常压室温等离子体快速诱变产油酵母的条件及其突变株的特性

采用新型常压室温等离子体射流诱变产油酵母,结合快速突变产油酵母操作条件及基于96孔板的高通量筛选手段,获得了一系列增殖速度和产油量发生变化的突变株。在等离子体对菌株致死率为99％的条件下获得的以突变株增殖速度为指标的正突变率达到27.2％。用含酵母粉 (10 g/L)、蛋白胨 (10 g/L) 及葡萄糖 (20 g/L) 的酵母膏胨葡萄糖培养基进行发酵实验表明,筛选得到的高产突变体产油量从对照株的1.87％ (W/W) 增加到4.07％ (W/W)。     

利用脂肪酸合成酶抑制剂和磷酸香草醛反应筛选高产油脂酵母菌

利用80 MeV/u碳离子束对产油菌株粘红酵母进行辐照诱变, 采用含有脂肪酸合成酶抑制剂cerulenin的培养基进行高产油脂突变株的初筛, 并通过磷酸香草醛反应和氯仿甲醇抽提法对初筛菌株油脂含量进行分析。结果表明, cerulenin对酵母细胞生长有较好的抑制作用, 浓度为8.96×10-6 mol/L时, 抑制率达98%以上, 可作为筛选浓度。通过磷酸香草醛反应法对初筛菌体油脂含量进行定量分析, 发现菌体油脂含量与该反应在530 nm处的光吸收成线性正相关, 初筛菌株的正突变率达65%以上。该方法快速方便, 是一种较为理想的产油酵母筛选方法。通过该方法, 筛选出了2株油脂含量高于对照90%以上的突变株。     

利用脂肪酸合成酶抑制剂和磷酸香草醛反应筛选高产油脂酵母菌

利用80 MeV/u碳离子束对产油菌株粘红酵母进行辐照诱变,采用含有脂肪酸合成酶抑制荆cerulenin的培养基进行高产油脂突变株的初筛,并通过磷酸香草醛反应和氯仿甲醇抽提法对初筛菌株油脂含量进行分析.结果表明,cerulenin 对酵母细胞生长有较好的抑制作用,浓度为8.96×10-6mol/L时,抑制率达98%以上,可作为筛选浓度.通过磷酸香草醛反应法对初筛茵体油脂含量进行定量分析,发现菌体油脂含量与该反应在530 nm处的光吸收成线性正相关,初筛菌株的正突变率达65%以上.该方法快速方便,是一种较为理想的产油酵母筛选方法.通过该方法,筛选出了2株油脂含量高于对照90%以上的突变株.     

YAG激光照射对高山被孢霉花生四烯酸产量的影响

花生四烯酸产生菌高山被孢霉的孢子在时间为8min,距离为10cm的YAG激光剂量照射下,其致死率为75％－80％,以该剂量反复进行照射及菌种筛选,得到其最高生物量28.6g/L,油脂11.04g/L,3.10g/L的突变株T105,比对照菌株的AA产量及油脂量分别提高了2．95倍和1．71倍。通过对突变株T105的形态,代谢情况以及继代稳定性进行分析,认为YAG激光诱变育种是获得茶花生四烯酸高产菌株的有效方法。     

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％。     

低场核磁共振技术快速检测小球藻油脂含量及其在高通量选育中的应用

为了建立一个高效的高产油微藻诱变育种流程,微藻中油脂含量快速和准确的测定在其中具有重要作用。在本研究中,首先利用低场核磁共振技术,建立了直接检测干藻粉和培养液中小球藻油脂含量的方法,其信号强度与细胞中油脂含量存在特异的线性关系,干藻粉和藻液中油脂含量与信号值拟合的R2均高于0.99,说明该方法用于小球藻油脂含量的检测是准确和可行的。同时该方法与传统油脂测量方法相比,具有快速、简便和准确等优点。但其通量不及尼罗红染色法,因此,我们开发了将尼罗红染色法用于初筛,低场核磁共振技术用于复筛的新型高通量藻种复合筛选方法,并将此筛选方法应用于一种异养高产油原壳小球藻的诱变育种过程中。首先从3 098株诱变藻种中初筛得到108株具有较高油脂含量的藻株,然后利用低场核磁共振技术复筛得到9株高产油性能的藻株,其中一株甘油三酯含量超过20%,比原始藻株提高1倍,培养168 h后培养液油脂浓度达到5 g/L,证明此诱变育种流程不仅提高了筛选的效率还可靠且可行。     

