微生物——铊相互作用的生物地球化学研究——以真菌（Fungus）为例

铊是一种有毒有害的重金属元素，已经引起了广泛的关注。本论文通过对黔西南铊矿区土壤和沉积物样品的菌株分离、铊高耐受性菌株的筛选、胞外吸附、富集、亚细胞水平区系分布、絮凝实验及ITS序列等实验研究分析，并结合铊的地球化学相关研究，较系统地阐述了真菌--铊的生物地球化学过程机理，得出以下结论： 1、与环境背景区相比，黔西南滥木厂铊矿区内的河流、土壤中铊的已有不同程度的积累，直接导致了当地微生物生物量在很大程度上的降低，微生物生物量与铊含量间有显著的负相关关系。研究区内的沉积物、土壤中的微生物区系结构和数量发生了明显变化，细菌、真菌及放线菌数量均出现显著降低，而且三大微生物对重金属污染的敏感性大小也不一样，即放线菌>细菌>真菌。从土壤样品中分离到的主要菌群仍为常见种属，如青霉属（Penicillium）、木霉属（Trichoderma）、拟青霉（Paecilomyces）等。 2、经过初筛菌株的铊耐受性实验，在1000 mg/L水平筛选得到九株高耐受性菌株。吸附实验表明：微生物菌株对铊的吸附效率在4.63～16.89%，且随着环境中铊浓度的上升而降低，这可能是因为铊浓度的升高加大了对微生物生长的抑制作用，所形成的菌丝体（或菌丝球）减少，表面积也相应减少，从而导致了吸附效率的下降。各种常量元素和铊的关系呈显著相关性，钙、钾和钠等常量元素也是微生物赖以维持生存的因子，可能由于微生物细胞对钙、钾的吸附方式与对铊的吸附方式类似。因此，随着铊处理浓度的上升，钙和钾的吸附量也随之减少，而钠则呈现相反的趋势。 3、富集实验表明，九株菌株对铊的富集量随着铊处理浓度上升而降低，其影响趋势与对生物量的影响趋势基本一致，最高可达到7189 mg/kg，最大富集系数为7.2。九株菌株对常量元素的富集与对铊的富集并无明显的相关性，但在考察铊处理浓度对常量元素的富集影响时发现，铊处理浓度的上升与对钙的富集量表现出较强的正相关；而对钾、钠、镁的富集影响并不明显。 4、亚细胞水平上的铊分布研究表明，铊的富集优先顺序为：细胞质>细胞壁>细胞器。亚细胞水平的区隔化作用是微生物对铊的主要耐受机制，细胞质是赋存铊的主要场所（53.83～79.45 %）。结合各亚细胞组分中常量元素与铊之间的相关性，并联系前人的研究，Tl+主要是通过细胞壁的Na+ -K+ ATPase和K+ -电位门通道进入细胞内的从而影响细胞的正常代谢的，而Ca2+的活化更有助于这一过程。 5、絮凝实验表明，培养三天后的发酵液对矿区废水中铊的去除率最高可达到70.49 %，最佳影响因子组合为：pH=8，温度为16℃，搅拌时间为4分钟。菌株的絮凝活性最高可达到57.32%，最佳影响因子组合为：pH=8，温度为14℃，搅拌时间为4分钟。 6、通过对九株铊高耐受性菌株的ITS序列分析及其在Gene Bank中的BLAST比对结果表明，五株菌株同属于木霉属（Trichoderma），两株菌株同属于青霉属（Penicillium）。这表明这两类真菌对铊的适应性较强，为以后寻找铊高耐受性菌株及其资源化利用提供了理论基础。

Rearing of Procypris rabaudi during early life-history stages

The early life-history of Chinese rock carp Procypris rabaudi was investigated during a 56-day rearing period: 318 artificially propagated P. rabaudi larvae were reared throughout metamorphosis in a small-scale recirculation system (345 L water volume, 10 x 18 L rearing tanks, 150 L storage and filter compartment with bioballs, 20-30 larvae L-1) at the Institute of Hydrobiology, Wuhan, Hubei Province, China. The newly hatched larvae had an initial total length of 8.93 +/- 0.35 mm SD (n = 10) at 3 days post-hatch and reached an average total length of 33.29 mm (+/- 1.88 mm SD, n = 10) 56 days after hatching. Length increment averaged 0.45 mm day(-1), resulting in a mean growth of 24.4 mm within the 56-day period. High mortality rates of up to 92% derived from an introduced fungus infection and subsequent treatment stress with malachite green. Our results indicate that Chinese rock carp can be raised successfully from artificially fertilized eggs. We therefore assume this species to be a candidate for commercial aquaculture.

