Production of poly-β-hydroxybutyrate (PHB) by <Emphasis Type="Italic">Alcaligenes latus</Emphasis> from maple sap

Maple sap, an abundant natural product especially in Canada, is rich in sucrose and thus may represent an ideal renewable feedstock for the production of a wide variety of value-added products. In the present study, maple sap or sucrose was employed as a carbon source to Alcaligenes latus for the production of poly-β-hydroxybutyrate (PHB). In shake flasks, the biomass obtained from both the sap and sucrose were 4.4 ± 0.5 and 2.9 ± 0.3 g/L, and the PHB contents were 77.6 ± 1.5 and 74.1 ± 2.0%, respectively. Subsequent batch fermentation (10 L sap) resulted in the formation of 4.2 ± 0.3 g/L biomass and a PHB content of 77.0 ± 2.6%. The number average molecular weights of the PHB produced by A. latus from maple sap and pure sucrose media were 300 ± 66 × 10³ and 313 ± 104 × 10³ g/mol, respectively. Near-infrared, ¹H magnetic resonance imaging (MRI), and ¹³C-MRI spectra of the microbially produced PHB completely matched those obtained with a reference material of poly[(R)-3-hydroxybutyric acid]. The polymer was found to be optically active with [α]²⁵ _D equaled to −7.87 in chloroform. The melting point (177.0°C) and enthalpy of fusion (77.2 J/g) of the polymer were also in line with those reported, i.e., 177°C and 81 J/g, respectively.     

Determination of poly-β-hydroxybutyrate (PHB) production by some <Emphasis Type="Italic">Bacillus</Emphasis> spp.

In this study, 29 strains of the genus Bacillus were isolated from different soil samples which were taken from grasslands of Ankara, Turkey and were identified as B. brevis, B. sphaericus, B. cereus, B. megaterium, B. circulans, B. subtilis, B. licheniformis and B. coagulans. Two strains, B. sphaericus ATCC 14577 and B. subtilis ATCC 6633 were also included in this study. Poly-β-hydroxybutyrate (PHB) production by these strains was determined by the spectrophotometric method, and it was found that PHB production ranged from 1.06–41.67% (w/v) depending on the dry cell weight. The highest PHB production and productivity percentage was found in B. brevis M6 (41.67% w/v).     

High-cell-density production of poly-β-hydroxybutyrate (PHB) from methanol by Methylobacterium extorquens: production of high-molecular-mass PHB

Methylobacterium extorquens ATCC 55366 was successfully cultivated at very high cell densities in a fed-batch fermentation system using methanol as a sole carbon and energy source and a completely minimal culture medium for the production of poly--hydroxybutyrate (PHB). Cell biomass levels were between 100 g/l and 115 g/l (dry weight) and cells contained between 40% and 46% PHB on a dry-weight basis. PHB with higher molecular mass values than previously reported for methylotrophic bacteria was obtained under certain conditions. Shake-flask and fermentor experiments showed the importance of adjusting the mineral composition of the medium for improved biomass production and higher growth rates. High-cell-density cultures were obtained without the need for oxygen-enriched air; once the oxygen transfer capacity of the fermentor was reached, methanol was thereafter added in proportion to the amount of available dissolved oxygen, thus preventing oxygen limitation. Controlling the methanol concentration at a very low level (less than 0.01 g/l), during the PHB production phase, led not only to prevention of oxygen limitation but also to the production of very high-molecular-mass PHB, in the 900–1800 kDa range. Biomass yields relative to the total methanol consumed were in the range 0.29–0.33 g/g, whereas PHB yields were in the range 0.09–0.12 g/g. During the active period of PHB synthesis, PHB yields relative to the total methanol consumed were between 0.2 g/g and 0.22 g/g. M. extorquens ATCC 55366 appears to be a promising organism for industrial PHB production.     

生物合成材料聚β-羟基丁酸(PHB)的研究进展

聚β-羟基丁酸(PHB)是原核微生物在碳、氮营养失衡的情况下,作为碳源和能源贮存在生物体内的一类热塑性聚酯.它作为微生物合成的可降解材料,除了具有与化学合成高分子相似的性质外,还具有一般化学合成高分子没有的性质,如光学活性好、透氧性低、抗紫外线辐射、生物可降解性、生物组织相容性、压电性和抗凝血性等,具有广阔的应用前景,越来越受到人们的关注.国内外的许多公司和科研机构纷纷开展可降解塑料的研发工作.着重介绍了PHB的理化性质、检测方法、生物合成、降解以及基因改良菌种方面的研究进展,同时对其应用、目前存在的问题以及可能的解决方案进行了讨论.     

