Control of acetic acid concentration by pH-stat continuous substrate feeding in heterotrophic culture phase of two-stage cultivation of Alcaligenes eutrophus for production of P(3HB) from CO2, H2, and O2 under non-explosive conditions

A-two stage culture method of hydrogen-oxidizing bacterium, Alcaligenes eutrophus, is used to produce poly-D-3-hydroxybutyrate, P(3HB) from CO2, O2, and H2 without using a very high oxygen transfer rate while maintaining the O2 concentration in gas phase below 6.9 (v/v)% to prevent detonation of the gas mixture. The two-stage method consists of a heterotrophic culture using fructose as carbon source for exponential cell growth and an autotrophic culture for P(3HB) accumulation. We investigated the use of acetic acid as a cheaper carbon source than fructose for the heterotrophic culture in the two-stage method. However, the acetate concentration in the culture system must be maintained at 1.0 g. dm-3 since its inhibitory effect on the cell growth is very strong. Then, high cell density cultivation of A. eutrophus was investigated by pH-stat continuous feeding of acetic acid to control acetate concentration. As a result, acetate concentration was automatically maintained around 1.0 g. dm-3 by using a feed with a composition in CH3COOH/CH3COONH4/KH2PO4 molar ratio of 5:1:0.084. Cell concentration increased to 48.6 g. dm-3 after 21 h of cultivation. The cell mass grown in the fed-batch culture on acetic acid was useful for P(3HB) production from CO2 in the subsequent autotrophic culture stage. Copyright 1999 John Wiley & Sons, Inc.     

Microbial production of poly-D-3-hydroxybutyrate from CO2

This short review covers the biotechnological aspects of the production of poly-D-3-hydroxybutyric acid, P(3HB), from H2, O2 and CO2 by autotrophic culture of the hydrogen-oxidizing bacterium, Ralstonia eutropha. Considering the efficiency of utilization of a gas mixture as substrate, a practical fermentation process using R. eutropha for the mass production of P(3HB) from CO2 should be designed on the basis of a recycled-gas, closed-circuit culture system. Also, maintaining the O2 concentration in a gas phase lower than 6.9% (v/v) is essential to prevent the gas mixture from exploding. Our study, using an explosion-proof fermentation bench plant and a two-stage culture system with a newly designed air-lift fermenter, demonstrated that very high P(3HB) yield and productivity could be obtained while the O2 concentration was maintained below 6.9%. However, a study on the continuous production of P(3HB) from CO2 by chemostat culture of R. eutropha revealed that the productivity and content of P(3HB) in the cells was considerably lower than by fed-batch culture. It is deduced that the use of the hydrogen-oxidizing bacterium, Alcaligenes latus, which accumulates P(3HB) even in the exponential growth phase, will be useful for the effective production of P(3HB) from CO2.     

Production of poly(D-3-hydroxybutyrate) from CO(2), H(2), and O(2) by high cell density autotrophic cultivation of Alcaligenes eutrophus

Hydrogen-oxidizing bacterium, Alcaligenes eutrophus autotrophically produces biodegradable plastic material, poly(D-3-hydroxybutyrate), P(3HB), from carbon dioxide, hydrogen, and oxygen. In autotrophic cultivation of the microorganism, it is essential to eliminate possible occurrence of gas explosions from the fermentation process. We developed a bench-plant scale, recycled-gas, closed-circuit culture system equipped with several safety features to perform autotrophic cultivation of A. eutrophus by maintaining the oxygen concentration in the substrate gas phase below the lower limit for a gas explosion (6.9%). The culture vessel utilized a baskettype agitator, resulting in a K(L) a value of 2970 h(-1). Oxygen gas was also directly fed to the fermentor separately from the other gases. As a result, 91.3 g . dm(-3) of the cells and 61.9 g . dm(-3) of P(3HB) were obtained after 40 h of cultivation under this oxygen-limited condition. The results compared favorably with those reported for mass production of P(3HB) by heterotrophic fermentation. (c) 1995 John Wiley & Sons, Inc.     

