厌氧产氢菌的分离筛选、诱变选育及其产氢特性的研究

本文从四川绵竹酒厂、成都市龙泉长安垃圾填埋场以及四川大学荷花池底的厌氧污泥中先后分离得到63株厌氧产氢菌，其中H-8、H-61、HC-10等16株产氢细菌产氢能力较高，HC-10的产氢能力最高，最大产氢量和最大产氢速率分别达到2840 ml H2/L培养基和25.39 mmol H2/g drycell·h，对HC-10进行生理生化鉴定和分子生物学鉴定，判定其为clostridium sp.，对HC-10的产氢条件进行了研究，结果表明，该菌的最适生长温度为35 ℃，最适生长初始pH为7，以葡萄糖为最佳碳源，以蛋白胨为最佳氮源，不利用无机氮源，其产氢发酵液相产物以乙醇和乙酸为主，其发酵类型属于乙醇型发酵。此外，以酒糟废液作为底物，进行了菌株HC-10的生物强化试验，研究表明，投加了HC-10的强化系统其产氢量比对照高出40.32%。 同时为了获得厌氧产氢菌的高效突变株，分别以产氢菌H-8和H-61为原始菌株进行微波诱变处理，对微波诱变参数进行了优化，考察了突变株的遗传稳定性、产氢特性及耐酸性。菌株H-8经过微波诱变得到5株高产氢突变株HW7、HW33、HW181、HW184、HW195，经多次传代表明HW195是稳定的高产突变株。突变株HW195具有较好的耐酸性，在pH值为2.8时仍能生长。通过间歇发酵实验，其最大产氢量和最大产氢速率分别达到2460 mL/L培养基和27.97 mmol H2/g drycell·h，比原始菌分别提高了50.75%和41.7%。菌株H-61经过微波诱变后选育得到的突变株HW-18，其最大产氢量和最大产氢速率分别达到2190 mL/L培养基和25.86 mmol H2/g drycell·h，比原始菌分别提高了23.03%和31.00%。 为了对比各种诱变方式对产氢菌产氢能力的影响，以厌氧产氢菌H-61为原始菌株，先后经亚硝基胍(NTG)、紫外(UV)诱变，选育得到1株高产突变株HCM-23。在葡萄糖浓度为10 g/L的条件下，其产氢量为3024 mL/L培养基，比原始菌株提高了69.89%；其最大产氢速率为33.19 mmol H2/g drycell·h，比原始菌株提高了68.14%。经过多次传代实验，稳定性良好。其发酵末端产物以乙醇和乙酸为主，属于典型乙醇型发酵。其最适产氢初始pH为6.5，最适生长温度为36 ℃，以蔗糖为最佳碳源。与原始菌株相比，突变株HCM-23的产氢特性发生了改变，如生长延滞期延长，可利用无机氮源等。 From anaerobic activated sludge, 16 strains of hydrogen producing bacteria were newly isolated. One of them named as HC-10 had the highest hydrogen producing capability, under the batch fermentative hydrogen production condition, the maximal hydrogen yield and hydrogen production rate was 2840 mL/L culture and 25.39 mmol H2/g drycell·h. It was identified as clostridium sp.HC-10 by 16S rDNA sequence analysis. Various parameters for hydrogen production, including substrates, initial pH and temperature, have been studied. The optimum condition for hydrogen producing of strain HC-10 were achieved as: initial pH 7.0, temperature 35 ℃, glucose as the favorite substrate, Moreover, using distiller's solubles wastewater as substrate, HC-10 strain was added in the biohydrogen producing system to research the bioaugmentation effection. The results showed that the hydrogen production of bioaugmentation system was 40.32% higher than the noaugmentation system. An anaerobic, hydrogen producing strain H-8 was irradiated by microwave to optimize the microwave mutagenesis condition, and to test the heredity, hydrogen-producing potential and aciduric of the mutants. An aciduric mutant named as HW195 with steady hydrogen-producing capability was obtained, which can grow at pH 2.8. Its capability of hydrogen production was tested in the batch culture experiments. The maximum hydrogen yield and hydrogen production rate was 2460 mL/L culture and 29.97 mmol H2/g drycell·h, which was 50.7% and 41.7% higher than those of the initial strain, respectively. When used the strain H-61 as original strain, a mutant named as HW18 was obtained. The maximum hydrogen yield and hydrogen production rate was 2190 mL/L culture and 25.86 mmol H2/g drycell·h, which was 23.03% and 31.00% higher than those of the initial strain, respectively. The results demonstrated that microwave mutagenesis could be used in the field of hydrogen producing microorganism. The hydrogen producing strain H-61 was used as an original strain which was induced by NTG and UV for increasing and the hydrogen production capability. One of the highest efficient H2-producing mutants was named as HCM-23 with its stable hydrogen production capability. which was tested in the batch culture experiments. With the condition of 10 g/L glucose, its cumulative hydrogen yield and hydrogen production rate was 3024 mL/L culture and 33.19 mmol H2/g drycell·h, 69.89％and 68.14％ higher than that of the original strain, respectively. The terminal liquid product compositions showed that the mutant HCM-23 fermentation was ethanol type, while the original strain H-61 fermentation was butyric acid type. Varieties of parameters of hydrogen production fermentation were studied, including time, carbon source, nitrogen source, glucose concentration, glucose utilization, initial pH and incubation temperature had been studied, indicated the optimum condition of hydrogen production for the mutant HCM-23 as initial pH6.5, temperature 36 ℃, and the favorite substrate was sucrose. The hydrogen production characters of the mutant and the original strain were different, such as, the growth lag phase and the utilization of inorganic nitrogen source, etc. This work shows a good application potential of NTG-UV combined mutation in the biohydrogen production. And the hydrogen production mechanism and metabolic pathway should be explored furthermore.

活性炭纤维(ACF)吸附法处理河流突发污染事故模拟研究

环境突发污染事故给人民生活、经济发展和生态环境造成重大影响，研究污染物泄漏造成河流突发污染事故的应急处理方法十分必要。本论文选取苯酚、苯胺和亚甲基蓝等典型污染物为实验对象，采用吸附容量大、密度与水接近的活性炭纤维(ACF)为吸附剂。在自制的河流模型中，研究了ACF以苯酚、苯胺和亚甲基蓝为典型污染物的吸附过程，考察了吸附剂投加量、污染物浓度、吸附剂比表面积、吸附剂投加方式、水流速度与水质等对吸附速率与吸附效果的影响。实验结果表明，ACF能以较快的速率吸附苯酚、苯胺和亚甲基蓝，吸附率都在95％以上； ACF投加量是影响吸附速率最重要的因素，当一次性投加ACF质量之比为 1:2:4时，吸附速率常数之比近似为1:2:4；污染物浓度对吸附速率的影响显著，浓度较低时吸附速率较高。苯酚初始浓度为7mg·L-1时，经过86分钟的吸附，处理后的浓度可以达到地表水Ⅴ类水中挥发酚的限值要求(0.1mg·L-1)；在吸附11分钟左右追加适量的ACF，能够明显提高吸附速率；河水流速和河流中的天然有机物、浊度、河水硬度对ACF吸附都不产生显著影响，这说明ACF作为河流突发污染事故应急处理的吸附剂，有广泛的适应性。在实际河水中，ACF对苯酚的吸附过程与在模拟河水中相似，吸附效果显著。实验结果还表明，ACF对苯酚的吸附是放热反应，符合Freundlich模型和Langmuir模型。事故应急处理后，应该及时将吸附了污染物的ACF打捞上来，有利于进行后续处理。 Emergency environmental pollution accidents pose significant impacts on our living, economic development and ecological environment. The study on the approach of emergency control for the contingency caused by leakage of pollutants in rivers is very necessary. In the experiment, phenol, aniline and methylene blue were selected as representative pollutant and activated carbon fiber (ACF) was selected as adsorbent, which has strong adsorption capacity and similar density to water. In the self-made river model, the effects of ACF dosage, pollutant concentration, ACF surface area, ACF adding ways, water flow rate and water quality on adsorption courses were investigated. The experimental results showed that ACF could adsorb pollutant quickly and effectively. The ACF dosage was the most important factor that affected adsorption rate .When the ACF dosage rate was 1:2:4, the constants of adsorption rate was approximately 1:2:4. The effect of pollutant concentrations on the adsorption rate was notable. Faster adsorption rates were achieved at low pollutant concentrations. Phenol concentration reached the limits of volatile phenol in Category Ⅴ surface water (0.1 mg·L-1) after 86 minutes of adsorption with initial phenol concentration of 7 mg·L-1. After 11 minutes of adsorption, certain amount of ACF was added and the adsorption rate was improved significantly. River flow rate and water quality have little effect on the adsorption rate. The adsorption results obtained in actual river water were comparable with that in simulating river water. The results also showed that, ACF on the absorption of phenol is exothermic reaction, witch matched with the Freundlich model and the Langmuir model. After emergency treatment, the ACF absorbed pollutants should be promptly salvaged for follow-up treatment.

