Production of poly(3-hydroxybutyrate) by high cell density fed-batch culture of Alcaligenes eutrophus with phospate limitation

High cell density fed-batch fermentation of Alcaligenes eutrophus was carried out for the production of poly(3-hydroxybutyrate) (PHB) in a 60-L fermentor. During the fermentation, pH was controlled with NH(4)OH solution and PHB accumulation was induced by phosphate limitation instead of nitrogen limitation. The glucose feeding was controlled by monitoring dissolved oxygen (DO) concentration and glucose concentration in the culture broth. The glucose concentration fluctuated within the range of 0-20 g/L. We have investigated the effect of initial phosphate concentration on the PHB production when the initial volume was fixed. Using an initial phosphate concentration of 5.5 g/L, the fed-batch fermentation resulted in a final cell concentration of 281 g/L, a PHB concentration of 232 g/L, and a PHB productivity of 3.14 g/L . h, which are the highest values ever reported to date. In this case, PHB content, cell yield from glucose, and PHB yield from glucose were 80, 0.46, and 0.38% (w/w), respectively. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 28-32, 1997.     

Production of poly(3-hydroxybutyric acid) by fed-batch culture of Alcaligenes eutrophus with glucose concentration control

Alcaligenes eutrophus NCIMB 11599 was cultivated to produce poly(3-hydroxybutyric acid) (PHB) from glucose by the automatic fed-batch culture technique. The glucose concentration of the culture broth was controlled at 10 to 20 g/L by two methods: using exit gas data obtained from a mass spectrometer and using an on-line glucose analyzer. The effect of ammonium limitation on PHB synthesis at different culture phases was studied. The final cell concentration, PHB concentration, and PHB productivity increased as ammonia feeding was stopped at a higher cell concentration. High concentrations of PHB (121 g/L) and total cells (164 g/L) were obtained in 50 h when ammonia feeding was stopped at the cell concentration of 70 g/L. The maximum PHB content reached 76% of dry cell weight and the productivity was 2.42 g/L h with the yield of 0.3 g PHB/g glucose.     

Poly-3-hydroxybutyrate production byAzotobacter chroococcum

Thirty-seven soil isolates and mutants ofAzotobacter chroococcum tested for poly-3-hydroxybutyrate (PHB) production using Sudan black B staining method were found to be positive. One mutant showed a higher number of PHB-producing cells and maximum number of granules per cell. Using 2% glucose and 15 mmol/L ammonium acetate, PHB production was found to be maximum at 36 and 48 h of growth under submerged cultivation and under stationary cultivation, respectively. PHB production was found to be higher on sucrose and commercial sugar (as carbon sources) as compared to glucose and mannitol. As commercial sugar is cheaper than sucrose it was selected as carbon source for PHB production, that being found to be maximum at 1% concentration. Inorganic nitrogen sources seemed to have no stimulatory effect on the production of PHB. However, ammonium acetate (15 mmol/L) was found to be best for PHB production. Peptone (0.2 %) gave a better yield of PHB under both growth conditions. Using all optimized conditions, PHB production was studied in ten selected strains. Two of them were found to be best PHB producers under both growth conditions, one producing 621 and 740 μg/g dry mass under submerged cultivation and under stationary cultivation, respectively, while the second one produced 589 and 733 μg/g.     

A robust fed-batch feeding strategy independent of the carbon source for optimal polyhydroxybutyrate production

A three-stage control strategy independent of the organic substrate was developed for automated substrate feeding in a two-phase fed-batch culture of Cupriavidus necator DSM 545 for the production of the biopolymer polyhydroxybutyrate (PHB). The optimal feeding strategy was determined using glucose as the substrate. A combined substrate feeding strategy consisting of exponential feeding and a novel method based on alkali-addition monitoring resulted in a maximal cell concentration in the biomass growth phase. In the PHB accumulation phase, a constant substrate feeding strategy based on the estimated amount of biomass produced in the first phase and a specific PHB accumulation rate was implemented to induce PHB under limiting nitrogen at different biomass concentrations. Maximal cell and PHB concentrations of 164 and 125 g/L were obtained when nitrogen feeding was stopped at 56 g/L of residual biomass; the glucose concentration was maintained within its optimal range. The developed feeding strategy was validated using waste glycerol as the sole carbon source for PHB production, and the three-stage control strategy resulted in a PHB concentration of 65.6 g/L and PHB content of 62.7% while keeping the glycerol concentration constant. It can thus be concluded that the developed feeding strategy is sensitive, robust, inexpensive, and applicable to fed-batch culture for PHB production independent of the carbon source.     

