1986).
From the results reported here, operation at a temperature of 65°c would
also appear desirable for an effective process.
-138-
The rate limiting step for particulate substrate biodegradation is the
solubilization of the organic matter, a fact recognised by Eastman and
Ferguson (1981) for aerobic sludge treatment. The optimisation of
temperature could thus be only necessary for enhancing the capacities of
those process microbes producing the yeast cell wall hydrolysing
enzymes, with the remaining components of the mixed population adopting
the role of opportunists.
The physical effects of increasing temperature in bioreactors are also
important. Higher temperatures increase the vapour pressures,
diffusivity and ionization of many compounds, and it decreases viscosity
and gas solubility (Zeikus, 1979). The influence of temperature on
diffusion coefficients for gases was expressed by Scheibel (1954) as
D= T/Fp
where T, is the absolute temperature, P• the liquid viscosity and F, the
diffusion factor. Hamer and Bryers (1985) showed that the oxygen
diffusion coefficient in water increases markedly with increasing
temperature and the increase largely compensates for the reduction in
oxygen solubility with increasing temperatures. Thus, both the
physiological properties of microbes and the variability of the physical
properties of their environment dictate the optimum conditions for
effective process operation in systems of a heterogenous nature.
-139-
References
Brock TD (1969) Microbial growth under extreme conditions. In: Meadow P and Pirt SJ (eds) Microbial growth. Proceedings 19th Symp Soc Gen Microbial. Cambridge University Press, Cambride, p 15.
Eastman JA and Ferguson JF (1981) Solubilization of particulate organic carbon during the acid phase of anaerobic digestion. J. Wat Poll Cont Fed 53: 352-366.
Gould MS and Drnevich RF (1978) Autothermal thermophilic aerobic digestion. Env Engng Div Proc of Am Soc Civ Engnrs 104: 259-270.
Hamer G and Zwiefelhofer HP (1986) Aerobic thermophilic hygienization -a supplement to anaerobic mesophilic waste sludge digestion. IChemE symposium series 96: 163-180.
Jewell WJ and Kabrik RM (1980) Autoheated aerobic thermophilic digestion with aeration. J Wat Poll Cont Fed 52: 512-523.
Mason CA, Hamer G, Fleischmann Th and Lang C (1986) Aerobic thermophilic biodegradation of microbial cells: 1. Some effects of dissolved oxygen concentration. Appl Microbial Biotechnol.
Sonnleitner B and Fiechter A (1983) Advantages of using thermophiles in biotechnological processes: expectations and reality. Trends in Biotechnol 1:74-80.
Wolinski WK (1985) Aerobic thermophilic sludge stabilisation using air. Wat Poll Cont 84: 433-445.
(OC) (d) -1 (mg l ) -1 (mg 1 ) -1 (mg l ) -1 (mg l ) -1 (mg 1 ) (mg -1 l )
60 1.5 2365 322 83 130 19 72
60 3.0 00 27 23 22 NFa NF
60 5.0 2700 347 84 138 NF NF
65 1.5 2813 846 158 164 79 130
65 3.0 1552 419 208 160 96 144
65 5.0 1300 521 183 144 85 235
70 1.5 1121 181 61 28 25 31
70 3.0 2869 410 168 44 53 47
(a - none found)
Tab1e 1. Carboxylic acid production during oxygen limited aerobic thermophilic digestion of
whole yeast cells as a function of temperature and bioreactor residence time.
Valerate -1 (mg l )
22
NF
NF
87
42 I
24 ..... ... 0 I
23
7
••
.. i :g
~ 30
i ... & IO
~ !!! ~
10
L , s
a
Figure 1.
a
Figure 2.
-141-
b c
Residence Time (d) Residence Time ldl
Total suspended solids concentration in feed (left hand bar
of each pair and in the bioreactor during aerobic thermophilic
biodegradation at a) 60°c, b) 65°c, c) 10°c.
b c
Dissolved organic carbon in feed yeast cells suspension (left
hand bar of each pair) and after solubilization/biodegradation
(right h~nd bar) at a) 60°c, b) 65°c, and c) 10°c.
a
..
Figure 3.
a
Figure 4.
-142-
b c
Residence Time (di Residence Time (di
+ NH4
- N concentration in the feed (left hand bar of each pair)
and after aerobic thermophilic biotreatment (right hand bar) at
a) Go0 c, bl 65°c, c) 10°c.
b c
Residence Time (di Rtsldenco Time (di Resi"- Time (di
Total suspended solids removal rate as a function of both residence
time and temperature during aerobic thermophilic treatment at
low oxygen concentrations. a) 60°c, b) 65°c, c) 10°c.
.. a i i Bo"° " ~ ~ •o ~ i ]~ .!!IO "' . ~
i·· ~ ~ 0
Figure 5.
00 a " .. " f .. ii5
i .. i l~ ~ !I .. ~ • J i 10
l
Figure 6.
-143-
b c
Residence Time (d) Residence Time (d) Residence Time (d)
Volatile suspended solids degraded as a percentage of the influent
(feed) volatile suspended solids concentration following
aerobic thermophilic biotreatment at a) 60°c, b) 65°c, c) 10°c.
b c
Reslda.C. Time (d) Residence Time (d)
Amount of total suspended solids solubilised/biodegraded
as a percentage of the feed yeast cell concentration during
aerobic thermophilic biotreatment at a) 60°c, b) 65°c and
c) 10°c.
-144-
CHAPTER 11
FUNDAMENTAL ASPECTS OF WASTE SEWAGE SLUDGE TREATMENT:
Abstract
MICROBIAL SOLIDS BIODEGRAOATION IN AN AEROBIC THERMOPHILIC SEMI-CONTINUOUS SYSTEM
The solubilisation and biodegradation of whole microbial cells by an aerobic thermophilic microbial population was investigated over a 72 h period. Various parameters were followed inlcuding total suspended solids reduction, changes in the dissolved organic carbon, protein and carbohydrate concentrations, and carboxylic acid production and utilisation. From the rates of removal of the various fractions a simple model for the biodegradation processes is proposed and verified with respect to acetic acid production and utilisation, and total suspended solids removal. The process is initiated by enzymic degradation of the substrate microbe cell walls followed by growth on the released soluble substrates at low dissolved oxygen concentration with concommitant carboxylic acid production. Subsequent utilization of the unbranched, lower molecular weight carboxylic acids allows additional energy supply following exhaustion of the easily utilisable soluble substrate from microbial cell hydrolysis.
-145-
Introduction
The enforcement , of increasingly stringent environmental legislation in
many countries has resulted in increased capacity for both municipal
sewage and industrial wastewater treatment by combinations of
mechanical, biological and physico-chemical process technology. The
major result of this situation is an ever increasing quantity of waste
sludge, which presents a serious ultimate disposal problem. Policies
which allow the dumping/spreading of untreated sludge both on land and
at sea are increasingly subject to criticism, necessitating the devel-
opment of effective sludge treatment technologies, Effective sludge
treatment requires the stabilization of biodegradable matter in the
sludge and the removal of potentially pathogenic organisms and toxic
chemicals from the sludge. Conventional waste sludge treatment involves
mesophilic anaerobic digestion, a technology which poses questions with
respect to its effectiveness for the removal of pathogens and some toxic
chemicals (1) • Microbes, including pathogens originally present in raw
sewage and wastewater, are a major constituent of waste sludge and the
biodegradation of such particulate matter is the key to effective
stabilization and/or hygienization. Aerobic thermophilic processes have
been proposed either as a pre-treatment stage prior to conventional
anaerobic mesophilic digestion (2) or as a complete sludge treatment
(3).
The work reported here concerns the process biology of a semi-con-
tinuous, laboratory-scale bioreactor in which microbial solids are
undergoing solubilization and biodegradation under aerobic thermophilic
conditions. In order to have a standardized feedstock, pressed bakers
yeast was used.
-146-
Theoretical
The dissolution of solids in agitated batch reactors has, since the
theoretical analysis (4) and experimental verification thereof (5) by
Hixson and Crowell, been considered to follow the cube root law. In the
case of cellulose particles, which are solubilized by enzymic
hydrolysis, Humphrey and co-workers (6, 7 ,8) have proposed a complex
shrinking-site model, which incorporates both inhibition and repression,
but ultima~ely predicts modified cube root law type dissolution. In the
case of the enzymic hydrolysis of microbial cells prior to their
utilization as carbon energy substrates by the microbes responsible for
their hydrolysis, the cube root law is inapplicable, because here the
enzymic hydrolysis involves either puncturing or bursting of the
substrate microbe cell walls, processes that depend on point attack and
point strength of the substrate microbes.
For purposes of modelling the system under investigation here, the
following processes are assumed to be occuring:
1. A feed cell population consisting of intact yeast cells is added to
a culture containing thermophilic process microbes.
2. The thermophilic process microbes produce extracellular enzymes which
are capable of cleaving the cell wall of the yeast. Enzymes attach to
the yeast cells in a non-specific distribution over the entire surface.
3. Cell wall lysis occurs at random locations on the yeast cells
resulting in release of the soluble cytoplasmic components of the
substrate particulates. Lysis results after a minimum of one site
cleavage in the wall but may require multiple site cleavage before the
wall is sufficiently weak to lyse.
4. Thermophilic process microbes utilise the released soluble substrates
as carbon and energy source and in so doing produce acetate as a result
of the low dissolved oxygen concentration. Further lysis of remainin9
-147-
whole yeast cells supplies a constant source of soluble nutrients for
the thermophilic process microbes. Enzymic degradation of the yeast cell
wall polymers supplements this supply.
5. After a significant period, the rate of supply of soluble substrates
from yeast cell lysis slows down and the thermophilic microbes begin to
utilize the now accumulated acetate to support growth and energy
requirements. Additionally, any remaining wall particulates are
hydrolysed to soluble energy substrates.
Mathematical description
The mathematics of this process are based on the following molar
stoichiometry.
Yeast Cells
Acetate
Thermophilic microbes
Thermophilic microbes
Acetate
The elemental composition of yeast cells is assumed to be the same as
that for thermophlic microbes and is expressed above on a nitrogen and
ash free basis. For the gramme yield coefficients a molecular weight of
21 g was assumed for l mole of cells inclusive of nitrogen and ash. Thus
from the above coefficients, yield values (g/g) of thermophlilic
microbes and acetate from yeast cells would be 0.3 and 0.52,
respectively. In order to describe growth of thermophilic microbes,
Monad kinetics were assumed for growth on yeast cells and Monod kinetics
with an inhibition term for growth on acetate, i.e.,
(2)
f (SS) µmax (Ss} • Ss Ks +Ss SS
µ(Ac) ~ Pmax(Ac) ' Ac.Ki (Ss+Ki) (Ac+Ksl\c)
-148-
The nomenclature is defined in Table 4.
(3)
(4)
The remaining rate expressions i.e., for yeast cell lysis, for process
microbe death/lysis, and for particulate hydrolysis, have been assumed
to be first order due to lack of information regarding the kinetics of
such processes. In the case of the particle hydrolysis rate constant the
use of first order kinetics can be justified for reasons discussed by
Eastman and Ferguson (9).
From this basic consideration, a stoichiometric matrix can be
constructed for the five parameters, x p (process microbes) x s (feed
yeast cells), Ss (soluble substrate concentration from lysed yeast
cells), P (particulate materials released from cells as a result of
lysis, e.g., cell wall fractions) and Ac (acetate) The stoichiometric
matrix is shown in Table 1.
From this, the following material balances can be described:
X : dX (5) p ____£.
dt
X : dX (6) s __ s_ dt
Ss: (7)
P: dP
dt
Ac: dAc
-149-
= Y .µ(Ss).X - p(Ac) .X Ac/Ss p p -y~~ y
Xp/Xs Xp/Ac
Materials and Methods
(8)
(9)
Bioreactor and operating conditions. A l.5 1 bioreactor (Bioengineering
AG, Wald, CH) with full measurement instrumentation, i.e. pH, dissolved
oxygen concentration, temperature, impellar speed monitoring, was used
for the experiments. Air (6 l/h) was sparged into the bioreactor which
was operated with an impellar speed of 900 rpm. The operating
temperature was maintained constant at 60°c and the working volume was
1300 ml. The pH was not controlled.
Aerobic thermophilic culture and feed. Aerobic thermophilic microbes
were obtained from an operating municipal waste sludge thermophilic
50 ml samples were fixed in glutaraldehyde (4% v/v) washed with
distilled water and frozen in a thin layer between two copper plates
according to the method of Miiller !! al (11). They were then fractured
at -1os0 c and dried at -ao0 c for 2 h in a Balzers BAF 300 freeze etching
device (Balzers AG, Lichtenstein) as described by Walther !! !! (12).
