Relations between gibberellin hyphae and secondary metabolic product GA3 by resting culture method

针对赤霉菌生长与产次级代谢产物赤霉素的关系进行摇瓶静息培养研究.在不同菌龄及不同底物浓度的条件下,菌丝产素能力有较大不同.实验数据表明:在菌龄96 h,葡萄糖浓度为5g/L时,菌丝静息培养72 h后,培养液中赤霉素含量最高,达1100 μg/mL;同时单位时间单位菌丝产率也最大,为7.1μg/mg·h;在该菌龄条件下,当葡萄糖浓度为1g/L时,赤霉素转化效率最高,为0.8484.该研究有助于进一步优化发酵工艺,降低赤霉素生产成本.     

赤霉素发酵溶氧优化调控研究

为了解溶氧对赤霉素发酵过程影响以及相应工艺优化,采用不同溶氧条件下藤仓赤霉菌Gibberella fujikuroi分批发酵生产赤霉素的过程进行菌丝浓度、残糖浓度和GA3产物浓度检测,并微分运算得出比生长速率与比产物合成速率随发酵时间变化,分析了溶氧对比生长速率与比产物合成速率以及得率的影响,进而提出Gibberella fujikuroi发酵高产的溶氧控制策略:在发酵初始阶段(0–50h)控制溶氧30%左右,以维持较高的菌体生长速率;发酵中后期(50–184h),溶氧控制在15%,以获取菌丝持续较高的GA3合成速率能力。采用这一优化溶氧控制策略,发酵过程中最大菌丝浓度19.24g/L、最终赤霉素浓度2 180mg/L和平均比产物合成速率0.616mg/(g·h),比未优化前发酵分别提高了8.33%、13.25%和4.58%,表明所采取的分阶段溶氧控制策略对促进GA3生产有效。     

静息培养条件下赤霉菌菌丝产素能力的研究

针对赤霉菌生长与产次级代谢产物赤霉素的关系进行摇瓶静息培养研究。在不同菌龄及不同底物浓度的条件下,菌丝产素能力有较大不同。实验数据表明：在菌龄96h,葡萄糖浓度为5gfL时,菌丝静息培养72h后,培养液中赤霉素含量最高,达1100μg／mg;同时单位时间单位菌丝产率也最大,为7．1μg/mg·h;在该菌龄条件下,当葡萄糖浓度为1dL时,赤霉素转化效率最高,为0．8484。该研究有助于进一步优化发酵工艺,降低赤霉素生产成本。     

利用特比萘芬抗性筛选赤霉素高产菌株及相关发酵特性的研究

【目的】诱变、筛选赤霉素高产菌株,并明确该菌遗传稳定性及部分发酵特性。【方法】利用亚硝基胍、60Co-γ射线以及复合诱变的方法,结合抑真菌剂-特比萘芬抗性筛选,定向选育赤霉素高产突变株;通过琼脂斜面传代实验确定遗传稳定性;发酵罐培养了解其部分发酵性状。【结果】藤仓赤霉菌单细胞悬液先后经过终浓度为300 mg/L亚硝基胍30 min和60Co-γ射线300戈瑞下复合诱变,得到抗120μg/L特比萘芬的赤霉菌突变株。摇瓶复筛,确定赤霉素突变菌株的赤霉素合成能力,其中ZNL13-3菌产赤霉素效价为2 215±35 mg/L,较之诱变前发酵效价平均提高了11.87%。ZNL13-3菌株经连续15次试管斜面传代考察,菌株赤霉素合成的稳定性较好,能维持原发菌株93.2%,具有良好的遗传稳定性和生产应用前景。5 L发酵罐培养,比较菌丝浓度和产物浓度随发酵时间变化趋势,ZNL13-3菌比生产速率优于原发菌株。【结论】赤霉菌抗特比萘芬的变异特性与赤霉素高产之间存在某种对应关系,为进一步定向优化筛选优良菌株提供参考。     

