深层液体培养法生产沼泽红假单胞菌

根据沼泽红假单胞菌 (Rhodopseudomonaspalustris)在好氧黑暗条件下也可生长的特性 ,采用液体深层培养方法 ,在通气式发酵罐中进行大规模生产。对培养基中的碳源、氮源、生长因子和微量元素进行了优化 ,用正交试验确定了主要培养基成分的添加量和接种量。在 10 0 0mL三角瓶和 2 5L全自动通气式发酵罐上进行了放大试验 ,在适宜工艺条件下培养 2 4h ,菌体浓度可达 6 0亿 /mL。     

沼泽红假单胞菌染色体的提取

近年来,由于工业产生的有机物对环境的污染越来越严重,推动了人们对能降解芳香物的沼泽红假单胞菌(Ｒ.ｐａｌｕｓｔｒｉｓ)的研究兴趣[1-8]。大多数芳香族化合物是严重危害人类健康的污染物质,为从基因水平研究沼泽红假单胞菌对芳香族化合物的生物降解机制,需要找到一种提取染色体的理想方法。泽沼红假单胞菌质膜构造比较特殊,且菌体富含蛋白质、色素、多糖等,给染色体提取造成一定难度。在本工作中采用了几种方法进行染色体的提取。1　材料与方法11　菌株来源:Ｙ6为本实验室分离、鉴定并保存的沼泽红假单胞菌。12　菌体培养121　沼泽红假单…     

沼泽红假单胞菌的研究进展

概述了沼泽红假单胞菌菌株的分离纯化,主要特性及应用研究方面的进展。     

沼泽红假单胞菌对微囊藻毒素的降解

以铜绿微囊藻为材料,通过固相萃取-高效液相色谱方法(SPE-HPLC),研究了沼泽红假单胞菌对微囊藻毒素MC-LR的降解作用。结果表明:沼泽红假单胞菌在厌氧、光照强度2000lx、35℃、pH7.0、乙酸钠为碳源、菌液初始浓度OD680为0.325和初始MC-LR为3mg.L-1时,6d降解率为36.5%,12d达到最高,降解率为78.7%。此降解条件和蓝藻水华爆发的环境条件基本一致,因此该菌株在水华爆发季节消除水中的微囊藻毒素方面具有应用潜力。     

沼泽红假单胞菌去除镉的研究

研究了沼泽红假单胞菌(Rhodopseudomonas palustris)S菌株的生长、Cd2 的去除与供氧光照、碳源、pH及温度的关系。结果表明:在好氧黑暗、苹果酸为碳源、pH7.0和30℃条件下,S菌株对初始浓度为25mg/L的Cd2 去除率最大,达到85%。在最佳条件下,Cd2 初始浓度为10mg/L~25mg/L时,去除率达85%,Cd2 浓度增加到150mg/L时,去除率仅为23%。测定Cd2 在菌体不同部位的分布发现菌体中富集的Cd2 63.5%在细胞壁上,33.5%在细胞原生质体内。同时,结合该菌体细胞超薄切片透射电镜观察证明了S菌株对Cd2 的去除主要在细胞壁上。     

沼泽红假单胞菌培养条件的初步研究

就沼泽红假单胞菌Ｐ３．９菌株的主要培养条件进行了初步研究。Ｐ３．９菌株在厌氧光照条件下能利用多种类型的有机碳源物质;其细胞生长的最适ｐＨ为６．５—８,最佳光照强度为２０００—３０００Ｌｘ;培养基中添加酵母膏能明显促进细胞生长。在适宜的培养条件下,菌液细胞数可高达４８亿个／ｍｌ。     

沼泽红假单胞菌偶氮还原酶新基因的克隆表达

前期实验证明沼泽红假单胞菌（Rhodopsedomonas palustris）对偶氮染料有较强的隆解能力,通过PCR扩增,从该菌粒DNA中扩增获得了一条未登录新基因PAR-1。将该基因构建到融合蛋白表达载体pGEX-4T-1,通过IPTG诱导,进行原核蛋白表达,SDS-PAGE电泳显示,有约48kD融合蛋白表达。对经诱导的大肠杆菌（BL21）进行偶氮染料脱色试验,检测到轻微脱色活性。     

