Increasing the cell viability and heterologous protein expression of Pichia pastoris mutant deficient in PMR1 gene by culture condition optimization

In this study, we assessed the potential of PMR1-disrupted Pichia pastoris (Pppmr1) expressing human serum albumin and interferon alpha2b fusion protein (HSA-IFN-alpha2b) in large-scale fermentation. The high osmotic pressure of standard basal salts medium (BSM) was detrimental to the growth and viability of Pppmr1. HSA-IFN-alpha2b was secreted into a supernatant with a concentration of up to 112 mg/L after 20 h of induction and then began to decline. In vitro stability tests indicated that the disappearance of HSA-IFN-alpha2b was ascribed to proteolytic degradation. Decreasing the salt concentration of BSM medium to one quarter of the original formula improved the growth and viability of Pppmr1. As a result of reduced cell lysis and protease release, HSA-IFN-alpha2b was stable in the supernatant, which enabled a longer production phase (30 h) and a higher expression level (215 mg/L). Lowering the culture temperature to 20°C increased the cell viability during carbon source transition and alleviated the oxygen and methanol limitation, which extended the production phase to 40 h and increased the expression level to 680 mg/L. The addition of 2% Soytone prolonged the production phase to 60 h and increased the expression level to 1,260 mg/L, which was more than tenfold higher than that of Pppmr1 cultured under the conditions recommended by Invitrogen.     

豚草种带内生真菌及其对种子发芽和幼苗生长的影响

内生真菌是一类共生于植物体内,能够不同程度影响宿主植物生态适应性和竞争能力的微生物。分析内生真菌在豚草种子中的分布、种群结构,以及内生真菌发酵液对种子发芽和幼苗生长的作用。结果显示:发生于6个地区的豚草种子均能分离获得内生真菌,分离率在19%—92.63%之间,不同地区之间差异极显著(P0.01)。内生真菌主要存在于种子的总苞部位,分离率达到65.52%。发生于福建省长乐市松下镇的豚草种带内生真菌种群包含5个属,以链格孢属(Alternaria)真菌为优势菌群,占82.26%;其次为镰孢属(Fusarium)真菌,占9.68%;其它3个属的真菌发生较少,均低于5%。内生真菌主要以水平传播方式在豚草不同世代之间传播。供试的7个内生真菌菌株的发酵液均不同程度地抑制豚草种子发芽,降低幼苗地上部高度、根长度、根数量和总生物量,但不同菌株发酵液之间抑制程度差异明显,显示不同菌株对豚草种子发芽和幼苗生长产生不同的影响。内生真菌发酵液处理后的种子仍然保持较高程度的活力;不同内生真菌发酵液处理后,有活力的种子维持在50%—87.5%之间,均高于(或等于)清水处理的种子,说明内生真菌代谢产物只是抑制种子的发芽,但并不导致种子的腐烂和死亡。这些研究结果初步显示种子携带的内生真菌可能在豚草入侵生物学中发挥重要的作用。     

Zymomonas mobilis cell viability: measurement method comparison

Comparison of three different cell viability methods: slide count, plate count and methylene blue staining techniques, applied onZymomonas mobilis cultures, was performed. The slide technique proved to be faster and more accurate than the plate count method, and both of them far more reliable than the standard methylene blue method which constantly overestimated theZymomonas cell viability. The slide technique is advantageous also because it gives information on the cell morphology changes, notably the abnormal cell elongation, in the ethanol fermentation.     

Enhanced production of acetoin and butanediol in recombinant Enterobacter aerogenes carrying Vitreoscilla hemoglobin gene

Microbial production of butanediol and acetoin has received increasing interest because of their diverse potential practical uses. Although both products are fermentative in nature, their optimal production requires a low level of oxygen. In this study, the use of a recombinant oxygen uptake system on production of these metabolites was investigated. Enterobacter aerogenes was transformed with a pUC8-based plasmid carrying the gene (vgb) encoding Vitreoscilla (bacterial) hemoglobin (VHb). The presence of vgb and production of VHb by this strain resulted in an increase in viability from 72 to 96 h in culture, but no overall increase in cell mass. Accumulation of the fermentation products acetoin and butanediol were enhanced (up to 83%) by the presence of vgb/VHb. This vgb/VHb related effect appears to be due to an increase of flux through the acetoin/butanediol pathway, but not at the expense of acid production.     