钩端螺旋体聚Beta羟基丁酸(PHB)生物合成途径分析

【目的】致病型问号钩端螺旋体(问号钩体, Leptospira interrogans)和腐生型双曲钩体(L. biflexa)能够大量合成菌体内贮藏物, 这可能是钩体在营养贫瘠环境中长时间存活的主要原因之一。本研究对钩体聚Beta羟基丁酸(PHB)贮藏物进行定性定量测定, 通过基因组分析补充定义PHB合成主要功能基因, 并采用分子生物学方法初步证明PHB合成途径的完整性, 为进一步研究PHB合成与钩体抗逆能力的关系奠定基础。【方法】采用脂类特异性尼罗红染色法和浓硫酸氧化-紫外分光光度计测定法, 对问号钩体和双曲钩体的PHB贮藏物进行定性定量测定; 采用生物信息学方法(BLAST和InterProscan/InterPro2Go), 通过同源性分析和功能结构域搜索寻找钩体基因组中的PHB合成相关基因; 最后采用克隆测序和定量RT-PCR技术检测相关基因表达情况, 初步验证生物信息学预测结果。【结果】尼罗红染色和氧化后比色定量实验证明钩体合成细菌常见贮藏物PHB, 问号钩体合成量为菌体干重的42%?45%, 双曲钩体合成量为64%?68%。尽管已公布的多个钩体基因组中均没有定义完整的PHB合成途径, 但本研究通过综合生物信息学分析, 在问号钩体和双曲钩体中鉴定了PHB合成途径的主要功能基因(phbC)。克隆测序和定量RT-PCR证实钩体转录表达大部分PHB合成相关基因(phbA/B/C), 说明钩体内该生物途径基本完整, 且部分高水平表达基因可能是钩体主要的PHB合成相关基因。【结论】问号钩体和双曲钩体均可合成PHB贮藏物, 且具有基本完整的PHB合成生物途径。     

生物降解塑料PHB的研究概况

论述了生物降解塑料PHB的生产状况、PHB的性能、生物降解性以及PHB在工农业生产特别是在医学领域中的应用。说明PHB在替代化学合成塑料、缓解环境危机和提供新型功能性生物医用材料等方面具有重要意义。     

活性污泥积累PHB (Poly-β-hydroxybutyrate)及其菌群多样性分析

【目的】通过调整活性污泥在驯化过程中碳、氮、磷比例以及供氧模式, 以提高其积累PHB的能力。应用PCR-DGGE定期对驯化期间菌群动态进行分析。【方法】以乙酸钠为碳源, 在SBR (Sequencing batch reactor) 内以厌氧/好氧 (A/O) 交替的驯化模式, 逐级提高碳源浓度, 限制氮源浓度, 人为创造营养不均衡条件来逐步提高活性污泥积累PHB的能力。【结果】当碳浓度逐步提高, COD升高至1 200 mg/L, COD/N/P为1 200/9.6/30时, 活性污泥中PHB积累量达到最大, 占细胞干重的64.2%。【结论】驯化过程中逐步提高COD负荷, 并增加COD/N的值有利于PHB积累, 利用苏丹黑和Albert法染色显示菌群内产PHB菌占很大比例, 透射电镜显示菌体胞内含有大量白色PHB颗粒。经DGGE菌群动态分析, 发现驯化过程中菌群种类出现较为明显的演替, 而在一个反应周期(6 h)内菌种数量也有一定改变。驯化获得的高产PHB的菌群中含Acinetobacter、Bacillus、Bacteroidetes、Chryseobacteria 及proteobacteria等5个属的微生物, 菌群多样性较为丰富。     

High production of poly-β-hydroxybutyrate (PHB) by an Azotobacter vinelandii mutant altered in PHB regulation using a fed-batch fermentation process