The specimens were rotary shadowed (5nm) with carbon and overlayed with
platinum carbon (5nm) and viewed in a Hitachi s-700 scanning electron
microscope (Nissai, Sangyo, J). As a consequence of this preparation
method some fracturing of surface cells occurred.
Chemicals. All chemicals used were of analytical grade and supplied by
either Fluka AG, (Buchs/SG, CH) or Merck AG (Darmstadt, D).
In this study, the biodegradation of particulate microbial solids by a
mixed thermophilic aerobic microbial culture was investigated. This
step, typical of the rate-limiting step in waste sludge biodegradation,
was followed in the semi-continuous operation of the process where whole
yeast cells were used as a stand<1rdized. substitute for the complex
biopolymeric particulates present in actual waste sludge feeds. The
thermophilic microbes in the bioreactor were preconditioned by
maintaining the same operating conditions for the immediate period prior
-152-
to the start of this investigation. At the start of the experiment 50\
of the reactor contents (650 ml) were removed and 650 ml of a yeast
suspension in the phosphate and trace element solution were added. The
addition was sufficiently slow so as to prevent any decrease in
temperature of the bioreactor. The temperature during the entire
experimental period, including the change over, was controlled at, and
never deviated from, 60°c. The pH was not controlled during the
experiment and remained constant for the first 24 hat pH 5.9 (Fig. 1).
However, after 24 h the pH increased and attained a final value of 7.68
after 72 h. Oxygen was supplied to the bioreactor in air but at a
sufficiently slow flow rate as to achieve a low dissolved oxygen
concentration. The amount of dissolved oxygen in the bioreactor (\
saturation, where saturation at was 3.03 g/l) during the
biodegradation process is shown in Figure l. The dissolved oxygen
concentration was initially very low (2\) and decreased further to an
undetectable level between 18 and 26.5 h. There followed a slight rise
to l\ after 48 h and a levelling out at 4% after 65 h.
Biodegradation was followed with respect to the decrease in total and
volatile suspended solids and the results are shown in Fig. 1. The total
suspended solid removal rates are shown in Table 2. The rate
approximately doubles after 21 h operation to 0.64 g/l.h, subsequently
decreasing to a lower value after 48 h. The quantity of solids removed
at the end of the experiment represents 77\ of the feed concentration
(41.4 g). Concommitant to the decrease in total and volatile suspended
solids, there was an increase in the amount of dissolved organic carbon
(DOC). This reached a plateau value of ca. 5.8 g/l between 24 and 48 h
(see Fig. 1) after which time the level declined.
Since the solids were being biodegraded at a fast rate, especially
during the priod between 18 and 26.5 h, it was of interest to look at
the fate of the particulate feed cells .f!!. ~ to see whether either
whole cells were being biodegraded or cell lysis was occurring leaving
behind partially intact ghosts composed primarily of cell walls. This
was conducted by carrying out light microscope direct counts of the
-153-
yeast cells. The results of these counts are shown in Fig. 2. Noticeable is the very sharp decline in yeast cell numbers during the lB to 26.5 h
period, paralleling the decrease in total and volatile suspended solids shown in Fig. 1. This decrease in whole yeast cell numbers occurred at a
high rate (Table 3) particularly between 18 and 26.5 h where rates 3.5 to 7 x the initial rate were observed. By the end of the experiment, 98\
of the whole yeast cell concentration in the feed had been either partially or totally biodegraded (Table 3) . This compares to a residual
total suspended solids concentration equivalent to 23\ of the substrate solids. This difference can be accounted for by the presence of
particulate lysed cell fractions and by the growth of the thermophilic aerobic microbes. Due to their size and thus ease of direct microscopic
quantification, yeast cell numbers are easily determined. On the other hand, bacterial cell numbers are not so easily determined due to their
morphological diversity and the imposition of a spectrum of nutrient specificities and temperature requriements for accurate quantification
(13). Therefore, a method was used which followed the activity of the
bacteria which, whilst being an indirect means of quantification,
nevertheless provides the more important information as to what is happening w,ithin the bioreactor. As shown in Fig. 2, the activity rises
significantly between 18 and 26.5 h and remains at or above this maximum value between 26.5 and 48 h until the yeast cells have been almost
totally biodegraded. The start of the rise in thermophilic microbial activity occurs simultaneous with the increases in the rate of solids removal and in pH (Fig. ll. The decline in activity after 48 h is reflected in the almost insignificant further removal of solids after
this time and is also accompanied by a decrease in dissolved organic carbon and an increase in dissolved oxygen concentration.
Direct examination of the biodegradation of yeast cells was carried out using scanning electron microscopy (Fig. 3). It should be noted that the
micrographs present qualitative rather than quantitative information as
a result of the concentration effects in their preparation procedure. Fig. 3(a) shows the substrate for the biodegradation process. This is a
discretely dispersed suspension of yeast cells with negligable bacterial
-154-
contamination. After introduction into the bioreactor, the cells are
mixed with the thermophilic aerobic microbes (Fig. 3b). The ensuing
degradation of yeast cells and growth of process microbes is shown in
Fig. 3 c-e. These electron micrographs indicate that a variety of
morphological types of microbes are present. Direct evidence for lytic
effects are difficult to discern with any degreee of confidence, but
whilst there is some suggestion that lysis is occurring (see Fig. 3e)
this implies that certainly some of the yeasts seen under the light
microscoE'Ej may be ghosts rather than intact cells. As time progresses in
the biodegradation process the yeast cells appear much more irregular
and shrunken.
If lysis is occurring it is unlikely to be as a result of direct contact
between the process microbes and the yeast cells: more likely is the
release of lytic enzymes from the thermophilic aerobic process microbes.
Therefore, it was of interest to examine the variation in the levels of
cellular and extracellular protein (Fig. 4). Cellular protein levels
(i.e. protein contained in both thermophilic process microbes and yeast
cells) decreases constantly during the course of the experiment, whilst
the level of extracellular protein remains effectively constant and even
exceeds the intracellular concentration after 72 h. The extracellular
protein was not differentiated into structural and enzymic fractions and
therefore direct evidence of enzymic activity was not obtained. However,
as the level of cellular protein decreases, - -
+ the amount of NH4 -N
released increases (Fig. 4). Both N02 -N and N03 -N concentrations were
below detection limits (1 mg/l).
The cell wall of bakers yeast (Saccharomyces cerevisiae) is composed of
approximately equal quantities of glucan and mannan, both of which are
carbohydrates which can often account for about 85\ of the dry weight of
the wall (14). Fig. 5 showe the levels of cellular and extra-cellular
carbohydrate during the biodegradation of yeast cells. In contrast to
the suspended solids and whole yeast cell numbers, the bulk of the
carbohydrate is degraded in the first 18 h (49\), with very little
activity during the 18 to 26.5 h period. Between 26.5 and 48 h further
-155-
degradation occurs resulting in a final carbohydrate concentration
equivalent to only 9' of the initial. Analogous to the extracellular
protein level, the extracellular carbohydrate remained effectively
constant during the time course.
In order to identify some of the components of the dissolved organic
carbon fraction, assays of low molecular weight carboxylic acids were
carried out during the biodegradation process. The results, shown in
Fig. 6, indicate that most of the DOC was in the form of acetate, which
reached a maximum concentration of 5.9 g/l after 26.5 h. From the data
shown in Fig. 6 it appears that the lower the molecular weight and the
less branched the molecule the earlier both production and
biodegradation occurs. Propionate also peaked at 26.5 h but at a lower
concentration (0.88 g/l). Similarly, butyrate was produced at a still
lower concentration and probably reached a maximum between 24 and 48 h,
but like acetate and propionate was fully biodegraded after 72 h. Both
iso-butyric and iso-valeric acids were present in significant quantities
after 72 h.
Discussion
The foregoing results clearly indicate the apparent complexity of the
process under consideration. They suggest a sequence of events:
1. Initial degradation of cell wall polymers resulting in the release
of soluble cell components, thus the initial slow disappearence in
whole yeast cells but fast rate of carbohydrate biodegradation.
2. Accumulation of carbo><ylic acids, particularly acetic acid, as a
result of the low dissolved oxygen concentration, and thus an
increase in the dissolved organic carbon concentration.
•156-
3. Once suitable soluble substrates become available, the process
thermophilic microbe activity is enhanced and soluble substrate is
preferentially utilized.
4. Exhaustion of the preferred substrate followed by utilisation of low
molecular weight carboxylic acids.
5. Hydrolysis of remaining cell wall fragments and utilisation of
higher molecular weight carboxylic acids.
If one simplifies this sequence of events by omitting the production of
all carboxylic acids other than acetic acid, it should be possible to
describe the process by the model presented earlier, provided
appropriate values of the constants and yield coefficients in the
material balances are selected. On the basis of the values shown in
Table 4, the predictions resulting from the model are shown in Figures 7
and 8 as complete lines. Experimental data where available, are
superimposed as points in these figures. As can be seen, a relatively
good fit is achieved, indicating that the simple model proposed is
capable of describing the overall process provided a slight degree of
simplification is introduced.
The tendancy for those carboxylic acids with higher molecular weights
than acetic acid to accumulate and subsequently to only degrade towards
the end of the batch cycle could be a disadvantage for a sludge
treatment process if used in isolation and without subsequent anaerobic
stabilization, because the presence of such carboxylic acids would
impart malodour to both treated sludge and associated supernatent.
-157-
Conclusions
Introduction of ·new technologies to achieve more efficient process
operation require a sound understanding of the fundamental aspects
involved. Process optimisation of the thermophilic aerobic sludge
treatment technology is now possible knowing the sequence of events involved in microbial solids destruction described here.
References
1. McEvoy, J.; Giger, W.: Determination of linear alkylbenzene-sulfonates in sewage sludge by high-resolution gas chromatography/mass spectrometry. Environ. Sci. Technol. 20 (1986) 376-383.
2. Hamer, G.; Zwiefelhofer, H.P.: Aerobic thermophilic hygienization -a supplement to anaerobic mesophilic waste sludge digestion. Inst. Chem. Eng. Symposium series 96 (1986) 163-180.
3. Smith, J.E. Jr.; Young, K.W.; Dean, R.B.: Biological oxidation and disinfection of sludge. Wat. Res. 9 (1975) 17-24.
4. Hixson, A.W.; Crowell, J.H.: Dependence of reaction velocity surface and agitation: I Theoretical consideration. Engng. Chem. 23 (1931) 923-931.
upon Ind.
5. Hixson, A.W.; Crowell, J ,H.: Dependence of reaction velocity upon surface and agitation: II - Experimental procedure in study of surface. Ind. Engng. Chem. 23 (1931) 1002-1009.
6. Humphrey, A.E.; Armiger, W.B.; Zabriskie, D.W.; Lee, S.E. 1 Moreira, A.; Joly, G.: Utilization of waste cellulose for the production of single cell protein. In: Continuous Culture 6: Applications and New Fields (A.C.R. Dean, D.C. Ellwood, C.G.T. Evans and J. Melling, Eds). (1976) 85-99.
7. Humphrey, A.E.; Moreira, A.1 Armiger, W.; Zabriskie, D.: Production of single cell protein from cellulose wastes. Biotechnol. Bioeng. Symp. 7 (1977) 45-64.
8. Moreira, A.R.; Phillips, J.A.; Humphrey, A.E.: Utilization of carbohydrates by Thermanospora Sp. grown on glucose, cellobiose and cellulose. Biotechnol. Bioengng. 23 (1981) 1325-1338.
-158-
9. Eastman, J.A.; Ferguson, J.F.: Solubilization of particulate organic carbon during the acid phase of anaerobic digestion. J. Wat. Poll. Cont. Fed. 53 (1981) 352-366.
10. Herbert, D. 1 Phipps, P.J; Strange, R.E.: Chemical analyisis of microbial cells. In: Methods in Microbiology 7B (J.R. Norris and D.W. Ribbons, eds) (1971) 209-344.
11. Mliller, M.; Meister, N.; Moor, H.: Freezing in a propane jet and its application in freeze-fracturing. Mikroskopie (Wien) 36 (1980) 129-140.
12. Walther, P.; Mliller, M.; Schweingruber, M.E.: The ultrastructure of the cell surface and plasma membrane of exponential and stationary phase cells of Schizosaccharomyces pombe, grown in different media. Arch. Microbiol. 137 (1980) 128-134.
13. Mason, C.A.; Hamer, G.; Bryers, J. D.: The death and lysis of microorganisms in environmental processes. FEMS Microbiology Reviews 39 (1986) 373-401.