自养氨氧化混合种群特性和共存机理初步研究

组特殊自养氨氧化混合种群，表现：无机环境种群生长迅速、生物量高；在一个完全无机的自养生长环境中，不仅保持高氨氧化速率，并出现丰富的异养微生物种群；该种群置于异养、厌氧环境中，迅速表现出产氢特征。对于这样一个特殊的生态体系，研究其共生机理，以及联接这些种群之间的碳源和能源问题，将具有非常重要意义。我们拟从种群特征、细胞表面分泌产物、游离体系产物多糖、蛋白和脂肪酸方面开展研究。 第一部分，自养氨氧化混合种群的基本特征。采用氨氧化培养基，进行种群氨氧化特征研究；采用扫描电镜观察自养混合种群的微观特征；沉降、离心去除微生物种群，分析水相中的总有机碳、糖类等物质；利用LB培养基进行种群的分离、纯化，并采用DGGE手段对微生物种群结构进行分析。结果表明，接入菌种后（2/5000（V/V）），培养液中氨（200mg/L）在3－5天内快速降解；亚硝酸盐与氨氮变化呈负相关趋势，仅有少量硝酸盐含量（< 30mg/L）。氨氧化种群的生物量增长与氨氧化趋势一致，初始生物量7.75 mg/L(蛋白含量)，3-5天后生物量快速增长，并达到最高63.06 mg/L（蛋白含量）。电镜图片显示，种群外包裹一层粘液。离心除去菌体后，检测培养液总有机碳和糖的含量，同样表现出与生物量增长相似的特征，分别由初始的3.73、2.35 mg/L，3－5内天迅速增加，并分别达到最大值35.19、27.45 mg/L。经初步分离、纯化并对纯化菌株进行测序，获得了10株异养微生物分别为布鲁氏菌科苍白杆菌属、纤维单孢菌、类芽孢菌属、黄杆菌属、无色杆菌、鞘脂单胞菌、嗜麦芽寡养单胞菌、噬氢菌属、硫红球菌、假单胞菌；DGGE显示，约有20分条离带，我们对其中的两条优势条带进行切割回收测序，鉴定为欧洲亚硝化单胞菌（Nitrosomonas eur）。 第二部分：混合种群自养-异养菌共生的可能机制。在对微生物种群特征初步分析基础上，针对胞外糖类组分可能被微生物代谢分解，我们重点对微生物细胞蛋白质与糖类进行分析。采用超声结合RIPA裂解液裂解，SDS-PAGE电泳分析混合种群总蛋白种类，并通过氨基酸分析仪及红外光谱法分析氨基酸组成及蛋白红外特征。采用超声破碎结合反复冻融对细胞样品进行处理，提取液采用醇沉、Sevage脱氮白，凝胶过滤方法脱盐和分级分离。对提取物的糖分析包括：紫外扫描，红外光谱，核磁共振，单糖组成分析；扫描电镜观察菌群破裂现象。SDS-PAGE分析结果表明：氨氧化种群不同生长阶段都显示出42kD蛋白表达量很高，d4时42kD蛋白表达已经很强，4-7d内一直持续这种过量表达，直到d8后表达开始减弱。说明42kD蛋白可能与氨氧化密切相关。红外光谱分析显示：细胞提取物的特征峰分布在3427.42cm-1、1718.18 cm-1和1681.72 cm-1、1160.07和1086.74 cm-1，分别对应为OH、 C=O、C－O－C基团，表明具有蛋白的典型特征；氨基酸分析显示蛋白中的Gly,Asp,Ala,Glu含量相对较高。 提取物中胞外多糖分离谱图得到不均一组分，共得到6个收集峰；紫外扫描在201－213 nm处有多糖吸收峰，同样表明多糖成分不均一性；多糖红外光谱特征峰主要分别在3400.49 cm-1、2920.28 cm-1、1154.54和1087.52 cm-1，对应OH、－CH2－ or CH 、C－O－H or C－O－C等多糖特征基团；多糖提取物核磁共振1H d4.3～5.9之间出现强吸收峰，这是1H中，多糖存在的明显证据，1H NMR中，其中O-乙酰基的甲基上的氢信号为d1.1～1.3之间。糖肟全苯甲酸酯衍生物的HPLC测定中，得到单一的单糖峰，由于时间问题，还未进行更深入的试验；电镜图片显示，种群中的细胞有大量的破裂现象。 实验表明，自养氨氧化混合种群显示出快速的氨氧化速率，氨氧化过程生物量和有机质的增加明显。微生物种群包裹粘液层，并分离纯化出大量的异养菌；去除菌体后的游离培养液中存在有机质（包括多糖）说明无机自养生长体系中存在异养菌生长、繁殖的二次碳源；细胞提取物中蛋白条带数目多、种类丰富；细胞多糖提取物具有明显的多糖特征，以及单糖的存在。结合种群的显微特征和游离体系中的有机质的检测结果，我们认为，无机自养生长体系中，种群细胞生长过程中发生的破裂现象可能是导致大量的蛋白、多糖释放到游离胞外，并成为其他异养菌生长的碳源和氮源。这可能是自养体系中，大量异养菌共生的可能机制，至于是什么原因引起种群生长过程中产生的破裂现象，还有待下一步深入研究。 A group of mixed autotrophic ammonia oxidizing populations, having much biological characteristic tested by concerned personnel for pilot test: Performed rapid population growth and obtained high biomass in inorganic environment; Not only maintained a high rate of ammoxidation, promoted a wealth of heterotrophic microbial populations growth in a totally inorganic and autotrophic growth environment; Placed in heterotrophic and anaerobic environment,had the performance characteristics that could rapidly produce hydrogen.For such a special ecological system, Study its symbiotic mechanism and the connection between these populations of carbon and energy issues, will have a very important significance. We intended from the characteristics of the population, the secretion product of cell surface, free substance in the liquid medium like polysaccharide, protein and fatty acids carrying out research. Part I: The basic features of mixed autotrophic ammonia oxidizing populations . Use inorganic liquid medium, processed study for ammonia oxidation characteristics of the population; we used scanning electron microscopy to get micro-features of autotrophic ammonia oxidizing populations .The medium was carried out settlement and centrifugal then removed the microbial populations, after all of that we analysis the water phase for total organic carbon(TOC), carbohydrate and other substances; Solid ammonia oxidizing medium was adopted to separation and purification of population, DGGE means was for structure analysis of microbial population. The results showed that after the inoculum of bacteria (2 / 5000 (V / V)), ammonia in the culture medium (200 mg / L) was rapid degradation in 3-5 days; ammonia and nitrite have the negative correlation between changes in the trend, then only a small amount of nitrate content (<30mg / L). The biomass growth of ammoxidation population in line with the trend of ammonia oxidation, the initial volume of it was 7.75 mg / L (protein content), in 3-5 days upto 63.06 mg / L (protein content). Electron microscope image showed, the populations were wrapped in a layer of mucus, including the a large number ruptted micorbe , Centrifuge to remove bacteria, then detected the medium for total organic carbon and sugar content, result took on the same characteristics with biomass growth, that were from the initial 3.73、2.35 mg / L respectively, in 3-6 days achieved rapid increase in the maximum to 35.19、27.45 mg / L respectively. After initial separation、 purification ,then processed sequencing to strains purified and got the result that there were 10 heterotrophic microorganisms : Brucella Branch pale bacillus, Cellu lomonas, Bacillus species category, a Flavobacterium, colorless Bacteria, Aeromonas sheath fat, little support maltophilia Aeromonas, macrophages species hydrogen, sulphur-MI, Pseudomonas bacteria spores; DGGE display, there were 20 separation bands approximately. Part II: Mixed populations that autotrophic - heterotrophic bacteria symbiotic mechanism. On the basis of preliminary analysis of microbial population characteristics, aiming at extracellular carbohydrate components might be decomposition by microbial, we focused on microbial cell protein and carbohydrate analysis. Using ultrasound combined with RIPA lysis cracking the cells, SDS-PAGE electrophoresis analysis the total protein species of the population, and through the amino acid analyzer studied the compositions of amino acid and infrared spectroscopy analysis of a protein infrared characteristics. Using ultrasound combined with repeatedly freezing and thawing to treated the cell sample, then took the means that alcohol precipitation, deproteinization by Sevage, gel filtration aimed at desalination and grade separation to deal with the lysates . The extraction of sugar analysis included: UV scanning, IR, NMR, single-sugar composition analysis. SDS-PAGE analysis showed that: 42 kD protein expression was very high at different growth stages of mixed autotrophic ammonia oxidizing populations , on the fourth day, 42 kD protein expression had been very strong, 4-7d, it had continued this excessive expression, then started to weaken after 7 days. 42 kD protein that might be closely associated with ammonia oxidation. Infrared spectral analysis showed that: cell extracts with the characteristic that the peak distribution in 3427.42 cm-1、1718.18 cm-1 and 1681.72 cm-1、1160.07 cm-1 and 1086.74 cm-1 corresponding to OH、C = O、C-O-C Groups which had the typical characteristics of protein; and analysis showed that amino acids including Gly, Asp, Ala, Glu ,the content in the protein is relatively high. Exopolysaccharide in the extracts had the separation map that it was uneven, received a total of six collection peaks by the detection mode of phenol-sulphruic acid method ; ultraviolet scan in the 201-213 nm department had polysaccharide absorbing peak, the same ingredients that polysaccharide heterogeneity; infrared polysaccharide spectral characteristics of the main peak at 3400.49 cm-1, 2920.28 cm-1, 1154.54 and 1087.52 cm-1, corresponding OH,-CH2-or CH, C-O-H or C-O-C;and other characteristics of polysaccharide group; 1H NMR of polysaccharide extract appeared absorption peak between d4.3 ~5.9, which is the apparent evidence of polysaccharide, In 1H NMR, the hydrogen signal of one of O-acetyl was between 1.1 to 1.3. The determination of Sugar oxime whole benzoate derivatives by HPLC, there was a single-sugar peak, as a matter of time, yet more in-depth test. Summary: Mixed autotrophic ammonia oxidizing populations show us that it had the ability in ammonia oxidizing and it was great, organic matter and biomass increased significantly in the process of ammonia oxidation. Microbial populations was wrapped up slime layer, the phenomenon of cell breakdown obviously, and there were a lot of separation and purification of the heterotrophic bacteria; a lot of organic matter (including polysaccharides)remined in the medium that removal of cell indicated the inorganic system existed secondary carbon sources that could be used by the heterotrophic bacteria ; there were a large number proteins bands of cell extract, rich variety; cell extracts of polysaccharide had obvious characteristics of polysaccharide, and the existence evidence of single-sugar. Combined population of microscopic characteristics and free of organic matter in the test results, we believe that the health of inorganic system, population growth occurred in the course of the breakdown of the phenomenon is likely to lead to a lot of protein and polysaccharide released into the extracellular free, And other heterotrophic bacteria use them to the growth as carbon and nitrogen. This may be autotrophic system, the large number of heterotrophic bacteria symbiotic mechanism.

