Co-Production of Fungal Biomass Derived Constituents and Ethanol from Citrus Wastes Free Sugars without Auxiliary Nutrients in Airlift Bioreactor

The potential of two zygomycetes fungi, Mucor indicus and Rhizopus oryzae, in assimilating citrus waste free sugars (CWFS) and producing fungal chitosan, oil, and protein as well as ethanol was investigated. Extraction of free sugars from citrus waste can reduce its environmental impact by decreasing the possibility of wild microorganisms growth and formation of bad odors, a typical problem facing the citrus industries. A total sugar concentration of 25.1 g/L was obtained by water extraction of citrus waste at room temperature, used for fungal cultivation in shake flasks and airlift bioreactor with no additional nutrients. In shake flasks cultivations, the fungi were only able to assimilate glucose, while fructose remained almost intact. In contrast, the cultivation of M. indicus and R. oryzae in the four-liter airlift bioreactor resulted in the consumption of almost all sugars and production of 250 and 280 g fungal biomass per kg of consumed sugar, respectively. These biomasses correspondingly contained 40% and 51% protein and 9.8% and 4.4% oil. Furthermore, the fungal cell walls, obtained after removing the alkali soluble fraction of the fungi, contained 0.61 and 0.69 g chitin and chitosan per g of cell wall for M. indicus and R. oryzae, respectively. Moreover, the maximum ethanol yield of 36% and 18% was obtained from M. indicus and R. oryzae, respectively. Furthermore, that M. indicus grew as clump mycelia in the airlift bioreactor, while R. oryzae formed spherical suspended pellets, is a promising feature towards industrialization of the process.     

氮离子注入选育高效发酵木糖生产L(+)-乳酸的米根霉

为了获得能够高效发酵木糖生产L( )-乳酸的菌株,采用氮离子注入诱变方法,对出发菌株米根霉RLC41-6进行改良,获得高产L( )-乳酸菌株RQ4012,发酵周期为72h,产酸能力达74.37g/L,产酸速率达1.03g/(L.h),比RLC41-6提高了1.6倍.经过多次传代实验证明该菌株具有较好的遗传稳定性.     

放线菌酮对米根霉积累L-苹果酸代谢途径的调控作用

实验考察了富马酸酶活抑制剂放线菌酮对米根霉发酵产L-苹果酸和富马酸的影响. 结果表明,发酵16 h加入浓度15 mg/L的放线菌酮使米根霉富马酸酶胞质同功酶比活力峰值降低48.7%,而苹果酸脱氢酶胞质同功酶活力却未有明显改变,生成的富马酸比对照样减少37.1%,而L-苹果酸积累量提高54.6%,达到21.7 g/L. 表明通过对米根霉富马酸酶胞质同功酶的抑制作用能减弱米根霉积累富马酸代谢途径中由L-苹果酸向富马酸的转化,从而促进米根霉积累L-苹果酸.     

根霉ZM-10脂肪酶的分离纯化和活性部位的化学修饰

根霉ZM-10脂肪酶经过硫酸铵盐析、DEAE-Sepharose FF阴离子交换层析、SephardexG100凝胶过滤层析得到电泳纯脂肪酶,分子量约为42 ku,纯化倍数15.3倍,酶活回收率22.2%。采用NBS、EDC、DEPC、CH-T、PMSF、DTNB等6种化学修饰剂对根霉ZM-10脂肪酶进行化学修饰和底物保护实验,研究了其分子中氨基酸侧链基团与酶活性中心的关系。实验结果表明,酸性氨基酸(天冬氨酸?谷氨酸)残基、组氨酸残基、丝氨酸残基、色氨酸残基为根霉ZM-10脂肪酶活性的必需基团。酶分子中酸性氨基酸(天冬氨酸?谷氨酸)残基、组氨酸残基和丝氨酸残基位于根霉ZM-10脂肪酶的活性中心部位,而色氨酸残基在保持脂肪酶活性中起到重要作用,但其不位于根霉ZM-10脂肪酶的活性中心部位。     