14. Rose, A.H.: Chemical Microbiology, 3rd Edn. (1976) Butterworths, London.
-159-
~ Stoichiornet r ic matrix for aerobic thermophilic biodegredat ion process.
xP x. Ss p Ac
~ Reaction Rate
l. Substrate Cell 0 -1 Y Ss/Xs YP/Xs KL.XS Lysia
2. Porticle 0 0 +l ·l Kh.P Hydrolysis
J. Growth on +l 0 -1 0 •Y Ac/SS ~(Ss).Xp
Lysis Products YXp/Ss YXp/Ss
4. Process Microbe -1 0 +YSs/Xp +YP/Xp Kd.Xp Oeath/Lysis
s. Growth on +l 0 0 -1 µ(Ac) .xP Acetate Yxp/Ac
Note:- As the stoichiometry is incomplete, i.e., co2 and o2 are not considered, the matrix
is not balanced. The symbols used are explained in Table 4.
~ Solids removal data.
Time after addition
of substrate cells
lh)
18
21
24
2(l.5
48
72
-160-
Solids removal
rate
lq/l.h)
0. 33
0. 38
0. 64
o. 64
0. 20
0. OJ
Notes l. Solids concentration at time Oh
Solids removed
\t 1 ll \feed 0
concentration
2712)
26 46
31 50
39 56
46 61
65 74
68 77
2. Reduction of solids due to dilution of feed by bioreactor contents
Table J. Yeast cell removal data.
'I'ime after addition Whole yeaHt cell Yeast cells Removed
of substrate cells removal rate
lh) 1106/1.h) \t ill % feed 0
28 12 )
18 1. 63 22 44
21 23. 02 33 52
24 50. 54 57 69
26. 5 lJ. 23 63 13
48 6. 38 85 89
72 o. 68 98 98
Notes:- I.Yeast cell number at time Oh
2. Reduct ion of yeast ce 11 number due to dilution of feed by bioreactor
contents.
-161-Table 4. Values for yield coefficients and kinetic constants used for
calculation using the mathematical model
Stoichiometric Coefficient or Kinetic Constant
Description
YSs/Xp
Pmax(Ss)
Yield process microbes on substrate yeast cells
Yield process microbes on acetate
Yield acetate produced by process microbes on substrate yeast cells growing
Yield soluble substrate from lysis of yeast cells
Yield soluble substrate from lysis of process microbes
Yield particulates from lysis of yeast cells
Yield particulates from,.lysis of process microbes
Maximum specific growth rate constant for ,growth of process microbes on soluble substrate
Maximum specific growth rate constant for growth of process microbes on acetate
Value
0.30
0.17
0.52
0.80
0.80
0.20
0.20
0.60
0.60
Saturation coefficient for growth of process 0.80 microbes on soluble substrate
K. 1
Saturation coefficient for growth of process microbes on acetate
Death/lysis rate constant for process microbes
Inhibition constant for growth of process microbes on acetate
Lysis rate constant for whole yeast cells
Hydrolysis rate constant for particulate biomass
0.01
0.02
0.00012
0.04
0.065
Units
g/g
g/g
g/g
g/g
g/g
g/g
g/g
g/l
g/l
-1 h
g/1
-1 h
-"' 35 8
30 7.5 <I)
0 ::::; 5l 25
0 w 0 z UJ 0.. <I)
20 7
iil 15
~ ~ g 0 z <
6.5
10
6 5
0 5.5
-162-
.... ~·p···········
0 24 Tl ME (HOURS)
46
..
z 0 I-< 0:: :::J
10 6
5 -"' z 0
4~ "" u u
':;;: 5 3 z <I) "" I.!) 02 /, ........ o ;;fl 0::
0
72
N 0
2
0 0
0 UJ > --' 0 <I) <I)
0
Firo>re 1. Changes in the concentration of total suspended solids (TSSJ, volatile suspended solids (VSS), dissolved organic carbon (DOC), pH and dissolved oxygen concentration during semi-continuous aerobic thermophilic biodegradation of microbial solids with a cycle time of 3 days.
10 2.5
ACTIVITY 2.0 B ' e ...... , "' ----..... E
!2 ........... c:
" 0
" 6 1.5 "' \ ~
0:: ~ z w I 0 IXl \ :E I-:::J 4 1.0 IXl z !>._ 0::
0 --' b....,..._,YEAST
<I) --' co UJ < u ........... .... ........... 0.5 "' ....... ....... < "O........, w >- .......... .........
0 ............ o-__
0 0 24 48 72
TIME {HOURS)
Figure 2. Changes in process thermophilic aerobic microbe activity and substrate yeast cell number during semi-continuous aerobic thermophilic biodegradation of microbial cells.
-163-
Figure 3a. Scanning electron micrograph of bakers yeast
cells used as the feed for the experimental
studies on the aerobic thermophilic treatment
process.
-164-
Figure 3b. Scanning electron micrograph of bakers yeast cells immediately after addition to a culture
containing aerobic thermophilic process bacteria
at 60°c.
-165-
Fi9ure Jc. Scanning electron micrograph of the aerobic
thermophilic solubilization/biodegradation
process 24 h after the addition of the substrate
microbes to the bioreactor.
-166-
Figure Jd. Scanning electron micrograph of the aerobic thermophilic solubilization/biodegradation process 48 h after the addition of the substrate microbes to the bioreactor. A significant quantity of cell debris can be seen, together with the various morphological forms of process thermophilic microbes present.
-167-
Figure Je. Scanning electron micrograph of the aerobic thermophilic solubilization/biodegradation process 72 h after the addition of the substrate microbes to the bioreactor. Most of the yeast cells now have the appearance of being "ghosts" whereby partially intact cell walls are still present but the contents have been lost by lysis.
-168-
Figure 3f. Scanning electron micrograph of the aerobic
Changes in cellular and ~xtracellular protein concentrations and of NH 4-N during semi-continuous aerobic therm~philic biodegradation of microbial solids. No NH 4-N was a~ded with the yeast cell substrate, released NH 4-N being derived from deamination of the particulate nitrogen containing fraction of the biomass.
Changes in cellular and extracellular concentration of carbohydrates during the solubilization and biodegradation of whole yeast cells by aerobic thermophilic microbes.
Fi~re 8. Predicted values for process microbe biomass(X ) and for particulate biomass (P). No comparison with exl:erimental values was made due to the lack of data. The contribution of these biomass fractions to the total suspended solids is also shown.
-1 72-
CHAPTER 12
BIOPARTICULATE SOLUBILIZATION AND BIODEGRADATION
IN SEMI-CONTINUOUS AEROBIC THERMOPHILIC DIGESTION
Abstract
The effects of charge size, added nitrogen on the degradation of microbial semi-continuous operation digestion process. Using
cycle time and the presence of extent of solubilization/bio-solids were investigated in of the aerobic thermophilic
a charge size of 50% of the bioreactor operating volume resulted in the removal of more than 59% of the feed total suspended solids concentration, and showed enhanced performance compared to 25% bioreactor volume charge size. The addition of a supplementary nitrogen source had pronounced effects on dissolved organic carbon concentrations, with the larger charge size but did not affect overall solids removal, whereas using the smaller charge size, addition of a supplementary nitrogen source resulted in ,improved process performance.
-173-
Introduction
Microbial growth theory and a significant amount of research in microbial biodegradation processes is only directly relevant to soluble
and/or miscible carbon energy substrates despite the fact that particulate biodegradable substrates are encountered in wastewaters and
waste sludges subjected to biotreatment and in many other situations. Waste sludge biodegradation processes require initial solubilisation of
the particulate matter present prior to biodegradation of the released soluble lysis products. Despite the fact that this constitutes the rate
limiting step in waste sludge biotreatment, relatively little research has been directed towards this aspect of the process.
Recently, several west European governments have shown concern about the
effectiveness of existing waste sludge treatment technologies, particularly with regard to the removal of pathogenic organisms and of
toxic chemicals. Sludge pre-treatment under aerobic thermophilic
operating conditions has been proposed and is being investigated as a
means of improving current technology, especially when such processes are operated in conjunction with conventional anaerobic mesophilic
sludge treatment. Aerobic operation in the first stage is preferable when toxic compounds which are essentially recalcitrant under anaerobic
conditions but which can be biodegraded under aerobic are present in waste sludges (McEvoy and Giger, 1986).
Several municipal aerobic thermophilic waste sludge treatment plants
operate in the semi-continuous mode (Zwiefelhofer, 1985; Wolinski, 1985) as opposed to continuous flow mode described by most researchers (Smith
.!!!:, al., 1975, Keller and Berninger, 1981, Kabrick and Jewell, 1982). The former have the advantage of a defined minimum retention time, a factor
of critical importance with respect to the hygienisation potential of
the process.
-174-
Our understanding of the solubilization/biodegradation of microbial
solids in aerobic thermophilic biotreatment systems remains scant. Kamer
and Mason (in press) have proposed a model for the biodegradation of
microbial solids in a semi-continuous system whereby growth of the
aerobic thermophilic population occurs on the released soluble compounds
contained in the cell cytoplasm following enzymatic degradation of a
part of the feed microbe cell wall. The cytoplasm is under a high
osmotic pressure such that lysis of the feed microbe cell wall easily
causes release of the cell contents. Growth on this soluble substrate
occurs simultaneously with the production of acetate when the system is
run under oxygen restricted conditions. As time progresses, the
remainder of the cell wall fractions are hydrolysed into utilisable
soluble substrates whilst the accumulated acetate is used as an
additional growth substrate. The effects of varying the charge size and
cycle time (minimum particle residence time) on the performance of this
process cycle has never been investigated in detail. Moreover, most
treatment plants operate only on weekdays whereby on Mondays to Fridays
a 24 h cycle is normal, whereas charging the reactor between Friday and
Monday allows a 3 day cycle time. Therefore, it was also of interest to
examine the effects of cycle time on process performance.
The final variable investigated in these experiments was the question as
to whether there was a requirement for addition of further nutrients.
Whilst phosphate is most unlikely to be limiting in such processes, the
availability of nitrogen could well be a problem. Nitrogen, when not
present in excess would have to be obtained from organic-N components of
the feed microbial cells. The differences in behaviour of process
systems in the presence of excess inorganic-N and under reduced availa-
bility of such sources were assessed.
-175-
Materials and Methods
Bioreactor and operating conditions. A l.5 l bioreactor (Bioengineering
AG, Wald, CH) with full measurement instrumentation, i.e. pH, dissolved
oxygen concentration, temperature and impellar speed monitoring, was
used for the experiments. Air (6 l h -l) was sparged into the bioreactor
which was operated with an impellar speed of 900 rpm. The operating
temperature was maintained constant at 6o0 c and the working volume was
1300 ml. The pH was not controlled.
Aerobic thermophilic culture and feed. Aerobic thermophilic microbes
were obtained from an operating municipal waste sludge aerobic
Figure l. Total suspended solids in the feed (first bar), after 7 h (middle bar) and after 24/72 h during semi-continuous aerobic thermophilic solubilization/biodegradation of microbial solids, as a function of cycle time, the presence (+N) or absence (-N) of inorqanic-N in the feed and of the charge size (25% and 50\ of bioreactor operating volume).
251 501 .. .. +N -N +N -N +N +N
" ••
..
.. "
Cycle Tim• (d)
-N
-N
Figure 2. The amount of total suspended solids biodegraded as a percentage of the concentration in the feed after 7 h (left hand bar, where data available) and after 24/72 h during semi-continuous aerobic thermophilic solubilization/biode9radation of microbial solids, as a function of cycle time, the presence (+N) or absence (-N) of inorganic-N in the feed and of the charge size (25\ and 50\ of bioreactor operating volume) .
..
J! ..
I r· i ..
Figure 3.
-183-
2U SOX .. -N +N -N +N -N +N -N
,. ,.,,
T :::::::
"'· I .. ·;:: f::i / .. :::~::
.... :. ·.-:• ::.~ .. ::~::: :·:·::· ~i ".'/
~~r .. ::·:. 0 .....
it .. .... ..... :~:::
•• I ~~~~~ ~~i~
~~ 71 •• .. ,
·~{~~ ~ ::i::
....,
,, TI' ... (d) Cycit ..
The amount of volatile suspended solids biodegraded as a percentage of the concentration in the feed after 7 h (left hand bar, where data available) and after 24/72 h during semi-continuous aerobic thermophilic solubilization/biode9radation of microbial solids, as a function of cycle time, the presence (+N) or absence (-N) of inorganic-N in the feed and of the charge size (25• and 50~ of bioreactor operating volume).
+N -N +N -N
Figure 4. Dissolved organic carbon concentration in the feed (first bar), after 7 h (middle bar) 'and after 24/74 h during semi-continuous aerobic thermophilic solubilization/biodeqradation of microbial aolids, as a function of cycle time, the presence (+N) or absence (-N) of inorganic-N in the feed and of the charge size (25\ and 50• of bioreactor operating volume).
