Microbial Production of Biodegradable Plastics ... - ACS Publications

Chapter 16

Microbial Production of Biodegradable Plastics from Carbon Dioxide and Agricultural Waste Material

Downloaded by UNIV OF SYDNEY on October 27, 2015 | http://pubs.acs.org Publication Date: May 1, 1997 | doi: 10.1021/bk-1997-0666.ch016

Ayaaki Ishizaki, Naohiko Taga, Toshihiro Takeshita, Toshikazu Sugimoto, Takeharu Tsuge, and Kenji Tanaka Department of Food Science and Technology, Faculty of Agriculture, Kyushu University, Hakozaki, Higashi-ku, Fukuoka 812, Japan

Fermentative production of biodegradable plastic material, poly-D-3hydroxybutyrate, P(3HB) from CO2 or agricultural wastes is expected to contribute to the solution of global environmental pollution problems. The practical cultivation systems to produce P(3HB) from CO2, H2 and O2 by hydrogen-oxidizing bacterium were developed by maintaining O2 concentration in gas phase below the lower limit for explosion. P(3HB) productivity was increased by improving gas mass transfer with the use of air-lift fermentor and the addition of 0.05 % C M C to the culture medium. P(3HB) was also produced from xylose via L-lactate by two-stage culture method using Alcaligenes eutrophus and Lactococcus lactis IO-1. P(3HB) productivity was increased by a pH-stat fed-batch culture method with feeding the substrate solution so as to control L-lactate concentration at very low level.

Polyhydroxyalkanoates, PHAs are potential raw materials for manufacturing biodegradable plastics(l). Alcaligenes eutrophus is a hydrogen-oxidizing bacterium that is able to grow autotrophically using H2, O2 and CO2 and heterotrophically using organic acids as substrate with the accumulation of poly-D-3-hydroxybutyric acid, P(3HB) in the cell under nutrient-limited conditions(Fig.l). The growth rate of this hydrogen-oxidizing bacterium is much higher than that of other autotrophs such as photosynthetic organisms and hence the bacterium has the potential to be used in industrial processes. Production of P(3HB) from CO2 or organic acids derived from agricultural wastes by A.eutrophus, could contribute to the solution of two environmental pollution problems of increased CO2 levels in the atmosphere and that of the disposals of non-biodegradable plastic waste. Here, we describe a strategy and system set-up for fermentative production of P(3HB) from CO2 and agricultural waste materials.

© 1997 American Chemical Society

In Fuels and Chemicals from Biomass; Saha, B., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1997.

16. ISHIZAKI ET AL.

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Microbial Production of Biodegradable Plastics


1. Strategy and system-set up for P(3HB) production from CO2 For practical application of autotrophic production of P(3HB) from CO2 by A.eutrophus, the loss of substrate gas utilization efficiency concomitant with the exhaustion of the gas from fermenter, and the potential of explosion of the substrate gas mixture are very serious problems to be solved. A recycled-gas closed-circuit culture system attains high gas utilization efficiency by reusing the exhausted gas. Figure 2 shows the gas-recycling culture system in bench scale. This system could also eliminate the potential for the gas mixture to explode by maintaining the oxygen concentration in the gaseous phase below the lower limit for explosion(6.9 %(v/v)) and introducing several other safety measures. Oxygen consumption by the cells in autotrophic synthesis of P(3HB) is very large as shown in the following equations, the decrease in the driving force for oxygen from the gas phase into the liquid phase then results in the serious decrease in P(3HB) productivity. Exponential cell growth; 21.36 H2 + 6.21 O2 + 4.09 CO2 + 0.76 NH3 -»

P(3HB) formation; 33 H 2 O + 12 O2 + 4

C O 2 ->

C4.09H7.1301.89N0.76 C4H6O2

+ 30 H 2 O 1

Hence, a doughnut-shaped agitation system to attain a KLa of 2,970 h" was used in the bench plant to compensate for the decrease in oxygen transfer. As a result, cell and P(3HB) concentrations increased to 91.3 g»dnr and 61.9 g»dnr respectively, under 02-limited condition after 40 h of cultivation (Fig.3). While the 62 concentration in the gas phase was maintained at very low level, the overall productivities of biomass and P(3HB) obtained in this cultivation were 2.28 g^dnr * h and 1.55 g-dnr^h" , respectively, which were much higher than those reported for other autotrophic cultivation of hydrogen-oxidizing bacteria (Table I). 3