A mixed fermentation strategy based on exponentially fed-batch cultures (EFBC) and nutrient pulses with sucrose and yeast extract was developed to achieve a high concentration of PHB by Azotobacter vinelandii OPNA, which carries a mutation on the regulatory systems PTSNtr and RsmA-RsmZ/Y, that negatively regulate the synthesis of PHB. Culture of the OPNA strain in shake flaks containing PY-sucrose medium significantly improved growth and PHB production with respect to the results obtained from the cultures with the parental strain (OP). When the OPNA strain was cultured in a batch fermentation keeping constant the DOT at 4%, the maximal growth rate (0.16 h⁻¹) and PHB yield (0.30 g_PHB g_Suc⁻¹) were reached. Later, in EFBC, the OPNA strain increased three fold the biomass and 2.2 fold the PHB concentration in relation to the values obtained from the batch cultures. Finally, using a strategy of exponential feeding coupled with nutrient pulses (with sucrose and yeast extract) the production of PHB increased 7-fold to reach a maximal PHB concentration of 27.3 ± 3.2 g L⁻¹ at 60 h of fermentation. Overall, the use of the mutant of A. vinelandii OPNA, impaired in the PHB regulatory systems, in combination with a mixed fermentation strategy could be a feasible strategy to optimize the PHB production at industrial level.     

一种新型生物塑料(PHB)的研究进展和开发前景

一种新型生物塑料（ＰＨＢ）的研究进展和开发前景郭秀君于昕（山东大学微生物学系塑料作为高分子聚合物成为现代社会不可缺少的重要材料。塑料制品在国民经济各部门发挥着重要作用。但这类化学合成的高分子不能被生物降解。所以一旦废弃,必然给环境造成极大危害,使环境恶化,生态平衡破坏,因此,为保护环境,防止污染,从长远考虑,发展生物可降解塑料十分必要。目前世界上许多国家如美国、西欧各国及日本、中国等都在积极...     

发酵生产PHB动力学模型的研究

用２Ｌ机械搅拌式发酵罐培养真养产碱杆菌９２０,得到菌体生长、基质消耗和产物ＰＨＢ合成的动力学曲线。通过分析建立了发酵生产ＰＨＢ的动力学模型,根据实验数据确定模型参数,将模型预测值与实验值进行比较,结果表明模型预测值与实验值较吻合。     

Effects of dietary poly-β-hydroxybutyrate (PHB) on microbiota composition and the mTOR signaling pathway in the intestines of <Emphasis Type="Italic">litopenaeus vannamei</Emphasis>

Poly-β-hydroxybutyrate (PHB) is a natural polymer of the short chain fatty acid β-hydroxybutyrate, which acts as a microbial control agent. The mammalian target of the rapamycin (mTOR) signaling pathway plays a crucial role in intestine inflammation and epithelial morphogenesis. In this study, we examined the composition of intestine microbiota, and mTOR signaling-related gene expression in Pacific white shrimp Litopenaeus vannamei fed diets containing different levels of PHB: 0% (Control), 1% (PHB1), 3% (PHB3), and 5% (PHB5) (w/w) for 35 days. High-throughput sequencing analysis revealed that dietary PHB altered the composition and diversity of intestine microbiota, and that the microbiota diversity decreased with the increasing doses of PHB. Specifically, dietary PHB increased the relative abundance of Proteobacteria and Tenericutes in the PHB1 and PHB5 groups, respectively, and increased that of Gammaproteobacteria in the three PHB groups. Alternatively, PHB decreased Alphaproteobacteria in the PHB3 and PHB5 groups. At the genus level, dietary PHB increased the abundance of beneficial bacteria, such as Bacillus, Lactobacillus, Lactococcus, Clostridium, and Bdellovibrio. The relative mRNA expression levels of the mTOR signaling-related genes TOR, 4E-BP, eIF4E1α, and eIF4E2 all increased in the three PHB treatment groups. These results revealed that dietary PHB supplementation had a beneficial effect on intestine health of L. vannamei by modulating the composition of intestine microbiota and activating mTOR signaling.     

