酿酒酵母INVSC1对油菜菌核病菌抑制作用的研究

以油菜菌核病菌为供试病原菌,在摇瓶发酵备件下,研究了酿酒酵母菌INVSC1在各种培养条件下对油菜菌核病菌的抑菌效果,确定了酿酒酵母的最佳培养条件：最佳培养温度为28℃,培养时间为20h,接种量为5％。酿酒酵母能抑制油菜菌核病可能与它发酵产乙醇有关。酿酒酵母抑制油菜菌核病的发现使生物防治又多了一类新的可供选择的生防菌。     

根瘤菌培养基的优化和剂型的比较研究*

以费氏中华根瘤菌 (Sinorhizobiumfredii)HN0 1和大豆慢生根瘤菌 (Bradyrhizobiumjaponi cum)USDA1 1 0作为供试菌 ,进行YMA、TY、SM、PA和BSE等 5种培养基的比较试验。结果表明 ,两种菌都在BSE中生长速度最快。对USDA1 1 0进行培养基的优化试验 ,筛选出一种能将生长速度提高 2倍左右的优化培养基。制作了供试菌的固体、液体与冻干菌剂 ,结果表明在 3种供试固体剂型载体中 ,草炭要优于蛭石 ,蛭石又优于珍珠岩。供试菌在两种液体剂型中的存活率和活菌数均较高 ,氮气或真空剂型对存活率的影响没有明显的差别。两种冻干菌剂的结果表明 ,供试菌在冻干过程中的死亡率较高 ,液氮冻干优于常规冻干。冻干菌剂在储存条件下的存活率为 : 2 0℃ >4℃ >室温     

玫烟色拟青霉最适液体培养条件的研究

通过对不同营养和不同培养条件下玫烟色拟青霉菌丝生物量和产孢量的研究,结果表明:葡萄糖为玫烟色拟青霉液体培养的最适碳源,蛋白胨为该菌生长的最适氮源,C/N为10∶1~20∶1最适于玫烟色拟青霉菌丝生长和产孢;25℃2、4 h全光照条件,对该菌生长和产孢均有利。接种后144~168 h时,菌丝生物量和产孢量均达到高峰,分别为31.72 mg/mL、24.62孢子/mL,为黑暗条件下的1.5倍和18.3倍,因此玫烟色拟青霉液体发酵终点应选择在接种后144~168 h为最好。     

单、双菌发酵淡豆豉不同发酵期纤溶酶活性

目的采用单菌种或双菌种发酵制备淡豆豉,对比分析不同发酵阶段发酵产物的纤溶酶活性。方法以黄豆和黑豆为发酵基料,采用枯草芽胞杆菌(1号菌)、伞枝梨头霉(2号菌)、米曲霉(3号菌)单菌种和双菌种发酵(包括前酵和后酵)。采用琼脂糖—纤维蛋白平板法测定不同发酵阶段发酵产物的纤溶酶活性。结果以黄豆为基料,以枯草芽胞杆菌单菌种37℃发酵7d,再经42℃后酵3d,其纤溶酶活性达到最高值804.61IU/g,显著高于其以黑豆为基料发酵的纤溶酶活性。1号菌单独发酵的淡豆豉纤溶酶活性高于2号和3号菌单独发酵及三者两两组合发酵淡豆豉的纤溶酶活性。结论以纤溶酶活性为考察指标,淡豆豉最佳发酵条件为:以黄豆为基料,以枯草芽胞杆菌单菌进行发酵,通过37℃前酵7d,再经42℃后酵3d其纤溶酶活性最高。     

三株散囊菌发酵茶叶的初步研究

散囊菌属真菌(Eurotium spp.)能赋予发酵茶独特的口感和香味。本研究利用前期从广西某六堡茶中筛选并鉴定的三株散囊菌属真菌Aspergillus chevalieri E2、Aspergillus chevalieri E3与Aspergillus cristatus E6,探讨在不同温度下以优化察氏液体培养基培养的生长状况,发酵前不同灭菌条件下的茶叶品质,以及所得茶汤中茶多酚含量、总抗氧化能力和DPPH·自由基清除能力。结果显示:三株真菌在优化的察氏液体培养基中31℃～34℃下都能良好生长。茶叶发酵温度为28℃,三株真菌在发酵初始含水量为20%以上生长良好,其中E3和混合发酵组的生长速度最快。E2在茶叶表面生长出大量金黄色子囊果以及大量浅绿色分生孢子梗; E3几乎只有浅绿色分生孢子梗; E6几乎只有金黄色子囊果。发酵茶叶制作的茶汤内茶多酚含量比未发酵低,抗氧化性指标也有所下降,说明本实验真菌发酵促进了茶内抗氧化物质的氧化。本研究对源于六堡茶不同散囊菌属真菌的茶叶发酵有重要参考价值。     

