海洋枯草芽孢杆菌3512A抗真菌脂肽的研究

芽孢杆菌 (Bacillus) 被认为是一种具有潜在的能有效抑制植物病原真菌并促进植物生长作用的有益菌种，它能产生许多种不同结构的抗菌物质，其中脂肽类抗菌物质是其中重要的一类。这些抗菌脂肽具有对动植物无害、对环境友好、不易产生交叉抗性等特点，因而在生物防治由真菌引起的病害中起着十分重要的作用。海洋中也存在着大量芽孢杆菌，并且由于海洋的特殊生存环境有可能开发出与陆生芽孢杆菌性质或功能不同的代谢产物。 本论文从108株海洋芽孢菌中筛选到一株抗真菌活性显著、脂肽产量高的海洋枯草芽孢杆菌3512A（Bacillus subtilis 3512A）。对该菌株产生的脂肽进行分离纯化，结合酸沉淀、Flash Chromatography、HPLC等现代色谱手段，从其发酵液中分离得到了 4 个纯化合物，分别是流分 4 中分离到的Comd.1 和Comd.2，以及流分 8 中分离到的ALP1 和 ALP3。流分 4 中的 Comd.1 和Comd.2 分子量分别为1036D和1050D，结合1D, 2D-NMR, MS 等结构分析后确定分子式分别为 C53H93N7O13 和 C54H95N7O13，Comd.2为Comd.1的甲基化产物，均属于surfactin 的同系物，其它组分还有待进一步测定分析。而流分 8 中得到的三个化合物 ALP1、ALP2 和ALP3，对ALP1 和ALP3进行鉴定，通过 TLC 原位酸水解实验、1H-NMR 图谱分析等推断 ALP1 和 ALP3也属于环状脂肽类化合物。对这两种脂肽化合物的氨基酸组成进行分析，发现这两种化合物的氨基酸组成相同，推测可能是一组同系物，且这两种化合物的氨基酸组成不同于目前已报道的脂肽类化合物的氨基酸组成，可能是一类新的脂肽类物质，其具体化学结构还有待于进一步分析鉴定。 考察3512A所产粗脂肽的热稳定性和pH稳定性，结果显示粗脂肽的热稳定性很好，即使在115 ℃、30 min, 活性只有部分损失；粗脂肽对酸的耐受性比较好，对碱有部分耐受性，pH10后活性降低显著。通过单因素法考查培养基组成和培养条件对3512A产脂肽能力的影响，研究结果表明，以蔗糖或葡萄糖作碳源，以硝酸铵为氮源，pH为8.0，每250 ml三角瓶装液量60 ml，以5% 的接种量28℃，180 r/min培养48 h，脂肽产量最高，达到1003 mg/L。