Excretion of pyruvate by mutants of Alcaligenes eutrophus,which are impaired in the accumulation of poly(β-hydroxybutyric acid) (PHB), under conditions permitting synthesis of PHB

Summary Mutants of Alcaligenes eutrophus, which are defective in the intracellular accumulation of poly(-hydroxybutyric acid), PHB, were cultivated in the presence of excess carbon source after growth had ceased due to depletion of ammonium, sulphate, phosphate, potassium, magnesium, or iron. Under these conditions all mutants excreted large amounts of pyruvate into the medium. Excretion of pyruvate occurred with lactate, gluconate or fructose as carbon sources; the highest rate of pyruvate excretion (8 mmol/1 per hour) was obtained with lactate. The rate of pyruvate excretion by strain N9A-PHB^-02-HB^-1 on gluconate amounted to 2.5–6.3 mmol/g protein per hour depending on the depleted nutrient. The ratios of the molar rates for the utilization of the substrates versus those for the excretion of pyruvate were 1.9, 1.0 or 0.7, respectively. Wild-type strains did not excrete even traces of pyruvate, but accumulated PHB. Depending on the limiting nutrient, strain N9A accumulated PHB at a rate of 0.21–0.49 g/g protein per hour. On a molar basis (-hydroxybutyrate monomers versus pyruvate) the ratios for the rates for accumulation of PHB in the wild-type and for excretion of pyruvate in PHB-negative mutants were 0.9–1.4. The fermentation enzymes alcohol dehydrogenase and lactate dehydrogenase were not synthesized in cells starved of a nutrient; they were only detectable in cells cultivated under conditions of restricted oxygen supply. The latter conditions caused accumulation of PHB in the wild-type and excretion of pyruvate by the PHB-negative mutant.     

Proteomic analysis of Bacillus thuringiensis ΔphaC mutant BMB171/PHB(-1) reveals that the PHB synthetic pathway warrants normal carbon metabolism

A phaC knockout mutant from Bacillus thuringiensis (Bt) strain BMB171, named BMB171/PHB(-1), was constructed. A physiological and metabolic investigation and a proteomic analysis were conducted for both ΔphaC mutant and its parent strain. Grown in peptone medium with 5 gram glucose per liter as sole carbon source, BMB171/PHB(-1) produced various organic acids. Here the excreted pyruvate, citrate, lactate, acetate and glutamate were quantitatively analyzed. Deletion of phaC gene from the BMB171 strain resulted in 1) growth delay; 2) higher consumption of dioxigen but lower cell yield; 3) stagnation of pH movement; 4) overproduction of organic acids; 5) rapid descent of cell density in the stationary phase; and 6) a sporulation-deficient phenotype. Our proteomic study with qPCR reconfirmation reveals that the absence of PhaC led to a metabolic turmoil which showed repressed glycolysis, and over-expressed TCA cycle, various futile pathways and amino acid synthesis during vegetative growth. It is thus thought that B. thuringiensis BMB171 effectively regulated its carbon metabolism upon the presence of the functional PHB synthetic pathway. The presence of this pathway warrants a PHB-producing bacterium better surviving under different environmental conditions.     

基于Petri网T不变量的PHB新陈代谢建模分析(英文)

从拓扑结构的角度分析生化反应网络是生物信息学研究中的一个热点问题。通过将两种传统的途径分析方法(基元模式和极端途径)与Petri网的T不变量分析进行了比较,结果表明:它们本质上是一致的,但是采用Petri网的T不变量分析更便捷。然后,利用Petri网技术构建了PHB代谢模型。对该模型作了结构分析,将计算得到的23个T不变量进行了分组:I组表示简单的可逆反应,II组表示循环的反应,III组可用于调控ATP/ADP比率,IV组是与PHB生产直接相关的反应,可用于代谢工程以提高PHB的产率。最后讨论了Petri网的T不变量分析在这个领域中的应用。     

Production and characterization of poly-β-hydroxybutyrate (PHB) polymer from Aulosira fertilissima

