ETHANOL PRODUCTION BY ANAEROBIC FERMENTATION IN GENETICALLY MANIPULATED ENTERIC BACTERIA By t s Hon-chiu Leung Thesis submitted as a partially fulfilment for the degree of MASTER OF PHILOSOPHY JUNE 1991 DIVISION OF BIOLOGY GRADUATE SCHOOL THE CHINESE UNIVERSITY OF HONG KONG 《::...
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ETHANOL PRODUCTION BY ANAEROBIC FERMENTATION IN ... · and infrared spectroscopy , Swings and De Ley (1977) concluded 4 different strains into a single specie Zymomonass mobilis with
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ETHANOL PRODUCTION BY ANAEROBIC FERMENTATION IN
GENETICALLY MANIPULATED ENTERIC BACTERIA
By
t s
Hon-chiu Leung
Thesis
submitted as a partially fulfilment
for the degree of
MASTER OF PHILOSOPHY
JUNE 1991
DIVISION OF BIOLOGY
GRADUATE SCHOOL
THE CHINESE UNIVERSITY OF HONG KONG 《::.. .
325421 •ft、从“ T F 5-93
I 2 I JUN W2 | \ 0 V-—~1» —J ^ I
Ur̂VERS.TY / /
會 : ¾ ^ . -一二 泸
Abstracts
Ethanol is in great demand for industrial uses such as organic solvents,
• «.•'' — __
germicide, antifreeze and fuel. An ethanol producing bacteria, Zymomonas
^obilis, has the ethanol fermentative pathway. This bacteria possesses the enzyme
pyruvate decarboxylase which converts pyruvate into acetaldehyde. Acetaldehyde is then converted to ethanol by the enzyme alcohol dehydrogenase.
The genes ̂ c (coding for the enzyme pyruvate decarboxylase) and adhB •i .
(coding for the enzyme alcohol dehydrogenase) had been cloned from Z mobilis
into a plasmid and then transformed into Escherichia coli. The transformant
changed into producing ethanol as the major fermentative end product.
In this study, I tried to transform the plasmids pZAN4 and pZAN2, which
harbouring pdc-adhB and pdc respectively, into Salmonella typhimurium strains.
The restriction defective, (rrn+) intermediate host JR502 was transformed first.
The modified plasmids were then isolated and transformed into wild type S.
typhimurium LTZ.pflR mutant of S. typhimurium HSK1124 was also transformed
with PZAN2 and PZAN4. “
A high production of ethanol was detected in JR502 harbouring PZAN4
(128 mM from 55 mM glucose), when compared with host itself ( 5.9 mM from
55 mM glucose), a 22 fold increase was observed. An fifteen fold increase of
ethanol production was also detected in wild type S. typhimurium harbouring
xi
pZAN4 (91 mM verse 6.1 mM in host itself). No significant increase of ethanol
production was detected in pflR mutant HSK 1124.
.“ Growth of strains of S. typhimurium harbouring plasmid PZAN4 was faster
than those harbouring pZAN2 which in turn was faster than the hosts themselves.
So the presence of these two plasmids promoted the growth of the strains of 5.
typhimurium. The plasmid pZAN4 was stably maintained inside the hosts up to
2 4 hours while pZAN2 was retained at 100 % level up to 9 hours only. LT2
tolerated 5 % ethanol (v/v) without a significant fall of cell density. JR502
decreased to an optical density of 0.3 in this ethanol concentration. HSK1124
showed a sudden drop in cell density at 5 % ethanol concentration. The attempt
to subclonepdc-adh using the broad-host-range plasmid pKT240 was unsuccessful.
A marine Vibrio, V. sp strain 60, was identified according to routine
identification processes as V, anguUlarum. Evidence was also provided from
systematic studies of V. species by Arbitrarily-primed Polymerase Chain Reaction
(AP-PCR). The optimal growth conditions of V. sp. strain 60 was found to be 2
% sodium chloride, P medium, pH 6 to 8 and 42 °C. A plasmid RP4 derived
broad-host-range plasmid pIOl was isolated which could be used as a shuttle
cloning vector. Ethanol production by fermentation of 50 mM sugars was
performed and the best substrate for ethanol production by fermentation was
mannitol (32.7 mM) while the second best was fructose (26.3 mM). K sp. strain
60 tolerated 3 % ethanol and 1 M sodium chloride. Transformation process by
參 •
electroporation in V. sp. strain 60 was developed, the optimal resuspending buffer
consisted of 272 mM sucrose, 15 % glycerol and 7 mM sodium phosphate at pH
7,the large plasmid pIOl was transformed back into strain 60 using this process.