Nisin高产菌株选育及应用的研究

本论文以牛奶为原料分离出42株球菌，经鉴定其中15株为乳链球菌（Streptococcus lactis）。对15株乳链球菌进行Nisin效价测定后，确定SN-21为诱变的出发菌株（598.1IU/ml,NO.95培养基），经过硫酸二乙酯和紫外线的多次诱变后，选育出一株Nisin高产菌株（1514IU/ml,CM培养基），命名为S. L. 21 (Streptococcus lactis 21)。通过对S. L. 21发酵条件的选优，其Nisin效价达到1862IU/ml。采用紫外吸收光谱法确定S. L. 21发酵产物中的抑菌物质为Nisin。在Nisin高产菌株选育的同时，开创了Nisin应用于蕨菜罐头的加工贮藏研究。通过在蕨菜罐头中添加0.1g/kg的Nisin，降低了蕨菜罐头的杀菌温度（100 ℃）和杀菌时间（15min），保证了蕨菜的品质，提高了蕨菜罐头的贮藏安全性，这为低酸易软烂野生蔬菜食品的加工贮藏提供了一条重要途径。100 ℃，15min杀菌条件下，在加工的蕨罐头中，未添加Nisin的处理和添加苯甲酸钾（1.0g/kg）的处理，均出现胀罐，爆罐现象。经分析确定蕨菜罐头胀罐原因是由污染的细菌产气引起的。通过分离污染菌优势类群得到8株芽孢杆菌，经系统的细菌学鉴定，4~#菌株为巨大芽孢杆菌（Bacillus megaterium），8~#菌株为短小芽孢杆菌（Bacillus pumilus），其余为枯草芽孢杆菌（Bacillus subtilis）。经进一步产气试验证明5~#菌株产气快，产气量大，是引起蕨菜罐头胀罐的污染优势种，经研究发现蕨菜罐头污染菌优势种是一种枯草芽孢杆菌的新变种，命名为枯草芽孢杆菌嗜热耐盐新变种（Bacillus sutilis n. var. sp.），这株新变种具有耐热（90 ℃），耐盐（10%NaCl），产生的特点。对蕨菜罐头污染菌优势种（5~#）进行防腐剂抑菌试验发现其对Nisin敏感（MIC 500ppm），而对苯甲酸钠（MIC 1.5%）和山梨酸钾（MIC 3%）不敏感，抑菌率试验进一步证明，在允许应用范围内，只有Nisin对蕨菜罐头防腐有良好的效果，而苯甲酸钠和山梨酸钾抑菌效果均不理想。在Nisin对污染菌优势种（5~#）的溶菌作用试验中，通过电镜可以观察到，Nisin的作用首先是破坏细胞壁，细胞膜，造成细胞内物质外流，严重溶菌结果，导致污染细菌死亡。以上关于Nisin应用于蕨菜罐头防腐，蕨菜罐头污染菌优势类群的分析，污染菌优势种的研究的试验结果均为国内外首次报导。总之，通过在蕨菜罐头中添加Nisin来抑制污染菌优势种的生长敏殖，进而达到降低杀菌强度的目的，对于保证蕨菜固有的品质及今后蕨菜等野生资源的开发具有重要的意义。

地衣芽孢杆菌GXN151纤维素酶基因的克隆与表达

从广西大学农场陈旧稻草堆、甘蔗渣堆、龙胜温泉等地采集不同的土样和水样，从中共分离到10株能降解结晶纤维素的细菌、放线菌和真菌，对它们的165RNA或185 rRNA基因序列进行了分析，其中从稻草堆中分离到的好氧细菌GXN 151具有耐中温、生长迅速、能降解天然纤维素的特点。运用生理生化和电镜观察进一步将其鉴定为地衣芽抱杆菌。用pUC18和pBluescript KS＋作载体，分别以CoR工和品u3AI部分酶切的GXN 151的总DNA作目的片段，在大肠杆菌中构建了地衣芽抱杆菌GXN151的2个基因文库。运用含狡甲基纤维素的平板筛选法，从GXN151的基因文库中共筛选到14个表达梭甲基纤维素酶（CMCase）活性的克隆，采用酶切分析、亚克隆、Southern杂交、DNA测序分析将这些克隆划分为3类不重叠克隆群。pGxNLI、pGXNLZ、pGxNL7、pGxNP12和pGxNPI～pGXNP6共10个克隆归为一类重叠克隆，测序分析了PGXNLZ的序列，其长度为3672bP，其上含有一个完整的长1626 bp的ORF（GenBonk索引号为AY291583），可编码一个含542个氨基酸的内切葡聚糖酶Ce15A，其预计分子量为59，625D娜Ce15A含有家族5糖基水解酶催化功能域和家族3碳水化合物结合组件（CBM3）。PCR 扩增了ceJSA的编码框并将其克隆到大肠杆菌表达载体pET-30a（＋）上，酶谱分析表明该基因在大肠杆菌JM109（DE3）和BL21（DE3） pLysS中均表达出具梭甲基纤维素酶活性的蛋白质产物。克隆pGxNLg测序共得5818bp，pGxNLg序列中含有一个完整的内切葡聚糖酶基因cel12A（GenBaok索引号为AY291066）和一个外切-Q-葡萄糖营酶基因amyA，cel12A长783 bp，可编码含261个氨基酸的蛋白质，预计分子量为29，035 Da，含有一个家族12糖基水解酶催化功能域。amyA为1680bP，推断其编码含560个氨基酸的蛋白质，预计分子量为65，121 Da。PCR扩增了cel12A基因的含催化功能域编码区的DNA序列并连接到表达载体pET30a（＋）上得表达质粒pGxN12A，pGXN 12A在大肠杆菌JM1O9（DE3）和BL21（DE3）pLysS中均J高效表达，并对表达条件进行了研究。克隆pGXNLS、pGXNPS和pGXNpn为一类重叠克隆，测序表明pGxNPll克隆的序列共为3406bP，它包括了一个完整的内切葡聚糖酶基因ce19A和一个不完整的纤维二糖水解酶基因ce148A，ce19A基因由1899bP组成，可编码一个含633个氨基酸的蛋白质，预计分子量为71，240Da。ce19A基因的产物Ce19A含有一个家族9糖基水解酶催化功能域和一个家族3碳水化合物结合组件（CBM3）。Ce148A属于糖基水解酶第4S家族，DNA杂交表明cel48A基因的未被克隆的下游序列位于一个约10kb的SaLI片段或4kb的EcoRI片段上。

