真养产碱菌利用甜菜糖蜜发酵产聚β－羟基丁酸的研究

探讨了以廉价原料甜菜糖蜜培养真养产碱菌（Ａｌｃａｌｉｇｅｎｅｓｅｕｔｒｏｐｈｕｓ）Ｈ１６生产聚β－羟基丁酸（ＰＨＢ）的可行性。对培养基优化试验表明,菌体产量可达２０ｇ／Ｌ,ＰＨＢ产量达９．８ｇ／Ｌ,糖转化率为２７．５％。用２升自控发酵罐进行验证,在良好的供氧条件和特定的ｐＨ值自控条件下发酵周期从４８小时缩短到４０－４２小时,菌体和ＰＨＢ量都有提高。菌体最高产量２６ｇ／Ｌ,ＰＨＢ最高产量１３ｇ／Ｌ,糖转化率为２０．４％。ＰＨＢ占细胞的含量,摇瓶和罐上结果都在５０％左右。     

高产稳产聚羟基烷酸的重组大肠杆菌的构建

重组大肠杆菌Ｅｓｃｈｅｒｉｃｈｉａ ｃｏｌｉＨＭＳ１７４（ｐＴＺ１８ＵＰＨＢ） 含有携带聚羟基烷酸（ＰＨＡ） 合成基因（ ｐｈａＣＡＢ）＊＊ 的质粒ｐＴＺ１８ＵＰＨＢ,是很有潜力的ＰＨＡ 生产菌,但存在着质粒不稳定和不能合成３羟基丁酸（３ＨＢ） 与３羟基戊酸（３ＨＶ） 共聚物［Ｐ（３ＨＢｃｏ３ＨＶ）］ 的缺陷。将ＲＫ２ 质粒上的ｐａｒ ＤＥ 基因引入ｐＴＺ１８ＵＰＨＢ 构成质粒ｐＪＭＣ２ ,该质粒可以在宿主Ｅ．ＣｏｌｉＨＭＳ１７４ 中稳定遗传。将培养基中的磷酸盐浓度降至１８ ｍ ｍｏｌ／Ｌ,发现Ｅ．Ｃｏｌｉ ＨＭＳ１７４（ｐＪＭＣ２） 能够以丙酸为前体合成Ｐ（３ＨＢｃｏ３ＨＶ） ,其中３ＨＶ 在共聚物中的含量为５ ％ ～８ ％ 。在５Ｌ自动发酵罐中分批补料培养Ｅ．Ｃｏｌｉ ＨＭＳ１７４（ｐＪＭＣ２） ,培养基初始磷酸盐浓度为１５ ｍ ｍｏｌ／Ｌ,３０ ｈ 后每升培养液中干菌体可达４２－５ ｇ,Ｐ（３ＨＢｃｏ３ＨＶ） 占干重的７０ ％ ,其中３ＨＶ 在共聚物中的含量为４－９ ％ 。     

真养产碱杆菌突变株65－7产聚－β－羟基丁酸的研究

本文对真养产碱杆菌突变株６５－７在台式２Ｌ发酵罐中利用葡萄糖积累ＰＨＢ进行了碳源和氮源补料分批培养的研究。结果表明７２ｈ发酵液中细胞干重达５０ｇ／Ｌ,ＰＨＢ占细胞干重的７７％,糖对ＰＨＢ的转化率为２５％。制得的ＰＨＢ产品纯度与Ｓｉｇｍａ公司的相当,熔点１７４℃。     

真养产碱杆菌突变株65—7产聚—β—羟基丁酸的研究

本文对真养产碱杆菌突变株６５－７在台式２Ｌ发酵罐中利用葡萄糖积累ＰＨＢ进行了碳源和氮源补料分批培养的研究。结果表明７２ｈ发酵液中细胞干重达５０ｇ／Ｌ,ＰＨＢ占细胞干重的７７％,糖对ＰＨＢ的转化率为２５％。制得的ＰＨＢ产品纯度与Ｓｉｇｍａ公司的相当,熔点１７４℃     

