ENVIRONMENTAL BIOTECHNOLOGY: THE TANDEM OF … · by a limited nulTlber of straightforward techniques, which tinuous oil spills and leaks ill pipelines and storage tanks are often

, :/BIOCATALYSIS-2000: FUNDAMENTALS k APPLICATIONS 15

ENVIRONMENTAL BIOTECHNOLOGY: THE TANDEMOF BIOCATALYTICAL AND ENGINEERING DEVELOPMENTS

S. V. Kalyuzhnyi

The paper gives several examples of integrated approaches based on the tandem of bio-catalytical and engineering developments in environmental biotechnology for treatmentof 3 main compartments of the environment-soil, water and gas phase. The first topic

i analyses the current situation with oil pollution of soils and water surfaces in Russiaand presents the results of field bioremediation trials on the basis of recently devel-oped biopreparation "Rhoder". The second topic discusses the recent findings aimingto extend an applicability of anaerobic wastewater treatm-ent at temperatures as low as4-10 DC. A performance of novel anaerobic-aerobic hybrid reactor is analysed in the thirdtopic with regard to treatment of recalcitrant azo dye wastewater. The latter 2 topicslye within a conventional function of environmental biotechnology (so-called "end of pipetreatment") while the fourth topic dealing with the development of biocatalytical technol-

!.. ogy of H 2 S removal and sulphur recovery from polluted gases highlights a transformationr I of this discipline into a new phase substantially contributing to resource conservation and

Ii sustainable production in the modern society.,.',I

Introduction losophy inside of environmelltal biotechnology should be. .. . . . . holistic and this requires both a detailed knowledge about

The d1sarmmgly slmple defimt1on or1gmated from Al- biocatalytic mechanisms involved and well designed engi-ber.~ Einste~n ("Th~ environment ~s anything, which isn:t neering systems.lllt: ) explams succmctly why soc1ety has so many enV1- Th . 1 1 f .

t t d. . . e paper glves severa examp es 0 m egra e ap-ronmental problems at the begmmng of the 3rd m1l1en- h b d th t d f b. t 1 t. 1 d .

. .. proac es ase on e an em 0 10ca a y 1ca an eng1-mum. Indeed, the enV1ronment 1S the "tragedy of the. ., .

" ' t b 1 b d d b d Th . neenng developments m env1ronmental b1otechnology forcommons -1 e ongs to no 0 y an to every 0 y. IS t t t f 3 . t t f th . t. ,rea men 0 mam com par men s 0 e enVlronmen -results m the fact that ample examples of ecologIcal prob- '

I t d h Th fi t h t 1 th. . SOl, wa er an gas p ase. e rs c ap er ana yses elem8 or even dIsasters are encountered, Treatment of enVl- t .t t. .th .1 11 t . f .1 d t" " curren Sl ua lon Wl 01 po u lon 0 SOl S an wa er sur-ronmental problems lS mamly based on blocatalytlcal meth- r ' R "r d th It f f 11 1 b. d. t '

, , . laces m ussla an e resu sou -sca e loreme la lonOU8 due to thelr relatlve cheapness and reasonably hlgh t ' 1 th b . f .1 d 1 d b. t '

, ,. . na s on e a.~lS 0 recen,. y eve ope loprepara lonefficlency. The whole subject area lS defined as enVlron- " Rl d " Th d 1 t t th t fi d., . . , 10 er, e secon clap er presen s e recen n mgsmental b1otechnology, wh1ch IS currelltly tht~ bIggest area of .. t t d 1. b ' l.t f b. t t..1 . 1 1. . f b. 1 .. h d 11 almmg 0 ex en an app lca 11 yo anacro 1C WM ewa ermuustna app lcatlon 0 10cata YS1S Wlt regar to overa t t t t t t 1 4 10°C A f. f . rea men a empera ures as ow as - . per or-

quantlty 0 processed matter. However, wlth respect to the .. . ..

t M. t 1b' t 1 1 " th h . h Id mance of novel anaeroblc-aeroblc hybrid reactor lS dlscussedernJ enVlronmen a 10 ec illO ogy e emp asls s ou,.. .

1 t "b." t 1 t 11 tl " t h 1 " In the thlrd chapter wlth regard to treatment of recalcltrant)(: pu on 10 a eas equa y a.~ on le ec no ogy ,.,th h ' th h. t ' 1 t ' tl t t t f azo dye wastewater, Fmally. blocatalytlcal technology ofoug m e lS onca perspec lve, Ie rea men 0 en- H S 1 d 1 h f 11 d '

. 1 bl .,. 2 remova an su p ur recovery rom po ute gases lSv1ronmenta pro ems was monopohsed by samtary engl- h. hI" h dneerillg. As a consequence, the "bio" component has until 19 Jg te ,

recently largely been ignored and dealt with stocha.~tical-Iy rather than Illechanistically, However, at present, we Spilled petroleum remediation in open waterare facing a nUlnber of formidable environmental problems aquatories, wetlands, and soils: using novel8uch as greenhouse effect, acid rain, depletion of ozone lay- biopreparation "Rhoder"er, enrichment of ground and surface waters with nutrientsand recalcitrant xenobiotics, dispo8al of mlmicipalsolid and Dlle to massive movemf~nts of petroleum from theanimal wastes etc. These problems can no longer be solved oil-producing countries to the major oil consumers and con-by a limited nulTlber of straightforward techniques, which tinuous oil spills and leaks ill pipelines and storage tanksare often a perfect illustration of Murphy's Law, i, e., they followed by.,pmoffs, approximately 35 million tons of oiltransform one problem into another often more intractable enters the sea per annum [1]. Since 1 ton of oil contami-problem. Examples: one cleans water by stripping the pol- nates 12 km 2 of water surface, it results in the fact thi\t

