发酵产氢菌株的筛选及影响因素研究

采用双层平板法从污泥池中筛选出一株产氢较高的发酵菌株,经生理生化鉴定表明,分离菌株初步鉴定为消化链球菌属(Peptostreptococcus).研究静态培养条件下葡萄糖、pH、温度及和酵母膏对菌株产氢的影响及不同发酵时间段的产氢情况.结果表明,在葡萄糖浓度20.0 g/L,pH 7.0,温度37 ℃和酵母膏2.0 g/L时,产氢量达21.07 mmol/L,为初始培养条件下的4.14倍.同时,在24～36 h时间段产氢率达到最高,为0.44 mmol/(L·h),并且在60 h时产氢量达到最大累计产氢量的89.2%.     

红酵母COS—5产胡萝卜素条件的研究

研究碳源、氮源和添加剂对红酵母（Rhodotorulasp．）COS－5产胡萝卜素的影响,并通过正交试验优化其产胡萝卜素的培养基组成。结果表明,COS－5产胡萝卜素的适宜培养基：蔗糖50g、蛋白胨5g、酵母膏5g、桔子皮15g、盐酸硫胺素0．002g、定容1L,pH6．0.250mL三角瓶装培养基30mL。在上述条件下28℃振荡培养96h,细胞生物量为27．5mg干重／mL发酵液,胡萝卜素含量达492．7μg／g干重细胞。COS－5胡萝卜素在474．4nm、338．6nm和310．2nm处有     

嗜热脂肪土芽孢杆菌CHB1产蛋白酶特性

目的：研究嗜热脂肪土芽孢杆菌CHBl产酶特性。方法：以蛋白酶活力为主要指标,考察温度、pH、接种量、装量等条件对CHB1产蛋白酶的影响。结果：CHB1适宜的产蛋白酶条件为：装量40mLt250mL,pH8．0,接种量5％,温度58℃,转速180r／min,时间36～44h,Tween-80对CHB1产蛋白酶具有抑制作用。结论：优化后蛋白酶产量有较大提高,最高酶活力达48U／mL,是所报道多数嗜热细菌产蛋白酶量的2—12倍。CHB1蛋白酶特性的研究及产蛋白酶量的提高,有利于揭示CHB1在堆肥化中的作用机理,提高堆肥效果。     

解脂亚罗酵母P12单倍体的制备

【背景】目前解脂亚罗酵母在实验研究和工业生产方面的应用越来越广泛,但相较于常规酵母而言,解脂亚罗酵母缺乏简便有效的遗传转化体系,致使其在基因表达调控方面存在较大困难。同时,酵母的染色体倍性也会对基因敲除效果产生影响,选择单倍体细胞作为功能基因改造的受体可以避免等位基因之间相互作用的影响,解决多倍体细胞基因敲除不完全的问题。【目的】以解脂亚罗酵母诱变菌株P12为研究对象,以不同方法分离得到单倍体菌株,建立解脂亚罗酵母单倍体的制备方法。【方法】分别采用固体和液体McClary产孢培养基诱导解脂亚罗酵母菌株产生子囊孢子,培养条件为30℃,固体7-14 d;液体200 r/min,2-4 d。以2%浓度的蜗牛酶33℃水浴裂解子囊孢子细胞壁3 h,通过染色镜检和PCR鉴定筛选单倍体细胞。【结果】镜检结果表明,解脂亚罗酵母在液体产孢培养基中产孢速度较快,相同视野下孢子数约为固体产孢培养基的3.7倍,在固体产孢培养基中产孢质量较好。初步探索并筛选得到6株解脂亚罗酵母P12 B型单倍体菌株。【结论】解脂亚罗酵母P12 B型单倍体菌株的获得可为后续继续开展基因工程操作奠定基础。     

黑曲霉产菊粉酶的发酵条件优化及诱变育种

对1株黑曲霉产菊粉酶的发酵条件进行了研究,确定了优化的发酵条件为菊粉2%,酵母膏2%,(NH4)H2PO40 5%,Na Cl0 5%,MgSO4·7H2O0 05%,ZnSO4·7H2O0 01%,初始pH6 5,接种量1 6%,装液量30ml,发酵5d,菊粉酶活最高可达26U/ml,I/S为1 15。经Co60诱变筛选出1株突变株C-32,在相同的发酵条件下菊粉酶活提高30%,I/S不变。     

