尼泊尔水东哥、水麻、木姜冬青和青荚叶的化学成分研究

活性筛选中发现尼泊尔水东哥 (Saurauia napaulensis DC.) 树皮95%乙醇提取物具有α-淀粉酶抑制活性、水麻(Debregeasia orientalis) 枝叶95%乙醇提取物显示血管紧张素转化酶(ACE)抑制活性、青荚叶(Helwingia japonica (Thunb.) Dieter.) 95%乙醇提取物的中小极性部分显示蛋白酪氨酸磷酸酯酶(PTP)1B抑制活性。为全面了解它们的成分及相关活性成份，主要运用硅胶柱层析方法从这三个植物分离得到39个化合物，通过波谱分析或与已知品对照的方法对其进行了鉴定。对木姜冬青(Ilex litseaefolia Hu et Tang)的成分做了进一步的研究，取得了如下结果。 1. 从尼泊尔水东哥树皮的95%乙醇提取物分离并鉴定12个化合物： auranamide、aurantiamide benzoate、齐墩果酸、β-谷甾醇、β-胡萝卜甙、乌苏酸、2α,3α-二羟基-12-烯-28-乌苏酸、2α,3β,24-三羟基-12-烯-28-乌苏酸、(2S,3S,4R,10E)-2-[(2'R)-2' -hydroxytetracosanoylamino] -10-octadecene -1,3,4-triol、 2α,3α,24-三羟基-12-烯-28-齐墩果酸、2α,3β-二羟基-12-烯-28-乌苏酸和2α,3α,24-三羟基-12-烯-28-乌苏酸。 2. 从水麻枝叶的95%乙醇提取物分离并鉴定了18个化合物：棕榈酸、二十烷酸、二十烷酸甲酯、β-谷甾醇、Monogynol A、桦木酸、Hederagenin、β-胡萝卜甙、18αH-19(29)-烯-3-酮-乌苏烷、3，4-开环-20(30)-烯-乌苏烷-3-酸、Pomolic acid，表儿茶素、儿茶素、槲皮素、槲皮素-3-O-β-D-吡喃葡萄糖苷、紫丁香苷、紫丁香酚苷和山萘酚-3-O-芸香糖。儿茶素、槲皮素和槲皮素-3-O-β-D-吡喃葡萄糖苷为具有ACE抑制活性的成分。 3. 从木姜冬青95%乙醇提取物的乙酸乙酯部分分离并鉴定了5个化合物： 2-O-β-D-吡喃葡萄糖-6,2´-二羟基-4,4´-二香草酰氧甲基-1,1´-二苯醚（冬青苷）和四个已知化合物：七叶内酯、香草酸、3,4-二甲氧基苯乙酸和vanilloylcalleryanin。冬青苷为新化合物。 4. 从青荚叶95%乙醇提取物的中小极性部分分离并鉴定了9个化合物：β-谷甾醇、β-胡萝卜苷、羽扇豆醇、桦木醇、桦木酸、棕榈酸甘油酯、桂皮酸、6αH-4-烯-3-酮-豆甾醇和6βH-4-烯-3-酮-豆甾醇。 5. 对1985-2006年间天然二苯醚类化合物及活性研究进展进行综述. The in vitro test indicated that the 95% ethanolic extract of the barks of Saurauia napaulensis DC showed α-amylase inhibitory activity, the 95% ethanolic extract of the whole plants of Debregeasia. orientalis showed angiotensin converting enzyme (ACE) inhibitory activity and some fractions of the 95% ethanolic extract of the aerial parts of Helwingia japonica showed protein tyrosine phosphatase (PTP)1B inhibitory activity. In order to investigate components and active compounds of the three plants, they were chemically studied mainly using. Thirty-nine compounds were isolated predominantly by column chromatography identified by spectral methods or comparing them with authentic samples. Further investigation of Ilex litseaefolia Hu et Tang was carried out. Major results are as follows: 1. Twelve compounds were isolation from the 95% ethanolic extract of the barks of S. napaulensis DC. They were identified as auranamide, aurantiamide benzoate, oleanolic acid, β-sitosterol, β-daucosterol, ursolic acid, 2α,3α-dihydroxyurs-12-en-28-oic acid, 2α,3β,24-trihydroxyurs-12-en-28-oic acid, (2S,3S,4R,10E)-2-[(2'R)-2'-hydroxytetracosanoyl amino]-10-octadecene-1,3,4-triol, 2α,3α,24 -trihydroxyolean-12-en-28-oic acid, 2α,3β-dihydroxyurs-12-en-28-oic acid, and 2α,3α,24-trihydroxyurs-12-ene-28-oic acid, respectively, by spectral methods or comparing them with authentic samples. 2. Eighteen compounds were isolation from the 95% ethanolic extract of the whole plants of D. orientalis. They were identified as palmitic acid, henicosanoic acid, henicosanoic acid methyl ester, β-sitosterol, monogynol, betulinic acid, hederagenin, β-daucosterol, 18αH-urs-20(30)-en-3-one, 3,4-seco-urs-20(30)-en-3-oic acid, pomolic acid, (-)-epicatechin, (+)-catechin, quercetin, quercetin 3-O-β-D-glucopyranoside, syringin, syringiaresinol digloside and kaempferol-3-O-rutinose. (+)-Catechin, quercetin and quercetin 3-O-β-D-glucopyranoside were the ACE inhibitory active components. 