川牛膝多糖的免疫活性及其水解活性片段分离研究

川牛膝多糖（CP）是从传统中药川牛膝（Cyathula officinalis Kuan）中提取的一种活性多糖，现代药理研究表明川牛膝多糖是川牛膝许多生物活性的物质基础。本实验室前期进行了川牛膝多糖的提取、分离、结构鉴定及其部分活性研究，发现川牛膝中多糖含量非常高，在对川牛膝多糖活性的初步研究中也证实了其具有免疫调节作用。我们为了进一步了解其免疫调节活性，并为构效关系的研究奠定基础，对其进行了如下研究： 1． 通过体外毒性检测、淋巴细胞增殖实验、NK细胞杀伤活性和腹腔巨噬细胞吞噬中性红活性测定，发现川牛膝多糖在10～300μg/mL浓度范围内，对细胞无毒性作用；能够促进LPS诱导的B淋巴细胞增殖(P<0.01)、增强NK细胞杀伤活性(P<0.05)和PMΦ吞噬中性红活性(P<0.01)，且随多糖浓度增高而增强；但其对ConA诱导的T淋巴细胞的增殖无促进作用(P>0.05)。 2． 通过正常小鼠体内淋巴细胞转化实验、迟发型变态反应分析、抗体生成细胞检测、碳粒廓清检测、腹腔巨噬细胞吞噬鸡红细胞活性和NK细胞活性测定，发现川牛膝多糖在适应性免疫方面能够促进SRBC免疫小鼠体内的抗体生成细胞的生成（P<0.01）和增强DNFB诱导的DTH（P<0.05），但对ConA诱导的脾淋巴细胞增殖无促进作用（P>0.05）；在固有免疫方面能够提高小鼠碳粒廓清速率（P<0.05），PMΦ吞噬 CRBC 活性（P<0.01）和NK细胞杀伤活性（P<0.05）。同时还发现其对由环磷酰胺(Cy)引起的白细胞数下降具有很好的抑制作用（P<0.01）。 3． 为了获得结构明确、均一的保留活性的川牛膝多糖片段，为其作用机制、构效关系研究提供关键研究材料，我们开展了“保留免疫活性的最小片段”的分离制备的初步研究。建立并优化了川牛膝多糖的酸水解条件，发现在6%的样品浓度，0.025mol/L的硫酸浓度，65℃的水解温度，水解时间为8min的条件下可以得到一系列连续的多糖片段；采用Bio-Gel P2 分子筛柱层析分离得到5个级分，通过体外淋巴细胞增殖实验、NK细胞活性测定、腹腔巨噬细胞吞噬中性红实验发现其中的一个片段仍保留较强的免疫活性，并测得其分子量约为2057Da，为保留免疫活性的最小片段的进一步分离奠定了基础。 Cyathula officinalis Kuan is a commonly-used Traditional Chinese Medicine (TCM) with a wide range of pharmacological activities. Modern pharmacological researches showed the polysaccharide extracted from it (CP) is an important component for many bioactivities of this TCM. In the previous studies, we found CP showed significant immuno-regulative activities. In order to evaluate this activity systematically and lay foundations for revealling its immuno-regulative machanisms and the Structure -Function relationship, we carried out the following research works: 1． The in vitro immunoactivities of CP were evaluated by using normal mice immunocytes with respects to cytotoxicity, lymphocytes proliferation, NK activity and the ability of peritoneal macrophage phagocytizing neutral red. The polysaccharide showed no cytotoxicity below the concentration of 300 μg/mL, and could promote B lymphocytes proliferation (P<0.01), enhance NK activity (P<0.05) and the ability of peritoneal macrophage phagocytizing neutral red (P<0.01) at the concentration of 10-300 μg/mL. The above effects were positively correlated with the concentration of the polysaccharides. But it could not promote T lymphocytes proliferation (P>0.05). 2． The in vivo immunoactivities of CP were observed on normal mice through the following indices: splenic lymphocyte transformation efficiency, delayed-type allergy, antibody-forming cells activity (AFC), rate of carbon clearance, rate of peritoneal macrophage phagocytizing chicken red blood cell (CRBC) and NK activity, and its influence on the decline of the mouse leucocyte count induced by Cy. The polysaccharide at medium-dose enhanced delayed-type allergy （P<0.05）and NK activity（P<0.05） and increased the rate of carbon clearance（P<0.05）, AFC activity（P<0.01） and the rate of peritoneal macrophage phagocytizing CRBC（P<0.01）. The polysaccharides also effectively resisted the decline of the mouse leucocyte count induced by Cy（P<0.01）. However, it couldn’t increase the splenic lymphocyte transformation efficiency（P>0.05）. 3． Attempting to isolate and prepare the minimal fragments retaining activity with identical structure for further studying on immuno-regulative mechanism and Structure-Function relationship, we carried out the study on hydrolysis of CP, isolation of hydrolysed fragments, and the activity evaluation of the isolated fragments. CP with concentration of 6% was hydrolysed at 65℃ for 8 min with sulfuric acid of 0.025 mol/L，then the hydrolysate was separated using Bio-Gel P2 chromatography, 5 portions of fragments were obtained. The immunoactivities of these fragments were evaluated by using normal mice immunocytes with respect to lymphocytes proliferation, NK activity and ability of peritoneal macrophage phagocytizing neutral red. One fragment with relative molecular mass of 2057Da was found retaining immunoactivity.

己酸乙酯酯化真菌筛选及发酵条件研究

本文主要研究了具有己酸乙酯酯化活性的真菌菌株的筛选和发酵条件优化。从大曲和糟醅样品中分离纯化获得79株产生透明圈的丝状真菌，菌落形态初步识别结果显示分离菌株包括红曲霉属、根霉属及曲霉属等菌株。其中菌株EM-56酯化酶活力最强，发酵获得的粗酶制剂酶活为172.36 u。根据显微形态、菌落形态及生理生化特征，初步鉴定该菌株为曲霉科红曲霉属紫色红曲霉（Monascus purpureus）。 在此基础上重点研究了菌株EM-56在不同培养基成分及不同培养条件下的产酶情况，确定了最佳培养基和培养条件。通过单因素实验确定在基础培养基中添加最佳碳源为葡萄糖，最佳氮源为蛋白胨。正交优化实验结果确定了最佳培养基组成：以麸皮为基础培养基，添加葡萄糖 2%，蛋白胨 0.3 %，KH2PO4.3H2O 0.05 %，MgSO4.7H2O 0.06 %。菌株EM-56在上述培养基中的最佳发酵条件为：初始pH 5.5，发酵温度为35°C，发酵时间7d，种龄48h，接种量8%，装瓶量50g / 瓶（500mL）。在最佳培养基和发酵条件下，菌株EM-56发酵获得的粗酶制剂酶活达到241.56 u，比优化前提高了40.15%。 In this paper, the research focuses on the selection of fungus with esterifying activity and optimization of fermentation conditions. We isolated 79 strains which had transparent zones from Daqu and fermented grains. The isolated strains contained Monascus、Rhizopus and Aspergillus through primary morphology analysis. The strain of EM-56 which produces strongest esterase was selected. The enzyme activity reached 172.36u. According to related literature, EM-56 was identified as Monascus purpureus through morphology analysis and biochemical determination. We also studied the effects of different medium and fermentation conditions on the esterase production of strain EM-56. The optimal medium and fermentation conditions were determined. Single factor experiment result shows that the optimal carbon source added is glucose and the optimal nitrogen source added is peptone. The optimal fermentation medium determined by orthogonal optimization test is as follows: wheat bran as substrate, glucose 2%, peptone 0.3%, KH2PO4.3H2O 0.05%，MgSO4.7H2O 0.06%. The optimal fermentation conditions are: initial pH 5.5, cultural temperature 35°C, cultural time 7d, seed age 48h, inoculation 8%, medium mass 50g / flask（500mL）. The esterse activity of EM-56 cultivated in the optimal medium and fermentation conditions reached 241.56u and increased by 40.15% compared with the original activity.

