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。     

盐单胞菌利用短链脂肪酸合成聚羟基脂肪酸酯

盐单胞菌(Halomonas)能够利用多种底物为碳源生长,由于其能在高盐条件下进行不灭菌的开放发酵,已被开发用作下一代生物技术的底盘细胞.包括乙酸、丙酸和丁酸在内的短链挥发性脂肪酸能够以生物质为原料制备,有望成为用于微生物发酵的新型碳源.利用10-50g/L浓度的丁酸为碳源对Halomonas sp.TD01和TD08...     

利用农业废弃物发酵生产D-乳酸

利用农业废弃物玉米芯酶解液替代葡萄糖作为碳源,棉籽粕替代酵母膏作为氮源发酵生产D-乳酸。结果表明:在初始还原糖质量浓度为100 g/L(葡萄糖88.5 g/L,木糖11.5 g/L)、棉籽粕3.5 g/L、每升发酵体积添加3 U的中性蛋白酶以及pH 6.5的情况下,采取补料发酵措施,菌株Sporolactobacillus sp.YBS1-5在90 h内产生了111.8 g/L的D-乳酸,糖酸转化率为87%,光学纯度达98%以上,生产强度达1.24 g/(L·h)。本文提供了一种利用农业废弃物发酵产D-乳酸的新途径。     

有机酸胁迫下厌氧污泥产氢效果

乙酸、丙酸、乳酸及丁酸是厌氧发酵产氢过程中4种主要的液相末端发酵产物,其积累对产氢过程有一定的抑制作用。实验利用多种有机酸胁迫提高污泥的酸耐受能力,并以污泥中脱氢酶活性为生化指标,对不同浓度酸胁迫下厌氧污泥活性变化进行了研究。通过对胁迫后污泥产氢量及末端产物进行对比。结果表明,酸胁迫后污泥产氢量有一定增加,其中乙酸和丁酸胁迫效果最好,较对照组提高了近一倍;末端产物分析研究表明,不同的有机酸胁迫后,其产量在发酵过程中都有一定的增加,而乙酸含量在酸胁迫后都有不同程度的提高。     

代谢工程改造大肠杆菌合成丁二酸及发酵罐放大工艺

【背景】Escherichia coli AFP111发酵生产丁二酸时大量副产乙酸,丁二酸得率低。【目的】代谢工程改造EscherichiacoliAFP111,提高丁二酸得率,降低副产物乙酸的生成,建立100 L规模的丁二酸发酵工艺。【方法】一步同源重组敲除乙酸合成途径关键酶基因,改造丁二酸合成途径关键酶启动子实现过表达;单因素优化5L发酵罐培养条件。【结果】敲除乙酸产生途径编码乙酸激酶和磷酸转乙酰酶的基因ackA-pta、苏氨酸脱羧酶和2-酮丁酸甲酸裂解酶的基因tdcDE获得SX02菌株,摇瓶发酵条件下其乙酸产量下降了53.42%,丁二酸得率提高9.85%。在SX02菌株基础上,经启动子改造过表达编码葡萄糖激酶的基因glk后获得菌株SX03,其Glk酶活性提高3.66倍,乙酸产量下降了31.62%,丁二酸得率提高8.28%。SX03菌株发酵生产丁二酸在5 L发酵罐进行放大,其乙酸产量为3.97 g/L,丁二酸得率为1.62 mol/mol葡萄糖,相比出发菌株的乙酸产量下降了75.76%,丁二酸得率提高19.12%。在5L发酵罐上对比研究了中和剂Na2CO3和NaOH混合液替换碱式MgCO3的发酵效果,并优化了发酵pH、搅拌转速和葡萄糖浓度,获得如下最适发酵条件:pH6.8,搅拌转速250r/min,葡萄糖100g/L,发酵结束时乙酸产量为2.24 g/L,丁二酸得率为1.66 mol/mol葡萄糖。中和剂替换优化后乙酸产量下降了20.65%,丁二酸得率提高2.47%。菌株SX03发酵工艺进一步在100 L发酵罐上实现放大,其乙酸产量为1.91 g/L,丁二酸得率为1.30 mol/mol葡萄糖。【结论】通过代谢工程改造的大肠杆菌,其副产物乙酸含量显著下降,丁二酸得率提高,并在5 L和100 L发酵罐上实现了工艺放大,展现出较大的工业化利用潜力。     

利用纤维床反应器固定化发酵生产丙酸

构建了一种纤维床反应器(FBB), 并将其应用于丙酸的生产。将棉纤维绕成桶状, 固定于反应器中, 即可用于丙酸固定化发酵。以40 g/L的葡萄糖为碳源, 与游离细胞相比, 利用FBB生产丙酸, 丙酸产量由14.58 g/L提高至20.41 g/L, 发酵时间由120 h缩短至60 h。研究了不同糖浓度条件下FBB生产丙酸情况, 并将补料策略应用于丙酸发酵中。结果表明: 补料发酵能够有效改善Propionibacterium freudenreichii CCTCC M207015在高糖条件下丙酸对葡萄糖转化率较低、副产物较多的问题。经补料发酵280 h, 丙酸产量达45.91 g/L, 丙酸质量约占有机酸总质量比例为72.31%。     