Plackett-Burman和Box-Benhnken Design实验设计法优化华根霉产糖化酶发酵培养基的研究

应用Plackett-Burman设计法对影响华根霉发酵的培养基组分进行筛选,所选取的11个相关因素为:葡萄糖、麦芽糖、蔗糖、橄榄油、蛋白胨、黄豆粉、酪蛋白胨、麦麸、MgSO4·7H2O、K2HPO4、KH2PO4。确定影响产糖化酶活的关键因素为橄榄油、酪蛋白胨、麦麸,接着进行最陡爬坡实验逼近3个关键因素的最大响应区域。在此基础上,采用Box-BenhnkenDesign实验设计法对发酵培养基组分进行优化,得出最佳条件。此时橄榄油为0.01%、酪蛋白胨为8.14%、麦麸为4.32%、糖化酶活为18.7U,比优化前提高81%。     

米根霉F1-ATPase活性降低突变株的筛选研究

本实验采用同步辐射软X射线Nk诱变米根霉,获得一株新霉素抗性突变株NA-2,该菌株F1-ATPase活性降低86.82%,胞内ATP含量下降18.99%,最终菌体浓度为出发菌株的79.22%,发酵周期缩短了12h,葡萄糖平均消耗速度和L-乳酸生产强度分别高出25.17%和32.57%,平均葡萄糖消耗比速(qs)、L-乳酸生成比速(qp)和细胞比生长速率(μ)分别提高86.42%、128.13%和60.0%,但胞内能荷变化不明显。进一步研究发现,突变株糖酵解途径中关键酶磷酸果糖激酶(PEK)、丙酮酸激酶(PK)和磷酸甘油醛激酶(GAPDH)的活性较原始菌株提高了58.34%、23.53%和11.29%,乙醇脱氢酶(ADH)活性降低了32.11%,乳酸脱氢酶(LDH)活性提高了42.27%。结果表明,降低米根霉F1-ATPase活性有效地提高了糖酵解关键酶活性而加速葡萄糖代谢,提高了L-乳酸生产强度。     

少根根霉发酵糖质原料积累

少根根霉Ｒ＿３０菌株摇瓶发酵过程中的酶活分析及间接实验表明：Ｌ－苹果酸积累的关键酶是苹果酸脱氢酶（Ｅ．Ｃ．１．１．１．３７），Ｒ＿３０菌株的Ｌ－苹果酸积累主要是通过草酰乙酸还原及丙酮酸羧化，ＴＣＡ循环及乙醛酸循环主要用于维持菌体的正常代谢．     

光学纯度L(+)-乳酸高产菌株的选育

从60份不同来源的土壤样品中筛选得到米根霉(Rhizopus oryzae)为出发菌株,通过紫外线和60Coγ射线诱变处理,运用推理育种技术,选育到一株有较高的乳酸脱氢酶活性和耐高渗的L( )-乳酸高产突变株Rhi3-2.该菌株产L( )-乳酸光学纯度高,最高产量为88.45g/L,较出发菌株提高91.24%,糖利用率提高17.87%,耗糖酸产率提高35.15%.菌株经8次传代培养,L( )-乳酸产量下降10.7%,是一株性状较稳定可深入研究开发的优良菌株.     

Production of active lipase by Rhizopus oryzae from sugarcane bagasse: solid state fermentation in a tray bioreactor

This study deals with production of lipase in solid state fermentation by Rhizopus oryzae from sugarcane bagasse. A tray bioreactor was designed for the extracellular enzyme production. Daily, lipase production was evaluated at several incubation temperatures. Furthermore, the influence of temperature and humidity of the cabinet, depth of solid bed, particle size, initial moisture content and supplementary substrate (olive oil) as carbon source was investigated. The obtained results showed that bioreactor temperature of 45 °C, humidity of 80%, solid bed depth of 0.5 cm, particle size in the range of 0.335–1 mm, substrate initial moisture content of 80% for the top tray and 70% for the middle tray and supplementary substrate of 8% (v/w) olive oil led to maximum lipase production. Under optimum fermentation conditions after 72‐h incubation, maximum lipase activities for the top, middle and bottom trays were 215.16, 199.36 and 52.64 U gds^?1, respectively.