结合缺陷型菌株显色法采用离子束诱变筛选高产L-精氨酸突变株

为快速高效筛选L-精氨酸高产突变株,建立一种缺陷菌株平板显色法并采用低能N+离子束对L-精氨酸生产用菌株钝齿棒杆菌SYPA5-5进行诱变处理,通过上述平板显色法筛选获得高产突变株.对突变株进行摇瓶发酵实验,最终选育出一株L-精氨酸产量较高且产酸性能比较稳定的突变菌株钝齿棒杆菌SYPA5-5-36.该菌株摇瓶发酵L-精氨酸产量可达35.85 g/L,比出发菌株提高了19.5％.因此,缺陷型菌株平板显色法可以用于快速、高效筛选高产L-精氨酸突变株.     

L-Gln高产突变菌株发酵条件的初步优化

将诱变实验筛选出的遗传稳定性高产突变株C.glutamicum N-U-6作为研究对象,采用单次单因子非统计优化技术确定了它在培养温度为30℃下250 mL的摇瓶培养条件为:150 g.L-1葡萄糖,最佳无机氮源及其浓度为:40 g.L-1NH4Cl;最佳有机氮源及其浓度:14 g.L-1尿素;玉米浆浓度:10 g.L-1,初始pH为7.2,装液量为30 mL,种龄为12 h,接种量为10%。谷氨酰胺产量达到37.21 g.L-1,比优化前的突变株(33.54 g.L-1)提高10.9%。     

作物秸秆水解条件优化及其对小球藻培养的影响

以小麦、玉米、谷子秸秆为原料，使用碱性双氧水对其进行预处理后再添加纤维素酶和木聚糖酶进行水解，以水解液中总糖含量为指标，评估水解效果并进行条件优化。将3种作物秸秆水解液作为有机碳源，探究其用于培养小球藻的效果。结果表明：3种作物秸秆最佳水解条件为固液比1∶15、温度30℃、处理时间12 h。在此条件下，玉米、谷子和小麦秸秆水解液总糖含量分别达1.677、1.412和1.211 g·L^-1。采用3种作物秸秆水解液培养小球藻，可显著提高小球藻的生物量和油脂含量。其中，玉米秸秆水解液效果最佳，小球藻生物量和油脂含量分别达到1.801 g·L^-1和30.1%。表明利用作物秸秆水解液为碳源，可显著提高小球藻的生物量并促进油脂富集。研究结果为秸秆木质纤维素原料的高效转化利用奠定了基础，为农业废弃物资源化利用提供了新思路，也为利用秸秆水解液高效培养小球藻提供了理论依据。     

高产DHA破囊壶菌Aurantiochytrium sp. PKU#SW7诱变株的筛选

【目的】对野生菌株Aurantiochytrium sp.PKU#SW7诱变育种,筛选高产DHA突变株。【方法】采用UV诱变和化学药物胁迫筛选方式,以菌株的生物量、油脂产量、DHA产量作为筛选指标,获得高产DHA突变株。【结果】经鉴定获得一株DHA高产突变株PKU#PM003,该菌株传代4次后仍保持较好的遗传稳定性。摇瓶发酵后,PKU#PM003生物量产量高达6.62 g/L,比原始菌株5.95 g/L提高了11.26%,脂肪酸含量高达4.01 g/L,比原始菌株3.18 g/L提高了26.1%,DHA在脂肪酸中所占比例由29.97%增加到33.43%,产量提高了41.01%,油脂突变效果显著。【结论】突变株PKU#PM003可作为性状优良的工业化发酵生产菌种,并在DHA产量提升上仍具有巨大的空间。     