Tourmaline ceramic balls stimulate growth and metabolism of three fermentation microorganisms

Effects of tourmaline ceramic balls on growth and metabolism of Saccharomyces cerevisiae, Lactobacillus acidophilus and Aspergillus oryzae were studied. Treatments with 3, 6, 9 or 12 g of tourmaline ceramic balls in a 50 ml culture showed significant stimulation of the growth of the three microorganisms. In optimal treatments with 12 g of tourmaline balls, the growth of S. cerevisiae, L. acidophilus, and A. oryzae was increased by 34, 32 and 10%, respectively. After 72 h fermentation of S. cerevisiae, total carbohydrate content in the culture medium was decreased by 65% and ethanol production was increased by 150%. Total carbohydrate content was decreased by 80% and the pH value was decreased by 0.3, as a result of organic acid production in the medium of L. acidophilus after 72 h fermentation. In the case of A. oryzae, enzyme activities of protease and amylase were increased by 90 and 31%, respectively, after 96 h fermentation. Results indicated that tourmaline stimulates initiation of growth in the early lag stage and increases production of metabolites at a later stage of fermentation. The strong stimulatory effect of tourmaline on growth, utilization of substrates and production of metabolites in the three microorganisms suggests a potential application in the fermentation industry.     

Evaluation of bacterial contamination in a fed-batch alcoholic fermentation process

Alcoholic fermentation by a commercial baker's yeast in a fed-batch process with cell recycling and high-test molasses as substrate was strongly inhibited by Lactobacillus fermentum CCT 1407 after a few recycles. When total acidity (mainly lactic acid) exceeded 4.8 g/l broth it seriously interfered with yeast bud formation and viability and above 6.0 g/l it decreased alcoholic efficiency.     

蝉拟青霉液体发酵条件优化

采用液体发酵蝉拟青霉,对蝉拟青霉的发酵条件进行优化,以提高蝉拟青霉胞外多糖产量及生物量。摇瓶发酵条件下,在单因素基础上设计正交实验确定各因素的最佳组合。优化后得最佳发酵培养基:蔗糖8%,牛肉膏0.75%,酵母膏0.125%,MgSO_4·7H_2O 0.3%,KH_2PO_4 0.2%,麸皮0.5%。该条件下胞外多糖产量为5.96 g/L,生物量为42 g/L,较优化前提高了1倍。采用发酵罐进行扩大培养,对分批发酵时的初糖浓度进行了优化,并分析了补料分批发酵对发酵过程的影响。发酵罐培养时最适初糖浓度为5%,此时生物量最高为38 g/L,多糖含量最高为5.5 g/L;采用补料分批发酵时,多糖产量最高为5.89 g/L,生物量最高为40 g/L,效果优于分批发酵。     

Growth and Fermentation Characteristics of a Strain of the Wine Yeast Kluyveromyces thermotolerans Isolated in Greece

Summary During the single culture fermentation of grape must K. thermotolerans, strain TH941, isolated in a wine-producing region in northern Greece, reached a very high cell concentration of 8.4 log (c.f.u ml⁻¹), followed by a rapid decline of the viable cells. The yeast produced 9.6 g L-lactic acid l⁻¹ during the growth phase, 7.58% v/v of ethanol and showed a limited degradation of L-malic acid as well as a low production of volatile acidity. In the presence of 3% v/v and 6% v/v of ethanol the K. thermotolerans isolate was able to grow. At 9% v/v of ethanol it could not grow but showed no loss of viability for 10 days.     

Kinetics and metabolic behavior of a composite culture of <Emphasis Type="Italic">Kloeckera apiculata</Emphasis> and <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> wine related strains

The kinetics and metabolic behavior of Kloeckera apiculata mc1 and Saccharomyces cerevisiae mc2 in composite culture was investigated. K. apiculata showed a higher viability through the fermentation; however the maximum cell density of both yeasts decreased. This behavior was not due to ethanol concentration, killer toxins production or competition for assimilable nitrogenous compounds between both yeasts. Despite the consistent production of secondary products by single culture of K. apiculata, an increase of these compounds was not observed in mixed culture. These results contribute to a better understanding of the behavior of non-Saccharomyces yeasts and their potential application in the wine industry.     