Attecfipt to clone pdc-adh into pIOl was, however, unsuccessful.
« 書 •
i n
Acknowledgement
I would like to thank cordially my supervisor, Dr. H. S. Kwan for his
patient supervision, numerous encouragement and critical comments throughout
the course of my study and preparation of this thesis.
I would like to express my sincere gratitude to Dr. P. K. Wong and Dr. J.
A. Buswell for serving as the members- of my Thesis Committee, and Prof. Ericka
L. Barrett for serving as the external examiner.
Thanks are also due to Dr. K. Y. Chan for his valuable suggestions and
numerous discussions. Finally, I would like to express my sincere thankfulness to
Dr. K. K. Wong and Mr. K. M. Pang for supplying me bacterial strains and
technical assistance.
xi
Dedication
TO MY PARENTS
‘• ” :•. •…. v . “ . ”i:
Table of Contents
Abstract i
Acknowledgement i.v
Dedication v
Table of Contents v i
Introduction 1
Literature Review 4
1) Ethanol production in bacteria
1.1) Zymomortas mobilis ‘
1.2) Clostridium species 7
1.3) Enterobacter,Klebsiella, Serritia and Erwinia sp. 9
1 . 4 ) Escherichia coli a n d Salmonella
typhimurium 10
2) Pyruvate decarboxylase of Z. mobilis
2.1) Enzyme properties 13
2.2) Cloning and expression of pdc gene 15
3) Alcohol dehydrogenase (o^/i) gene
3.1) Cloning, chararterization and expression of adh
genes 17
4) Gene transfer systems in Vibrio species
5) Rationale and objectives of this study 22
xi
Part I) Ethanol Production in terrestrial enteric bacteria
A) Introduction 24
B) Materials and Methods
1) Bacterial strains and plasmids 25
2) Media 26
3) Solutions 27
4) Isolation of plasmids
4 . 1 ) S m a l l S c a l e I s o l a t i o n of
plasmids 30
4 . 2 ) L a r g e S c a l e I s o l a t i o n of
32 plasmids
5) Construction of a broad-host-range
plasmid harbouring Zymomonas mobilis genes. 35
6) Transformation 35
7 ) H i g h P e r f o r m a n c e L i q u i d
Chromatography of Organic Acids 36
8) Maintenace of plasmids harbouring the genes of
Zymomonas mobilis genes. 3 8
9) Ethanol tolerance of S. typhimurium strains 38
參 •
V l l
C) Results
1) Construction of Salmonella typhimurium strains
harbouring Z. mobilis genes 3 9
2) Fermentative end products in culture medium 48
3) Growth of hosts and transformants 61
4) Ethanol tolerance of & typhimurium strains 65
5) Maintenance of plasmids 67
6) Cons t ruc t i on of b r o ad - ho s t - r a nge
plasmid harbouring Z. mobilis genes 69
D) Discussions
1) Comparison of ethanol production m Escherichia
coli and Salmonella typhimurium 72
2) Ethanol tolerance of 5. typhimurium strains 74
3) Maintenance of plasmids 76
4) Cons t ruc t i on of b r o ad -ho s t - r a nge
plasmids harbouring Z mobilis genes 78
%
、 , • •. v i i i
Part II) Ethanol Production in marine enteric bacteria
A) Introduction 79
, B) Materials and Methods
, 1) Bacterial strains and plasmids 80
2) Media 30 v
$ “ > 3) Solutions 80
4) Routine Identification Processes 81
5) Systematic studies by Arbitrarily-
Primed Polymerase Chain Reaction 86
6) Optimal growth conditions S8
7) Isolation of broad-host-range plasmid pIOl (64
kb) 89
8) Transformation of Vibrio sp. strain 60 9 0
9) Production of ethanol using different carbon
sources in fermentation 91
C) Results
1) Identification of Vibrio sp. strain 60 92
2) Optimal growth conditions 101
3) Isolation of high molecular weight plasmid 105
4) Ethanol production from different carbon sources 1 Q 7
Lane 1: \Hind IE 2 and 3: Uncut pZAN4 4 and 5: Uncut pZAN2 6: Host only 7: Xffindm
47
2) Fermentative end products in culture medium.