-184-
25% 50%
-N
• Figure 5. NH4
-Nin the feed (first bar), after 7 h (middle bar) and after 24/72 h during semi-continuous aerobic thermophilic solubilization/biodegradation of microbial solids, as a function of cycle time, the presence (+N) or absence (-N) of inorganic-N in the feed and of the chaxge size (25% and 50% of bio~eactor operating volume}~
Time (h) Variation in pH during aerobic thermopbilic stabilization/• biodeqradation of microbial solids, as a function of the cycle time and of the presence (+N) or absence (·N) of inorganic-N in the feed using a charge size of 25• of the bioreactor operating volume.
!!l!!!!..1• Variation in pll during aerobic thermophilic stabilization/-biod•tradation ot llticrobia1 •olida, •• a function of the cycle time and of the preaenca (+Nl or absence (-Nl of inorganic-N in the feed uaing a char9e size of SO• of the bioreactor operating volume.
-186-
CHAPTER 13
SOME FUNDAMENTAL ASPECTS OF TWO STAGE WASTE SEWAGE
SLUDGE TREATMENT WITH AEROBIC THERMOPHILIC PRETREATMENT
ANO ANAEROBIC STABILIZATION
Synopsis
The design and effective operation of aerobic thermophilic waste sewage sludge pretreatment processes requires a detailed understanding of the process microbiology. In this paper the mechanisms of microbial solids biodegradation are defined in relatively simple terms and a realistic process model is developed. The problems associated with pathogen survival are examined in detail and the facets involved in both the reinfection and regrowth of pathogens in treated sludges are discussed.
-187-
J.. Introduction
Increasingly stringent environmental legislation in most West European
countries has resulted in increased capacity for municipal sewage and
industrial wastewater treatment by combinations of mechanical,
biological and physico-chemical process technology. The major by-product
of both mechanical and aerobic biological treatment processes is waste
sludge, a putrefactive, aqueous suspension of biodegradable, partially
biodegradable and essentially non-biodegradable solids and similarly
degradable dissolved and sorbed matter. Waste sludge presents a serious
disposal problem, particularly in regions remote from the sea, where
frequently the policy for ultimate disposal involves spreading treated
sludge on agricultural land so as to cause minimum nuisance.
and then slowly decreases with increasing residence times.
-196-
(iv) Total suspended solids (feed and process microbes plus
particulate matter) decreases gradually with increasing
residence times.
(v) Soluble substrate (residual lytic products from feed and
process microbes) concentration initially drops
precipitously and remains at a very low level with
increasing residence times.
(vi) Acetate concentration first increases and then decreases
with increasing residence times.
These predictions have been experimentially verified in a model process
system, discussed elsewhere (12), where an aerobic thermophilic mixed
process culture was grown on a standardized microbial solids substrate
(bakers yeast) to simulate secondary sludge in a completely mixed,
aerated bioreactor maintained at 6S0 c and operated at various residence
times. Some results for total suspended solids and acetate
concentrations from this investigation are shown as points in Figure 3.
These experimentally determined values confirm the trends indicated by
the theoretical analysis, but the fit between theory and experiment is
by no means perfect; a feature which undoubtedly results frOlll the high
degree of simplification particularly ignoring all cartioxylic acids
other than acetic acid, used to describe such a highly complex process.
It should also be noted that the values attributed to two constants
involved in the process model, the lysis rate constant for process
microbes and the inhibition constant for acetate utilization, markedly
affect the predicted level of acetate accumulation.
The predictions clearly indicate that aerobic thermophilic treatment
processes are best used as pretreatment processes. To achieve complete
stabilization of microbial solids solely by aerobic thermophilic
treatment would require residence times well in excess of S ~ys, and it
-197-
seems probable that such processes would not compete effectively, on
either technical or economic grounds, with conventional anaerobic
mesophilic stabilization processes. However, the predictions clearly
indicate the high levels of activity that occur in such processes when
operated with residence times of less than two days, particularly where
high rates of biodegradation are coupled with effective hygienization
which is so when they are used as pretreatment processes. Acetate
accumulation during pretreatment can only be regarded as beneficial for
the rate of the subsequent anaerobic stabilization process.
6. Sludge eygienization
The objectives of all sludge hygienization processes is the destruction
of pathogenic bacteria, viruses, worm eggs and protozoa which can
potentially result in serious diseases in humans and animals after
disposal of treated waste sewage sludge.
Pathogenic organisms have optimum temperatures for growth below 45°c and
most are either inactivated or "killed" at temperatures in excess of
so0 c, hence the susceptibility of pathogens to thermophilic treatment
processes.
Bacteria of enteric origin such as Escherichia ££!.!:. and Klebsiella
pneumoniae are used as indicators of more serious pathogens. Thus, ~·
coli and K. eneumoniae, which are both themselves potentially
pathogenic, are monitored together with other coliforms to indicate the
possible presence of more seriously pathogenic microbes such as
Salmonella spp. and Vibrio spp. such an approach is embodied in the
Swiss Federal Ordinance concerning sewage sludge treatment and disposal
(2), as mentioned earlier. The ability of bacteria to survive heat
treatment is a function of the exposure time and the temperature used.
However, the physiological state of the bacteria is also significant.
-198-
The realization of effective sewage sludge hygienization involves two
major facetsi the death/irreversible inactivation of pathogens and
prevention of subsequent reinfection with and regrowth potential of
pathogens. In order to investigate these facets, experiments haVE< been
undertaken in which the bacterium !· pneumoniae was grown aerobically at
35°c and pH 6.80 under steady state conditions in a bioreactor operated
as a chemostat and the spent culture fed to a second bioreactor where
the bacteria were subjected to heat treatment under asceptic conditions,
constant pH and aeration. The experimental system ill shown
diagrammatically in Figure 4.
1· Bacterial survival
The survival of!· pneumoniae cells exposed to temperatures of 35°, 41°,
49°, 55° and 60°c with a mean residence time of 32 h in the second
bioreactor was determined using measurement of metabolic activity in an
INT [2-(p-iodophenyl)-3-(p-nitropnenylJ-S-phenyltetrazolium chlorid~
reduction assay (13), rather than bY means of inac:;curate and
inappropriate agar plate count techniques. A significant decrease in
overall metabolic activity was found between microbes growin9 in the
chemostat at 35°c and those present in the second bioreactor when
maintained at 3S0c. Increasing the temperature of the second bioreactor
resulted in further decreases in metabolic activity at 41° and 49°c,
although the levels found were significant in that they indicated the
bacteria were still capable of metabolic reactions despite the
super-optimal temperatures. At 55° and 60°c the measured metabolic
activity was extremely low, but not zero. Thus, despite temperatures in
excess of the minimum for effective deactivation, metabolic activity
could still be detected.
-199-
This low level of metabolic activity found after apparently prolonged
heat treatment at either 55° or 60°c in a continuous flow bioreactor
suggests a potential for regrowth under conditions where short
circuiting of feed in the bioreactor can occur and where appropriate
substrates and nutrients are available, as would be the case in sewage
sludge treatment processes where overall stabilization was incomplete.
In sewage sludge treatment, the heterogeneity of the process feed could
enhance short circuiting in continuous flow hygienization processes.
8. Reinfection and R!growth
In order to investigate reinfection and regrowth after heat treatment of
K. pneumoniae cells at 49°, 55° and 60°c, the flow of spent culture
fluid from the chemostatically operated bioreactor was stopped, but the
operating temperature of the second bioreactor was maintained. After 4 h
the temperature of the second bioreactor was reduced to 35°c and a flow
of fresh sterile medium added such that the mean residence time was 12.5
h. The ensuing washout and regrowth of the culture in the second
bioreactor was monitored and the results are shown in Figure 5, where
regrowth after a distinct lag, can clearly be seen after treatment at
all three temperatures. Checks were carried out to confirm that the
growing bacteria were in fact, K. pneumoniae. The most plausible
explanation for the regrowth is reinoculation of the cooled culture by
head space splash drainage rather than survival of bacteria in the
liquid culture, particularly as the bioreactor head space would never
have reached the heat treatment temperature. Wall growth is, of course,
commonly encountered in the head space of continuous flow bioreactors
(14).
The consequences of this, is that in any incompletely filled bioreactor,
the head space will contain a source for reinoculation, by head space
drainage, even under conditions where careful control is carried out.
-200-
!· Concluding Relnarks
Aerobic thermophilic pretreatment processes of fer a realistic technology
for achieving effective hygienization of waste sewage sludges. However,
the experimental results presented clearly indicate that ~th process
design and operating strategies must be based on an adequate
understanding of the process microbiology of such systems. The results
also suggest that thermophilic hygienization processes must be coupled
with fully effective stabilization processs if treated sludge is to meet
stringent standards prior to ultimate disposal on agricultural land,
It is unlikely that the use of aerobic thermophilic processes for
complete sewage sludge stabilization, as a single stage process, will be
able to effectively compete with conventional anaerobic mesophilic
treatment processes, on either an economic or a technological basis,
although their use as a pretreatment process, could, in addition to
achieving effective hygienization, also increase process ratee during
subsequent stabilization.
-201-
References
1. Giger, W., Brunner, P.H. and Schaffner, c. (1984). 4-Nonylphenol in sewage sludge: Accumulation of toxic metabolites from nonionic surfactants.· Science, 225, 623-625.
3. Husmann, W. and Malz, F. (1959). Untersuchung zur biologischen Abwasserreinigung auf aerober thermophiler Grundlage. GWF-Wasser/Abwasser !.Q.2. 189-193.
4. Bull, A.T. and Quayle, J.R. (1982). New dimensions in microbiology: an introduction. Phil. ~· .!'!£l· ~· ~· ~· ~ 297, 447-457.
5. Eastman, J.A. and Ferguson, J.F. (1981). Solubilization of particulate organic carbon during the acid phase of anaerobic digestion. l· .!!!!!· ~· ~· ~· 22_, 352-366.
6. Hixson, A.W. and Crowell, J.B. (1931). Dependence velocity upon surface and agitation: I consideration. Ind. Engng. ~· 23, 923-931.
of reaction Theoretical
7. Hixson, A.W. and Crowell, J .H. (1931). Dependence of reaction velocity upon surface and agitation: II- Experimental procedure in study of surface. ~· Engng. Chem. !]_, 1002-1009.
8. Humphrey, A.E., Armiger, W.B., Zabriskie, D.W., Lee, S.E., Moreira, A. and Joly, G. (1976). Utilization of waste cellulose for the production of single cell protein. In: Continuous Culture 6: Applications and New Fields (A.C.R. Dean, D.C. Ellwood, C.G.T. Evans and J. Melling, Eds), 85-99.
9. Humphrey, A.E., Moreira, A., Armiger, w. and Zabriskie, o. (1977).
10.
Production of single cell protein from cellulose wastes. Biotechnol. Bioeng. !r.!!!f· z, 45-64.
Moreira, A.R., Phillips, J.A. and Humphrey, Utilization of carbohydrates by Thermanospora glucose, cellobiose and cellulose. Biotechnol. 1325-1338.
A. E. (1981). Sp. grown on Bioengni. !]_,
11. Harrison, D.E.F. and Loveless, J.E. (1971). The effect of growth conditions on respiratory activity and growth efficiency in facultative anaerobes grown in chemostat culture. l· ~· Microbiol. ~· 35-43.
-202-
12. Mason, C.A., Hamer, G., Fleischtmlnn, Th. and Lanc,J. c. Aerobic thermophilic biodegradation of micr0bial cells; 2. Some effects of temperature (Manuscript submitted for publication).
13. Lopez, J.M., KooJ?1114n, B. and Bitton, G. (1986). INT~dehydroqenase
test for activated sludge process control. Biotechnol. Bioen9. ~· 1080-1085.
14. Hamer, G. (1972). Discussion used for the cultivation Biotechno1. Bioen9. 14, 1-12.
on entrained droplets in fermentere of single-celled microorganisms.
Nomenclature
YXp/Xs
YA<;/Ss
Ks Ss
Ks Ac
Yield process thermophile microbes on feed microbial cells
Yield process microbes on acetate
Yield acetate produced by process thermophiles growing on feed microbial cells
Yield soluble substrate from lysis of feed microbes
Yield soluble substrate from lysis of process thermophile microbes
Yield particulates from lysis of feed microbes
Yield particulates from lysis of process thermophile microbes
Maximum specific growth rate con-stant for growth of process microbes on soluble substrates
Maximum specific growth rate con-stant for growth of process microbes on acetate
saturation coefficient for growth of process microbes on soluble substrate
Saturation cqef ficient for growth of process microbes on acetate
-203-
l(d Death/lysis rate constant for process T-1 microbes
I(. Inhibition constant for growth of -1 1 process microbes on acetate ML
KL Lysis rate constant for feed microbes T -l
l(h Hydrolysis rate constant for parti- -1 culate biomass T
D Dilution rate T -1
x So Microbial cell concentration in feed ML -1
F, F2 Hydraulic inflow and outflow rates LT -l
x,x• Steady state bacterial concentrations ML-1
-204-
Table l. - StoichiQMetric matrix for aerobic ther1110phi lie b iode9radation proceaa.