芸香苷对家蚕谷胱甘肽-S-转移酶部分基因的诱导表达

谷胱甘肽-S-转移酶(glutathione-S-transferases, GSTs) 是对一种机体的解毒代谢起重要作用并可以被诱导的酶系。为了从分子水平上探究家蚕Bombyx mori GSTs 基因与植物次生物质芸香苷代谢的关系, 本实验采用双跟踪标定定量PCR (dual-spike-in qPCR)方法, 用 5×10^-1, 5×10^-2, 5×10^-3 ng/μL 3 个浓度芸香苷溶液处理家蚕5龄幼虫, 并对各组织中 GSTs Epsilon 家族不同基因的转录水平进行检测。结果显示, 浓度为 5×10^-2 ng/μL 的芸香苷溶液能诱导GSTs 基因的转录水平发生显著变化。在中肠中, BmGSTe1, BmGSTe2 和 BmGSTe6的诱导转录水平较高, 在诱导后24 h达到最大值; 在脂肪体中BmGSTe1, BmGSTe6和BmGSTe7的转录水平相对较高且分别在诱导后 2 h, 2 h和4 h 达到最大值; 而GSTs基因在马氏管中表达量均很低或者检测不到表达。与 5×10^-2 ng/μL 浓度相比, 5×10^-1 ng/μL 的芸香苷溶液诱导后各基因的转录水平上升的幅度较小并且达到峰值的时间不同, 而 5×10^-3 ng/μL 浓度的芸香苷溶液并不能使GSTs基因的诱导转录水平发生变化。结果提示, 家蚕 GSTs Epsilon 家族基因对芸香苷的代谢具有重要作用。     

调控pH提高分批发酵赤霉素GA3产量

为高效率发酵生产GA₃,对藤仓赤霉菌发酵过程pH进行优化调控研究。采用5L全自动发酵罐,在pH 3.0-5.0条件下,对藤仓赤霉菌菌丝生长及GA₃产量的影响进行了考察,实验数据表明：在pH 4.0条件下,菌比生长速率可获最大值,为0.395/h;而pH 3.0条件下,GA₃比生成速率最大,达到4.43mg/(g?h)。基于不同pH条件下,对菌比生长速率、得率、GA₃比生成速率的影响,提出GA₃分批发酵过程中的pH调控策略,即：0-20h,pH自然;20-50h,pH 4.0;50-80h,pH 3.0-3.5;80h后控制pH为3.5-4.0。在此控制模式下,经过196h发酵GA₃的终产量达到2 224mg/L,GA₃产率44.5mg/g,GA₃生产强度0.242mg/(L?h),分别比不控制pH条件下发酵的数值增长了7.75%、7.74%、8.04%,表明该pH控制策略能增进GA₃发酵生产效率。     

鲁西北平原冬小麦田臭氧浓度变化特征及对产量的潜在影响和机理分析

近地层高浓度臭氧(O₃)对农作物生长和产量形成有明显的影响。利用在中国科学院禹城综合试验站(山东省)冬小麦(Triticum aestivum)农田生态系统上观测的O₃浓度及微气象资料, 分析了鲁西北平原冬小麦农田生态系统O₃浓度的日变化和季节变化规律, 在此基础上初步分析了O₃浓度与CO₂通量(F_c)的关系, 并用欧洲和美国科学家在实验室得到的O₃浓度-冬小麦产量关系模型估算了O₃对冬小麦产量的潜在影响。结果表明: O₃浓度存在明显的日变化规律, 日最小值和最大值分别出现在7:00和16:00左右。整个观测期间(2011年3-5月)平均O₃浓度为(30.4 ± 20.1) nL·L ^-1(平均值±标准误差); 30 min平均浓度的最大值为93.1 nL·L ^-1。在冬小麦春季生长季节, O₃浓度日平均值呈现逐步增加的趋势, O₃浓度日均增加约为0.17 nL·L ^-1·d ^-1; 白天7 h和12 h平均浓度(M7和M12)分别为45.7和43.1 nL·L ^-1; O₃浓度超过40 nL·L ^-1的3个月累积值(AOT40)为9.8 μL·L ^-1·h; 超过60 nL·L ^-1的O₃浓度累积值(SUM06)为12.6 μL·L ^-1·h; 经过权重修正的O₃污染指标W126为10.1 μL·L ^-1·h。在高浓度O₃ (>60 nL·L ^-1)情况下, CO₂通量与O₃浓度呈现负相关关系, 鲁西北平原O₃对冬小麦光合作用影响的阈值取60 nL·L ^-1比较合适, 该值高于欧洲国家普遍采用的40 nL·L ^-1。基于以上结果, 初步估算得出: 在目前的O₃浓度水平下, 鲁西北平原近地层O₃可能会使冬小麦产量减少5.2%-8.8%。     

广西龙眼主栽品种丰产园果实及叶片的营养状况

对广西三个龙眼主栽品种"大乌圆"、"石硖"和"储良"进行果实性状和营养成分、叶片N、P、K含量分析,结果表明:各品种果实营养成分之间及其与叶片N、P、K含量之间有一定的相关性;初步提出广西丰产龙眼叶片N、P、K含量范围:"大乌圆"1.5×10^-2~1.9×10^-2, 0.08×10^-2~0.12×10^-2,0.48×10^-2~0.64×10^-2;"石硖" 1.5×10^-2~1.7×10^-2,0.09×10^-2~0.10×10^-2,0.39×10^-2~0.56×10^-2; "储良"1.4×10^-2~1.8×10^-2,0.08×10^-2~0.11×10^-2,0.38×10^-2~0.62×10^-2。     