3

3

_1

1

2. Two-stage culture method for P(3HB) production under low O2 condition The use of a fermenter with very high KLa is not practical in industrial-scale fermentation process because power consumption for agitation will be uneconomically large. Hence, we developed a new culture method using a conventional-type fermenter under low O2 conditions. In this method, heterotrophic cultivation using fructose as carbon source was first carried out for exponential cell growth. After the fructose in the medium was exhausted, the culture broth was centrifuged. The harvested cells were suspended in sterilized mineral medium and autotrophic cultivation for P(3HB) accumulation was performed by feeding a substrate gas mixture in which the O2 concentration was below 6.9%(second culture stage). This method is referred as a two-stage culture method. The cell and P(3HB) concentrations were 26.3 g»dnr and 21.6 g»dnr , respectively after 40 h of autotrophic cultivation. The average productivity of P(3HB) in the autotrophic stage was about 0.56 g»dm- »h . According to previous reports (9-20) on the fermentative production of P(3HB), the productivity of P(3HB) was 0.08 - 4.00 g-dm" *^ in heterotrophic cultures and 0.04 -1.54 g-dm- ^" in autotrophic cultures. Although the KLa of the fermenter used in this experiment was 340 h and the O2 3

3

3

-1

3

3

1

-1


1

296

FUELS AND CHEMICALS FROM BIOMASS

R O I II O-CH-CH2-C . n y


Poly-(D-3-hydroxybutyrate)

^ CH3 |

CHa OW CH2 I II I O - C H - C H 2 —C f \0-CH-CH2

O II -C

Poly-(D-3-hydroxybtyrate-co-D-3-hydroxyvalerate)

Figure 1

Chemical structure of poly-D-hydroxybutyric acid.

1,02 cylinder; 2, H2 cylinder; 3, CO2 cylinder; 4, gas reservoir; 5, differential-pressure type flowmeter; 6, DO controller; 7, alkali feeding pump; 8, pH controller; 9, NH3 solution; 10, fermentor; 11, circulating pump; 12, mixing pump; 13, internal pressure sensor; 14, saline replacement; 15, flowmeter

Figure 2

Schematic diagram of explosion-proof type recycled-gas closedcircuit culture system.


Microbial Production of Biodegradable Plastics 297

16. ISHIZAKI ET AL.

concentration in the gas mixture was very low, a relatively high P(3HB) production rate was obtained. In this culture method, CO2 is evolved from fructose during the heterotrophic stage. However, the two-stage culture works as a CO2 absorption system because the amount of CO2 consumed during the autotrophic stage is about twice that of the heterotrophic stage.


3. Development of pH-stat batch culture with continuous feeding substrate solution to obtain protein-rich cell mass for two-stage culture Fructose is an expensive carbon source in the fermentation industry, hence we next investigated the application of other economical carbon sources for the heterotrophic culture in the two stage method. We used acetic acid as the carbon source and developed a pH-stat continuos substrate-feeding method for the culture. Flask culture experiments showed the optimum concentration of acetic acid for cell growth was very low (ca. 1.0 g»dnr ) and the growth was seriously inhibited by a slight increase in acetic acid concentration. It was, therefore, necessary to control the acetate concentration around this level in high cell density cultivation. The ratio of consumption of acetic acid to that of ammonium by A.entrophus cells was determined by a standard-type batch culture experiment to be about 10 (mol-acetic acid/molammonium). It was therefore expected that the acid-base equilibrium in culture system would be balanced by feeding the substrate solution in which the C/N ratio was 10 (mol/mol) so as to maintain the culture pH at a constant level, in order for acetate concentration in the fermentor to be also controlled at low level. However, in batch culture with such a feeding, acetate concentration of the culture liquid increased after cell concentration reached approximately 5 g«dnr . The increase in acetate concentration was thought to be due to the depletion of mineral nutrients. Hence, the mineral concentrations in the medium was increased 5 times as that of the basal medium (this was referred as 5-fold medium). As a result, acetate concentration was controlled around 1 g*dnr and cell concentration reached about 25 g«dm after 18 h. Acetate concentration increased after that due to the depletion of phosphate. A pHstat batch culture was hence carried out by feeding a solution in which the C/P ratio was 118.4 (mol-C/mol-P). Acetate concentration was maintained around 1 g dnr , and cell and protein concentrations increased to 48.6 g*dnr and 35.0 g»dnr , respectively after 21 h of cultivation (Fig.4). Autotrophic cultivation for P(3HB) accumulation was then performed using the protein-rich cell mass obtained from a pH-stat batch culture into which the modified substrate solution was fed. When the cell concentration reached to about 5 g dnr , feeding of the substrate solution was stopped and autotrophic cultivation was performed by feeding a substrate gas mixture into the fermentor. P(3HB) accumulated in the cells up to about 60 % by dry cell weight. This feeding method can therefore be used in fermentation process where the cell growth or P(3HB) accumulation is inhibited by high concentrations of the substrate such as propionate, formate, or lactate, etc. 3