长白山阔叶红松林土壤氮转化微生物作用的研究

自长白山北坡自然保护区采集阔叶红松林 A_0 和 A_1 层土壤。进行了微生物数量、土壤酶活性、微生物生物量、土壤速效氮及土壤中群体微生物氮转化测定；分离并鉴定了芽孢杆菌36株、产荧光假单胞菌34株，并对各优势菌株进行了氮转化活性的测定。A_0 层土壤在微生物数量、土壤酶活性、微生物生物量、土壤速效氮 (NH_4~+-N) 等方面明显高于A_1层。氨化作用、硝化作用速率也得到同样的结果；硝酸盐还原、固氮作用及同化作用速率两次采样测定的结果不同。而土壤速效氮 (NO_3~－N)是 A_1 高于 A_0层。芽孢杆菌的优势种是 B. megaterium 和B. cereus，产荧光假单胞菌的优势种是 P. fluorescens－F。所有分离到的芽孢杆菌及产荧光假单胞菌均能活跃地进行氨化作用。所有芽孢杆菌都能进行反硝化作用。而能进行反硝化作用的 P. fluorescens－F及 P. fluorescens－C其作用能力均高于 Bacillus S的20倍以上，同化作用是这两类菌的共同特征。不同种之间以及同种异株间进行氮转化速率的比较。有的差异很大。讨论了这两类菌的数量分布与土壤氮转化速率的关系。建立了反映土壤内部氮转化的模型。

Aspergillus sp136抗细菌耐药活性成分研究

基于广谱抗细菌耐药性这一思路，本研究中心建立了一套抗细菌耐药性化合物的筛选方法。由此从3000多种西南地区特殊生境的微生物和植物样品提取物中筛选获得17个抗细菌耐药性活性样品。对其中一株来自峨嵋山土样的微生物（Aspergillus sp136）进行了深入研究。通过TLC自显影等方法从其发酵产物中追踪分离得到抗耐药有效成分，并鉴定为烟曲霉酸。 采用多种方法对烟曲霉酸的体外抗细菌耐药活性进行评价。在平板扩散法中，烟曲霉酸表现出对青霉素（β-内酰氨抗生素）的协同抗耐药能力，其活性大约3倍于克拉维酸。在MIC的测试实验中，烟曲霉酸表现出对青霉素（β-内酰氨抗生素）以及非β-内酰氨抗生素如红霉素、四环素、氯霉素、链霉素、卡那霉素、庆大霉素的抗耐药能力。在棋盘格杀菌以及时间致死曲线的研究中，烟曲霉酸也表现出对青霉素、红霉素、四环素的协同抗细菌耐药活性。 在广泛的活性筛选中发现烟曲霉酸对LDLR基因具有上调活性，表明烟曲霉酸可能具有降血脂的活性。 在研究中发现，同空白对照相比，烟曲霉酸使耐药菌（Bacillus cereus NCPF63509）细胞外β-内酰胺酶酶活大幅度下降，而细胞内β-内酰胺酶酶活仅略有上升，这表明烟曲霉酸对β-内酰胺酶分泌过程具有抑制作用。 综述了β-内酰胺酶的研究进展。 A two-step agar diffusion method was established to screen wide spectrum synergistic antibacterial agents. By using this method, 17 active samples against antibiotic resistance were discovered from more than 3000 plants and microbes, which were collected from southwest china. One isolate Aspergillus sp136 collected from E-mei mountain area was selected for further studies. From the metabolites of this strain, a synergistic antibacterial compound was isolated by bioautographic TLC assay-guided fractionation and identified as helvolic acid. The synergistic effect of helvolic acid was confirmed by several methods in vitro. The synergistic effect of helvolic acid with penicillin (β-lactam antibiotics) was about 3 times as that of clavulanic acid with penicillin in agar diffusion assay. In MIC studies, helvolic acid exhibited synergistic effects with β-lactam antibiotics such as penicillin and non β-lactam antibiotics such as erythromycin, tetracycline, kanamycin, streptomycin and gentamycin. In checkerboard and time-kill studies, helvolic acid also exhibited synergistic effects with penicillin, erythromycin and tetracycline. In general screen of bioactivities, helvolic acid upregulate LDLR gene, which was indirectly determined by the activity of fluorescent enzyme. Therefore, helvolic acid might have the ability to lower lipid in blood. Compared with blank control, the extracellular β-lactamase activity decrease significantly and the intracellular β-lactamase activity increase slightly in Bacillus cereus NCPF63509 in the presence of helvolic acid, indicating that the secretion of β-lactamase was inhibited by helvolic acid. The research of β-lactamase was reviewed.