丙酮酸发酵过程中光滑球拟酵母过程功能的强化

对不同葡萄糖浓度下光滑球拟酵母分批发酵生产丙酮酸的动力学模型分析发现, 葡萄糖浓度是影响光滑球拟酵母发酵生产丙酮酸过程功能的关键因素。在发酵初始阶段, 低浓度葡萄糖可维持较高的菌体比生长速率; 对数生长中前期, 葡萄糖快速进料使菌体浓度接近最大值, 并实现碳流从菌体生长转向丙酮酸积累; 对数生长后期葡萄糖浓度控制在33.4 g/L以维持高丙酮酸对葡萄糖产率系数 (0.71 g/g)。采用奇异控制的葡萄糖流加方式, 在7 L发酵罐上控制不同发酵阶段葡萄糖浓度处于最佳水平以强化光滑球拟酵母过程功能, 丙酮酸产量 (83.1 g/L)、产率 (0.621 g/g)、生产强度[1.00 g/(L·h)]与分批发酵对比, 分别提高了21.3%、21.6%和29.9%。     

粗毛栓菌和蜡状芽孢杆菌及其共固定菌对Pb2+的吸附

将粗毛栓菌菌丝球与蜡状芽孢杆菌共固定为共固定菌.以粗毛栓菌菌丝球、蜡状芽孢杆菌和共固定菌为研究对象,测定不同吸附时间、初始pH、吸附剂浓度和Pb²⁺浓度对3种生物吸附剂吸附Pb²⁺的影响,并将3种生物吸附剂吸附Pb²⁺前后的红外吸收光谱进行分析比较.结果表明： 在吸附剂浓度为2 g·L^-1、pH为5.0、Pb²⁺浓度为50 mg·L^-1条件下对Pb²⁺吸附1 h效果良好,其吸附率分别为71.7%、91.0%和96.9%.生物吸附剂红外光谱主要由蛋白质、碳水化合物和含硫、磷酸基团的吸收带组成,表明对Pb²⁺吸附起主要作用的官能团是羟基、羧基、磷酸基和含硫基团.     

Itaconic acid production by immobilizedAspergillus terreus from xylose and glucose

Summary Aspergillus terreus NRRC 1960 spores were entrapped in calcium alginate gel beads or alternotely the fungal mycelium was immobilized either on Celite R-626 or in agar gel cubes, and the biocatalyst was employed both in repeated batch and in continuous column reactors to produce itaconic acid from D-xylose or D-glucose. The highest itaconic acid yield obtained in a submerged culture batch fermentation was 54.5% based on total initial glucose (55 g/l) with a volumetric productivity of 0.32 g/l h, and 44.8% from xylose (67 g/l) with a productivity of 0.20 g/l h. In a repeated batch fermentation mycelium immobilized in agar gel had a productivity of 0.112 g/l h, and mycelium grown from spores immobilized in calcium alginate gel 0.06 g/l h, both from xylose (60 g/l). With the best immobilized biocatalyst system used employing Celite R-626 as a carrier, volumetric productivities of 1.2 g/l h from glucose and 0.56 g/l h from xylose (both at 60 g/l) were obtained in continuous column operation for more than 2 weeks.     

Biotechnological production of xylitol in a three-phase fluidized bed bioreactor with immobilized yeast cells in Ca-alginate beads

Cells of Candida guilliermondii entrapped in Ca-alginate beads were used for xylitol production, from concentrated hemicellulose hydrolyzate of sugarcane bagasse, in a fluidized bed bioreactor (FBR). The maximum xylitol concentration 28.9 g xylitol/L was obtained at a high aeration rate of 600 mL/min after 70 h of fermentation, indicating that the use of high aeration rate in this system is favored for better oxygen transfer into the immobilized cells. The specific xylitol productivity and the xylitol yield were of 0.4 g xylitol/L.h and 0.58 g xylitol/g xylose respectively. The immobilization efficiency at the end of the fermentation was of 65 %. After 90 h of fermentation xylitol productivity and yield decreased to 0.25 g xylitol/L.h and 0.47 g xylitol/g xylose respectively, indicating the beginning of xylitol consumption by the yeast. The use of FBR system with immobilized cells presented high xylitol yield and productivity.     

