嗜热子囊菌利用短链有机酸生产角质酶

以嗜热子囊菌(Thermobifida fusca WSH03-11)发酵生产角质酶为模型,研究微生物利用市政污泥厌氧酸化所产短链有机酸为碳源发酵生产高附加值产品的可能。发现：(1)以丁酸、丙酸和乙酸为碳源时,有机酸和氮元素浓度分别为8.0 g/L和1.5 g/L有利于角质酶的生产;而以乳酸为碳源时,最适有机酸和氮源浓度分别为3.0 g/L和1.0 g/L;(2)改变诱导物角质的浓度,以丁酸、丙酸、乙酸和乳酸为碳源,分别比优化前提高了31.0%、13.3%、43.8%和73.2%;(3)在四种有机酸中,T. fusca WSH03-11利用乙酸的速率最快,平均比消耗速率是丙酸的1.3倍,丁酸的2.0倍及乳酸的2.2倍;以丁酸为碳源时的酶活(52.4 U/mL)是乳酸的1.7倍、乙酸的2.5倍和丙酸的3.2倍;角质酶对乳酸的得率(12.70 u/mg)分别是丁酸的1.4倍、丙酸的3.0倍和乙酸的3.8倍;(4)以混合酸为碳源生产角质酶,T. fusca WSH03-11优先利用乙酸,而对丁酸的利用受到抑制。进一步研究发现,混合酸中0.5 g/L的乙酸将导致丁酸的消耗量降低66.7%。这是首次利用混合酸作碳源发酵生产角质酶的研究报道。这一研究结果进一步确证了利用市政污泥厌氧酸化所产有机酸为碳源发酵生产高附加值产品的可行性,为以廉价碳源生产角质酶奠定了良好的基础。     

嗜热子囊菌利用短链有机酸生产角质酶

以嗜热子囊菌(Thermobifida fusca WSH03-11)发酵生产角质酶为模型,研究微生物利用市政污泥厌氧酸化所产短链有机酸为碳源发酵生产高附加值产品的可能。发现：(1)以丁酸、丙酸和乙酸为碳源时,有机酸和氮元素浓度分别为8.0 g/L和1.5 g/L有利于角质酶的生产;而以乳酸为碳源时,最适有机酸和氮源浓度分别为3.0 g/L和1.0 g/L;(2)改变诱导物角质的浓度,以丁酸、丙酸、乙酸和乳酸为碳源,分别比优化前提高了31.0%、13.3%、43.8%和73.2%;(3)在四种有机酸中,T. fusca WSH03-11利用乙酸的速率最快,平均比消耗速率是丙酸的1.3倍,丁酸的2.0倍及乳酸的2.2倍;以丁酸为碳源时的酶活(52.4 U/mL)是乳酸的1.7倍、乙酸的2.5倍和丙酸的3.2倍;角质酶对乳酸的得率(12.70 u/mg)分别是丁酸的1.4倍、丙酸的3.0倍和乙酸的3.8倍;(4)以混合酸为碳源生产角质酶,T. fusca WSH03-11优先利用乙酸,而对丁酸的利用受到抑制。进一步研究发现,混合酸中0.5 g/L的乙酸将导致丁酸的消耗量降低66.7%。这是首次利用混合酸作碳源发酵生产角质酶的研究报道。这一研究结果进一步确证了利用市政污泥厌氧酸化所产有机酸为碳源发酵生产高附加值产品的可行性,为以廉价碳源生产角质酶奠定了良好的基础。     

化工上的应用

利用AIcaligenes eutrophus ATCC17699以4一经丁酸、4一氯丁酸、1,4一丁二醇和7一丁内醋为碳源生产出新的3一经丁酸盐和4一经丁酸盐的共聚多酷.共聚多酷中4一经丁酸盐的量从0一37mol%不等,取     

一株乳酸利用、丁酸产生菌的分离与鉴定及代谢特性的初步研究

利用改进型Hungate技术从猪粪中分离到一株乳酸利用、丁酸产生双重功能菌株LB01。常规生化检测表明菌株LB01为革兰氏阳性、严格厌氧菌,能利用葡萄糖、果糖、麦芽糖和乳酸等碳源,并产生大量的气体;16S rRNA序列比对表明其与GenBank中的Megasphaera hominis与Uncultured rumen bacterium 3c3d-18的同源性最高,同源性高达99%。菌株LB01可以利用乳酸,并将其主要转化为丁酸和丙酸,在有葡萄糖的情况下,菌株LB01尚能够利用乙酸并生成丁酸。与乳杆菌K9共培养时,菌株LB01有效地利用了乳杆菌K9代谢过程中产生的乳酸,减缓了由于乳酸积累而造成的pH值下降,并且将乳酸转化为丁酸和丙酸。这些代谢特征表明菌株LB01是一株具有潜在应用价值的肠道益生菌,它能够利用乳酸和乙酸(补充额外能量),能有效地防止乳酸和乙酸的积累,同时生成包括丁酸在内有益的短链脂肪酸,调控后肠道pH,营造着微酸的环境。     