Pilot scale production of poly(3-hydroxybutyrate-co-3-hydroxy-valerate) by fed-batch culture of recombinantEscherichia coli

Production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB/V)], by fed-batch culture of recombinantEscherichia coli harboring a plasmid containing theAlcaligenes latus polyhydroxyalkanoate (PHA) biosynthesis genes, was examined in two pilot-scale fermentors with air supply only. In a 30 L fermentor having aK _La value of 0.11 s⁻¹, the final P(3HB/V) concentration and the P(3HB/V) content obtained were 29.6 g/L and 70.1 wt%, respectively, giving a productivity of 1.37 g P(3HB/V)/L-h. In a 300 L fermentor having aK _La of 0.03 s⁻¹, the P(3HB/V) concentration and the P(3HB/V) content were 20.4 g/L and 69 wt%, respectively, giving a productivity of 1.06 g P(3HB/V)/L-h. These results suggest that economical production of P(3HB/V) is possible by fed-batch culture of recombinantE. coli in a large-scale fermentor having lowK _La value.     

Inhibitory effect of carbon dioxide on the fed-batch culture of Ralstonia eutropha: evaluation by CO2 pulse injection and autogenous CO2 methods

In order to see the effect of CO(2) inhibition resulting from the use of pure oxygen, we carried out a comparative fed-batch culture study of polyhydroxybutyric acid (PHB) production by Ralstonia eutropha using air and pure oxygen in 5-L, 30-L, and 300-L fermentors. The final PHB concentrations obtained with pure O(2) were 138.7 g/L in the 5-L fermentor and 131.3 g/L in the 30-L fermentor, which increased 2.9 and 6.2 times, respectively, as compared to those obtained with air. In the 300-L fermentor, the fed-batch culture with air yielded only 8.4 g/L PHB. However, the maximal CO(2) concentrations in the 5-L fermentor increased significantly from 4.1% (air) to 15.0% (pure O(2)), while it was only 1.6% in the 30-L fermentor with air, but reached 14.2% in the case of pure O(2). We used two different experimental methods for evaluating CO(2) inhibition: CO(2) pulse injection and autogenous CO(2) methods. A 10 or 22% (v/v) CO(2) pulse with a duration of 3 or 6 h was introduced in a pure-oxygen culture of R. eutropha to investigate how CO(2) affects the synthesis of biomass and PHB. CO(2) inhibited the cell growth and PHB synthesis significantly. The inhibitory effect became stronger with the increase of the CO(2) concentration and pulse duration. The new proposed autogenous CO(2) method makes it possible to place microbial cells under different CO(2) level environments by varying the gas flow rate. Introduction of O(2) gas at a low flow rate of 0.42 vvm resulted in an increase of CO(2) concentration to 30.2% in the exit gas. The final PHB of 97.2 g/L was obtained, which corresponded to 70% of the PHB production at 1.0 vvm O(2) flow rate. This new method measures the inhibitory effect of CO(2) produced autogenously by cells through the entire fermentation process and can avoid the overestimation of CO(2) inhibition without introducing artificial CO(2) into the fermentor.     

Synthesis of short-/medium-chain-length poly(hydroxyalkanoate) blends by mixed culture fermentation of glycerol

Glycerol was used as a substrate in the bio-production of poly(hydroxyalkanoates) (PHAs) in an effort to establish an alternative outlet for glycerol and produce value-added products. Pseudomonas oleovorans NRRL B-14682 and Pseudomonas corrugata 388 grew and synthesized poly(3-hydroxybutyrate) (P3HB) and medium-chain-length PHA (mcl-PHA) consisting primarily of 3-hydroxydecanoic acid (C(10:0); 44 +/- 2 mol %) and 3-hydroxydodecenoic acid (C(12:1); 31 +/- 2 mol %), respectively, from glycerol at concentrations up to 5% (v/v). Cellular productivity maximized at 40% for P. oleovorans in 5% (v/v) glycerol and 20% for P. corrugata in 2% (v/v) glycerol after 72 h. Increasing the glycerol media concentration from 1% to 5% (v/v) caused a 61% and 72% reduction in the molar mass (M(n)) of the P3HB and mcl-PHA polymers, respectively. Proton-NMR analysis of the glycerol-derived P3HB revealed that the M(n) decrease was the result of esterification of glycerol onto the polymer in a chain terminating position. However, no evidence of glycerol-based chain termination was present in the mcl-PHA. The growth patterns of P. oleovorans and P. corrugata on glycerol permitted their use as mixed cultures to produce natural blends of P3HB and mcl-PHA. By incorporating a staggered inoculation pattern and varying the duration of the fermentations, P3HB/mcl-PHA ratios were achieved that varied from 34:66 to 96:4.     

