嗜热厌氧纤维素降解细菌的分离、鉴定及其系统发育分析

利用纤维素降解细菌和纤维素粘附的方法分别从新鲜牛粪、高温堆肥和本实验室保存的纤维素降解富集物中分离得到4株嗜热厌氧纤维素降解细菌。分离菌株为革兰氏染色阴性,直的或稍弯曲杆菌,菌体大小为0.4μm～0.6μm×3μm～15μm,严格厌氧,不还原硫酸盐,形成芽孢。多数芽孢着生于菌体顶端。分离菌株能利用纤维素滤纸、纤维素粉Whatman CFII、微晶纤维素、纤维素粉MN300和未经处理的玉米秆芯、甘蔗渣、水稻秸杆。分离菌株在pH6.2～8.9、温度45℃～65℃范围内利用纤维素,最适pH为7.0～7.5,最适温度为55℃～60℃,发酵纤维素产生乙醇、乙酸、H2和CO2。分离菌株还可利用纤维二糖、葡萄糖、果糖、麦芽糖、山梨醇作为碳源。部分长度的16S rDNA序列分析表明,分离菌株EVAI与Clostridium thermocellum具有99.8%相似性。     

Engineering and Two-Stage Evolution of a Lignocellulosic Hydrolysate-Tolerant Saccharomyces cerevisiae Strain for Anaerobic Fermentation of Xylose from AFEX Pretreated Corn Stover

The inability of the yeast Saccharomyces cerevisiae to ferment xylose effectively under anaerobic conditions is a major barrier to economical production of lignocellulosic biofuels. Although genetic approaches have enabled engineering of S. cerevisiae to convert xylose efficiently into ethanol in defined lab medium, few strains are able to ferment xylose from lignocellulosic hydrolysates in the absence of oxygen. This limited xylose conversion is believed to result from small molecules generated during biomass pretreatment and hydrolysis, which induce cellular stress and impair metabolism. Here, we describe the development of a xylose-fermenting S. cerevisiae strain with tolerance to a range of pretreated and hydrolyzed lignocellulose, including Ammonia Fiber Expansion (AFEX)-pretreated corn stover hydrolysate (ACSH). We genetically engineered a hydrolysate-resistant yeast strain with bacterial xylose isomerase and then applied two separate stages of aerobic and anaerobic directed evolution. The emergent S. cerevisiae strain rapidly converted xylose from lab medium and ACSH to ethanol under strict anaerobic conditions. Metabolomic, genetic and biochemical analyses suggested that a missense mutation in GRE3, which was acquired during the anaerobic evolution, contributed toward improved xylose conversion by reducing intracellular production of xylitol, an inhibitor of xylose isomerase. These results validate our combinatorial approach, which utilized phenotypic strain selection, rational engineering and directed evolution for the generation of a robust S. cerevisiae strain with the ability to ferment xylose anaerobically from ACSH.     

多级串联悬浮床反应器系统中自絮凝颗粒酵母乙醇连续发酵耦合废糟液直接全循环使用的研究

在一套四级串联悬浮床生物反应器系统中,以双酶法制备的玉米粉糖化液为底物,进行了废糟液全循环条件下自絮凝颗粒酵母乙醇连续发酵的实验研究。在实验中,每隔5 d将从末级反应器收集到的发酵液集中精馏处理,得到的废糟液直接用于玉米粉调浆制糖。系统连续运行了120d,共进行了24批次实验,数据分析表明系统达到了平衡状态。在平均发酵时间为20h条件下,发酵终点乙醇浓度平均为11.7%（V/V）,残还原糖浓度平均为7.9g/L,装置运行平稳。这些工作为自絮凝颗粒酵母乙醇发酵耦合废糟液直接全循环使用、实现污染物源头减废、清洁生产奠定了理论和实验基础。     

The Intracellular pH of Clostridium paradoxum, an Anaerobic, Alkaliphilic, and Thermophilic Bacterium

