小克银汉霉γ-亚麻酸高产菌株的快速筛选和发酵条件研究

以小克银汉霉C0为出发菌株,经过5-氟尿嘧啶和紫外线复合诱变,采用抗失水苹果酰肼与抗低温(15℃)相结合的筛选方法,获得一株生产性能比出发菌株显著提高的突变株C23。采用5L全自动发酵罐对小克银汉霉C23发酵生产γ-亚麻酸的pH值控制和补料工艺进行研究,发现将发酵液pH值维持在5.5,当发酵进行到60h、84h、108h时,分别补糖15g/L,发酵192h后收获,结果生物量、油脂产量和γ-亚麻酸产量分别达到49.88g/L、21.93g/L、2.69g/L。     

C．shehatae TZ8耐乙醇菌株的筛选及发酵性能研究

以C.shehataeTZ8为出发茵株,利用1％溶壁酶和1％蜗牛酶酶解1．5h,制备成C.shehataeTZ8原生质体,并对原生质体进行紫外诱变,以含不同浓度乙醇的木糖液体培养基培养进行初筛和复筛,获得一株遗传性能稳定、耐乙醇能力达5．5％（v／v）的蕾株C．shehataeTZ8-4,比初始菌株耐乙醇能力提高了2％。对突变株C．shehataeTZ8-4发酵性能的研究结果表明：C．shehataeTZ8-4发酵糖能力从80g/L（葡糖糖和木糖比为2：1）提高到120g／L,最大乙醇产量从27．41g／L提高到43．12g／L。     

培养条件对钝顶螺旋藻(Sp)NS-90020脂肪酸组成和含量的影响

研究了不同培养条件对钝顶螺旋藻（Sp）NS－90020脂肪酸合成的影响。随着温度升高,其不饱和脂肪酸,γ一亚麻酸（GLA）相对含量降低,总脂肪酸含量升高,当温度为40℃时总脂肪酸和γ-亚麻酸绝对含量都达到最大值,分别为73．4mg／g干重和11．9mg／g干重;当培养基中NaCl浓度高于0．017mol／L时,其GLA相对含量降低,但低于0．0017mol／L时,对其脂肪酸组成无显著影响;氨水使其脂肪酸和GLA绝对含量升高,并在50mgN（NH3·H2O）时达到最大值,分别为67．96mg／g干重和13．63mg／g千重;暗处理92h使其总脂肪酸和GLA绝对含量升高;缺乏Fe2 或Mg2 或Mo2 时,其总脂肪酸和GLA绝对含量降低,而缺乏PO43-时,其总脂肪酸和GLA绝对含量略有升高。     

环境条件及摇瓶补糖策略对谷胱甘肽发酵的影响

研究了酵母摇瓶发酵中pH、装液量、初糖浓度、碳氮磷比和补糖方式对谷胱甘肽(GSH)发酵的影响。结果表明,GSH发酵适宜的初始pH和装液量分别为6 0和500ml锥形瓶内装液量60m1。初糖浓度对GSH发酵有较大的影响,超过12g／l,的初糖浓度将减少胞内GSH含量。应用计算得出的一种以控制比生长速率为目的的摇瓶补糖策略,在总糖浓度为26．2g／L下发酵12h,最终细胞浓度和胞内GSH含量分别达到8.78g／L和13．6mg／g,发酵液内GSH总量达到119．4mg／L,细胞对糖产率达到0.335g／g。     

拉曼被孢霉产γ-亚麻酸的发酵条件优化

对影响发酵产γ-亚麻酸的因素（温度、N源、P源等）进行单因素考察。结果发现,各因素对拉曼被孢霉（Mortierella ramanniana NBRC 8187）产γ-亚麻酸均有不同程度的影响。通过正交试验设计对KH2PO4、NaNO3、酵母膏和温度进行了L9（34）试验设计,以γ-亚麻酸产量为优化目标,直观分析得出影响产量的因素大小顺序为酵母膏、温度、KH2PO4、NaNO3,最终确定最佳的培养条件：酵母膏5 g/L、25℃、KH2PO41 g/L、NaNO33 g/L。在此条件下,检测得出在第5天γ-亚麻酸产量达到0.968 g/L,相比优化前（0.583 g/L）提高了66%。     