环境对沼泽红假单胞菌基因型的影响

<正>紫色光合细菌是不产氧光合细菌的一个重要分支,包括紫色非硫细菌和紫色硫细菌。其中紫色非硫细菌具有极其丰富的代谢模式,可进行光能异养、光能自养和化能异养生长,其代谢的多样性使得它们广泛存活于不同的生态系统中,如土壤、湖泊、海洋及底泥等[1-2]。沼泽红假单胞菌(Rhodopseudomonas palustris)是紫     

沼泽红假单胞菌对健康小鼠肠道微生物的影响

探讨不同剂量沼泽红假单胞菌对健康小鼠生理功能及肠道微生物结构的影响。

将48只C57/BL6小鼠随机分为对照组(无菌生理盐水)以及沼泽红假单胞菌低剂量组(5×10⁸ CFU/mL)、中剂量组(5×10⁹ CFU/mL)、高剂量组(5×10¹⁰ CFU/mL), 每组12只; 不同剂量灌胃4周后, 测定小鼠胃泌素(Gas)、生长抑素(SS)含量, 观察肠道形态, 并应用Illumina-MiSeq测序平台进行16S rRNA基因V3-V4区测序, 分析其肠道微生物群落结构、多样性及组间差异。

与对照组相比, 低剂量组对促进小鼠Gas分泌差异具有统计学意义(t=6.426, P < 0.01), 高剂量组对抑制小鼠SS分泌差异具有统计学意义(t=3.711, P < 0.05);对照组小鼠回肠肠绒毛高度差异无统计学意义(P > 0.05), 且无肠道炎症刺激反应。沼泽红假单胞菌干预后细菌丰富度和多样性增加, 在门水平上, 与对照组相比, 实验组小鼠肠道菌群拟杆菌门丰度显著降低, 厚壁菌门丰度显著增加(均P < 0.05);在属水平上, 与对照组相比, 实验组小鼠肠道菌群Lachnospiraceae NK4A136 group、乳杆菌属、拟杆菌属、理研菌属和Prevotellaceae UCG-001相对丰度显著增加(均P < 0.05)。

沼泽红假单胞菌能够维持胃肠激素稳态和肠道形态结构, 且对健康小鼠肠道菌群的丰富度和组成产生一定的影响, 维持肠道平衡与稳定。

     

沼泽红假单胞菌乙酸光合放氢研究

依据光合细菌生长代谢特性和有机废水降解主要产物类型,11种有机物被用于沼泽红假单胞菌（Rhodopseudomonas palustris）Z菌株的光合产氢研究,其中,乙酸反应体系产氢活性最高。在此基础上,研究了该菌株的生长与产氢动力学行为,探求了影响该菌株光合放氢的主要限制性影响因素。结果表明,该菌株产氢与生长部分相关。种子培养基和菌龄对产氢活性有明显影响。细胞最适产氢和生长所需要的光照强度和温度基本一致。当种子来源于硫酸铵高菌龄预培养物或谷氨酸钠对数期预培养物时,该菌株产氢活性显著增加,产氢延滞期明显缩短。氧浓度和接种量对产氢活性也有显著影响。供氢体和氮源浓度直接决定细胞的生长与光放氢活性。在低于70 mmol/L乙酸钠和15 mmol/L谷氨酸钠时,产氢活性随底物浓度的增加而增强。谷氨酸钠浓度高于15mmol/L时,由于游离NH₄⁺的出现,产氢活性受到抑制,但却明显刺激细胞的生长。在标准状况下,该菌株的最大产氢速率可达19.4 mL·L^-1·h^-1。     