luti\nts into the air or removes organics from water which 30% of the World Ocean surfi\ce are already covered hy~re then dumped in the soil. Hence, a particular type of oil film [2]. Meantime, 1 I of oil eliminates xygen fromwaste can not anymore be treated without considering all 40 m 3 of water and kills 100 million of fish larvae. Eventhe consequellces for the environment. For instance, acti- low concentrations of oil such as 0.1 mg/l exert the deathvated sludge treatment now not only refers to the W'clter of juvenile forms of marine animals after several days ofcomponent, but also to the biosolids produced and volatile exposition and substantially inhibit the growth of microal-organic compounds and odour generated. Thus, the phi- gae [3], The toxic effects of hydrocarbons to all forms of life

Department of Chemical Enzymology, Chemistry Faculty, Mo..cow State Univer..ity. 119899 Moscow. Russia.

. V..t.ok "himi)!"

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was recognised long ago and is usually ascribed to the oil I ration "Rhoder" was certificated in 1999 for production,

dissolving the lipid portion of the cytoplasmic membrane, I delivery and application on territory of Russia (Certificate

thus allowing cell contents to escape [4]. On the basis of No. 77.99.11.515.P.4865.8.99 issued 17.08.99 by the Russian

the facts presented above. oil should be considered as one of Ministry of Health). The delivery form of "Rhoder" usual-

the most dangerous pollutants for the environment taking ly includes a concentrated wet suspension of cells of both

into account both its high toxicity and enormous scale of bacteria (1: 1 w/w) with a titre of hydrocarbon-degrading

invasion into biosphere. bacteria of 109-1010 cells/mi. The working solution is pre-

Russia occupies the 3rd place (after Saudi Arabia and pared on site by dilution of concentrated suspension with

Iraq) with regard to oil reserves (62.7 billion tons) and water followed by addition of some nutrients and biostim-

: the total oil extraction accounted for 295 million tons in ulators.: 1999 [5]. Due to systematic accident spills, an annual re- Sites and remediation methods used. The follow-

lease of oil into the environment in Russia accounts for ing oil polluted sites were used for field testing of bio-25 millions tonnes according to the estimations (may be a preparation "Rhoder" in 1995-1999: bay of river Cher-little bit exaggerated) of "Greenpeace" [2]. Among a vari- naya (Lukhovitsy, Moscow region), lakes and wetland (Vyn-ety of approaches proposed for elimination of these spills [1], gayakha, Western Siberia), lake and wetland (Ural, West-three main methods (mechanical, physico-chemical and mi- ern Siberia), marshy peat soil (Nizhnevartovsk, Western

~ crobiological ones being applied both separately and in var- Siberia). Some characteristics of these sites are listen in~ ious combinations) are currently considered as the most Table 1. When necessary and possible, preliminary me-

perspective methods for Russian conditions [6]. Each of chanical collection (PMC) of spilled oil on the site was un-these methods has its advantages and drawbacks. Un- dertaken before application of bioremediation technology.der fresh and abundant spills, the mechanical methods The latter include a spraying of the working solution on theof oil collection are usually applied as a principal treat- polluted areas using pump equipment. Usually the treat-ment. However, oil pollution is not eliminated completely. ment with biopreparation was repeated twice or triple withThe physico-chemical methods using special reagents (de- a time interval of 2 weeks. The impact of activity of indige-tergcnts, emulsifiers, solidifiers, adsorbents, etc.) can effi- no us hydrocarbon-degrading bacteria (HDB) was assessedciently concentrate oil pollution, but frequently they them- by a spraying of the working solution lacking "Rhoder" onselves are not fairly irreproachable from the ecological point the control areas having a similar oil pollution level. Theof view, e. g., collection of oil-saturated adsorbents as well generalised results of field tests are presented in Table 1.as their subsequent utilisation becomes sometimes trouble- Bioremediation of open water aquatories. Fromsome. The microbiological methods using both external Table 1, it is seen that Rhoder has demonstrated a veryintroductions of oil-degraders cultivated ex situ and stim- high efficiency for treatment of aquatories, especially at lowulation of indigenous microorganislIlS (if they are present initial oil level (IOL) as in the case of bay of river Chernaya.in necessary concentrations) are usually quite efficient for It should be noted that the residual oil level (ROL) aftertreatment of low polluted water surfaces and soils. How- 4 weeks of bioremediation of this site was only 0.04 mg/l,ever. their effects are frequently not so pronounced at a i. e., lower than the Russian PLOP (0.05 mg/l). The con-high level of oil pollution. Besides, the low average an- centrations..pf HDB and heterotrophic bacteria firstly in-nual temperatures on the overwhelming majority of terri- creased by 1-2 orders of magnitude at day 14 and thentory, especially where the principal oil fields are located, returned back to the initial level after an exhaustion ofis another critical bottleneck for application of these meth- organic substrates in the river water. Thus, an additionods in Russia because bacterial oil-degrading activity drops of the external bacteria seemed not to result in substantialdramatically under temperatures below 10°C. Despite the changes of microbial community existing in the river water.above-mentioned limitations, microbiological methods are Analogously, the initial dosage of nutrients was chosen indrawing more and more attention in our country, especially such a way that the residual level of phosphate and nitrateas post-treatment or polishing steps, due to their economic after treatment was low enough to prevent a possible eu-attractiveness and ability to fulfil with the stringent legisla- trophication of this bay. The both lakes in Vyngayakhation requirements concerning a permissible level of oil pollu- had a high IOL (Table 1) and the thick (till 1-2 cm) oiltion (PLOP). In this chapter, the experience accumulated film was clearly seen on their surfaces. In spite of ratherin 1994-1999 with application of recently developed bio- tough conditions, the triple treatment with Rhoder accom-preparation "Rhoder" for spilled petroleum bioremediation panied by unusual warm weather in that period resulted inin open water aquatories, wetlands and soils is summarised. an almost complete elimination of oil pollution-the ROLs