Growth-associated production and characterization of poly (3-hydroxybutyric acid) of Azotobacter beijerinckii DAR-102

Production of poly (3-hydroxybutyric acid) [P(3HB)] by Azotobacter beijerinckii DAR-102 isolated in this laboratory has been optimized under batch-culture. The accumulatad polymer attained 58% of cell dry mass during mid-stationary phase with an yield of 0.58 g/l when grown in nitrogen-free medium. The optimum concentration of glucose and fructose for P(3HB) production was 3% (w/v) and 2% (w/v) respectively while that of casamino acid and tryptose was 0.1% (w/v). Phosphate at a concentration suboptimal for growth and limitation of oxygen in the medium favoured P(3HB) accumulation. The production of P(3HB) was maximum with an inoculum dose of 4% (v/v). The accumulated polymer was isolated by direct chloroform extraction of the dry cell mass and purified by precipitation with diethyl ether. The purified polymer has been characterized in terms of its solubility properties, melting temperature, and UV-, IR- and NMR-spectroscopic analyses.     

Modeling of poly(3-hydroxybutyrate) production by high cell density fed-batch culture of R<Emphasis Type="Italic">alstonia eutropha</Emphasis>

High cell density culturing has been conducted for the production of poly(3-hydroxybutyrate) fed-batch cultures ofRalstonia eutropha with phosphate limitation. It was found that a high glucose concentration inhibited the synthesis of P(3HB) in the high cell density culture ofR. eutropha. Although a low glucose concentration can trigger the synthesis of P(3HB) in a manner similar to that of phosphate limitation, it also limited both the P(3HB) synthesis and the cell growth, and led to a low P(3HB) productivity because glucose is the sole carbon source in this reaction. An unstructured model was proposed for predicting the cell growth and P(3HB) synthesis in high cell density cultures ofR. eutropha, where the phosphate concentration played a key role in the accumulation of P(3HB) and in cell growth. Good agreements were found between the experimental data and model predictions. The results of simulation showed that the final P(3HB) concentration would decrease more than 25% when the glucose was concentration increased to 40 g/L, and indicated that the optimal glucose concentration for P(3HB) production by high cell density cultures ofR. eutropha was around 9 g/L.     

Recovery of poly(3-hydroxybutyrate) from high cell density culture of Ralstonia eutropha by direct addition of sodium dodecyl sulfate

A simple and effective method for the recovery poly(3-hydroxybutyrate) [P(3HB)] directly from high cell density culture broth with no pretreatment steps has been developed. This method consists of direct addition of sodium dodecyl sulfate (SDS) to the culture broth, shaking, heat treatment, and washing steps. When the SDS/biomass ratio was higher than 0.4, the purity of recovered P(3HB) was over 95% for various cell concentrations of 50–300 g dry cell l^–1, with the highest value of 97%. The recovery of P(3HB) was over 90% regardless of cell concentration and SDS dosage (SDS/biomass ratios, 0.1–0.7). One g SDS digests 0.72 g non-P(3HB) cell materials. The reduction in molecular weight, due to degradation of P(3HB) by SDS, was negligible.     

The production of poly(3-hydroxybutyrate) [P(3HB)] by a newly isolated Bacillus sp. ST1C using liquid waste from biodiesel production

A newly isolated poly(3-hydroxybutyrate) [P(3HB)] producing strain, ST1C, was identified as Bacillus aryabhattai based on its morphological, biochemical and molecular characteristics. It synthesized and accumulated relatively high amounts of P(3HB). The aim of this work was to establish if it could convert an inexpensive liquid waste product from the production of biodiesel, biodiesel liquid waste (BLW), to P(3HB). Using a mineral salt medium (MSM) containing 2.0 % (v/v) glycerol present in the BLW and both normal batch and a draw and fill culture method, B. aryabhattai ST1C produced a maximum P(3HB) content and biomass concentration of 72.31 % dry cell weight (DCW) and 7.24 g/L, respectively, over a 24 h cultivation period in the draw and fill cultivation method. From 24 h to the end of cultivation at 72 h both the P(3HB) content and the biomass concentrations continuously reduced. Concentrations of glycerol in the BLW in this MSM above 3.0 % (v/v) or from pure glycerol (PG) or with an added NaCl concentration of greater than 3.0 % significantly reduced both the maximum P(3HB) content and the biomass concentrations.     