鲜甘薯快速乙醇发酵条件的初步研究

本文对不同菌种（酵母菌和运动发酵单胞菌）快速生产燃料乙醇的条件进行了研究，实现了鲜甘薯快速转化为燃料乙醇。全文分为两部分： 第一部分：酵母菌快速生产燃料乙醇的条件研究。通过单因素试验，酵母菌快速生产燃料乙醇的条件为：发酵方式采用边糖化边发酵（SSF），蒸煮温度为85 ℃，料水比2:1（初始糖浓度 210 g/kg），糖化酶用量0.75 AGU/g 鲜甘薯，接种量10%（v/w）。在最优条件下，经过24 h发酵，乙醇浓度可达97.44 g/kg, 发酵效率为92%，发酵强度为4.06 g/kg/h。由于采用了低温蒸煮和SSF，可以大大节约能耗，从而降低乙醇生产的成本。同时，利用摇瓶优化的条件，进行了10 L，100 L，500 L发酵罐的放大试验，由于发酵罐初期可以人为通氧，使菌体能迅速积累，发酵时间缩短2 h，发酵效率在90%以上。 第二部分：运动发酵单胞菌快速生产燃料乙醇条件研究。通过单因素试验和正交试验获得了发酵的最佳参数：初始pH值6.0-7.0，硫酸铵5.0 g/kg，糖化酶量1.6 AUG/kg淀粉，初始糖浓度200 g/kg，接种量12.5%（v/w）。经过21 h发酵，乙醇浓度为95.15 g/kg，发酵效率可达94%。同时对不灭菌发酵也进行了研究，发酵效率可达92%。为鲜甘薯运动发酵单胞菌燃料乙醇的工业化生产打下基础。 对发酵结束后的残糖进行了研究。通过薄层层析和葡萄氧化酶测定证明：无论是酵母菌还是运动发酵单胞菌发酵结束后的发酵液中都不含葡萄糖。经过HPLC进一步分析残糖说明：发酵液中已没有葡萄糖成分；经糖化酶水解后仍没有葡萄糖出现；但经酸水解后又出现了葡萄糖，说明结束后的残糖是一些低聚糖结构。有关残糖的结构需要进一步研究。可以通过开发高效的低聚糖水解酶来降低发酵液的残糖，提高原料的利用率。 A new technology for rapid production fuel ethanol from fresh sweet potato by different microorganisms (Saccharomyces cerevisiae and Zymomonas mobilis) was gained in this research. The paper involved two parts: Part 1: The study on fuel ethanol rapid production from fresh sweet potato by Saccharomyces cerevisiae. The following parameters of Saccharomyces cerevisiae was investigated by a series of experiments: fermentation models, cooking temperature, initial sugar concentration and glucoamylase dosage. The results showed that SSF (simultaneous saccharification and fermentation) not only reduced the fermentation time (from 30 to 24h) but also enhanced the ethanol concentration (from 73.56 to 95.96 g/kg). With low-temperature-cooking (85 ℃) using SSF, the Saccharomyces cerevisiae was able to produce ethanol 97.44 g/kg which the fermentation yield could reach to 92% and ethanol productivity 4.06 g/kg/h from sweet potato enzymatic hydrolysis. Furthermore, the savings in energy by carrying out the cooking (85 ℃) and saccharification (30 ℃) step at low temperature had been realized. The results were also verified in 10 L, 100 L and 500 L fermentor. The fermentation yield was no less than 90%. The fermentation time of fermenter was shorter than Erlenmeyer flask. This may be that the aeration in the early fermentation period is available, which lead to the rapidly commutations of biomass. Part 2: The technology of ethanol rapid production with simultaneous saccharification and fermentation ( SSF ) by Zymomonas mobilis，using fresh sweet potato as raw material was studied. The effects of various factors on the yield of ethanol were investigated by the single factor and the orthogonal experiments. As a result, the optimal technical conditions were obtained from those experiments：initial pH value 6.0-7.0, nitride 5.0 g/kg，(NH4)2SO4, glucoamylase 1.6 AUG/kg starch, inoculums concentration 12.5% (v/w). The Zymomonas mobilis was able to produce ethanol 95.15 g/kg, with 94% of the theoretical yield, from fresh sweet potato after 24 h fermentation. The fermentation efficiency of non-sterilized was also reach to 92%. We also analyzed the final fermentation residual sugars of Saccharomyces cerevisiae and Zymomonas mobilis. When the residual sugars were analyzed by thin-layer chromatogram and glucose oxidase, there was no glucose. The analysis of reducing sugars by HPLC showed that there was no glucose existed in the fermentation liquor. However, the glucose appeared after being hydrolyzed by acid. It is indicated that the residual sugars in the final fermentation liquor were the configuration of oligosaccharide, which was linked by the special glycosidic bonds. It was feasible for reducing residual sugars to develope the enzyme that can degradation the oligosaccharide.

毛壳菌产抗真菌活性物质菌株筛选及种间原生质体融合研究

毛壳菌属很多种类具有重要生防价值，其生防机理包括对植物病原真菌的重寄生作用、诱导植物产生抗病性、产生抗真菌活性的次生代谢产物等。迄今，学界对毛壳菌的研究主要集中在毛壳菌的生防机理，毛壳菌活性次生代谢产物的分离等方面。本研究致力于产抗生素的毛壳菌的种间原生质体融合，从产抗生素毛壳菌菌株的筛选开始，进而对产抗生素的角毛壳菌进行诱变选育，最终用产不同抗生素的角毛壳菌与球毛壳菌进行种间原生质体融合。主要有以下五方面研究结果。 1、毛壳菌抗真菌活性物质产生菌株的筛选：不同毛壳菌菌株发酵液采用琼脂扩散法对植物病原真菌进行抑菌活性试验，结果显示，菌株CH08和CH23的发酵液对芒果炭疽、苹果炭疽和马铃薯晚疫菌具有抑制作用。菌株CH16和CH17的发酵液对芒果炭疽菌、苹果炭疽菌有抑制作用。菌株CH21发酵液对辣椒炭疽菌和西瓜枯萎菌有抑制作用。经形态学研究，菌株CH08、CH16、CH17和CH23鉴定为球毛壳菌，菌株CH21鉴定为角毛壳菌。对角毛壳菌与球毛壳菌菌株发酵液抑菌谱比较，发现角毛壳菌与球毛壳菌发酵液具有明显不同的抑菌谱，表明角毛壳菌与球毛壳菌产生不同的抗真菌活性物质。 2、角毛壳菌（CH21）和球毛壳菌（CH08）原生质体制备和再生条件研究：考察了菌龄、酶浓度、稳渗剂及其浓度、酶解温度、酶解时间及再生培养基对原生质体制备和再生的影响。用菌龄为生长54 h的角毛壳菌菌丝，以0.06 M磷酸缓冲液（pH6.0）配制成含蜗牛酶15 mg/ml、溶壁酶10 mg/ml、蔗糖0.6 mol/L的酶解液，30℃酶解1.5 h，原生质体释放量2.02×107个/g；以PDA为再生培养基，0.7 mol/L的蔗糖再生稳渗剂，再生率可达51.45%。用菌龄为生长48 h的球毛壳菌菌丝，以0.06 M磷酸缓冲液（pH6.0）配制成含蜗牛酶15 mg/ml、溶壁酶10 mg/ml、蔗糖0.6 mol/L的酶解液，30℃酶解1 h，原生质体释放量达1.57×108个/g；以PDA为再生培养基，0.7 mol/L的蔗糖为再生稳渗剂，再生率可达41.48%。 3、角毛壳菌（CH21）原生质体紫外诱变选育：以CH21为出发菌株，制备原生质体进行紫外诱变，诱变条件为：15 w紫外灯，距离30 cm，照射90 s，致死率80%~85%。建立了诱变菌株初筛的双层平板筛选模型。经平板初筛和摇瓶复筛，获得一株突变菌株CH21-I-402，其发酵液抑菌活性较出发菌株提高18.3%。 4、抗性标记菌株的获得：菌株CH21-I-402和CH08抗生素药敏试验表明， CH21-I-402菌株对潮霉素有抗性、对G418（Geneticin）敏感，菌株CH08对潮霉素和G418都敏感。根癌农杆菌EHA105介导的新霉素磷酸转移酶基因转化球毛壳菌，经PCR检测，新霉素磷酸转移酶基因成功转化进菌株CH08-GR70，CH08-GR120。转化子对G418抗性提高3～4倍，对潮霉素仍然比较敏感。 5、以G418和潮霉素抗性为筛选标记的原生质体融合与融合菌株AFLP分析：制备角毛壳菌CH21-I-402和球毛壳菌CH08-GR70原生质体，以35％的PEG6000为助融剂进行原生质体融合，以65 μg/ml的潮霉素和60 μg/ml G418为抗性筛选标记，获得46个再生菌株。再生菌株连续传代5代后，再生菌株表现出多种形态类型。利用AFLP技术对再生菌株及亲本菌株基因组DNA分析表明，再生菌株PF1、PF26为融合菌株。抑菌活性测试表明，融合菌株PF26发酵液对芒果炭疽菌和苹果轮纹菌有强的抑制作用，且抑菌活性比亲本球毛壳菌明显提高。 Chaetomium spp. have great potentials as biocontrol agents against a range of plant pathogens on the basis of its mycoparasitism, induced plant disease resistance, production of antifungal metabolites, and so on. Previous researches on C. spp. mostly focused on the mechanisms of its biocontrol and the isolation of secondary metabolites. In this study, screening antifungal C. spp., mutation breeding of C. cupreum and interspecies protoplast fusion between C. cupreum and C. globosum were carried out, respectively. The corresponding results are as follows: Firstly, among more than 40 C. spp., the strains produced anti-fungal antibiotics were screened by agar diffusion experiments. Results showed that both CH08 and CH23 had inhibition against Colletotrichum gloeosporioides, Cladosporium fulvum, and Phytophthora infestans. Both CH16 and CH17 had inhibition against Colletotrichum gloeosporioides and Cladosporium fulvum. In addition, CH21 exhibited anti-fungal activity against Fusarium oxysporum f. sp niveum and Colletotrichum capsici. Furthermore, CH08, CH16, CH17 and CH23 were identified as C. globosum, CH21 was proved to be C. cupreum based on morphology. The comparison of the anti-fungal spectrum between C. cupreum and C. globosum, showed they could produce different antibiotics. Secondly, specified protocols for preparing and regenerating protoplasts from mycelia of C. cupreum CH21 and C. globosum CH08 were studied. The effects of the age mycelia, the concentration of enzyme, digestion temperature and time, kinds of osmotic stabilizer and regeneration medium on protoplasts preparation and regeneration were all optimized, respectively. In one protocol, with 15 mg/mL snailase, 10 mg/mL lywallzyme, 0.6 M sucrose, in 0.06 M phosphate buffer (pH6.0), and digested for 1.5 h at 30 ºC, 2.02×107 protoplasts from each gram mycelia were obtained from cultures of C. cupreum CH21 grown in potato dextrose broth (PDB) medium for 54 h. And when 0.7 M sucrose was used as osmotic stabilizer in the regeneration medium OPDA (potato dextrose agar with osmotic stabilize), the regeneration efficiency of protoplasts was 51.45%. In another protocol, with 15 mg/mL snailase, 10 mg/mL lywallzyme, 0.6 M sucrose, in 0.06 M phosphate buffer (pH6.0), and digested for 1 h at 30 ºC, 1.57×108 protoplasts from each gram mycelia were obtained from cultures of C. globosum CH08 grown in PDB for 48 h. And when 0.7 M sucrose was used as osmotic stabilizer in the regeneration medium OPDA, the regeneration efficiency of protoplasts was 41.48%. Thirdly, the mutagenesis conditions and secondary screening model of C. cupreum CH21 were explored. An 80% to 85% death rate could be achieved when the protoplasts of C. cupreum CH21 were irradiated by 15 w UV lamp from 30 cm distance for 90 s. In addition, the doublelayer plate’s method for the primary screening of high-producing antibiotics strains was established. A high yielding antibiotic mutant CH21-I-402 was obtained through the primary screening on plate and the secondary selection in Erlenmeyer flask, compared to the original CH21 strain, the antifungal activity of the mutant CH21-I-402 was increased by 18.3%. Fourth, the sensitivity to antibiotics of both C. cupreum CH21-I-402 and C. globusm CH08 was detected. Results showed C. cupreum CH21-I-402 was sensitive to G418 (Geneticin) (Gs) and resistant to Hygromycin B(Hr), and C. globusm CH08 was sensitive to both G418 (Geneticin) (Gs) and Hygromycin B(Hs). At the same time, neomycin phosphotransferase II (npt II) gene was transformed into C. globusm CH08(Gs, Hs) mediated by Agrobacterium tumefaciens EHA105, and the npt II gene was verified by polymerase chain reaction in resistance to G418 strains CH08-GR70 and CH08-GR120. The transformants still showed sensitive to Hygromycin B(Hs). Finally, a selection system for hybrids was set up by interspecies protoplast fusion between C. cupreum and C. globusm using dominant selective drug resistance markers. At first, protoplasts of C. cupreum CH21-I-402 (Hr, Gs) and C. globusm CH08-GR70 (Hs, Gr) were prepared, then the protoplasts were fused in the presence of 35% polyethylene glycol 6000 and regenerated on OPDA medium with 65 μg/ml Hygromycin B and 60μg/ml G418, at last 46 colonies with Hr and Gr were obtained. Even after 5 generations’ subculture, most of the colonies displayed significant difference in taxonomic characteristics with their parental strains. Regenerated strains PF1 and PF26 were confirmed as fusants by amplified fragment length polymorphisms analysis with the genomic DNA as the model. PF26 showed higher inhibitory activity against Colletotrichum gloeosporioides and Macrophoma kuwatsukai than that of the parental strain C. globusm.