产酸丙酸杆菌生长特性及5L罐发酵动力学研究

论文在摇瓶水平对产酸丙酸杆菌基本生长特性（温度、pH、摇床转速、接种量、种龄等）、碳源、氮源利用情况、产物抑制及5 L罐发酵动力学进行了研究。结果表明,该菌在32℃,初始pH 6．5,摇床转速150 r/min,接种24 h的种子液,接种量为5％条件下,产酸丙酸杆菌生长及产酸水平达最高值;该菌可利用碳源十分广泛,但对氮源要求比较高,只可利用有机氮源;在不同初始葡萄糖浓度下,产酸丙酸杆菌生长及产酸水平差异不大,无明显底物抑制现象;在2g/L的初始丙酸盐浓度下,该菌生长受到明显抑制;在5L发酵罐中,初始葡萄糖浓度为58．8 g/L,发酵72 h,葡萄糖消耗完全,丙酸终浓度达22．4 g/L,丙酸得率和产率分别达0．381 g/g和0．295 g/（L·h）,丙酸占总酸比例达72．10％。     

4种木霉菌对棉花黄萎病菌抑制作用的测定

以绿色木霉(Trichoderma viride)、康氏木霉(Trichoderma koningii)和二株未知木霉菌株(Trichoderma spp.)为供试木霉菌株,采用对峙培养法测定了不同温度处理下对棉花黄萎病菌(Verticilliam dahliae)的拮抗作用。结果表明,木霉在不同温度下对棉花黄萎病菌的抑制作用不一,其中以30℃和25℃黑暗条件下木霉对棉花黄萎病菌菌丝生长的抑制作用最强,对峙培养5d后,绿色木霉、康氏木霉和二株未知木霉菌在30℃条件下对棉花黄萎病菌的抑制率分别达到67.3%、65.9%、56.8%、65.9%,25℃条件下的抑制率分别为65.9%、72.9%、65.9%、78.8%;20℃下木霉菌株对棉花黄萎病菌的抑制作用次之,10℃、15℃和35℃条件下木霉菌丝扩散速度较慢,且孢子产生量少,不能有效地抑制棉花黄萎病菌菌丝的扩展,表明环境温度过高或过低对木霉菌丝的生长及分生孢子的产生均有较大影响。该研究为筛选棉花黄萎病菌更为有效的生防木霉菌株提供理论依据。     

VC三菌种一步发酵方法的构建与研究

就维生素C微生物一步发酵方法进行了探索,构建了酮古龙酸杆菌、氧化葡萄糖酸杆菌和芽孢杆菌三菌混菌一步发酵的方法。研究发现,植物内生芽孢杆菌可以与酮古龙酸杆菌配合,促进酮古龙酸杆菌生长和产酸。在有山梨醇存在的条件下酮古龙酸杆菌及其伴生菌能够快速地生长增殖,植物内生芽孢杆菌在发酵的10h中不断消耗山梨醇。5L的发酵罐中,酮古龙酸杆菌、氧化葡萄糖酸杆菌和植物内生芽孢杆菌三菌混菌一步发酵在恒定的30℃温度,600r/min搅拌速度和1.5vvm通气条件下,补料发酵过程中醇酸质量转化率达到了81.89%,在分批发酵过程中,醇酸质量转化率达到了87.90%,进一步优化了维生素C生产工艺。     

调控pH提高分批发酵赤霉素GA3产量

为高效率发酵生产GA₃,对藤仓赤霉菌发酵过程pH进行优化调控研究。采用5L全自动发酵罐,在pH 3.0-5.0条件下,对藤仓赤霉菌菌丝生长及GA₃产量的影响进行了考察,实验数据表明：在pH 4.0条件下,菌比生长速率可获最大值,为0.395/h;而pH 3.0条件下,GA₃比生成速率最大,达到4.43mg/(g?h)。基于不同pH条件下,对菌比生长速率、得率、GA₃比生成速率的影响,提出GA₃分批发酵过程中的pH调控策略,即：0-20h,pH自然;20-50h,pH 4.0;50-80h,pH 3.0-3.5;80h后控制pH为3.5-4.0。在此控制模式下,经过196h发酵GA₃的终产量达到2 224mg/L,GA₃产率44.5mg/g,GA₃生产强度0.242mg/(L?h),分别比不控制pH条件下发酵的数值增长了7.75%、7.74%、8.04%,表明该pH控制策略能增进GA₃发酵生产效率。     