虎杖对植物病原真菌的杀菌活性及有效成分研究

植物源杀菌剂的开发应用以及从植物中寻找杀菌活性物质作为先导化合物，是目前杀菌研究领域的热点之一。本文以蓼科植物虎杖（Polygonum cuspidatum Sieb.et Zucc.) 为材料，研究了虎杖提取物的杀菌活性和作用机理，确定了虎杖中的有效杀菌活性成分，并以此为先导化合物进行了衍生物合成与结构活性关系的研究。 不同溶剂提取物制备与杀菌活性测定结果表明，乙醇适合作为虎杖植物杀菌剂的提取溶剂，提取率高，对多种植物病原真菌具有广谱的杀菌和抑菌活性，除对黄瓜白粉病（Sphaerotheca fuliginea）表现出很好的防治效果外，对苹果腐烂病菌（Valsa mali）、玉米小斑病菌（Helminthosporium maydis）、葡萄炭疽病菌（Colletotrichum gloeosporioides）、小麦赤霉病菌（Fusarium graminearum）、油菜菌核病菌（Sclerotinia sclerotiorum）、水稻纹枯病菌（Rhizoconia solani）等也具有很好的抑制作用。虎杖回流提取物对黄瓜白粉病的杀菌作用以保护作用为主，兼具一定的治疗作用，并且具有一定的内吸活性，持效期约为4-7 d。温室试验结果表明，虎杖乙醇回流提取物10%可溶性液剂对黄瓜白粉病的EC90值为172.83 mg/L，田间小区试验表明该制剂在800-1600 mg/L的浓度下，对黄瓜白粉病的防效达到76.3-93.4%，具有较好的应用前景。 对苹果腐烂病菌的抑菌作用机理表明，虎杖乙醇提取物对该病原菌有明显的抑制作用，能够抑制蛋白质、葡萄糖等菌体细胞内物质的合成，从而使病菌代谢速度减慢，抑制其生长。虎杖提取物还能够使几丁质酶和β-1,3葡聚糖酶这两种细胞壁相关水解酶的活性升高，降解细胞壁而破坏菌体结构，使菌体自溶。 过测定虎杖乙醇回流提取物对黄瓜体内等一些防御酶和病程相关蛋白活性的影响，表明在40 mg/L和400 mg/L浓度下，虎杖乙醇提取物能够使黄瓜叶片内的过氧化物酶（POD）、多酚氧化酶(PPO)、苯丙氨酸解氨酶(PAL)、几丁质酶等不同程度的升高，从而在一定程度上提高植物对病原真菌的抗病能力。 通过生物活性跟踪测定以及pH梯度提取法确定了虎杖中的主要杀菌活性成分为蒽醌类化合物大黄素（emodin）和大黄素甲醚（physcion），结构通过了HPLC-MS和1H NMR确认，并且通过HPLC确定了虎杖乙醇回流提取物中二者的含量分别为3.28%和1.11%。 以虎杖中的有效成份之一的大黄素为原料，通过羟基的甲基化反应合成了包括已知物大黄素甲醚在内的11个大黄素衍生物，其中5个化合物为首次报道，并进行了初步结构活性关系研究。结果表明通过对大黄素3-OH位置以短直链烷基取代，其衍生物对黄瓜白粉病的活性大大提高，其中以甲基取代的衍生物大黄素甲醚的活性为母体大黄素的16.7倍，而以取代苄基修饰的衍生物的活性没有明显提高。一些目标化合物的活性明显优于三唑酮。研究中还意外发现大黄素的甲基化衍生物三甲氧基大黄素在4000 mg/L时能够明显抑制甜菜夜蛾幼虫的取食与生长发育，而大黄素和大黄素甲醚则无此作用。

乙草胺、甲胺磷及其复合对黑土真菌的生态效应

农药对土壤微生物区系结构和功能的影响以及潜在的生态风险成为人们关注的热点之一。本文以中科院海伦生态站农田黑土作为实验土壤，采用室内模拟的方法，利用传统的（CFU和ergosterol）及分子微生物生态学技术（DGGE，real-time PcR，clolle library）研究了乙草胺、甲胺磷及其复合对黑土真菌的生态效应，并得出以下结果：经8周处理，中、高浓度乙草胺（150和250mgkg-1）对土壤真菌数量、生物量和可培养真菌种群多样性具有长期抑制效应。乙草胺处理8周后可培养真菌和土壤固氮微生物n州基因的种群结构不能得到恢复，而总的真菌结构可基本恢复。甲胺磷对土壤可培养真菌数量和生物量具有促进作用，以高浓度（250mgkg~(-1)）尤为显著。高浓度甲胺磷（25omgkg~(-1)）对nifH基因多样性有长期抑制效应。甲胺磷处理8周后可培养真菌种群结构不可恢复，而总的真菌和n积基因种群结构可部分恢复。两者复合后对真菌数量，生物量，多样性及n担基因多样性的影响无论是促进还是抑制其作用强度都大于单因子。处理8周后可培养真菌、总的真菌和n州基因三者的种群结构均不可恢复。克隆测序分析发现乙草胺、甲胺磷及其复合可明显促进植物致病真菌（colletolrichum；truncatum，Rhizoctonia zeae，Fusarium oxysporum）的生长，同时使土壤中常见的青霉菌数量减少，使农药处理后具有潜在的植物病害爆发的风险。本试验结果表明，乙草胺、甲胺磷及其复合对土壤真菌数量、结构、多样性和功能基因nifH的多样性及其种群组成有不同程度的影响，甚至产生某些不可逆的长期生态效应。复合处理对土壤真菌的影响要大于两个单因子作用，表现了明显的复合生态效应。一般来说受到午扰的真菌种群结构不容易自然恢复，因此建议在施用这两种农药过程中要避免大量、频繁的单独或复合施用。