3. Further phytochemical investigation of the ethyl acetate parts of 95% ethanolic extract of the whole plant of I. litseaefolia afforded 2-O-β-D-glucopyranose-4,4´-di-vanilloyloxymethyl-2,6´-dihydroxy-1,1´-diphenyl ether (ilexiside), esculetin, vanillic acid, 3,4-dimethoxybenzylacetic acid and vanilloylcalleryanin. Ilexiside was new compound. 4. Nine compounds were isolation from the 95% ethanolic extract of the whole plant of H. japonica: β-sitosterol, β-daucosterol, lupeol, betulin, betulinic acid, glycerol monopalmitate, cinnamic acid, stignast-4-en-6β-3-one and stignast-4-en-6α-3-one 5.Diphenyl ether compounds from nature between 1985-2006 were summarized.

五枫藤、小驳骨和川西茶藨的化学成分研究

八月瓜属植物五枫藤(Holboellia latifolia Wall.)和驳骨草属植物小驳骨(Gendarussa vulgaris Nees)均为药用植物, 前者化学成分研究不深入, 后者的化学成分未见报道。川西茶藨(Ribes takare D. Don)为茶藨子属植物, 没有化学成分的报道。本论文对三个植物的化学成分和活性成分进行了研究, 主要通过色谱方法分离得到了48 个化合物, 采用波谱分析或与已知标准品对照等手段鉴定了它们的结构, 其中有1 个新的原小檗碱类化合物和3 个新的联苯类化合物,发现了具有细胞毒活性和α-葡萄糖苷酶抑制活性的化合物。1、从五枫藤地上部分的95%乙醇提取物中分离得到了12 个化合物: 五加苷K (1)、hederagenin 3-O- α-L-rhamnopyranosyl-(1→2)- α-L-arabinopyranoside (2)、β-萘乙酸(3) 、3-O-α-L-rhamnopyranosyl-(1→2)-[β-D-glucopyranosyl-(1→3)]-α-L-arabinopyranosyl oleanolic acid 28-O-α-L-rhamnopyranosyl-(1→4)-β-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl ester (4) 、3-O- α-L-rhamnopyranosyl-(1→2)-O- β- D-glucopyranosyl-(1→2)- α-L-arabinopyranosyl oleanolic acid (5) 、3-O-( β-D-glucopyranosiduronic acid)-oleanolic acid 28-O- β-D-glucopyranoside (6)、lup-20(29)-en-3-one (7)、lupeol (8)、β-谷甾醇(9)、齐墩果酸(10)、乌苏酸(11)、β-胡萝卜苷(12)。化合物1 对Lu-06、N-04 和Bre-04 癌细胞株的GI50 分别是0.77µg/mL、1.26 µg/mL 和1.55 µg/mL, 化合物2 对N-04 癌细胞株的GI50 为2.44 µg/mL。2、从小驳骨地上部分的95%乙醇提取物中分离得到了1 个原小檗碱类新化合物13-hydroxyl gusanlung A (25), β-谷甾醇(9)、齐敦果酸(10)、β-胡萝卜苷(12)、棕榈酸(1-)甘油酯(13)、棕榈酸(14)、阿苯哒唑(15)、阿苯哒唑砜(16)、阿苯哒唑亚砜(17)、aurantiamide acetate (18)、华良姜素(19)、芫花素(20)、(-)-丁香树酯醇(21)、gusanlung B (22) 、eupteleasaponinsⅤ acetate (23)、gusanlungA (24)、刺五加苷E (26)、岩白菜素(27)、咖啡酸(28)。化合物25 对肝癌细胞株(HepG2) 的GI50 为2.08 µg/mL。3、从川西茶藨地上部分的95%乙醇提取物中分离鉴定了22 个化合物: β-谷甾醇(9) 、β- 胡萝卜苷(12) 、O-acetyloleanolic aldehyde (29),4,7,8-trimethoxy-2,3-methylenedioxydibenzofuran (30) 、3', 5-dimethoxy-3, 4-methylenedioxybiphenyl (31) 、桦木醇(32) 、6,7-dimethoxy-1-methyl-3,4-dihydroquinolin-2-one (33)、3'-hydroxy-5-methoxy-3,4-methylenedioxybiphenyl (34) 、7-hydroxy-4,8-dimethoxy-2,3-methylenedioxydibenzofuran (35)、桦木醛(36)、没食子酸(37) 、6β- 羟基-4- 烯-3- 酮- 豆甾醇(38) 、5α, 8α-epidioxy-(22E,24R)-ergosta-6, 22-dien-3β-ol (39)、verrucofortine (40)、6-methoxycalpogoniumisoflavone A (41)、2-羟基二苯甲酮(42)、桦木酸(43), 3, 5-二甲氧基苯甲酸-4-O-β-D-吡喃葡萄糖苷(44)、洋芹素(45)、刺槐素(46)、水杨酸(47)、洋芹素-5-O- β-D-葡萄糖苷(48), 化合物30、31 和35 为新的联苯化合物。化合物30的α-葡萄糖苷酶抑制率为10.2% (1.00 mg/mL); 化合物35 的抑制率为17.2% (1.00mg/mL)。4、综述了1960 年以来原小檗碱类化合物药理活性研究进展。 