利用核糖体工程选育丙酮丁醇菌提高丁醇产量

利用核糖体工程技术对丙酮丁醇梭菌Clostridium acetobutylicum L7进行诱变筛选,以获得丁醇高产菌株。使用链霉素诱变C.acetobutylicum L7并结合设计的平板转接逐次提高链霉素浓度的筛选路线,获得丁醇产量较高的菌株S3。结果表明,S3丁醇产量为(12.48±0.03)g/L,乙醇产量为(1.70±0.07)g/L,相对于原始菌分别提高了11.2%及50%;丁醇/葡萄糖转化率由原始菌的0.19提高到0.22,丁醇生产率达到0.24 g/(L.h),相比提高30.5%;耐受丁醇浓度由原始菌的12 g/L提高到14 g/L;发酵液粘度下降到4 mPa/s,同比降低了60%,利于后续分离工作的进行,降低发酵成本。进一步研究工作表明,S3菌株遗传稳定性良好。因此,核糖体工程技术是一种选育丁醇高产菌株的有效方法。     

高产不饱和油脂海鞘真菌桔青霉Asc-2-4的诱变选育

诱变育种是获得高产菌株,实现微生物工业化生产油脂的重要措施。以前期获得的高产不饱和油脂菌株桔青霉（Penicillium citrinum）Asc-2-4为出发菌株,利用丙二酸建立快速筛选高产不饱和脂肪酸突变菌的方法,通过紫外线 氯化锂复合诱变得到1株高产油脂突变菌Asc-2-4-1,油脂含量比出发菌株提高了92.98%。经过初步的培养基无机盐优化,其油脂得率和不饱和脂肪酸产量达到了7.10 g/L和3.84 g/L,与Asc-2-4相比,分别提高了84.42%和77.78%。结果表明,通过复合诱变选育技术可选育出高产突变菌株,选育的Asc-2-4-1可望作为产油微生物被开发利用。     

响应面法优化木薯淀粉发酵生产单细胞油脂工艺

采用响应面分析法对发酵性丝孢酵母菌株以木薯淀粉水解液为原料合成微生物油脂的培养条件进行优化。首先利用Plackett-Burman试验设计确定影响油脂产量的主要因素,在此基础上再利用Box-Behnken试验设计及响应面分析法对其进行条件优化。结果表明,发酵温度、C/N、pH对油脂产量具有显著影响,产油脂的最佳发酵条件为:发酵温度28.78°C、C/N 126.18、pH 6.69,油脂产量达到14.88g/L,比优化前提高了28.6%。同时,气相色谱分析表明,微生物油脂脂肪酸组成成分主要包括棕榈酸、硬脂酸、油酸、亚油酸酯等,是优良的生物柴油制备原料。     

Impact of legume versus cereal root residues on biological properties of West African soils

Many microbial turnover processes in acidic sandy subtropical soils are still poorly understood. In a 59-day pot and a 189-day laboratory incubation experiment with two West African continuous cereal soils, the effects of 2 mg g^?1 root residues were investigated on growth of sorghum seedlings, soil microbial biomass and activity indices, using cowpea, groundnut, pearl millet, maize and sorghum. The effects of root residues were compared with mineral P or mineral P + N treatments and with a non-fertilized control treatment. On the Alfisol (Fada, Burkina Faso), shoot dry mass was always significantly higher than on the Ultisol (Koukombo, Togo). Highest shoot dry mass was observed after application of mineral P + N on the Alfisol and after mineral P alone on the Ultisol. The application of legume root residues led to small and non-significant increases in dry mass production compared to the non-amended control, whereas the application of cereal root residues led to a decline, regardless of their origin (millet, maize or sorghum). Contents of microbial biomass C, microbial biomass N and ergosterol were 75 to 100% higher in the Alfisol than in the Ultisol, while ATP was only 36% higher. Organic amendments increased ergosterol concentrations by up to 145% compared to the control and mineral P application. Microbial biomass C and microbial biomass N increased by up to 50% after application of root residues, but ATP only up to 20%. After application of legume root residues, cumulative CO₂ production was similar in both soils with an average of 370?µg CO₂-C g^?1 over 189 days. After application of cereal root residues, cumulative CO₂ production was higher in the Alfisol (530?µg g^?1) than in the Ultisol (445?µg g) over 189 days.     