模拟酸雨对马尾松菌根影响研究

本实验表明：外生菌根真菌彩色豆马勃、劣味乳菇、丝膜菌对PH的适应范围较广，最适生长BH呈酸性。模拟酸雨对马尾松幼苗菌根的外部形态和内部结构有明显影响。在温室栽培中，模拟酸雨（PH2.0）显著抑制菌根侵染率，在田间实验中，对菌根侵染率有一定的影响。菌根PH和土壤PH值随模拟酸雨PH下降而逐渐降低，接种菌根菌可略提高菌根PH和土壤PH值。菌根真菌过氧化氢酶对培养基中PH的变化不敏感，模拟酸雨对菌根过氧化氢酶活性影响也不明显。但沙培中，模拟酸雨（PH2.0）显著抑制菌根过氧化氢酶活性。模拟酸雨（PH2.0）显著刺激菌根过氧化物酶活性，接种菌根菌可以降低菌根过氧化物酶活性。不同PH的培养基对菌体硝酸还原酶活性有明显影响，而且菌体生长速度与硝酸还原酶活性呈正相关。模拟酸雨（PH2.0）显著抑制菌根硝酸还原酶活性，而接种菌根菌明显提高根系硝酸还原酶活性。菌体酸性磷酸酶活性对培养基中PH变化不敏感，同样菌根酸性磷酸酶活性对模拟酸雨的影响也不明显，但是接种菌根菌可明显提高根系酸性磷酸酶活性。模拟酸雨对马尾松幼苗茎的高生长影响不显著。但是对幼苗茎、根系的干重和侧根总长度有显著抑制作用。轻度酸雨（PH4.5-3.0）对马尾松幼苗生长有促进作用，接种菌可提高幼苗生长。从菌根形态结构和生理活性上看，接种菌根菌可减轻模拟酸雨对马尾松幼苗根系的危害，增强对模拟酸雨的抗性。4dThe result of experiment showed that ectomycorrhizal fungi Pisolithus tinctorins. Lactarius insulsus. Cortinarius russus can be growth in broad PH rang in pure culture, the optimum growth PH is acidity. The external morphology and internal structure of ectomycorrhiza of P. massoniana are affected with simulated acid rain. In greenhouse, simulated acid rain (PH2.0) treatment caused significant decrease in the percent infection, but it's not marked in field. The PH of mycorrhizal and soil are reduced with reducing rainfall PH. These PH are slight higher for inoculation with ectomycorrhizal fungi. Catalase activity of ectomycorrhizal fungus is not sensitive to medium with different PH. Mycorrhiza catalase activiyt is not affected significantly with simulated acid rain, but it's inhibited significantly with simulated acid rain (PH2.0) in the sand culture. Peroxidase atcivity of mycorrhiza is enhanced significantly with simulated acid rain (PH2.0), but it's universally lower for inoculation with ectomycorrhizal fungus. Ectomycorrhizal fungus nitrate reductase activity is affected significantly to medium with differdnt PH, the rates of these fungi growth and nitrate reductase activity is significant correlation. Nitrate reductase activity of mycorrhiza is inhibited significantly with simulated acid rain (PH 2.0), but it's increased significantly for inocnlation with mycorrhizal fungi. Ectomycorrhizal fungas acid phosphatase activity is not affected to medium with different PH, Mycorrhiza acid phosphatase activity is not affected with simulated acid rain too, the acid phosphatase activity of roots inoculated with mycorrhizal fungas is increased significantly. The highest acidity level simulated rain reduced signhficantly root system biomass and the dry weight of stem. Iower acidity level simulated rain can stimulated the growth of P. massoniana, the growth of seedling inocnlated with mycorrhizal fungus can be increased.