Synergism among lactic acid, sulfite, pH and ethanol in alcoholic fermentation of Saccharomyces cerevisiae (PE-2 and M-26)

Summary The industrial production of ethanol is affected mainly by contamination by lactic acid bacteria besides others factors that act synergistically like increased sulfite content, extremely low pH, high acidity, high alcoholic content, high temperature and osmotic pressure. In this research two strains of Saccharomyces cerevisiae PE-2 and M-26 were tested regarding the alcoholic fermentation potential in highly stressed conditions. These strains were subjected to values up to 200 mg NaHSO₃ l⁻¹, 6 g lactic acid l⁻¹, 9.5% (w/v) ethanol and pH 3.6 during fermentative processes. The low pH (3.6) was the major stressing factor on yeasts during the fermentation. The M-26 strain produced higher acidity than the other, with higher production of succinic acid, an important inhibitor of lactic bacteria. Both strains of yeasts showed similar performance during the fermentation, with no significant difference in cell viability.     

少根根霉产孢能力、变种与发酵产物的相关性

[目的]探究少根根霉不同生态环境、不同地理位置、不同产孢能力和不同分类学变种的发酵产物多样性及相关性,为工业生产提供指导.[方法]代表性的68株少根根霉菌株于糯米培养基中进行液态发酵,利用高效液相色谱法测定各种发酵产物浓度,计算发酵产物间的Pearson相关系数,通过多因素方差分析和主成分分析解析各类菌株与发酵特性的相...     

贝莱斯芽孢杆菌GDND-2对烟草镰孢根腐病菌生长发育的影响

【背景】尖孢镰孢(Fusariumoxysporum)引起的烟草根腐病在世界烟区普遍发生,严重影响烟草产量和质量,化学农药无法有效防治病害,利用生防菌防治该病成为研究热点。【目的】明确贝莱斯芽孢杆菌(Bacillusvelenzensis)GDND-2对尖孢镰孢生长发育的抑制作用。【方法】制备含10%和20%GDND-2发酵滤液的PDA培养基,涂在玻片上,接种尖孢镰孢分生孢子,观察滤液对尖孢镰孢孢子萌发、菌丝生长、孢子形成和色素产生的影响,应用扫描和透射电镜观察菌体超微结构的变化。【结果】贝莱斯芽孢杆菌GDND-2发酵滤液延迟孢子萌发2h以上,造成芽管膨大畸形,促使菌丝提早分枝,抑制菌丝延伸,使菌丝产生畸形球状结构,10%和20%发酵滤液对菌丝生长抑制率达53.41%和61.58%。滤液延迟病菌产孢,显著抑制产孢量,影响孢子形态,刺激病菌产生色素。10%和20%发酵滤液延迟产孢20h和28h,对产孢量的抑制率为52.11%和78.85%,滤液促使病菌形成小型分生孢子。观察尖孢镰孢的超微结构,部分菌丝膨大、畸形,细胞壁变薄,细胞膜消失,细胞质渗出,胞内呈空腔结构;部分菌丝严重皱缩、扭曲、...     

Immobilization of Lactobacillus rhamnosus in polyvinyl alcohol/calcium alginate matrix for production of lactic acid

Immobilization of Lactobacillus rhamnosus ATCC7469 in poly(vinyl alcohol)/calcium alginate (PVA/Ca-alginate) matrix using “freezing–thawing” technique for application in lactic acid (LA) fermentation was studied in this paper. PVA/Ca-alginate beads were made from sterile and non-sterile PVA and sodium alginate solutions. According to mechanical properties, the PVA/Ca-alginate beads expressed a strong elastic character. Obtained PVA/Ca-alginate beads were further applied in batch and repeated batch LA fermentations. Regarding cell viability, L. rhamnosus cells survived well rather sharp immobilization procedure and significant cell proliferation was observed in further fermentation studies achieving high cell viability (up to 10.7 log CFU g⁻¹) in sterile beads. In batch LA fermentation, the immobilized biocatalyst was superior to free cell fermentation system (by 37.1%), while the highest LA yield and volumetric productivity of 97.6% and 0.8 g L⁻¹ h⁻¹, respectively, were attained in repeated batch fermentation. During seven consecutive batch fermentations, the biocatalyst showed high mechanical and operational stability reaching an overall productivity of 0.78 g L⁻¹ h⁻¹. This study suggested that the “freezing–thawing” technique can be successfully used for immobilization of L. rhamnosus in PVA/Ca-alginate matrix without loss of either viability or LA fermentation capability.