鸭跖草生防菌叶点霉F-3菌株生物学特性的研究

主要研究影响鸭跖草生防菌叶点霉菌株菌丝生长和孢子萌发的条件。研究表明,PDA培养基和鸭跖草培养基是该菌生长的适宜培养基,该菌生长的温度范围为20~35℃,最适温度为25℃,分生孢子萌发的温度范围为25~35℃,最适温度为30℃;pH为6时生长最快,偏酸偏碱有利于孢子的萌发,在pH为10时萌发最好。F-3菌株在供试的6种碳源中生长速度相差不大,在不同氮源中差异显著,以硝酸铵为氮源生长最好。     

The Anaerobic Fungus Neocallimastix sp. Strain L2: Growth and Production of (Hemi)Cellulolytic Enzymes on a Range of Carbohydrate Substrates

The anaerobic fungus Neocallimastix sp. strain L2, isolated from the feces of a llama, was tested for growth on a range of soluble and insoluble carbohydrate substrates. The fungus was able to ferment glucose, cellobiose, fructose, lactose, maltose, sucrose, soluble starch, inulin, filter paper cellulose, and Avicel. No growth was observed on arabinose, galactose, mannose, ribose, xylose, sorbitol, pectin, xylan, glycerol, citrate, soya, and wheat bran. The fermentation products after growth were hydrogen, formate, acetate, ethanol, and lactate. The fermentation pattern was dependent on the carbon source. In general, higher hydrogen production resulted in decreased formation of lactate and ethanol. Recovery of the fermented carbon in products at the end of growth ranged from 50% to 80%. (Hemi)cellulolytic enzyme activities were affected by the carbon source. Highest activities were found in filtrates from cultures grown on cellulose. Growing the fungus on inulin and lactose yielded the lowest cellulolytic activities. Highest specific activities for avicelase, endoglucanase, β-glucosidase, and xylanase were obtained with Avicel as the substrate for growth (0.29, 5.9, 0.57, and 13 IU · mg⁻¹ protein, respectively). Endoglucanase activity banding patterns after SDS-PAGE were very similar for all substrates. Minor differences indicated that enzyme activities may in part be the result of secretion of different sets of isoenzymes. Received: 10 July 1996 / Accepted: 22 July 1996     

Experiments on the microbiology of cellulose decomposition in a municipal sewage plant

Summary Analyses of sewage solids show cellulose to be one of the chief components. Culture counts of cellulolytic bacteria in a primary anaerobic sewage digestor show them to be present in numbers as high as 1 million per ml. The tendency of cellulolytic bacteria to cling to cellulose fibers makes it highly probable that the number of cellulolytic cells is much larger. All 10 cellulolytic strains isolated in pure culture show better growth in solid than in liquid media, and for some of them agar possesses growth promoting properties. For some strains, phytone and trypticase can replace the agar but other strains could not be grown in media containing no agar. Hydrogen, carbon dioxide, ethanol, formic acid, acetic acid, and lactic acid have been identified as fermentation products and glucose shown to be a product of cellulose digestion. Cellobiose, starch, dextrin, and maltose were fermented by 5 tested strains, inulin and esculin by one of them, but none of 17 other carbohydrates, including glucose, were attacked. The rate of cellulose fermentation by a mixed culture of aClostridium sp. and a cellulose decomposer is much greater than the rate by the latter alone. The rate of fermentation by a pure culture is not affected by acetate concentrations up to 5000 parts per million. It is postulated that the rate of fermentation of cellulose may be the factor limiting the rate of sewage fermentation though more evidence regarding rates of fermentation of other constituents of sewage is needed before final conclusions can be drawn. This investigation was supported in part by a research grant from the National Institute of Health, U.S. Public Health Service.     