五种毛壳霉属和曲霉属真菌代谢产物及细胞毒活性

毛壳霉属(Chaetomium)和曲霉属(Aspergillus)真菌产生多种具有生物活性的化合物。为系统阐明两属微生物的次生代谢物，对三种毛壳霉、两种曲霉真菌分别进行固态发酵，以色谱和波谱技术研究发酵物中的成分，分离鉴定了51个化合物，其中23个为新化合物，测试了部分化合物对肿瘤细胞的活性。 1、从螺卷毛壳霉（C. cochloides）固态发酵物中分离鉴定了11个化合物，3个新化合物为螺卷毛壳霉素A~C（1~3）。化合物1、3及dethio-tetra (methylthio) chetomin（4）对Bre-04、Lu-04和N-04细胞株生长抑制的GI50值为0.05~7.0 μg/mL。 2、从印度毛壳霉（C. indicum）固态发酵物中鉴定的三个异喹啉生物碱印度毛壳霉素A~C（12~14）代表两类骨架新颖的异喹啉生物碱。 3、从巴西毛壳霉（C. brasiliense）固态发酵物中鉴定了11个化合物，其中Mollicellins I~J（15~16）、2-Hydroxymethyl-6-methylmethyleugenin（19）为新化合物。化合物16和Mollicellin H（18）对Bre-04、Lu-04、N-04细胞株生长抑制的GI50值在2.5~8.6 μg/mL。 4、从土曲霉（A. terreus）固态发酵物中鉴定了18个化合物。5个为新化合物为Terretonin A~D（24~27）和Asterrelenin（28），24~27为二倍半萜化合物，28为吲哚生物碱。 5、从杂色曲霉(A. versicolor）固态发酵物中鉴定了16个化合物。9个新的化合物Brevianamides K~N (40~43)、Averins A~C (44~46)和Glyanphenines A~B (47~48)代表三种类型的生物碱。 6、综述了1997－2007年间新的二倍半萜的研究进展。 The fungi of the genera Chaetomium and Aspergillus produce various secondary metabolites with biological activities. In order to systematically study the secondary metabolites, the solid-state fermented rice culture of three species of Chaetomium and two of Aspergillus were chemically studied. By the means of chromatograhy and spectroscopy, 55 compounds were isolated and identified, among of them 23 were new ones. The biological activities of some compounds were investigated. 1. From the fungus C. cochliodes, three new epipolythiodioxopiperazines, chaetocochins A-C (1-3) were isolated, together with 8 known ones (4-11). Compounds 1, 3 and 4 showed growth inhibitory effects against cancer cell lines Bre-04, Lu-04 and N-04 with GI50 values from 0.05 to 7.0 μg/mL. 2. Three novel isoquinolines Chaetoindicins A-C (12-14) were isolated and identified from the fungus C. indicum. Chaetoindicin A, Chaetoindicins B-C represented two classes of novel carbon skeletons. 3. Three new compounds, Mollicellins I-J (15-16), and 2-hydroxymethyl-6-methylmethyleugenin (19), were isolated from C. brasiliense. Compound 16 and Mollicellin H (18) showed growth inhibitory effects against cancer cell lines Bre-04, Lu-04 and N-04 with GI50 values from 2.5 to 8.6 μg/mL. 4. Eighteen compounds were isolated from the fungus A. terreus. Terretonin A-D（24 - 27）and Asterrelenin（28） are new compounds belonging to sesterterpoids and indole-ralated alkaloid, respectively. 5. From the fungus A. versicolor, sixteen secondary metabolites, including nine new ones, Brevianamides K-N (40-43), Averins A-C (44-46), and Glyanphenines A-B (47-48), were isolated and identified. Brevianamides K-N (40-43), Averins A-C (44-46), and Glyanphenines A-B (47-48) represented three classes of alkaloids. 6. New sesterterpenes and their bioactivities reported from 1997 to 2007 were summarized.