酵母产γ-氨基丁酸发酵培养基的优化

目的：提高酵母产γ-氨基丁酸的能力。方法：采用单因素及正交设计实验对酵母产γ-氨基丁酸（GABA）的培养基进行优化。结果：确定最适碳源为葡萄糖,最佳氮源为蛋白胨和硫酸铵复合氮源,合适的无机盐为KH2PO4;最佳发酵培养基为3%葡萄糖,3%蛋白胨,0.3%（NH4）2SO4和0.1%KH2PO4。在此培养条件下,摇瓶发酵可以获得1.690g.L-1的GABA产量。结论：发酵培养基的优化,提高了菌株产γ-氨基丁酸的能力。     

酵母菌产麦角固醇发酵条件的研究

为了提高酵母菌麦角固醇的产量,采用摇瓶培养法,对筛选出的一株酵母菌YN2产麦角固醇发酵条件进行了研究。结果表明,酵母菌YN2产麦角固醇适宜的培养基配方为：酵母粉1％,牛肉膏2．5％,葡萄糖8％,K2HPO4 0．3％,MgSO4 0．15％,该菌株产麦角固醇最适培养条件为：培养温度28℃,起始pH6．5,发酵时间72h。在优化的实验条件下,麦角固醇含量可达2．2％,100ml发酵液中麦角固醇产量达25．30mg。     

一株产纤维素酶细菌的筛选鉴定

目的：从青贮饲料中分离筛选产纤维素酶的细菌。方法：用刚果红染色法和羧甲基纤维素酶活力测定法对分离所得的细菌进行筛选。结果：筛选到1株产纤维素酶能力较强的菌株,编号为ws-6。对该菌进行形态观察、生理生化鉴定和16S rDNA序列测定,鉴定为地衣芽孢杆菌（Bacillus licheniformis）。结论：该菌最适生长pH 5．0—7．0,最适生长温度35℃,产CMC酶活力达2．55U／mL。     

酵母中性脂快速检测及积累动态分析

以油红O、苏丹黑B为染料,结合酸或碱热破壁萃取,建立了通过比色快速检测酵母中性脂含量的方法。其中油红O萃取效果较好,比色结果更加准确,苏丹黑B也可以作为一种替代的中性脂含量检测的染料。并用该方法初步研究了两种中性脂肪甘油三酯代谢调控的关键基因磷脂酸磷酸酶(PAH1)和甘油三酯脂酶(triglyceride lipase,TGL)敲除突变体中性脂的变化。结果显示,前者甘油三酯显著降低,但总中性脂肪含量不变,后者甘油三酯及总中性脂肪含量均明显积累;同时,用该方法监测了酵母中性脂含量随生长周期的动态变化,表明指数生长期后有明显的积累。     

嗜热菌Anoxybacillu sp.产淀粉酶培养基优化及产酶条件研究

目的：对产淀粉酶嗜热菌Anoxybacillu sp.菌株进行培养基优化及产酶条件研究,以便提高菌株的产酶能力,并为下一步菌 株的诱变育种研究提供基础。方法：常规方法液体培养菌株,用平板初筛和DNS法复筛选择产淀粉酶能力较高的菌株;单因素筛 选培养基最适的碳源、氮源、Ca2+浓度和Mg2+浓度,对单因素筛选的最佳碳源、氮源、Ca2+和Mg2+的三个较佳浓度进行四因素三 水平正交试验优化培养基;对培养基不同pH值及不同培养温度进行培养条件研究。结果：产淀粉酶菌株筛选结果显示：六株菌中 淀粉酶酶活力值最大的是菌株Anoxybacillu sp.DL4,差异有统计学意义（P＜0.05）。培养基单因素筛选结果显示：最适碳源为麦芽糖、最适氮源为 硝酸铵、最适Ca2+、Mg2+浓度均为0.02%,差异有统计学意义（P＜0.05）。培养基优化结果显示：C 源0.1 %,N源0.2 %,Mg2+ 0.04%, Ca2+ 0.04 %为最佳的培养基成分组合。产酶条件筛选结果显示：培养基pH 值为6、培养温度为55 ℃时菌株产酶水平最高,差异有 统计学意义（P＜0.05）。结论：培养基的优化及最适的产酶条件能提高嗜热菌Anoxybacillu sp.DL4 产淀粉酶能力,Ca2+、Mg2+离子 对菌株产淀粉酶有促进作用。     

