Optimization of Culture Medium for Sporulation and Biomass Production of a Nematophagous Fungus: Consideration of Nutritional and Environmental Conditions

This study reports the effects of various nutritional and environmental factors on sporulation and biomass of Paecilomyces lilacinus IPC‐P. These factors included carbon and nitrogen sources, carbon‐to‐nitrogen ratios, mineral elements and vitamins together with water potentials, temperatures, dark/light cycles and pH. On the basis of these results, together with a ‘two‐step’ cultivation and orthogonal method, the culture conditions for sporulation of this fungus were optimized. The spore suspension was inoculated on a basal medium (sucrose 19.00 g/l, soy peptone 4.06 g/l, K₂HPO₄ 1.00 g/l, KCl 0.50 g/l, MgSO₄ 0.50 g/l, FeSO₄ 0.01 g/l, agar 13.00 g/l) for 4 days, before being transferred to a sporulation medium (dextrin 2.27 g/l, urea 2.13 g/l, CaCl₂ 3.00 g/l, ZnSO₄·7H₂O 0.01 g/l, agar 13.00 g/l) for a further 4 days under the following environmental conditions: ?3.9 MPa/pH 7/light 24 h/temperature 29°C; these conditions were altered to ?0.3 MPa/pH 6/light 24 h/temperature 23°C in order to obtain better biomass yields. The data presented provide information on the nutrient and environmental requirements of this fungus, which will be essential for its commercial production.     

Optimization of Low-Cost Culture Media for the Production of Biomass and Bacteriocin by a Urogenital Lactobacillus salivarius Strain

The aim of this work was to formulate a culture medium of lower cost than conventional laboratory media, in order to simultaneously obtain high amounts of both biomass and bacteriocin of vaginal Lactobacillus salivarius CRL 1328. The growth assays under different culture conditions were performed by using a 2^8?2 central composite experimental design, with a central point and sixteen additional points. The factors taken into consideration were glucose, lactose, yeast extract, tryptone, ammonium citrate, sodium acetate, MgSO₄ and MnSO₄. The simultaneous presence of a carbon source (mainly glucose), a nitrogen source (mainly yeast extract) and salts (mainly MnSO₄, MgSO₄ and sodium acetate) allowed the highest cell biomass and bacteriocin levels to be reached in the experimental design. Through the application of the desirability function, several optimal medium compositions to achieve efficient production of biomass and bacteriocin were predicted. The optimized growth media allow a cost reduction of around 25 to 40% compared with conventional broths. The results obtained represent an advance in the search of the most suitable strategies for the production of bioactive compounds for pharmaceutical products to prevent or treat female urogenital infections.     

白腐菌木质素降解酶及其在木质素降解过程中的相互作用

木质素是一类不易降解的生物物质,在自然界中,白腐真菌对木质素的降解能力最强.白腐真菌降解木质素主要依靠分泌的三种酶:木质素过氧化物酶(Lip)、锰过氧化物酶(MnP)和漆酶(Lac).对白腐真菌分泌的三种木质素降解酶在性质、分布等方面进行了比较,系境地介绍三种木质素降解酶的催化作用,并阐述其在木质素降解过程中的相互作用.     

Continuous Culture Used for Media Optimization

A technique is described in which continuous culture is used for the optimization of media in terms of growth-supporting ability. The technique consists of identifying growth-limiting nutrients by observing the reaction of the continuous system to injection of suspected growth-limiting nutrients into the growth vessel.     

Identification of a Degradation Intermediate of the Momilactone A Rice Phytoalexin by the Rice Blast Fungus

We have already shown that major rice diterpene phytoalexin, momilactone A, was detoxified by Magnaporthe oryzae. We report here the identification by NMR, MS, and chemical synthesis of 3,6-dioxo-19-nor-9β-pimara-7,15-diene (1) as the degradation intermediate. Compound 1 exhibited similar antifungal activity to that of momilactone A, indicating 1 to be a precursor of possible detoxified compounds.     

一株纤维素分解菌的筛选、鉴定及其对玉米秸秆的降解效果

为了筛选具有高效分解玉米秸秆纤维素能力的菌株,采集玉米秸秆还田土样作为样品,并于20℃条件下进行富集培养。利用以玉米秸秆纤维素为唯一碳源的固体分离培养基和刚果红染色法进行初筛,再将筛选到的菌株进行液体发酵培养并取上清液测定酶活,最终获得1株产纤维素酶能力较强的真菌SY-403。结合形态学特征与分子生物学鉴定结果得知,菌株SY-403为蓝状菌属（Talaromyces stollii）。对其所产纤维素酶酶学性质进行初步研究,结果表明,该酶最适反应pH为6.0,最适反应温度为20℃。在模拟室外条件（15℃）下进行秸秆降解试验,玉米秸秆经菌株SY-403处理40 d时,秸秆失重率及纤维素分解率分别达到42.67%、55.26%。利用傅里叶变换红外（Fourier transform infrared,FTIR）光谱技术对降解过程中官能团的变化进行分析,结果表明,纤维素相关谱峰（1 052~1 054 cm-1）相对强度减弱,而羟基相关谱峰（1 328~1 330 cm-1）相对强度增强,这说明纤维素已被分解为可利用的短链结构,即菌株SY-403可用于降解玉米秸秆。     