When the extracellular pH was increased from 7.6 to 9.8, Clostridium paradoxum, a novel alkalithermophile, increased its pH gradient across the cell membrane ((Delta)pH, pH(infin) - pH(infout)) by as much as 1.3 U. At higher pH values (>10.0), the (Delta)pH and membrane potential ((Delta)(psi)) eventually declined, and the intracellular pH increased significantly. Growth ceased when the extracellular pH was greater than 10.2 and the intracellular pH increased to above 9.8. The membrane potential increased to 110 (plusmn) 8.6 mV at pH 9.1, but the total proton motive force ((Delta)p) declined from about 65 mV at pH 7.6 to 25 mV at pH 9.8. Between the extracellular pH of 8.0 and 10.3, the intracellular ATP concentration was around 1 mM and decreased at lower and higher pH values concomitantly with a decrease in growth rate.     

Purification and Properties of a Thermostable Pullulanase from a Newly Isolated Thermophilic Anaerobic Bacterium, Fervidobacterium pennavorans Ven5

Extremely thermophilic anaerobic fermentative bacteria growing at temperatures between 50 and 80(deg)C (optimum, 65 to 70(deg)C) were isolated from mud samples collected at Abano Terme spa (Italy). The cells were gram-negative motile rods, about 1.8 (mu)m in length and 0.6 (mu)m in width, occurring singly and in pairs. Cells commonly formed spheroids at one end similar to Fervidobacterium islandicum and Fervidobacterium nodosum. The new isolate differs from F. nodosum by the 7% higher G+C content of its DNA (40.6 mol%) but is similar to Fervidobacterium pennavorans and F. islandicum in its G+C content and phenotypic properties. The phylogenetic dendrogram indicates that strain Ven5 belongs to the order Thermotogales and shows the highest 16S ribosomal DNA sequence similarity to F. pennavorans, F. islandicum, and F. nodosum, with similarities of 99.0, 98.6, and 96.0%, respectively. During growth on starch the strain produced a thermostable pullulanase of type I which preferentially hydrolyzed (alpha)-1,6 glucosidic linkages. The enzyme was purified 65-fold by anion-exchange, gel permeation, and hydrophobic chromatography. The native pullulanase has a molecular mass of 240,000 Da and is composed of three subunits, each with a molecular mass of 77,600 Da as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Optimal conditions for the activity and stability of the purified pullulanase were pH 6.0 and 85(deg)C. At pH 6.0, the half-life of the enzyme was over 2 h at 80(deg)C and 5 min at 90(deg)C. This is the first report on the presence of pullulanase type I in an anaerobic bacterium.     

自絮凝酵母SPSC01在组合反应器系统中酒精连续发酵的研究

建立了一套由四级磁力搅拌发酵罐串联组成、总有效容积4000mL的小型组合生物反应器系统 ,其中一级罐作为种子培养罐。以脱胚脱皮玉米粉双酶法制备的糖化液为种子培养基和发酵底物 ,进行了自絮凝颗粒酵母酒精连续发酵的研究。种子罐培养基还原糖浓度为100g L ,添加 (NH₄)₂HPO₄ 和KH₂PO₄ 各 20g L ,以0.017h^-1 的恒定稀释速率流加 ,并溢流至后续酒精发酵系统。发酵底物初始还原糖浓度 220g/L ,添加 (NH₄)₂HPO₄ 15g/L和KH₂PO₄2 5g/L ,流加至第一级发酵罐 ,稀释速率分别为 0.017、0.025、0.033、0.040和0.05 0h^-1。实验数据表明 ,自絮凝颗粒酵母在各发酵罐中呈部分固定化状态 ,在稀释速率0.040h^-1 条件下 ,发酵系统呈一定的振荡行为 ,其他四个稀释速率实验组均能够达拟稳态。当稀释速率不超过 0 0 33h^-1 ,流出末级发酵罐的发酵液中酒精浓度可以达到 12 % (V/V)以上 ,残还原糖和残总糖分别在 0 11%和 0 35 % h^-1,流出末级发酵罐的发酵液中酒精浓度可以达到12%（V/V）以上,残还原糖和残总糖分别在0.11%和0.35%（W/V）以下。在稀释速率为0.033h^-1时,计算发酵系统酒精的设备生产强度指标为3.32（g·L^-1·h^-1）,与游离酵母细胞传统酒精发酵工艺相比,增加约1倍。     