Optimized growth kinetics of Pichia pastoris and recombinant Candida rugosa LIP1 production by RSM

A predictive model for Pichia pastoris expression of highly active recombinant Candida rugosa LIP1 was developed by combining the Gompertz function and response surface methodology (RSM) to evaluate the effect of yeast extract concentration, glucose concentration, temperature, and pH on specific responses. Each of the responses (maximum population densities, specific growth rate (mumax), protein concentration, and minimum lag phase duration) was determined using the modified Gompertz function. RSM and 4-factor-5-level central composite rotatable design (CCRD) were adopted to evaluate the effects of growth parameters, such as temperature (21.6-38.4 degrees C), glucose concentration (0.3-3.7%), yeast extract (0.16-1.84%), and pH (5.3-8.7) on the responses of P. pastoris growth kinetics.Based on ridge maximum analysis, the optimum population density conditions were: temperature 24.4 degrees C, glucose concentration 2.0%, yeast extract 1.5%, and pH 7.6. The optimum specific growth rate conditions were: temperature 28.9 degrees C, glucose concentration 2.0%, yeast extract 1.1%, and pH 6.9. The optimum protein concentration conditions were: temperature 24.2 degrees C, glucose concentration 1.9%, yeast extract 1.5%, and pH 7.6. Based on ridge minimum analysis, the minimal lag phase conditions were: temperature 32.3 degrees C, glucose concentration 2.1%, yeast extract 1.1%, and pH 5.4. For the predicted value, the maximum population density, specific growth rate, protein concentration, and minimum lag phase duration were 15.7 mg/ml, 3.4 h(-1), 0.78 mg/ml, and 4.2 h, and the actual values were 14.3 +/- 3.5 mg/ml, 3.6 +/- 0.6 h(-1), 0.72 +/- 0.2 mg/ml, and 4.4 +/- 1.6 h, respectively.     

Medium components and culture conditions affect the thermotolerance of aerial conidia of fungal biocontrol agent Beauveria bassiana

AIMS: To produce more thermotolerable conidia of Beauveria bassiana, a well-known fungal biocontrol agent, by optimizing the medium components and culture conditions. METHODS AND RESULTS: The conidia produced on media including 0.5-6% glucose, sucrose or starch as carbon source and 50-300-microg ml(-1) Cu2+, Zn2+, Mn2+ or Fe3+ as additive to Sabouraud dextrose medium at 15-30 degrees C, pH 4-8 or KCl-adjusted water availabilities were exposed to 30-min wet heat stress at 48 degrees C. The medium components for conidial production with greatly enhanced thermotolerance included 4% glucose as optimum or 1% starch as alternative for the carbon source and < or =50-microg ml(-1) Mn2+ for the metal additive. The culture conditions were optimized as 25 degrees C and pH 5-6. Conidial thermotolerance decreased remarkably when sucrose and Fe3+ or Cu2+ were used in the cultures, but altered slightly when 50-200-microg ml(-1) Zn2+ were included. CONCLUSIONS: The tolerance of B. bassiana conidia to the thermal stress was significantly affected by the medium composition and culture conditions under which the conidia were produced. SIGNIFICANCE AND IMPACT OF THE STUDY: Proper treatment of small grains as mass production substrates for more glucose release and supplement of glucose or 50-microg ml(-1) Mn2+ are possible means to enhancing conidial thermotolerance and field persistence for improved insect control.     