沼泽红假单胞菌J001产辅酶Q10摇瓶发酵条件优化

本试验对沼泽红假单胞菌J001菌株250ml摇瓶发酵辅酶Q10条件进行了优化。结果表明其最佳初始pH值为6.5-7.5,温度为28-31℃,摇床转速100 r min-1,摇瓶装液量为200ml,接种量达10%时可直接进入对数生长期;碳源以NaAc较好,氮源以(NH4)2SO4和NH4Cl较好,磷源用量对考察指标影响不明显;用中心组合设计响应曲面法对碳氮源用量进行了优化,当NaAc浓度为5.39（g l-1）,(NH4)2SO4浓度为0.385（g l-1）即碳氮比为14/1时,对菌胞生长最有利;当NaAc浓度为5.70（g l-1）,(NH4)2SO4浓度为0.365（g l-1）即碳氮比为15.6/1时,对辅酶Q10产量最有利。     

营养条件和流加发酵对放射型根瘤菌(Rhizobium radiobacter)产辅酶Q10的影响

利用放射型根瘤菌WSH2 6 0 1(RhizobiumradiobacterWSH2 6 0 1)重点考察了葡萄糖、蔗糖、玉米浆和蛋白胨、添加物以及流加发酵对细胞生长和产辅酶Q1 0 的影响 ,结果表明 ,葡萄糖和蔗糖适合于生产辅酶Q1 0 的最佳浓度分别为 30g L和 40g L ;辅酶Q1 0 发酵时玉米浆和蛋白胨的最适浓度分别为 11g L和 16g L ;添加蕃茄汁、玉米浆能提高发酵液的生物量 ,玉米浆、异戊醇、L 甲硫氨基酸等能促进辅酶Q1 0 的积累 ;与分批发酵相比 ,在 7L罐上流加蔗糖其细胞生物量 (DCW)和辅酶Q1 0 积累量增加 ,若在流加蔗糖的同时流加适当浓度的玉米浆能显著提高辅酶Q1 0 的产量 ,最大产量达到 5 2 .4mg L ;最大生物量 (DCW)和胞内辅酶Q1 0 含量 (C B值 )分别达到 2 6 .4g L和 2 .38mg g DCW ,比不流加的分批发酵分别提高 5 3 %和 33% ,比只流加蔗糖分别提高 2 4%和 2 6 %。     

辅酶Q10产生菌的抗性筛选及发酵条件优化

以根癌土壤杆菌(Agrobacterium tumefaciens)WSHAT12为出发菌株,通过硫酸二乙酯诱变,获得遗传稳定性好的抗L-乙硫氨酸(Eth)突变株WSH-E01,通过进一步的诱变处理,获得L-乙硫氨酸和维生素K3(VK3)双抗性突变株WSH-V01,以双抗性突变株WSH-V01为出发菌株,再进行诱变处理,获得一株X-gal利用能力提高的突变株WSH-X01,与出发菌株WSHAT12相比,突变株WSH-X01的辅酶Q10产量提高幅度达50.6%,同时,对突变株WSH-E01的发酵条件进行优化。出发菌株WSHAT12、突变株WSH-E01、WSH-V01和WSH-X01在优化后的发酵条件下辅酶Q10产量分别达到23.1mg/L、26.8mg/L、29.5mg/L和34.8mg/L。     

Use of wood vinegar to enhance 5-aminolevulinic acid production by selected Rhodopseudomonas palustris in rubber sheet wastewater for agricultural use

