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(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.     

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.     

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.     

营养条件对光滑球拟酵母发酵生产丙酮酸的影响

丙酮酸是多种氨基酸、维生素及其它有用物质的重要前体,广泛应用于化工、制药及农用化学品工业。能够直接发酵生产丙酮酸的菌种主要有Ａｃｉｎｅｔｏｂａｃｔｅｒ[1],Ｅｎｔｅｒｏｂａｃｔｅｒ[2],Ｅｎｔｅｒｏｃｏｃｃｕｓ[3],Ｅｓｃｈｅｒｉｃｈｉａ[4],Ａｇａｒｉｃｕ?..     

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.     

Enhancing polyhydroxybutyrate production from high cell density fed-batch fermentation of Bacillus megaterium BA-019

This study demonstrated the improved polyhydroxybutyrate (PHB) production via high cell density cultivation of Bacillus megaterium BA-019 with balanced initial total sugar concentration and carbon to nitrogen (C/N) weight ratio. In the 10 L stirred fermentor operated at 30 °C, pH 7.0, 600 rpm, and 1.0 vvm air, with the initial total sugar concentration of 60 g/L and urea at the C/N weight ratio of 10:1, 32.48 g/L cell biomass with the corresponding PHB weight content of 26.94 % and volumetric productivity of 0.73 g/L h were obtained from batch cultivation. Continuing cultivation by intermittent feeding of the sugarcane molasses along with urea at the C/N weight ratio of 12.5:1 gave much improved biomass and PHB production (90.71 g/L biomass with 45.84 % PHB content and 1.73 g/L h PHB productivity). Similar biomass and PHB yields were obtained in the 90 L stirred fermentor when using the impeller tip speed as the scale-up criterion.     

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.     

Production of poly(3-hydroxybutyrate) from whey by cell recycle fed-batch culture of recombinant Escherichia coli

A new fermentation strategy using cell recycle membrane system was developed for the efficient production of poly(3-hydroxybutyrate) (PHB) from whey by recombinant Escherichia coli strain CGSC 4401 harboring the Alcaligenes latus polyhydroxyalkanoate (PHA) biosynthesis genes. By cell recycle, fed-batch cultivation employing an external membrane module, the working volume of fermentation could be constantly maintained at 2.3 l. The final cell concentration, PHB concentration and PHB content of 194 g l^–1, 168 g l^–1 and 87%, respectively, were obtained in 36.5 h by the pH-stat cell recycle fed-batch culture using whey solution concentrated to contain 280 g lactose l^–1 as a feeding solution, resulting in a high productivity of 4.6 g PHB l^–1 h^–1.     

真养产碱杆菌合成3-羟基丁酸与3-羟基戊酸共聚物发酵条件研究

在摇瓶条件下,对真养产碱杆菌（Ａｌｃａｌｉｇｅｎｅｓｅｕｔｒｏｐｈｕｓ）的３羟基丁酸与３羟基戊酸共聚物（ＰＨＢＶ）发酵过程中ＨＶ组分的前体物质———丙酸的加入时间和加入量进行了研究,结果表明,ＰＨＢＶ中ＨＶ组分含量与丙酸的加入时间和加入量有密切的关系,丙酸的最佳加入时间为菌体生长阶段结束后的多聚物合成初期;尽管高浓度丙酸下可获得较高的ＨＶ组分含量,但会明显抑制菌体的生长和产物的合成。通过对２Ｌ小罐中ＰＨＢＶ合成阶段流加不同糖／酸比混合液所得的发酵结果的比较,并在综合考虑ＰＨＢＶ浓度、ＨＶ组分含量、生产强度和生产成本等基础上,提出了在ＰＨＢＶ合成期流加液的糖／酸比应随菌体对丙酸利用能力的下降而不断增加的流加策略,在此条件下,细胞干重、ＰＨＢＶ浓度和ＰＨＢＶ含量和ＨＶ摩尔分率分别达到５２１ｇ／Ｌ、４０８ｇ／Ｌ、７８３％和１６２ｍｏｌ％,ＨＶ组分对丙酸的产率系数为０５ｇ／ｇ,ＰＨＢＶ的生产强度达到０７４ｇ／（Ｌ／ｈ）。     