I /1 '· ·-.. -.::::_ · ---.................. rss I I / ...... ~.~·-... ·:·-1·---. ....... ·. -··· I : -.. ·. Parr-··· I l : ·-.. ..... \ tcu1a1es-,.....-·-
Figure J. Steady state solution of mathematical model for aerobic thermophilic sludge tretment. Stoichiometric and kinetic constants used: YX /X , 0.3 g g-1 ,
-1 p -J: -1 Pmax(Ss)' 0,6 h ; Ksss' 0.8 g l ; Kd, 0.004 h ;
-1 -1 Y~~/Ac' 0.17g g ; ~Tax(Ac)' 0.6 h_
1; Ki, 0.00012 g
1-1 KsAc' 0,01 :11 i KL, 0.02S h -i YSs/Xs' 0.8 g g-l Kh' 0.06S h ; Y§!/Xp' 0.8 g g -i YP/~s' 0.2 g 9 YAc/Xs' 0.52 g g ; x50 , 40 g l • Points represent experimental values for total suspended solids (TSS) (A) and for carboxylic acid concentrations (o).
Figure 4. Two continuous flow stirred tank bioreactors in series showing steady state operating conditions. The outflow from the first bioreactor was used as the sole feed to the second bioreactor which was maintained at temperatures of 35°, 41°, 49°, 53° and 6o0 c with a residence time of 32 hours.
-208-5 4 A
E 3 c
<.O 2 -.:!' t.n c 0 :.;:: c.. 1 ... 0 U)
D <(
E c;
<.O ...;:t c.o c 0 :.;::; c.. ... 0 U) .D <(
E c:: <.O ...;:t t.n c:: .2 -c.. ._ 0 U) .D <(
81 0.7 0.6 0.5
4
3
2
1 o.9 0.8 0.7 0.6 0.5
4
3
2
1 09 0.8 0.7 0.6 0.5
-4
8
c
-2 0 2 4 6 Time(h)
8 10 12
~egrowth of ~· pneumqniae cu1turee at 35°c after heat-treatment
at; A, 49°ci B, ss0c and c, 60°c. Tl\e e~pected washout curves are
also shown(---). Before initiating a flow of carbQn and
nutrients, the culture was ""'intained at the elevated temperature
for four hours without a flow of fresh cells from the first
bioreactor.
-209-
CHAPTER 14
GENERAL DISCUSSION
Whilst each chapter contains
overall discussion presented
consequences and implications
Cldditional information, where
drawn.
discussion of the results presented, the
here concentrates on the more general
of the work and further, provides
necessary, to enforce the conclusions
Microbial death, lysis and "cryptic" growth - Fundamental Aspects
Microbial solids destruction, whether in technical-scale wastewater
treatment processes, during optimised growth, during unbalanced growth
or as a result of externally applied stresses is a topic which has been
largely ignored and is, therefore, little understood when compared with
the available knowledge concerning microbial growth and growth related
phenomenon. That microbial growth and microbial death/lysis are
processes which effectively occur coincidentally in most real systems is
often overlooked, and has frequently led to either over simplification
or to erroneous data interpretation.
Much of the data concerning microbial death is found in the food and
pharmaceutical literature where contaminating microbes are subjected to
E!Xtreme stresses so that products meet public health safety
requirements. However, as mentioned in the conclusions to chapter two,
microbes are neither immortal nor are they the infallable machines that
they are often made out to be. Even under idealised growth conditions,
mistakes can occur - resulting in death/lysis phenomena.
-210-
At the start of the experimental programme, it was hypothesised that the
processes of death, lysis and "cryptic" growth were all events which
could occur in a growing culture of microbes. To test this hypothesis,
major problems were encountered in both defining what was meant by these
terms and in the selection of satisfactory methods which could be used
to measure the rates of the respective processes.
As a result of the model presented in chapter three and the use of the
INT-reduction assay as a means of quantifying active microbe
concentrations (see appendix for structure of INT) it became clear that
certainly in bacterial mono-cultures, death and lysis are synonymous, in
that death is more likely to occur by lysis than by failure of metabolic
processes. Even in situations where a gradual decline in the metabolic
activity of individual cells occurs, because of the finely balanced
control mechanisms required to maintain the integrity of the cell wall
and membrane structure, and hence, the individual entity, lysis is
likely to precede total metabolic shutdown due to an imbalance in
homeostatic mechanisms. In multicellular organisms the definition ol:
death presents additional problems since a part of the organism might
still maintain metabolic potential whilst the whole fails to function as
an integrated entity. However, such problems in multicellular organisms
are outside the scope of this discussion and, the theories presented.
here are therefore, only applicable to unicellular microbes. In
bacteria, the absence of compartmentalisation allows death to be defined
as a total absence of metabolic activity in an otherwise intact cell.
The situation pertaining to laboratory cultures of microorganisms may
also apply to natural environments where complete shutdown of metabolic
activity, resulting in the production of intact dead cells may be a rare
event such that it is likely that here also actual death is preceded by·
lysis.
Lysis has been a recognised fate of bacteria for a long time. Stolp and
Starr (1965) described the fate of an individual bacterium undergoing
lysis as a kind of death which has no parallel in higher organisms. They
suggested that, beyond the loss of vital functions (metabolism, growth
-211-
and reproduction) death by lysis is characterised by the concurrent and
abrupt dissolution of the individual. Involuntary autolysis, or the
spontaneous lysis of a cell without the aid of an exogenous agent can
occur whenever the metabolism of the cell is drastically disturbed. For
example, in cultures of Bacillus subtilis growing exponentially,
autolysis can be induced either by a sudden interruption of oxygen
supply in aerated cultures or by the addition of metabolic inhibitors
such as KCN, NaN3 or dinitrophenol (Meyer, 1985). In the natural
environment, opportunists, microbes which are capable of inducing
bacterial lysis, have evolved and are able to grow on microbial lysis
products. This is typical of the behaviour of the Myxobacterium spp.
which are commonly found in soil and in waste sludge. Attempts have been
made to harness such behaviour by using these bacteria, and others with
similar potential, in the biological control of cyanobacterial blooms in
fresh surface waters (Burnham, 1981; Wright and Thompson, 1985). Such
processes have also been shown to apply to bacterial growth on the lysis
products of green algae in the epilimnion of lakes and could thus be of
importance in nutrient cycling in such environments (Uehlinger, 1986).
Chapman and Grey (1986) recently discussed the importance of the lysis
of dying microbes with respect to recycling of nutrients in the soil
environment via the process of "cryptic" growth, and "cryptic" growth
has also been recently recognised as a feature in animal. cell culture
where the effects of shear in bioreactors containing hybridoma cells can
result in the lysis of these fragile, shear-sensitive, wall-less cells
thus providing aditional substrate availability (D.F. Ollis, Personal
Communication) .
The model presented in chapter three can be simplified if all the
processes resulting in the production of dead cells are omitted from the
scheme. The result describes a culture in which the viable biomass is
reduced predominantly as a result of lysis phenomena, but with a
hitherto unproven loss by genetic inactivation. This latter process is
certainly relevant when a culture is subjected to super-optimal but not
growth inhibitory temperatures and capitalisation of the sustained
metabolic potential of such cells could have benefits in
-212-
biotechnological production processes requiring high product yields with
concommitant low biomass yield coefficients. Similar applications of
this concept could be achieved by selective irradiation of a culture
such that low level genetic damage results, thereby preventing cell
replication but not inhibiting metabolic activity. Obviously in
continuous culture processes such action could be directed towards a
significant fraction of a culture, simultaneously ensuring that a
sufficient fraction of "replication-positive" cells remains to prevent
washout. Experimentation must follow the development of techniques which
can distinguish DNA injury with respect to replication, such as by
specific enzyme assays for DNA polymerase, or by flow cytometry.
Since lysis is the most likely cause of biomass reduction in continuous
cultures, two product classes result; soluble organic material i.e.,
proteins, nucleic acids, carbohydrates etc. and particulate organic
material. Whilst measurement of the dissolved fraction is relatively
simple, experimental quantification of the particulate fraction is more
difficult. Differentiation between particulate matter and soluble matter
is usually artificial, i.e., when does particulate matter cease to be
particulate and be considered soluble? In the experimental procedure
described in chapter three, a particulate was artificially defined as
being anything that fails to pass through a 0.4pm membrane filter. To
demonstrate that this definition is not necessarily adequate, the
following experiment was conducted:
From a steady state chemostat culture of !· pneumoniae (D=0.077 h-1),
200 ml culture broth was removed and centrifuged at 15 1 000 x g for 10
min. The supernatant was decanted and filtered through a series of
different pore-size filters and the DOC in the resulting filtrates
determined. The results are shown below in Table 1.
A 14% difference in the amount of DOC present can be demonstrated when
the DOC in the filtrates are compared after filtration through very
small and very large pore size filters. The quantification of
particulate species in a heterogenous liquid environment is, therefore,
-213-
complex and detection may require the application of highly
sophisticated techniques, e.g., immunofluorescence microscopy for the
detection of cell wall fragments, to clearly differentiate between
soluble and particulate matter.
Table 1. The effect of filter pore size on particulate
retention.
Filter DOC
Pore Diameter
(pml -1 (mg 1 )
1.0 79
0.8 79
0.6 78
0.4 79
0.2 74
0.1 74
0.05 69
0.015 68
The experimental results for the soluble organic lytic fraction, when
compared with the computer predictions (Chapter 3, Fig.5) are
unsatisfactory. Unfortunately during the preparation of the data for
this publication, the carbon content of the lytic products was not
considered, such that the predicted line shown in this figure is
incorrect. Additional experimental points were obtained after submission
of the paper and these are shown below in Figure l together with the
recalculated data for the predicted DOC concentration assuming that 50'
I O>
c: 0 ..c ,_ <U u 0 c: <U O> ,_
0
"C (I.> > 0 (/) (/)
0
-214-
of s 2 (dissolved organic lytic product) is carbon. The carbon present in
EDTA and in polypropylene glycol have been taken into account in the
calculation of the experimental data points. The trend appears from the
additional data points to be effectively constant with increasing
dilution rate. The model predicts, more or less, the same general trend
but disagrees with respect to the absolute quantities. One possible
reason for this discrepancy is the assumption made in the model that all
the soluble lysis products released are indeed biodegradable. The
experimental data for DOC su9gest that this might not be the case and
that a part might be either non-biodegradable or only very slowly
biodegraded.
150
0 120
0
90 0 0 0
0 0 00 0 0 0 0 0 0 0
~ 0 0 60 0 0
30
0 0 0.2 0.4 0.6 0.8
Dilution Rate ( h-1)
Figure 1. Steady state concentrations of dissolved organic carbon (points) and the computer prediction (line) from the model described in chapter 3, and corrected for the carbon content of the released lytic product.
-1.0
-215-
To investigate this, and to determine experimentally the lysis rate
constant, it would be necessary to characterise the lysis products of K.
pneumoniae and to identify one component which is either recalcitrant or
slowly biodegraded. It would then be possible to determine lysis
kinetics by quantifying this fraction at different dilution rates.
The computer programme used for the mathematical calculations of the
model is shown in the appendix (A2). To examine the sensitivity of the
model to the choice of constants and coefficients a sensitivity analysis
was carried out whereby the effects of increasing and decreasing
individual constants were determined on the predictive response of the
model. The following coefficients and constants were particularly
sensitive:
Yield of non-viable cells by enzymic conversion of primary
substrate. Clearly whilst the non-viable cells are incapable of
replication they could, from incorrect choice of this coefficient,
conceivably continuously increase in mass. The only mechanisms limiting
this are lysis and washout from the system. When this constant was
increased the model predicted that the overall biomass would increase,
particularly that of the viable cell fraction. The reverse was the case
for a decrease in the value of this coefficient.
p• 2
: The maximum specific growth rate constant an dissolved organic
carbon. This constant was also sensitive, affecting most markedly, the
quantity of DOC accumulating in the system. Thus, after examining the
data in Fig. l above, a change in this constant could result in better
agreement between model and experiment. The value for this coefficient
was selected at random. However, the results describing "cryptic" growth
kinetics in chapter four clearly show that the value used in the
original model was unsatisfactory.
K4 , K8 and K3 : The death rate constant of viable cells, the lysis rate
constant of viable cells and the inactivation rate constant of viable
cells were also sensitive parameters affecting the predictions of the
model. It would probably have been justified, in the light of the
results, to have had a single term for K4 and K8
when K7 (the lysis rate
constant of dead cells) is assumed to be unity.
-216-
The modified model, similar to that described above, is presented in
chapter four where particular attention is focussed on "cryptic" growth.