Production of poly-β-hydroxybutyrate by fed-batch culture of recombinantEscherichia coli

Summary A recombinantEscherichia coli strain harboring the PHB biosynthesis genes fromAlcaligenes eutrophus was used to produce poly--hydroxybutyrate (PHB) by pH-stat fedbatch culture. Initial glucose concentration for optimal growth was found to be 20g/L from a series of flask cultures. A final PHB concentration of 88.8 g/L could be obtained after 42 hrs of cultivation.     

Application of self-cycling fermentation technique to the production of poly-beta-hydroxybutyrate

The production of poly-beta-hydroxybutyrate (PHB) by Alcaligenes eutrophus DSM 545 in a cyclone bioreactor was compared using various culture methods: batch, fed-batch, and self-cycling fermentation (SCF) with and without extended periods of nutrient deprivation. SCF is a semi-continuous method that results in a nutrient limitation for every successive generation of cells and, therefore, may have advantages for products whose formation follow secondary metabolite kinetics. Use of the SCF technique without extended nutrient deprivation produced a PHB concentration of 1.2 g L(-1) as 40% of the biomass dry weight. With nitrogen deprivation for 4 or 6 h, the concentration of PHB decreased when compared to the standard SCF technique. However, nitrogen deprivation periods of 8 h resulted in an increase in PHB concentration to 2.7 g L(-1) or 59% of the biomass dry weight. The nutrient cycling may act to repress PHB accumulation during periods of nitrogen deprivation, unless a time threshold has been reached, after which PHB accumulation occurs as in normal batch culture. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 815-820, 1997.     

Control of glucose feeding using exit gas data and its application to the production of PHB from tapioca hydrolysate byAlcaligenes eutrophus

Summary A method to estimate the glucose concentration in the culture broth using CO₂ evolution rate (CER) data from a mass spectrometer was developed.Alcaligenes eutrophus was cultivated to produce poly(3-hydroxybutyric acid) (PHB) from tapioca hydrolysate using this method. Thek value (g glucose/mol CO₂), defined as the glucose consumption per CO₂ evolution, decreased with culture time and was automatically changed using CER data. The glucose concentration in the culture broth could be controlled at 10 to 20 g/L. A final cell concentration of 106 g/L, PHB concentration of 61 g/L. and PHB content of 58 % of dry cell weight were obtained after 59 h of cultivation.     

Effect of single nutrient limitation of poly-beta-hydroxybutyrate molecular weight distribution in alcaligens europhus

The effect of magnesium and phosphate limitation on the molecular weight distribution of poly-beta-hydroxybutyrate (PHB) in Alcaligens europhus in cotinuons culture has been stuied. Conditions of nitrogen limitation both with glucose excess (above ca. 20 g/L) and without excess were investigated Under N-limitation and glucose excess, M(w) decreases when the magnesium content is decreased below 50% (19.7 mg/L) of the basal medium content; this also results in a broadenng of molecular weight distribution (M(w)/M(n)) from 2 to 5 and a decrease in M(w) fron 2 x 10(6) to 0.9 x 10(6). Below 20% of the basal content of magnesium (7.9 mg/L) these two trends were reversed. This behaviour was not observed in the absence of glucose excess, phshate had virtually no effect on PHB M(w) or its distribution, whereas wih no (or little) glucose excess M(w) of the PHB decreased with phosphate concentrations below 50% of the basal level (0.705 g/L). Hence, in continuous or fed-batch cultures, in addition to nitrogen limitation to alklow for PHB accumulation, it is necesary to control both the addition of glucose (no excess) and also to maintain magnesium limitation (ca. 25% of basal medium level, 9.9 mg/L) and phosphate above 50% of he basal level (0.705 g/L). Thus, when broadening of molecular weight destribution (increase in M(w)/M(n)) is observed at the end of fed-batch culture it is probably caused by phosphate limitation and/or glucose excess. (c) 1995 John Wiley & Sons, Inc.     