羟基脂肪酸聚酯制备与热性质研究

不同的碳源条件下,真养产碱杆菌可在胞内积累聚羟丁酸(PHB)或含羟基戊酸单体(HV)比例不等的聚羟丁戊酸共聚物(PHBv)。利用次氯酸钠,氯仿混合液体系提取上述羟基脂肪酸聚酯(PHA),提取率为85％,纯度达97．O％。以差示扫描热分析法对PHB和PHBV材料进行热性质研究,发现材料中的HV组分逐渐增加．材料的熔点Tm,熔化焓Hm逐渐下降。热分解峰值逐渐向低温区偏移。但含HV为6lmol％的PHBV材料有关热性质出现回升现象。     

互营烃降解菌系M82的脂肪酸降解特性

【目的】通过分子生态学手段筛选适合互营烃降解菌Syntrophus sp.生长的非烃碳源。【方法】利用实验室驯化获得的正十六烷烃降解产甲烷菌系M82为接种物,添加不同碳源(正十二烷二元酸、正十四烷二元酸、正十六烷烃、十六烷酸钠、乳酸钠和丙酸钠)传代培养,通过PCR-DGGE和qPCR技术研究不同碳源条件下Syntrophaceae科细菌的丰度与变化趋势;利用T-RFLP方法分析古菌群落结构。【结果】菌系M82可以利用多种脂肪酸生长并产生甲烷,但是细菌群落结构发生了变化,只在添加正十二烷二元酸和正十四烷二元酸的培养液中检测到了代表Syntrophaceae细菌的条带,并且每毫升菌液中Syntrophaceae细菌的log丰度分别达到7.4和7.6,比添加其它几种非烃碳源的实验组丰度高2-3个单位。古菌群落结构主要由乙酸营养型产甲烷古菌(Methanosaeta)和氢营养型产甲烷古菌(Methanoculleus)组成。【结论】Syntrophus sp.细菌可以利用正十二烷二元酸和正十四烷二元酸这两种非烃碳源生长,这为我们定向分离互营烃降解菌和研究起始烃降解机制和代谢机理提供了依据。     

嗜水气单胞菌合成含3-羟基戊酸单体的聚羟基脂肪酸共聚酯的研究

分别利用葡萄糖或葡萄糖酸钠与十一碳酸、月桂酸与十一碳酸为混合碳源进行嗜水气单孢菌 (Aeromonashydrophila)菌株 4AK4的摇瓶培养 ,实现了含有 3 羟基戊酸 (3HV)单体的聚羟基脂肪酸酯的微生物合成。当使用葡萄糖或葡萄糖酸钠与十一碳酸为混合碳源时 ,野生型A .hydrophila 4AK4及含有 3 羟基丁酸辅酶A合成基因phaA和phaB的重组A .hydrophila 4AK4 (pTG01)能够合成-3-羟基丁酸(3HB)与-3HV的共聚物 ,且葡萄糖或葡萄糖酸钠与十一碳酸比例为 1∶1时最利于细胞生长和PHA的积累。当使用月桂酸和十一碳酸为混合碳源时 ,A .hydrophila4AK4能够合成-3HB、3HV与 β-羟基己酸 (3HHx)的共聚物 ,且随着混合碳源中十一碳酸的含量增加 ,A .hydrophila4AK4合成的PHA中-3HV的比例增加 ,而-3HB和-3HHx的比例降低.     

培养条件对海洋假单胞菌脂肪酸的影响

研究了不同温度条件下一株海绵附生假单胞菌(Pseudomonassp.)在不同碳源培养基中的生长情况及脂肪酸变化.结果表明,该海洋菌生长最适温度为30℃,在以淀粉作为外加碳源的培养基中生长最好;实验菌含13种脂肪酸,主要是c16:1(n7)、c15:0、c16:0、c17:0、c18:1(n6)、c18:1(n9)、9,10cp c17:0和其同分异构体.在30℃温度条件下,不饱和脂肪酸的相对含量急剧减少.在有外加碳源(葡萄糖和淀粉)的培养基中生长的细菌,奇数脂肪酸和环丙基脂肪酸含量远比未外加碳源的低.聚类分析结果表明,两种环境因子中,温度比碳源的影响更明显.     