The fermentative end products were resolved by High Performance Liquid
Chromatography. The column was first equilibrated by injecting different
fermentative end products at different concentrations. The retention time and the
peak area of a particular product were then determined (Figure 7). A calibration
curve was constructed which shown the linear relationships of concentration of
product and the peak- area resolved in HPLC. The products included succinate,
lactate, acetate, acetaldehyde and ethanol. The calibration curves were showirin
the Figure 8 and 9.
• " r ‘.
48
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„ ,-. o-j ,
g. !.
^ J J J • r- ' .
A V i ?- 2 1
1 p ^ .
. . P -2-4. ^5. 96
:~~ •‘ “ B
: — . . , . . 、、 -
i?. 13 . •
饭 . P lO
r ^ “ ' . . . . + •
( . ‘ ,
l - 2- 50 * . /̂ "4. ?c- . ..
_ , , 1 . . ,.. 3 . . , . , - : … • . f . &6 .
___.. . . ‘ …丨丨 I • • •、:丨…--, • • • — : ' .
C ) 1 7 . 2 4
I . . . . . . • -J , / . . . ; 、 ,
. t ^ • . :. . . : ‘ • . .
: »:•. rr- “丨h r. :-:. * . _
—. . ' • • • • • “ " : ‘ .:…1 iTy 二|7_ • 二 “ •
. . • . d .. : ±?; i i .
20. 71 cr n ; ; ; ; E “
; :— : :~~Z$To2 ‘~~‘
f . . ‘ -. . Cja ‘• - ‘ _ . ‘ . . ‘
• • . . . • “ •' '•*•*•- . • • ... •
Figure^ 7—Calibration — of ethanol concentration by High Performance Liquid Chromatography. Ethanol was retained at about 24 min. A) LB 1 % glucose medium
+ 1 % ethanol. B) LB 1 % glucose medium + 2 % ethanol. C) LB 1 % glucose
medium + 3 % ethanol. D) LB 1 % glucose medium + 5 % ethanol.
Among the tests, there were differences in the rate of reaction, despite of
these minor differences, three species possessed same characteristics in all tests.
K cmguillarum ATCC 19264 hydrolysed starch at the fastest rate while K sp.
strain 60 was the slowest starch hydrolysing strain. The growth of V. cmguillarum
ATCC 19264 and 14281 on SIM agar occurred mainly on surface while V. sp.
strain 60 occurred both at surface and inferior of SIM agar. The red color
developed deeper in V. anguillarum ATCC19264 and 14281 than in K sp, strain
60,so ATCC 19264 and 14281 reduced nitrate to larger extent did than V. sp.
strain 60.
Based on the results of these test, V. sp. strain 60 was suggested as V.
anguillarum according to the routine identification key in Berge/s manual, the
similarity in most tests of V. sp. strain 60 with other two strains of V, anguillarum
support the evidence that V. sp. strain 60 might be K cmguillarum.
/
95
1.2) Strain identification by Arbitrarily Primed Polymerase Chain Reaction(AP-
PCR)
In this study, chromosomes were prepared in small scale from V.
anguillarum ATCC19264, ATCC14281, V. alginolyticus, K parahaemofyticus, K
splendidus and V. vulnificus. The electrophoretic diagram of chromosomes of the
species of Vibrio were shown in Figure 22,
After 2 cycles of low stringency of amplification with low Taq: DNA
polymerase concentrated, the reaction mixture were subjected to 10 cycles of94°C
for 1 min., then 50。C for 1 min. and 72。C for 2 min. The final reaction mixture
was added with normal amount ol Taq DNA polymerase and subjected to 30 to
40 normal cycles of amplification. The AP-PCR products were revealed in 3 %
agarose gel electrophoresis, the fingerprints were shown in Figure 23 and Figure
24.
V. anguillarum A1CC 19264 and ATCC 14281 showed similar pattern with
V. sp. strain 60. The fingerprints of 3 species matched even in other independent \
studies. Major bands were observed with homology up to 75 % among these three
stains. Minor band differences implied that they are distinctively different strains.
Other genus of Vibrio showed a totally different fingerprint pattern in all trials.
So AP-PCR supported the evidence that V. sp. strain 60 was V. anguillarum.
96
取 > — , a— •
一 t - -— — — I •
_ ~ _ — 、 = a r
麵 M ^ —
sa-— B-*- . —H*- S r
mm — — -mm ! ! mmm tm-^ mt* :
; •• S F ^ ; 圓
Figure 21. Schematic diagram of Arbitrarily-primed Polymerase Chain Reaction (AP-PCR).