3

3

-3

#

3

#

3


3

3


298


Figure 3

so

Cultivationtime(h)

Figure 4

Time course of pH-stat batch culture with feeding acetic acid and inorganic nutrients solution. The C/N and C/P ratios in the feed solution were 10 and 118.4(mol/mol), respectively.



H2/O2/CO2

H2/O2/CO2

H2/O2/CO2

Alcaligenes eutrophus Alcaligenes

Pseudomonas

42

48

Batch

Fed-batch

40

Batch

Methanol

Protomonas

Methanol

Sucrose

Glucose 1)

50

Fed-batch

5.57

__

7.89

142.0 250.0

1.80

20

19 1.85 90.4

18

4.0 68.4

17

16

15

130 2)

1.12

136.0

3.28

1.84

164.0

223.0

2.42

2.11

89.0

121.0

3

0.60

36.0

2.79

117.0

60.0

1.00

14

---

--

0.33 3.00

Alcaligenes Glucose* Fed-batch 50 eutrophus valerate 1)The inoculum cell size was 13.7 g-dm . 2)The product was poly(hydro.\ybutyrate-co-hydrox> valerate). 3

70

Fed-batch 18

Fed-batch

Fed-batch 121

bacterium organophilum

Methylo-

latus

Alcaligenes

eutrophus

Alcaligenes

extroquens

E.coli

Glucose

Recombinant

12 13

11

-0.23

-16.0

0.26

18.0

0.40

5

--

10

61.9

2.28

-

9

Ref.

1.55

3

0.50

3

24.0

3

91.3

1

Culture Cultivation Cell Cell P(3HB) P(3HB) method time concentration productivity concentration productivity (h) (g-dm) (g-dm-h') (g-dm) (g-dm-h') 1.00 25.0 Batch 25 --

Alcaligenes H 2 / O 2 / C O 2 Continuous eutrophus Alcaligenes H2/O2/CO2 Batch 70 eutrophus H16 Alcaligenes H 2 / O 2 / C O 2 Continuous hydrogenophilus Pseudomonas H 2 / O 2 / C O 2 Continuous h ydrogenothermophila Alcaligenes H2/O2/CO2 Batch 60 eutrophus