利用纤维床反应器固定化发酵生产丙酸

构建了一种纤维床反应器(FBB), 并将其应用于丙酸的生产。将棉纤维绕成桶状, 固定于反应器中, 即可用于丙酸固定化发酵。以40 g/L的葡萄糖为碳源, 与游离细胞相比, 利用FBB生产丙酸, 丙酸产量由14.58 g/L提高至20.41 g/L, 发酵时间由120 h缩短至60 h。研究了不同糖浓度条件下FBB生产丙酸情况, 并将补料策略应用于丙酸发酵中。结果表明: 补料发酵能够有效改善Propionibacterium freudenreichii CCTCC M207015在高糖条件下丙酸对葡萄糖转化率较低、副产物较多的问题。经补料发酵280 h, 丙酸产量达45.91 g/L, 丙酸质量约占有机酸总质量比例为72.31%。     

流加发酵对重组Bacillus subtilis发酵生产角质酶的影响

为实现基因工程菌Bacillus subtilis WSHB06-07生产角质酶的高产,在3L发酵罐中考察了不同初糖浓度对菌体生长和产酶的影响,并在选择38 g/L初始蔗糖浓度的基础上,进行碳源的分批流加和恒速流加,结果表明发酵16 h开始流加碳源,采用总补糖量60g/L,蔗糖平均流速为4g/(L·h)的恒速补料方式,角质酶酶活在31h可达到最大545.87U/ml,比分批发酵酶活提高67.8%,并获得较高的角质酶生产强度,满足工业化生产要求。     

利用五碳糖产高纯度L-乳酸的大肠杆菌基因工程菌的构建

[目的]本研究以已敲除多个产杂酸酶基因的大肠杆菌(Escherichia coli)乙醇工程菌SZ470(△frdBC △ldhA △ackA △focA-pflB △pdhR::pflBp6-pflBrbs-aceEF-lpd)为起始菌株,进一步敲除其乙醇脱氢酶(alcohol dehydrogenase,ADH)基因,同时插入带有自身启动子的乳酸片球菌(Pediococcus acidilactici)的L-乳酸脱氢酶(L-lactate dehydrogenase,LLDH)基因,构建可利用五碳糖同型发酵L-乳酸重组大肠杆菌.[方法]利用λ噬菌体Red重组系统构建乙醇脱氢酶基因(adhE)缺失菌株Escherichia coli JH01,并克隆P.acidilactici的ldhL基因,利用染色体插入技术将其整合到JH01基因组,构建产L-乳酸大肠杆菌基因工程菌Escherichia coli JH12,利用无氧发酵15 L发酵罐测定重组菌株L-乳酸产量.[结果]工程菌JH12在15 L发酵罐中以6％的葡萄糖为碳源进行发酵,发酵到36 h的过程中葡萄糖的消耗速率为1.46 g/(L·h),乳酸生产强度为1.14 g/(L·h),乳酸的产量达到41.13 g/L.发酵产物中未检测到琥珀酸、甲酸的生成,仅有少量乙酸生成,L-乳酸纯度达95.69％(L-乳酸在总发酵产物的比率).工程菌JH12以6％的木糖为碳源进行发酵,发酵到36 h的过程中葡萄糖的消耗速率为0.88 g/(L·h),乳酸生产强度为0.60 g/(L·h),乳酸的产量达到34.73 g/L.发酵产物中杂酸少,乳酸的纯度高达98％.[结论]本研究通过基因敲除、染色体插入及无氧进化筛选获得一株产L-乳酸的大肠杆菌工程菌JH12,该菌株不需利用外源质粒,稳定性好,可利用五碳糖进行发酵,发酵产物中杂酸少,L-乳酸的纯度高.本研究为L-乳酸大肠杆菌工程菌的构建提供一定的技术支持,同时也为大肠杆菌L-乳酸的工业化生产提供了参考依据.     