利用甘蔗糖蜜酒精发酵液生产腐植酸的菌种鉴定及发酵条件研究

从土壤中筛选出一株适合用甘蔗糖蜜酒精发酵液生产腐植酸的菌株H812。单因素实验和正交实验结果表明,该菌株培养的最适酒精发酵液浓度为16°Bx,最适培养条件为:时间8d、温度34℃、摇床转速200r/min、初始pH7.0、接种量12%和装液量50ml/250ml,其中温度对发酵产品影响显著。在优化的条件下,腐植酸产量为38.12 g/L,较优化前提高了148.34%。对H812菌株进行形态特征分析以及ITS序列分析,推测该菌株为曲霉属真菌。     

Constituents of Fusel Oils Obtained Through the Fermentation of Corn,Barley and Sweet Molasses

The components of the fusel oils obtained through the fermentation of corn, barley and sweet molasses were separated by fractional distillation and adsorption chromatography. Each of the components was analyzed by gas chromatography and infrared spectrometry. Newly isolated components were as follows: methyl heptenone, fatty acids having odd number of carbon atoms, acetic esters of higher aliphatic alcohols ranging from C₆ to C₁₁, benzyl alcohol, ethyl phenylacetate, phenylethyl propionate, acetophenone, limonene, and α-ionone. Moreover, an unknown ketone C₁₀H₁₆O were isolated from corn fusel oil, and trans-neroridol from sweet molass fusel oil.     

Molecular Cloning and Structure of the Gene for Esterase from a Thermophilic Bacterium,Bacillus stearothermophilus IFO 12550

Isoezymes of aspartate aminotransferase (EC 2.6.1.1, AAT) were purified to . homogeneity and crystallized from bran of rice (Oryza sativa cv. Koganemasari). The native molecular weights of AAT-1 and AAT-2 isoenzymes were 88,000 and 94,000 with the subunit molecular weights of 44,000 and 47,000, respectively, indicating that the lloloenzymes of the isoencymes are dimers. The isoelectric points of AAT-1 and AAT-2 were pH 6.5 and 5.0, respectively: both isoenzymes have no subform. The isoenzymes showed · similar K_ms for four. natural substrates, with the exception that AAT-1 had a higher affinity for L-glutamate than AAT-2. Amino donor and acceptor specificities of the isoenzymes were almost identical and fairly high. Amino acid compositions of the isoenzymes were similar but not the same. The isoenzymes contained one mole of pyridoxal 5′-phosphate (PLP) per subunit and showed characteristic absorption spectra of PLP enzymes. Polyclonal antibodies raised against AAT-1 selectively cross-reacted with AAT-1 but not with AAT-2. Conversely, the antibody raised against AAT-2 selectively cross-reacted with AAT-2 but not with AAT-1.     