白腐菌选择性降解木质素的研究

对15株白腐真菌进行了以玉米秸秆为基质的初步筛选，从中获得一株选择性系数较高的菌株Y10，并对其降解玉米秸秆的情况进行了研究。结果表明，在30天的培养过程中菌株Y10对玉米秸秆降解的选择性系数都大于1，第15天选择性系数最高为3.88。对未经降解和降解过的玉米秸秆分别作了紫外光谱和红外光谱分析，结果表明，经该菌降解后玉米秸秆的化学成分发生了很大变化，且木质素的降解程度要大于纤维素的降解程度。对菌株Y10进行了ITS-5.8S rDNA序列鉴定，初步判定其为Cerrena sp.。 为了考查不同的外源添加物对菌株Y10降解玉米秸秆的影响，在以玉米秸秆为基质的固态发酵培养基中分别添加了7种金属离子、8种碳源、6种氮源。结果显示，这7种金属离子均能促进木质素的降解，并且一定浓度的某些离子明显抑制纤维素的降解；其中添加0.036%的MnSO4·H2O和0.36%的MgSO4·7H2O对纤维素降解的抑制作用比较强，降解率分别为0.96%和1.31%，木质素的选择性系数分别达到了34.40和20.17。8种碳源中除麦芽糖外都能促进木质素的降解，除微晶纤维素外都明显促进纤维素的降解。6种氮源中酒石酸铵、硫酸铵、草酸铵和氯化铵的添加都会使该菌生长变慢，而且氮源浓度越高菌丝生长越慢。外加碳源和金属离子对半纤维素降解和选择性系数的影响不大。 同时对菌株Y10在液态培养下产木质素降解酶的条件和培养基做了优化。结果表明，在初始产酶培养基中，菌株Y10的漆酶酶活在第10d达到最高，锰过氧化物酶酶活在第11d达到最高，基本上检测不到木质素过氧化物酶。菌株Y10产漆酶的最适温度为32℃，最适PH为6.0；产锰过氧化物酶的最适温度为32℃，最适PH为6.5。菌株Y10产漆酶的最佳碳源为甘露糖，最佳氮源为酒石酸铵，最适诱导剂VA浓度为3 mmol/L，最适表面活性剂TW-80浓度为1%。 利用响应面法对其产漆酶的培养基进行优化，优化后的培养基配方为葡萄糖10.00 g/L，酒石酸铵0.50 g/L，大量元素296.50 ml/L，微量元素100.00 ml/L，NTA 1.40 g/L，VA 5.00 mmol/L，吐温-80加入量为0.10%。进行了菌株Y10产漆酶的验证实验，实测酶活为5282.56 U/L，与预测酶活5162.73 U/L接近。在优化后培养基中，菌株Y10在第14 d达到生长的最高峰，第20 d时，漆酶酶活最高，为11325.00 U/L；第16 d时，锰过氧化物酶酶活最高，为30.77 U/L。 对菌株Y10的漆酶酶学性质做了初步的研究，结果显示，酶反应的最适温度为40℃-65℃，最适PH为3.0。在40℃，PH=3.0时，漆酶催化ABTS反应的米氏方程为 。 Fifteen white-rot fungi based on corn stalk were screened. One white-rot fungus Y10 with high selectivity value was obtained. The degradation of corn stalk was initially studied. The results indicated that the selectivity value was above 1 during the 30 day-cultivation and the highest was 3.88 after 15 days. The composition of untreated and treated stalk was analyzed through ultraviolet spectroscopy and infrared spectroscopy. It was found that the composition of treated stalk was greatly altered and the degree of the degradation of lignin is greater than the cellulose. Y10 was identified as Cerrena sp. by ITS -5.8S rDNA sequence analysis. The influence of metal ions, carbon sources and nitrogen sources on corn stalk degradation by white-rot fungus was studied. While all seven metal ions could promote lignin degradation, the cellulose degradation was best inhibited at certain ion concentrations. Notably, when 0.036% MnSO4·H2O and 0.36% MgSO4·7H2O were added into the medium, the cellulose degradation was restrained to the extents that the coefficients of lignin selectivity rose to 34.40 and 20.17 respectively. It was also found that all carbon sources except maltose can promote lignin degradation. The addition of carbon sources other than microcrystalline cellulose significantly promoted cellulose degradation. The addition of the nitrogen sources, ammonium tartrate, ammonium sulfate, oxalate, ammonium chloride, resulted in remarkable inhibition to mycelium growth; the larger the concentrations of nitrogen sources are, the slower the mycelium grew. The addition of carbon sources and metal ions had less impact on the degradation of hemicellulose and selectivity value. Meanwhile, we optimized the conditions and culture medium of the lignin-degrading enzyme production of strain Y10. The results showed that in the initial culture medium, the Lac activity was highest at the 10th day, the MnP activity was highest at the 11th day and the LiP could not be detected. The optimum condition of Lac was at temperature 32 and PH =6.0 and the optimum condition of MnP was at temperature 32 and PH =6.5. The optimum carbon source for Lac was seminose, the optimum nitrogen source was ammonium tartrate, the optimum content of VA was 3 mmol/L, the optimum content of TW-80 was 1%. PB and RSM were used to optimize the culture medium of laccase by white-rot fungus Y10. The optimum culture medium was consist of glucose 10.00 g/L, ammonium tartrate 0.50 g/L, macro elements 296.50 ml/L, trace elements 100.00 ml/L, NTA 1.40 g/L, VA 5.00 mmol/L, TW-80 0.10%. Under the optimal conditions, the activity of laccase was 5282.56 U/L and the experimental value agreed with the predicted value 5162.73 U/L. The biomass was highest at the 14th day, the Lac activity was highest at the 20th day, the MnP activity was highest at the 16th day. The results of the studies on the characteristics of Lac showed that the optimum temperature for Lac activity is 40℃-65℃ ; the optimum PH for Lac activity is 3.0 and under 40℃，PH=3.0, the Michaelis-menten equation of Lac catalized ABTS oxidation was .

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.