Cottonseed Meal Bioactive Peptides as an Alternative to Antibiotic Growth Promoters in Broiler Chicks

The aim of this experiment was to evaluate the effects of bioactive peptides derived from enzymatic hydrolysis of cottonseed meal (CSBP) compared with zinc bacitracin, as an antibiotic growth promoter (AGP), on productive traits, serum lipid profile, and ileal microbial population in broiler chickens. A total number of 240-day-old broiler chicks (Ross 308) were allocated into 4 treatments, replicated 5 times based on a completely randomized design. The dietary treatments included a basal diet serving as control group, basal diet?+?40 mg/kg zinc bacitracin as AGP group, and the basal diet supplemented with 15 or 20 g/kg CSBP substituting equal quantity of maize and soybean meal. Performance traits, including daily weight gain, feed intake, feed conversion ratio (FCR), and livability were recorded. At the end of the study, serum lipid parameters, ileal microbial population, and economical indices were determined. The results indicated that feed intake and FCR increased (P?<?0.05) in birds receiving 20 g/kg CSBP over the entire period (1–35 days), but there was no significant effect of CSBP on body weight, although numerically higher than the control group. However, the antibiotic group showed a significant increase (P?<?0.05) in body weight and feed intake. Livability, European Production Efficiency Factor (EPEF), and European Broiler Index (EBI) significantly improved in broiler chickens fed antibiotic and 15 g/kg CSBP supplement (P?<?0.05). Adding 20 g/kg of CSBP to the diet significantly increased serum triglycerides and decreased low-density lipoproteins (LDL) compared to the control and antibiotic groups on day 35. The relative weight of abdominal fat and LDL to HDL ratio were significantly lower for CSBP and antibiotic treatments than the control group (P?<?0.05). Supplementation of antibiotic and both graded levels of CSBP decreased the ileum population of Escherichia coli (P?<?0.05). The current findings suggest that including CSBP in broiler diets may benefit production through improving growth rate of broilers and balancing gut microbiota population. In addition, CSBP could be considered as a potential alternative to antibiotics in an AGP free production system.

     

Exploring low-cost carbon sources for microbial lipids production by fed-batch cultivation of <Emphasis Type="Italic">Cryptococcus albidus</Emphasis>

Volatile fatty acids (VFAs), acetic acid, acetates, and ethanol were used as carbon sources for the production of microbial lipids using Cryptococcus albidus in batch cultures. C. albidus utilized organic acids less than glucose in the production of lipids, resulting in a lipid yield coefficient on VFAs of 0.125 g/g. In a two-stage batch culture, the lipid content increased to 43.8% (w/w) when VFAs were used as the sole carbon source in the second stage, which was two times higher than that of the batch culture. Furthermore, a 192 h, two-stage fed-batch cultivation of C. albidus produced a dry cell weight, lipid concentration, and lipid content of 26.4 g/L, 14.5 g/L, and 55.1% (w/w), respectively. The fed-batch culture model used in this study featured pure VFA solutions, with intermittent feeding, under oxygen-enriched air supply conditions. This study investigated several alternative carbon sources to reduce the cost of microbial lipids production and proved the feasibility of using VFAs as the carbon source for the provision of a high lipid content and productivity.     

Screening and characteristics of a butanol-tolerant strain and butanol production from enzymatic hydrolysate of NaOH-pretreated corn stover

As a gasoline substitute, butanol has advantages over traditional fuel ethanol in terms of energy density and hydroscopicity. However, solvent production appeared limited by butanol toxicity. The strain of Clostridium acetobutylicum was subjected to mutation by mutagen of N-methyl-N'-nitro-N-nitrosoguanidine for 0.5?h. Screening of mutants was done according to the individual resistance to butanol. A selected butanol-resistant mutant, strain 206, produced 50?% higher solvent concentrations than the wild-type strain when 60?g glucose/l was employed as substrate. The strain was also able to produce solvents of 23.47?g/l in 80?g/l glucose P2 medium after 70?h fermentation, including 5.41?g acetone/l, 15.05?g butanol/l and 3.02?g ethanol/l, resulting in an ABE yield and productivity of 0.32?g/g and 0.34?g/(l?h). Subsequently, Acetone-butanol-ethanol (ABE) production from enzymatic hydrolysate of NaOH-pretreated corn stover was investigated in this study. An ABE yield of 0.41 and a productivity of 0.21?g/(l?h) was obtained, compared to the yield of 0.33 and the productivity of 0.20?g/(l?h) in the control medium containing 52.47 mixed sugars. However, it is important to note that although strain 206 was able to utilize all the glucose rapidly in the hydrolysate, only 32.9?% xylose in the hydrolysate was used after fermentation stopped compared to 91.4?% xylose in the control medium. Strain 206 was shown to be a robust strain for ABE production from lignocellulosic materials and has a great potential for industrial application.     

