产氢产乙酸菌ZR-1 的分离鉴定及产酸特性

采用改良的亨盖特厌氧操作技术, 从有机废水污泥中分离到一株耐低温高效产氢产乙酸菌ZR-1。经过对其形态学观察、生理生化特征研究及16S rRNA 序列比对, 初步鉴定为梭状芽胞杆菌属的乙二醇梭菌(Clostridium glycolicum)。通过单因子实验, 在厌氧条件下对该菌株的培养温度、pH、最适底物、金属离子的影响等产酸条件进行了优化。结果表明该菌株最适生长温度37 °C,最佳培养基初始pH 值8.5, 最适发酵底物丁酸盐, Mn²⁺对其产酸有一定的激活作用。最适培养条件下丁酸盐降解率达到12.7%, H₂ 含量达到了28.73%。     

初始底物浓度及pH对丁酸梭菌T4发酵木糖产氢的影响

本研究采用间歇培养方式对丁酸梭菌T4发酵木糖进行产氢研究,考察初始pH和初始底物浓度对其产氢特性的影响。结果表明,菌株T4在初始pH5.0～8.5及初始底物浓度5～40g/L时均可以产氢,其累积产氢量和最大比产氢速率随着pH及底物浓度的增加均呈现先增加后减少的趋势。在pH6.5和底物浓度20g/L时,比产氢速率和累积产氢量达到最大,分别为4.26L/L和18.86mmol-H2/hg-DCW,而后随着pH或者底物浓度的增加二者均呈现减少的趋势;在pH6.5和底物浓度15g/L时,得到最大值比产氢量为2.17mol/mol-木糖。而在不同的pH下,发酵产生的液态产物主要是乙酸和丁酸,其中在pH小于6.0时,有少量的丙酸生成,而在pH大于6.0时,则有乙醇生成。     

厌氧细菌Acetanaerobacterium elongatum从葡萄糖的产氢特性研究

为了了解影响厌氧发酵产氢细菌Acetanaerobacterium elongatumZ7产氢效率的因素,采用生理学方法对其进行了研究。结果表明:乙醇型发酵菌A.elongatumZ7的最适产氢温度为37℃,最适产氢的起始pH为8.0。该菌发酵葡萄糖和阿拉伯糖产氢的能力较强,氢气产率分别为1.55mol H2/mol葡萄糖和1.50mol H2/mol阿拉伯糖。酵母粉是菌株Z7生长和产氢所必须的生长因子;pH影响菌株的生长和葡萄糖利用率;氢压则影响电子流的分配,从而改变代谢产物乙酸和乙醇的比例;当产氢菌与甲烷菌共培养以维持发酵体系低的氢压时,可使氢的理论产量提高约4倍;培养基中乙酸钠浓度>60mmol/L明显抑制产氢。另外,一个只利用蛋白类物质的细菌能够促进菌株Z7对葡萄糖的利用,进而提供氢产量,为生物制氢的工业化生产提供理论参考。     

污泥厌氧消化产酸发酵过程中乙酸累积机制

[目的]研究污泥厌氧消化产挥发性脂肪酸(VFA)过程中的有机物碳流的转化机制,阐明乙酸累积机理。[方法]研究溴乙烷磺酸盐(BES)和氯仿(CHCl3)抑制模型下中间代谢产物和气体的累积,检测各产乙酸功能菌群数量,推断污泥产酸发酵过程中的有机物碳流方向和乙酸累积机理。[结果]BES模型乙酸浓度达27 mmol/L,fhs基因拷贝数比对照组高2-3倍,产氢产乙酸菌略有下降。CHCl3模型乙酸浓度达22 mmol/L,fhs基因拷贝数比BES组低一个数量级,产氢产乙酸菌下降明显。[结论]BES特异性较高,除产甲烷菌外对其他厌氧产酸细菌没有影响,乙酸浓度增加并且其主要来源于水解发酵产酸以及同型产乙酸过程。氯仿除抑制产甲烷菌外,对同型乙酸菌和产氢产乙酸菌也有强烈的抑制作用。     