亚硝化—厌氧氨氧化处理猪场废水研究

猪场废水COD浓度高、氨氮浓度高、悬浮物浓度高，已成为农村面源污染的主要来源，并严重威胁到农村饮用水安全。猪场废水氨氮浓度高、处理难度大，如何采用经济高效的方法，去除氨氮使其达到排放标准，一直是猪场废水处理中面临的重要难题。 厌氧氨氧化是近年受到国内外水处理研究者广泛关注的新型生物脱氮技术，具有不需要外加有机碳源、节省供氧量、降低能耗等优点。虽然国内外研究者对厌氧氨氧化过程的脱氮机理、厌氧氨氧化菌的生理生化特性等进行了多方面的研究，但已有的报道大多以模拟废水为研究对象，以猪场废水为研究对象的报道，在国内外文献中极少有报导。 本论文以猪场废水为主要研究对象，考察了猪场废水的亚硝化过程、厌氧氨氧化的启动过程，并对亚硝化和厌氧氨氧化联合用于猪场废水脱氮进行了探索。 1．论文首先研究了猪场废水的亚硝化过程，考察了废水水质和主要运行条件对亚硝化过程的影响。实验表明：（1）亚硝化阶段反应时间为8到10h时，出水中氨氮和亚硝酸盐浓度比可达到1：1～1：1.23，满足厌氧氨氧化反应对二者比例的要求；达到前述要求时，氨氮去除率达到58.3～65.6 %，亚硝化率在整个过程均保持在97 %以上，COD去除率在59.2～68.6 %；（2）曝气量（溶解氧）对亚硝化过程影响显著，随着曝气量增大，达到厌氧氨氧化要求的氨氮与亚硝酸盐氮浓度比例所需水力停留时间τ越短，pH出现明显下降的时间越短；（3）τ对应的pH在7.8～8.1之间，无需进行pH调节即可满足厌氧氨氧化反应对pH的要求；（4）氨氮和COD降解过程遵循一级反应动力学，氨氮和COD降解的速率常数分别为0.0656～0.0724 1/h和0.0491～0.0664 1/h。 2．在进行亚硝化过程研究的同时，以模拟废水为试验对象，进行厌氧氨氧化启动研究。以反硝化污泥和养殖厂储水池厌氧底泥的混合污泥作为接种污泥，历时大约100天，培育出具有厌氧氨氧化活性的污泥，氨氮和亚硝酸盐氮最高进水浓度分别为223.8 mg/L和171.4 mg/L，去除率最高分别达48%和41.5%，此时二者消耗比例为1.33：1。 3．在猪场废水的亚硝化研究完成和厌氧氨氧化过程初步启动成功后，在模拟废水中逐步加入猪场废水的亚硝化处理出水，逐步实现亚硝化和厌氧氨氧化的组合。亚硝化出水添加到厌氧反应器后，厌氧氨氧化反应仍可继续进行，且去除效率逐步提高。研究发现添加的亚硝化出水中携带的亚硝化细菌在厌氧氨氧化菌膜外层生长并累积，增加了厌氧氨氧化反应基质的传质阻力，妨碍了厌氧氨氧化效率的提高。 4．亚硝化－厌氧氨氧化实际工程应用探索中，生物接触氧化池可在有效去除废水中的有机物的同时实现亚硝化，出水中氨氮和亚硝酸盐比例平均为1.10，可满足后续厌氧氨氧化的要求；在适宜的进水浓度和温度下，ABR池出现了厌氧氨氧化启动的迹象；研究同时发现，水质的波动和气温的变化是工程中影响厌氧氨氧化菌活性的重要因素。 论文的主要创新点在于：（1）以猪场废水为研究对象，以实现厌氧氨氧化为目标，对亚硝化过程进行了比较详细的考察，获得了亚硝化出水满足厌氧氨氧化要求的工艺条件，通过对其COD和氨氮降解过程的考察，得出亚硝化阶段COD降解和氨氮去除的动力学模型；（2）对亚硝化－厌氧氨氧化处理猪场废水进行了探索，确立了影响其污染物去除率稳定的重要因素。 论文的上述研究成果，为厌氧氨氧化技术的实用性研究提供理论依据。 Piggery wastewater, which is characterized by high concentration of COD、ammonium and suspend substance, has become a most important source of non-point source pollution and also severely threats drinking water security in rural area. How to discharge piggery wastewater with the ammonium concentration meeting standard by economical and effective method? This is the most urgent problem in piggery wastewater treatment. As a new biological nitrogen removal technology, Anammox process has been paid more and more attention by researchers all over the world. Anammox has advantages of no need of organic carbon addition, low oxygen consumption and energy consumption. Plenty of investigations have been carried out to the mechanism, physiological and biochemical characteristic of bacteria about Anammox. Most of researches focused on synthetic wastewater, there is rare report about its application in piggery wastewater. In this paper，experimental studies were performed to investigate Sharon process in treatment of piggery wastewater，the start up process of Annammox using synthetic wastewater were studied, the feasibility of applying Sharon-Anammox process in the nitrogen removal of piggery wastewater was evaluated. 1. Sharon process of piggery wastewater was firstly investigated to analyze the effects of water quality and main running parameters, which meet the NH4+-N to NO2--N ratio requirement of successive Anammox. Results showed: (1)During Sharon Process，after 8～10 hours’ reaction the NH4+-N to NO2--N ratio in effluent reached 1:1.0～1:1.23, when the removal percentage of NH4+-N was 58.3～65.6 %, a semi-nitration rate of above 97 % was achieved during the process; meanwhile 59.2～68.6 % of the COD was also removed. (2)The aeration rate（oxygen） had obvious effect on the hydraulic retention time(τ) which met the NH4+-N to NO2--N ratio requirement of Anammox. As aeration rate increased, the hydraulic retention time(τ) was shortened. (3) The pH corresponding to τ was between 7.8 and 8.1, thus it needed no artificial adjustment. (4) The reduction of ammonia and COD followed the first-order reaction kinetics. The velocity constants of ammonia and COD were 0.0656～0.0724 1/h and 0.0491～0.0664 1/h, respectively. 2. The startup of Anammox process using the artificial wastewater was performed simultaneously with Sharon. The aim was to investigate the running parameters of Anammox and make foundation for the combination stage. By using the mixture of denitrifying sludge and anaerobic sludge in tank of the breeding factory, sludge of Anammox activity was cultivated in UASB after 100 days. The removal percentage of NH4+-N and NO2-N were up to 48% and 41.5%, respectively, when the NH4+-N and NO2-N influent concentration were 223.8 mg/L and 171.4 mg/L, respectively, the NH4+-N and NO2-N removal rate was 1.33:1. 3. After investigation of Sharon and startup of Anammox, effluent of Sharon process was added into the synthetic wastewater to combine Sharon and Anammox step by step. It took some time after the addition of Sharon effluent that Anammox reaction continued and the removal rate kept increasing. It indicated that nitrifying bacteria were carried by the Sharon effluent cumulated in the outer layer of Anammox. This enhanced transfer resistance of Anammox reaction and the increasing removal rate was restrained. 4. In the bio-contact oxidation pond of practical project, Sharon process were carried out successfully and organic compounds were removed effectively. An average NO2-N/ NH4+-N rate of 1:1.0 was achieved in the effluent, which met the requirement of successive Anammox. Under condition of suitable influent concentration and temperature, there was evidence that Anammox could start up in ABR. The variety of wastewater and temperature had great affects on Anammox activity in practical engineering. Innovation of this paper: (1) The Sharon process for treating piggery wastewater was discussed in details. Technological parameters that met requirement of Anammox were obtained. The dynamic models of COD and ammonium removal in the process were educed. (2) Sharon-Ananmmox for treatment of piggery wastewater was investigated, and the primary influencing factors was studied. This paper could be a theoretical consult for research of Anammox utility.