     

Controlled formation of volatile components in cider making using a combination of Saccharomyces cerevisiae and Hanseniaspora valbyensis yeast species

The effect of pure and mixed fermentation by Saccharomyces cerevisiae and Hanseniaspora valbyensis on the formation of major volatile components in cider was investigated. When the interaction between yeast strains of S. cerevisiae and H. valbyensis was studied, it was found that the two strains each affected the cell growth of the other upon inoculation of S. cerevisiae during growth of H. valbyensis. The effects of pure and mixed cultures of S. cerevisiae and H. valbyensis on alcohol fermentation and major volatile compound formation in cider were assessed. S. cerevisiae showed a conversion of sugar to alcohol of 11.5%, while H. valbyensis produced alcohol with a conversion not exceeding 6%. Higher concentrations of ethyl acetate and phenethyl acetate were obtained with H. valbyensis, and higher concentrations of isoamyl alcohol and isobutyl were formed by S. cerevisiae. Consequently, a combination of these two yeast species in sequential fermentation was used to increase the concentration of ethyl esters by 7.41–20.96%, and to decrease the alcohol concentration by 25.06–51.38%. Efficient control of the formation of volatile compounds was achieved by adjusting the inoculation time of the two yeasts.     

酱香型白酒发酵中地衣芽孢杆菌前噬菌体的鉴别

【背景】噬菌体是微生物群落的重要组成部分,但传统白酒发酵中噬菌体的分类和存在尚不清楚。【目的】通过检测公共数据库和酱香型白酒发酵中地衣芽孢杆菌(Bacillus licheniformis)基因组中的前噬菌体整合区域,探究传统酱香型白酒发酵中关键功能菌株的前噬菌体分类和侵染情况。【方法】使用未培养(细菌全基因组分析)和可培养(菌株筛选和特异性PCR反应)技术对不同环境来源和来自酱香型白酒发酵的地衣芽孢杆菌前噬菌体的分类和存在进行解析。【结果】细菌全基因组分析显示,30株来自不同环境的地衣芽孢杆菌基因组中共注释到165个前噬菌体,其中63.6%(105/165)为完整前噬菌体序列。97.1%感染地衣芽孢杆菌的噬菌体属于长尾噬菌体科(Siphoviridae),2.9%属于肌尾噬菌体科(Myoviridae),53.0%完整前噬菌体的基因功能未知。在来自酱香型白酒发酵的B. licheniformis MT-B06中检测到7个前噬菌体整合序列,其中57.1%(4/7)为完整前噬菌体序列,来自酱香型白酒发酵的地衣芽孢杆菌存在多种不同前噬菌体的共感染。来自酱香型白酒发酵的地衣芽孢杆菌前噬菌体存在来自细菌基因组上相邻CotD孢子外壳蛋白(CotD family spore coat protein)基因的水平基因转移。在26株来自酱香型白酒发酵的地衣芽孢杆菌中,69.2%(18/26)存在噬菌体编码主要衣壳蛋白的基因,100.0%(26/26)存在噬菌体编码CotD孢子外壳蛋白的基因。【结论】来自不同环境的地衣芽孢杆菌和酱香型白酒发酵的地衣芽孢杆菌中存在高水平的前噬菌体整合,来自酱香型白酒发酵的地衣芽孢杆菌前噬菌体中广泛存在来源于宿主的CotD孢子外壳蛋白基因的水平基因转移。本研究为首次对传统发酵白酒中噬菌体的分类和存在进行探究,有助于对发酵微生物群落中噬菌体-细菌相互作用加深理解。     

Synergism of cellulase enzymes in mixed culture solid substrate fermentation

Sugar cane bagasse was subjected to a mixed culture, solid substrate fermentation with Trichoderma reesei QM9414 and Aspergillus terreus SUK-1 to produce cellulase and reducing sugars. The highest cellulase activity and reducing sugar amount were obtained in mixed culture. The percentage of substrate degradation achieved employing mixed culture was 26% compared to 50% using separate cultures of the two molds. This suggests that the synergism of enzymes in mixed culture solid substrate fermentation have lower synergism than in pure culture.     

八棱海棠种子超低温保存中含水量对糖代谢的影响

含水量是影响种子超低温保存效果的关键因素，而其作用机制尚不完全清楚。为探讨含水量对种子超低温保存生活力的影响途径，该研究以八棱海棠种子为材料，通过硅胶干燥法获得不同含水量的种子，测定超低温保存后种子生活力、糖含量及相关酶指标的变化并分析相关性。结果表明：(1)超低温保存15 d后，不同含水量种子生活力不同，随着种子含水量的降低，种子生活力呈现先升高后降低的趋势，含水量为9.02%的八棱海棠种子生活力最高，为53.33%;超低温保存120 d后，种子生活力随着含水量下降一直升高，含水量为6.40%生活力最高，为27.78%。这表明八棱海棠种子含水量对超低温保存后的生活力有明显影响，但受液氮保存时间影响，随着液氮保存时间的延长，最适含水量降低。(2)相关分析显示，超低温保存后种子含水量与生活力呈极显著负相关(r=-0.82);与果糖和蔗糖含量、酸性转化酶、果糖激酶呈显著负相关，而种子萌发率与这些指标呈显著正相关。这表明种子含水量通过影响酸性转化酶活性而影响蔗糖和果糖含量，进而影响蔗糖代谢，响应低温和脱水胁迫，最终导致生活力差异。种子生活力还受到介导果糖激酶的果糖代谢影响。此外，海藻糖也是种...     