一株高产脯氨酸的嗜醋酸棒杆菌的选育及发酵条件优化

以嗜醋酸棒杆菌为出发菌株,经过片段化全基因组体外诱变、重组和连续的磺胺胍抗性筛选,获得一株L-脯氨酸的高产菌株。摇瓶发酵优化结果表明,葡萄糖、生物素和硫胺素的最适用量分别为16%、300μg/L、400μg/L,最适pH为6.8~7.0,装液量为25ml/500ml摇瓶,发酵培养72h后L-脯氨酸产率高达到75.6g/L,与对照相比提高了5%。考察了50L发酵罐中细胞生长对L-脯氨酸产量的影响,补料分批发酵结果表明(比生长速率分别为0.06/h、0.08/h和0.1/h),比生长速率在0.08/h左右时L-脯氨酸的产率最高,L-脯氨酸的比生产速率Q_P达到0.091 g/(g.h),产率高达82.1 g/L,比优化前提高了14%。     

High production of D-β-hydroxyisobutyric acid from methacrylic acid by Candida rugosa and its mutant

Production of D-β-hydroxyisobutyric acid (D-HIBA) from methacrylic acid (MA) was investigated using Candida rugosa IFO 0750 and its mutant. Cell growth decreased as the MA concentration increased and was inhibited at D-HIBA concentrations higher than 30 g/l. Optimal MA concentration for D-HIBA production was in the range of 10–20 g/l. It was also noted that cell growth and D-HIBA production were inhibited by higher concen-trations of Na⁺, K⁺, and NH₄ ⁺, which were required for pH control during cultivation. With a suitably designed feeding mode of MA, the parent strain produced 65 g/l of D-HIBA after 120?h of fed-batch cultivation, but molar conversion yield of D-HIBA was less than 40%. The mutant, unable to assimilate propionic acid, produced as high as 70 g/l of D-HIBA in the same culture period with a molar conversion yield of more than 70%.     

Influence of 12-week nicotine treatment and dietary copper on blood pressure and indices of the antioxidant system in male spontaneous hypertensive rats

Nicotine treatment and copper (Cu) deficiency have been associated with an increased production of reactive oxygen species that may contribute to the development and/or progression of cardiovascular diseases (CVD). The present study investigated the influence of dietary Cu intake on the response to chronic nicotine treatment in spontaneous hypertensive rats (SHR) with respect to tissue trace mineral levels, several components of the oxidant defense system, and lipid peroxidation rates. SHR weighing 100–110 g were fed a Cu deficient diet (?Cu) (0.5 μg Cu/g) for 14 d prior to nicotine treatment. SHR were inserted with tablets that released nicotine at a rate of 75 μg/h or placebo (control). Following tablet insertion, rats were fed a control diet (+Cu) (12.0 μg Cu/g) or the ?Cu diet. Nicotine treatment lasted for 12 wk. Blood pressure (BP) was higher in nicotine-treated SHR than in control SHR at wk 3; BP was unaffected by diet. BP was higher in +Cu nicotine-treated SHR at wk 6 compared to ?Cu nicotine and control rats. BP was not affected by nicotine or diet at wk 2. Liver, heart, and brain Cu levels and liver, heart, and red cell CuZn superoxide dismutase and plasma ceruloplasmin oxidase activities were lower in the ?Cu SHR than in the +Cu SHR. Liver Fe levels were higher and plasma Fe levels were lower in the ?Cu rats than in the +Cu rats. Liver selenium-dependent-glutathione peroxidase (Se-GSH-Px) activity was lower in the ?Cu rats than in the +Cu rats; heart and thoracic aorta Se-GSH-Px activity was unaffected by ?Cu diet. Thoracic aorta, liver, and heart GSH-reductase activities were unaffected by treatments. Plasma thiobarbituric acid reactive substances (TBARS) were higher in the ?Cu than in the +Cu SHR. Liver and heart TBARS production was similar among the groups. These data show that nicotine can exacerbate the development of high BP in susceptible individuals; Cu deficiency did not exacerbate the effects of nicotine.