白腐菌在两种固体基质上的漆酶产量及同工酶活性比较研究

一株新分离的白腐菌Z1可在两种农业废弃物(麸皮和稻草秸杆)基质上生长并分泌漆酶,其中在麸皮基质上的漆酶产量比在稻草秸杆基质上的漆酶产量高出10倍左右,同时活性电泳显示Z1菌在麸皮基质上可产生3条漆酶同工酶,比在稻草秸杆基质上多1条,其活性也比在稻草秸杆基质中的漆酶活性高。     

The Effect of Fungal Morphology on Ligninolytic Enzyme Production by a Recently Isolated Wood-Degrading Fungus Trichophyton Rubrum LSK-27

The morphology and ligninolytic enzyme production of a recently isolated wood-degrading fungus Trichophyton rubrum LSK-27 was investigated. In submerged cultures, the organism appeared to be an efficient manganese peroxidase (MnP) producer. When grown in baffled and unbaffled shake flasks with three different working volume/total volume ratios (WV/TV 10, 25 and 50%), the organism displayed notable morphological differences, with variations in pellet shape and size. Cultivation in baffled flasks with 25% WV/TV resulted in higher MnP and also laccase production as well as an earlier appearance of these enzymes in culture broth. However, oxygen limitation conditions inhibited MnP and laccase production and resulted in considerable changes in the morphology of this fungus.     

侧耳菌与粗毛栓菌在麦草培养基中产生木质纤维素降解酶的研究

草本植物,包括农作物秸杆的木质素主要是由松柏醇、芥子醇和对香豆醇的脱氢聚合物和对香豆酸组成[1,2],是结构复杂、稳定、多样的生物大分子物质.虽难于被一般微生物降解,但自然界中仍存在一些可降解木质素的微生物种类,白腐真菌是最重要的一类,它们通过分泌漆酶(Laccases,Lac)、木质素过氧化物酶(Lignin peroxidases,LiP)、锰过氧化物酶(Manganese-dependent peroxidases,MnP)、纤维素酶(Cellulas-es,Cel)和半纤维素酶(Hemicellulases,Hcel)等降解植物生物质.由于白腐菌在造纸工业中的生物制浆和纸浆生物漂白、环境保护等方面[4]有着很好的应用前景,因此倍受关注. 本研究选用在液体培养基中产酶能力强且产酶较快的白腐真菌侧耳sp2和粗毛栓菌[5]进行固体培养,研究它们产生木质纤维素降解酶类和降解植物生物质的能力.研究结果报道如下.     

Combining Genotype Improvement and Statistical Media Optimization for Isoprenoid Production in E. coli

Isoprenoids are a large and diverse class of compounds that includes many high value natural products and are thus in great demand. To meet the increasing demand for isoprenoid compounds, metabolic engineering of microbes has been used to produce isoprenoids in an economical and sustainable manner. To achieve high isoprenoid yields using this technology, the availability of metabolic precursors feeding the deoxyxylulose phosphate (DXP) pathway, responsible for isoprenoid biosynthesis, has to be optimized. In this study, phosphoenolpyruvate, a vital DXP pathway precursor, was enriched by deleting the genes encoding the carbohydrate phosphotransferase system (PTS) in E. coli. Production of lycopene (a C40 isoprenoid) was maximized by optimizing growth medium and culture conditions. In optimized conditions, the lycopene yield from PTS mutant was seven fold higher than that obtained from the wild type strain. This resulted in the highest reported specific yield of lycopene produced from the DXP pathway in E. coli to date (20,000 µg/g dry cell weight). Both the copy number of the plasmid encoding the lycopene biosynthetic genes and the expression were found to be increased in the optimized media. Deletion of PTS together with a similar optimization strategy was also successful in enhancing the production of amorpha-1,4-diene, a distinct C15 isoprenoid, suggesting that the approaches developed herein can be generally applied to optimize production of other isoprenoids.     

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.     