结冷胶固定化保加利亚乳杆菌及在玉米秸秆连续发酵d-乳酸中的应用

秸秆生物炼制化学品是解决秸秆资源利用附加值低、减轻秸秆焚烧带来的环境污染的主要方法之一。本研究制备了结冷胶固定化保加利亚乳酸杆菌(Lactobacillus bulgaricus)T15凝胶珠(结冷胶-T15凝胶珠),并对其性质进行表征,建立了结冷胶-T15凝胶珠固定化细胞循环连续发酵产D-乳酸发酵工艺。结冷胶-T15凝胶珠的断裂应力为(91.68±0.11) kPa,较海藻酸钙固定化T15凝胶珠(海藻酸钙-T15凝胶珠)提高了125.12%,表明结冷胶-T15凝胶珠的强度更强。以结冷胶-T15凝胶珠为出发菌株,葡萄糖为发酵基质,10批次循环(720h)发酵,其D-乳酸最高批次产量为(72.90±2.79)g/L,较海藻酸钙-T15凝胶珠提高了33.85%,较游离T15提高了37.70%。将葡萄糖更换为玉米秸秆酶解液,使用结冷胶-T15凝胶珠进行10批次循环(240 h)发酵,D-乳酸生产强度可达(1.74±0.79)g/(L·h),远高于游离菌。10批次循环发酵后结冷胶-T15凝胶珠磨损率小于5%,表明结冷胶是一种细胞固定化的良好载体,可广泛应用于细胞固定化工业发酵领域。本研究为细胞...     

Continuous production of gibberellic acid in a fixed-bed reactor by immobilized mycelia of Gibberella fujikuroi in calcium alginate beads

Summary The continuous production of gibberellic acid with immobilized mycelia of Gibberella fujikuroi was maintained over a hundred days in a tubular fixed-bed reactor. Free mycelium at the beginning of the storage phase was harvested from G. fujikuroi shake-flask culture and was immobilized by ionotropic gelation in calcium alginate beads.The continuous recycle production system consisted of a fixed-bed reactor, a container in which the culture medium was heated, stirred and aerated, and valves for sample withdrawal or reactant addition during the first 1320 h (55 days). A two-phase continuous extractor was then added for the last 960 hours (40 days). Free and immobilized mycelium shake-flask cultures with the same strain used in the continuous culture system were also realized to compare growth, maintenance and production parameters. The results show about the same gibberellic acid productivity in both free and immobilized mycelium shakeflask cultures: 0.384 and 0.408 mgGA₃·gBiomass^-1 ·day^-1, respectively, whereas in the continuous system the gibberellic acid production is about twice as large for a similar biomass: 0.768 mgGA₃·gBiomass^-1·day^-1. Several factors affecting the overall productivity of the immobilized systems were found to be: the quality and the quantity of mycelia in the biocatalyst beads and the immobilization conditions.     

Kinetics of Gibberella fujikuroi growth and gibberellic acid production by solid-state fermentation in a packed-bed column bioreactor

In this work the growth of Gibberella fujikuroi and gibberellic acid (GA3) production were studied using coffee husk and cassava bagasse as substrates in a packed-bed column bioreactor connected to a gas chromatograph for exit gas analysis. With the respirometric data, a logarithmic correlation between accumulated CO2 and biomass production was determined, and the kinetics of the fungal growth was compared for estimated and experimental data. The solid medium consisted of coffee husk (pretreated with alkali solution), mixed with cassava bagasse (7:3 dry weight basis), with a substrate initial pH of 5.2 and moisture of 77%. Cultivation was carried out in glass columns, which were packed with preinoculated substrate and with forced aeration of 0.24 L of air/[h (g of substrate)] for the first 3 days, and 0.72 L of air/[h (g of substrate)] for the remaining period. The maximum specific growth rate (microm) obtained was 0.052 h(-1) (between 24 and 48 h of fermentation). A production of 0.925 g of GA3/kg of substrate was achieved after 6 days of fermentation.     