原生质体紫外诱变选育可利用廉价碳源的高产油新菌株

【目的】研究并建立利用原生质体紫外诱变技术选育可利用廉价碳源发酵的高产油新菌株的方法。【方法】采用1.5%蜗牛酶和1.0%纤维素酶混合液水解去除细胞壁得到2A00015(近平滑假丝酵母,Candida parapsilosis)的原生质体,将其放于紫外灯下诱变及再生壁培养,筛选获得可利用廉价碳源发酵的高产油酵母,并采用气相色谱质谱联用法(GC-MS)测定其脂肪酸组成。【结果】突变效果最好的突变菌株2A00015/25用葡萄糖发酵培养7 d后,其生物量、油脂产率和产油量分别为17.77 g/L、58.12%和10.32 g/L,较原始菌株分别提高了12.45%、23.32%和38.68%;利用废糖蜜发酵培养,其生物量、油脂产率和产油量分别为18.54 g/L、49.44%和9.17 g/L,较原始菌株分别提高了9.09%、21.16%和32.18%。利用废糖蜜培养其产油效率虽低于利用葡萄糖培养,但从环境保护及原材料成本的角度考虑,用废糖蜜作为碳源发酵培养产生油脂更具优势。诱变菌株利用废糖蜜发酵后产生油脂经检测含有8种脂肪酸,其脂肪酸组成与植物油近似,其中不饱和脂肪酸含量占脂肪酸总量的82.4%。【结论】通过利用原生质体紫外诱变技术,成功选育出一株新的可利用廉价碳源的高产油海洋菌株,产油率达到49.4%,提高了21.2%。     

拉曼被孢霉F_5发酵生产γ—亚麻酸的研究

对拉曼被孢霉突变株F5发酵生产γ—亚麻酸的最适碳源、氮源、发酵时间及温度、无机盐离子添加、最适碳源浓度及补加碳源时间等发酵条件进行了研究探讨。最适发酵培养基组成为 (g/L) :葡萄糖 1 0 0 ,酵母浸出粉 4 ,蛋白胨 1 ,K2 HPO4 1 ,CaCl2 1× 1 0 - 2 ,MgSO4 5× 1 0 - 2 ,FeSO4 1× 1 0 - 2 ,ZnSO4 7.5× 1 0 - 3,CuSO4 0 .5× 1 0 - 3,MnSO4 2× 1 0 - 3,pH 6.0。培养温度为 2 5℃ ,1 4 0r/min振荡培养 1 0天 ,培养 8天后 (即收获前 2天 )补加 5 %葡萄糖。发酵结果为 :DC 2 4 .5 9g/L ,TL 1 0 .84g/L ,TL/DC 4 4.0 9% ,GLA/TL 1 0 .67% ,GLA产量为 1 1 5 6.63mg/L。GLA产量较初始结果提高 1 5 6.1 5 %。该菌株已达到工业化生产菌株要求     

粗壮假丝酵母(Candida valida 20231)产脂肪酶发酵条件的研究

对粗壮假丝酵母(Candida valida 20231)产脂肪酶所需的培养基成分和发酵条件进行了优化。结果表明,粗壮假丝酵母在接种后3 h即进入对数期,18 h后转入稳定期,66 h后进入衰退期;发酵36 h达到最大酶活,至72 h仍然无明显下降趋势。最适合的培养基成分为:4.0%大豆粉,2.0%菜籽油,0.3%葡萄糖,0.3%NH_4NO_3,1.2%K_2HPO_4,0.42%NaH_2PO_4,0.4%MgSO_4·7H_2O。使用优化的培养基,最佳产酶的条件为:装液量80 mL(250 mL摇瓶),初始pH 6,接种量为2.0%,种子菌龄为12 h。在最佳产酶条件下,脂肪酶活力达到32.6 U/mL。     

嗜碱芽孢杆菌XE22—4—1碱性弹性蛋白酶发酵条件的研究

从西藏天然碱湖中筛选到一株产碱性弹性蛋白酶（alkaline elastase)菌株,最适生长pH为10．0,经鉴定为Bacillus sp.,编号XE22－4－1.该菌产酶最适碳源为2％葡萄糖,氮源为0．25％酵母粉,豆饼粉对发酵产酶有促进作用。2L发酵罐实验表明,溶解氧是影响该菌株产酶的重要因素。通过提高通气量和改变搅拌速度,该菌株可在发酵48h内达到产酶高峰,酶活力最高达266u/mL。     

Interactions between P-limitation and different C conditions on the fatty acid composition of an extremophile microalga