Combined Application of Rice Straw and Fungus Penicillium Chrysogenum to Remediate Heavy-Metal-Contaminated Soil

The search for cheap and environmentally friendly materials is essential for remediation of heavy-metal-contaminated agricultural soils. A pot experiment was undertaken to evaluate the application of rice straw and filamentous fungus Penicillium chrysogenum (P. chrysogenum) on the fractionation of copper (Cu) and cadmium (Cd), soil microbial properties, and Cu and Cd uptake by romaine lettuce (Lactuca sativa) in a contaminated agricultural soil. Rice straw was applied at three rates (0, 7.8, and 11.7 g kg^?1 soil), and in combinations with P. chrysogenum (1.0 × 10⁶ spores g^?1 soil). It was found that the combined treatment of rice straw and P. chrysogenum significantly decreased the acid-extractable Cu and Cd by 15.4–25.1% and 20.2–27.3%, and increased the oxidizable Cu and Cd by 16.1–18.0% and 72.1–98.4%, respectively. Soil microbial biomass and fresh weight of lettuce were also remarkably enhanced after rice straw plus P. chrysogenum addition. Rice straw combined with P. chrysogenum was more effective in reducing Cu and Cd uptake by lettuce than rice straw alone. The joint application of rice straw and P. chrysogenum remarkably reduced Cu and Cd concentrations in lettuce shoots by 13.6–21.9% and 32.9–41.7%, respectively. These results indicate that the combined application of P. chrysogenum and rice straw is a promising method to alleviate the bioavailability of metals, and to improve soil microbial properties and plant yield in heavy-metal-polluted agricultural soils.     

Production of Vesicular-Arbuscular Mycorrhizal Fungus Inoculum in Aeroponic Culture

Bahia grass (Paspalum notatum) and industrial sweet potato (Ipomoea batatas) colonized by Glomus deserticola, G. etunicatum, and G. intraradices were grown in aeroponic cultures. After 12 to 14 weeks, all roots were colonized by the inoculated vesicular-arbuscular mycorrhizal fungi. Abundant vesicles and arbuscules formed in the roots, and profuse sporulation was detected intra-and extraradically. Within each fungal species, industrial sweet potato contained significantly more roots and spores per plant than bahia grass did, although the percent root colonization was similar for both hosts. Mean percent root colonization and sporulation per centimeter of colonized root generally increased with time, although with some treatments colonization declined by week 14. Spore production ranged from 4 spores per cm of colonized root for G. etunicatum to 51 spores per cm for G. intraradices. Infectivity trials with root inocula resulted in a mean of 38, 45, and 28% of bahia grass roots colonized by G. deserticola, G. etunicatum, and G. intraradices, respectively. The germination rate of G. etunicatum spores produced in soil was significantly higher than that produced in aeroponic cultures (64% versus 46%) after a 2-week incubation at 28°C. However, infectivity studies comparing G. etunicatum spores from soil and aeroponic culture indicated no biological differences between the spore sources. Aeroponically produced G. deserticola and G. etunicatum inocula retained their infectivity after cold storage (4°C) in either sterile water or moist vermiculite for at least 4 and 9 months, respectively.     

Statistical Optimization of Culture Conditions for Milk-Clotting Enzyme Production by Bacillus Amyloliquefaciens Using Wheat Bran-An Agro-Industry Waste

In order to improve the production of the milk-clotting enzyme under submerged fermentation, two statistical methods were applied to optimize the culture conditions of Bacillus amyloliquefaciens D4 using wheat bran as nutrient source. First, initial pH, agitation speed, and fermentation time were shown to have significant effects on D4 enzyme production using the Plackett–Burman experimental design. Subsequently, optimal conditions were obtained using the Box–Behnken method, which were as follows: initial pH 7.57, agitation speed 241 rpm, fermentation time 53.3 h. Under these conditions, the milk-clotting enzyme production was remarkably enhanced. The milk-clotting enzyme activity reached 1996.9 SU/mL, which was 2.92-fold higher than that of the initial culture conditions, showing that the Plackett–Burman design and Box–Behnken response surface method are effective to optimize culture conditions. The research can provide a reference for full utilization of wheat bran and the production of milk-clotting enzyme by B. amyloliquefaciens D4 under submerged fermentation.     

腺苷蛋氨酸发酵条件及发酵培养基的优化

腺苷蛋氨酸具有转甲基、转硫和转氨丙基等重要生理作用 ,已成为治疗疾病的重要药物。研究了腺苷蛋氨酸发酵条件及发酵培养基的优化。结果表明 :发酵培养基的最适初始pH值为5.0 ;最佳发酵温度为 30℃ ;最适底物浓度为 1 % ;8%蔗糖为最佳碳源 ;0.5 %的NH4NO3 为最佳氮源 ;无机离子的最佳组合为 0.01 %MgSO4·7H2 O、0.03%CaCl2 、0.04%FeSO4·7H2 O和 0.01%LiCl。     

A Method for the Production of Pre-reduced Anaerobically Sterilized Culture Media

A simplified method for the production of pre-reduced anaerobically sterilized media for the cultivation of strict anaerobes is described.     