Ethanol fermentation in a magnetically fluidized bed reactor with immobilized Saccharomyces cerevisiae in magnetic particles

Ethanol fermentation by immobilized Saccharomyces cerevisiae cells in magnetic particles was successfully carried out in a magnetically stabilized fluidized bed reactor (MSFBR). These immobilized magnetic particles solidified in a 2 % CaCl(2) solution were stable and had high ethanol fermentation activity. The performance of ethanol fermentation of glucose in the MSFBR was affected by initial particle loading rate, feed sugar concentration and dilution rate. The ethanol theoretical yield, productivity and concentration reached 95.3%, 26.7 g/L h and 66 g/L, respectively, at a particle loading rate of 41% and a feed dilution rate of 0.4 h(-1) with a glucose concentration of 150 g/L when the magnetic field intensity was kept in the range of 85-120 Oe. In order to use this developed MSFBR system for ethanol production from cheap raw materials, cane molasses was used as the main fermentation substrate for continuous ethanol fermentation with the immobilized S. cerevisiae cells in the reactor system. Molasses gave comparative ethanol productivity in comparison with glucose in the MSFBR, and the higher ethanol production was observed in the MSFBR than in a fluidized bed reactor (FBR) without a magnetic field.     

利用基因工程菌HC01固定化细胞转化生产D-对羟基苯甘氨酸

对一菌两酶工程菌HC01转化底物DL-对羟基苯海因(DL-HPH)的最适条件及其细胞固定化进行了研究,HC01游离细胞转化DL-HPH的最适条件为40°C、pH7.5。通过对固定化细胞酶活力测定,确定细胞固定化的最优条件为海藻酸钠浓度2.5%、细胞浓度0.029g/mL、钙离子浓度3%。固定化HC01的热稳定性比游离细胞高5°C,二价金属离子Mn2+、Mg2+、Cu2+、Co2+和Ni2+在浓度为0.1mmol/L时对固定化细胞中D-海因酶(HYD)和N-氨甲酰-D-氨基酸酰胺水解酶(CAB)两酶的活力无显著影响,Mn2+和Mg2+可分别使游离细胞中CAB活力提高至原来的2.1和2.7倍。在氮气保护下,当初始pH为9.0、转化温度为40°C、转速为80r/min,利用固定化HC01转化30g/L的DL-HPH时,36h后转化率可达97%左右,产物D-HPG经纯化后光学纯度达到99.7%,得率可达85%。     

铁改性沸石对Clostridium acetobutylicum XY16固定效率及发酵性能的影响

考察4种无机铁盐改性沸石对丁醇生产菌Clostridium acetobutylicum XY16的固定效率及其发酵产丁醇性能的影响。结果表明:铁改性沸石对菌体的固定效率均优于未改性沸石,而Fe3+改性效果优于Fe2+,经FeCl3改性的沸石对菌体具有良好的吸附作用,当Fe3+-zeolite用量为180 g/L时,细胞的固定效率达到87%。在此基础上,比较了沸石负载的铁离子量对丁醇发酵性能的影响,沸石负载的铁离子量为6.0 mg/g时可显著提高丁醇发酵性能,当葡萄糖质量浓度为60 g/L时进行发酵,丁醇产量为13.5 g/L,总溶剂可达20 g/L,总溶剂的生产速率为0.385g/(L.h),比游离细胞发酵分别提高了9.5%、10.3%和40%。     

Immobilized growing cells of Gibberella fujikuroi P-3 for production of gibberellic acid and pigment in batch and semi-continuous cultures

Summary The performance of immobilized growing cells of Gibberella fujikuroi P-3 was affected by the immobilization agent used, nature and age of cells, mycelial cell density, size of beads and inclusion of linseed oil. The beads, prepared by using standardized procedures, could be used for 8 cycles without affecting productivity in semicontinuous culture. The rate of production of gibberellic acid was 0.58–0.66 mg/l/h. An inverted conical fluidized bioreactor, based on the design employed in continuous plant cell culture, was adopted. This bioreactor offers many advantages. The pigment produced by the fungus is not similar to bikaverin, norbikaverin and O-dimethylanhydrofusarubin, the known polyketides.     

Production of tylosin and nikkomycin by immobilized Streptomyces cells

Summary Mycelia of Streptomyces sp. T 59-235 and Streptomyces tendae Tü 901 (producing the antibiotics tylosin and nikkomycin, resp.) were immobilized in different carriers. With both organisms best antibiotic production was observed in calcium alginate gel.Influence of aeration, cell density and flow rate on antibiotic production was investigated in batch fermentation and in a continuous system (air-bubbled reactor).In batch fermentation, immobilization prolongued the production phase from 72 to 120 h (Streptomyces T 59-235) and from 72 to 96 h (S. tendae). The relative productivity of immobilized cells was 40 to 50% compared to that of free mycelia in both cases.In continuous tylosin fermentation highest production rate was observed in a medium nearly saturated with oxygen.Nikkomycin production by immobilized S. tendae could be maintained for longer than 350 h in a continuous system. The production rate depended on cell density and flow rate of the medium. The maximum specific productivity was 100% compared to that of free mycelium in batch culture.