不同碳源生物转化合成L-亮氨酸的代谢计量分析

目的:建立黄色短杆菌利用不同碳源生物合成L-亮氨酸的代谢网络模型,并进行代谢网络计量分析.方法:通过对所构建的L-亮氨酸代谢网络模型进行途径分析,确定以果糖、葡萄糖、蔗糖或木糖为碳源时L-亮氨酸生物合成的基元模型、最大理论产率和不同模型的呼吸熵.结果:通过途径分析得到了L-亮氨酸生物合成的基元模型.以果糖、葡萄糖、蔗糖和木糖为碳源时L-亮氨酸的最大理论产率均为66.7%,其对应的最大呼吸熵分别为18、16、19、18.结论:L-亮氨酸理论得率与碳源种类无关;呼吸熵增加,能够有效提高L-亮氨酸合成代谢流,限制菌体量的过量生成.与其他碳源相比,蔗糖能够避免碳架溢流出现,合成L-亮氨酸能量代谢需求低;而葡萄糖能够较好地满足菌体生长和产酸的需求.     

响应面法优化枯草芽孢杆菌产脂肪酶的合成培养基

对枯草芽孢杆菌(Bacillus subtilis)CICC20034利用合成培养基液体发酵产脂肪酶的条件进行了优化。首先采用单因子实验筛选出最适诱导剂为三丁酸甘油酯,氮源为尿素,碳源为葡萄糖,无机盐为MgSO4。在此基础上,利用Plackett-Burman设计对影响产酶因素的效应进行评价,筛选出具有显著效应的三丁酸甘油酯、尿素、KH2PO4和培养基起始pH值4个最显著的因素。用最陡爬坡路径逼近最大产酶区域后,利用响应面中心组合设计对显著因素进行优化,获得最适合成培养基组分为:葡萄糖8g/L,尿素8.57g/L,三丁酸甘油酯2.62%,KH2PO42.59g/L,MgSO4.7H2O0.5g/L,TritonX-1000.5g/L,pH9.47。优化后的B.subtilis CICC 20034胞外脂肪酶活力达0.483U/ml,比初始酶活力0.072U/ml提高了6.7倍。     

Biosynthesis of poly(3-hydroxybutyrate-Co-3-hydroxyvalerate) by a mutant of Sphaerotilus natans

The copolyester of 3-hydroxybutyrate and 3- hydroxyvalerate was synthesized from the combined carbon sources of glucose and sodium propionate by a filamentaion-defective mutant of Sphaerotilus natans, which is a typical filamentous bacterium often found in activated sludge. The 3-hydroxyvalerate content in the produced polymer increased with increasing concentrations of propionate. Cell growth and polyester synthesis were observed even when 0.6% sodium propionate was added to the medium, when the 3-hydroxyvalerate content in the polymer produced was about 60 mol%. The monomer composition of the copolymer was also varied by aeration conditions, time of propionate feeding, and cultivation time. This strain flocculated in accordance with cell growth, allowing rapid and convenient separation of the biomass from the culture fluid.     

The effect of carbon source supplementation on the production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) by Cupriavidus necator

The objective of the present study was to investigate the ability of Cupriavidus necator to produce poly-(3-hydroxybutyrate-co-3-hydroxyvalerate) on various carbon sources in batch cultivation. These results show that C. necator produces poly-3-hydroxybutyrate from single carbon sources. The highest poly-3-hydroxybutyrate (P3HB) content was achieved at growth on fructose in the exponential growth phase. The maximum yield of the P3HV content was obtained when fructose was mixed with acetate. The highest content P3HB-co-3HV was also achieved by C. necator when we supplied C-excess and N- and P-normal conditions. These results indicate that C. necator accumulates high polyhydroxyalkanoates (PHA) content by depleting these elements in the culture medium. Nitrogen and phosphorus limitation has no significant effect on the PHA production, whereas C-excess leads to an increase in PHA formation of up to 92% PHAs of cell dry weight after growth on 5 g/L acetate and 40 g/L fructose.     