Biopolymers such as polyhydroxyalkanoates (PHAs) are a class of secondary metabolites with promising importance in the field of environmental, agricultural, and biomedical sciences. To date, high-cost commercial production of PHAs is being carried out with heterotrophic bacterial species. In this study, a photoautotrophic N₂-fixing cyanobacterium, Aulosira fertilissima, has been identified as a potential source for the production of poly-β-hydroxybutyrate (PHB). An accumulation up to 66% dry cell weight (dcw) was recorded when the cyanobacterium was cultured in acetate (0.3%) + citrate (0.3%)-supplemented medium against 6% control. Aulosira culture supplemented with 0.5% citrate under P deficiency followed by 5?days of dark incubation also depicted a PHB accumulation of 51% (dcw). PHB content of A. fertilissima reached up to 77% (dcw) under P deficiency with 0.5% acetate supplementation. Optimization of process parameters by response surface methodology resulted into polymer accumulation up to 85% (dcw) at 0.26% citrate, 0.28% acetate, and 5.58?mg?L^?1 K₂HPO₄ for an incubation period of 5?days. In the A. fertilissima cultures pre-grown in fructose (1.0%)-supplemented BG 11 medium, when subjected to the optimized condition, the PHB pool boosted up to 1.59?g?L^?1, a value ～50-fold higher than the control. A. fertilissima is the first cyanobacterium where PHB accumulation reached up to 85% (dcw) by manipulating the nutrient status of the culture medium. The polymer extracted from A. fertilissima exhibited comparable material properties with the commercial polymer. As compared with heterotrophic bacteria, carbon requirement in A. fertilissima for PHB production is lower by one order magnitude; thus, low-cost PHB production can be envisaged.     

聚-β-羟基丁酸(PHB)在细菌建立感受态中的作用

介绍了PHB作为细胞膜上的物质运输通道成分在细菌感受态中的结构、分布、分析方法以及其可能的作用 ,阐明了PHB的合成与细菌感受态建立的关系和PHB在感受态细胞摄取外源DNA时的作用方式 ,同时也对控制生物体合成PHB的相关基因和酶作了简要介绍 ,并探讨了研究PHB的实际意义。     

生物技术生产生物可降解塑料PHB的前景

生物技术生产生物可降解塑料ＰＨＢ的前景谢安勇,宋艳茹（中国科学院植物研究所,北京１０００４４）ＰＲＯＳＰＥＣＴＯＦＰＲＯＣＵＣＩＮＧＰＨＢ,ＡＢＩＯＤＥＧＲＡＤＡＢＬＥＰＬＡＳＴＩＣＢＹＵＳＩＮＧＢＩＯＴＢＣＨＮＯＬＯＧＹ￥ＸｉｅＡｎ－ｙｏｎｇ;Ｓｏ...     

生物技术生产生物可降解塑料PHB的研究进展

本文综述了ＰＨＢ的合成和降解途径及其关键酶的基因克隆,比较了细菌发酵法及遗传吃力菌生产ＰＨＢ的优缺点并讨论了发酵法生产及ＰＨＡｓ的前景。随着近年来植物基因工程的发展,用植物大规模生产生物可降解塑料已成为可能,但然后存在许多障碍,本文进一步探讨了转基因植物生产ＰＨＢ的限制因素和克服方法。     

High production of Poly-β-hydroxybutyrate (PHB) from Methylobacterium organophilum under potassium limitation

Summary Mass production of Poly(-hydroxybutyrate) (PHB) from Methylobacterium organophilum under potassium-limited condition was carried out using a microcomputeraided automatic fed-batch culture system. The concentration of methanol was kept within the range of 2 – 3 g/l which did not show any inhibitory effect on cell growth. The PHB accumulation was stimulated when potassium concentration in the culture broth fell below 25 mg/l. After 70 hours of cultivation, the concentrations of cell mass and PHB were obtained to be 250 g/l and 130 g/l, respectively, which corresponded to a volumetric productivity of 1.8 – 2.0 g-PHB/1-hr. PHB contents ranged from 52% to 56% of dry cell weight with a yield factor (Y_P/S) of 0.19 g-PHB/g-methanol.     

植物生产新型生物聚合物PHB的研究进展

聚-β-羟基丁酸酯(polyhydroxybutyrate,PHB)是一种可完全生物降解和具有良好生物相容性的高分子材料,可作为传统塑料的替代品,在塑料、化学药品和饲料市场上具有巨大的商业应用价值。近年来,用植物生产新型生物聚合物PHB的技术取得一定进展,可望通过农业生产提供可再生的工业原料,这使得它具有广泛的发展前景。采用转基因方法可以大幅度提高用植物生产PHB的产量,包括控制启动子来驱使转基因表达,减少内源性酶在竞争性代谢途径中的活性,通过插入的基因来增加聚合物的碳含量。这些研究成果增加了我们对碳的可获取性的了解和区分不同的植物细胞器、细胞和器官类型的能力,推动了用植物生产PHB及其他产物的进展。