γ-亚麻酸产生菌Mucor sp．EIM-10的筛选及分子鉴定

为了获得高产γ-亚麻酸（γ-linolenic acid,GLA）的菌株,利用苏丹黑染色法筛选获得1株GLA产生菌EIM-10,通过摇瓶培养,其生物量可达11．882g／L,菌丝体油脂含量可达18．86％。气相色谱-质谱（GC—MS）分析表明其γ-亚麻酸质量分数（占总脂肪酸）高达27．68％。为进一步鉴定该菌株,克隆测定了该菌18SrRNA基因序列,并对其进行系统进化树分析,结果表明该菌属于毛霉属,与Mucor racemosus、Mucor plumbeus、Mucor ramosissimus与Mucor circinelloides同属一个分支。     

被孢霉W_（A－90）发酵生产γ－亚麻酸的研究

本文研究了由被抱霉变株ＭＡ－９０生产了γ－亚麻酸的发酵条件．确定了最适碳源、复源及Ｃ／Ｎ．初步建立了生产γ－亚麻酸的工艺条件。葡萄糖、蔗糖及天冬酰胺和尿素为最适碳、氮源。在Ｃ／Ｎ为２０／１,葡萄糖浓度为８０ｇ／Ｌ的条件下．油脂产量和油脂中γ－亚麻酸的含量分别为８．２ｇ／Ｌ和１４．１３％,γ－亚麻酸产量及生物量分别为１．１５７ｇ／Ｌ和３１．２ｇ／Ｌ。后期适当降低培养温度和良好的通气条件均有利于γ－亚麻酸的积累。     

被孢霉MA—90发酵生产γ—亚麻酸的研究

本文研究了由被孢霉变株ＭＡ－９０生产了γ－亚麻酸的发酵条件,确定了最适碳源、氮源及Ｃ／Ｎ,初步建立了生产γ－亚麻酸的工艺条件．葡萄糖、蔗糖及天冬酰胺和尿素为最适碳、氮源。在Ｃ／Ｎ为２０／１,葡萄糖浓度为８０ｇ／Ｌ的条件下,油脂产量和油脂中γ－亚麻酸的含量分别为８．５ｇ／Ｌ和１４．１３％,γ－亚麻酸产量及生物量分别为１．．１５７ｇ／Ｌ和３１．２ｇ／Ｌ。后期适当降低培养温度和良好的通气条件均有利于γ－     

液体悬浮培养促进铁皮石斛原球茎高效诱导、增殖的研究

用正交设计方法对铁皮石斛原球茎具有高效增殖影响的激素（BA,NAA,KT,马铃薯汁）以及配比进行研究,结果表明：以茎段为外植体在培养基Ms＋BA2．0mg／L＋NAA2．0mg／L＋马铃薯汁10％＋糖3％,具有很好的原球茎诱导作用,50d后诱导率达95．20％;原球茎在1／2MS＋BA2．0mg／L＋NAA1．0mg／L＋KT0．5mg／L＋糖3％的培养基上,以液体悬浮培养,原球茎增殖达49．032g／50d。     

高效利用木糖产油的氮离子注入Mortierella alpina诱变选育研究

木糖的有效利用是木质纤维素全利用的基础。为了获得高效利用木糖产油的高山被孢霉菌株,通过多轮氮离子束诱变,筛选出一株有效利用木糖的产油高山被孢霉1502．8（MortiereUaalpina1502—8,MAIS02．8）,并研究了以葡萄糖／木糖（W／W,5／3）组成的混合糖为碳源,突变株生长和油脂积累的特性。采用单因子和正交实验对培养基组成和发酵条件进行了优化,结果表明,诱变菌在温度28oC,pH8．0,接种量8％,装液量30％,蛋白胨0．76％和豆粕1％,分批补糖的最优发酵条件下发酵9d,生物量和菌体油脂积累量分别达29．8g／L和11．7g／L,较出发菌提高了2．59倍和2．05倍,同时原料糖利用率达到99．4％。     

Synthesis of biologically active lipids by fungus Mucor lusitanicus 306D grown on media with various composition

Growth and lipogenesis of fungus Mucor lusitanicus 306 D producing gamma-linolenic acid was studied under various regimes of nitrogen and carbon nutrition. Media containing food industry wastes such as maize extract, molasses, and protein hydrolysate were used. Content of gamma-linolenic acid was higher when using carbohydrates such as glucose and molasses as carbon sources and urea as a nitrogen source. At high glucose concentration (100 g/l), fed batch cultivation provided high content of gamma-linolenic acid in lipids (1 g/l). After extraction of lipids, fungus biomass contained 42% proteins with all essential amino acids. Defatted biomass was shown to be effectively assimilated by minks.     