产酸丙酸杆菌耐酸菌株的选育及其应用

为了解除微生物发酵生产丙酸过程中代谢产物(丙酸)对菌体生长的抑制作用,以实验室保藏的产酸丙酸杆菌(耐30g/L丙酸)为出发菌株P-0,通过丙酸压力筛选获得了一株耐10g/L丙酸的产酸性能良好的菌株P-10,降低了发酵过程中丙酸对菌体生长的抑制作用。菌株P-10做摇瓶发酵,发酵周期168h,丙酸浓度为49.66g/L,产酸速率为0.30g/(L·h),较出发菌株P-0提高了53.04%;7L发酵罐实验表明,菌株P-10发酵周期168h,丙酸浓度为55.63g/L,产酸速率0.33g/(L·h)。同时对菌株P-10做二次接种实验,结果表明,84h为二次接种最适时间段,且84h进行二次接种时,丙酸浓度提高了17.77%,二次接种实验不但有利于有机酸的积累,而且可以提高菌株的产酸能力和耐酸能力;经过选育的菌株P-10具有优良的产酸稳定性,有利于菌种的工业化生产和应用,同时对后续的发酵分离耦合具有重要意义。     

Production of PHA from starchy wastewater via organic acids

Polyhydroxyalkanoate (PHA) was produced from a starchy wastewater in a two-step process of microbial acidogenesis and acid polymerization. The starchy organic waste was first digested in a thermophilic upflow anaerobic sludge blanket (UASB) reactor to form acetic (60-80%), propionic (10-30%) and butyric (5-40%) acids. The total volatile fatty acids reached 4000 mg l(-1) at a chemical oxygen demand (COD) loading rate of 25-35 g l(-1) day(-1). A carbon balance indicates that up to 43% of the organic carbon in the starchy waste went to the organic acids and the rest to biogas, volatile suspended solids and residual sludge accumulated in the reactor. The acid composition profile was affected by COD loading rate: a medium rate around 9 g l(-1) day(-1) gave a high propionic acid content (29% wt) and a high rate around 26 g l(-1) day(-1) led to a high butyric acid content (34% wt). The acids in the effluent solution after microfiltration were utilized and polymerized into PHA by bacterium Alcaligenes eutrophus in a second reactor. Fifty grams of PHA was produced from 100 g total organic carbon (TOC) utilized, a yield of 28% based on TOC, which is comparable with 55 g PHA per 100 g TOC of pure butyric and propionic acids used. PHA formation from individual acids was further investigated in a semi-batch reactor with three acid feeding rates. With a limited nitrogen source (80-100 mg NH(3) per liter), the active biomass of A. eutrophus, not including the accumulated PHA in cells, was maintained at a constant level (8-9 g l(-1)) while PHA content in the cell mass increased continuously in 45 h; 48% PHA with butyric acid and 53% PHA with propionic acid, respectively. Polyhydroxybutyrate was formed from butyric acid and poly(hydroxybutyrate-hydroxyvalerate) formed from propionic acid with 38% hydroxyvalerate.     

Characterization of anaerobic microbial culture with high acidogenic activity

The mixed cultures which were used were isolated from municipal sludge digesters, and the production of organic acids (acetic, propionic, butyric, etc.) from carbohydrates was tested. The behavior of the reference population (culture R) obtained directly from the sewage treatment plant, is compared to that obtained after three months in a plug-flow reactor (Gradostat fermentor) without pH control (culture A) and after six months with pH control (culture B). For culture B, the specific rate of acid production is related to the cell growth rate by (1/X)r_p= 17 µ + 1.6 with a maximal acid concentration of 40 g/liter. The batch culture yields are improved from 0.36g/g for the initial culture (R) to 0.72 g/g for culture B after six months in continuous culture, and 0.8 g/g in plug-flow continuous culture. The productivity of organic acids reaches 1.7 g/liter·hr. It is suggested that the acidogenic fermentation, the first step of methanogenesis, is a potential process to produce acetic, propionic, and butyric acids.     

Enhanced cutinase production of Thermobifida fusca by a two-stage batch and fed-batch cultivation strategy

In this study, cutinase production by Thermobifida fusca WSH03-11 was investigated with mixed short-chain organic acids as co-carbon sources to demonstrate the possibility of producing high value-added products from organic wastes. T. fusca WSH03-11 was cultured with different combinations of butyrate, acetate, and lactate with a purpose of increasing cutinase activity. The optimum proportion of butyrate, acetate, and lactate was 4:1:3. In batch cultivation, acetate and lactate were consumed quickly, while the consumption of butyrate was depressed in the presence of acetate with a concentration higher than 0.5 g/L. Based on these results, a two-stage batch and fed-batch cultivation strategy was proposed: a batch culture with acetate and lactate as the co-carbon sources in the first 10 h, and then a fed-batch culture with a constant butyrate feeding rate of 12 mL/h during 11∼20 h. By this two-stage cultivation strategy, cutinase activity, dry cell weight, and consumption rate of butyrate were increased by 70%, 103.4%, and 4.3-fold, respectively, compared to those of the batch cultivation. These results provided a novel and efficient way to produce high value-added products from organic wastes.     