The extremophilic microalga Chlamydomonas acidophila inhabits very acidic waters (pH 2-3.5), where its growth is often limited by phosphorus (P) or colimited by P and inorganic carbon (CO(2)). Because this alga is a major food source for predators in acidic habitats, we studied its fatty acid content, which reflects their quality as food, grown under a combination of P-limited and different carbon conditions (either mixotrophically with light + glucose or at high or low CO(2), both without glucose). The fatty acid composition largely depended on the cellular P content: stringent P-limited cells had a higher total fatty acid concentration and had a lower percentage of polyunsaturated fatty acids. An additional limitation for CO(2) inhibited this decrease, especially reflected in enhanced concentrations of 18:3(9,12,15) and 16:4(3,7,10,13), resulting in cells relatively rich in polyunsaturated fatty acids under colimiting growth conditions. The percentage of polyunsaturated to total fatty acid content was positively related with maximum photosynthesis under all conditions applied. The two factors, P and CO(2), thus interact in their effect on the fatty acid composition in C. acidophila, and colimited cells P-limited algae can be considered a superior food source for herbivores because of the high total fatty acid content and relative richness in polyunsaturated fatty acids.     

羰基还原酶产生菌Candida ontarioensis制备(R)-2-氯-1-(3-氯苯基)乙醇

从实验室保藏的菌株中筛选获得Candida sp.PT2A,并通过18S rRNA鉴定为安大略假单胞菌Candida on-tarioensis。对C.ontarioensis不对称还原合成(R)-2-氯-1-(3-氯苯基)乙醇的发酵产酶条件和转化条件进行优化,确定了最适的发酵产酶条件和转化条件:温度30℃,初始pH 6.5,摇床转速180 r/min,菌体质量浓度200 g/L。采用2-氯-1-(3-氯苯基)乙酮质量浓度为10 g/L时,还原反应72 h,(R)-2-氯-1-(3-氯苯基)乙醇的e.e.值为99.9%,产率为99%;底物质量浓度提高至30 g/L时,产率下降为84.3%。采用十六烷基三甲基溴化铵(CTAB)对C.ontarioensis细胞进行通透性处理(CTAB g/L,4℃下处理20 min),在30 g/L底物下反应24 h,产物的e.e.和产率分别达到99.9%和97.5%。     

2-氧代-4-苯基丁酸乙酯还原酶产生菌筛选及产酶条件

研究了利用生物催化不对称还原的方法制备(R)-2-羟基-4-苯基丁酸乙酯[(R)-HPBE]。以2-氧代-4-苯基丁酸乙酯(OPBE)为底物,通过对实验室保藏菌株进行筛选,得到一株产物立体选择性较高的菌株G2ndida krusei SW2026,并对其发酵产酶条件进行研究。其最适的发酵培养基组成为4.5%葡萄糖,3%蛋白胨,1.5%牛肉膏,0.05%Mn~(2+);适宜的产酶发酵条件为初始pH 6.0,温度28℃,摇床转速180 r/min,发酵周期48 h。将此条件下发酵培养的菌体用于OPBE的不对称还原反应,产物(R)-HPBE的对映体过量值(e.e.)可达97.33%,产率最高达到72.54%。     

Glycerol production by a novel osmotolerant yeast Candida glycerinogenes

Candida glycerinogenes, an osmotolerant yeast isolated from a natural sample in an environment of high osmotic pressure, had a modest sugar-tolerance and an extremely high glycerol productivity. The optimum conditions for glycerol formation by C. glycerinogenes were a temperature of 29-33 degrees C and a pH of 4-6. The optimum medium for glycerol production consisted of 230-250 g glucose/l, 2 g urea/l and 5 ml corn steep liquor/l (55-65 mg phosphates/l); the pH was not adjusted. The highest yield of glycerol was 64.5% (w/w) based on consumed glucose from 240 g glucose/l, and the highest concentration of glycerol was 137 g/l from 260 g glucose/l. These results were obtained by using a 30-l agitated fermentor under optimal fermentation conditions. In ten batch-fermentations carried out in a 50,000-l airlift fermentor, an average yield of glycerol of 50.67% (w/w) and an average glycerol concentration of 121.9 g/l were obtained from an average 240.6 g glucose/l.