In this model only K1 , the lysis rate of viable cells is sensitive, any
variation in this constant radically changes the quantitative prediction
of the model. Thus, it is absolutely essential and unfortunate that it
was not possible to adequately assess this rate constant. The computer
programme for calculating the predictions for this modified model is
given in appendix A3,
Some limited attention has been focussed on this question in the
literature. For example, Drozd .:.!: !_!. (1978) reported on the lysis of a
Methylococcus sp. in continuous culture. Since this bacterium is
incapable of assimilating lytic products, steady state measurements of
the dissolved organic carbon concentration in the culture supernatant
could be directly related to the specific lysis rate, the metabolic
pathway in this obligate methanotrophic bacterium resulting in the
production of co2 and biomass only. Drozd and co-workers found that
there was a direct relationship between dilution rate and the specific
lysis rate, with the rate of lysis increasing with increasing dilution
rate.
Meyer and Wouters (1984) examined the lysis of Bacillus subtilis var
niger WM in continuous culture. They were able to induce an autolytic
response in this bacterium by interfering with energy generation, and
could measure the extent of lysis by monitoring the decline in optical
density. In contrast to the work by Drozd and co-workers, Meyer and
Wouters found no relationship between lysis rate and growth rate nor
between lysis rate and growth limitation when comparing cells derived
from potassium-, phosphorous-, carbon- and magnesium-limited chemostat
cultures.
These two papers clearly demonstrate different facets of lysis. The
paper by Drozd .:.!: al., is relevant to in vivo lysis whilst Meyer and
Wouters are more interested in the potential activity of autolytic
enzymes when induced. Whether the two are necessarily related remains to
-217-
be shown. Thus, there is some justification, until more data becomes
available, to assume that the in vivo lysis rate is dilution rate
dependent as was assumed in the case in the model describing "cryptic"
growth.
The levels of dissolved organic carbon found in chemostat cultures of ~·
pneumoniae were lower in the experiments reported here, where the pH was
maintained at 6.80 and the temperature at 35.o0 c, than the
concentrations found in cultures of the same organism when cultured in
oxygen saturated, glucose limited chemostat cultures at pH 6.50 and at
30.o0 c. Harrison and Loveless (1971) report a DOC concentration of 255 ! 50 mg 1-l at a dilution rate of 0. 2 1 h-l which is much higher than
values obtained under the different culture conditions used in the
experiments reported here. Thus, there is suggestion of the potential
for increased lytic activity on changing from optimised to non-optimum
growth conditions.
It is possible also that in this current series of experiments the
observed DOC could have arisen either as a result of mechanical damage
or as a result of shear stresses from the impeller. These possibilities
were discounted after quantifying proportionately similar levels of DOC
accumulation at slower impeller speeds, when using lower concentrations
.~of glucose in the feed to ensure oxygen sufficient operation with lower
impeller speeds.
In the fundamental section of this thesis, there has been some comments
on the concept of endogenous metabolism with respect to starvation
survival. Such concepts are now relatively old. Lamanna (1963) described
a starving cell as one in which a gradual decline in the amount of
substance occurs such that it gradually wastes away and dies. He
suggests that it would be much more efficient if the cell, when deprived
of external resources, could automatically cease endogenous metabolism
and rest in a state of suspended animation. Clearly some 23 years later,
whilst knowledge concerning endogenous metabolism has advanced little,
it is now also known that some cells can ,exist in either a resting or a
-218-
dormant state (Novitsky and Morita, 1983; Stevenson, 1978).
Nevertheless, some of the current concepts are also somewhat odd. For
example, in an article by Kjelleberg et al. (1983), the behaviour of a
Vibrio sp. and of a Pseudomonas sp. were examined at interfaces
following the induction of starvation. These investigators found that
the initial events could be characterised by rapid fragmentation of the
cells followed by a continuous size reduction. The fragmentation gives
rise to an dramatic increase in the number of cells during the first 1
to 2 h, and the subsequent dwarfing apparently occurs over the first 4
to 5 h, during which time there is no loss in viability and measurable
metabolic activity. A similar sequence of events for another microbe has
been reported by Morita (1982). This series of stages of adaptation to
starvation prompts several questions; i.e., does this rapid division of
a mother cell into two daughter cells at the onset of starvation occur
such that a full compliment of DNA is present in each daughter cell? If
so, why does the bacterium invest valuable energy into rushed completion
of DNA replication instead of using the partially formed DNA as a source
of energy? How does the microbe "know" that it is going to have to
survive for a long period in the absence of exogenous nutrients such
that it initiates this sudden replication and rapid dwarfing effects
seen at the onset of starvation?
Clearly our understanding of starvation-survival is at present very
limited and many conflicting ideas continue to co-exist. However, whilst
these fundamental studies were undertaken to provide a framework to
understanding the effects involved in the destruction of microbes in
aerobic thermophilic sludge treatment, attention was directed towards
the question of microbial decline in general and the last paper in this
section has specific relevence to heat stress as well as starvation and
shows some of the problems involved in data interpretation in
starvation-type studies.
-219-
Microbial death, lysis and •crypt.ic• growth - applied aspects.
The utilisation of particulate substrates by microorganisms has been
neglected in the development of microbial growth theory and in much of
the work concerned with understanding the processes involved in waste
sewage sludge treatment. It is unfortunate that many reports concerning
anaerobic treatment processes depict it as being a sequence of stages
begining with simple soluble substrates e.g., proteins, lipids,
carbohydrates, and ignore the process stages involved in the production
of these simple substrates from more comlex polymeric and particulate
matter.Therefore, it was of considerable importance in this study that
the same deficiency should not occur. This was the main reason for using
a defined reproducable feed composed of whole yeast cells for the
process research programme. As a consequence, it was possible, through a
series of fundamental studies, to propose a mechanism for the
biodegradative process and thus appreciate the efects of varying
parameters such as temperature, pH, dissolved oxygen etc. on overall
process performance. The results clearly show that an ideal set of
conditions for microbial solids degradation would consist of low
dissolved oxygen conditions, a temperature between 6o0 c and 65°c and a
pH between 6. 5 and 7. 5. At present, where this process is used in
practise, only pH and temperature are routinely examined, but
measurement and control of dissolved oxygen concentration is rare.
Nevertheless, of the treatment plants which have been looked at in
experimental studies, the often high levels of carboxylic acids produced
indicate that they must operate at low dissolved oxygen concentrations
(Hamer and Zwiefelhofer, 1986). That this process can function optimally
with repect to both hygienisation and stabilisation has been shown very
nicely in practise at a sewage treatment plant in Marstatten, Kanton
Thurgau, Switzerland, where, despite the fact that the sludge storage
tank is open to the atmosphere, no recontamination or regrowth of
Enterobacteriacae has been found to occur during prolonged storage of
the treated sludge (H.P Zwiefelhofer, Personal Communication).
-220-
The Swiss Federal Ordinance of 1981, which states the hygiene
requirements of treated sludge makes no reference to the survival of
viruses. However, in a project conducted by Wyler and colleagues at the
University of Zilrich, the question of viral susceptability in the
aerobic thermophilic sludge treatment process has been examined
(Traub,1985). A technique was used which involved entrapping viruses
between polycarbonate membranes and suspending this in an aerobic
thermophilic bioreactor. It was found that viral inactivation occurred
under the thermophilic aerobic process conditions. However, some
viruses, notably Parvovirus, were resistant to short term exposure
( < 2h) at 60°c and would thus be more susceptable under semi-continuous
process operation than under continuous flow operation where washout
from the bioreactor could occur before deactivation. The pH was also
shown to be of relevence for the rate of viral inactivation with alkali
pH values, i.e., pH 8.0 being inducive for viral destruction. However,
the results of the present investigation into the effects of pH in this
study showed that operation at such high pH values would not necessarily
be conducive to optimum solids removal.
Similar results with respect to viral inactivation were described
recently by Dizer ~ al. (1986) and by Filip ~ al. (1986) where they
also investigated the fate of Enterobacteriaceae and of various other
groups of bacteria during aerobic and anaerobic thermophilic sludge
treatment processes. ~· coli and Salmonella spp. were effectively
destroyed at temperatures above 50°c under both aerobic and anaerobic
conditions, whilst some bacteria, notably sulphate-reducing bacteria and
Clostridium spp., were not destroyed even at 60°c under anaerobic
thermophilic conditions. No results were reported for the survival of
this group of bacteria during aerobic thermophilic treatment. The
possibility that these were thermophilic strains was not discussed
although it was suggested that the Clostridium spp. were probably spore
forming strains.
-221-
The presence of pathogenic spore-forming bacteria in feeds to aerobic
thermophilic waste sludge pre-treatment processes is a potential problem
for effective hygienisation. Representatives of this group are both
anaerobic and aerobic, e.g., £· botulinum and Bacillus anthracis, and
whilst spores are typically resistant survival structures to stresses
such as temperature, the use of either aerobic thermophilic or anaerobic
thermophilic treatment is unlikely to have any effect on such organisms.
In this context, it is probable that effective sludge stabilisation,
i.e., the degradation of all the potentially biodegradable matter
present, is as important for sludges containing spore-forming pathogenic
bacteria as with respect to the possible regrowth of Enterobacteriaceae.
Should stabilisation be inadequate, both aerobic and anaerobic
spore-forming bacteria could conceivably revert to their vegetative
forms and propagate in large numbers during the subsequent process
stages, a feature of definite concern with respect to the practise of
sludge disposal on agricultural land. It might be advantageous to
undertake routine counts of spore-forming bacteria in a similar manner
as for Enterobacteriaceae in the evaluation of safety for disposal of
the treated sludge on agricultural land.
Whilst the results and discussions presented in this dissertation are
part of a process research programme designed to investigate the
mechanisms involved in particulate degradation in aerobic thermophilic
sludge pre-treatment processes, there remains the question as to its
applicability to real feeds. Investigations to understand this problem
remain to be carried out both under controlled laboratory conditions and
during small pilot-scale operation using a feed of microbial cells
together with hydrocarbons, cellulose and lipids. This would be a
natural progression for this work, and only then could reliable
predictions be made with respect to the effective treatment of real
waste sludges .• What this work has shown, is the basis for efficient and
effective operation. Many of the results have already been seen to have
parallels under real process operating conditions. Without further
controlled investigations, i.e., using a supplemented feed, the effects
of feed variability under real process conditions cannot be either
properly predicted or controlled.
-222-
With the increasing availability of sensitive and reliable tests for
bacterial activity assessment, e.g., the INT-reduction assay, it would
be of interest to monitor the activity of the thermophilic process
microbes under different process operating conditions. Particularly in
the case of semi-continuous process operation, optimisation of cycle
times can be investigated with respect to the variation in activity of
the process microbes. Thus it should be possible to define conditions
whereby microbial activity can be maintained high and solids removal
maximized by operating at a particular cycle time. Investigations of
this kind should also provide information as to the effects of weell.-
day /week-end operation particularly with respect the length of time
required for the activity to recover after week-end operation.
Only very limited microbiological investigations were conducted on the
aerobic thermophilic system, but these showed that a mixed culture was
certainly present. Whilst these microbes were selectively enriched under
laboratory conditions, it remains to be seen as to whether the relative
numbers of the different species are the same as found in the
technical-scale plant from which the laboratory culture was originally
derived. However, the culture showed a considerable degree of robustness
as judged by the fact that the physical conditions, e.g., temperature,
Clearly aerobic thermophilic treatment is inappropriate as a complete
treatment process and effective removal of the biodegradable matter from
waste sludges requires a second treatment stage. Nevertheless, an
aerobic thermophilic process stage conditions the sludge for faster and
more effective subsequent mesophilic anaerobic digestion and thus offers
advantages over conventional single stage mesophilic anaerobic treatment
in both this respect and in exhibiting higher hygienization potential.
References
Burnham,J.C. (1981) Entrapment and lysis of the cyanobacterium Phormidum luridium by aqueous colonies of Myxococcus ~- Arch. Microbiol. 129, 285-295.
Chapman,S.J., and Gray,T.R.G. (1986) Importance of cryptic growth, yield factors and maintenance energy in models of microbial growth in soil. Soil Biol. Biochem. 18, 1-4.
Dizer,H., Leschber,R., Lopez Untersuchungen zur anaerob-thermophile 703-709.
Pila,J.M. and Seidel,K. (1986) Entseuchung von Klarschlamm durch
Behandlung. Korrespondenz Abwasser JJ,
Drozd,J.W., Linton,J.D., Downs,J. and Stephenson,R.J. (1978) An in situ assessment of the specific lysis rate in continuous cultures of Methylococcus sp. (NCIB 11083) grown on methane. FEMS Microbiol. Lett. 4, 311-314.
Filip,Z., Dizer,H., Leschber,R. and Seidel,K. (1986) Untersuchungen zur Entseuchung von Klarschlamm durch aerob-thermophile Behandlung. Zbl. Bakt. Hyg. B 182, 241-253.