Engineering Halomonas TD01 for the low-cost production of polyhydroxyalkanoates

The halophile Halomonas TD01 and its derivatives have been successfully developed as a low-cost platform for the unsterile and continuous production of chemicals. Therefore, to increase the genetic engineering stability of this platform, the DNA restriction/methylation system of Halomonas TD01 was partially inhibited. In addition, a stable and conjugative plasmid pSEVA341 with a high-copy number was constructed to contain a LacI^q-P_trc system for the inducible expression of multiple pathway genes. The Halomonas TD01 platform, was further engineered with its 2-methylcitrate synthase and three PHA depolymerases deleted within the chromosome, resulting in the production of the Halomonas TD08 strain. The overexpression of the threonine synthesis pathway and threonine dehydrogenase made the recombinant Halomonas TD08 able to produce poly(3-hydroxybutyrate-co-3-hydroxyvalerate) or PHBV consisting of 4–6 mol% 3-hydroxyvalerate or 3HV, from various carbohydrates as the sole carbon source. The overexpression of the cell division inhibitor MinCD during the cell growth stationary phase in Halomonas TD08 elongated its shape to become at least 1.4-fold longer than its original size, resulting in enhanced PHB accumulation from 69 wt% to 82 wt% in the elongated cells, further promoting gravity-induced cell precipitations that simplify the downstream processing of the biomass. The resulted Halomonas strains contributed to further reducing the PHA production cost.     

Degradation of poly (3-hydroxybutyrate) and its copolymer poly (3-hydroxybutyrate-co-3-hydroxyvalerate) by a marine Streptomyces sp. SNG9

A marine Streptomyces sp. SNG9 was characterized by its ability to utilize poly(3-hydroxybutyrate) (PHB) and its copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate P (3HB-co-HV). The bacterium grew efficiently in a simple mineral liquid medium enriched with 0.1% poly(3-hydroxybutyrate) powder as the sole carbon source. Cells excreted PHB depolymerase and degraded the polymer particles to complete clarity in 4 days. The degradation activity was detectable by the formation of a clear zone around the colony (petri plates) or a clear depth under the colony (test tubes). The expression of PHB depolymerase was repressed by the presence of simple soluble carbon sources. Bacterial degradation of the naturally occurring sheets of poly(3-hydroxybutyrate) and its copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) was observed by scanning electron microscopy (SEM). Morphological alterations of the polymers sheets were evidence for bacterial hydrolysis.     

Production of polyhydroxyalkanoates from methanol by a new methylotrophic bacterium Methylobacterium sp. GW2

A new bacterial strain, isolated from groundwater contaminated with explosives, was characterized as a pink-pigmented facultative methylotroph, affiliated to the genus Methylobacterium. The bacterial isolate designated as strain GW2 was found capable of producing the homopolymer poly-3-hydroxybutyrate (PHB) from various carbon sources such as methanol, ethanol, and succinate. Methanol acted as the best substrate for the production of PHB reaching 40 % w/w dry biomass. PHB accumulation was observed to be a growth-associated process, so that there was no need for two-step fermentation. Optimal growth occurred at 0.5 % (v/v) methanol concentration, and growth was strongly inhibited at concentration above 2 % (v/v). Methylobacterium sp. strain GW2 was also able to accumulate the copolyester poly-3-hydroxybutyrate-poly-3-hydroxyvalerate (PHB/HV) when valeric acid was supplied as an auxiliary carbon source to methanol. After 66 h, a copolymer content of 30 % (w/w) was achieved with a PHB to PHV ratio of 1:2. Biopolymers produced by strain GW2 had an average molecular weight ranging from 229,350 to 233,050 Da for homopolymer PHB and from 362,430 to 411,300 Da for the copolymer PHB/HV.     

Development of Halomonas TD01 as a host for open production of chemicals

Genetic engineering of Halomonas spp. was seldom reported due to the difficulty of genetic manipulation and lack of molecular biology tools. Halomonas TD01 can grow in a continuous and unsterile process without other microbial contaminations. It can be therefore exploited for economic production of chemicals. Here, Halomonas TD01 was metabolically engineered using the gene knockout procedure based on markerless gene replacement stimulated by double-strand breaks in the chromosome. When gene encoding 2-methylcitrate synthase in Halomonas TD01 was deleted, the conversion efficiency of propionic acid to 3-hydroxyvalerate (3HV) monomer fraction in random PHBV copolymers of 3-hydroxybutyrate (3HB) and 3HV was increased from around 10% to almost 100%, as a result, cells were grown to accumulate 70% PHBV in dry weight (CDW) consisting of 12 mol% 3HV from 0.5 g/L propionic acid in glucose mineral medium. Furthermore, successful deletions on three PHA depolymerases eliminate the possible influence of PHA depolymerases on PHA degradation in the complicated industrial fermentation process even though significant enhanced PHA content was not observed. In two 500 L pilot-scale fermentor studies lasting 70 h, the above engineered Halomonas TD01 grew to 112 g/L CDW containing 70 wt% P3HB, and to 80 g/L CDW with 70 wt% P(3HB-co-8 mol% 3HV) in the presence of propionic acid. The cells grown in shake flasks even accumulated close to 92% PHB in CDW with a significant increase of glucose to PHB conversion efficiency from around 30% to 42% after 48 h cultivation when pyridine nucleotide transhydrogenase was overexpressed. Halomonas TD01 was also engineered for producing a PHA regulatory protein PhaR which is a robust biosurfactant.     