Biopreparation. The biopreparation known under were 5 and 190 mg/l in lakf~s 1 and 2, respectively (Ta-commercial name "Rhoder" and recently developed in ble 1). Moreover, after bioremediation both these lakesAll-Russian Research Institute of oii and Gas together were certificated by the local ecological authorities as "thewith Moscow State University [7] consists of two bacteria objects almost free of oil pollution". During the treatment- Rhodococcus ruber and Rhodococcus erythropolis reveal- of the lake;.}n Ural (Table 1), a majority of oil pollution

ing a synergistic action on hydrocarbon degradation un- was removed by mechanical collection (90%), i. e., the oilder a joint application. The individual strains were iso- contamination level decreased from 11 to 1.01 g/l after thislated from oil-water mixture originating from Bondyuzh- step. The subsequent treatment by Rhoder (twice) led toskoye oil field (Tatarstan, Russia), and the corresponding the residual oil contamination of 0.43 g/l resulting in anpure cultures were then deposited to the All-Russian Col- overall treatment efficiency of 96% (Table 1). A relativelylection of Microorganisms (ARCM indexes are 1513-D and high level of residual contamination could be mainly relat-1514-D, respectively) and patented [8-9]. The bioprepa- ed to the presence of oil polluted sediments accumulated in

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.. BIOCATALYSIS-2000: FUNDAMENTALS & APPLICATIONS "'. ~'

Table 1,~,~:'1 Bioremediation results of "Rhoder" field tests [6]

Initial oil pollution TreatmentSite Area m2 . (Pre )-treatment ffi . e.. m the upper layer (10cm). gll e clenCY.7.

River Chernaya 100 0.44 "Rhoder" (twice) >99.9

Vyngayakha:lake 1 5.000 15.1 "Rhoder" (triple) >99.9I lake 2 5.000 19.1 "Rhoder" (triple) 99! wetland 10.000 24.3 "Rhoder" (triple) 65

Ural: .lake 1.900 11.0 PMC. + "Rhoder" (twice) 96wetland 2.000 10.5 PMC. + "Rhoder" (triple) 94

Nizhnevartovsk:marshy peat soil 1.000 758-828 (g/kg) ploughing + "Rhoder" (triple) 14-24

. PMC is the preliminary mechanical collection of free oil.

this lake. These sediments served as a continuous source of to apply pre-treatment, the possible strategy can includfoil emission to the lake water. multiple microbiological treatment with ploughing, pH ad.

Bioremediation of wetlands. Relatively inferior re- justing and supplementing by nutrients throughout severa:suIts of remediation of the wetland in Vyngayakha(Table 1) years. On the aged spills (> 5 years), the oil-degradin~can be attributed to the fact that due to specific local ge- activity of indigenous microflora is usually high enough t(ological conditions it was hardly possible to apply a PMC omit an addition of biopreparations produced ex-situ. ThEof free oil on this site. However, taking into account a economically reasc:>nable strategy can include a stimulatioI

high 101 (> 24 gjl) and age of spill (4 years old), the re- of indigenous HDB already adapted to the site environment

suIts look quite satisfactorily - approximately 65% removal b. t Idf .1 t . t. (T bl 1) 0 th t 1. Anaero IC wastewater treatmen at co0 01 con amma Ion a e . n e con rary, app Ica- 0tion of PMC of spilled oil (75% removal) followed by triple temperatures (4-10 C)

treatment with Rhoder has resulted in much higher overall Anaerobic treatment has several well known advantreatment efficiency (94%) in the case of remediation of the tages in comparison with aerobic treatment, especially fOJwetland in Ural (Table 1). treatment of high-strength wastewater - no energy needl