废弃活性污泥中产聚羟基脂肪酸酯菌株Bacillus sp. PB-3的发酵条件优化

通过尼罗红染色法结合荧光显微镜镜检,从废弃活性污泥中分离得到1株高产聚羟基脂肪酸酯(PHAs)的菌株Bacillus sp.PB-3,经气相色谱法鉴定该菌株胞内产物为聚β-羟基丁酸酯(PHB)。对培养基成分及发酵条件优化后,获得最佳培养方案:12 g/L的葡萄糖为C源,2 g/L的牛肉膏为N源,初始pH 7.5,培养基装液量80 mL,转速为200 r/min,37℃培养48 h,PHB质量分数可达菌体干质量的32.09%,比优化前提高30%。     

Production of poly-β-hydroxybutyrate on molasses by recombinant Escherichia coli

Beet molasses successfully replaced glucose as sole carbon source to produce poly--hydroxybutyrate by a recombinant Escherichia coli strain (HMS174/pTZ18u-PHB). The fermentation with molasses was cheaper than with glucose. The final dry cell weight, PHB content and PHB productivity were 39.5 g/L, 80% (w/w) and 1 g/Lh, respectively, in a 5 L stirred tank fermenter after 31.5 h fed-batch fermentation with constant pH and dissolved O2 content. © Rapid Science Ltd. 1998     

Oscillatory behavior of Saccharomyces cerevisiae in continuous culture: I. Effects of pH and nitrogen levels

The appearance of sustained oscillations in bioreactor variables (biomass and nutrient concentrations) in continuous cultures of Saccharomyces cerevisiae indicates the complex nature of microbial systems, the inadequacy of current growth kinetic models, and the difficulties which may arise in bioprocess control and optimization. In this study we investigate continuous bioreactor behavior over a range of operating conditions (dilution rate, feed glucose concentration, feed ammonium concentration, dissolved oxygen, and pH) to determine the process requirements which lead to oscillatory behavior. We present new results which indicate that high feed ammonium concentrations may eliminate oscillations and that under oscillatory conditions ammonium levels are generally low and oscillatory as well. The effects of pH are complex and oscillations were only observed at pH values 5.5 and 6.5; no oscillations were observed at a pH of 4.5. Under our nominal operating conditions (feed glucose concentration 10 g/L, dilution rate 0.145 h(-1), feed ammonium concentration 0.0303M, dissolved oxygen level 50%, pH 5.5, and T = 30 degrees C) we found two possible final bioreactor states depending on the transient used to reach the nominal operating conditions. One of the states was oscillatory and characteristic of oxidative metabolism and the other was nonoscillatory and fermentative.     

Comparison of recombinant Escherichia coli strains for synthesis and accumulation of poly-(3-hydroxybutyric acid) and morphological changes

A stable high-copy-number plasmid pSYL105 containing the Alcaligenes eutrophus polyhydroxyalkanoic acid (PHA) biosynthesis genes was constructed. This plasmid was transferred to seven Escherichia coli strains (K12, B, W, XL1-Blue, JM109, DH5alpha, and HB101), which were subsequently compared for their ability to synthesize and accumulate ploy- (3-hydroxybutyric acid) (PHB). Growth of recombinant cells and PHB synthesis were investigated in detail in Luria-Bertani (LB) medium containing 20 g/L glucose. Cell growth, the rate of PHB synthesis, the extent of PHB accumulation, the amount of glucose utilized, and the amount of acetate formed varied from one strain to another. XL1-Blue (pSYL105) and B (pSYL105) synthesized PHB at the fastest rate, which was ca. 0.2 g PHB/g true cell mass-h, and produced PHB up to 6-7 g/L. The yields of cell mass, true cell mass, and PHB varied considerably among the strains. The PHB yield of XL1-Blue (pSYL105) in LB plus 20 g/L glucose was as high as 0.369 g PHB/g glucose. Strains W (pSYL105) and K12 (pSYL105) accumulated the least amount of PHB with the lowest PHB yield at the lowest synthesis rate. JM109 (pSYL105) accumulated PHB to the highest extent (85.6%) with relatively low true cell mass (0.77 g/L). Considerable filamentation of cells accumulating PHB was observed for all strains except for K12 and W, which seemed to be due either to the overexpression of the foreign PHA biosynthesis enzymes or to the accumulation of PHB. (c) 1994 John Wiley & Sons, Inc.     