Plants Holboellia latifolia Wall and Gendarussa vulgaris Nees, are used as folkmedicine. Ribes takare D. Don belongs to the genus Ribes. The three plants have notbeen chemically studied in detail. Chemical and bioactive study of three plants led tothe isolation of 48 compounds by chromatography. Their structures were elucidatedon the basis of spectroscopic evidence or comparison with authentic samples. Amongthe 48 componds isolated one protoberberine alkaloid and three biphenyls are newones. Cytotoxic and α-glucosidase inhibitory compounds had been found.1. Twelve compounds were isolated from the 95% ethanol extract of the aerial partof H. latifolia Wall. They were characterized as fellow: eleutheroside K (1),hederagenin-3-O- α-L-rhamnopyranosyl-(1→2)- α-L-arabinopyranoside (2),2-naphthyl acetic acid (3),3-O-α-L-rhamnopyranosyl-(1→2)-[β-D-glucopyranosyl-(1→3)]-α-L-arabinopyranosyl oleanolic acid 28-O-α-L-rhamnopyranosyl-(1→4)-β-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl ester (4), 3-O- α-L-rhamnopyranosyl-(1→2)-O- β- D-glucopyranosyl-(1→2)- α-L-arabinopyranosyl oleanolic acid (5),3-O-( β-D-glucopyranosiduronic acid)-oleanolic acid-28-O- β-D-glucopyranoside (6),lup-20(29)-en-3-one (7), lupeol (8), β-sitosterol (9), oleanolic acid (10), ursolicacid (11), and β-daucosterol (12). Compound 1 showed moderate cytotoxicity againstLu-06 (GI50, 0.77 µg/mL), N-04 (GI50, 1.26 µg/mL) and Bre0-4 (GI50=1.55 µg/mL)and compound 2 showed moderate cytotoxicity against N-04 (GI50=2.44 µg/mL).2. A new protoberberine alkaloid, 13-hydroxyl gusanlung A (25), was isolated fromthe aerial part of Gendarussa vulgaris Nees, together with β-sitosterol (9), oleanolicacid (10), β-daucosterol (12), glycerol monopalmitate (13), palmific acid (14),albendazole (15), albendazole sulphone (16), albendazole sufloxide (17), aurantiamideacetate (18), kumatakenin (19), genkwanin (20), (-)-syringaresinol (21), gusanlung B(22), eupteleasaponinsⅤ acetate (23), gusanlung A (24), eleutheroside E (26),bergenin (27) and caffeic acid (28). Compound 25 showed cytotoxicity against HepG2 cells (GI50, 2.08 µg/mL).3. Phytochemical study of the Ribes takare D. Don led to the isolation of three newbiphenyls, 4,7,8-trimethoxy-2,3-methylenedioxydibenzofuran (30), 3', 5-dimethoxy-3,4-methylenedioxybiphenyl (31) and 7-hydroxy-4,8-dimethoxy-2,3-methylenedioxydibenzofuran (35), along with nineteenknown compounds, β-sitosterol (9), β-daucosterol (12), O-acetyloleanolic aldehyde(29), betulin (32), 6,7-dimethoxy-1-methyl-3,4-dihydroquinolin-2-one (33),3'-hydroxy-5-methoxy-3, 4-methylenedioxybiphenyl (34), betulinic aldehyde (36),gallic acid (37), stigmast-4-en-6β-ol-3-one (38), 5α, 8α-epidioxy-(22E, 24R)-ergosta-6,22-dien-3β-ol (39), verrucofortine (40), 6-methoxycalpogonium isoflavone A (41),2-hydroxybenzophenone (42), betulinic acid (43), 3,5-dimethoxygallic acid-4-O- β-D-glucopryranoside (44), apigenin (45), acacetin (46), salicylic acid (47) andapigenin-5-O- β-D-glucopryranoside (48). α-Glucosidase inhibitory rates ofcompound 30 and 35 were respectively 10.2% and 17.2% at a concentration of 1.00 mg/mL).4. Pharmacological activities of protoberberines were summarized.