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.

捷安肽素对采后柑桔青绿霉菌抑制效果及作用机理研究

捷安肽素是一种由枯草芽孢杆菌（Bacillus subtilis）ZK 产生的抗真菌多肽。本文以柑桔青霉菌（Penicillium italicum）和绿霉菌（Penicillium digitaum）为供试真菌，研究了捷安肽素的抑菌性能及作用机理，为捷安肽素开发为有效的生物杀菌剂提供理论依据。全文共分两部分：第一部分：捷安肽素对柑桔青霉菌和绿霉菌抑制效果研究。采用琼脂扩散法测定捷安肽素对柑桔青霉菌和绿霉菌的抑菌活性。53.9 µg/mL 捷安肽素对绿霉菌和青霉菌的抑菌圈直径分别为26.7mm 和24.1mm。结果表明捷安肽素能够抑制柑桔青绿霉菌的生长，柑桔绿霉菌比青霉菌对捷安肽素敏感。在柑桔果实上，研究了不同浓度、不同接入时间的捷安肽素对柑桔青霉病和绿霉病的防治效果，并与常用化学杀菌剂抑霉唑、咪鲜胺、甲基硫菌灵和多菌灵作比较。53.9 µg/mL捷安肽素处理柑桔果实，柑桔青霉病和绿霉病发病率分别为5.0 %和5.3 %,比对照低95.0 %和94.7 %；柑桔青霉病和绿霉病的病情指数分别为1.87 和2.18，比对照低73.73 和97.82。结果表明，捷安肽素能够有效地防治柑桔青绿霉病。与对照相比，捷安肽素先于或后于柑桔青绿霉菌接入时，对柑桔青绿霉菌均有抑制作用，但抑制效果随接入间隔时间的增长而降低。第二部分：捷安肽素对绿霉菌作用机理研究。首先在光学显微镜和透射电镜下观察捷安肽素处理后绿霉菌菌丝表面形态结构与菌丝体内超微结构的变化。形态观察发现，捷安肽素处理24h以内，绿霉菌菌丝结构无变化。捷安肽素作用36h后，绿霉菌菌丝不规则缢缩和膨大。48h后，在绿霉菌菌丝顶端、中部、末端的多处细胞均可发生畸形的球状结构，这种畸变结构随处理的延长而增加，致使细胞成为捻珠状。处理72 h后，畸变球形细胞开始断裂离解。处理96h后，镜下几乎无完整菌丝，成单个的球状细胞，部分细胞出现破裂。而对照菌丝表面光滑，结构完整。通过透射电镜观察发现，与对照相比，捷安肽素处理后，绿霉菌细胞壁、细胞膜轮廓模糊不清，细胞质外泄。推测捷安肽素能够使绿霉菌细胞膜通透性发生改变。进一步实验利用紫外-可见分光光度计检测捷安肽素作用后绿霉菌胞外液紫外吸光度的变化，表明捷安肽素作用于绿霉菌菌丝后，细胞内蛋白质、核酸缓慢泄漏。通过Atomscan Advantage单道扫描等离子体发射光谱仪(ICP)测定捷安肽素作用后菌丝体内K+浓度的改变，结果表明捷安肽素作用于柑桔绿霉菌1h内，菌丝体内K+含量迅速下降，为对照绿霉菌K+含量的37.53 %，1 h后菌丝体内K+含量变化趋于平缓。K+的迅速泄漏，以及蛋白质、核酸的泄漏表明捷安肽素通过迅速改变绿霉菌细胞膜通透性，使绿霉菌菌丝生长受到抑制。Jiean-peptide produced by Bacillus subtilis ZK has broad-spectrumresistance to plant pathogens. In this study, we investigated the antifungal propertyand the possible antifungal mechanism of jiean-peptide against two commonphytopathogenic fungi of citrus fruits: blue molds (P. italicum) and green molds (P.digitatum).The paper involved two parts:Part 1 is the study of the antifungal property of jiean-peptide against blue moldsand green molds of citrus fruits. The in vitro inhibition effect of jiean-peptide againstblue molds and green molds was detected by agar diffusion method. The diameters ofinhibition zones of green molds and blue molds are 26.7mm and 24.1mm respectivelyby treating with 53.9 µg/mL jiean-peptide. It shows that jiean-peptide effectivelyinhibits the both phytopathogenic fungi, and it is more effective for inhibiting greenmolds than blue molds. The effectiveness of jiean-peptde to inhibit green molds andblue molds in vivo was investigated compared with four conventional fungicides thatare imazalil, prochloraz, carbendazin and methylthiophanate. The result is that the incidences of the blue mold disease and green mold disease are 5.0 % and 5.3 %, thedisease