This study aimed to produce inexpensive 5-aminolevulinic acid (ALA) in a non-sterile latex rubber sheet wastewater (RSW) by Rhodopseudomonas palustris TN114 and PP803 for the possibility to use in agricultural purposes by investigating the optimum conditions, and applying of wood vinegar (WV) as an economical source of levulinic acid to enhance ALA content. The Box–Behnken Design experiment was conducted under microaerobic-light conditions for 96 h with TN114, PP803 and their mixed culture (1:1) by varying initial pH, inoculum size (% v/v) and initial chemical oxygen demand (COD, mg/L). Results showed that the optimal condition (pH, % inoculum size, COD) of each set to produce extracellular ALA was found at 7.50, 6.00, 2000 for TN114; 7.50, 7.00, 3000 for PP803; and 7.50, 6.00, 4000 for a mixed culture; and each set achieved COD reduction as high as 63%, 71% and 75%, respectively. Addition of the optimal concentration of WV at mid log phase at 0.63% for TN114, and 1.25% for PP803 and the mixed culture significantly increased the ALA content by 3.7–4.2 times (128, 90 and 131 μM, respectively) compared to their controls. ALA production cost could be reduced approximately 31 times with WV on the basis of the amount of levulinic acid used. Effluent containing ALA for using in agriculture could be achieved by treating the RSW with the selected ALA producer R. palustris strains under the optimized condition with a little WV additive.     

代谢工程改造大肠杆菌生产辅酶Q10

辅酶Q10(CoQ10)是一种脂溶性抗氧化剂,具有提高人体免疫力、延缓衰老和增强人体活力等功能,广泛应用于制药行业和化妆品行业。微生物发酵法能可持续性生产辅酶Q10,具有越来越多的商业价值。本研究首先将来自类球红细菌的十聚异戊二烯焦磷酸合成酶基因(dps)整合到大肠杆菌ATCC 8739染色体上,敲除内源的八聚异戊二烯焦磷酸合成酶基因(ispB),使内源的辅酶Q8合成途径被辅酶Q10合成途径取代,得到稳定生产辅酶Q10的菌株GD-14,其辅酶Q10产量达0.68 mg/L,单位细胞含量达0.54 mg/g DCW。随后用多个固定强度调控元件在染色体上对MEP途径的关键基因dxs和idi基因以及ubiCA基因进行组合调控,将辅酶Q10单位细胞含量提高2.46倍(从0.54到1.87 mg/g)。进一步引入运动发酵单胞菌Zymomonas mobilis的Glf转运蛋白代替自身的磷酸烯醇式丙酮酸:碳水化合物磷酸转移酶系统(PTS),使辅酶Q10产量进一步提高16%。最后,对高产菌株GD-51进行分批补料发酵,辅酶Q10产量达433 mg/L,单位细胞含量达11.7 mg/g DCW。这是目前为止文献报道的大肠杆菌产辅酶Q10最高菌株。     

Optimal production of sulphuric acid by Thiobacillus thiooxidans using response surface methodology

Response surface methodology was applied to determine the optimal medium composition for sulphuric acid production by an indigenous Thiobacillus thiooxidans. With a view to reducing the number of experiments and obtaining the mutual interactions between the variables, a 2⁴ full-factorial central composite design was employed for experimental design and analysis of the results. Four independent variables, KH₂PO₄, (NH₄)₂SO₄, MgSO₄ and elemental sulphur, in the growth medium were tested. Elemental sulphur was found to exhibit positive correlation with the sulphuric acid yield, whereas the other three variables showed no significant effect. No mutual interactions were found between elemental sulphur and each of the other three variables. The optimal composition of the growth medium to achieve the optimal production of sulphuric acid was determined as follows (g/l): KH₂PO₄=3.5, (NH₄)₂SO₄=4.9, MgSO₄=0.7 and elemental sulphur=23.7. The corresponding sulphuric acid production and dry cell weight were 38 662 ppm and 0.7 g/l, which are about 2.6- and 1.75-fold increase, respectively, compared with those using the conventional T. thiooxidans optimum growth medium.     

响应面法优化普通小球藻混合营养培养基组成生产生物质

利用响应面法优化了混合营养培养普通小球藻生产生物质的培养基组成.首先采用Plackett-Burman设计对11个相关营养因素的效应进行了评价,并筛选出影响小球藻细胞生长的3个主要因素为KNO3、葡萄糖和NaC1;然后结合Box-Behnken设计建立了以小球藻浓度为响应值的二次回归方程模型,获得优化的培养基组成为KNO31.64g/L、葡萄糖45g/L、NaC1 1.57g/L;模型预测的最大浓度为5.28g/L,验证值为5.68g/L;验证结果表明,所建立模型预测精度较好,可用于优化小球藻的混养培养基组成.优化条件下混养小球藻细胞的蛋白质和色素含量较优化前降低,而可溶性糖和油脂含量提高,脂肪酸以棕榈酸和油酸为主;细胞组分分析结果显示,混养培养所得小球藻生物质具有作为生产微藻生物能源原料的潜力.     