被孢霉γ-亚麻酸高产菌株选育

利用紫外线复合氯化锂诱变高山被孢霉SM96,筛选出一株高产γ-亚麻酸的菌株TM11,产量比出发菌株(25mg/L)提高了近3倍。对影响其多不饱和脂肪酸产生的因子Mg2+,VB6,营养盐溶液,磷源,碳源,氮源酵母膏进行了正交试验,选出TM11最佳的培养基配方:MgSO4·7H2O1.0g,VB6150mg/L,酵母膏6g/L,葡萄糖100g/L,营养盐溶液1mL/L,K2HPO42g/L。在上述最佳培养基中TM11的生物量达18.0213g/L,总脂达10.8128g/L,GLA量达668mg/L。     

Citric Acid Production from Hydrolysate of Pretreated Straw Cellulose by Yarrowia lipolytica SWJ-1b Using Batch and Fed-Batch Cultivation

In this study, crude cellulase produced by Trichoderma reesei Rut-30 was used to hydrolyze pretreated straw. After the compositions of the hydrolysate of pretreated straw were optimized, the study showed that natural components of pretreated straw without addition of any other components such as (NH₄)₂SO₄, KH₂PO₄, or Mg²⁺ were suitable for citric acid production by Yarrowia lipolytica SWJ-1b, and the optimal ventilatory capacity was 10.0 L/min/L medium. Batch and fed-batch production of citric acid from the hydrolysate of pretreated straw by Yarrowia lipolytica SWJ-1b has been investigated. In the batch cultivation, 25.4 g/L and 26.7 g/L citric acid were yields from glucose and hydrolysate of straw cellulose, respectively, while the cultivation time was 120 hr. In the three-cycle fed-batch cultivation, citric acid (CA) production was increased to 42.4 g/L and the cultivation time was extended to 240 hr. However, iso-citric acid (ICA) yield in fed-batch cultivation (4.0 g/L) was similar to that during the batch cultivation (3.9 g/L), and only 1.6 g/L of reducing sugar was left in the medium at the end of fed-batch cultivation, suggesting that most of the added carbon was used in the cultivation.     

Production of <Emphasis Type="SmallCaps">l</Emphasis>-lactic acid from a mixture of xylose and glucose by co-cultivation of lactic acid bacteria

The production of optically pure lactic acid in a high yield from xylose or a mixture of xylose and glucose, which is a model hydrolysate of lignocellulose, is described. In a single cultivation, Enterococcus casseliflavus produced 38 g/l of lactic acid with an optical purity of 96% enantiomeric excess (ee) and 6.4 g/l of acetic acid from 50 g/l of xylose when MRS medium was used. When a mixture of 50 g/l of xylose and 100 g/l of glucose was used as the carbon source in a cultivation of E. casseliflavus alone, glucose was converted to lactic acid in the early phase of the cultivation but xylose was hardly consumed. In a co-cultivation where E. casseliflavus and Lactobacillus casei specific for glucose were simultaneously inoculated, little or no lactic acid was produced after the glucose was almost consumed. A co-cultivation with two-stage inoculation (in which E. casseliflavus was added at a cultivation time of 40 h after L. casei cells were inoculated) resulted in complete consumption of 50 g/l of xylose and 100 g/l of glucose. In the co-cultivation, 95 g/l of lactic acid with a high optical purity of 96% ee was obtained at 192 h. Such a co-cultivation using two microorganisms specific for each sugar is considered to be one promising cultivation technique for the efficient production of lactic acid from a sugar mixture derived from lignocellulose.     

Control of glucose feeding using exit gas data and its application to the production of PHB from tapioca hydrolysate byAlcaligenes eutrophus

Summary A method to estimate the glucose concentration in the culture broth using CO₂ evolution rate (CER) data from a mass spectrometer was developed.Alcaligenes eutrophus was cultivated to produce poly(3-hydroxybutyric acid) (PHB) from tapioca hydrolysate using this method. Thek value (g glucose/mol CO₂), defined as the glucose consumption per CO₂ evolution, decreased with culture time and was automatically changed using CER data. The glucose concentration in the culture broth could be controlled at 10 to 20 g/L. A final cell concentration of 106 g/L, PHB concentration of 61 g/L. and PHB content of 58 % of dry cell weight were obtained after 59 h of cultivation.     