不同预处理条件对海水养殖废弃物发酵产氢的影响

【目的】利用海水养殖场有机废弃物厌氧发酵产氢,可在减少有机污染物的同时获取氢气。【方法】以海水养殖场有机废弃物为底物,比较嗜热酶(S-TE)、酸、碱、灭菌、微波不同预处理方法对厌氧发酵产氢效果的影响,并对发酵过程中底物性质变化[SCOD、可溶性蛋白质、可溶性糖、pH、VFAs(挥发性脂肪酸)和乙醇]进行探讨。【结果】灭菌预处理产氢效果最好,产氢率为22.0 mL/g VSS,酸处理的效果最差,产氢率为7.6 mL/g VSS。可溶性糖大量消耗之后,氢气不再产生。接种S-TE预处理污泥的底物能更多地释放营养物质,并在整个发酵过程中保持较为稳定的pH值。发酵过程中产生的VFAs主要成分是乙酸,在发酵后期出现乙醇。【结论】灭菌预处理是海水养殖场有机废弃物厌氧发酵产氢的最佳预处理方法,可溶性糖为这一过程主要的营养来源。     

一株新型同型产乙酸菌Clostridium sp.的自养和异养生长特性

【背景】同型产乙酸菌是一类利用乙酰辅酶A途径固定CO_2合成自身细胞物质并生成乙酸、乙醇等代谢产物的厌氧菌群,其分布广泛、种类繁多且代谢多样。深入研究同型产乙酸菌菌株的代谢能力及特性,对探索该种群的生理生化特性及其环境作用至关重要。【目的】研究一株同型产乙酸菌Clostridium sp. BXX的最适培养条件及其自养与异养生长特性。【方法】设置BXX菌株培养温度10-55°C、初始pH 6.0-9.0、NaCl浓度0-2.0%、不同氮源,测定菌体细胞含量和产物生成浓度,确定菌株最适培养条件。研究BXX菌株分别以H_2/CO_2、合成气、CO、葡萄糖、1,2-丙二醇、甲酸钠、乙二醇甲醚、甘油、丙酮酸和乳酸为底物时的底物消耗、产物生成、菌体细胞含量和pH等,探究其自养和异养生长特性。【结果】BXX菌株的最适培养温度为30°C,初始pH为7.0,NaCl浓度为1.0%,氮源为酵母粉。BXX菌株能以H2/CO2、合成气、葡萄糖、1,2-丙二醇、甲酸钠、乙二醇甲醚和甘油为底物生长,不能以CO、丙酮酸或乳酸为底物生长。【结论】BXX菌株既能自养生长产乙酸,又能异养生长产乙醇。BXX菌株是乙酸发酵的优良菌种资源,有较好的工业应用潜力。     

厌氧细菌Acetanaerobacterium elongatum从葡萄糖的产氢特性研究

为了了解影响厌氧发酵产氢细菌Acetanaerobacterium elongatum Z7产氢效率的因素,采用生理学方法对其进行了研究。结果表明：乙醇型发酵菌A. elongatum Z7的最适产氢温度为37℃, 最适产氢的起始pH为8.0。该菌发酵葡萄糖和阿拉伯糖产氢的能力较强,氢气产率分别为1.55mol H2/mol葡萄糖和1.50mol H2/mol阿拉伯糖。酵母粉是菌株Z7生长和产氢所必须的生长因子;pH影响菌株的生长和葡萄糖利用率;氢压则影响电子流的分配,从而改变代谢产物乙酸和乙醇的比例;当产氢菌与甲烷菌共培养以维持发酵体系低的氢压时,可使氢的理论产量提高约4倍;培养基中乙酸钠浓度> 60mmol/L明显抑制产氢。另外,一个只利用蛋白类物质的细菌能够促进菌株Z7对葡萄糖的利用,进而提供氢产量,为生物制氢的工业化生产提供理论参考。     

一个新的高温产氢菌及产氢特性的研究

利用Hungate滚管技术从西藏山南地区热泉淤泥中分离到一株高温产氢的厌氧发酵细菌T42。菌株T42革兰氏染色反应为阴性,但KOH裂解试验证实其为革兰氏阳性杆菌。菌体大小为0.7μm~0.9μm×3.2μm~7μm,不运动,不产芽孢。其生长温度范围为32℃~69℃,最适生长温度为60℃~62℃,生长pH范围为5.0~8.8,最适生长pH为7.0~7.5,代时30min。有机氮源是T42菌株的必需生长因子。菌株T42利用淀粉、纤维二糖、蔗糖、麦芽糖、糊精、果糖、糖原和海藻糖等底物生长并发酵产氢,发酵葡萄糖的终产物为乙酸、乙醇、H2和CO2。G C含量为31.2mol%。系统发育分析表明菌株T42与Thermobrachium celere和Caloramator indicus位于同一分支,生理生化特征也表明菌株T42应是Thermobrachium属的一个新菌株,在中国普通微生物菌种保藏中心的保藏号为AS1.5039。菌株T42的最佳产氢初始pH为7.2,最佳产氢温度为62℃,其氢转化率为1.06mol H2/mol葡萄糖,最大产氢速率为24.0mmol H2/gDW/h。20mmol/L的Mg2 和2mmol/L的Fe2 可分别提高菌株T42的产氢量20%和23.3%,而Ni2 对其产氢无明显的作用。当菌株T42和热自养甲烷热杆菌(Methanothermobacter thermautotrophicus)Z245共培养时,由于降低了氢分压,使其葡萄糖利用率和氢产量分别提高1倍和2.8倍,发酵产物乙酸和乙醇的比例也从1提高到1.7。     