Bioremediation of soils. The inferior results of for aeration (on the contrary, generation of energy in thE-Rhoder" bioremediation field tests obtained on the marshy form of biogas), substantially reduced nutrient require-peat soils in Nizhnevartovsk (Table 1) were not surprising ments, high organic loading rates (01R) etc. [10]. Howti\king into account an extremely high 101 (> 750 gjkg ever, an implementation of (;onventional anaerobic treat.of dry matter) and age of spill (6 years). Since an over- ment (especially in the countries with a cold climate sud-:vhelming majorit~ of the spilled oil was adsorbed by pea.t, as Russial!s often hindered by the necessity of maintainin!It was not economically reasonable to apply a PMC of 011. an operation temperature-mesophilic (30-37°C) or ther.The pre-treatment used included only a ploughing of up- mophilic (55-60°C) which is significantly higher than amper layer of contaminated area accompanied by addition of bient temperatures. This chapter discusses the recent findlime (to increase pH) and nitrogen and phosphorous fertilis- ings [11-12] aiming to extend an applicability of anaerobi,ers. An average (for 3 lots) reduction of oil pollution was wastewater treatment at psychrophilic temperatures as lov19% (Table 1) under application of "Rhoder", while with- as 4-10°C.out "Rhoder" addition it was 13% (data not shown). The Since low temperatures usually lead to a sharp delatter fact manifested about a high activity of indigenous crease of the biocatalytical activity of methanogenic miHDB already developed on the contaminated site during 6 crobial consortium involved in anaerobic digestion, a stratyears and substantially stimulated by pH adjusting and nu- egy in maintaining a reasonable efficiency of wastewatetrient addition. This supposition was further confirmed by treatment should include an increase (as much as possibledirect counts of MPN of HDB from the lot without Rhoder of concentration of biocatalysts inside the reactor orjancaddition, which were 103 and almost 106 cellsjml in the a gradual adaptation of the consortium to psychrophilibeginning and in the end of experiments, respectively (data conditions. Both these approaches were combined in th,not shown). present study by using granular mesophilic sludge havin:

Summarising the results presented in this chapter, one rather high methanogenic activity and up-flow anaerobican say the following. Field tests showed a very high effi- sludge bed (UASB) reactor promoting self-immobilisatio:ciency of biopreparation "Rhoder" for remediation of aqua- (and thus accumulation inside the reactor) of the cells ctories moderately contaminated by oil « 20 gjl). Howev- methanogenic consortium in the form of well-settled graner, for treatment of heavy polluted aquatories (thickness ules. Raw vinasse obtained by distillation of low qualitof oil film> 3 mm) as well as oil spills on wetlands and wines and diluted by tap water was us~d as feeding influgrounds, the best strategy should include a preliminary me- ent. The other details of experimental study are presentelchanical collection of free oil, or application of adsorbents, in works [11-12]. The perfornlance data of long-term treator other pre-treatment methods followed by microbiolog- ment of vinasse under psychrophilic conditions are generical polishing step. If for some reasons it is impossible alised in Table 2.

.. V.,'nik Kh;m;ya

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18 VESTNIK MOSKOVSKOGO UNIVERSITETA. KHIMIYA. 2000. Vol.41, No.o. ;,upplell,c"L... .~ ,.&...& , T"ble 2

'1 Performance data of long-term treatment of vinasse under psychrophilic conditions(average values are given in brackets) [11-12].

C Temperature, "C, Parameters! 9-10 7-8 3-51 .i Single UASB reactor

Run Run la. Run lb. Run 2a Run 2b Run 3Run days 0-67 68-158 159-185 197-236 251-273Recycle ratio 1:2.6 1:2.6 1:2.6 1:11.6 1:11.6OLR, g COD/lId 0.3-5.1(2.7) 1.4-7.3(4.7) 3.2-4.6(3.7) 2.3-3.5(3.0) 1.1-2.7(1.7)HRT, days 0.8-5.1(1.9) 0.5-1.6(0.9) 0.85-0.87 0.9-1.3(1.1) 1.14-1.17

'"" Influent COD, g/l 3.6-5.2(4.0) 1.2-9.9(4.2) 2.7-4.0(3.2) 3.0-3.6(3.2) 1.3-3.1(2.0)Effluent COD, g/l 0.3-2.7(1.0) 0.5-3.6(1.8) 0.8-1.9(1.0) 0.9-1.5(1.2) 0.6-1.5(0.8)COD removal, % 48-92(72) 48-92(60) 52-79(68) 48-70(60) 15-72(57)

. Two UASB reactors in series

Run days 0-63 82-107 122-147Reactor Rl R2 Rl R2 Rl R2Recycle ratio 1:1 1:18 1:1 1:18 1:1 1:18OLR, g COD/lId 3.2-5.5(4.4) 0.8-4.0(2,5) 2.3-4.2(3.5) 1.2-3.0(2.2) 2.0-2.7(2,5) 1.5-2.2(1.7)HRT, days 0.8-1.3(1.0) 0.8-1.2(1.0) 1.0-1.1(1.0) 1.0-1.1(1.0) 0.8-1.0(0.9) 0.8-1.0(0.9)Influent COD, g/l 3.1-5.4(4.3) 1.6-3.9(2.5) 2.5-4.2(3.5) 1.4-3.1(2.3) 1.9-2.6(2.4) 1.1-1.9(1.5)Effluent COD, g/l 1.0-3.9(2.4) 0.4-2.8(1.2) 1.3-3.1(2.3) 0.4..1.9(1.0) 1.1-1.9(1.5) 0.3-1.2(0.7)

, COD removal. % 16-76(46) 24-80(58) 19-52(37) 29-78(61) 25-52(37) 43-74(53)

Combined system (Rl+R2)

OLR, g COD/lId 1.6-2.8(2.2) 1.2-2.1(1.8). 1.0-1.4(1.3)HRT, days 1.6-2.5(2.0) 2.0-2.2(2.0) 1.6-2.0(1.8)COD removal, % 36-91(78) 42-89(76) 60-86(71). Run la-non-preacidified influent; run lb-preacidified influellt.