利用重组大肠杆菌产聚-4-羟基丁酸的研究

重组大肠杆菌 E.coli XL-1 Blue（pKSSE5.3）携带Ralstonia　eutropha　H16的 PHA聚合酶基因（phaC）和Clostridium kluyveri的4-羟基丁酸:CoA转移酶基因（orfZ）,可以利用葡萄糖和4-羟基丁酸为碳源合成均聚的聚-4-羟基丁酸[P（4HB）]。优化培养基和培养条件后,进行了补料分批培养。结果表明,经68h左右培养,E.coli XL-1 Blue（pKSSE5.3）的发酵液中菌体干重达13g/L,P（4HB）的密度达5g/L,P（4HB）百分含量为36％。从收获的冻干细胞中提纯得到40g均聚的P（4HB）,为进一步分析检测P（4HB）生物、理化、加工特性及其应用价值成为可能。     

Optimization of culture conditions in CO2 fixation for succinic acid production using Actinobacillus succinogenes

The culture conditions in CO(2) fixation by Actinobacillus succinogenes for succinic acid production were investigated by a model of available CO(2) in a 3-l fermentor. The results from the model analysis showed that the available CO(2) for succinic acid production in the fermentation broth is the sum of HCO(3) (-), CO(3) (2-), and CO(2) influenced by external culture conditions such as medium components, CO(2) partial pressures, and temperature. The optimized conditions for CO(2) supply in a 3-l fermentor were determined as follows: CO(2) partial pressure and stirring speed were maintained at 0.1 MPa and 200 r min(-1), respectively, with a pH of 6.8 and a temperature of 37°C; 0.15 mol l(-1) NaHCO(3) was added. Under the optimized conditions, a CO(2) fixation rate of 0.57 g l(-1) h(-1) was obtained, and a succinic acid concentration of 51.6 g l(-1) with a yield of 75.8% was reached. These results suggest that optimized conditions of CO(2) supply are effective in high succinic acid production and thus have potential applications in succinic acid production and CO(2) fixation.     

Enterotoxin production by Vibrio cholerae and Vibrio mimicus grown in continuous culture with microbial cell recycle

We have examined the effect of complete cell recycle on the production of cholera toxin (CT) by Vibrio cholerae and CT-like toxin by Vibrio mimicus in continuous culture fermentations. Complete cell recycle was obtained by filtering culture fluids through Amicon hollow fibers with an exclusion limit of 100,000 daltons (H1P100-20) and returning the concentrated cell slurry to the fermentor. A single 1-liter laboratory fermentor system modified with this recycle loop was capable of producing over 20 liters of cell-free culture filtrate per day. Toxin production in this system was compared with yields obtained in traditional continuous cultures and in shake flask cultures. Yields of CT from V. cholerae 569B in the recycle fermentor were highest at the highest dilution rate employed (1.0 vol/vol per h). The use of complete cell recycle dramatically increased yields over those obtained in continuous culture and equaled those obtained in shake flasks. The concentration of CT in the filtrate was slightly less than half of that measured in culture fluids sampled at the same time. Similarly, V. mimicus 61892 grown in the presence of 50 micrograms of lincomycin per ml produced 280 ng of CT per ml in the recycle fermentor, compared with 210 ng/ml in shake flasks under optimal conditions. The sterile filtrate from this fermentation contained 110 ng/ml.     

Degradation of microbial polyester poly(3-hydroxybutyrate) in environmental samples and in culture

Poly(3-hydroxybutyrate) [P(3HB)] test-pieces prepared from the polymer produced by Azotobacter chroococcum were degraded in natural environments like soil, water, compost and sewage sludge incubated under laboratory conditions. Degradation in terms of % weight loss of the polymer was maximum (45%) in sewage sludge after 200 days of incubation at 30°C. The P(3HB)-degrading bacterial cultures (36) isolated from degraded test-pieces showed different degrees of degradation in polymer overlayer method. The extent of P(3HB) degradation increases up to 12 days of incubation and was maximum at 30°C for majority of the cultures. For most efficient cultures the optimum concentration of P(3HB) for degradation was 0.3% (w/v). Supplementation of soluble carbon sources like glucose, fructose and arabinose reduced the degradation while it was almost unaffected with lactose. Though the cultures degraded P(3HB) significantly, they were comparatively less efficient in utilizing copolymer of 3-hydroxybutyrate and 3-hydroxyvalerate [P(3HB-co-3HV)].     