pH值和产甲烷抑制剂对两相耦合系统污泥发酵定向产乙酸的影响

采用产氢产乙酸/同型产乙酸两相耦合工艺对剩余污泥进行了半连续式厌氧发酵,主要研究了pH值和产甲烷抑制剂2-bromoethanesulphonate(BES)对耦合系统定向产乙酸的影响.结果表明:碱性pH(pH=10.0)和添加BES都能促进A相乙酸的积累,提高乙酸的产率,同时碱性pH比添加BES更有利于污泥的水解.当...     

颗粒厌氧污泥中的产氢产乙酸细菌研究

本文报道颗粒厌氧污泥中产氢产乙酸细菌的含量及存在方式。在正常运行状态,随着颗粒污泥的培养和生长、产氢产乙酸细菌含量维持在10⁷-10⁸个/ml.一旦厌氧反应器“酸化”,颗粒污泥性能变差,产氢产乙酸细菌急剧下降,减小到约10⁵个/ml.比正常状态低2-3个数级,说明细菌生长受到了不可逆抑制。电镜观察表明,产氢产乙酸细菌的分布不是随机的,它们以微菌落方式存在并排列有序。除了与甲烷短杆菌互营共生外,还发现了一种和甲烷丝菌间的新型互营共生关系,分析     

果蔬废弃物两相厌氧发酵产己酸的效能研究

利用厌氧菌群生物合成己酸被认为是一种非常有潜力的新型废弃物资源化技术,但是其合成效能的提高是目前亟待解决的关键问题。本研究以实际果蔬废弃物为原料,对两相厌氧发酵产己酸的效能进行了研究。首先优化接种比以提高酸化相的水解转化效率;在此基础上通过调控醇酸比和pH以强化产己酸相的发酵效能。结果显示,果蔬废弃物厌氧产酸的最佳接种比为2∶1,此时水解率和酸化率分别可达到98.1%和83.2%,乙酸和丁酸产量分别达到5.4 g/L和3.3 g/L。合理控制醇酸比和pH对提高产己酸相的发酵效能非常关键。当醇酸比和pH控制为4∶1和7.5时,己酸生成量可达14.9 g/L,约占液相总COD的80.84%;而低醇酸比和低pH易造成丁酸的累积,从而降低了己酸产量。己酸发酵过程属于非生长偶联型,己酸菌(Clostridium kluyveri)指数增长期伴随着丁酸的生成,而己酸合成主要发生在生长中后期。此外,己酸菌对于pH变化较为敏感,适当提高pH有助于减轻有机酸毒性,提高生物量;但是碱性环境会严重抑制己酸菌的生长繁殖。研究表明,通过分别对酸化相和产己酸相进行优化和调控,两相发酵策略更有利于提高己酸合成效能。     

Physiological adaptations of anaerobic bacteria to low pH: metabolic control of proton motive force in Sarcina ventriculi.

Detailed physiological studies were done to compare the influence of environmental pH and fermentation end product formation on metabolism, growth, and proton motive force in Sarcina ventriculi. The kinetics of end product formation during glucose fermentation in unbuffered batch cultures shifted from hydrogen-acetate production to ethanol production as the medium pH dropped from 7.0 to 3.3. At a constant pH of 3.0, the production of acetate ceased when the accumulation of acetate in the medium reached 40 mmol/liter. At a constant pH of 7.0, acetate production continued throughout the entire growth time course. The in vivo hydrogenase activity was much higher in cells grown at pH 7.0 than at pH 3.0. The magnitude of the proton motive force increased in relation to a decrease of the medium pH from 7.5 to 3.0. When the organism was grown at pH 3.0, the cytoplasmic pH was 4.25 and the organism was unable to exclude acetic acid or butyric acid from the cytoplasm. Addition of acetic acid, but not hydrogen or ethanol, inhibited growth and resulted in proton motive force dissipation and the accumulation of acetic acid in the cytoplasm. The results indicate that S. ventriculi is an acidophile that can continue to produce ethanol at low cytoplasmic pH values. Both the ability to shift to ethanol production and the ability to continue to ferment glucose while cytoplasmic pH values are low adapt S. ventriculi for growth at low pH.     