钩藤和蹄叶橐吾的化学成分研究

本论文由三章组成。第一、二章分别报道钩藤、蹄叶橐吾的化学成分研究工作。从两种药用植物中共分离和鉴定了37 个化合物，其中有1 组6 个新的骨架相似的鞘糖脂类同系物及5 个新的倍半萜类化合物。第三章概述了艾里莫芬烷内酯类化合物的研究进展。 第一章报道钩藤(Uncaria rhynchophylla)带钩茎枝乙醇提取物的化学成分。采用正、反相硅胶柱层析等分离方法，运用NMR、MS 等波谱技术共分离鉴定得到22 个化合物，分属于五环三萜类化合物、鞘糖脂类化合物等，其中有1 组6个新的鞘糖脂类化合物同系物，另外有7 个化合物首次从该植物中分离得到。 第二章报道蹄叶橐吾(Ligularia fischeri)根部乙醇提取物化学成分的分离纯化和结构鉴定。通过正、反相硅胶柱层析等分离纯化和MS、NMR 等波谱解析，共分离鉴定得到18 个化合物，其中5 个是新化合物。它们的结构分别确定为3β-acetyl-6β, 8α, 10β-trihydroxyeremophila-7(11)-en-8, 12-olide (23), 3β-acetyl-8β,10β-dihydroxy-6β-(2-methylbutyryloxy)-eremophilenolide (24), 3β-acetyl-6β, 10β-dihydroxyeremophila-7(11), 8(9)-dien-8, 12-olide (25), 3β-acetyl-6β, 10β-dihydroxyeremophila- 7(11), 8(9)-dien-8, 12-olide (26), (3aR, 4R, 5S, 7aS)-2-acetyl-7ahydroxy-3a, 4, 5, 6, 7, 7a-hexahydro-1H-inden-5-yl acetate (27)。这5 个化合物经体外生物活性测试，结果表明化合物23 具有抑制酪氨酸磷酸酯酶的活性, 其IC50 值为1.30 μg/ml。 第三章概述了近二十年来艾里莫芬烷内酯类化合物的研究进展，包括其结构及药理活性两个方面，列举了从23 种植物中分到的114 个艾里莫芬烷内酯类化合物，同时也对其主要生物活性进行了总结。 This dissertation consists of three chapters. The first and second chapters elaborate the phytochemical investigation of Uncaria rhynchophylla and Ligularia fischeri. Thirty-seven compounds, including six new ones, were isolated and identified by spectroscopic methods and X-ray diffraction experiments. The third chapter is a review on the study progress of Eremophilanolides. The first chapter focus on the isolation and identification of chemical constituents from Uncaria rhynchophylla. Twenty-two compounds were isolated from the roots of U. rhynchophylla by repeat column chromatography over normal and reversed phase silica gel. Those compounds mainly belonged pentacyclic triterpenoids and sphingolipids. Among them, a series homologues of six sphingolipids were new compounds and seven compounds were firstly reported in this plant. The second chapter is about the phytochemical investigation of L. fischeri. Eighteen compounds were isolated and identified. Among them, four new erem-ophilanolides and a new dinoreremophilane derivative were characterized as 3β-acetyl-6β, 8α, 10β-trihydroxyeremophila-7(11)-en-8, 12-olide (23), 3β-acetyl-8β, 10β-dihydroxy-6β-(2-methylbutyryloxy)-eremophilenolide (24), 3β-acetyl-6β, 10β-dihydroxyeremophila-7(11), 8(9)-dien-8,12-olide (25), 3β-acetyl-6β, 10α-dihydroxyeremophila- 7(11), 8(9)-dien-8,12-olide (26), (3aR,4R,5S,7aS)-2-acetyl-7a-hydroxy-3a, 4, 5, 6,7, 7a-hexahydro-1H-inden-5-yl acetate (27) by spectroscopic analysis and confirmed by X-ray crystallography analysis. It was revealed that compound 23 has the ability to inhabit the PTP1B in vivo. And its IC50 is 1.30 μg/ml. The third part is a review on the study progress of Eremophilanolides.