Applicability of pectate-entrapped <Emphasis Type="Italic">Lactobacillus casei</Emphasis> cells for <Emphasis Type="SmallCaps">l</Emphasis>(+) lactic acid production from whey

Lactic acid is a versatile organic acid, which finds major application in the food, pharmaceuticals, and chemical industries. Microbial fermentation has the advantage that by choosing a strain of lactic acid bacteria producing only one of the isomers, an optically pure product can be obtained. The production of l(+) lactic acid is of significant importance from nutritional viewpoint and finds greater use in food industry. In view of economic significance of immobilization technology over the free-cell system, immobilized preparation of Lactobacillus casei was employed in the present investigation to produce l(+) lactic acid from whey medium. The process conditions for the immobilization of this bacterium using calcium pectate gel were optimized, and the developed cell system was found stable during whey fermentation to lactic acid. A high lactose conversion (94.37%) to lactic acid (32.95 g/l) was achieved with the developed immobilized system. The long-term viability of the pectate-entrapped bacterial cells was tested by reusing the immobilized bacterial biomass, and the entrapped bacterial cells showed no decrease in lactose conversion to lactic acid up to 16 batches, which proved its high stability and potential for commercial application.     

Production of hydrogen from marine macro-algae biomass using anaerobic sewage sludge microflora

Hydrogen was produced from various marine macro-algae (seaweeds) through anaerobic fermentation using an undefined bacterial consortium. In this study, anaerobic fermentation from various marine macro-algae for Ulva lactuca, Porphyra tenera, Undaria pinnatifida, and Laminaria japonica was studied. From this analysis Laminaria japorica was determined to be the optimum substrate for hydrogen production. When L. japornica was used as the carbon source for enhanced hydrogen production, the optimum fermentation temperature, substrate concentration, initial pH, and pretreatment condition were determined to be 35°C, 5%, 7.5, and BT120 (Ball mill and thermal treatments at 120°C for 30 min), respectively. In addition, hydrogen production was improved when the sludge was heat-treated at 65°C for 20 min. Under these conditions, about 4,164 mL of hydrogen was produced from 50 g/L of dry algae (L. japonica) for 50 h, with a hydrogen concentration around 34.4%. And the maximum hydrogen production rate and yield were found to be 70 mL/L·h and 28 mL/g dry algae, respectively.     

Effect of n-dodecane on Crypthecodinium cohnii fermentations and DHA production

The potential use of n-dodecane as an oxygen vector for enhancement of Crypthecodinium cohnii growth and docosahexaenoic acid (DHA) production was studied. The volumetric fraction of oxygen vector influenced the gas–liquid volumetric mass transfer coefficient k _L a positively. The k _L a increased almost linearly with the increase of volumetric fraction of n-dodecane up to 1%. The stirring rate showed a higher influence on the k _L a than the aeration rate. The effects of this hydrocarbon on C. cohnii growth and DHA production were then investigated. A control batch fermentation without n-dodecane addition (CF) and a batch fermentation where n-dodecane 1% (v/v) was added (DF) were carried out simultaneously under the same experimental conditions. It was found that, before 86.7 h of fermentation, the biomass concentration, the specific growth rate, the DHA, and total fatty acids (TFA) production were higher in the CF. After this fermentation time, the biomass concentration, the DHA and TFA production were higher in the DF. The highest DHA content of biomass (6.14%), DHA percentage of TFA (51%), and DHA production volumetric rate r _DHA (9.75 mg l⁻¹ h⁻¹) were obtained at the end of the fermentation with n-dodecane (135.2 h). The dissolved oxygen tension (DOT) was always higher in the DF, indicating a better oxygen transfer due to the oxygen vector presence. However, since the other C. cohnii unsaturated fatty acids percentages did not increase with the oxygen availability increase due to the n-dodecane presence, a desaturase oxygen-dependent mechanism involved in the C. cohnii DHA biosynthesis was not considered to explain the DHA production increase. A selective extraction through the n-dodecane was suggested.