利用甘蔗糖蜜发酵生产花生四烯酸的研究

通过培养高山被孢霉利用糖蜜来发酵生产花生四烯酸(ARA),研究了不同甘蔗糖蜜预处理方法对ARA发酵生产的影响。研究表明:H2SO4法是最利于ARA发酵生产的糖蜜预处理方法。利用预处理的甘蔗糖蜜发酵生产ARA,通过单因素实验设计,确定了最优的培养条件,包括初始还原糖80 g/L,N源6 g/L,接种量20%,初始pH6.0和培养温度25℃,在此条件下发酵,干细胞质量、油脂含量、ARA产量和糖利用率分别达到28.5 g/L、11.7g/L、3.68 g/L和94.5%。     

A two-stage cultivation process for the growth enhancement of Chlorella vulgaris

This study proposes a two-stage cultivation process with an autotrophic growth followed by a mixotrophic process. The results indicated that a two-stage cultivation process using a daily dose of 3 g/L of glucose could achieve 7.4 g/L of biomass, which was about a 64 % increase over simple autotrophic cultivation. In the second stage of mixotrophic cultivation, glucose was regarded as a better carbon source for cell growth, than was glycerol. Linoleic acid (C18:2) would be the primary component in the two-stage cultivation as in the autotrophic cultivation. Even carbon source was provided in the second stage of mixotrophic cultivation; lower light intensity limited the mixotrophic growth, which indicated that photosynthesis still plays an important role in the second stage of mixotrophical cultivation. The final biomass was higher after this two-stage cultivation process, which made it suitable for application in the production scale-up of algal biomass.     

High-cell-density fed-batch cultivation of the docosahexaenoic acid producing marine alga Crypthecodinium cohnii

The heterotrophic marine alga Crypthecodinium cohnii is known to produce docosahexaenoic acid (DHA), a polyunsaturated fatty acid with food and pharmaceutical applications, during batch cultivation on complex media containing sea salt, yeast extract, and glucose. In the present study, fed-batch cultivation was studied as an alternative fermentation strategy for DHA production. Glucose and acetic acid were compared as carbon sources. For both substrates, the feed rate was adapted to the maximum specific consumption rate of C. cohnii. In glucose-grown cultures, this was done by maintaining a significant glucose concentration (between 5 and 20 g/L) throughout fermentation. In acetic acid-grown cultures, the medium feed was automatically controlled via the culture pH. A feed consisting of acetic acid (50% w/w) resulted in a higher overall volumetric productivity of DHA (r(DHA)) than a feed consisting of 50% (w/v) glucose (38 and 14 mg/L/h, respectively). The r(DHA) was further increased to 48 mg/L/h using a feed consisting of pure acetic acid. The latter fermentation strategy resulted in final concentrations of 109 g/L dry biomass, 61 g/L lipid, and 19 g/L DHA. These are the highest biomass, lipid, and DHA concentrations reported to date for a heterotrophic alga. Vigorous mixing was required to sustain aerobic conditions during high-cell-density cultivation. This was complicated by culture viscosity, which resulted from the production of viscous extracellular polysaccharides. These may present a problem for large-scale industrial production of DHA. Addition of a commercial polysaccharide-hydrolase preparation could decrease the viscosity of the culture and the required stirring.     

Biosensor online control of citric acid production from glucose by Yarrowia lipolytica using semicontinuous fermentation

Our study aimed at the development of an effective method for citric acid production from glucose by use of the yeast Yarrowia lipolytica. The new method included an automated bioprocess control using a glucose biosensor. Several fermentation methodologies including batch, fed‐batch, repeated batch and repeated fed‐batch cultivation were tested. The best results were achieved during repeated fed‐batch cultivation: Within 3 days of cycle duration, approximately 100 g/L citric acid were produced. The yields reached values between 0.51 and 0.65 g/g and the selectivity of the bioprocess for citric acid was as high as 94%. Due to the elongation of the production phase of the bioprocess with growth‐decoupled citric acid production, and by operating the fermentation in cycles, an increase in citric acid production of 32% was achieved compared with simple batch fermentation.