severities are 1.87 and 2.18 respectively when citrus are inoculated with 53.9µg/ml jiean-peptide. The decay incidences and disease severities were significantlyreduced by treating with jiean-peptide compared with the control. The results indicateJiean-peptide is effective for controlling blue molds and green molds on citrus. Theoptimized inoculation time was also investigated. When inoculated with jiean-peptideat 0 h, 6 h, 12 h, 24 h and 48 h before or after pathogens’ inoculation, Jiean-peptidecan suppress the occurrence of blue molds and green molds compared with the control, but the effect of later inoculation decreases compared with the inoculation at the sametime.In Part 2, we investigated the possible antifungal mechanism against greenmolds of citrus. At first, we observed the exterior morphological changes andultrastructural changes of blue molds under light microscopy (LM) and transmissionelectron microscopy (TEM). Compared with untreated control cells which aregenerally uniform in shape, the appearances of treated hyphae change obviously. Itshows that some cells of hyphae irregularly shrink or enlarge when cultured for 36h.When the treating time of jiean-peptide increases, the aberrance of the hyphaebecomes more obvious, and hyphae exhibit the moniliform appearances. Finally, thereis no intact hypha leaved except only single cells, and some of which appear fractured.By transmission electron microscopy (TEM) observation, we find that the outline ofthe cell wall and the cell membrane of hyphae are blurry, and the cytoplasma oozesout. The observation result under LM and TEM suggests that jiean-peptide mightchange the permeability of the cell membrane. So we conducted further experiment todetect the change of permeability when the cells of blue molds were treated withjiean-peptide. And the effect of jiean-peptide on non-growing cells of blue molds wastested. By the spectrophotometer measurement, we found that compounds with lightabsorption at 260 nm and 280 nm were released and amounts increased within 12 hcompared with the control. Moreover, by the ICP measurement, the leakage of K+occurred immediately in the presence of jiean-peptide within 1 h, but with nearly nofurther change after 1 h. All these results indicate that jiean-peptide could change themembrane permeability of blue molds immediately and result in leaking nucleotides,proteins and K+ from cells.