One stage U ASB psychrophilic treatment. Dtlr- above-mentioned supposition about the existence of massing run la (10°C), when non-preacidified influent wa.'J treat- transfer limitations inside the psychrophilic sludge bed.

t ed. an OLR was increl\8ed stepwise to 4-5 g COD/lid with Decrease of temperature during run 2a to 7 °C did nota total chemical oxygen demand (COD) removal of around result in deterioration of reactor performance though the70%. (Table 2). A significant presence of propionate (pre- OLRs were somewhat lower (around 4 g COD/lid) thandominant component) and acetate was observed in the efflu& those applied during run 1b (Table 2). In order to decreaseents. However, only traces of sugars, ethanol and butyrate mass transfer limitations, the recycle ration was increasedwere detected in the reactor liquor, while the headspace gas during run 2b (days 197-236, Table 2). As expected, anhydrogen concentration was negligible. These facts clear- almost 4 times increase of Vup resulted in a better VFAly demonstrate that low temperatures affect the various removal though a total COD removal efficiency slightly de-

. stages of anaerobic digestion differently, with propionate ~reased compared to run 2a. This was mainly due to anconversion becoming the rate-limiting step [13]. It should mcreased.~ludge washout beca~se small sludge ~g~regate~be also noted that a substantial increase (- 20%) of sludge were c~ntmu?usly accumulated m the effluent ~eclplent ves-bed height had occurred over this run that was primari- sel dunng this run. A further decrease of workmg tempera-ly due to a substantial growth of acidogens in the reac- tur~ to 4 °C was accompanied by a decrease of OLR imposedtor bec use fl ff t 1 . th 1 dunng run 3 (days 251-273, Table 2). In general, the over.

, a a u y ou er ayer covenng e granu es was .. .d . . b t . f th 1 d all reactor performance was slmllar to that dunng run 2b

seen un er microscopiC 0 serva Ions 0 e s u ge aggre- .. A sludge washout was also ob8erved but It tended to deceasE

gates. Smce such types of aggregates can provoke sludge d . thO It b ' ' t f fi 1 d. . . , unng IS run. was ecause a maJon y 0 ne s u gfflotation and create mass transfer hmltatlons for substrates t 1 d 1. . t d f th t d .

. , .. .. aggrega es were a rea y e lmma e rom e reac or urm~of proplonate-degradmg and acetlcla.'Jtlc bactena which are 2b M. . b t. f th 1 d t k t. . run. lcroscoplC 0 serv,~ Ion 0 e s u ge a en ausually located m the central part of aggregates, It was th d f 3 h d h 1 . d . . .. . e en 0 run s owe an overw e mmg pre ommance 0

decided to apply preacldlficatlon of wastewater in order fl ff 1 t (4 5 ) .th . 1 f d ' Ih. b . , u y arge aggrega es - mm WI lrregu ar orms an II to ac. i~ve e~ter COD removal. Howev~r, feedmg wI~h looked like flocculent one. Such evolution of the sludge car.

preacidlfied vmasse (run 1b, Table 2) dId not result m be attributed to the fact that the reactor influent was nol! a~y enhancement ?f COD removal with the effluent pro- completely acidified by preacidification procedure applied

pionate concentratIons o:te~ ex~eeded 1.5 g COD/I, In e. g., sometimes quite noticeable concentrations of ethanoo.rder. to have a deep~~ mslght mto the processes o~cur- (till 2 g COD/I) and sugars (till 0.6 g COD/I) entered t<rmg m the psychrophllic UASB reactor, the sludge kmet- the reactor stimulating a development of fluffy acidogenilic ch~~acteristics were assessed in situ, i. e., under reactor biomass which deteriorated a sludge quality. Thus, a concondItions (days 120-138). Apparent half saturation con- trol of preacidification efficiency seems to be essential fo..,tants Km for all the substrates tested were found (data a stable p;~treatment proceS8 of winery wastewater at 10'not shown) to be greater than 1.0 g COD/l at the imposed temperatures.up-flow liquid velocity (Vup ) of 0.1 m/h, which supports the Two stage U ASB psychrophilic treatment. In or

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. BIOCATALYSIS-2000: FUNDAMENTALS & A1'1'Ll\';AJ.lV!~"

der to control preacidification of wastewater with the aim erties of azo dyes dictate the anaerobic-aerobic sequence in_I to enhance a COD removal two UASB reactors were com- designing an efficient biomineralisation process. Two sep-

- bined in series. Reactor R1 ~ainlY served a.~ preacidificator arate treatment steps are usually applied for this purpose.

to generate VFA for feeding reactor R2. High recycle ratio In order tC1~ptimise the t~eatment process and ove.rall eco-(1: 18) was applied in reactor R2 in order to decrease mass nomics of the correspondmg technology, we combIned thetransfer limitations while recycle ratio in reactor Rl was anaerobic and aerobic phases into one single unit called thekept at low level (1: 1) because diffllsionallimitations are anaerobic-aerobic hybrid reactor (AnAHR) in this studynot very important for fast acidogenic step. The sludge (Fig. 1 a ). The advantages of this innovative design includefrom run 3 consisting predominantly of fluffy large aggre- reduced aeration costs and lower space requirements whilegates (see above) was used as a seed for both the reactors. offering substantial mitigation of a broad spectrum ofrecal-Analysing the results obtained during two-stage UASB pre- citrant xenobiotic contaminants (not only azo dyes) foundtreatment (Table 2), one can say the following. A combined in industrial wastewaters. This chapter discusses the per-system with two reactors in series has demonstrated higher formance of the mesophilic (30°C} AnAHR using the azo- ~ removal efficiencies and significantly better operation sta- dye Siriusgelb (Fig. Ib) and ethanol as donor of reductive