97
1 2 3 4 5 a
i l ^ f l l H I — D N A
RNA
Figure 22. Electrophoretic d i a g r a m of chromosomal DNA of 、 Vibrio species.
Lane 1: K vu_cus 5广„〜‘ 2; 7. splendidus ^ V. alginolyticus
3: V. parahemolyticus (QE Hospital) 4: V. parahemolyticus
\
98
a7。
2—BSHBB-。.啦
Figure 23. Fingerprint of AP-PCR using M13 sequencing primer (24mer). ‘ °
Lane 1: XBstE E 2: Vibrio anguillarum ATCC 19264 3: V. anguillarum ATCC 142S1 4: V. sp. strain 60 5: V. alginolyticus 6: V. fluvialis 7: V, parahemolyticus 8: V. parahemolyticus^ from Queen EHzabeth Hospital) 9: V. splendidus 10: V:
vulnificus 11: pBR322 Mspl marker
99
1 2 3 4 5 6 7 8 9 10 . T l .
1.264
P P B B f e k B H —a 7 0 2
Figure 24. Fingerprint of AF-PCR using M13 reverse sequencing primer (24mer).
Lane 1: \BstB JL 2: Vibrio anguillarum ATCC 19264 3: V. anguillarum ATCC 14281
T
4: V. sp. strain 60 5: V. alginolyticus 6: V. fluvialis 7: V. parahemolyticus 8: V. parahemolyticus、from Queen Elizabeth Hospital) 9: V. splendidus K
10: V. vulnificus
100
2) Optimal Growth conditions
Wild type V. sp. strain 60 was grown in different media of different
substrates, different salt concentration, different temperature and pH value. One
percent (v/v) overnight culture of V. sp. strain 60 was added to the media and
the growth was measured as optical density at time intervals. The shortest
generation time corresponded to the fastest growth rate.
Among the salt concentration range of 1 % (w/v) to 3 %, the generation
time of V. sp. strain 60 was the shortest at 2 % sodium chloride. No growth was
observed when tbe salt was omitted. So all media used to grow V. sp. strain 60 in
this study contained 2 % (w/v) sodium chloride.
The growth curves of K sp. strain 60 in different media were shown in
Figure 25 and Figure 26. The generation times of V. sp. strain 60 in different
media and temperature were summarized in Table 8.
Generally, a shorter generation time was found when V. sp. strain 60 was
grown in P medium indicating that Bacto-peptone was a better substrate to this
bacterium than Bacto-tryptone. The fact that Bacto-peptone contained 14 % total
nitrogen but Bacto-tryptone contained only 12.7 % total nitrogen might contribute
to the difference in growth rate. The optimal pH range was 6 to 8. So in order to
grow V. sp. strain 60 under optimal condition》the optimal medium was 2 %
sodium chloride, pH 6 to pH 8,P medium ( 10 % Bacto-peptone, 5 % yeast
comparisons, phage sensitivity (Selander et al” 1987) and more recently, DNA
based methods,particularly polymerase chain reaction (Atlas and Bej, 1990). In
this study, strains could be distinguished by comparing polymorphisms in genomic
fingerprints using AP-PCR. In AP-PGR, a single primer was used, two cycles of
DNA amplification in low stringency followed by normal cycles of high stringency
was employed to generate different banding patterns from chromosomes of
different bacteria. The advantages of AP-PCR are: l)no prior sequence
information is required, 2) simple and reproducible fingerprints of complex
genomes can be generated ( Welsh and McClelland, 1990).
The AP-PCR fingerprints of different strains of Vibrio anguillarum revealed
that they had common bands; only 20 % of the bands were different in migration
rates in 3 % agarose gel electrophoresis, this implied minor genomic difference
among same species but different strains of bacteria. The band pattern was
determined mainly by the amplification of template DNA in the first two cycles
of amplification under low stringency condition. If the annealing temperature was
too high to select pairing of partially complementary base sequence, no clear
product was formed. The annealing temperature possessed a tolerable range that
116
hardly any bands was formed if above this range. Smears will be formed which
revealed a highly random priming if the annealing temperature is below the
range. This method was repeated and related strains always showed similar •
banding patterns. Although this is an unorthodoxical method, it was faster than
hybridization methods by restriction fragment length polymorphism.