Production of poly-(beta-hydroxybutyric-co-beta-hydroxyvaleric) acids

Alcaligenes latus, Alcaligenes eutrophus, Bacillus cereus, Pseudomonas pseudoflava, Pseudomonas cepacia, and Micrococcus halodenitrificans were found to accumulate poly-(beta-hydroxybutyric-co-beta-hydroxyvaleric) acid [P(HB-co-HV)] copolymer when supplied with glucose (or sucrose in the case of A. latus) and propionic acid under nitrogen-limited conditions. A fed-batch culture of A. eutrophus produced 24 g of poly-beta-hydroxybutyric acid (PHB) liter-1 under ammonium limitation conditions. When the glucose feed was replaced with glucose and propionic acid during the polymer accumulation phase, 17 g of P(HB-co-HV) liter-1 was produced. The P(HB-co-HV) contained 5.0 mol% beta-hydroxyvaleric acid (HV). Varying the carbon-to-nitrogen ratio at a dilution rate of 0.15 h-1 in a chemostat culture of A. eutrophus resulted in a maximum value of 33% (wt/wt) PHB in the biomass. In comparison, A. latus accumulated about 40% (wt/wt) PHB in chemostat culture under nitrogen-limited conditions at the same dilution rate. When propionic acid was added to the first stage of a two-stage chemostat, A. latus produced 43% (wt/wt) P(HB-co-HV) containing 18.5 mol% HV. In the second stage, the P(HB-co-HV) increased to 58% (wt/wt) with an HV content of 11 mol% without further addition of carbon substrate. The HV composition in P(HB-co-HV) was controlled by regulating the concentration of propionic acid in the feed. Poly-beta-hydroxyalkanoates containing a higher percentage of HV were produced when pentanoic acid replaced propionic acid.     

Production of poly-(beta-hydroxybutyric-co-beta-hydroxyvaleric) acids.

Alcaligenes latus, Alcaligenes eutrophus, Bacillus cereus, Pseudomonas pseudoflava, Pseudomonas cepacia, and Micrococcus halodenitrificans were found to accumulate poly-(beta-hydroxybutyric-co-beta-hydroxyvaleric) acid [P(HB-co-HV)] copolymer when supplied with glucose (or sucrose in the case of A. latus) and propionic acid under nitrogen-limited conditions. A fed-batch culture of A. eutrophus produced 24 g of poly-beta-hydroxybutyric acid (PHB) liter-1 under ammonium limitation conditions. When the glucose feed was replaced with glucose and propionic acid during the polymer accumulation phase, 17 g of P(HB-co-HV) liter-1 was produced. The P(HB-co-HV) contained 5.0 mol% beta-hydroxyvaleric acid (HV). Varying the carbon-to-nitrogen ratio at a dilution rate of 0.15 h-1 in a chemostat culture of A. eutrophus resulted in a maximum value of 33% (wt/wt) PHB in the biomass. In comparison, A. latus accumulated about 40% (wt/wt) PHB in chemostat culture under nitrogen-limited conditions at the same dilution rate. When propionic acid was added to the first stage of a two-stage chemostat, A. latus produced 43% (wt/wt) P(HB-co-HV) containing 18.5 mol% HV. In the second stage, the P(HB-co-HV) increased to 58% (wt/wt) with an HV content of 11 mol% without further addition of carbon substrate. The HV composition in P(HB-co-HV) was controlled by regulating the concentration of propionic acid in the feed. Poly-beta-hydroxyalkanoates containing a higher percentage of HV were produced when pentanoic acid replaced propionic acid.     