利用数学统计方法快速优化木聚糖酶液体发酵培养基

木聚糖是植物中半纤维素的主要成分,含量仅次于纤维素的可再生多糖资源。木聚糖酶的使用可以有效提高半纤维素的利用,降低原料成本,减少污染,保护生态环境。应用快速有效的数学统计方法对木聚糖酶生产菌黑曲霉（A8pergillus niger）FS418液体发酵培养基进行优化。首先采用Plackett-Burman设计对培养基中8因素进行筛选,获得影响产酶3个最重要的营养组分：蛋白胨、麸皮、甘蔗渣;然后采用响应面分析法对3个因素进行3水平的优化,得到它们的最佳组合：麸皮6.0％、蛋白胨0.2％、甘蔗渣0.4％。优化后液体发酵水平比初始设计有了较大提高,最终产酶水平达到5147.29U／mL、实测值与模型的预测值只有0.8％的误差。     

Marc-145细胞无血清培养液组分的优化筛选

目的：对Marc-145细胞无血清培养液组分及浓度进行优化筛选。方法：采用Plackett-Burman设计和中心组合旋转设计对不同的营养成分进行优化筛选。结果：经Plackett-Burman设计,利用20组实验对14种不同营养成分进行考察,发现孕酮、抗氧化试剂、乙醇胺、维生素B12、维生素B12和脂肪酸复合剂对Marc-145细胞无血清悬浮生长的比生长速率有显著影响;针对该6种因素进行中心组合旋转实验设计,利用53组实验筛选出它们的最优使用浓度分别为孕酮5．5mg／L、抗氧化试剂250μL/L、乙醇胺2．1mL／L、维生素B12 2．86mg／L、维生素B6 44μg/L和脂肪酸复合剂215μL/L。结论：经2种实验设计方法优化,确定了Marc-145细胞无血清培养液组分及最适浓度,为无血清悬浮培养Marc-145细胞及增殖猪繁殖及呼吸综合征病毒提供了实验基础。     

海洋红树林内生真菌产抗肿瘤活性化合物1403C发酵条件的优化

旨在考察pH、溶解氧和剪切力对Halorosellinia sp.( No.1403)发酵过程的影响.结果表明,在发酵中后期及整个发酵过程中控制发酵液的pH恒定对菌体的生长影响不明显,但在不同发酵时期控制不同的恒定pH值对1403C的产量影响较大.仅在发酵的前24h控制恒pH7.0,有利于菌丝的生长,但抑制1403C的生物合成,菌体干重为对照组的159.1％,1403C的产量仅为对照组的29.4％;从48 h开始控制恒pH7.0,有利于1403C的生物合成,菌体干重量为对照组的94％,1403C的产量为对照组的123.2％;适量的溶解氧和剪切力有利于1403C的生物合成;从48 h开始提高剪切力可获得较高的1403C产量,是对照组的151.8％.综上所述,在发酵至48 h时开始控制恒pH7.0,并适当增大剪切力,在整个发酵过程中控制适当充足的溶解氧有利于1403C的合成.     

Optimization of a MRC-5 Cell Culture Process for the Production of a Smallpox Vaccine

A cell culture process adaptable to produce smallpox vaccine at large scale has been developed. To achieve this, Design of Experiments (DOE) was applied to identify and optimize critical cell culture process parameters for MRC-5 cell growth and recovery during cell expansion. For cell growth, a 2^5?1 partial factorial (two level, five factor, 16 conditions) study was designed to evaluate the effects of basal media, seeding density, culture volume, feeding frequency and serum concentration on population doubling level (PDL) after 6–7 days in adherent T-flask cultures. Results indicated that lowering the cell density to 1×10⁴ cells/cm², increasing the culture volume to 0.5 ml/cm² and increasing serum concentration to 20% significantly improved cell expansion. These findings correlated with PDLs above 2.0 and cell densities above 1×10⁵ cells/cm² at the end of the study period. For cell recovery at passaging, a similar DOE was used to evaluate the effect of trypsin concentration, solution temperature, duration of treatment, incubation temperature and duration of standing time between quenching and reseeding. By increasing the trypsin treatment duration to 60 min and lowering the standing time between quenching and reseeding to within 1 h, the recovery of the MRC-5 cells was greatly improved. By using these newly defined conditions, a two-fold improvement in cell expansion was consistently achieved in both roller bottles and 10 layer Nunc^® Cell Factories (Cell Factories). Application of these new conditions for current Good Manufacturing Practices (cGMP) production of MRC-5 cell banks and clinical material demonstrated predictably high cell expansion as well as significantly higher production of vaccinia virus, thus providing the basis for manufacturing vaccinia virus at large scale. These findings demonstrate the need for cell culture optimization and the effectiveness of DOE to rapidly define processes suitable for cGMP manufacturing of a smallpox vaccine or other viral vaccine products.