The biodegradation of poly-3-hydroxyalkanoates,PHAs, with long alkyl substitutents byPseudomonas maculicola

Utilizing a quantitative clear zone technique, the activity of an extracellular depolymerase system fromPseudomonas maculicola was investigated. Polymer degradation was influenced by the amount and availability of secondary carbon sources, with a simultaneous utilization of both sources. The initial carbon source in the liquid preculture also affected the eventual colony growth and polymer degradation. The enzyme solution was determined to readily degrade poly-3-hydroxyalkanoates (PHAs) with relatively long alkyl substituents at the 3 position: poly-3-hydroxyoctanoate (PHO), poly-3-hydroxynonanoate (PHN), and their copolymers (P[HO-co-HN]) and poly-3-hydroxyundecanoate (PHU). However, the system was unable to degrade either PHAs with shorter alkyl groups, including poly-3-hydroxybutyrate (PHB) and the copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (P[HB-co-HV]) or PHAs with unusual substituents such as poly(3-hydroxy-5-phenylvaleric acid) (PHPV). It is proposed that degradation of these more bulky side chain polymers was prevented by the inability of the bacteria to assimilate their monomeric components, which inhibited the successful utilization of secondary carbon sources and thus inhibited colony growth.     

Polyhydroxybutyrate: An intriguing biopolymer

The microbial polymer poly-3-hydroxybutyrate (PHB) and related poly-hydroxyalkanoates, such as poly-3-hydroxyvalerate and poly-3-hydroxyoctanoate, are unique biodegradable thermoplastics of considerable commercial importance. The structure, properties and regulation of synthesis and degradation of PHB are reviewed and the microbial production of copolymers of 3-hydroxybutyrate and 3-hydroxyvalerate, with properties varying according to copolymer composition, is discussed.     

Synthesis of biodegradable polyesters by Gram negative bacterium isolated from Malaysian environment

A locally isolated Gram negative bacterium, Cupriavidus sp. USMAA9-39 was able to produce various types of biodegradable polyesters through a two-step cultivation process. These are copolymer poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)], copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] and terpolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate-co-4-hydroxybutyrate) [P(3HB-co-3HV-co-4HB)]. These polymers were synthesized by this bacterium when grown with a combination of some carbon sources. The biosynthesis of P(3HB-co-4HB) was achieved by using carbon sources such as γ-butyrolactone or 1,4-butanediol or by a combination of oleic acid with either γ-butyrolactone or 1,4-butanediol. Meanwhile, poly(3-hydroxybutyrate-co-3-hydroxyvalerate) was produced using 1-pentanol or valeric acid or by a combination of oleic acid with either 1-pentanol or valeric acid. When γ-butyrolactone or 1,4-butanediol with either valeric acid or 1-pentanol were used as mixed carbon sources, P(3HB-co-3HV-co-4HB) terpolymer were produced. The presence of 3HB, 3HV or/and 4HB monomers were confirmed by gas chromatography and nuclear magnetic resonance (NMR) spectroscopy.     

Synthesis of poly-(3-hydroxybutyrate-co-3-hydroxyvalerate) by recombinant Escherichia coli

Several recombinant Escherichia coli strains, including XL1-Blue, JM109, HB101, and DH5alpha harboring a stable high-copynumber plasmid pSYL105 containing the Alcaligenes eutrophus polyhydroxyalkanoate (PHA) biosynthesis genes were constructed. These recombinant strains were examined for their ability to synthesize and accumulate poly-(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] copolymer from glucose and either propionate or valerate. All recombinant E. coli strains could synthesize the P(3HB-co-3HV) copolymer in the medium containing glucose and propionate. However, only the homopolymer poly-(3-hydroxybutyrate) [P(3HB)] was synthesized from glucose and valerate. The PHA concentration and the 3HV fraction could be increased by inducing with acetate and/or oleate. When supplemented with oleate, the 3HV fraction increased by fourfold compared with that obtained without induction. Induction with propionate resulted in lower PHA concentration due to the inhibitory effect, but an 3HV fraction of as high as 33.0% could be obtained. These results suggest that P(3HB-co-3HV) can be efficiently produced from propionate by recombinant E. coli by inducing with acetate, propionate, or oleate. (c) 1996 John Wiley & Sons, Inc.