Biosynthesis of Poly-beta-Hydroxyalkanoates from Pentoses by Pseudomonas pseudoflava

The potential of Pseudomonas pseudoflava to produce poly-beta-hydroxyalkanoates (PHAs) from pentoses was studied. This organism was able to use a hydrolysate from the hemicellulosic fraction of poplar wood as a carbon and energy source for its growth. However, in batch cultures, growth was inhibited completely at hydrolysate concentrations higher than 30% (vol/vol). When P. pseudoflava was grown on the major sugars present in hemicelluloses in batch cultures, poly-beta-hydroxybutyric acid (PHB) accumulated when glucose, xylose, or arabinose was the sole carbon source, with the final PHB content varying from 17% (wt/wt) of the biomass dry weight on arabinose to 22% (wt/wt) of the biomass dry weight on glucose and xylose. Specific growth rates were 0.58 h on glucose, 0.13 h on xylose, and 0.10 h on arabinose, while the specific PHB production rates based on total biomass ranged from 0.02 g g h on arabinose to 0.11 g g h on glucose. PHB weight-average molecular weights were 640,000 on arabinose and 1,100,000 on glucose and xylose. The absolute amount of PHB in the cells decreased markedly when nitrogen limitation was relaxed by feeding ammonium sulfate at the end of the PHB accumulation stage of the arabinose and xylose fermentations. Copolymers of beta-hydroxybutyric and beta-hydroxyvaleric acids were produced when propionic acid was added to shake flasks containing 10 g of glucose liter. The beta-hydroxyvaleric acid monomer content attained a maximum of 45 mol% when the initial propionic acid concentration was 2 g liter.     

Production of poly-3-hydroxybutyrate by Bacillus sp. JMa5 cultivated in molasses media

A strain of Bacillus sp. coded JMa5 was isolated from molasses contaminated soil. The strain was able to grow at a temperature as high as 45°C and in 250 g/l molasses although the optimal growth temperature was 35–37°C. Cell density reached 30 g/l 8 h after inoculation in a batch culture with an initial concentration of 210 g/l molasses. Under fed-batch conditions, the cells grew to a dry weight of 70 g/l after 30 h of fermentation. The strain accumulated 25–35%, (w/w) polyhydroxybutyrate (PHB) during fermentation. PHB accumulation was a growth-associated process. Factors that normally promote PHB production include high ratios of carbon to nitrogen, and carbon to phosphorus in growth media. Low dissolved oxygen supply resulted in sporulation, which reduced PHB contents and dry weights of the cells. It seems that sporulation induced by reduced supply of nutrients is the reason that PHB content is generally low in the Bacillus strain.     

Formation of aerobic granules and their PHB production at various substrate and ammonium concentrations

Aerobic granular sludge rich in polyhydroxybutyrate (PHB) was cultivated in a sequencing batch reactor (SBR) by seeding anaerobic granular sludge. The PHB content in aerobic granules was investigated and the experimental results reveal that both influent chemical oxygen demand (COD) and ammonium concentrations had a significant effect on the morphological characteristics and the PHB production of the aerobic granular sludge. At a COD and ammonium concentration of 750 mg/L and 8.5mg/L, respectively, the PHB content of the granules reached 44%, but their poor settling ability, as evidenced by a high sludge volume index, was observed. This was attributed to the outgrowth of filamentous bacteria on the granule surface. However, an increase in the ammonium concentration resulted in an elevated sludge concentration and a decrease in the PHB content in the granules. In this case, the aerobic granular sludge with a regular and compact structure was formed. The results suggest that, through controlling the COD and ammonium concentrations in the influent, the PHB-rich aerobic granular sludge with good settling ability could be cultivated.     

Ethanol production by Zymomonas mobilis in fed-batch fermentations

The production of ethanol by Zymomonas mobilis NRRL B-4286 was studied in fed-batch cultures. Initial percent (w/v) glucose, rate of feed, and quantity of 50%; (w/v) glucose feed were varied. Glucose inhibition of growth rate occurred at concentrations greater than 8% (w/) Feed was begun after 4 h incubation. Feed volume was ca. 36%; of starting batch volume to get ca. 10%; (w/v) ethanol at harvest. The range of feed rates studied varied from 16%; batch volume/h (glucose concentration increased to an inhibitory level) to 4%; batch volume/h (glucose concentration dropped rapidly to zero and was limiting). Increasing feed volume to 46%; of starting volume at the best feed rate (ca. 10%; feed volume/h) increased final ethanol concentration to 11.3%; (w/v). However, the resultant increase in fermentation time from ca. 21 to 29 h decreased ethanol volumetric productivity from 5.2 to 4.6 g/L h.     