真养产碱杆菌二级连续培养系统中稀释率对聚β—羟基丁酸合成的影响

在研究真养产碱杆菌ＷＳＨ３一级连续培养动力学的基础上,采用二级连续培养系统对不同稀释率下聚β羟基丁酸的生产进行了研究。结果表明：在一级连续培养系统中,当稀释率为０２１ｈ－１时,细胞干重最大值达２７１ｇ／Ｌ;二级培养系统中,稀释率为０１４ｈ－１时细胞干重最大值为４７６ｇ／Ｌ;在稀释率为０１２ｈ－１时,ＰＨＢ的生产强度达到最大值为２５０ｇ／（Ｌ·ｈ）,但胞内ＰＨＢ含量仅为４７６％;在稀释率为００７５ｈ－１时,产物对基质的转化率达到最大值为０３８ｇ／ｇ,此时ＰＨＢ的生产强度达２１４ｇ／（Ｌ·ｈ）和胞内ＰＨＢ含量±７２１％;随着细胞比生长速率的增长,细胞中ＰＨＢ含量和单位菌体合成ＰＨＢ的量不断下降。     

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）。     

真养产碱杆菌突变株65－7产羟基丁酸与羟基戊酸共聚物的研究

研究了真养产碱杆菌突变株６５－７,以葡萄糖为主原料,添加丙酸或戊酸,采用二步发酵积累共聚物聚β－羟基丁酸－β－羟基戊酸（ＰＨＢＶ）。摇瓶总发酵时间为５０ｈ,细胞干重达７－１１ｇ／Ｌ,共聚物含量占细胞干重的７０％以上,其中β－羟基戊酸（３ＨＶ）含量占ＰＨＢＶ的１０－７２％,主要取决于不同碳源的组成,丙酸和戊酸对ＨＶ的转化率分别为０．４１－０．６３ｇＨＶ／ｇ丙酸和０．４０－０．７４ｇＨＶ／ｇ戊酸,制得的ＰＨＢＶ产品纯度９９％以上,分子量６．９×１０^{５相似文献}   

芽孢杆菌M_(50)产生β甘露聚糖酶的条件研究

从土壤中分离到９ 株产生β甘露聚糖酶的芽孢杆菌（ Ｂａｃｉｌｌｕｓ ｓｐ ．） 。Ｂａｃｉｌｌｕｓｓｐ ． Ｍ５０２５０ｍ Ｌ三角瓶摇瓶培养试验,以４ ％ 的魔芋粉为碳源,１－０ ％ （ ＮＨ４）２ＳＯ４ 为氮源,０－３５ ％Ｎａ２ＣＯ３ ,３０ ～３４ ℃培养６０ｈ 产酶达到高峰。酶活力为１８０ ～２００ｕ／ｍ Ｌ。１００Ｌ 罐发酵,在３０ ～３２ ℃,１∶０ ．７５ｖｖｍ 通气量,２００ｒ／ｍｉｎ 条件下,发酵液酶活力高达３３０ｕ／ｍ Ｌ。酶的最适反应温度和ｐＨ 分别为５０ ℃和６－０ ,低于５０ ℃,ｐＨ５ ．０ ～７ ．０ 酶稳定。Ｆｅ^３＋ 、Ａｌ^３＋ 、ＥＤＴＡ、Ｈｇ^２＋ 对酶有抑制作用,而Ｂａ^２＋ 、Ｍｎ^２＋ 对酶有激活作用。发酵粗酶液对苎麻精干麻精练,显示对精干麻的半纤维素残胶具有降解作用。     

聚β—羟基丁酸产生菌Z5—GⅡ发酵条件的研究

对聚β－羟基丁酸（ＰＨＢ）产生菌Ｚ５－ＧⅡ的发酵培养基及发酵条件进行优化研究;结果表明：该菌株在蔗糖１％,酵母粉０．３％,酵母浸汁０．３％,Ｋ２ＨＰＯ４０．２％;ｐＨ７．２￣７．４的优化发酵培养基中,接种量８％,种２８ｈ,发酵培养３６ｈ,细胞干重为６．８７ｇ／Ｌ,ＰＨＢ产率可达的细胞干重的６１．８６％。该菌株还可利用葡萄糖生产废液为碳源生产ＰＨＢ,具有实现工业化生产的潜力。     