hydrogenovora

eutrophus

Substrate

Strains

fermentaive production of P(3HB) using various microorgansims and substrates

Table I. List of autotrophic cultivations of hydrogen-oxidizing bacteria and


300



4. Application of air-lift type fermenter and improvement of gas mass transfer by changing medium rheology Air-lift fermentors have often been used instead of the traditional stirred-tank fermentor for production of penicillin(21), a-amylase(22), xanthan(23) and single cell protein(24). As the air-lift fermentor does not require mechanical agitation, the energy consumption is lower than that of a stirred-tank fermentor. The effect of the change in medium viscosity by adding carboxymethylcellulose (CMC) in an air-lift type fermenter on gas-hold up, bubble formation, flow pattern and mass transfer of oxygen has been reported by many researchers(25-27). We investigated the application of an air-lift type fermentor and the effect of change in medium viscosity by addition of C M C . Figure 5 shows the air-lift type fermentor used in this study, which was assembled as described by Okabe et al(28). To obtain high mass transfer achieved through the formation of small bubbles, a sintered stainless steel sparger (pore size, 10 mm; diameter, 12 mm; length, 20 mm) was installed at the bottom of the reactor. The feeding rate of the substrate gas mixture in the air-lift fermentor was 2 dm •nun , which is equivalent to a superficial gas velocity of 2.62 cm^S". Figure 6 shows the changes in medium viscosity and gas hold-up at various concentrations of C M C in the air-lift fermentor. Gas hold-up increased in proportion to the increase in C M C concentration up to 0.1% (w/v) but the gas hold-up decreased above 0.1% (w/v) of C M C . Deducing from this result, addition of C M C up to 0.1% (w/v) was expected to increase oxygen transfer rate with resultant increase in P(3HB) productivity. Figure 7 shows the time courses of autotrophic culture of A. eutrophus in the air-lift fermentor with addition of various concentrations of C M C . The productivity of P(3HB) in the culture with addition of 0.05 %(w/v) C M C (shown in Fig.7b) was increased to twice as that of the control culture with no addition of CMC(shown in Fig.7a). In the culture with addition of 0.1 % C M C , P(3HB) productivity was about 1.5 times higher than that of the control culture(Fig.7c). However, there was no apparent effect of the addition of C M C on the productivity of P(3HB) in the cultivation using the stirred-tank fermentor. A comparison was made for the effect of C M C addition on the mass transfer of oxygen in the air-lift and stirred-tank fermenters. When measurements were done by the static method, maximum KLa value for the air-lift fermenter was obtained at 0.05% of C M C concentration. The values of KLa for the air-lift fermentor measured by the sulfite oxidation method was observed to decrease with an increase in C M C concentration. For stirred-tank fermentor on the other hand, there was no increase in KLa values by addition of C M C into the culture medium. In the measurement by the sulfite oxidation method, the KLa of the stirred-tank fermenter was lowest at 0.1 % CMC. It is generally known that some kinds of surfactants, such as C M C , affect the sulfite-oxidation reaction. P(3HB) production rate observed in the fermentation experiments using the air-lift fermentor, correlated to the KLa measured by the static method but did not correlated to the KLa measured by the sulfite oxidation method. The KLa value measured by sulfite oxidation method was larger than those measured by the static method. These results mean that the static method is more reliable for the measurement of KLa than the sulfite oxidation method in autotrophic culture for P(3HB) production using air-lift fermentor. However, the relationship between the 3

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ISHIZAKI ET AL.


Condenser-

pH electrode

DO electrode


Cylinder.

Silicone stopper

Dimensions of the air-lift fermentor. Reactor: total volume total length Cylindrical part: length inner diameter diameter of draft tube length of draft tube Sparger: pore size length diameter

350 ml 350 mm 300 mm 40 mm 30 mm 100 mm 10 nm 20 mm 10 mm

Dimensions of the stirred-tank fermentor. Draft tube Liquid filter for HPLC Stainless-steel tube Figure 5

Reactor: total volume length inner diameter Silicone stopper Magnetic stirrer-bar: length diameter

200 ml 112 mm 50 mm 40 mm 5 mm

Schematic diagram of the air-lift type fermenter.

0.2

0.3

0.4

CMC (wt%) Figure 6

The effect of C M C concentration on viscosity and gas-hold up of culture medium.


301


302


Cultivation time (h)

Figure 7



Time course of autotrophic cultivation of A.eutrophus in air-lift type fermenter under various concentrations of CMC. (a): no addition of C M C , (b): 0.05 %(w/v) C M C , (c): 0.1 %(w/v) C M C .


16. ISHIZAKI ET AL.


303

KLa measured by the static method and the gas hold-up was not close, especially for the case where 0.1% CMC was used. This cannot be easily explained at present.