齐墩果酸的微生物转化

齐墩果酸（OA）是一个分布广泛、含量丰富的天然三萜化合物，常以皂苷元的形式广泛存在于植物中,具有多种重要生物活性。但是OA许多活性较弱，且生物利用度低，限制了其在临床上的应用。一是OA水溶性差;二是抗癌活性仍与临床应用的抗癌药物相差比较大。 真菌在微生物转化中具有种类多、培养条件比较简单等特点，为了寻找到具有转化OA能力的菌株，采取一步发酵的方法，在18株实验室保藏真菌菌株中筛选到5株目的菌株,TLC分析显示有转化效果。 随后采用二步发酵的方法作为复筛，验证5株菌株转化能力，波谱分析结果表明5株菌株对OA确实有转化作用。 选择5株菌种中代号1F-2 2菌株作为放大实验菌株，分离转化产物，得到OA衍生物108（相对分子量414m/z）和1010（相对分子量340 m/z），分离出的产物用于活性检测。寻找到产物108的RP-HPLC分离条件，质谱得出二者相对分子质量。 为验证OA转化产物抗肿瘤活性，首次研究了OA对卵巢癌细胞株IGROV1和人乳腺癌细胞株MDA-MB-231作用，通过细胞增殖抑制实验、用MTT法检测细胞活性，结果表明齐墩果酸可降低卵巢癌细胞株IGROV1和乳腺癌细胞MDA-MB-231细胞增殖能力并呈剂量依赖性，对肿瘤细胞株的半数有效抑制浓度化IC50 分别为36.58μg/mL和38.8μg/mL (P<0．01)。OA能抑制肿瘤细胞活性，并且OA对卵巢癌细胞株IGROV1抑制活性高于乳腺癌细胞MDA-MB-231。 在此基础上，转化产物108和1010对卵巢癌细胞株IGROV1和人乳腺癌细胞株MDA-MB-231的抑制作用也进行研究，MTT实验结果表明，转化产物对两株癌细胞也有抑制活性（P<0．01）。 总之，本文工作为进一步开展齐墩果酸类化合物结构改造和抗肿瘤活性的研究奠定了基础。 Oleanolic acid (OA) is a triterpenoid widely distributed in the nature which possesses various important bioactivities. OA also serves as aglycon of many natural saponins. However, the relatively weak activities and poor bioavailability hinder its clinical use. Firstly, poor water-solubility results in worse bioavailability. Secondly, compared with clinical antitumor drug, the antitumor effect of OA has a great difference, it is worse. Many fungi have ability to transform nature products into a variety of derivatives, and transformation conditions of fungi are simple. Attempt to obtain fungi strains able to biotransform OA, we carried out the following experiments: To investigate the biotransformation 0f OA by strains supplied firstly, we used one-step fermentation method to screen the aimed strains from 18 fungus strains stored in our laboratory. On the basis of the initial screening experiments, we found 5 aimed strains. The TLC results showed that the 5 fungi strains could transform OA into other components derivatives. Then we used two-step fermentation method as secondly screening. We repeated the five strains to do the experiments, analytical data of the results proved the transformation indeed. In the followed experiments work, we chose 1F-2 2 strain as large-scale transformation fungus from the aimed fungi. We got two biotransformation products of OA by 1F-2 2, and named those derivatives 108 and 1010. We found RP-HPLC separation conditions of product 108. The two products were characterized by ESI-MS. To verify the anti-tumor activity of biotransformation products of OA, we studied the inhibition effect of oleanolic acid on the ovarian carcinomas IGROV1 and breast cancer cell line MDA-MB-231 firstly. With an assay based on a tetrazolium dye (MTT), the effects of various concentrations of oleanolic acid on ovarian carcinomas IGROV1 and breast cancer cell line MDA-MB-231 were studied. MTT method was used to measure the tumor cells viability. Compared with the control group, oleanolic acid can significantly inhibit the viability of the ovarian carcinoma cells IGROV1 and MDA-MB-231 breast cancer cell line (P<0．01), IC50 values were 36.58μg/mL or 38.8μg/mL. Oleanolic acid can inhibit the malignant tumor cells viability, and inhibitory activity of OA to ovarian carcinomas IGROV1 was higher than to breast cancer cell line MDA-MB-231. On this basis, we studied the anti-tumor activity of the two derivatives of OA [called 108 (414 m/z) and 1010(340 m/z)]. It came to the conclusion that the two derivatives also showed potent inhibitory effect on the growth of these tumor cells（P<0．01）. Therefore, the results of studies will benefit the further investigating on the relationships of structures and antitumor activities of OA.