bility compared to : single U:A.SB t~ea~ment at ~empera- equivalents (Fig. 2) [16]. The concentrations of Siriusgelbtures as low as 4-10 C. Any difficultIes m a combmed sys- and ethanol were 0.3 and 0.82 g COD/I, respectively. Ittern performance including sludge lifting or heavy washout should be noted that throughout the entire experimentalhave no~ been observed at all. It should be not~d, however, run, only traces (if any) of ethanol and acetate (very rarely)i ' that a sIngle UASB reactor was operated at hIgher OL~ d t t d I' n the UPp er Part of the anaerobic zone of. . . . were e ec e(but wIth ~reacldlfied wast~water) than t.h~ OL~ Imposed the AnAHR. This suggests that the conversion of ethanol

ri ?n a combm~d system treatmg non-preacldlfied wastewater to methane was already complete in this zone and the mea-;" If one takes litO acc~unt. the overall volume of both reac- sured COD content of the slLmples taken from the upper~ tors. Thus, an apphcatlon of two UASB reactor system

t f th '" b . t t 11 as those of the' . .

h Id par 0 e anaero IC com par men as we

'0 Imphes hIgher capItal and operatIonal costs whIch s ouR ffl ' d 1 d d d d df . ... .

f 1 AnAH e uent represente on y non- egra e azo ye an!; be taken Into account under possIble ImplementatIon 0 ow" b. t t t Fr th th I d its breakdown products.~' temperature anaero IC pre rea men. om e 0 er Ian, .

a single UASB reactor operating at psychrophilic temper- During the first 18 days, when the azo dye loadIng ratEatures seems to need at least a partial preacidification of (ADLR) was 0.09 g COD/I/day using a HRT (hydrauli<wastewater in order to ensure its more-or-less stable oper- retention time) of ~pproximILte~y 3.4 days (Fi.g. 2a), az(

I ation. dye treatment efficIency (TE) m the anaerobIc compart.~ Concluding this chapter, it should be ~oted that anaer- ment was 51% and the overall TE of the AnAHR Wal~ obic treatment in high rate reactors like UASB reactors is 71% (Fig. 2b). After an in(:rease of ADLR to an aver.

feasible at temperatures as low as 4-10°C. However, sub- age value of 0.18 g COD/l/dILY for the period from day 1~stantial mass-transfer limitations for the soluble substrates to day 32 (Fig. 2a), azo dye anaerobic and overall TElinside the reactor sludge bed were encountered. Therefore, dropped slightly and were on average 50 and 64%, re,an application of higher recycle rations is essential for en- spectively (Fig. 2b). In the final stage of this experi.hancement of UASB treatment under psychrophilic condi- ment (day 33 onwards), the ADLR was further increased t<tions. The produced anaerobic effluents were shown to be 0.3 g COD/I/day keeping the HRT around 1 day (Fig. 2a)efficiently post-treated aerobically-final effluent COD con- This resulted in a further drop of both TEs-44 and 56Ojcentrations were around 0.1 ~/l [12]. A successful operation (on average) for the anaerobic compartment and the enof the UASB reacto~s at qUIte l~w t~mpera~ures (4-10°C) tire AnAHR, respectively (Fig. 2b). Negligible absorbanop~ns good perspectlve.s for apphcatlon of l11gh~rate anaer- cies at 37:5. nm (maximum absorbance of Siriusgelb) werO?IC treatme~t at ambIent temperatures, e. g. m south re- observed in the reactor effluent throughout the entire exglons of RussIa. perimental run indicating complete decomposition of thi

B . , 1° t ' f d . ° t . azo dye. However, complete decolouration of the efflulomlnera Isa Ion 0 azo yes In Innova Ive.bo

b o h b o d t ent dId not occur, rather, It remamed shghtly rose r

anaero Ic-aero IC y rl reac or ...

colour compared to the IntensIve brownish-yellow colou--',- , Aro-d-ye9'-represent a major grOtlp of all the dyes pro- .. - . "-. --- ~"

duced world-wide [14]. Approximately 10-15% of over- :,'all production is released into the environment mainly viawastewater [15]. This is very dangerous because some of theazo dyes or their breakdown products have a strong toxic,mutagenic or carcinogenic influence on the living organisms;therefore, the corresponding wastewaters should be treat- Azo dye Efnuented before discharge. However, a majority of azo dyes are Fig. 1 a. The experimental set-lIp for biomineralisation of azo

quite resistant to biodegradation under aerobic conditions dyes in the AnAHR.and easily pass through conventional aerobic wastewater OONtreatment systems. On the other hand, azo dyes are read- COONs (}ily decolourised by splitting the azo bond(s) in anaerobic f '\ -environments. In turn, the anaerobic breakdown products 011 h ""..-o-NHC-mrQ-N'N- J H(are more susceptible to biodegradation under aerobic condi- V - 0 -tions rather than under anaerobic conditions. These prop- Fig. lb, The structural formula of azo dyeSiriusgelb GG.

'° V.,tn;k Kh;m;y.

-

.. 20 VESTNIK MOSKOVSKOGO UNIVERSITETA. KHIMIYA. 2000. Vol.41, 1'40.1>. :,upplemenlO-- ~~-~ LRazOLRtOt ~RT Ef-an ...'Ef-AnAHR~ I 5, - 4 TE-an TE-AnAHR}, .,..'. I,. I 0,6 100* ~' . , . ,. ' ~ ; (/), ~ /'4),:f\>:< 0 t -0 .",.,', ~1 3 =>- 0 80 ~ , ---' , -- 0-.'" ~

, ;;;;;. 1, I -0 U 04 ~~ I ~ , 60 E-O a '., 2 ~ ODU . --, ~ ~ Q)

b =>-

~O,5 "',-', ,~, :I: ~02 . "'"~., 40-0~ ::::~:~ , JXX~:\;.:.'. - ~ , .. I=" . . " . ."' - ~, ' ~.- X ..-r-'.~ ,-, .' .