The characteristics of a new non-spore-forming cellulolytic mesophilic anaerobe strain CM126 isolated from municipal sewage sludge

A new mesophilic anaerobic cellulolytic bacterium, CM126, was isolated from an anaerobic sewage sludge digester. The organism was non-spore-forming, rod-shaped, Gram-negative and motile with peritrichous flagella. It fermented microcrystalline Avicel cellulose, xylan, Solka floc cellulose, filter paper, L-arabinose, D-xylose, β-methyl xyloside, D-glucose, cellobiose and xylitol and produced indole. The % G + C content was 36. Acetic acid, ethanol, lactic acid, pyruvic acid, carbon dioxide and hydrogen were produced as metabolic products. This strain could grow at 20–44·5°C and at pH values 5·2–7·4 with optimal growth at 37–41·5°C and pH 7. Both endoglucanase and xylanase were detected in the supernatant fluid of a culture grown on medium containing Avicel cellulose and cellobiose. Exoglucanase could not be found in either supernatant fluid or the cell lysate. When cellulose and cellobiose fermentation were compared, the enzyme production rate in cellobiose fermentation was higher than in cellulose fermentation. The optimum pH for both enzyme activities was 5·0, the optimum temperature was 40°C for the endoglucanase and 50°C for the xylanase. Both enzyme activities were inhibited at 70°C. Co-culture of this organism with a Methanosarcina sp. (A145) had no effect on cellulose degradation and both endoglucanase and xylanase were stable in the co-culture.     

Sensitivity of various yeasts to crude cellulolytic enzyme complexes fromTrichoderma reesei

Summary Twenty-six yeast strains, representative of different yeast genera, were tested for their sensitivity to crude extracellular cellulolytic enzyme complexes obtained from the fungusTrichoderma reesei QM 9414 and its mutants M 6 and MHC 22 (Microcrystalline cellulose was the sole carbon source.) Practically all the yeast strains tested were found to be sensitive, exhibiting signs of cellwall weakening and lysis during prolonged incubation with the emzymes fromTrichoderma. Under growth conditions, the effect of cellulolytic enzymes on yeast cells and their growth rates was much less pronounced. However, at increased cellulase concentrations (5 mg/ml) in the growth medium, lysis of stationary phase yeast cells was observed.     

Interactions among cellulolytic bacteria from an anaerobic digester

High cellulolytic activity of particular strains did not cause dominance of one, or a few, species of fiber-digesting bacteria in a cattlewaste anaerobic digester. The population contained a large number of species and varieties with different cellulolytic and fiber-digesting activities. Although mixed cultures of some of these bacteria showed no intereffects, with others, cellulolysis was less or in some cases greater than that shown by individual components of the cultures. The interactions were probably related to effects on growth of the bacteria rather than on activities of components of the cellulase enzyme complex, and culture filtrates of two of the more numerous cellulolytic species ofClostridium affected growth of other cellulolytic bacteria. The inhibitory factor(s) appeared to be of bacteriocin type, but the stimulatory factor(s) was unknown. It was suggested that these interactions are localized or short-lived in the digester, and so the population remains in a dynamic steady state.Some inhibitions of growth of rumen cellulolytic bacteria were caused by the digester bacteria, but it was suggested that factors other than these inhibitions are responsible for the absence of rumen bacteria from anaerobic digesters.     

Effect of the carbohydrate growth substrate on polysaccharolytic enzyme formation by anaerobic fungi isolated from the foregut and hindgut of nonruminant herbivores and the forestomach of ruminants

The effect of the carbohydrate growth substrate on polysaccharide-degrading enzyme formation by anaerobic fungi was examined using four strains of Piromyces isolated from hindgut fermenters, three Piromyces isolates from the pre-peptic forestomach of macropodid marsupials, and two ruminal isolates of Neocallimastix spp. The range of enzymes formed by the nine isolates was similar although, under the growth conditions examined, one hindgut isolate did not form amylolytic enzymes. The cellulolytic and xylanolytic enzyme profiles were the same: inter-strain differences in the levels of enzymic activity were apparent, but they were not related to either the genus or intestinal origin of the isolates. Pectin degrading enzymes were not detected in any of the isolates. The cellulolytic and xylanolytic enzymes were formed constitutively during growth on mono-, di- and polysaccharidic carbohydrates but the specific activities were both strain-and substrate-dependent. The activities were considerably lower in glucose-grown preparations of three of the fungi (one each from the hindgut, foregut and rumen) indicating that enzyme synthesis was repressed by glucose; enzyme formation by the other isolates studied was not controlled by catabolite regulatory mechanisms.     