Enterotoxin production by Vibrio cholerae and Vibrio mimicus grown in continuous culture with microbial cell recycle.

We have examined the effect of complete cell recycle on the production of cholera toxin (CT) by Vibrio cholerae and CT-like toxin by Vibrio mimicus in continuous culture fermentations. Complete cell recycle was obtained by filtering culture fluids through Amicon hollow fibers with an exclusion limit of 100,000 daltons (H1P100-20) and returning the concentrated cell slurry to the fermentor. A single 1-liter laboratory fermentor system modified with this recycle loop was capable of producing over 20 liters of cell-free culture filtrate per day. Toxin production in this system was compared with yields obtained in traditional continuous cultures and in shake flask cultures. Yields of CT from V. cholerae 569B in the recycle fermentor were highest at the highest dilution rate employed (1.0 vol/vol per h). The use of complete cell recycle dramatically increased yields over those obtained in continuous culture and equaled those obtained in shake flasks. The concentration of CT in the filtrate was slightly less than half of that measured in culture fluids sampled at the same time. Similarly, V. mimicus 61892 grown in the presence of 50 micrograms of lincomycin per ml produced 280 ng of CT per ml in the recycle fermentor, compared with 210 ng/ml in shake flasks under optimal conditions. The sterile filtrate from this fermentation contained 110 ng/ml.     

Continuous culture in combined backmix-plug-flow-tubular-loop fermentor configurations

Several combinations of backmix, tubular-loop, and plug-flow fermentors with and without culture recycle were studied by computer simulations. The steady-state concentrations of cell mass in a continuous culture were calculated as a function of dilution rate using Monod growth kinetics. It was found theoretically and verified for one case experimentally that the maximum dilution rate, over which microbial cells were washed out from the fermentor, could be elevated well beyond the maximum specific growth rate if a particular fermentor combination was used. A combination of two backmix fermentors has been analyzed previously by Sinclair and Brown. Application of this type of fermentor combination as a seed tank for performing continuous culture of microbes in a plug-flow reactor was shown with special reference to fermentation production using the kinetics proposed by Luedeking and Piret, van Dedem and Moo-Young, and Brown and Vass.     

The effect of carbon dioxide on the production of an extracellular nuclease of Staphylococcus aureus

The production of the extracellular nuclease secreted by Staphylococcus aureus (Foggi strain) was studied in a fermentor in an attempt to improve yield and allow large-scale production of the enzyme. In shake flask cultures, 600 units/mL of the enzyme were produced routinely. However, only 450 units/mL of the enzyme at best were obtained in a small-scale fermentor (3 L). The supplementation of the air supply to the fermentor with carbon dioxide [20% (v/v)] increased levels of enzyme in the culture medium to 770 units/mL. Subsequently, this result was reproduced in larger fermentors (10 and 150 L). The possible mechanisms of the effect of carbon dioxide upon the growth of Staphylococcus aureus (Foggi) and the production of the enzyme are discussed.     

Production of poly(3-hydroxybutyric acid-co-4-hydroxybutyric acid) and poly(4-hydroxybutyric acid) without subsequent degradation by Hydrogenophaga pseudoflava