Mass production of poly-β-hydroxybutyric acid by fed-batch culture with controlled carbon/nitrogen feeding

Summary The effect of the ratio of methanol to ammonia, in the feeding solution the C/N ratio, on microbial PHB production was investigated. A constant C/N ratio regulated both the PHB content and the specific rate of PHB production. The results indicated that to produce the maximum PHB effectively in a short time the C/N ratio should be controlled automatically according to the increase in PHB content. Variation of the PHB content was estimated by tracing the timecourse of CO₂ concentration in exhaust gas. When the cell concentration reached 70 g/l, C/N ratio was gradually increased from the initial C/N ratio of 10 (mol methanol/mol ammonia). At 121 h, concentration of PHB reached 136 g/l, which was the maximum level so far obtained. The reaction time was considerably shortened compared with our previous study (175 h). Furthermore, PHB concentration reached 149 g/l at 170 h and total cell concentration became 233 g/l. PHB yield from methanol was 0.20 (g PHB/g methanol), which was also superior to the previous result of 0.18.     

Enhancement of poly-3-hydroxybutyrate (PHB) productivity by the two-stage supplementation of carbon sources and continuous feeding of NH4Cl

Gluconate and glucose were selected as the carbon substrates in the production of poly-3-hydroxybutyrate (PHB). Gluconate was utilized to maximize the specific growth rate during the first stage of cell growth, whereas glucose was used to maximize PHB biosynthesis during the second stage of PHB accumulation. The sequential feeding of gluconate and glucose resulted in a 50% enhancement of PHB productivity as compared to the cultures cultivated on glucose alone. In conjunction with secondary glucose uptake, the presence of a trace amount of ammonium increased the rate of PHB biosynthesis during the stage of PHB accumulation. Via the feeding of 0.03 mmol/h of NH₄Cl solution prior to the exhaustion of the initial amount of NH₄Cl, PHB productivity was significantly enhanced as compared to the cultures raised on glucose alone. The glucose-grown culture evidenced a higher level of NADPH during the NH₄Cl-exausted PHB accumulation stage than was observed in the gluconate-grown culture, which reflects that the reason of higher PHB production observed when glucose was used as a carbon source. NH₄Cl feeding following the depletion of initial NH₄Cl resulted in elevated levels of both ATP and NADPH, which increased the PHB biosynthesis rate, and also in a decrease in the level of NADH, which reflected the alleviation of the inhibitory effects on the cells caused by nitrogen depletion. Electronic Supplementary Material  Supplementary material is available for this article at and is accessible for authorized users.     

Production of Bacillus thuringiensis spores in total cell retention culture and two-stage continuous culture using an internal ceramic filter system

The production of Bacillus thuringiensis spores was investigated in a bioreactor incorporating a ceramic membrane filter to improve spore concentration and volumetric productivity. Two cultivation methods were used in this study: a total cell retention culture (TCRC), and a two-stage continuous culture with partial cell bleeding. In the TCRC, fed by 50 g/L of glucose, a spore concentration of 1.6 x 10(10) CFU/mL was obtained with a spore percentage of greater than 95% and a maximum cell mass of 82.2 g/L. The volumetric productivity was four times higher than that obtained from batch cultivation. In the two-stage continuous culture with partial cell bleeding spore concentration was strongly dependent on the bleed ratio. The spore concentration of 1.8 x 10(9) CFU/mL and the spore percentage of 70% were obtained at the second stage when a bleed ratio of 0.33 and a dilution rate of 0.23 h(-1) were used. (c) 1993 John Wiley & Sons, Inc.     