5. P(3HB) production from xylose via L-lactate fermentation For practical application of PHAs on commercial scale, consideration has to be given to the origin of carbon source used as well as the reducing the cost of production through the use of economical carbon sources. Carbon source such as 4hydroxyvaleric acid derived from fossil fuel is often used as raw material for producing PHAs. However, it is known that the consumption of a large amount of fossil fuel results in the increase in CO2 concentration in atmosphere. Waste materials such as lignocellulose is desirable from the viewpoints of utilization of unexplored resources and solution of the green house effect. Xylose is one of main components of hemicellulose contained in wood waste. At present, few types of commercial products have been produced from xylose. Young et. al. reported that Pseudomonas cepacia could produce P(3HB) from xylose and lactose, but the productivity was very low(29). Lactococcus lactis 10-1 isolated in our laboratory is able to produce L-lactic acid and acetic acid at a high production rate from xylose (30,31). L-Lactate also has the potential to be used as raw material in the manufacture of a biodegradable plastic, poly(L-lactate). A.eutrophus cannot utilize xylose but utilize lactate as carbon source. We therefore developed a culture method for the production of P H A from xylose employing these two bacteria. This culture method consisted of an initial fermentative production of L-lactate from xylose employing L.lactis 10-1 and a conversion of L-lactate into PHA by A.eutrophus. Flask culture experiment showed that the growth rate of A.eutrophus decreased according to the increase in L-lactate concentration in the medium and the cells could not grow above 30 g»drrr of L-lactate. In pH-controlled batch fermentations, a maximum specific growth rate of 0.6 h" was obtained when 5 g»drrr of L-lactate was used. The growth of microorganisms is generally inhibited by the presence of lactate, however, the specific growth rate of A.eutrophus when using L-lactate was higher than when other types of carbon source were used. According to our study for A.eutrophus, the maximum specific growth rate with using fructose was about 0.2 h" and the maximum specific growth rate in autotrophic condition was 0.42 h . Such growth characteristic of A.eutrophus on L-lactate is favorable for production of P(3HB) by the two-stage method. The accumulation of P(3HB) by A.eutrophus was next investigated using the culture supernatant containing L-lactate and acetate converted from xylose by Llactis IO-1. A pH-controlled batch culture of L.lactis 101 was first anaerobically carried out using 30 g»dnr of xylose as carbon source. When xylose in the culture was completely consumed, the culture broth was aseptically centrifiiged and the supernatant was returned to the fermenter. A.eutrophus cells was then inoculated and the second stage cultivation for P(3HB) accumulation was aerobically carried out. The initial L-lactate concentration in the second stage culture was adjusted to 10 g»drrr . After 24 hours of cultivation, 8.5 g-drrr of cells were produced. The final percentage of P(3HB) in the cell reached 55 %(w/w) without nitrogen source on medium being limited (32). The growth of A.eutrophus is inhibited by high lactate concentrations, therefore high cell density cultivation can be achieved by pH-stat batch culture with substrate feeding to control 3

1

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_1

3

3

3



304


A:C/N=10 B:C/N=23


Figure 8


Time course of pH-stat batch culture with feeding L-lactic acid and inorganic nutrients solution. The C/N ratio in the feed solution was changed from 10 to 23.3 (mol/mol) after 12 of cultivation.


16. ISHIZAKI ET AL.

Microbial Production of Biodegradable Plastics 305

L-lactate concentration in medium at low level. As the C/N ratio for the consumption of L-lactate and ammonium by the cells was determined to be 10 (mol/mol) by a standard-type batch culture, the feed solution in which the C/N ratio was prepared to 10, was first used in the pH-stat batch culture as feed substrate. However, it was impossible to control L-lactate concentration at a constant level by using this feed solution. It was observed that the microorganism accumulated P(3HB) in the cell even during exponential growth phase and excreted a small amount of an unknown organic acid, then the acid-base equilibrium was not balanced in the culture system. The C/N ratio in the feed solution was, therefore, changed from 10 to 23.3(mol/mol) after 12 h of cultivation and phosphate and other organic nutrients were also supplied. As a result, cell concentration increased to 102 g»dnr (Fig. 8). The P(3HB) content in the cells reached about 60 %(w/w) although nitrogen source in culture medium was not limiting. We are now investigating substrate feeding strategy to increase P(3HB) accumulation.