Conditions for forming ethanol during bioconversion of cellulose-containing raw material

Several natural associations composed by thermophilic anaerobic bacteria capable of utilizing various cellulose materials at 60 +/- 2 degrees C and pH 6.0-7.0 were isolated from the sludge of Kamchatka geothermal springs. The rate of ethanol production (up to 1.7 g/l per day) and the concentration of ethanol in the medium (up to 1.2%), as well as the fermentation period (10-15 days) were determined under anaerobic conditions in the presence of cellulose, coniferous sawdust, newsprint, or paper pulp as a carbon source. Microorganisms were found that inhibited the production of ethanol. The initial pH value was found to influence both the ethanol production rate and ethanol/acetate ratio. A pH decrease from 7.0 to 5.0 led to 6.7-fold increased the ethanol production and caused a 23.8-fold increase in the ethanol/acetate ratio.     

13C NMR studies of butyric fermentation in Clostridium kluyveri

The fermentation of 13C-labeled ethanol and acetate into butyrate and caproate by Clostridium kluyveri has been studied by using 13C NMR. The pathway involves the conversion of both ethanol and acetate into acetyl coenzymes A, two of which condense to form CoA-linked precursors of butyrate. If butyryl-CoA is involved in the condensation, caproate is the ultimate product. ATP is produced from acetyl-CoA via the reactions catalyzed by phosphotransacetylase and acetate kinase with acetate, a required carbon source, as a co-product. In spectra of whole cells incubated with the labeled carbon sources, label from ethanol appears rapidly in acetate, which then reaches a lower, steady-state concentration due to its re-entry into the pathway. The rapid initial production of acetate indicates equally rapid production of ATP. Label from acetate appears in ethanol only if ethanol is already present, indicating that this process is one of isotopic equilibration rather than net synthesis of ethanol from acetate. The ratio of butyrate to caproate produced depends strongly on the initial ratio of ethanol to acetate in the medium. The relative rates of utilization of ethanol and acetate vary as the fermentation proceeds. 13C-13C coupling in the butyrate and caproate produced from [1-13C]ethanol and [2-13C]acetate can be used to determine if the acetyl-CoA molecules arising from ethanol and acetate enter the same pool or if they remain separated. The data are consistent with random mixing of the acetyl-CoA produced from the two carbon sources.     

Influence of the pH on (open) mixed culture fermentation of glucose: a chemostat study

Catabolic products from anaerobic fermentation processes are potentially of industrial interest. The volatile fatty acids and alcohols produced can be used as building blocks in chemical processes or applied directly as substrates in a mixed culture process to produce bioplastics. Development of such applications requires a predictable and controllable product spectrum of the fermentation process. The aim of the research described in this paper was (i) to investigate the product spectrum of an open mixed culture fermentation (MCF) process as a function of the pH, using glucose as substrate, and (ii) to relate the product spectrum obtained to generalized biochemical and thermodynamic considerations. A chemostat was operated under carbon and energy limitation in order to investigate the pH effect on the product spectrum in a MCF process. A transition from CO(2)/H(2) production at lower pH values to formate production at higher pH values was observed. The ratio of CO(2)/H(2) versus formate production was found to be related to the thermodynamics of formate dehydrogenation to CO(2)/H(2). This transition was associated with a shift in the catabolic products, from butyrate and acetate to ethanol and acetate, likely due to a decrease in the oxidation state of the electron carriers in the cell. The product spectrum of the MCF process as a function of the pH could largely be explained using general biochemical considerations.     

Glycerol fermentation by (open) mixed cultures: a chemostat study

Glycerol is an important byproduct of bioethanol and biodiesel production processes. This study aims to evaluate its potential application in mixed culture fermentation processes to produce bulk chemicals. Two chemostat reactors were operated in parallel, one fed with glycerol and the other with glucose. Both reactors operated at a pH of 8 and a dilution rate of 0.1 h(-1). Glycerol was mainly converted into ethanol and formate. When operated under substrate limiting conditions, 60% of the substrate carbon was converted into ethanol and formate in a 1:1 ratio. This product spectrum showed sensitivity to the substrate concentration, which partly shifted towards 1,3-propanediol and acetate in a 2:1 ratio at increasing substrate concentrations. Glucose fermentation mainly generated acetate, ethanol and butyrate. At higher substrate concentrations, acetate and ethanol were the dominant products. Co-fermentations of glucose-glycerol were performed with both mixed cultures, previously cultivated on glucose and on glycerol. The product spectrum of the two experiments was very similar: the main products were ethanol and butyrate (38% and 34% of the COD converted, respectively). The product spectrum obtained for glucose and glycerol fermentation could be explained based on the general metabolic pathways found for fermentative microorganisms and on the metabolic constraints: maximization of the ATP production rate and balancing the reducing equivalents involved.     