补料培养提高真养产碱杆菌产3-羟基丁酸和3-羟基戊酸共聚物生产强度的研究

对Alcaligenes eutrophus进行高密度培养,研究表明在发酵过程中进行有效控制,可以较大幅度地提高3－羟基丁酸和3－羟基戊酸共聚物[P(3HB-co-3HV)]的生产强度。实验中选择使用限氮的方法积累P(3HB-co-3HV),分别采用丙酸和戊酸为3HV前体,对摇瓶种子生长状态,停氮时机对菌体生产P(3HB-co-3HV)的影响以及补酸（3HV前体）策略进行了研究,在6．6L罐中,以葡萄糖为碳源,以丙酸为3HV前体培养50h,细胞干重,PHA产量,PHA含量分别达到149．9g/L,149.9g/L,83.3%(其中3HV组分占PHA的12．4mol%),生产强度达到2．50（g.h^-1.L^-1);以戊酸为3HV前体培养45h,细胞干重,PHA产量,PHA含量分别达到160．2g/L,119.0g/L,74.2%（其中3HV组分占PHA的17．7mol%)生产强度达到2．64（g.h^-1.L^-1)。     

Production of poly(3-hydroxybutyrate) by fed-batch culture of filamentation-suppressed recombinant Escherichia coli.

Recombinant Escherichia coli XL1-Blue harboring a high-copy-number plasmid containing the Alcaligenes eutrophus polyhydroxyalkanoate synthesis genes could efficiently synthesize poly(3-hydroxybutyrate) (PHB) in a complex medium containing yeast extract and tryptone but not in a defined medium. One of the reasons for the reduced PHB production in a defined medium was thought to be severe filamentation of cells in this medium. By overexpressing an essential cell division protein, FtsZ, in recombinant E. coli producing PHB, filamentation could be suppressed and PHB could be efficiently produced in a defined medium. A high PHB concentration of 149 g/liter, with high productivity of 3.4 g of PHB/liter/h, could be obtained by the pH-stat fed-batch culture of the filamentation-suppressed recombinant E. coli in a defined medium. It was also found that insufficient oxygen supply at a dissolved oxygen concentration (DOC) of 1 to 3% of air saturation during active PHB synthesis phase did not negatively affect PHB production. By growing cells to the concentration of 110 g/liter and then controlling the DOC in the range of 1 to 3% of air saturation, a PHB concentration of 157 g/liter and PHB productivity of 3.2 g of PHB/liter/h were obtained. For the scale-up studies, fed-batch culture was carried out in a 50-liter stirred tank fermentor, in which the DOC decreased to zero when cell concentration reached 50 g/liter. However, a relatively high PHB concentration of 101 g/liter and PHB productivity of 2.8 g of PHB/liter/h could still be obtained, which demonstrated the possibility of industrial production of PHB in a defined medium by employing the filamentation-suppressed recombinant E. coli.     

Kinetics and effect of nitrogen source feeding on production of poly-β-hydroxybutyric acid by fed-batch culture

Summary A kinetic study of the production of poly--hydroxybutyric acid (PHB) by a fed-batch culture of Protomonas extorquens showed that a nitrogen source was necessary even in the PHB production phase. The effect of ammonia feeding on PHB production was consequently investigated. The nitrogen source (ammonia water) was supplied at a low constant feeding rate after the growth phase in which cell mass concentration reached 60 g/l. Feeding with a small quantity of ammonia resulted in a more rapid increase in intracellular PHB content than was the case without ammonia feeding. Excessive feeding of ammonia, however, caused not only degradation of accumulated PHB but also reduction of microbial PHB synthetic activity.     