PVA降解菌的产酶条件及特性研究

目前对PVA生物降解研究重点逐渐转移到对PVA降解菌和PVA降解酶的研究开发上，随着对PVA降解高效新菌株的不断发现和PVA降解酶作用机理和分泌机制的深入了解，利用高效微生物或酶法治理PVA这类高聚物的污染将具有较大的应用潜力。本论文研究工作正是基于这种客观条件下进行的，对本实验室前期分离的PVA降解菌株P1、共生菌B1+B2、Pa、Pb为研究对象，重点研究了菌株P1和共生菌B1+B2的产酶条件和产酶特性，验证找出了影响菌株P1产酶的生长因子，论证了菌株B1+B2的产酶特性，优化得出了菌株B1+B2的最佳产酶条件；然后对共生菌B1+B2的PVA降解酶的稳定性进行了研究；最后研究了最佳组合菌的产酶特性和最佳产酶条件。主要研究结果如下： 1 通过对菌株P1产酶因子的研究，找出了核黄素是菌株P1产酶的必须因子，在以淀粉为碳源时，核黄素只是产酶的必须因子，而不是菌体生长的必须因子；在以PVA为碳源时，核黄素既是生长的必须因子，也是产酶的必须因子，是菌株P1的生长因子。 2 对共生菌B1+B2的产酶条件和产酶特性进行了研究，并通过正交实验找出了影响菌株产酶的主要条件和菌株产酶的最佳条件。 3 对共生菌PVA降解酶的稳定性进行了研究，确定了影响酶稳定性的主要理化条件。 4 通过对菌株降解性能的比较，确定菌株Pa、Pb、共生菌、P1的作为组合菌的组成菌，然后通过复配实验确定出菌株的最佳组合为菌株Pa、P1、共生菌，最后通过正交实验确定最佳组合菌的最佳配比。 5对影响组合菌产酶的因素进行了研究，通过正交实验确定了影响组合菌产酶的主要因素和最佳产酶条件。 本文通过对PVA降解菌株产酶条件和特性的研究，旨在为PVA降解菌生产酶制剂及进一步优化PVA降解菌在PVA废水治理中的应用提供理论和应用依据。 Now the PVA-degrading bacteria and polyvinyl alcohol-degrading enzyme are the key studies on the PVA biological degradation. It has great application potential using special bacteria and enzyme to treat pollution of PVA, with some high efficient Strain and enzyme were found. The study of this paper was based on that objective condition. The stain P1, symbiotic bacteria B1+B2, stain Pa and strain Pb were studied .The conditions of enzyme production and enzyme production characteristic of stain P1, symbiotic bacteria B1+B2 were our key study, we tested and verified the growth factor which effected enzyme production of strain P1, demonstrated enzyme production characteristic of symbiotic bacteria B1+B2, optimized and obtained the optimum conditions of enzyme production; then we studied the stability of polyvinyl alcohol-degrading enzyme of strain B1+B2; last the enzyme characteristic and the optimum conditions of alcohol-degrading enzyme production of optimum combination stains were studied. The main study results are below: 1. Through the study of enzyme production factor we found that lactoflavin is the necessary factor in strain P1 enzyme production. When we used starch to be carbon energy, lactoflavin is only the necessary factor of enzyme production, but not growth factor. When we used PVA to be carbon energy, lactoflavin was not only the necessary growth factor ,but also the necessary enzyme production factor.So it was the growth factor of strain P1 2. The enzyme production conditions and enzyme production characteristic of symbiotic bacteria B1+B2 were studied. Through the orthogonal experimental design, the main conditions which effected the enzyme production and the optimum conditions of enzyme production were obtained 3. Through the study of the stability of polyvinyl alcohol-degrading enzyme, the main physical and chemical conditions which effected the enzyme stability were 4. The stain P1,symbiotic bacteria B1+B2, stain Pa and strain Pb were selected to form combination bacteria. The stain P1,symbiotic bacteria B1+B2,stain Pa were the optimum combination through duplication experiment. Then the optimum ratio was obtained through orthogonal experiment. 5. Studied the factors which effected the polyvinyl alcohol-degrading enzyme activity, then through orthogonal experiment, the main factors and condition of enzyme production which effected the combination bacteria were achieved. The result of the study was valuable for the ferment of the PVA-degrading enzyme and the optimization of the PVA-degrading performance in the PVA wastewater.

厌氧好氧一体化生物反应器处理发酵废水的中试研究

一体化反应器由于投资少、占地小、管理运行方便等优点而备受青睐。但现有的一体化反应器大都适用于处理中低浓度废水，耐受负荷普遍偏低。本课题研制出新型高效的厌氧好氧一体化生物反应器，旨在通过反应器结构优化、高效微生物载体研制、配合高效微生物菌剂技术处理中高浓度有机废水，实现高效和低耗，降低设备造价，提高反应器运行稳定性。 首先开展了菌剂对废水的适配试验。采用15种不同的微生物菌剂，以葡萄糖配水、中药提取废水、啤酒废水、氨氮配水为基质，分别测定了微生物菌剂的耗氧速率和厌氧比产甲烷速率，以其为指标比较了各菌剂对废水的适配性。根据结果选择活性高的14＃、8＃、10＃菌剂，在试验室进行了菌剂对废水的连续处理试验，取得良好的处理效果，为菌剂在厌氧好氧一体化生物反应器的小试、中试中的应用奠定了基础。 经小试研究后，又对厌氧好氧一体化生物反应器进行了处理发酵废水的中试研究。试验结果表明，反应器启动快，系统有机负荷2.72 kgCODm-3d-1时整个反应器去除率保持在84.5％～93.19％，在30多天内一次启动成功。冲击负荷试验中，系统总有机负荷最高可达到8.88 kgCODm-3d-1，系统去除率稳定在88.10％～96.88%，说明反应器处理效率高，抗冲击能力强。稳定运行期间，COD去除率可达90％以上，各项指标都能达到国家排放标准。 此外，对反应器配套系统高效菌剂、高分子复合颗粒载体进行了研究。结果显示，菌剂与反应器适配良好，各功能区形成了丰富、高活性的微生物，厌氧区颗粒污泥TS高达83.9 gL-1，VS/TS为56.9％～57.4％，比产甲烷活性为280～350 mLCH4 gvss-1d-1；好氧区固定化微生物TS高达1.921 gL-1，VS/TS为94.02～94.30％。对载体性能的研究表明，此高分子复合颗粒载体密度适中，易于流化，不易流失；粗糙多空，易于挂膜；且无生物毒害作用，稳定安全，是一种优良的生物载体。反应器各功能区对废水的降解过程分析，说明了反应器、菌剂、载体适配良好，在其协同作用下，实现了污染物的高效降解。 The integrated reactors were popular because of their characteristics such as little investment, small occupation of land, convenient of manage and running etc. But the present integrated reactors were mostly applied for treating wastewater of low concentration, the load tolerance was generally on the low side. A new type integrated anaerobic-aerobic bio-reactor was developed, which was conducted to treating organic wastewater of middle or high concentration by optimization of reactor structure, development of efficient microbe carrier and adaptation of high active microbial blends, to achieve high efficiency and low consume, reduce equipment cost, enhance running stabilization of reactor. The adaptability test of microbial blends on different wastewater was carried on firstly. Oxygen consumption rate and anaerobic specific activity of methane producing of 15 different microbial blends were measured separately taking glucose artificial wastewater, Chinese herb extracting wastewater, brewery wastewater and ammonia nitrogen artificial wastewater as substrate, by which the adaptabilities of different microbial blends to wastewater were compared. According to the results high active microbial blends 14#, 8# and 10# were selected and used in the continuous treatment of wastewater in the laboratory and had obtained good effect, which had laid a foundation for application microbial blends to small scale test and pilot test of integrated anaerobic-aerobic bio-reactor. After the small scale test, the pilot test of the integrated anaerobic-aerobic bio-reactor treating fermentation wastewater was carried on. The test results showed fast initiation of the reactor. When system organic load reached 2.72 kgCODm-3d-1the COD removal rate of the reactor was stable between 84.5%～93.19% and it initiated successfully in more than 30 days at a time. In the load shock test the maximum organic load of system could reach to 8.88 kgCODm-3d-1 and the COD removal rate could be stable between 88.10%~96.88% which indicated that the reactor was efficient for treating wastewater and had strong resistance to shock load. At stable running period the COD removal rate of the reactor was over 90% and each index of wastewater could reach to the national discharge standards. In addition, the high active microbial blends and the macromolecule compound granule carrier, the matching system of the reactor was studied. It showed that the microbial blends adapted well to the reactor and abundant and high active microbes were formed in each functional field. The TS of granule sludge in anaerobic field was as high as 83.9 gL-1, the VS/TS was 56.9%~57.4%, the specific activity of methane producing was 280~350 mLCH4 gvss-1d-1. And the TS of immobilized biological granule was as high as 1.921 gL-1, the VS/TS was 94.02%~94.30%. Study on the carrier showed that the self-made macromolecular compound granule carrier was moderate of density, easy of fluidization, unease of running off, rough and porous, easy of films fixation, no bio-toxic, stable and safe, was a kind of superior carrier. Analysis of degradation process in each functional field confirmed the reactor, microbial blends and carriers were in good adaptation and wastewater was decontaminated by their cooperation.