Conditions for Ethanol Production during Bioconversion of Cellulose-Containing Raw Material

Several natural associations composed of thermophilic anaerobic bacteria capable of utilizing various cellulose materials at 60 ± 2°C and pH 6.0–7.0 were isolated from the sludge of Kamchatka geothermal springs. The rate of ethanol production (up to 1.7 g/l per day) and the concentration of ethanol in the medium (up to 1.2%), as well as the fermentation period (10–15 days), were determined under anaerobic conditions in the presence of cellulose, coniferous sawdust, newsprint, or paper pulp as a carbon source. Microorganisms were found that inhibited the production of ethanol. The initial pH value was found to influence both the ethanol production rate and ethanol/acetate ratio. A pH decrease from 7.0 to 5.0 led to a 6.7-fold increase in ethanol production and caused a 23.8-fold increase in the ethanol/acetate ratio.     

Production and pH-Dependent Bactericidal Activity of Lactocin S, a Lantibiotic from Lactobacillus sake L45

The amount of lactocin S activity in a growing culture depends on the growth stage of the bacteria, the pH of the medium, the presence of ethanol, and the aeration of the culture. We observed the highest levels of bacteriocin activity in the early stationary growth phase of cultures at 30 deg C. When Lactobacillus sake L45 was grown in a fermentor at pH 5, it produced 2,000 to 3,000 bacteriocin units per ml, which represented an 8- to 10-fold increase in bacteriocin production compared with production during batch culture fermentation. Less than 10% of this level of bacteriocin activity was observed during fermentation at pH 6.0. When 1% ethanol was included in the growth medium, a two- to fourfold increase in the bacteriocin yield was observed. Aerating the culture during growth almost completely eliminated the production of active bacteriocin. Our results also showed that lactocin S-mediated killing of target cells depended on the pH of the culture. The pH had to be less than 6 in order to obtain a bactericidal effect with lactocin S-sensitive cells. At pH values greater than 6, lactocin S had no apparent effect on sensitive cells.     

Indirect monitoring of acetate exhaustion and cell recycle improve lactate production by non-growing Escherichia coli

A two-phase, lactate fermentation by Escherichia coli ALS974 generates succinate and ethanol anaerobically from acetate. These by-products can be minimized by monitoring acetate concentration indirectly with dissolved O₂ (DO) during the initial aerobic cell-growth phase. Without DO monitoring, 3 g succinate/l and 1 g ethanol/l were generated while, with monitoring, less than 1 g succinate/l and no detectable ethanol were generated with 130 g lactate/l being produced. Furthermore, using a cell-recycle fermentation with ultrafiltration prolonged the anaerobic lactate production phase from 22 to 34 h, thereby achieving a lactate productivity of 4.2 g/l h, nearly 20% greater than the productivity of the fed-batch process.     

Anaerobic acidogenesis of a complex wastewater: I. The influence of operational parameters on reactor performance

The influence of operational, parameters, such as hydraulic retention time, organic loading rate, influent substrate concentration, pH, and temperature, on the performance of the first phase of anaerobic digestion has been investigated. A complex substrate based on beef extract was used, and six series of experimental runs were conducted, each one showing the effect of one operational variable. The predominant fermentation products were always acetic and propionic acid, independent of the values of the operational parameters. For initial COD concentrations and hydraulic retention times above the critical values identified as 3 g/L and 6 h, respectively, the degree of acidification achieved was between 30 and 60%. The degree of acidification was found to increase with the hydraulic retention time and decrease with the influent substrate concentration and organic loading rate, while the opposite held true for the rate of product formation. Furthermore, it has been demonstrated that acidification is primarily determined by the hydraulic retention time and the rate of product formation by the influent substrate concentration. The concentration of the acetic acid produced was found to depend on the operational parameters. However, the concentration of propionic acid produced depended only on the substrate availability with a consistent proportion of 8% initial COD converted to it. The optimum pH and temperature were 7 and 40 degrees C, respectively. The percentage of acetic acid as a proportion of the total volatile fatty acids produced was found to increase with increasing pH and temperature, while the percentage of propionic acid seemed to decrease accordingly. Finally the effect of the temperature on the rate of acidification followed an Arrhenius type equation with an activation energy equal to 4739 cal/mol.