20 <t:-' ~ - - . ,'.~ " - ~ 'f.

0 ~o 0,0 0

0 10 20 30 40 50 0 10 20 30 40 50

Time, days Time, days

Fig. 2. Operation performance of AnAHR treating a Siriusgelb (0.3 g COD/I) synthetic wastewater supplemented with

ethanol (0.82 g COD/I).

of influent. A transient accumulation of intermediate of reactions in a liquid phase, physical absorption and directSiriusgelb decomposition-5-aminosalycilic acid (5-ASA)- chemical conversion have several evident drawbacks: highwas detected in the anaerobic compartment of the AnAHR reagent consumption, equipment corrosion, application ofbut not in the effluent. The 5-ASA concentrations peaked high temperature and pressure, etc., resulting in high pro-(till 0.06 g COD/I) immediately after increases of ADLR cess costs-250-750$/ton of removed sulphur [19]. Thisand then gradually decreased if the ADLR was kept con- chapter highlights the development of substantially cheap-stant. This observation suggests a stepwise adaptation of er alternative-biocatalytical technology for treatment ofanaerobic sludge for decomposition of 5-ASA. As can be H 2 S polluted gases. The schematic representation of theseen in Fig. 2b, a majority of the azo dye COD was removed process proposed is shown ill Fig. 3 [18]. Briefly, In thein the anaerobic compartment and the aerobic section had scrubber, the H2 S containing gas comes into contact witha relatively minor impact on the overall TE. The aerobic a slightly alkaline (pH 8.0-8.5) scrubbing solution where ab-removal, as a percentage of the influent, varied between sorption of..If 2 S takes place. Scrubbing liquor then passes20 and 30%. Such low TEs achieved in the aerobic step to the bioreactor containing immobilised bacteria of genusas well as effluent colouring can be attributed to the fact Thiobacillus where a soft oxidation of sulphide into elemen-that the breakdown products of anaerobic Siriusgelb de- tal sulphur accompanied by regeneration of alkalinity pro-composition (5-ASA and 1,4-phenilenediamine) are readily ceeds. Solid sulphur is removed and the liquid is returnedautooxidised to coloured polymeric products upon exposure to the scrubber for absorption of the next portion of H2 S.to air [17]. These autooxidation products are often complex Since the success of elegant technological scheme pre-humic compounds that are non-biodegradable. Incomplete sented in Fig. 3 is determined (in major extent) byefficien-recovery of ammonia (data not shown) also supports the cy ofbioreactor, significant efforts were put on optimisationabove-mentioned supposition about the inclusion of gener- of its construction and produ(:tivity [18]. The crucial pointated aromatic amines in these persistent polymeric prod- is that the bacteria of genus Thiobacillus used in the pro-ucts. cess oxidise sulphide not only into sulphur but also into

Thus, an innovative reactor construction where the sulphate:anaerobic and aerobic phases were combined in one sin-gle unit called an AnAHR is proposed for the treatment HS- + 0.502 ~ SO + OH-, (1)

of azo dyes as well as other aerobically persistent xenobi- HS- + 202 ~ S02- + H+. (2). . 4otIC contamInants. The performance of the AnAHR wastested with a synthetic wastewater containing Siriusgelb Obviously, the reaction (2) is highly undesirable for theand ethanol as co-substrate at 30°C. Almost complete de- process under development because it leads to expenditurecolouration of the influent and 56% removal of the azo dye of alkalinity of the liquid phase and formation of hard-COD was achieved using a HRT of 1 day and volumet- ly removable dissolved product (sulphate). To suppressric loading rate of 0.3 g azo dye COD/I/day. The efflu- this rea(:tion, oxygen-limiting conditions and high sulphideent contained no ethanol or acetate and its COD content loading rates should be imposed on the system [18]. Incould be attributed to the presence of non-biodegradable an engineering context, the various reactor constructionsautooxidation products of Siriusgelb breakdown intermedi- (conventional CSTR, reactor with external aerated loop,ates. Further research is needed to assess the feasibility of gas-lift) were tested on the laboratory level [18]. Currentlythis reactor concept for treatment of industrial wastewater the best construction consists of an automated close (withcontaining persistent compounds. respect to gas-phase) gas-lift reactor equipped with on-line

sensors for measuring dissolved oxygen, sulphide and pH.Biocatalytical technology for H 2 S removal and The electric signals from these sensors were transferred tosulphur recovery from polluted gases a programmable data logger system. A personal computer

programmed to function as a terminal emulator was usedBiogas usually contains around 1 vol. %, of H2 S, while to communicate with the data logger and to control the

natural gas can contain till 15 vol. % of H2 S [18]. Conven- feeding pumps. Using this highly controlled reactor andtional technologies for treating such gases based on chemical pure oxygen (instead of air), 94-98% efficiency of sulphide

BIOCATALYSIS-2000: FUNDAMENTALS & APPLICATIONS 21Gas free of HIS . I

~

S c rub b e r 0 H-Microaerobic

bioreactor

H S + OH- -- HS- .HS-+ 0.502 -- S,o..+ OH-I .. Sulphur

H S- + H I 0 ..