链霉菌S227抗耐药菌活性成分的研究

本论文以从四川峨嵋山森林土壤中分离筛选获得的一株产抗耐药性活性化合物的链霉菌S227为材料，对发酵液中活性物质的分离纯化及抗耐药性活性进行了研究。 建立了抗耐药性活性的定性、定量检测方法。建立的管蝶法活性定量检测的标准回归方程为：D=4.8229Ln(C)+3.6326 R=0.9972 ；纸片法活性定量检测的标准回归方程为：D=5.5Ln(C)-12.794 R=0.999。 根据建立的样品活性的检测方法，测定了发酵液的初始活性。实验证明活性物质的温度、pH稳定性好。 通过活性的定性、定量追踪方法，分别利用等体积的石油醚、乙酸乙酯、正丁醇在不同的pH梯度下萃取，确定了pH3条件下正丁醇能最大程度的萃取活性物质，说明活性物质极性很大。对正丁醇萃取相经过两次硅胶柱层析及薄层层析分离得到具有抗耐药菌活性的纯化样品S227-4。 经过核磁共振氢谱、碳谱数据分析初步确定S227-4为四聚糖，通过糖的水解实验初步确定S227-4由葡萄糖和半乳糖组成。 纸片法活性检测表明S227-4具有抗耐药菌活性。采用MIC测定法对该样品抗耐药活性进行研究。在证明该样品本身不具有抗菌活性的基础上，以临床分离的耐药性金黄色葡萄球菌为指示菌，考察了该样品与抗生素联合使用时对耐药菌抗生素MIC（最小抑菌浓度）值的影响，结果表明在不影响菌体生长的浓度条件下，该样品能明显降低多株耐药菌对多种抗生素的MIC值，不同程度地恢复所测试耐药菌对相应抗生素的敏感性。如S227-4与青霉素钠联用可以使S. aureus 12352的MIC降低8倍，而与红霉素联用可以使S. aureus 12334的MIC降低128倍。 The purification process and the activity of the anti bacterial drug resistance compounds produced by Streptomyces S227 isolated from the forest soil sample of the Mountain E’MEI in Sichuan Province were studied in this thesis. Quantitative and qualitative activity assay methods of the active compounds were established. The regression equation of the tube method was D=4.8229Ln(C)+3.6326, R=0.9972. The regression equation of the paper method was D=5.5Ln(C)-12.794, R=0.999. According to the established activity assay method, the incipient activity of the broth was evaluated. And it was proved that the stability of the active compounds was good. By quantitative and qualitative activity tracing method, petroleum ether, ethyl acetate and butanol were used to extract the active compounds at different pH. The result showed that butanol was the most effective agent for active component recovery at pH3. From the butanol extraction a purified sample, S227-4, was isolated by silica gel column chromatography and thin-layer chromatography . S227-4 was proved to be a tetra- saccharide by 1H-NMR and 13C-NMR. And its monosaccharides include glucose and galactose by hydrolysate analysis. The anti-drug resistant activity of S227-4 was tested in vitro by MICs assay using different drug resistant Staphylococcus aureus strains isolated clinically. The sample itself showed no anti-microbial activity in growth inhibitory experiment, but when it was used together with different antibiotics, it could remarkably decrease the MICs of different clinically isolated drug-resistant bacterial strains to these antibiotics. For example, when S227-4 was used with penicillin, the MIC of S. aureus 12352 decreased 8 times compared with that when penicillin was used alone. Meanwhile when it was used with erythromycin the MIC of S. aureus 12334 deceased 128 times compared with erythromycin alone.

微生物法生成辅酶Q10的研究

本文根据我们实验室建立的发酵产物中辅酶Q10定性定量检测方法，筛选得到一株可以代谢产生较多辅酶Q10的野生菌株放射形土壤杆菌(Agrobacterium radiobacter No.50)。 为了提高放射形土壤杆菌的辅酶Q10的产量，本实验利用液体培养研究了单因素对菌株辅酶Q10产量的影响，并用正交法确定了最佳液态发酵条件。最佳发酵培养基是：葡萄糖20g,蔗糖40g, 硫酸铵10g,玉米浆30g, 酵母膏3g，K2HPO4 3g,MgSO4.7H2O 1g，蒸馏水1000mL,pH 7.0-7.2。最佳发酵条件是：转接斜面菌种到种子培养基, 转速220r/min、温度28。C培养24h后，转入发酵培养基(250mL三角拼装液量为50mL，pH 7.0), 接种量为10％，转速220r/min、温度28。C，培养120h。在此条件下，菌体湿重约为50g/L，辅酶Q10含量约为20mg/L。 本文以放射形土壤杆菌为出发菌株进行诱变育种，以期获得辅酶Q10高产菌。根据微生物育种原理、参照辅酶Q10的代谢调控机制，以野生型放射形土壤杆菌(Agrobacterium radiobacter No.50)为出发菌株，采用紫外线和亚硝基胍复合诱变技术，依次筛选得到菌体提取物M抗性菌ARM－7、烟草提取物T抗性菌株ARMT-26、Vk3抗性菌株ARMTV-25、链霉素抗性菌株ARMTVS-32，菌株ARMTVS-32产量达到了36.8mg/L，与原始出发菌株相比，产量提高了77％。 研究了茄尼醇、对羟基苯甲酸、橘子皮提取物D、胡萝卜提取物E、烟草提取物对ARMTVS-32合成辅酶Q10的影响，结果表明这些物质对菌体合成辅酶Q10有一定促进作用，添加0.2g/L茄尼醇时，辅酶Q10含量提高了17%，达到了40.7mg/L；添加1.2g/L橘子皮提取物D时，辅酶Q10含量提高了13.8%，达到了39.6mg/L；添加0.5g/L胡萝卜提取物E时，辅酶Q10含量提高了25.3% ，达到了43.6mg/L；添加8g/L烟草提取物时，辅酶Q10含量提高了12.6%，达到了39.2mg/L。 Production of Coenzyme- Q10 (CoQ10) by fermentation is considered as a process with broad prospects.Quantitative Analysis of CoQ10 in the culture of microbe by TLC—UV spectrophotometry was developed, by using this method we got the strain Agrobacterium radiobacter,which was isolated from forest soil of southwest of China. The effect of the single factor on CoQ10-production ability of the strain was examined by liquid cultured, and its best optimum cultivation conditions were established by orthogonal method. The results showed that the optimum fermentation conditions were as following: carbon sources glucose 20g/L,sucrose 40g/L; nitrongen sources (NH4)2SO4 10g/L,maize liquid 30g/L;yeast extract 3g; K2HPO4 3g/L,MgSO4.7H2O 1g/L; initial pH was 7 and volume of medium(medium volume vs flask volume) was 50mL/500mL, incubating for 120h on a rotary shaker at 220 rpm and 28℃.Under these conditions, the biomass and CoQ10 concentration reached 50g/L and 20mg/L respectively. According to the biosynthesis mechanism of CoQ10 and breeding theory, CoQ10 over-production strains were screened by UV--NTG. mutation using Agrobacterium radiobacter No.50 as parent strain. A microbe-juice resistant mutant ARMTVS-32, which also could resist tobacco-juice, VK3 and streptomycin, was screened out from an agar plate. The CoQ10 content of ARMTVS-32 reached 36.8mg/L, which was 77% higher than the initial strain. In addition, We discussed the effects of some organic substrates on the synthesis of CoQ10 in ARMTVS-32. The results showed that solanesol, orange juice D, carrot juice E and tobacco juice could promote the CoQ10 accumulation in the cells. The CoQ10 content of ARMTVS-32 reached 40.7mg/L when added 0.2g/L solanesol,it reached 39.6mg/L when added 1.2g/L orange juice D，it reached 43.6mg/L when added 0.5g/L carrot juice E. it reached 39.2mg/L when added 8g/L tobacco juice.

