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 with High Productivity and High Polymer Content by a Fed-Batch Culture of Alcaligenes latus under Nitrogen Limitation

Alcaligenes latus has been known to produce poly(3-hydroxybutyrate) (PHB) in a growth-associated manner even under nutrient-sufficient conditions. However, the PHB content obtained by fed-batch culture was always low, at ca. 50%, which makes the recovery process inefficient. In this study, the effect of applying nitrogen limitation on the production of PHB by A. latus was examined. In flask and batch cultures, the PHB synthesis rate could be increased considerably by applying nitrogen limitation. The PHB content could be increased to 87% by applying nitrogen limitation in batch culture, which was considerably higher than that typically obtainable (50%) under nitrogen-sufficient conditions. In fed-batch culture, cells were first cultured by the DO-stat feeding strategy without applying nitrogen limitation. Nitrogen limitation was applied at a cell concentration of 76 g (dry cell weight)/liter, and the sucrose concentration was maintained within 5 to 20 g/liter. After 8 h of nitrogen limitation, the cell concentration, PHB concentration, and PHB content reached 111.7 g (dry cell weight)/liter, 98.7 g/liter, and 88%, respectively, resulting in a productivity of 4.94 g of PHB/liter/h. The highest PHB productivity, 5.13 g/liter/h, was obtained after 16 h.     

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     

Experimental optimization of fed-batch culture for poly-beta-hydroxybutyric acid production

The optimal feed rate profiles of glucose and ammonium hydroxide were calculated using a proposed model, and implemented for the production of poly-beta-hydroxybutyric acid (PHB) by Alcaligenes eutrophus. By implementing these optimal feed rates with a high glucose feed concentration of 700 g/L and an ammonium hydroxide concentration of 7%(w/w), it was possible to achieve a high final cell concentration of 141 g/L and a high PHB concentration of 105 g/L in 40 h of fed-batch operation. The PHB productivity was as high as 2.63 g/(L hr). (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 697-705, 1997.     

Unsterile and continuous production of polyhydroxybutyrate by Halomonas TD01

An unsterile and continuous fermentation process was developed based on a halophilic bacterium termed Halomonas TD01 isolated from a salt lake in Xinjiang, China. The strain reached 80 g/L cell dry weight containing 80% poly(3-hydroxybutyrate) (PHB) on glucose salt medium during a 56 h fed-batch process. In a 14-day open unsterile and continuous process, the cells grew to an average of 40 g/L cell dry weight containing 60% PHB in the first fermentor with glucose salt medium. Continuous pumping of cultures from the first fermentor to the second fermentor containing the nitrogen-deficient glucose salt medium diluted the cells but allowed them to maintain a PHB level of between 65% and 70% of cell dry weight. Glucose to PHB conversions were between 20% and 30% in the first fermentor and above 50% in the second one. This unsterile and continuous fermentation process opens a new area for reducing the cost in polyhydroxyalkanoates production.     

Effect of phosphate supply and aeration on poly-β-hydroxybutyrate production in Azotobacter chroococcum

The effects of phosphate supply and aeration on cell growth and PHB accumulation were investigated in Azotobacter chroococcum 23 with the aim of increasing PHB production. Phosphate limitation favoured PHB formation in Azotobacter chroococcum 23, but inhibited growth. Azotobacter chroococcum 23 cells demonstrated intensive uptake of orthophosphate during exponential growth. At the highest phosphate concentration (1·5 g/litre) and low aeration the amount of intracellular orthophosphate/g residual biomass was highest. Under conditions of fed-batch fermentation the possibility of controlling the PHB production process by the phosphate level in the cultivation medium was demonstrated. A 36 h fed-batch fermentation resulted in a biomass yield of 110 g/litre with a PHB cellular concentration of 75% dry weight, PHB content 82·5 g/litre, PHB yield Y_P/S = 0·24 g/g and process productivity 2·29 g/litre·h.     

Optimization of microbial poly(3-hydroxybutyrate) recover using dispersions of sodium hypochlorite solution and chloroform

Optimization was carried out for the recovery of microbiol poly(3-hydroxybutyrate) (PHB) from Alcaligenes eutrophus. This process involved the use of a dispersion made of sodium hypochlorite solution and chloroform. The dispersion enabled us to take advantage of both differential digestion by hypochlorite and solvent extraction by chloroform. The PHB recovery (%) from cell powder was maximized using a 30% hypochlorite concentration, a 90-min treatment time, and a 1:1 (v/v) chloroform-to-aqueous-phase ratio. Under these optimal conditions, the recovery was about 91% and the purity of recovered PHB was higher than 97%. The number average molecular weight, M(n) of recovered PHB was about 300,000 and the weight average molecular weight M(w) was about 1,020,000, compared to the original M(n) of 530,000 and M(w) of 1,272,000. The moderate decrease in both M(n) and M(w) might be ascribed to the shielding effect of chloroform. In addition, the relatively small decrease in M(w) probably resulted from the loss of short PHB chains which might be water soluble. The crystallinity of recovered PHB was in the range of 60 to 65%although a slightly higher crystallinity was observed when the dispersion was used. (c) 1994 John Wiley & Sons, Inc.     