一株丝状真菌对半纤维素利用的初步研究

本文筛选出一株能利用木糖产乙醇的丝状真菌Z7，对其利用木糖和半纤维素水解产物产乙醇的发酵条件进行了研究，并对Z7 利用玉米芯产木聚糖酶的条件进行了优化。全文分为三部分： 第一部分：目标微生物筛选、纯化及系统发育分析。以木糖为唯一碳源，采用梯度稀释和平板化线法从高温、中温酒曲中分离到16 株能利用木糖良好生长的丝状真菌；通过发酵试验复筛，获得一株能产乙醇的丝状真菌Z7；综合形态学和ITS 序列分析，初步鉴定为Aspergillus flavus。 第二部分：Z7 的乙醇发酵条件研究。以木糖为碳源，通过单因素试验确定最佳氮源和发酵温度；通过正交试验及SPSS 软件分析得到了不同N、P、K 成分对乙醇、残糖和菌体干重的影响。获得最佳的发酵条件为：（g/L）木糖50，尿素1, NH4NO3 1, K2HPO4 2 , KCl 0.5 , MgSO4.7H2O 0.5 , NaNO3 1 , pH 自然，培养温度33 ℃。以玉米芯半纤维素稀酸水解液为底物进行乙醇发酵，根据稀酸水解的单糖释放量和乙醇产量，确定115 ℃，1 h 为最佳玉米芯预处理条件；结合最佳发酵条件，添加1 g/L 的吐温20 能获得最大的乙醇浓度8.31 g/L。因此，Aspergillus flavus Z7 能利用半纤维素水解产物产乙醇，其中木糖的利用率80%以上。 第三部分：Z7 利用玉米芯产木聚糖酶条件优化。Aspergillus flavus Z7 在具有产乙醇能力的同时还具有产木聚糖酶的能力。本文通过单因素和正交试验得到最佳产酶培养基组分为：(g/L)玉米芯20，尿素2, 酵母膏2.5, K2HPO4 5，NaNO31, MgSO4.7H2O 1。单因素试验表明，用纱布代替塑料布密封摇瓶封口能显著提高产酶量；Z7 在碱性条件下具有更强的产酶性能。在最优条件下发酵，能产生最大木聚糖酶活122.23IU/mL。通过薄层分析，验证了Z7 产生的木聚糖酶具有水解木聚糖生成木糖及木寡糖的能力。 A strain of filamentous fungus which can produce ethanol by using the xylose was isolated in this research. The ethanol fermention conditions from xylose and dilute-acid hydrolyzate of the corn core were studied. The conditions of xylanase production by Z7 were also optimized. The paper involved three parts. Part1: Isolation, purification and phylogenetic analysis of the microbe. By using xylose as the single carbon source and the pla te streaking method, several filamentous fungi were isolated from the wine starter; through the fermentation test, a filamentous fungus Z7 which can produce ethanol was further recognized; furthermore, according to the morphologic observation and ITS seque nces analysis, Z7 was identified as Aspergillus flavus at the first step. Part2: Research on the condition of ethanol fermentation by Z7. By single factor experiment, the optional nitrogen resource and temperature of the fermentation were fixed; meanwhile, through the orthogonal array tests and the analysis of statistic software SPSS, the optional component of the culture medium and the fermentation condition were organized as follows: (g/L) xylose 50, urea 1, NH4NO3 1, K2HPO4 2, KCl 0.5 , MgSO4.7H2O 0.5, NaNO31, pH nature, temperature 33℃. Based on these optimal parameters, the fermentation of dilute-acid hydrolyzate of the corn core was carried on by Z7. According to the quantities of released sugar monomers and content of the ethanol, 115℃ in 1h is the best pretreatment condition; the maximal ethanol content can be obtained when 1g/L Tween 20 was added to. Therefore, the filamentous fungus Aspergillus flavus can use the hydrolysate of hemicellulose to produce ethanol, and the rate of xylose utilization was over 80%. Part3: Optimization of Z7’s xylanase producing condition from corn core. Aspergillus flavus Z7, which can utilize xylose or the hydrolysate of hemicellulose to produce ethanol, also had the ability of xylanase production. The optional component of the culture medium were fixed by the single factor experiment and the orthogonal array tests, and they were organized as follows: (g/L) corn core 20, Urea 2, Yeast extract 2.5, K2HPO4 5, NaNO31, MgSO4.7H2O 1; it was testified by the single factor experiment that sealing the shaking flasks with pledget other than plastic paper can obviously increase the xylanase activity; moreover, Z7 showed better xylanase production ability when in the alkali environment. Under the optional fermentation condition, the maximal xylanase activity 122.23IU/mL was proved. Through the analysis of thin- layer chromatography (TLC), the ability of xylanase from Z7, which can hydrolyze xylan to xylose monomer and oligomer, was vividly displayed.