117
2) Isolation of High Molecular Weight Plasmid
pIOl was a derivative plasmid of broad-host-range plasmid RP4. The host ..
range of RP4 included genera of Enterobacteriaceae 3nd Pseudomonas sp. (Olsen
and Shipley, 1973). There was not a plasmid isolation system developed for
marine vibrio, but it was found that K sp. strain 60 was able to conjugate with
Escherichia coli and Salmonella typhimurium (Ichige et al” 1989) and the
megaplasmid isolation system for 五.co/f was well established which could be used
to isolate plasmids up to 200 Kb (Hansen and Olsen,1978).. Thus p IO l was-first
transferred form V. sp. strain 60 to E. coli HB101. Transconjugant were selected
on EMB agar plate supplemented with kanamycin and carbenicillin. Thus only E.
coli harbouring the plasmid pIOl could form colonies.
The preparative method of pIOl by agarose gel electrophoresis was not
satisfactory simply because of low recovery in electroelution, so cesium chloride
density gradient ultracentrifagation was employed. However, the buoyant density
of the DNA of this plasmid in the gradient was very closed to that of
chromosomal DNA, the separation by swinging bucket formed two bands that
were only 1 mm apart, one could hardly draw the plasmid DNA without the
contamination of chromosomal DNA. If one used a vertical rotor, since the
diameter of the tubes was 25 mm, and the copy number of pIOl was very low, so
DNA lysate for 1 litre culture could only form a faint band in the centrifuge tube.
Finally, fixed angle tubes was the choice because it possessed a small diameter
(16mm) but an adequate length to develop a gradual gradient. Chromosomal
DNA and plasmid DNA were separated by nearly 1 cm that two clear bands
118
could be observed.
Sma I digestion of pIOl generated 5 fragments and one of which possess
the origin of replication of pIOl, one could fish out the region by cloning it into
a selectable marker plasmid and then transformed the recombinant plasmid into
different bacteria, thus a collection of shuttle vectors could be formed. Similar
approach was used to construct a broad-host-range plasmid (Ditta et al., 1980)
pRK290 with the addition of tetracycline resistant marker. The stability of the
constructed plasmid will be the prerequisite: of using this shuttle vector.
119
3) Ethanol Production of Vibrio sp. strain 60
A variety of carbon sources was tested in growing K sp. strain 60 in order
to determine the conversion ratio of these sugars by strain 60. The conversion
ratio of glucose was 16.8 % in V. sp. strain 60,compared with 5.39 % in S.
typhimurium JR502, 5.56% in LT2 and 3.37 % in HSK1124, V. sp. strain 60
produced ethanol to a larger extent than S. typhimurium without the help from
P d c and adhB genes of Z. mobilis. Intuitively, more ethanol will be produced if
P d c and adhB genes could be transformed into strain 60. This bacterium may be
used in industry to produce ethanol. Current industrial fermentation plants usually
require large volumes of freshwater for culture and cooling purpose, and the cost
of production includes the transportation cost of the raw materials form their
place of origin to the plant. In these two aspects, industrial fermentation using
marine microorganisms has the advantages of : 1) fermentation industries using
seawater which can support good growth of marine microorganisms can be
established in countries and island states lacking freshwater systems for water
supply to the fermentation plant and 2) industrial plants using marine
microorganisms in the fermentation processes could therefore be built along shore
using seawater as culture medium and for cooling and with minimum effluent
disposal costs. Alternately, a coastal tanker equipped as a fermentation plant
involving processes carries out by marine microorganisms could load fermentable
raw materials at various local seaports and carry out the fermentation in situ
during its voyage, therefore reducing the costs of transporting both raw materials
and final products. Since K sp. strain 60 could ferment a variety of sugars, by
providing low cost fermentation substrates, such as molasses and by making
120
mobile links between sources of biomass and potential markets, industrial
fermentation involving marine microorganisms would be economically attractive.
Moreover, Vibrio sp. strain 60 tolerate up to 1.19 M sodium chloride, which
exceeds the usual value of sea water, so Vibrio sp. strain 60 may grow in any
source of sea water to undergo fermentation.
Since ethanol at a certain level changes the cell membrane, enzymes and
cell wall structure ( Rose and Tempest, 1984), so the ceil density of V. sp. strain
60 decreased when the ethanol concentration was 5 %. Despite of ethanol
tolerance level, V. sp. strain 60 grew at the fastest rate at 42°C in P medium, such
an elevated temperature will enhance not only the growth of vibrio cells but also
enhances downstream processing, for example, evaporation and then
condensation of ethanol by fractional distillation. So the production per unit time
was increased and the time spent in downstream processing was decreased.
121
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