Optimizing conditions for poly(β-hydroxybutyrate) production by <Emphasis Type="Italic">Halomonas boliviensis</Emphasis> LC1 in batch culture with sucrose as carbon source

Halomonas boliviensis LC1 is able to accumulate poly(β-hydroxybutyrate) (PHB) under conditions of excess carbon source and depletion of essential nutrients. This study was aimed at an efficient production of PHB by growing H. boliviensis to high cell concentrations in batch cultures. The effect of ammonium, phosphate, and yeast extract concentrations on cell concentration [cell dry weight (CDW)] and PHB content of H. boliviensis cultured in shake flasks was assayed using a factorial design. High concentrations of these nutrients led to increments in cell growth but reduced the PHB content to some extent. Cultivations of H. boliviensis under controlled conditions in a fermentor using 1.5% (w/v) yeast extract as N source, and intermittent addition of sucrose to provide excess C source, resulted in a polymer accumulation of 44 wt.% and 12 g l⁻¹ CDW after 24 h of cultivation. Batch cultures in a fermentor with initial concentrations of 2.5% (w/v) sucrose and 1.5% (w/v) yeast extract, and with induced oxygen limitation, resulted in an optimum PHB accumulation, PHB concentration and CDW of 54 wt.%, 7.7 g l⁻¹ and 14 g l⁻¹, respectively, after 19 h of cultivation. The addition of casaminoacids in the medium increased the CDW to 14.4 g l⁻¹ in 17 h but reduced the PHB content in the cells to 52 wt.%.     

Utilization of agricultural residues for poly(3-hydroxybutyrate) production by Halomonas boliviensis LC1

Aims: Utilization of cheap and readily available agricultural residues as cheap carbon sources for poly(3‐hydroxybutyrate) (PHB) production by Halomonas boliviensis. Methods and Results: Wheat bran was hydrolysed by a crude enzyme preparation from Aspergillus oryzae NM1 to provide a mixture of reducing sugars composed mainly of glucose, mannose, xylose and arabinose. Growth of H. boliviensis using a mixture of glucose (0·75% w/v) and xylose (0·25% w/v) in the medium led to a PHB content and concentration of 45 wt% and 1 g l^?1, respectively, after 30 h. A similar PHB concentration was attained when H. boliviensis was grown on wheat bran hydrolysate but with a lower PHB content, 34 wt%. In a batch cultivation mode in a fermentor, using 1·8% (w/v) reducing sugars, the maximum PHB accumulation by H. boliviensis was attained in 20 h, but was reduced to about 30 wt%. By adding butyric acid (0·8% v/v), sodium acetate (0·8% w/v) and decreasing the reducing sugars concentration to 1·0% w/v in the medium, PHB accumulation and concentration were increased to 50 wt% and 4 g l^?1, respectively, after 20 h. Butyric acid and sodium acetate for PHB production could also be provided by anaerobic digestion of solid potato waste. Conclusions: Cheap and readily available agricultural residues can be used as substrates to produce PHB. The production of PHB by H. boliviensis using wheat bran hydrolysate as source of carbon is expected to reduce the production cost and motivates further studies. Significance and Impact of the Study: Large‐scale commercial utilization of PHB is mainly hampered by its high production cost. Carbon source for PHB production accounts up to 50% of the total production costs. Thus, the use of waste agricultural residues can substantially reduce the substrate cost (and in turn even provide value to the waste), and can downsize the production costs. This improves the market competitiveness. Studies on PHB production by moderate halophiles were recently initiated with H. boliviensis and findings show that it has potential for commercial exploitation. PHB production by H. boliviensis using wheat bran and potato waste is hence interesting.