Harrison,O.E.F., and Loveless,J.E. (1971) The effect of growth conditions on respiratory activity and growth effeiciency in facultative anaerobes grown in chemostat culture. J. Gen. Microbiol. 68, 35-43.
Kjelleberg,s., Humphrey,B.A. and Marshall,K.C. (1983) Initial phases of starvation and activity of bacteria at surfaces. Appl. Environ. Microbiol. 46, 978-984.
Lamanna,c. (1963) Studies on endogenous metabolism in bacteriology. Ann. NY. Acad. Sci. 102, 517-520.
-226-
Meyer,P.D. (1985) Cell wall autolysis and turnover in Bacillus subtilis. PhD thesis, University of Amsterdam.
Meyer,P.D. and Wouters, J.T.M. (1984) Is autolysis of Bacillus subtilis subject to phenotypic variation? In: Microbial Cell Wall synthesis and Autolysis. (C. Nombela, Ed.) pp 219-223, Elsevier Science Publishers, Amsterdam.
Morita,R. Y. (1982) starvation-survival of heterotrophs in the marine environment. Adv. Micro. Ecol. 6, 171-198.
Novitsky,J.A. and Morita,R.Y. (1977) Survival of a psychrophilic marine vibrio under long term nutrient starvation. Appl. Environ. Microbial. 33, 635-641.
Stevenson,L.H. (1978) A case for bacterial dormancy in aquatic systems. Microb. Ecol. 4, 127-133.
Stolp,H. and Starr,M.P. (1965) Bacteriolysis. Annu. Rev. Microbial. 19, 79-104.
Traub,F. (1985) Virusinaktivierung bei der Klarschlammbehandlung. Verband Schweizerischer Abwasserfachleute Bericht Nr 298.
Uehlinger,U. (1986) Bacteria and phosphorous regeneration in lakes. An experimental study. Hydrobiologia 135, 197-206.
Wright,S.J.L. and Thompson,R.J. (1985) Bacillus volatiles antagonise cyanobacteria. FEMS Microbial. Lett. 30, 263-267.
-227-
SUMMARY
The processes involved in and the factors affecting death, lysis and
"cryptic" growth have received little or no attention in microbiological
research. An understanding of these processes is particularly necessary
in research programmes designed to investigate the mechanisms involved
in and optimisation of microbial solids destruction in waste sludge
treatment processes.
In the work presented here, research at the fundamental level was
undertaken to investigate the processes of death, lysis and "cryptic"
growth in the absence of any form of stress, i.e., under optimal growth
conditions. The results suggest that death only results from either
genetic injury or by the application of stresses e.g., heat. Biomass
reduction during cultivation is suggested to occur as a result of lysis
events rather than through inactivation of metabolic functions. As a
result of the release of cellular components following lysis, the
process of "cryptic" growth, i.e., the utilisation of the lytic products
by members of the same population as carbon, energy, or other nutrient
sources can occur. Whilst this results in a complex series of events
during the growth process, a mathematical model is derived to aid in the
understanding of these various reactions and raises questions as to the
validity of using poorly-defined concepts such as maintenance energy to
describe physiological events in dynamic culture systems.
The application of heat is frequently used in the deactivation of
microorganisms. In a simple study, the physiological changes which occur
to a population of bacteria as a result of mild starvation together with
heat treatment were examined and also evaluated with respect to the
effects on individual cells. At temperatures slightly above the optimum
for growth, anomalous changes with respect to macromolecular
constituents per cell were obtained suggesting that the concepts of
endogenous metabolism alone are inappropriate to describe starvation or
other stress effects when changes in physical properties, e.g., wall and
membrane permeability are not simultaneously considered.
-228-
In the applied part of this study, the mechanisms involved in and
factors affecting the destruction of microbial solids in aerobic
thermophilic waste sludge treatment were investigated. This process is
designed to supplement conventional anaerobic mesophilic digestion, such
that beth pathogen and toxic chemical removal efficiencies are enhanced.
Whilst most work conducted on sludge digestion has concentrated either
on soluble substrate utilisation or on the degradation of substrates of
non-microbial origin, this study was designed to investigate the
degradation of microbial solids. This fraction represents a major
constituent of the total suspended solids in sludges and is derived from
both primary and secondary wastewater treatment processes. The effects
of physical process parameters such as temperature, pH, dissolved oxygen
concentration and bioreactor residence time were examined together with
effects of nutrient supplementation. The process proved to be highly
effective with respect to microbial solids destruction at pH 6.5, 6S0 c and when the air flow into the bioreactor was regulated such that the
residual dissolved oxygen concentration was very low.
The mechanism of microbial solids destruction was investigated in a
batch type experiment where the fate of particular components of the
feed microbial solids such as protein, carbohydrates and cell number
were monitored during the degradative process. Coupling this with
scanning electron microscopy allowed the proposal of a mechanism for the
biodegradation process whereby the cell walls of the feed microbes are
attacked by exo-enzymes produced by the thermophilic process microbes.
Lysis of the cell wall results in the release of readily utilisable
substrates whilst residual wall biodegradation occurs more slowly. Under
conditions where the dissolved oxygen concentration is low, considerable
amounts of carboxylic acids are produced which are readily utilised when
the aerobic thermophilic process stage is used as a pre-treatment to
anaerobic mesophilic digestion. Moreover the rate of anaerobic
mesophilic digestion is likely to be enhanced as a result of the
extensive particle solubilization in the pre-treatment process,
-229-
ZUSAMMENFASSUNG
Prozesse im Zusammenhang mit Tod, Lyse und kryptischem Wachstum haben in
der bisherigen mikrobiologischen Forschung wenig bis keine Beachtung
gefunden. Ein gesichertes Wissen liber diese Vorglfoge ist besonders
wichtig im Hinblick auf Programme, die eine Untersuchung und Optimierung
des mikrobiellen Abbaus von Feststoffen im Klarschlamm zum Ziele haben.
In der vorliegenden Arbei t wurden die grundlegenden Prozesse von Tod,
Lyse und kryptischem Wachstum in Abwesenheit von Stressfaktoren, d.h.
unter optimalen Wachstumsbedingungen, untersucht. Die Resultate deuten
darauf bin, dass Tod nur infolge genetischer Schaden oder durch den
Einfluss von Stress, z.B. Hitze, eintritt. Eine Reduktion der Biomasse
wahrend der Kultivation scheint das Resultat von Lyse zu sein und nicht
auf einer Inaktivierung der metabolischen Funktionen zu beruhen. Als
Folge der Freisetzung von Zellkomponenten durch Lyse kann kryptisches
Wachstum auftreten, d. h. die Verwendung von Lyseprodukten als
Kohlenstoff-, Energie- oder sonstige Nahrstoffquelle durch Vertreter
derselben Population. Daraus resultiert eine komplexe Folge von
Vorgangen wahrend des Wachstums. Zurn besseren Verstandnis wurde daflir
ein mathematisches Model! entwickelt. Es stellt sich in der Folge die
Frage, in wie we it ungeniigend definierte Begriffe wie "maintainance
energy" brauchbar sind zur Beschreibung der physiologischen Vorgi!nge in
einem Kultursystem.
Zur Inaktivierung von Mikroorganismen wird oft Hitze eingesetzt. Die
physiologischen Veranderungen einer Bakterienkultur als Folge einer
leichten Aushungerung zusammen mit einer Hitzebehandlung, wurden in
einer weiteren Studie untersucht bei gleichzeitiger Beobachtung von
Einzelzellen. Bei Temperaturen wenig oberhalb der optimalen wachstums-
temperatur wurden Veranderungen in den makromolekularen Inhaltsstoffen
von Zell en beobachtet. Das bedeutet, dass das Konzept des endogenen
Metabolismus alleine nicht geniigt um den Hungerzustand oder andere
Stressfolgen zu erklaren. Es mlissen gleichzeitige Veranderungen in den
physikalischen Eigenschaften der Zelle, wie z.B. bei der Permeabilitat
der Zellwand und der cytoplasmatischen Membran, angenommen werden.
-230-
Im angewandten Teil der Arbeit wurden die Faktoren, welche den
mikrobiellen Abbau von Feststoffen beeinflussen, wie auch der
Abbaumechanismus als solcher in aerob thermophilen Behandlungsreaktoren
von Kliirschlamm untersucht. Dieser Prozess dient als Zusatz zur
mesophilen biologischen Faulungsstufe und soll garantieren, dass der
Kliirschlamm am Ausgang der Anlage frei von pathogenen Organismen ist.
Gleichzeitig wird auch eine Verbesserung in bezug auf die Entfernung
umwelttoxischer Substanzen erwartet. Wiihrend der grosste Teil von
Arbeiten Uber den Abbau von Kliirschlamm sich entweder auf die Entfernung
geloster Stoffe oder auf den Abbau von Feststoff en nicht
mikrobiologischen Ursprungs beschriinkte, wurden in der vorliegenden
Arbeit der Abbau der mikrobiellen Feststoffe untersucht. Diese stellen
den Hauptteil der suspendierten Feststoffe im Kliirschlamm der ersten,
wie auch der zweiten Abwasserbehandlungsstufe dar. Der Einfluss der
physikalischen Prozessparameter wie Temperatur, pH, Konzentration des
gelosten Sauerstoffs und Aufenthaltszeit im Bioreaktor wurden zusammen
mit den Veriinderungen, die durch Zugabe von weiteren Niihrstoffen
hervorgerufen wurden, gepriift. Es zeigte sich, dass der Abbau
mikrobieller Feststoffe sehr effizient erfolgte bei einem pH-Optimum von
6.5 und einer Reaktortemperatur von 65°c sowie einer Beliiftungsrate, die
zu sehr kleinen Konzentrationen an gelostem Sauerstoff fiihrte.
Der Abbau von mikrobiellen Feststoffen wurde in 'Batch'-Versuchen
abgekliirt, so dass die einzelnen partikuliiren Komponenten wie Protein,
Kohlenwasserstof f e und Zellzahl wiihrend der Abbauprozesse verfolgt
werden konnte. Dies, kombiniert rnit Rasterelektronen-Mikroskopischen
Aufnahrnen erlaubt den Vorgang wie folgt zu charakterisieren: zuerst
werden durch Exoenzyme der thermophilen Organisrnen die Zellwl!nde der
gefUtterten Mikroben angegriffen, die darauf folgende Lyse setzt leicht
verwertbare Substrate frei, wiihrend der biologische Abbau der
Restzellwand langsamer erfolgt. Bei kleiner Konzentration an gelostem
Sauerstoff werden beachtliche Mengen von Carbonsiiuren produziert. Diese
konnen in einer anschliessenden, mesophilen, anaeroben Faulstufe leicht
verwertet werden. Zudem wird der anaerobe Abbau dieser Faulstufe
verbessert durch den Aufschluss der partikull!ren Stoffe irn
vorbereitenden therrnophilen Prozess.
-231-APPENDIX
Al
Iodonitrotetrazolium Chloride (INT)
Properties
Ivory white crystalline powder, m.p. 24S0 c. Slightly soluble in methanol
and water, very slightly soluble in acetone,ethyl acetate and
tetrahydrfuran, insoluble in ethyl ether. Reduction potential about
-0.09 volts. Yields a red violet monoformazan pigment in the presence of
dehydroqenases (LOH, SOH, etc.). Monoformazan melts at 185.6°C.
Uses
As a histochemical and cytochemical reagent for detection or
determination of dehy?roqenases, NAO and NAOP.
(Reference: Fluka Technical Bulletin).
-232-
APPENDIX
A2.