, :: A'ir

Gas with H:SFig, 3. Schematic representation of biocatalytical reagent less method for H 2 S removal from polluted gases.

conversion into elemental sulphur under sulphide loading 3. Murygina, V.P., Arinbasarov, M. U.j and Kalyuzhnyi, S. V.rates as high as 15 g S/l/day was achieved [18]. (1999) Ecology and industry of Russia, No.8, 16-19.

Finally, the manifest advantages of the proposed tech- 4. Currier, H.B. and Peoples, S.A. (1954) Hilgardia, 23,nology compared to the conventional methods should be 155-174.underlined: practically reagent less character (some salts are 5. Goskomstat RF (2000) Russia in figures p.OOO: officialnecessary for bacteria); cheapness; practically closed cycle statistics, Gosko~stat RF Press, Moscow.and minimum of wastewater; sole process product (sulphur) 6. Mlll"ygina, V., Arinbasarov M., and Kalyuzhnyi S. (2000) incan be readily re-used (for sulphuric acid production); am- Proc. of the 4tl1 Intern. Symp. on Environmental Biotech-bient temperature and pressure for the process making it rlology (Hartmans, S. and Lens, P., eds.), Noordwijkerhout,safe. the Netherlands, pp. 319-322.

7. Patent RF No. 2090697 (1997).Concluding remarks 8. Patent nr No. 2069492 (1996).

The presented examples clearly demonstrate that by 9. Pat.ent RF No. 2069493 (1996).creating optimal growth conditions for microorganisms in 10. Lettinga, G. (1996) Wat. Sci. Technol., 33(3), 85-98.proper designed engineering systems, conversion rates can 11. Kalyuzhnyi, S. V., Gladchel1ko, M.A., Sklyar, V.I., Kurako-be significantly increased resolving many problems of en- va, O. V., and Shcherbakov, S.S. (2000) Environ. Technol.,

vironmental biotechnology. Moreover, during last decade 21,919-925.this discipline has matured from its conventional function 12. Kalyuzlulyi, S. V., Gladcllenko, M.A., Sklyar, V.I., Kiz-(so-called "end of pipe treatment") to a new phase substan- imenko, Ye.S., and Shcherbakov S.S. (2000) Appl. Biochem.tially contributing to resource conservation and sustai~able Biotcchnol (submitted for publicati~n).production in the modern society. The discussed above 13. Rebac, S. (1998) Psychropl1ilic anaerobic treatment of lowbiocatalytical technology for H2 S removal and sulphur re- strength wastewaters, Ph. D. thesis, Wageningen Agricul-covery from polluted gases is a typical example of this. tural University, The Netherlands.

14. Carliell, C.M., Barclay, S.J., Naidoo, N., Buckley, C.A.,Acknowledgements Mulholland, D.A., and Senior, E. (1995) Water SA, 21(1),The financial supports of INTAS (Grants 96-1809 and 61-69. .

96 2045) N th I d R h 0 . t . (G t 97 15. Tan, N.C.G., Bor~er, A., Slenders, P., Svltelskaya, A.,- ,e er an s esearc rgamsa ion ran - . . ..

29925) IPP f US D t t f E (G t \ Lettmga, G., and FIeld, J.A. (1999) m ProceedIngs of, program 0 epar men 0 nergy ran IAWQ C f "W M... t . d E d f P.325733A G2) R . F d t . f B . R h on erence aste Inlmlsa Ion an n 0 Ipe- - , USSlan oun a ion 0 aslC esearc Tr . Ch . I d P h . I r d ."(G t 99 15 96064) R . M.. fS . d T h eatment In emlca an etroc emlca In ustnes

ra~ - - , USSlan m~stry 0 c!ence an ec - (BuitroI1j" G. and Macarie, H., eds.), Merida, Mexico,

nologles (G~ant 99-419), Luk?ovltskaya oll-!>ro~~ct st?r- pp. 227-234.age enterprise (Moscow provmce), compames LUKoll- 16 Kal I . S d SkI V (2000) W t S " ", h IU I f "" b """. . yuz UlYl, . an yar,. a. CI. J.ec no.,

ra ne tegas, Noya rskneftegas, Green (Nlzhnevar- 41, No. 12, 23-30.tovsk) and "Orenburggasprom" are gratefully acknowl- 17 R FI E (1997) B . t f t " d b d dd d . azo- ores,. . 10 rans orma Ion an 10 egra a-

e ge . Lion of N-.,ubstituted aromatics in methanogenic granular

References slt£dge, Ph. D. Thesis, Wageningen Agricttlture University,1. Rosenberg, E. and Ron, E.Z. (1996) in Bioremediation: The Netherlands.

principles and applications (Crawford, R.L. and Craw- 18. Kalyuzhnyi, S. V. and Fedorovich, V. V. (2000) Ecology andford, D.L., eds.), Cambridge University Press, New York, industry of Russia, No.2, 33-36.USA, pp. 100-124. 19. Lagas, J .A. (2000) in Environmental technologies to treat

2. Zhanovich, A.V., Gridin, O.M., and Gridin, A.O. (1995) sulfur P9'lution: principles and engineering (Lens, P. andin Proc. of the All-Russian Conference on Ecological Prob- Hulshoff Pol, L., eds.), IWA Publishing, London, pp. 237-lems, Moscow, pp. 3-6. 264.

11 V.,tD;k Kb;m;YD