产低温脂肪酶非极端细菌的筛选、产酶发酵及粗酶性质研究

目的：筛选产低温脂肪酶非极端细菌菌株,扩大脂肪酶的应用范围。方法：利用维多利亚蓝B平板显色法和摇瓶发酵法,从土壤中筛选产脂肪酶菌株,通过菌落形态和菌体特征观察初步对菌种进行鉴定,并对该菌株的产酶发酵培养基进行了优化。结果：得到一株产低温脂肪酶非极端细菌菌株sybc—li一1,该菌株适宜产酶培养基（％）为淀粉1、牛肉浸膏1、NaNO3 0．08、CaCl2 0．04、MgSO4 0．04、橄榄油2和OP1;初始DH8、30℃、200r／min培养72h,脂肪酶活力可高达到30．2U／mL;所产脂肪酶粗酶最适作用温度20℃,最适pH9．5,0℃时仍能保持70％的酶活性,属于低温酶;该酶与目前报道的低温脂肪酶相比,有较好的热稳定性,粗酶在pH8．5、70℃条件下保温60mla,酶活力损失30％。结论：该菌株为自然环境中筛选的非极端细菌,所产脂肪酶为低温脂肪酶,在开发应用上有良好的前景。     

Continuous Synthesis and Excretion of the Compatible Solute Ectoine by a Transgenic, Nonhalophilic Bacterium

The compatible solute 1,4,5,6-tetrahydro-2-methyl-4-pyrimidinecarboxylic acid (ectoine) acts in microorganisms as an osmotic counterweight against halostress and has attracted commercial attention as a protecting agent. Its production and application are restricted by the drawbacks of the discontinuous harvesting procedure involving salt shocks, which reduces volumetric yield, increases reactor corrosion, and complicates downstream processing. In order to synthesize ectoine continuously in less-aggressive media, we introduced the ectoine genes ectABC of the halophilic bacterium Chromohalobacter salexigens into an Escherichia coli strain using the expression vector pASK-IBA7. Under the control of a tet promoter, the transgenic E. coli synthesized 6 g liter⁻¹ ectoine with a space-time yield of 40 mg liter⁻¹ h⁻¹, with the vast majority of the ectoine being excreted.     

Urea Hydrogen Peroxide Reduces the Numbers of Lactobacilli, Nourishes Yeast, and Leaves No Residues in the Ethanol Fermentation

Urea hydrogen peroxide (UHP) at a concentration of 30 to 32 mmol/liter reduced the numbers of five Lactobacillus spp. (Lactobacillus plantarum, L. paracasei, Lactobacillus sp. strain 3, L. rhamnosus, and L. fermentum) from ~10⁷ to ~10² CFU/ml in a 2-h preincubation at 30°C of normal-gravity wheat mash at ~21 g of dissolved solids per ml containing normal levels of suspended grain particles. Fermentation was completed 36 h after inoculation of Saccharomyces cerevisiae in the presence of UHP, even when wheat mash was deliberately contaminated (infected) with L. paracasei at ~10⁷ CFU/ml. There were no significant differences in the maximum ethanol produced between treatments when urea hydrogen peroxide was used to kill the bacteria and controls (in which no bacteria were added). However, the presence of L. paracasei at ~10⁷ CFU/ml without added agent resulted in a 5.84% reduction in the maximum ethanol produced compared to the control. The bactericidal activity of UHP is greatly affected by the presence of particulate matter. In fact, only 2 mmol of urea hydrogen peroxide per liter was required for disinfection when mashes had little or no particulate matter present. No significant differences were observed in the decomposition of hydrogen peroxide in normal-gravity wheat mash at 30°C whether the bactericidal agent was added as H₂O₂ or as urea hydrogen peroxide. NADH peroxidase activity (involved in degrading H₂O₂) increased significantly (P = 0.05) in the presence of 0.75 mM hydrogen peroxide (sublethal level) in all five strains of lactobacilli tested but did not persist in cells regrown in the absence of H₂O₂. H₂O₂-resistant mutants were not expected or found when lethal levels of H₂O₂ or UHP were used. Contaminating lactobacilli can be effectively managed by UHP, a compound which when used at ca. 30 mmol/liter happens to provide near-optimum levels of assimilable nitrogen and oxygen that aid in vigorous fermentation performance by yeast.     