Mesophilic cellulolytic clostridia from freshwater environments

Eight strains of obligately anaerobic, mesophilic, cellulolytic bacteria were isolated from mud of freshwater environments. The isolates (C strains) were rod-shaped, gram negative, and formed terminal spherical to oval spores that swelled the sporangium. The guanine plus cytosine content of the DNA of the C strains ranged from 30.7 to 33.2 mol% (midpoint of thermal denaturation). The C strains fermented cellulose with formation primarily of acetate, ethanol, CO(2), and H(2). Reducing sugars accumulated in the supernatant fluid of cultures which initially contained >/=0.4% (wt/vol) cellulose. The C strains resembled Clostridium cellobioparum in some phenotypic characteristics and Clostridium papyrosolvens in others, but they were not identical to either of these species. The C strains differed from thermophilic cellulolytic clostridia (e.g., Clostridium thermocellum) not only in growth temperature range but also because they fermented xylan and five-carbon products of plant polysaccharide hydrolysis such as d-xylose and l-arabinose. At 40 degrees C, cellulose was degraded by cellulolytic mesophilic cells (strain C7) at a rate comparable to that at which C. thermocellum degrades cellulose at 60 degrees C. Substrate utilization and growth temperature data indicated that the C strains contribute to the anaerobic breakdown of plant polymers in the environments they inhabit.     

The characteristics of a new non-spore-forming cellulolytic mesophilic anaerobe strain CM126 isolated from municipal sewage sludge

A new mesophilic anaerobic cellulolytic bacterium, CM126, was isolated from an anaerobic sewage sludge digester. The organism was non-spore-forming, rod-shaped, Gram-negative and motile with peritrichous flagella. It fermented microcrystalline Avicel cellulose, xylan, Solka floc cellulose, filter paper, L-arabinose, D-xylose, beta-methyl xyloside, D-glucose, cellobiose and xylitol and produced indole. The % G + C content was 36. Acetic acid, ethanol, lactic acid, pyruvic acid, carbon dioxide and hydrogen were produced as metabolic products. This strain could grow at 20-44.5 degrees C and at pH values 5.2-7.4 with optimal growth at 37-41.5 degrees C and pH 7. Both endoglucanase and xylanase were detected in the supernatant fluid of a culture grown on medium containing Avicel cellulose and cellobiose. Exoglucanase could not be found in either supernatant fluid or the cell lysate. When cellulose and cellobiose fermentation were compared, the enzyme production rate in cellobiose fermentation was higher than in cellulose fermentation. The optimum pH for both enzyme activities was 5.0, the optimum temperature was 40 degrees C for the endoglucanase and 50 degrees C for the xylanase. Both enzyme activities were inhibited at 70 degrees C Co-culture of this organism with a Methanosarcina sp. (A145) had no effect on cellulose degradation and both endoglucanase and xylanase were stable in the co-culture.     

Lignocellulolytic enzyme production from submerged fermentation of paddy straw

Five strains of cellulolytic bacteria and four strains of Phanerochaete chrysosporium were evaluated for the lignocellulolytic enzyme production during submerged fermentation (SmF) of paddy straw. Extra-cellular enzyme assay for CMCase, FPase, Cellobiase, Xylanase, Lignin peroxidase and Laccase enzymes was performed after 7 and 15 days of submerged fermentation. Cellulomonas cellulans MTCC 23, Cytophaga hutchinsonii NCIM 2338 and Phanerochaete chrysosporium MTCC 787 were found to produce higher lignocellulolytic enzyme activities than rest of the cultures after 15 days of fermentation.     

Effect of coculture of anaerobic fungi isolated from ruminants and non-ruminants with methanogenic bacteria on cellulolytic and xylanolytic enzyme activities

Neocallimastix strain N1, an isolate from a ruminant (sheep), was cocultured with three Methanobacterium formicicum strains, Methanosarcina barkeri, and Methanobrevibacter smithii. The coculture with Methanobacterium formicicum strains resulted in the highest production of cellulolytic and xylanolytic enzymes. Subsequently four anaerobic fungi, two Neocallimastix strains (N1 and N2) from a ruminant and two Piromyces species from non-ruminants (E2 and R1), were grown in coculture with Methanobacterium formicicum DSM 3637 on filter paper cellulose and monitored over a 7-day period for substrate utilisation, fermentation products, and secretion of cellulolytic and xylanolytic enzymes. Methanogens caused a shift in fermentation products to more acetate and less ethanol, lactate and succinate. Furthermore the cellulose digestion rate increased by coculture. For cocultures of Neoallimastix strains with Methanobacterium formicicum strains the cellulolytic and xylanolytic enzyme production increased. Avicelase, CMCase and xylanase were almost completely secreted into the medium, while 40–60% of the -glucosidase was found to be cell bound. Coculture had no significant effect on the location of cellulolytic and xylanolytic enzymes.