PYNEO 10 ON ERROR GO TO 2000 SO REH PROGRAM BY G,A,H, KINGr J, ECOL. MODELLING Sr 259-268 (1978) 100 REH CHEMICAL EQUATIONS CHEMED 110 REH PREPARE LIST OF CHEMICAL SPECIES 120 REH FIRST SPECIES IS A BLANK 'BOX' 130 REH PREPARE LIST OF EQUATIONS 140 REH CONVERT EQUATIONS TO NUMBER FORM 150 REH LIST RATE COEFFICIENTS 160 REH LIST INITIAL CONCENTRATIONS FOR SPECIES 170 REH CONCENTRATION OF FIRST SPECIES IS PUT TO ONE 180 REH SPECIES AND EQUATIONS DIMENSIONED TO 20 190 REH ---------------------------------------------200 DIH E140r4lrRl40r3loVl40lrWl40l 201 DIH J(8lrNC4014lrEOl40)rEll40lrE2140l 202 DIH RICISlrR211SlrRSl15) 203 DIH Pl4) 204 FS='' 205 GOSUB 1100 207 INPUT 'INTEGRATION CRITERION='rX 210 PRINT'ENTER NUMBER OF VARIABLES -ENTRY PT I' 220 INPUT NI 230 PRINT'ENTER NUMBER OF PROCESSES -ENTRY PT 2' 240 INPUT II 250 PRINT'ENTER EQUATIONS - HAX. 8 NUMBERS -ENTRY PT 3' 251 PRINT'INPUT NlrV1rN2rV2r••••' 252 PRINT'Vl,,V4 = VARIABLESr Nl,,N4 =STOICHIOMETRIC FACTORS' 253 PRINT'NI<O FOR CONSUHPTIONr NI>O FOR PRODUCTION OF VARIABLE' 260 FOR I=I TO II 262 HAT J=ZER 264 HAT INPUT J 266 Kl=I 268 K2=INTC0,5+Kl/2l 270 NCirK2>=JIKI) : EIIrK2l=JCKl+I> 273 IF K2>=4 GOTO 277 274 IF JIK1+3><>0 THEN Kl=Kl+2 : GOTO 268 277 E11Il=K2 280 NEXT I 290 PRINT 300 PRINT'ENTER PROCESS FUNCTIONS -ENTRY PT 4' 301 INPUT'DO YOU WANT LIST IYES/N0l'IL$ 305 L$=LEFTIL$1IX) 310 IF L$<>'N' THEN GOSUB 1000 311 R8=0 312 PRINTlPRINT'PROCESS t'r'TYPE t' 315 FOR I=I TO II 320 PRINT I1 325 INPUT K : EOIIl=K : E21I>=R21K) 326 IF EllI><RllKl THEN PRINT'NOT ENOUGH VARIABLES FOR PROCESS'lGOTO 320 328 NEXT I 330 PRINT'ENTER RATE CONSTANTS -ENTRY PT S' 332 FOR I=I TO II 334 K=E21Il : PRINT'PROCESS 'III' NEEDS 'IKI' CONSTANTISl'I 336 HAT J=ZER : HAT INPUT J 337 FOR Kl=I TO K : RIIrKl)=J(KI) : NEXT Kl 338 IF EO<I>=8 THEN R8=0,0S*K3 339 NEXT I 340 PRINT'ENTER INITIAL CONCENTRATIONS -ENTRY PT 6' 350 FOR N=I TO NI 360 PRINT No : INPUT VIN) 370 NEXT N
-233-
380 T9~0 390 PRINT'SPECIFY WHICH VARIABLES PRINTED <HAX,4) -ENTRY PT 7' 392 HAT J=ZER : HAT INPUT J I P1=1 395 IF JCPll•O GOTO 403 397 PIP1l•JIP1> : P1•P1+1 : GOTO 395 403 Pl•Pl-1 : IF P1<1 GOTO 390 405 IF P1>4 THEN PRINT'TOO HANY OUTPUT-VARIABLES'! GOTO 390 410 PRINT'SPECIFY PRINT INTERVALL AND LIMIT -ENTRY PT 8' 420 INPUT T1 • T2 421 IF Ft<>"' THEN PRINT'DATA FILE OPEN ALREADY' I GOTO 955 422 PRINT'OUTPUT ON FILE (YES/NOlT -ENTRY PT 9' 424 INPUT Xt : XS•LEFTCXl1lX> 426 IF XS•'N' GOTO 432 428 INPUT 'NAHE OF FILE'I Fl 430 OPEN Ft FDR OUTPUT AS FILE 432 PRINT!PRINTIPRINT 440 PRINT'T'• 441 FOR P2=1 TD Pl l PRINT P<P2lr NEXT P2 443 IF P1<3 THEN PRINT 450 REH 460 T•T9 470 T4=0 474 PRINT Tr 475 IF P1=1 GOTO 495 480 FOR P2•1 TO Pl-1 483 KcP(P2l 485 PRINT VIK l 1
490 NEXT f'2 495 K•P1 : PRINT V<Kl 512 IF X••'N' GOTO 520 514 PRINT11rT'r'TO'•'I 515 IF N1s1 GOTO 518 S16 FOR N=l TO Nl-1 517 f'RINTtlrV!Nl'r'I l NEXT N 518 f'RINTl1rVIN1l 520 IF T>•T2 THEN 940 540! BEGIN LOOP <TO LINE 900)
·541 HAT W•ZER 550 FOR I•l TO I1 560 ON EO!Il GOTO 610r620r630164016S0r660r670r680 610!PROCESS 11 612 Y•R<I, 1) 615 GOTO 800 620!PROCESS t2 623 V1•E1Ir1l 625 Y•R(Ir1l*V<V1) 627 GOTO 800 6JO!PROCESS U 632 Vl•EIIrll 635 Y•R(I,1l*VIV1!*VIV1l 637 GOTO 800 640!PROCESS 14 642 Vt-E<Ir1) : V2•E(I,2) 645 Y•R<Ir1l*VIV1l*V(V2l 648 GOTO 800
650!PROCESS tS 652 V!=E<I.tl
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655 Y=R<I•l>*V(Vlll<R<Ir2ltV(VI)) bSB GOTO BOO 660!PROCESS 16 662 Vl=E(lrll : V2=E<I•2> 66S Y=R<l•1l*V(Vl)*V(V2)/(R{lr2)tV{V1)) 667 GOTO BOO 670!PROCESS 17 672 Vl=E<I•ll : V2=E!It2l 675 Y=RCl•1l*V<V1l*V<V2l/CRCl•2>+V<Vll)/(R(It3ltV(V2ll 670 GOTO BOO 6BO!PROCESS IB 692 Y=R(l,ll+RCit2l*SIN<6.283105*T/R!l•3>> 604 GOTO aoo 800 FOR Kl=! TO EICil BOS K=ECltKU BIO W<Kl=WCKltN<I•Kll*Y BIS NEXT Kl 820 NEXT I BSO!CALCULATE TIHE STEP 051 TO=! 955 FOR N=I TO NI 060 IF W<Nl=O OR V(Nl=O GOTO 075 965 TB=A9SCX*V(Nl/W<N>> 870 IF TB<TO THEN TO=TB B75 NEXT N 880 IF TO><Tl-T4l THEN TO=Tl-T4 BBi IF RB>O AND TO>RB THEN TO=RB B92 T=T+TO : T4=T4tTO BBS FOR N=I TO NI 890 V(Nl=V<Nl+TO*W<N> 900 NEXT N 910 IF T4>=TI GOTO 470 920 GOTO 540 940 T9=T 942 IF X$='N' GOTO 970 944 ~ NEXT 5 LINES VALID FOR TONY ONLY ~===s==••=~==•••••••••=~•m•• 945- T2=T2+100 946 PRINT : INPUT 'D='•D : PRINT 947 IF D=O THEN GO TO 955 948 R<I•l>=D FOR I=12 TO 10 949 GOTO 540 955 INPUT'CLOSE DATA FILE CYES/NOTl'IXS 960 Xt=LEFT<XS11Xl 969 IF Xt='Y' THEN PRINTll•CHRt<26) l CLOSE 1 : FS='' 970 INPUT'CHOOSE ENTRY PT FOR NEXT RUN - USE 0 TO END SET'IH 900 IF H<O OR H>9 GOTO 970 985 IF H=O GOTO 2000 990 ON H GOTO 210.230,2so.300.330,J40.390.410•421 lOOO!LIST OF PROCESS EOUATIONS 1001!************************* 1010 I9=8 101S Rf(ll='Kl' 1016 RS<2l='Kl*VI' 1017 R$(3l='K1•V1*V1" 1016 Rt(4l='K1*Vl*V2' 1019 Rt(5)•'K1*V1/(K2+V1)' 1020 RS!6l="K1*Vl*V2/!K2tV1l" 1021 R$<7l='K1*CV1/(~2tV1ll*CV2/(K3+V2ll 1022 Rt(Sl='K1tK2*SIN<2*PI*T/K3l" 1050 PRINTlPRINT'TYPES OF EQUATIONS AREl' 105S PRINT'NUHBER'
1060 FOR 1=1 TO 19 1065 PRINT loR$CI) 1070 NEXT I 1075 PRINT 1080 RETURN
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1100'HINIHUM NUM8ER OF VARIA~LES AND NUH8ER OF COEFFICIENTS FOR DIFFERENT 11011TYPES OF RATE EQUATIONS 1102!******************************************************************** 1110 HAT Rl=CON l MAT R2=CON 1115 RJC4)=2 1116 R2<5)=2 1117 R1C6l=2 R216l=2 1118 RIC7l=2 R2<7l=3 1119 R2(8)=3 1190 RETURN 2000 ENI.I
0 0 0 [) 0 $J INITIAL CONC Sl S2 Xv Xnv Xd p $1 PRINTED VARIABLES 1.2,J,4 $I INTERVAL /\NU LIMIT ••OELT , .. TENO $1 OUTPUT y ****.OUT
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$I NEX'r LINES INTRODUCE NEW VALUES FOR 0 AS MANY TIMES AS DESIRED $I JUS1' REPEA'l' ON NEW LINE, ONCE ONLY f'OR EACH CYCLE. ••o ••o 0 y 0 $RUN $HELDE BA'l'CH TONY r'INISHEO $EOJ
10 PRINT 20 PRINT 'MATHEMATICAL MODEL FOR GROWTH IN CONTINUOUS CULTURE' 30 PR INT 40 PRINT ·===•=:===~==a=====~====~,=~===·==·=~~·=·=======•s•~a==~====~~u~·
:;o f'P.INT 60 Pf<HIT 70 PR INT SO F'f:INT 90 EXTENL> 100 l•IH FEEDl 4) 110 HAT NUE=ZER(4r41 120 INPUT 'OUTPUT ON DATA FILE IYES/NOI 130 IF X$•'N' DOTO 190 140 INPUT 'NAHE OF DATA FILE 150 OPEN A$ FOR OUTPUT AS FILE 160 PRINT 170 INPUT 'DH HIN !SO INPUT 'DH HAX 190 INPUT 'DH STEP 200 INPUT 'OUTPUT ON SCREEN 210 ITCRIT•0,0001 220 F'flINT 230 INPUT 'YIELDl <YIELD 240 INPUT 'YIELD2 <YIELD 330 INF'UT 'HUE! 340 INPUT •KS! 350 INPUT 'HUE2 CXv-S21 360 INPUT 'KS2 130 !NF'UT 'KH 480 INPUT 'SO(! I 485 FOR !tH•!IHH!N TO [tHHAX 4ro K3•0.4*DH+0.02
Xv FROM Sil XV FROH S2l =· diUE 1
=' 11\Sl •"HUE2 :• 1KS:! = 1 rK4
'tSO(I) S TEF' STEPI•H
506 !F DH•O THEN GOTO 32767 510 NUEC!r!l•-1/Yl 520 NUEC!13)•1 540 NUE(3r3l•-l 550 NUE(3,2)=0.95*DH~O.l5 560 NUEC3r4l=!-NUE<3•2l 570 NUE<2r21•-l/Y2 580 NUE(2,3>=1 sro NUEC4r4l=-t 600 NUE(4r31=1 610 FOR !=1 TO 4 620 FEEDIIl•-SOCll*DH 630 NEXT I 640 Ll=HUEl/KS! 650 L2•11UE2/KS2
• rAf
'•DHMIN • 1DHl'!AX '' STEF'DH
•rot
840 I --------------------------------------------------------------------850 ! BEGIN OF ITERATION LOOP FOR STEADY STATE 860 ! --------------------------------------------------------------------870 LOOF'•() 880 HAT s~zER<1·4> 890 FOR I•! TO 4 900 S<I•ll•Ll*NUE!l•Il 910 S(J,2}2L2t.NUE(2,r> ?20 SCl•3l•K3*NUE<31Il 930 SCir4l•K4*NUEl41Il
960 HEY.T I 970 FOR I~l TO 4 980 S<I•ll•SII•I>-DH 990 NEXT I 1001 HAT S•INVISI 1010 HAT CSS•S*FEED 1020 L10Lll•L1 1030 L20Lll•L2
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1060 L1•HUE1*CSS<Zl/CKS!tCS511)1 1070 L2~HUE2*CSS(J)/CKS~+CSSC2JJ 1100 LOOP•LOOP+l 1110 IF LOOP>2000 THEN PR!NT 'WASHOUT ! !••: GOTO 32767 1120 IF A8S(L10LD/L1-11>ITCRIT OR A8S<L20LO/L2-1l>ITCRIT 1170 RT•1/DH 1180 Y08S•CSSl3)/IS0(1)-CSS<lll 1190 as•ItH/YOBS 1200 IF Ot•'N' GOTO 1440 1210 PRINT 'NUHSER OF ITERATION LOOPS •'•LOOP 1220 PRINT 1230 PRINT 'STEADY STATE IS ' 1~40 PRINT •===~===========· 1250 PRIHT 1260 PRINT USIHG "DILUTION RATE • ttttttt,tttt'oDH 1270 PRINT 1260 PRINT USING 'X 1290 PRINT USING 'S 1295 PRINT USING 'SL 1340 PRINT USING 'P 1350 PRINT