Production of Poly(3-hydroxybutyrate) from waste potato starch

There has been a considerable interest in using low cost carbon substrates for the production of Poly(3-hydroxybutyrate) (PHB). We have shown that saccharified waste potato starch can be used as a viable alternative carbon source in high cell density PHB production. Using Ralstonia eutropha NCIMB 11599 with phosphate limitation, 179 g/l biomass, 94 g/l PHB, Y(biomass/starch) = 0.46 g/g, Y(PHB/starch) = 0.22 g/g, and PHB productivity = 1.47 g/(l*h) were achieved. Residual maltose accumulated in the fed-batch reactor but caused no noticeable inhibition. Performance with saccharified starch was virtually identical to that with glucose.     

Increased PHB productivity by high-cell-density fed-batch culture of Alcaligenes latus, a growth-associated PHB producer

Alcaligenes latus, a growth-associated PHB producer, was cultivated by a pH-stat modal fed-batch culture technique to attain high PHB productivity. Both sucrose solution and inorganic medium were fed in conjunction with the supply of ammonia solution which serves as a nitrogen source and as a means of pH control. Compositions of the inorganic medium were formulated by elemental analysis of A. latus cell mass. The effect on inoculum size was examined to reduce culture time. High concentrations of cell (142 g/L) and PHB (68.4 g/L) were obtained in a short culture time (18 h) with an inoculum size of 13.7 g/L. The PHB content and the PHB productivity at the end of the fed-batch culture were 50% of dry cell weight and 4.0 g PHB/(L . h), respectively. (c) 1996 John Wiley & Sons, Inc.     

Production of poly (3-hydroxybutyrate) from starch by Azotobacter chroococcum

Production of poly (3-hydroxybutyrate) (PHB) from starch was investigated in flask, batch, and fed-batch cultures of Azotobacter chroococcum. In flask culture, PHB content increased up to 74% of dry cell wt with increasing culture volume. In batch culture, PHB content increased to 44% with O2 limitation. In fed-batch culture, cell concentration of 71 g/l with 20% PHB was obtained without O2 limitation, whereas cell concentration of 54 g/l with 46% PHB was obtained with O2 limitation.     

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.     

Production of poly-β-hydroxybutyrate by Azotobacter chroococcum H23 in chemically defined medium and alpechin medium

M.V. MARTINEZ-TOLEDO, J. GONZALEZ-LOPEZ, B. RODELAS, C. POZO AND V. SALMERON. 1995. Azotobacter chroococcum H23 is able to produce large amounts of poly-β-hydroxybutyrate (PHB) during growth in chemically-defined medium (N-free or with NH⁺₄) and alpechin (wastewater from olive oil mills) medium. Polymer production was not dependent of the nutrient limitation. Strain H23 was capable of accumulating PHB up to 70% of the cell dry weight after 24 h incubation in chemically-defined media containing 1% glucose, fructose, mannitol, saccharose or starch. Azotobacter chroococcum grown on NH⁺₄-medium supplemented with alpechin formed PHB up to 50% of the cell dry weight after 24 h, suggesting that these wastes could be utilized by Azotobacter as a cheap substrate for producing PHB.     

Poly(3-hydroxybutyrate) synthesis in fed-batch culture of Ralstonia eutropha with phosphate limitation under different glucose concentrations

Poly(3-hydroxybutyrate) (PHB) was produced by fed-batch cultures of Ralstonia eutropha with phosphate limitation under different glucose concentrations. When glucose was kept at 2.5 g l^–1, cell growth and PHB synthesis were limited due to the shortage of carbon source but a higher PHB content occurred in the cell-growth stage. This shows that a low glucose concentration is favorable for PHB accumulation in R. eutropha. PHB obtained with glucose at 9 g l^–1 is 1.6 times that obtained with 40 g l^–1. When glucose was in the range of 9 to 40 g l^–1, PHB concentration and productivity decreased significantly with the increase of glucose concentration. The highest PHB productivity was obtained with glucose at 9 g l^–1.     