功能菌剂的混合发酵研究

随着化工行业的发展，大量有毒有害难降解有机物随工业废水的排放进入环境，这些物质能够在环境中长期存在、积累和扩散，通过食物链对动植物的生存及人类的健康造成不良影响。本文以苯酚、对氯硝基苯、氯苯和十六烷为模拟污染物，以前期研制的功能菌剂为对象，经过紫外线线诱变筛选出优于出发菌株的功能菌，对诱变后功能菌的理化性能进行了研究，对菌种进行了鉴定，在此基础上，就其相互之间的微生态关系进行研究，为混合发酵提供理论基础，并就其最佳发酵条件及发酵参数进行了研究，最后对发酵产品的性能进行了检测。目前，国内外有关功能菌剂的研究还存在多方面的不足，主要包括：①由于多菌种混合发酵过程较为复杂，各菌之间存在复杂的相互作用，影响因素较多，关于菌种之间的相互关系研究得很少，环境功能菌剂的发酵方法大多采用单独发酵后混合的方式。单独发酵对原材料、设备和能源的利用率较低，对于多菌种制剂发酵，在设备、能源和原材料的方面造成的浪费更大，将会大幅增加菌剂的生产成本，影响多菌种功能菌剂的发展；②功能菌剂生产过程的质量控制方面研究得较少；③功能菌剂产品的稳定性、抗冲击性能研究得较少，对环境微生物制剂的研究主要集中在菌种选育和培养条件优化方面。 通过本论文研究，得到以下主要结论。 （1）在紫外线诱变处理中，用紫外线对发生一定程度退化的出发菌株进行诱变处理后，六株具有高效降解性能的菌株被筛选出来，诱变筛选出的菌株形态和ERIC-PCR指纹图谱与出发菌株相比发生了明显改变；而且诱变后的菌株对目标难降解底物的降解能力均得到改善，其中，FPN、FCB、F14、FEm对目标底物的降解率提高了20%以上；诱变后菌株经过7次连续传代接种后，对目标难降解底物的降解率无显著变化，具有一定的遗传稳定性。并对诱变后的功能菌进行了初步的鉴定，这6株菌都分别是芽孢杆菌。 （2）对诱变后的功能菌相互之间的微生态关系进行了研究，通过抑菌实验、生长量以及基质消耗量的比较，确定它们之间的生长关系是无害共栖关系，可以进行混合发酵。 （3）对该功能菌剂进行发酵培养条件研究，结果表明发酵培养基的最佳成分（g/L）：葡萄糖 31.0g/L、玉米粉10.0g/L、磷酸氢二钾1.0g/L、硫酸铵1.1g/L、硫酸镁0.55g/L。通过研究不同的培养条件对菌体生长和降解性能的影响，确定了最佳培养条件：培养基初始pH7.5；最适温度32℃；培养基装液量125mL（250 mL三角瓶），以及培养时间对降解性能的影响，培养20 h的产物对降解最为有利。通过研究添加不同目标污染物对菌体生长和降解性能的影响，确定了添加目标污染物的最佳量以及最佳时间：苯酚投加量：1.125 g/L，对氯硝基苯投加量：0.1 g/L；最佳投加时间为发酵培养开始后4 h。 （4）以摇瓶分批发酵最优条件为基础，对FPN、F10、FCB、FNa、F14 和 FEm进行了摇瓶分批发酵试验。以摇瓶分批发酵试验数据为依据，对功能菌剂分批发酵动力学进行了研究，建立了菌体生长和基质消耗的动力学模型，拟合模型能较好的反映功能菌剂分批发酵过程。 （5）功能菌剂和活性污泥协同作用，可以提高系统的生物降解能力，功能菌剂投加量为2%，新鲜活性污泥3500 mg/L，降解24 h条件下，功能菌剂和活性污泥的协同作用对COD的去除率和对照组相比，最多的提高了36.8%。功能菌剂和活性污泥协同作用以及活性污泥的单独作用，其生物降解过程均符合一级反应动力学过程，功能菌剂和活性污泥协同作用的生物降解动力学方程为：，相关系数97%。采用SBR运行方式，引入功能菌剂的SBR系统明显能够改善和提高生物降解的效率。与仅有活性污泥的系统相比，系统对COD的平均去除率可以提高27.1%，同时，系统的耐负荷冲击以及耐毒害冲击的性能比仅有活性污泥的SBR系统强，特别是负荷冲击对引入功能菌剂的SBR系统影响很小。仅有活性污泥的SBR系统经过负荷冲击和毒害冲击之后，不能恢复到冲击之前的水平，而且系统有效作用时间的周期比引入功能菌剂的SBR系统相比大大缩短，而引入功能菌剂的SBR系统处理效果较为稳定，恢复能力很强。 Along with the development of industries, many recalcitrant organic chemicals have been discharged into natural environments together with wastewaters and can exist in waters, soil and sediments for a long time without degradation. These haz-ardous substances, their byporducts and metabolizabilities can be highly toxic, mu-tagenic and carcinogenic, thereby threatening animals, plants and human health through food chain. Consequently the removal of these compounds is of significant interest in the area of wastewater treatment. In this dissertation, the phenol, hydro-quinone, chlorobenzene and hexadecane treated as the model pollutants, the func-tional microorganism agent was used as the starting strains, they treated with ultra-violet light, and then the mutant strains with high degradation ability were screened out and identified primarily, the relationship between these stains were studied, the medium composition and fermentation conditions were optimized, the degradation ability of the fermented production was tested. The literature survey indicates that the study of the microorganism agent is far from complete and more information is re-quired on following problems. 