Response Surface Models To Describe the Effects of Temperature, pH, and Ethanol Concentration on Growth Kinetics and Fermentation End Products of a Pectinatus sp

Growth curve data which had been fitted by use of the Gompertz and logistic functions have permitted the development of mathematical models to describe the growth of a Pectinatus sp. by several variables, namely, temperature, pH, and ethanol concentration. The activation energy of this microorganism was lower at 26 to 35(deg)C than at 15 to 22(deg)C. On the basis of the Arrhenius law, growth rate, maximum population density, and cell yield models have been developed by introducing the different activation energy (E(infa)) values. According to the model, optimal conditions were 35(deg)C, pH 6.5, and 0% (vol/vol) ethanol for the growth rate. For cell density and cell yield, optimal conditions were 32(deg)C, pH 6.0, and 1% (vol/vol) ethanol. No growth was observed for ethanol concentrations above 8% and pH values below 4.0. Other equations have also been made to describe the major end products fermented during fermentation by a Pectinatus sp. The synthesis of propionate and acetate is maximal at 28(deg)C at pHs of 5.5 and 6.25, respectively. This model completes the model suggested by Membre and Tholozan (J. Appl. Bacteriol. 77:456-460, 1994), which includes only one variable, i.e., the glucose concentration.     

Effect of Ethanol,Sulfur Dioxide and Glucose on the Growth of Wine Spoilage Yeasts Using Response Surface Methodology

Response surface methodology (RSM) was used to study the effect of three factors, sulfur dioxide, ethanol and glucose, on the growth of wine spoilage yeast species, Zygosaccharomyces bailii, Schizosaccharomyces pombe, Saccharomycodes ludwigii and Saccharomyces cerevisiae. Seventeen central composite rotatable design (CCRD) trials were designed for each test yeast using realistic concentrations of the factors (variables) in premium red wine. Polynomial regression equations were fitted to experimental data points, and the growth inhibitory conditions of these three variables were determined. The overall results showed Sa. ludwigii as the most resistant species growing under high ethanol/free sulfur dioxide concentrations, i.e., 15% (v/v)/20 mg L^-1, 14% (v/v)/32 mg L^-1 and 12.5% (v/v)/40 mg L^-1, whereas other yeasts did not survive under the same levels of ethanol/free sulfur dioxide concentrations. The inhibitory effect of ethanol was primarily observed during longer incubation periods, compared with sulfur dioxide, which showed an immediate effect. In some CCRD trials, Sa. ludwigii and S. cerevisiae showed growth recovery after a short death period under the exposure of 20–32 mg L^-1 sulfur dioxide in the presence of 11% (v/v) or more ethanol. However, Sc. pombe and Z. bailii did not show such growth recovery under similar conditions. Up to 10 g L^-1 of glucose did not prevent cell death under the sulfur dioxide or ethanol stress. This observation demonstrates that the sugar levels commonly used in wine to sweeten the mouthfeel do not increase wine susceptibility to spoilage yeasts, contrary to the anecdotal evidence.     

Expression of the Escherichia coli pntA and pntB Genes, Encoding Nicotinamide Nucleotide Transhydrogenase, in Saccharomyces cerevisiae and Its Effect on Product Formation during Anaerobic Glucose Fermentation

We studied the physiological effect of the interconversion between the NAD(H) and NADP(H) coenzyme systems in recombinant Saccharomyces cerevisiae expressing the membrane-bound transhydrogenase from Escherichia coli. Our objective was to determine if the membrane-bound transhydrogenase could work in reoxidation of NADH to NAD⁺ in S. cerevisiae and thereby reduce glycerol formation during anaerobic fermentation. Membranes isolated from the recombinant strains exhibited reduction of 3-acetylpyridine-NAD⁺ by NADPH and by NADH in the presence of NADP⁺, which demonstrated that an active enzyme was present. Unlike the situation in E. coli, however, most of the transhydrogenase activity was not present in the yeast plasma membrane; rather, the enzyme appeared to remain localized in the membrane of the endoplasmic reticulum. During anaerobic glucose fermentation we observed an increase in the formation of 2-oxoglutarate, glycerol, and acetic acid in a strain expressing a high level of transhydrogenase, which indicated that increased NADPH consumption and NADH production occurred. The intracellular concentrations of NADH, NAD⁺, NADPH, and NADP⁺ were measured in cells expressing transhydrogenase. The reduction of the NADPH pool indicated that the transhydrogenase transferred reducing equivalents from NADPH to NAD⁺.     