3

6. Conclusion The practical cultivation systems for hydrogen-oxidizing bacterium, A.eutrophus to produce a biodegradable plastic, P(3HB) from CO2 and xylose were developed and P(3HB) accumulation was improved by incorporating new strategies. The application of such culture systems should contribute to the solution of the global environmental pollution problems caused by increased CO2 level in atmosphere, disposal of non-degradable plastics and utilization of industrial waste materials. For practical application of biodegradable plastics, obviously considerable technological challenges must be overcome, especially in the reduction in production cost and improvement in extraction and refining process of the product. We are tackling this difficult problem and also investigating the conversion of various types of industrial waste materials to other useful compounds. Literature Cited 1. Anderson, D.; Dawes, E.A. Microbiol. Rev., 1990, 54, 450-472. 2. Ishizaki, A.; Tanaka, K. J. Ferment. Bioeng., 1990, 69, 170-174. 3. Ishizaki, A.; Tanaka, K. J. Ferment. Bioeng., 1991, 71, 254-257. 4. Tanaka, K.; Ishizaki, A.; Stanbury, P.F. J. Ferment. Bioeng., 1992, 74, 288291. 5. Tanaka, K.; Ishizaki., A.; Kanemaru., T.; Kawani, T.; Biotechnol. Bioeng., 1995, 45, 268-275. 6. Takeshita, T.; Tanaka, K.; Ishizaki, A.; Stanbury, P.F. J. Fac. Agric., Kyushu Univ., 1993, 38, 55-64. 7. Takeshita, T.; Tanaka, K.; Ishizaki, A.; Stanbury, P.F. J. Ferment. Bioeng., 1993, 76, 148-150. 8. Tanaka, K.; Ishizaki, A. J. Ferment. Bioeng., 1994, 77, 425-427. 9. Repask, R.; Mayer, R. Appl. Environ. Biotechnol., 1976, 32, 592-597. 10. Goto, E.; Suzuki, K.; Kodama, T.; Minoda, T. Agric., Biol. Chem., 1977, 41, 521-525.



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11. Morinaga, Y.; Yamanaka, S.; Ishizaki, A.; Hirose, Y. Agric. Biol. Chem., 1978, 42, 439-444. 12. Sonnleitner, B.; Heinzle, E.; Braubegg, G.; Lafferty, R.M. Eur. J. Appl. Microbiol. Biotechnol., 1976, 7, 1-10. 13. Miura, Y.; Okazaki, M.; Ohi, K.; Nishimura, T.; Komemushi, S. Biotechnol. Bioeng., 1982, 24, 1173-1182. 14. Igarashi, Y. Nippon Nougeikagaku kaishi (in Japanpsase) 1986, 60, 935-941. 15. Kim, B. S.; Lee, S. Y.; Chang, H. N. Bietechnol. lett., 1992, 14, 811-816. 16. Suzuki, T.; Yamane, T.; Shimizu, S. Appl. Microbiol. Biotechnol., 1986, 23, 322-329. 17. Kim, B. S.; Lee, S.C.; Lee, S.Y.; Chang, H.N.; Chang, Y.K.; Woo, S.I. Bietechnol. Bioeng., 1993, 43, 892-898. 18. Yamane, T.; Fukunaga, M.; Lee, Y.W. Biotechnol. Bioeng., 1996, 50, 197-202. 19. Kim, S.W., Kim, P., Lee, H.S., Kim, J. H. Biotechnol. lett., 1996, 18, 25-30. 20. Lee, I.Y.; Kim, M.K.; Kim, G.J.; Chang, H.N.; Park, Y.H.Biotechnol.lett., 1995, 17, 571-574. 21. Möller, J.; Niehoff, J.; Hotop, S.; Dors, M.; Schügerl, K. Appl. Microbiol. Biotechnol, 1992, 37, 157-163. 22. Chevalier, P.; Noüe, J. Enzyme Microbiol. Technol., 1988, 10, 19-23. 23. Suh, I.S.; Schumpe, A ; Deckwer, W.D. Biotechnol. Bioeng., 1992, 39, 85-94. 24. Westlake, R. Chem. Ing. Technol., 1986, 58, 934-937. 25. Deckwer, W.D.; Schumpe, A. Chem. Eng. Sci., 1993, 48, 889-911. 26. Kennard, M.; Janekeh, M.Biotechnol.Bioeng., 1991, 38, 1261-1270. 27. Schumpe, A ; Deckwer, W.D. bid. Eng. Chem. Process Des. Dev., 1982, 21, 706-711. 28. Okabe, M.; Ohta, N.; Park, Y.S. J. Ferment. Bioeng., 1993, 76, 117-122. 29. Young, F.K.; Kastner, J, Y.; May, S. W. Appl. Environment. Microbiol., 1994, 60, 4195-4198. 30. Ishizaki, A.; Ueda, T.; Tanaka, K.; Stanbury, P.F. Biotechnol. lett., 1992, 14, 599-604. 31. Ishizaki, A.; Ueda, T.; Tanaka, K.; Stanbury, P.F. Biotechnol. lett., 1993, 15, 489-494. 32. Tanaka, K.; Katamune, K.; Ishizaki, A. Can. J. Microbiol., 1995, 41, 257-261.


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