1, Because of the complexity of relationship in mixed fermentation and the complicated factors, the study is hardly to process.2, There is a lack of information on the quality control of the producing process .3, And there is a lack of information on the stability about the microorganism agent. In this dissertation, the main results of the present study could be summarized as follows: (1)The degenerate starting strains were treated with the ultraviolet light, and six mutant strains with high biodegradation ability were screened out by using the me-dium with selective pressure of model pollutants. The mutant strains had great changes in colonialmorphology and ERIC-PCR fingerprinting. And the mutant strains got obvious advantages over the starting strains in degradation ability and over 20% improvement of removal rates was achieved for FPN、FCB、F14 and FEm. The de-gradation ability of the mutant strains was stable after seven generations. After that, the mutant strains were primarily identified as bacillus respectively. (2) The relationship between these mutant strains was studied. By the compari-son of antibiosis effect, biomass and consumption of substrate, the relationships were neutralism and they could be mixed fermented. (3) The optimized cultivation conditions were as follows: glucose 31.0 g/L, corn power 10 g/L, K2HPO4 1.0 g/L, (NH4)2SO4 1.1 g/L, MgSO4 0.55 g/L, initial pH7.5, temperature 32℃, working volume 125 mL/250 mL, and cultivation time 20h (con-sidering the time effect on degradation ability), adding pollutants phenol (1.125 g/L) and hydroquinone (0.1 g/L) into the broth at 4 h after cultivation. (4) Based on the above optimum condition, the batch fermentation was per-formed with strains FPN, F10, FCB, FNa, F14 and FEm in shake flask. The batch fermentation kinetics was studied based on the experimental data. Two kinetic models were constructed which could reflect the regularity of growth and substrate consump-tion in the process of batch fermentation. (5) The co-operation of functional microorganism agent and activated sludge could raise biodegradation of system by adding some microorganism agent and 3500 mg/L fresh activated sludge. Bioaugumentation by the addition of high effective deg-radation culture enhanced the treatment effect of SBR system and the COD removal rate was increased by 20%-36.8%. Its biodegradation matched first-order dynamical reaction equation, and the reaction equation was ln0.2327.391ct=−+. The micro-organism agent had the effect of optimization to activated sludge micro-ecosystem. The SBR system adding 2% microorganism agent, the average COD removal rate of that was increased by 27.1% and stronger anti-shock ability to load and toxicant were achieved (compared with SBR system just adding activated sludge). Especially the load-shock has barely effect to the SBR system adding microorganism agent. After the load and toxicant shock, the SBR system just adding activated sludge couldn’t come back to original level and the activated sludge micro-ecosystem was frustrated. The applying of microorganism agent increased biological activity and system’s re-sistance ability to load shock and toxicant shock.