真养产碱杆菌突变株65—7产羟基丁酸与羟基戊酸共聚物的研究

研究了真养产碱杆菌突变株６５－７,以葡萄糖为主原料,添加丙酸或戊酸,采用二步发酵积累共聚物聚β－羟基丁酸－β－羟基戊酸（ＰＨＢＶ）。摇瓶总发酵时间为５０ｈ,细胞干重达７－１１ｇ／Ｌ,共聚物含量占细胞干重的７０％以上,其中β－羟基戊酸（３ＨＶ０含量占ＰＨＢＶ的１０－７２％,主要取决于不同碳源的组成,丙酸和戊酸对ＨＶ的转化率分别为０．４１－０．６３ｇＨＶ／ｇ丙酸和０．４０－０．７４ｇＨＶ／ｇ戊酸,制得     

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.     

A tunable switch to regulate the synthesis of low and high molecular weight microbial polyesters

The addition of poly(ethylene glycol) (Mn = 200 g/mol) (PEG-200) to the fermentation media of Alcaligenes eutrophus and Alcaligenes latus at various stages of growth resulted in the synthesis of poly(3-hydroxybutyrate) (PHB) with bimodal molecular weight distributions. The presence of 2% w/v-PEG-200 did not have deleterious effects on PHB volumetric yields and cell productivity. In general, the Mn values of the high (H) and low (L) fractions showed little variability as a function of the time at which PEG-200 was added to the cultures. By this approach, the H:L ratios (w/w) of the PHB synthesized by A. eutrophus and A. latus were varied from 9:91 to 76:24 and from 16:84 to 88:12, respectively. It is believed that the H fractions were formed prior to the addition of PEG-200 to the cultures. Also, once PEG-200 was made available to the cells, PEG-200 acted as a switch so that the reduced molecular weight fraction was formed. In addition, a necessary requirement for the above is that the frequency of transesterification reactions during polymer synthesis was small. The efficiency that PEG-200 reduced the molecular weight of the PHBs formed by both bacteria appears similar. Indirect evidence suggests that the PHB L fractions formed by A. latus subsequent to PEG-200 addition consist primarily of chains that have PEG terminal groups. This terminal chain structure was not observed for PHB formed by A. eutrophus.     

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(3-hydroxybutyrate) by fed-batch culture of recombinant Escherichia coli with a highly concentrated whey solution

Fermentation strategies for the production of poly(3-hydroxybutyrate) (PHB) from whey by recombinant Escherichia coli strain CGSC 4401 harboring the Alcaligenes latus polyhydroxyalkanoate (PHA) biosynthesis genes were developed. The pH-stat fed-batch cultures of E. coli CGSC 4401 harboring pJC4, a stable plasmid containing the A. latus PHA biosynthesis genes, were carried out with a concentrated whey solution containing 280 g of lactose equivalent per liter. Final cell and PHB concentrations of 119.5 and 96.2 g/liter, respectively, were obtained in 37.5 h, which resulted in PHB productivity of 2.57 g/liter/h.     

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.     

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.     

Removal of endotoxin during purification of poly(3-hydroxybutyrate) from gram-negative bacteria.

Poly(3-hydroxybutyrate) (PHB) was produced by cultivating several gram-negative bacteria, including Ralstonia eutropha, Alcaligenes latus, and recombinant Escherichia coli. PHB was recovered from these bacteria by two different methods, and the endotoxin levels were determined. When PHB was recovered by the chloroform extraction method, the endotoxin level was less than 10 endotoxin units (EU) per g of PHB irrespective of the bacterial strains employed and the PHB content in the cell. The NaOH digestion method, which was particularly effective for the recovery of PHB from recombinant E. coli, was also examined for endotoxin removal. The endotoxin level present in PHB recovered by 0.2 N NaOH digestion for 1 h at 30 degrees C was higher than 10(4) EU/g of PHB. Increasing the digestion time or NaOH concentration reduced the endotoxin level to less than 1 EU/g of PHB. It was concluded that PHB with a low endotoxin level, which can be used for various biomedical applications, could be produced by chloroform extraction. Furthermore, PHB with a much lower endotoxin level could be produced from recombinant E. coli by simple NaOH digestion.     

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.     

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.