Occurrence of Poly-β-Hydroxybutyrate in the Azotobacteriaceae

Poly-beta-hydroxybutyrate (PHB) from various representative strains of the genera Azotobacter, Beijerinckia, and Derxia was isolated and characterized. During growth in shake culture, with glucose as a carbon and energy source, and molecular nitrogen as a nitrogen source, increase in dry weight appeared linear, and PHB formed a constant percentage of the dry weight. In a medium containing 1% (w/v) glucose, PHB declined with the onset of the stationary phase of growth; with 2% (w/v) glucose, an increase in PHB content during stationary phase was noted in the case of some strains, before a subsequent decline. The decrease in PHB as a percentage of dry cellular weight (not of total amount present in the culture) during growth of some strains with 2% as opposed to 1% (w/v) glucose may be ascribed to a greater production of capsular polysaccharide. PHB content could not be used as a taxonomic criterion. Strain differences were as great as or greater than species differences. The only strain of Beijerinckia fluminensis obtained contained PHB, but it could not be grown on the nitrogen-free medium used. Two species of the genus Azotomonas, reported to be aerobic, nonsymbiotic nitrogen-fixers, did not grow on the nitrogen-free medium used and did not produce PHB during growth with a combined nitrogen source.     

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.%.     

Protein-free fed-batch culture of non-GS NS0 cell lines for production of recombinant antibodies

Presented is a novel antibody production platform based on the fed-batch culture of recombinant, NS0-derived cell lines. A standardized fed-batch cell culture process was developed for five non-GS NS0 cell lines using enriched and optimized protein-free, cholesterol-free, and chemically defined basal and feed media. The process performed reproducibly and scaled faithfully from the 2-L to the 100-L bioreactor scale achieving a volumetric productivity of > 120 mg/L per day. Fed-batch cultures for all five cell lines exhibited significant lactate consumption when the cells entered the stationary or death phase. Peak and final lactate concentrations were low relative to a previously developed fed-batch process (FBP). Such low lactate production and high lactate consumption rates were unanticipated considering the fed-batch culture basal medium has an unconventionally high initial glucose concentration of 15 g/L, and an overall glucose consumption in excess of 17 g/L. The potential of this process platform was further demonstrated through additional media optimization, which has resulted in a final antibody concentration of 2.64 +/- 0.19 g/L and volumetric productivity of > 200 mg/L per day in a 13-day FBP for one of the five production cell lines. Use of this standardized protein-free, cholesterol-free NS0 FBP platform enables consistency in development time and cost effectiveness for manufacturing of therapeutic antibodies.     

Production of poly(3-hydroxybutyrate) from inexpensive substrates

Two inexpensive substrates, starch and whey were used to produce poly(3-hydroxybutyrate) (PHB) in fed-batch cultures of Azotobacter chroococcum and recombinant Escherichia coli, respectively. Oxygen limitation increased PHB contents in both fermentations. In fed-batch culture of A. chroococcum, cell concentration of 54 g l⁻¹ with 46% PHB was obtained with oxygen limitation, whereas 71 g l⁻¹ of cell with 20% PHB was obtained without oxygen limitation. The timing of PHB biosynthesis in recombinant E. coli was controlled using the agitation speed of a stirred tank fermentor. A PHB content of 80% could be obtained with oxygen limitation by increasing the agitation speed up to only 500 rpm.     

Poly(β-hydroxybutyrate) production by a moderate halophile,Halomonas boliviensis LC1

The moderate halophile Halomonas boliviensis, isolated from a Bolivian saline soil sample, was able to accumulate poly(β-hydroxybutyrate) (PHB) when grown under conditions of nutrient limitation and excess carbon source. The concentration of sodium chloride in the medium influenced the cell-growth, -size, and rate of PHB accumulation. Cultivation in shake flasks led to a PHB accumulation of about 54 wt.% with respect to cell dry weight at 4.5% (w/v) NaCl in a medium with butyric acid and sodium acetate as carbon sources. The production of PHB was substantially improved to a maximum value of 88 wt.% during cultivation under controlled conditions of pH and oxygen concentration in a fermentor. The use of glucose and sucrose, respectively, as carbon source could also lead to the production of PHB at an average level of 55 wt.%.     

Process optimization and production of polyhydroxybutyrate using palm jaggery as economical carbon source by marine sponge-associated Bacillus licheniformis MSBN12

The Polyhydroxybutyrate (PHB) producer, Bacillus licheniformis MSBN12 was isolated from the marine sponge Callyspongia diffusa. The PHB production of B. licheniformis MSBN12 was optimized using a four-factor Box-Behnken design to find the interactive effects of variables such as palm jaggery, wheat bran, seawater, and incubation temperature. The maximum yield of PHB (6.38 g/L) was achieved through response surface methodology-based optimization and the optimized conditions were further used for the batch and fed-batch fermentation. Maximum biomass was reached at 48 and 36 h of incubation with PHB accumulation of 62.91 and 67.16 % (w/w of dry cells) for batch and fed-batch process. The production of PHB under fed-batch process with B. licheniformis MSBN12 was increased threefold over shake flask culture when palm jaggery as sole carbon source. The ¹H NMR data was extrapolated with peaks of the PHB reference standard and confirmed as PHB analog.