三孢布拉氏霉发酵产β-胡萝卜素的研究

本论文研究了利用三孢布拉氏霉(Blakeslea trispora)发酵产β-胡萝卜素的培养条件。主要包括：发酵培养基的确定，发酵条件的优化。还考察了发酵菌丝体中β-胡萝卜素的提取方法及薄层层析等。 首先研究了培养基成分对三孢布拉氏霉发酵产β-胡萝卜素的影响。确立了玉米淀粉作为碳源，黄豆粉（热榨）作为氮源，棉籽油作为植物油的发酵培养基配方，其成分为：玉米淀粉 3％，黄豆粉（热榨） 2％，棉籽油 3％，KH2PO4 0.2%，MgSO4·7H2O 0.2%，维生素B1 0.002％，pH值6.0。 其次，通过比较不同的发酵影响因子，分别得到最适的条件：如三孢布拉氏霉正负菌接种比例为1.3：0.7，培养基pH值为7.0（灭菌后），发酵促进因子为Triton X-100。并采用正交试验法，确定其最佳发酵条件为正负菌接种比例1.3/0.7，发酵培养基pH为7.0，在培养基中添加表面活性基Triton X-100 0.08％。使该菌株产β-胡萝卜素的量达到0.73g/L，较初始发酵条件提高了3.3倍。 研究中还找到一个简便有效的对β-胡萝卜素的提取方法，选用盐酸-热处理法进行细胞破壁，并选用沸程为60～90℃的石油醚进行萃取。 用三孢布拉霉菌丝体内类胡萝卜索的石油醚提取液点样于硅胶G板，以丙酮：石油醚（5：95）为展开剂能将β-胡萝卜素与其它类胡萝卜索分离。该方法简便快速，并有一定实用价值。 The fermentative conditions of β-carotene by Blakeslea trispora have been investigated. These conditions include fermentation medium, the optimization of some fermentation factor. The extracting methods and the TLC of carotenoids were also researched. Firstly, the effects of composition of fermentation medium on the yield of β-carotene were studied. the results showed that the best fermentation medium was corn starch 3％，soybean power 2％，cottonseed oil 3％，KH2PO4 0.2%，MgSO4·7H2O 0.2%，vitamin B1 0.002％，pH value 6.0. Secondly, through compared some factors, such as different proportion of plus and minus strains, pH value, nonionic surfactants, respective best values have been obtained. The best proportion of plus and minus strains is 1.3:0.7, pH value of fermentation medium (sterilized) is 7.0, fermentation accelerant which acts as surfactants is Triton x-100. Farther on, the fermentative conditions were optimized through orthogonal experiment, the optimization showed that proportion of plus and minus strains is 1.3:0.7，pH value is 7.0, content of Triton x-100 is 0.08％. And the yield of β-carotene reached 0.73g/L, which was up to 3.3 times through the fermentation. In the extracting study, it has showed hydrochloric acid-heat treatment is a simple, convenient and effective extracting methods is which was used to destroy the cell wall, and the extracting organic solvent is petroleum ether whose boiling range is 60~90 ℃. In the TLC experiments, extracting contents in the petroleum ether were spotted in the silicagel plate, and the mixed liquor of acetone and petroleum ether (5：95) is developping agent, which can distinguish β-carotene from other carotenoids. It is a simple and quick technique.

生物催化的Baeyer-Villiger氧化反应研究

Baeyer-Villiger氧化反应是一种很重要的化学反应，产生的许多中间体或产物可以被用来生产多种化学产品和药物。此反应具有多功能性，可以氧化多种羰基化合物，但是化学方法中的必需反应物——氧化剂在生产、储存、运输、反应的过程中都存在很多的不安全因素，反应的立体选择性也不强，而生物转化则具有底物选择性、立构选择性、化学选择性、对映选择性等一般化学反应中不具备的优点，在精细化工中占有很大的优势。在工业生物催化中有很好的应用前景。 为了研究生物催化的Baeyer-Villiger反应，我们从本实验室保藏菌种中分离筛选出一株能够以环己酮作为唯一碳源的菌株，进行初步研究并对其产物进行GC/MS定性，探讨了pH，装液量，底物浓度，培养时间，温度以及转速等条件对细菌生长的影响，并进一步研究了细菌的底物广谱性。 此菌株经鉴定属于邻单胞菌属Plesiomonas sp.)， 根据正交试验，确定了菌的最佳生长条件：底物浓度为1mL/L，底物浓度过高对菌株生长有抑制作用，转速为150 rpm ，温度为30℃ ，pH为7.0； 此菌株转化环己酮的产物通过GC/MS检测含有内酯，表明此菌株能够催化Baeyer-Villiger氧化反应；此菌株还能够以与环己酮有相似结构的环己烷，环戊酮等作为唯一碳源生长，说明此菌株底物利用范围比较广，用途比较广泛。 Baeyer-Villiger oxidation is an important chemical conversion, its products and intermediates can be used to produce a lot of medicine and fine chemicals. Its success is largely due to its versatility: a variety of carbonyl compounds can be oxidized, a large number of functional groups are tolerated, the regiochemistry is highly predictable and so on, but the oxidants that the traditional chemistry way needs have a number of problem in their production, storage, transportation and reaction, Chemistry way has not a high stereochemistry yet. However, biotransformations have many attractive characters, such as substrate-, stereo-, chemo- and enantioselectivity, so it has a great advantage in the fine chemical industry and has a bright prospect in the industrial biological catalysis. In order to study Baeyer-Villiger oxidation, we isolated a strain which can utilize cyclohexanone as sole carbon source and had a primary research on it. Its product was identified by GC/MS. Effects of pH, volume, concentration of cyclohexanone, cultivating time, temperature and rotate speed on the growth of bacteria were discussed, and the other organic substrates were also studied. The strain was identified as Plesiomonas sp.. The result of orthogonal test made it sure that the best growth condition of the strain is: rotate speed 150 rpm, temperature 30℃, pH7.0, concentration of cyclohexanone1ml/L. There is caprolactone in the product of the fermentation with cyclohexanone as substrate by GC/MS,which indicated that the strain can catalyse Baeyer-Villiger oxidation.In addition,the strain can utilize other organic substrates having the similar structure with cyclohexanone such as cyclohexane, cyclopentanone, Swertiamarin as sole carbon source.So the strain can be applied extentively.

一株掷孢酵母利用红薯淀粉酶解液积累油脂研究

本论文以红薯淀粉的双酶法水解液为碳源，从19 株红色酵母中筛选出一株油脂含量较高的菌株掷孢酵母（Sporobolomyces reseus）As.2.618。为了提高掷孢酵母（S.reseus）As.2.618 的油脂产量，考察了培养基组成对该菌生长情况及油脂积累的影响。用均匀设计法对培养基组成进行了优化，由DPS软件得出的优化结果为：还原糖103g/L、酵母粉11.5g/L、磷酸二氢钾0.3g/L、硫酸镁0.15g/L。生物量可达19.23 g/L,油脂含量为3.875 g/L。研究了添加二价离子对该菌的生长及油脂积累的影响,结果表明Zn2+对该菌生长和油脂积累都有显著促进作用。研究了发酵条件以及添加氧载体正十二烷对该菌发酵的影响，表明添加正十二烷有利用于该菌生长与油脂积累。得出最佳发酵条件是：在还原糖103g/L、酵母粉11.5g/L、磷酸二氢钾0.3g/L、硫酸镁0.15g/L。添加30mg/L 硫酸锌,接种量为5％，在24h 后添加2g/L 的碳酸钙和2%(v/v)正十二烷，pH6.0 培养温度为27℃，转速为200r/min，培养时间为7 天的条件下，该菌生物量干重可达35.05g/L,油脂含量也达11.98g/L。Lipid is one of the basic material for life-sustaining activities andimportant industrial materials. As lipid resources mainly come from the animal andthe plant, the problem of lipid lack is encountered at times. The lipid frommicroorganisms is the substitute and superior to the above lipid with a short period ofproduction and much cheaper fermentation materials such as agricultural and sidelineproducts or wastes of crop.Thus large scale production and broad application ofmicrobial lipid will be efficient not only in substitute of the animal and the plant lipidfor food and industrial field , but also inducing a new way leading to solve the energyproblem.For the purpose of exploring the characteristics of lipid production of redyeasts from sweet potato starch hydrolysates. 19 red yeasts are screened for thecapability of lipid producing and one strain Sporobolomyces reseus As.2.618 withsuperior performance is sellected.To improve the Sporobolomyces reseus As.2.618’s capability of lipidaccumulation , the components of the medium, which may influence the growth of thestrain and the lipid yield have been studied. To get the optimum mediumcomponents ,the “uniform design” was used .The DPS software gave the optimummedium component is: reducing sugar 103 g/L、yeast extract 11.5 g/L、KH2PO4 0.3g/L、MgSO4 0.15 g/L. The biomass could reach up to 19.23 g/L and lipid yield 3.87g/L with the above composition of fermentation medium.Furthermore the fermentation conditions , addition of the divalent metal ionsand the oxygen vector to increase the strain’s lipid producing capability are tested.The optimum condition is : reducing sugar 103 g/L、yeast extract 11.5 g/L、KH2PO40.3 g/L、MgSO4 0.15 g/L，Adding 30mg/L ZnSO4,and adding 2g/L CaCO3 2％(v/v)n-dodecane after 24h’s fermentation. the optimal fermentation condition were asfollow :30ml medium in the 500ml flask with initial pH 6.0,the flasks with 5%inoculation volume were at 200r/min shaking speed for 7d’s fermentation at27 .Under this kind of condition the high biom ¡æ ass which reach to 35.05 g/L could begot ,the yield of lipid also could reach to 11.98g/L.