The growth and nutrient utilization of the insect cell line Spodoptera frugiperda Sf9 in batch and continuous culture

The growth of the Spodoptera frugiperda cell line Sf9 was studied in batch and continuous culture. The results of batch cultivations showed that glucose was the preferred energy and carbon source limiting the cell density in both TNM-FH and IPL-41 media. Continuous culture using IPL-41-based feeding medium with different glucose (2.5, 5 and 10 g l⁻¹) and yeast extract concentrations (4, 8 and 16 g l⁻¹) showed that in serum-supplemented medium the maximum cell density was limited by glucose and yeast extract concentration. The transition to glucose limitation caused a decrease in growth rate and viability. A high cell density culture (18 × 10⁶ ml⁻¹) was obtained using a glucose concentration of 10 g l⁻¹ and a yeast extract concentration of 8 g l⁻¹ in the feeding medium. A yeast extract concentration of 16 g l⁻¹ inhibited growth. Unlike mammalian cell cultures, lactate, alanine and ammonia were not involved in growth inhibition. Lactate did not accumulate under aerobic conditions. Ammonia accumulation, if observed, was insignificant. The level of alanine synthesized and excreted into the culture medium never reached an inhibitory level. During glucose limitation alanine did not accumulate and ammonia was released. However, even in the presence of glucose significant amounts of Asp, Glu, Gln, Asn, Ser, Arg and Met were utilized for energy production. The amino groups of these amino acids were transferred to pyruvate or used for nucleic acid synthesis and excreted in the form of alanine into the culture medium. The consumption of His, Lys, Thr, Gly, Val, Leu, Phe, Tyr, Trp and Ile by growing Sf-9 cells was almost equal to their concentration in the biomass.     

利用糖蜜发酵生产生物降解塑料PHB的研究

由Ａｌｃａｌｉｇｅｎｅｓｌａｔｕｓ经单菌落分离,从中筛选到一株高效利用糖蜜产聚羟基丁酸（ＰＨＢ）的优良菌株１０１８。在６Ｌ台式发酵罐（Ｎ.Ｂ.Ｓ）中,进行了该菌利用甜菜糖蜜和甘蔗糖蜜积累ＰＨＢ的分批补料培养的研究。结果表明,培养５４ｈ左右发酵液中细胞干重达７０～８５ｇ／Ｌ,ＰＨＢ含量占细胞干重的６０％～７０％,生产强度１．０ｇＰＨＢ／Ｌ／ｈ以上;发酵液经非有机溶剂提取制得ＰＨＢ产品,纯度９５％,提取收率８０％以上。     

氮源流加对Alcaligenes eutrophus 积累聚β-羟基丁酸的影响

以真养产碱杆菌（Ａｌｃａｌｉｇｅｎｅｓ ｅｕｔｒｏｐｈｕｓ）为聚β－羟基丁酸９ＰＨＢ）的生产菌株,在分析了ＰＨＢ发酵过程参数变化的基础上,进一步探讨了ＰＨＢ合成期不同的硫酸铵流加速度对ＰＨＢ合成的影响。研究结果表明,在ＰＨＢ合成阶段,培养基中氮源的完全缺乏。导致细胞合成ＰＨＢ能力的下隆;在ＰＨＢ合成期,不同的氮源流加速率对ＰＨＢ合成过程存在着显著的影响,当流加速率较小时,尽管最终胞内ＰＨＢ含量很高     

基于pH的反馈补料方法在谷氨酸发酵中的应用

为简化谷氨酸发酵补料工艺,提出了一种新型的基于pH的补料方式。考察谷氨酸发酵过程中氨消耗量 (x) 和糖消耗量 (y) 发现,两者之间存在较好的线性关系 (y=7.4744x,R2=0.9989),以此为pH反馈补料工艺中补料液中葡萄糖与氨的混合比例,能较好地将谷氨酸发酵过程中葡萄糖浓度稳定在12~21 g/L。比较恒定葡萄糖浓度补料工艺与pH反馈补料工艺发现,采用pH反馈补料工艺进行发酵,葡萄糖转化率、谷氨酸产酸速率分别提高了9.06％和17.5％左右,同时发酵周期缩短2 h以上。