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.

3株细菌对土壤中芘和苯并芘的降解及其动力学

研究了3株多环芳烃(PAHs)高效降解菌对土壤中芘和苯并芘(BaP)的降解动态,用Michaelis-Menton和Monod动力学模型对结果进行拟合.结果表明,3株细菌对芘和BaP的降解率有显著性差异.芽孢杆菌(Bacillus sp.SB02)42 d对芘和BaP的降解率均最高.当土壤中芘和BaP的初始浓度为50 mg/kg时,芽孢杆菌(Bacillus sp.SB02)、动胶杆菌(Zoogloea sp.SB09)、黄杆菌(Flavobacterium sp.SB10)42 d对芘的降解率分别为42.69%、32.88%、25.07%,对BaP的降解率分别为33.04%、25.39%、22.02%.3株细菌对芘和BaP的降解速率也存在显著性差异.芽孢杆菌(Bacillus sp.,SB02)最快,1周可降解20.88%芘和12.6%的BaP,动胶杆菌(Zoogloea sp.SB09)次之,黄杆菌(Flavobacterium sp.SB10)降解速率最慢.

Study on bioactivity of extracts from marine sponges in Chinese Sea

The bioactivity screening of fractions from two inter-tidal sponges collected from the north of China Yellow Sea and one sponge collected from the South Chinese Sea was reported in this study. In sponge Hymeniacidon perleve there were 9 fractions out of 15 from CHCl3 extract with anti Staphylococcus aureus activity, 9 fractions out of 19 from BuOH extract with anti Escherichia coli activity, and three fractions from CHCl3 extract which had moderate to strong activity in inhibiting Bacillus subtilis, Candida albicans, and Aspergilus niger. The fractions of Reniochalina sp. showed bioactivity against bacteria and fungi. The fractions of Acanthella acuta Schmidt showed bioactivity against S. aureus and fungi. One compound from H. perleve obtained by the bioactively directing isolation was tested for bioactivity against the human hepatoma cell line Qgy7701 (IC50 10.1 mug/ml), Burkitt's lymphoma cell line Raji (IC50 9.76 mug/ml) and chronic myelogenous leukemia K562 (IC50 1.90 mug/ml). (C) 2003 Elsevier B.V. All rights reserved.

渤海红鳍东方豚体内产毒细菌的微生态分布及B3B菌株的生物学特性

对我国渤海红鳍东方豚(Fugurubripes)体内产毒细菌的微生态分布进行了考察.结果表明,渤海红鳍东方豚体内的各部位都普遍存在着产毒细菌,从其卵巢、肝脏等组织中分离到了19株可产强毒的菌株,对其中B3B菌株进行进一步研究鉴定,生理生化试验及16SrDNA序列测定结果表明,该菌属于芽孢杆菌属(Bacillus).同时,对其发酵产物进行分离精制后,通过小鼠试验、薄层层析试验及质谱分析,初步认定发酵产物中含有河豚毒素(Tetrodotoxin,TTX).

柴油降解菌的筛选及其降解特性

以市售0#柴油为惟一碳源对菌种进行筛选,得到2株高效降解柴油菌种Y1和Y2.经形态及生理生化特征分析,初步鉴定Y1为芽孢杆菌属(Bacillus),Y2为黄杆菌属(Flavobacterium).并对其生长曲线进行测定,为菌种的固定化提供了一定依据,以进一步对两株菌降解特性进行研究.结果表明:初始油质量浓度为150 mg/L、菌种Y1和Y2接种量为10%的条件下,经过48 h批培养实验,Y1和Y2的除油率分别为79.25%和77.23%,并随初始油质量浓度的增加而降低;同时观察到pH值显著影响两株菌的生理性质.

产纤溶酶海洋微生物B5815菌株的筛选及鉴定

采用酪素平板、纤维蛋白平板的初筛和摇瓶复筛的方法从205株海洋微生物中筛选得到4株纤溶酶活性较强的菌株,其中菌株B5815产纤溶酶活性最高,平均达258 IU/mL。通过对菌株B5815的形态特征、生理生化特性的测定及16S rDNA序列分析,综合鉴定其为短小芽胞杆菌(Bacillus pumilus)。

高效石油降解菌的筛选及其降解特性

从辽河油田和大庆油田石油污染土壤中分离筛选出两株高效石油降解菌L10和D6菌株,经形态观察、生理生化反应,确定此两株菌分别为芽孢杆菌属中的枯草芽孢杆菌(Bacillus subtilis)和地衣芽孢杆菌(Bacillus licheniform is)。采用室内盆栽培养方法,研究了石油烃的浓度和性质对两菌株降解活性的影响。结果发现,土壤中石油烃的含量和处理时间均影响微生物的降解效果,在处理10d时,石油烃的去除率随着污染强度的增加而降低;随着处理时间的延长,微生物适应环境后,在石油烃含量为0.5%～2.0%时,石油烃的去除率随着浓度的增加而升高,在石油烃含量为2.0%～10.0%时,石油烃的去除率随着浓度的增加而降低;石油烃的性质影响菌株的生物活性,L10和D6两菌株对稀油的去除效果明显高于对稠油的去除效果,各组分的去除率依次为烷烃>芳烃>胶质沥青质,两菌株对不同性质的石油烃中的烷烃、芳烃和胶质沥青质的去除率不同。