A Hydrogenophaga pseudoflava strain was able to synthesize poly(3-hydroxybutyric acid-co-4-hydroxybutyric acid) [P(3HB-co-4HB)] having a high level of 4-hydroxybutyric acid monomer unit (4HB) from gamma-butyrolactone. In a two-step process in which the first step involved production of cells containing a minimum amount of poly(3-hydroxybutyric acid) [P(3HB)] and the second step involved polyester accumulation from the lactone, approximately 5 to 10 mol% of the 3-hydroxybutyric acid (3HB) derived from the first-step culture was unavoidably reincorporated into the polymer in the second cultivation step. Reincorporation of the 3HB units produced from degradation of the first-step residual P(3HB) was confirmed by high-resolution 13C nuclear magnetic resonance spectroscopy. In order to synthesize 3HB-free poly(4-hydroxybutyric acid) [P(4HB)] homopolymer, a three-stage cultivation technique was developed by adding a nitrogen addition step, which completely removed the residual P(3HB). The resulting polymer was free of 3HB. However, when the strain was grown on gamma-butyrolactone as the sole carbon source in a synthesis medium, a copolyester of P(3HB-co-4HB) containing 45 mol% 3HB was produced. One-step cultivation on gamma-butyrolactone required a rather long induction time (3 to 4 days). On the basis of the results of an enzymatic study performed with crude extracts, we suggest that the inability of cells to produce 3HB in the multistep culture was due to a low level of 4-hydroxybutyric acid (4HBA) dehydrogenase activity, which resulted in a low level of acetyl coenzyme A. Thus, 3HB formation from gamma-butyrolactone is driven by a high level of 4HBA dehydrogenase activity induced by long exposure to gamma-butyrolactone, as is the case for a one-step culture. In addition, intracellular degradation kinetics studies showed that P(3HB) in cells was completely degraded within 30 h of cultivation after being transferred to a carbon-free mineral medium containing additional ammonium sulfate, while P(3HB-co-4HB) containing 5 mol% 3HB and 95 mol% 4HB was totally inert in interactions with the intracellular depolymerases. Intracellular inertness could be a useful factor for efficient synthesis of the P(4HB) homopolymer and of 4HB-rich P(3HB-co-4HB) by the strain used in this study.     

Effects of yeast extract and glucose on xanthan production and cell growth in batch culture of Xanthomonas campestris

Although available kinetic data provide a useful insight into the effects of medium composition on xanthan production by Xanthomonas campestris, they cannot account for the synergetic effects of carbon (glucose) and nitrogen (yeast extract) substrates on cell growth and xanthan production. In this work, we studied the effects of the glucose/yeast-extract ratio (G/YE) in the medium on cell growth and xanthan production in various operating modes, including batch, two-stage batch, and fed-batch fermentations. In general, both the xanthan yield and specific production rate increased with increasing G/YE in the medium, but the cell yield and specific growth rate decreased as G/YE increased. A two-stage batch fermentation with a G/YE shift from an initial low level (2.5% glucose/0.3% yeast extract) to a high level (5.0% glucose/0.3% yeast extract) at the end of the exponential growth phase was found to be preferable for xanthan production. This two-stage fermentation design both provided fast cell growth and gave a high xanthan yield and xanthan production rate. In contrast, fed-batch fermentation with intermittent additions of glucose to the fermentor during the stationary phase was not favorable for xanthan production because of the relatively low G/YE resulting in low xanthan production rate and yield. It is also important to use a moderately high yeast extract concentration in the medium in order to reach a high cell density before the culture enters the stationary phase. A high cell density is also important to the overall xanthan production rate. Received: 30 September 1996 / Received revision: 21 January 1997 / Accepted: 10 February 1997     

Growth, auxin requirement, and indole-3-acetic acid content of cultured crown-gall and habituated tissues of tobacco

Abstract. We used radioimmunoassay to compare the indole-3-acetic acid (IAA) concentration in cultured auxinautonomous tissues of Nicotiana tabacum L. cv.'Havana 425'that differ in growth rate, auxin requirement, capacity for organogenesis, and proximal cause of transformation. Lines HT37 and HB6 were crown-gall tissues transformed by the T37 and B6 strains of Agrobacterium tumefaciens . The tissue line 156AH, like the crown-gall lines, was auxin and cytokinin autotrophic, but arose spontaneously in culture and did not result from crown-gall transformation. The IAA content of the lines was variable and between 10⁻⁷ and 10⁻⁶ moles kg⁻¹ fresh weight. This same range of IAA concentration was found in growing leaf tissues on the plant. IAA concentrations were 2–5 fold higher in HB6 than HT37 during the first few days of culture, and exogenous auxin induced the teratomatous HT37 tissue to grow in an unorganized fashion suggesting that auxin plays a role in regulating tumor morphology. The major difference between genetically transformed crown-gall tissues and the auxin-cytokinin autotrophic tissue line was that net IAA production continued to rise for several weeks of culture in tumor tissues but only for the first few days of culture in the autotrophic tissue.