Utilization of l-alanine by Rhodopseudomonas acidophila

Rhodopseudomonas acidophila strain 7050 can satisfy all its nitrogen and carbon requirements from l-alanine. Addition of 100 M methionine sulfoximine to alanine grown cultures had no effect on growth rate indicating that deamination of alanine via alanine dehydrogenase and re-assimilation of the released NH ₄ ⁺ by glutamine synthetase/glutamate synthase was an insignificant route of nitrogen transfer in this bacterium. Determination of aminotransferase activities in cell-free extracts failed to demonstrate the presence of direct routes from alanine to either aspartate or glutamate. The only active aminotransferase involving l-alanine was the alanine-glyoxylate enzyme (114–167 nmol·min^–1·mg^–1 protein) which produced glycine as end-product. The amino group of glycine was further transaminated to yield aspartate via a glycineoxaloacetate aminotransferase (117–136 nmol·min^–1 ·mg^–1 protein). No activity was observed when 2-oxoglutarate was substituted for oxaloacetate. The formation of glutamate from aspartate was catalysed by aspartate-2-oxoglutarate aminotransferase (85–107 nmol·min^–1·mg^–1 protein). Determinations of free intracellular amino acid pools in alanine and alanine+100 M methionine sulfoximine grown cells showed the predominance of glutamate, glycine and aspartate, providing further evidence that in alanine grown cultures R. acidophila satisfies its nitrogen requirements for balanced growth by transamination.Abbreviations ADH alanine dehydrogenase - GDH glutamate dehydrogenase - GS glutamine synthetase - GOGAT glutamate synthase - MSO methionine sulfoximine - GOT glutamate-oxaloacetate aminotransferase - GPT glutamate-pyruvate amino-transferase - AGAT alanine-glyoxylate aminotransferase - GOAT glycine-oxaloacetate aminotransferase - GOTAT glycine-2-oxoglutarate aminotransferase - AOAT alanine-oxaloacetate aminotransferase     

响应面法优化黑曲霉产果胶酶培养基中无机盐成分的研究

利用SAS软件中的二水平设计和响应面分析方法较系统地研究了发酵培养基中无机盐组分对黑曲霉(Aspergillus niger)JW-1菌株产果胶酶的影响.得到了在一定条件下果胶酶随无机盐组分的变化规律,并根据分析结果优化了产酶培养基.最终确定KH_2PO_4,FeSO_4·7H_2O,CaCl_2·2H_2O的最优浓度分别为0.85mg/mL,1.86mg/mL和2.52mg/mL,此时果胶酶活力可达5054.6U·g~(-1),为该菌株今后的研究和应用奠定了基础.     

Isolation and characterization of Aspergillus sp. for the production of extracellular polysaccharides by response surface methodology

In this study, Aspergillus sp. was isolated for the production of extracellular polysaccharide. The process parameters were initially optimized by traditional methods. The cheap substrate, wheat bran was used for the production of extracellular polysaccharide in solid state fermentation. Supplementation of (1%, w/w) maltose, gelatin enhanced EPS production (5.36?mg/g). The salts such as, Cu²⁺ (4.9?mg/g), Ca²⁺ (3.5?mg/g), Zn²⁺ (2.9?mg/g), Mn²⁺ (3.4?mg/g) and Mg²⁺ (1.8?mg/g) stimulated EPS production. In two level full factorial experimental designs, the EPS yield varied from 3.18 to 11.65?mg/g wheat bran substrate with various combinations of the components supplemented with wheat bran substrate. Among these selected factors in central composite design, maltose significantly influenced on extracellular polysaccharide production.