Anaerobic digestion: microbial and biochemical aspects of volatile acid production

Summary The production of organic acids has been tested with bacterial flora selected from a municipal sludge digestor. In order to elucidate the basic mechanisms by which glucose is converted to volatile fatty acids, the examination of non-methanogenic bacteria was attempted. Both lactate-producers and lactate-utilizers were found among these bacteria. When mixed isolates were used as the inoculum, the accumulation of lactic acid and its further conversion to propionic and butyric acids was demonstrated at a carbon conversion rate of about 0.75. It is therefore suggested that this metabolic sequence may occur as a normal process in acidogenic fermentation, which is the first step in anaerobic digestion.     

Efect of the volatile fatty acids on phosphate uptake parameters by pure cultures of Acinetobacter

E.RUSTRIAN, J.P. DELGENES AND R. MOLETTA. 1996. Experiments were performed to examine the effect of volatile fatty acids(VFA) as carbon source, on the phoshate uptake parameters in four Acinetobacter strains. Acetic and butyric acids were equally good carbon sources for phosphate removal, while propionic acid was the least efficient substrate. The best ratios of assimilated phosphate vs VFA consumed were 0-178 wit acetic acid by Ac.calcoaceticus NRRL 4270, 0.21 with propionic acid by Ac.calcoaceticus NRRL 4270 AND 0.187 with butyric acid by Acinetobacter sp.SUCT 5.     

Assessment of toxicity of volatile fatty acids to Photobacterium phosphoreum

The toxicity of four volatile fatty acids (VFAs) as anaerobic digestion (AD) intermediates was investigated at pH 7. Photobacterium phosphoreum T3 was used as an indicator organism. Binary, ternary and mixtures of AD intermediates were designated by letters A (acetic acid + propionic acid), B (acetic acid + butyric acid), C (acetic acid + ethanol), D (propionic acid + butyric acid), E (propionic acid + ethanol), F (butyric acid + ethanol), G (acetic acid + propionic acid + butyric acid), H (acetic acid + propionic acid + ethanol), I (acetic acid + butyric acid+ ethanol), J (propionic acid + butyric acid + ethanol) and K (acetic acid + propionic acid + butyric acid + ethanol) to assess the toxicity through equitoxic mixing ratio method. The IC₅₀ values of acetic acid, propionic acid, butyric acid and ethanol were 9.812, 7.76, 6.717 and 17.33 g/L respectively, displaying toxicity order of: butyric acid > propionic acid > acetic acid > ethanol being additive in nature. The toxic effects of four VFAs could be designated as synergistic and one additive in nature.     

The investigation of effect of organic carbon sources addition in anaerobic–aerobic (low dissolved oxygen) sequencing batch reactor for nutrients removal from wastewaters

The effect of addition of organic carbon sources (acetic acid and waste activated sludge alkaline fermentation liquid) on anaerobic–aerobic (low dissolved oxygen, 0.15–0.45 mg/L) biological municipal wastewater treatment was investigated. The results showed that carbon source addition affected not only the transformations of polyhydroxyalkanoates (PHA), glycogen, nitrogen and phosphorus, but the net removal of nitrogen and phosphorus. The removal efficiencies of TN and TP were, respectively, 61% and 61% without organic carbon source addition, 81% and 95% with acetic acid addition, and 83% and 97% with waste activated sludge alkaline fermentation liquid addition. It seems that the alkaline fermentation liquid of waste biosolids generated in biological wastewater treatment plant can be used to replace acetic acid as an additional carbon source to improve the anaerobic–aerobic (low dissolved oxygen) municipal wastewater nutrients removal although its use was observed to cause a slight increase of effluent BOD and COD concentrations.     

Enhanced cutinase production with Thermobifida fusca by two-stage pH control strategy

A mutant of Thermobifida fusca ATCC 27730 was used for cutinase production. Acetate was the most suitable carbon source for cell growth and cutinase production compared with others. The pH was one of the most important factors affecting cutinase yield and productivity. Batch cutinase fermentations by mutant Thermobifida fusca WSH04 at various pH values ranging from 7.0 to 7.9 were studied. Based on the effects of different pH values on the specific cell growth rate and specific cutinase formation rate, a two-stage pH control strategy was developed, in which the pH was set at 7.3 for the first 20 h, and switched to 7.6 afterwards. By applying this two-stage pH control strategy for cutinase fermentation, the maximal cutinase activity reached 19.8 U/mL.     

Effect of organic acid on gastrointertinal motility of rat invitro

The quantity of organic acids ( lactic acid, acetic acid, propionic acid and butyric acid ) in the content of the gastrointestinal tract of germ-free and conventional rats and the $\text{in}$$\text{vitro}$ effects of the organic acid on the motility of the gastrointestinal tract of rats were investigated.Organic acids were detected only in the gastrointestinal contents of conventional rats but not in those of germ-free rats.Lactic acid detected in the stomach of rats stimulated the motility of both small and large bowel while acetic acid, propionic acid and butyric acid found in the cecum stimulated the motility of the large bowel but not of small bowel.