泸州老窖古酿酒作坊空气微生物分布特征研究

本文主要研究了泸州老窖古酿酒作坊内外环境空气真菌和空气细菌的群落结构和分布特征。结果如下： 作坊内外环境空气微生物浓度差别显著，并随季节变换而变化，春、夏季微生物浓度较高，秋、冬季较低，空气真菌在夏季达到最高，细菌在春季最高。 古作坊内外环境检测到的真菌均为16 属，但优势菌属不同，作坊外的优势菌属为青霉属(Penicillium)、曲霉属(Aspergillus)、无孢菌(non-sporing)、枝孢霉属(Cladosporium)和链格孢属(Alternaria)；而作坊内优势菌属为曲霉属、青霉属、酵母菌(Yeast)、无孢菌，作坊内还含有较高浓度的根霉属（Rhizopus）、毛霉属（Mucor）、短梗霉属（Aureobasidiu），枝孢霉属和链格孢属等，曲霉属、酵母菌、根霉属、毛霉属为古酿酒作坊重要的酿酒真菌，青霉属、链格孢属为酿酒不利菌群。对古作坊内曲霉属进行了初步鉴定，主要是小冠曲霉（A.cristatellus）、米曲霉（A.oryzae）、黑曲霉（A.niger）和白曲霉（A.cadidus）。 空气细菌10 属21 种，作坊内外环境的优势菌属均为芽孢杆菌属（Bacillus）、微球菌属(Micrococcus)、葡萄球菌属(Staphylococcus)、假单胞菌属(Pseudomonad)，其中芽孢杆菌属在作坊内占有绝对的优势，浓度比在40℅以上，是古酿酒作坊重要的酿酒细菌，另外还检测到较高浓度的乳酸杆菌（lactobucillus），这类菌容易使酒味发涩发苦，为酿酒不利菌。 作坊内外环境空气微生物表现出明显的交流现象。作坊内，青霉属、枝孢霉属、链格孢属、葡萄球菌属等杂菌占有一定比例；而在作坊外,芽孢杆菌属、曲霉属、根霉属(Rhizopus)、酵母菌等处于相对较高水平，绿化环境较好的营沟头作坊内的短梗霉属，枝孢霉属和链格孢属等杂菌含量低于什字头和新街子作坊。 The community structure and distribution characteristic of airborne microbes was investigated in ancient brewage workshops of luzhoulaojiao. The results are as follows: The concentration of airborne microbes was different in interior and exterior environment of ancient workshops, and also varied by seasons. microbial concentration was higher in spring and summer, and lower in fall and winner. The highest levels of airborne bacteria was in spring, but the fungal’s in summer. The identified genus of fungi were 16 in interior and exterior environment of the ancient workshops. But the dominant genus were different , The advantage genus in the interior were Aspergillus, Yeasts, Penicillum and Nonsporing and in the exterior were Penicillum, Nonsporing, Cladosporium, Aspergillus and Aureobasidiu. Rhizopus ,mucor, Aureobasidiu, Cladosporium, Alternaria and all also were at a higher level. Among these, Aspergillus, Yeasts, Rhizopus ,mucor are important vintage flora . Penicillum, Alternaria do harm to vintage. Aspergillus of ancient workshops was identified , the preponderant aspergillus species were A.cristatellus, A.oryzae, A.niger and A.cadidus in ancient brewage workshops. 10 genus 21 species bacteria were identified, the advantage genuses among the interior and exterior of the three workshops were bacillus, microccus, Staphylococcus Pseudomonas. Bacillus, which account for beyond 40℅ of the total bacteria concentration in all sampling pots, was the most dominant genus. Lactobacillus was identified at a high level in ancient workshops, it makes spirit taste bitter and astringent. So it is not a kind of good bacterium for vintage. The fungus in the interior and exterior atmosphere characterized intercommunion phenomenon. Obviously, the concentration of profitless fungus such as Penicillum, Cladosporium, Alternaria appeared in the interior, and the fungus such as Bacillus, Aspergillus, Rhizopus and Yeasts in the exterior were at a relatively high level. the harmfull fungus in yinggoutou workshops such as Aureobasidiu, Cladosporium, Alternaria and all were lower than shenzitou and xinjiezi workshops.

固定化微生物降解土壤中菲和芘的研究

Immobilized with PVA,sodium alginate and activated carbon,both Zoogloea sp. and Fusarium sp.strains could degrade phenanthrene and pyrene efficiently.The optimal carrier was made of 100ρ·g -1 L PVA,5 sodium alginateρ·g -1 L and 50 activated carbon ρ·g -1 L.The degradation rates of phenanthrene and pyrene in 10 days were 87.48% and 75.34% by the immobilized bacterium,37.04% and 20.85% higher than those by the free bacterium,and the rates in 15 days were 84.36% and 74.87% by the immobilized fungus,5.35% and 11.23% higher than those by the free fungus.

Antifungal Activity of Quaternized Carboxymethyl Chitosan

Quaternized carboxymethyl chitosan (QCMC) were synthesized and their antifungal activities against Alternaria Solani (A. Solani) and Physalospora piricola Nose (P. piricola Nose) were investigated. The results indicated that the quaternized carboxymethyl chitosan derivatives had better inhibitory effects than CMC, and the antifungal activities should be affected by the cation in these compounds.

黄土塬区果园套种对土壤微生物及酶活性的影响

研究了黄土塬区塬面不同套种作物的幼树苹果园土壤微生物的分布和酶活性。结果表明，果园土壤中微生物的分布在果树行与套种行中是不均匀的，细菌和真菌多为果树行多于套种行；土壤的过氧化氢酶，蔗糖酶，脲酶以及中性磷酸酶活性在套种小麦的果园中以套作行高于果树行，而套种烤烟则使脲酶和中性磷酸酶活性有所降低。套种豆类有利于土壤肥力的均衡发展。

牛蒡根际土壤致害菌Fusariumsolani分离鉴定

目的:探索牛蒡根际镰刀菌与牛蒡之间的相互关系。方法:从全国30个地区采集牛蒡根际土壤样品,进行了根际土壤真菌数量和群落的生态学研究,测定镰刀菌发酵液对牛蒡幼苗和牛蒡子萌发的影响,并对其中毒性最强的两株镰刀菌F130和F131进行了形态学和分子生物学的鉴定。结果:镰刀菌为牛蒡根际的最优势类群,贡献率为34.297%,其次为木霉,贡献率为22.519%;绝大多数镰刀菌对牛蒡有明显的毒性作用,其中F130和F131被鉴定为Fusarium solani。结论:Fusarium solani是牛蒡根际土壤中的致害菌。