Comparison of the Bacterial Communities in Anaerobic, Anoxic, and Oxic Chambers of a Pilot A2O Process Using Pyrosequencing Analysis

A₂O process is a sequential wastewater treatment process that uses anaerobic, anoxic, and oxic chambers for nitrogen and phosphorus removal. In this study, the bacterial communities among these chambers were compared, and the diversity of the bacteria involved in nitrogen and phosphorus removal was surveyed. A pilot-scale A₂O process (50 m³ day^?1) was operated for more than 6 months, and bacterial 16S rRNA gene diversity was analyzed using pyrosequencing. A total of 7,447 bacterial sequence reads were obtained from anaerobic (1,546), anoxic (2,158), and oxic (3,743) chambers. Even though there were differences in the atmospheric condition and functionality, no prominent differences could be found in the bacterial community of the three chambers of the pilot A₂O process. All sequence reads, which were taxonomically analyzed using the Eztaxon-e database, were assigned into 638 approved or tentative genera. Among them, about 72.2 % of the taxa were contained in the phyla Proteobacteria and Bacteroidetes. Phosphate-accumulating bacteria, Candidatus Accumulibacter phosphatis, and two other Accumulibacter were found to constitute 3.1 % of the identified genera. Ammonia-oxidizing bacteria, Nitrosomonas oligotropha, and four other phylotypes in the same family, Nitrosomonadaceae, constituted 0.2 and 0.9 %, respectively. Nitrite-oxidizing bacteria, Nitrospira defluvii, and other three phylotypes in the same family, Nitrospiraceae, constituted 2.5 and 0.1 %, respectively. In addition, Dokdonella and a phylotype of the phylum Chloroflexi, function in nitrogen and/or phosphate removal of which have not been reported in the A₂O process, constituted the first and third composition among genera at 4.3 and 3.8 %, respectively.     

Properties and Role of Glyceraldehyde-3-Phosphate Dehydrogenase in the Control of Fermentation Pattern and Growth in a Ruminal Bacterium, <Emphasis Type="Italic">Streptococcus bovis</Emphasis>

To clarify the control of glycolysis and the fermentation pattern in Streptococcus bovis, the molecular and enzymatic properties of NAD⁺-specific glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were examined. The GAPDH gene (gapA) was found to cluster with several others, including those that encode phosphoglycerate kinase and translation elongation factor G, however, gapA was transcribed in a monocistronic fashion. Since biochemical properties, such as optimal pH and affinity for glyceraldehyde-3-phosphate (GAP), were not very different between GAPDH- and NADP⁺-specific glyceraldehyde-3-phosphate dehydrogenase (GAPN), the flux from GAP may be greatly influenced by the relative amounts of these two enzymes. Using S. bovis JB1 as a parent, JB1gapA and JB1ldh, which overproduce GAPDH and lactate dehydrogenase (LDH), respectively, were constructed to examine the control of the glycolytic flux and lactate production. There were no significant differences in growth rates and formate-to-lactate ratios among JB1, JB1gapA, and JB1ldh grown on glucose. When grown on lactose, JB1ldh showed a much lower formate-to-lactate ratio than JB1gapA, which showed the highest NADH-to-NAD⁺ ratio. However, growth rates did not differ among JB1, JB1gapA, and JB1ldh. These results suggest that GAPDH is not involved in the control of the glycolytic flux and that lactate production is mainly controlled by LDH activity.