N-烷基氮杂糖及其衍生物的合成

多羟基哌啶类化合物通常称为氮杂糖，由于与糖结构的相似性，亚胺基环醇表现出强的糖苷酶和糖基转移酶抑制活性，可调控在生物识别及酶结构控制中起到重要作用的糖蛋白的生物合成与水解。因此这类抑制剂有望成为与糖代谢紊乱有关的疾病的治疗药物，如：抗糖尿病、抗肿瘤、抗溶酶体贮积症及抗病毒感染（包括艾滋病）等药物。正是由于氮杂糖的重要生物活性及诱人的药用开发前景，近年来，有关氮杂糖及其衍生物的合成、生物活性及应用研究备受关注。 本论文探索了一系列的作为潜在的迈克加成中间体1-C-乙酰甲基/甲氧羰基甲基-5-N-取代呋喃核糖碳苷衍生物在碱的作用下先发生β-消除反应，接着发生分子内的迈克加成反应生成1-C-乙酰甲基-N-取代氮杂吡喃糖碳苷衍生物及1-C-甲氧羰基甲基-N-取代氮杂吡喃糖碳苷衍生物的方法，该转变过程为先通过β-消除得到非环状的α/β不饱和共轭酮或酯的中间体，接着5-N-取代氨基与分子内的α/β不饱和共轭酮或酯发生分子内的1,4-亲核加成，其中，2'-酯的环加成立体选择性的得到β型1-C-乙酰甲基-N-取代氮杂吡喃糖碳苷衍生物，而2'-酮的环加成得到立体异构体1-C-乙酰甲基-N-取代氮杂吡喃糖碳苷衍生物。此外，该类N-取代氮杂吡喃糖碳苷衍生物进一步脱除保护基，得到了一系列新的N-取代氮杂吡喃糖衍生物，拓展了氮杂吡喃糖碳苷分子库。 中间体1-C-(2'-oxoalkyl)-5-N-alkylated glycoribofuranoside的合成是由核糖为原料，通过对其结构修饰，在C-5氮原子上先引入不同的取代基，在C-1上引入乙酰甲基或甲氧羰基甲基。C-5取代氨基的引入通过两种方法：(a) 5-取代链状脂肪氨基可由链状的伯胺直接与5-甲磺酰基发生SN2亲核取代得到；(b) 5-取代芳香氨基可通过芳香醛与C-5氨基缩合再由硼氢化钠还原得到。2'-酰基的引入通过烯丙基氧化得到：2'-酮羰基由醋酸汞和琼斯试剂氧化得到；2'-酯基由高锰酸钾氧化再碘甲烷的作用下得到。 The polyhydroxylated piperidines, commonly be called azasugars. Iminocyclitols and their derivatives have exhibited remarkable biological activity to inhibit glycosidase-processing enzymes, with resulting potential chemotherapeutic applications against diabetes, cancer, lysosomal storage disorders and viral infections including AIDS. Recently, because of the important biological activity and excellent foreground on pharmaceutical application, great attention has been attracted to the synthesis of the new derivatives and analogues. In this dissertation, 1-C-(2'-oxoalkyl)-5-N-substituted-glycoribofuranosides, which used as latent substrates for intramolecular hetero-Michael addition, were converted to 2-ester and 2-ketone aza-C-glycopyranosides by base treatment. The transformation was achieved through β-elimination to an acyclic α/β-conjugated ketone or ester, followed by an intramolecular hetero-Michael addition by the 5-N-alkylated amino group. The 2-ester cycloaddition was highly stereoselective in favor of an equatorial 1-C-substitution while the 2-ketone cycloaddition was produced a pair of stereoisomers of 2′-ketonyl aza-C-glycoside. Additionally, the resultant different N-alkylated aza-C-glycopyranosides could be further prepared for various azasugar library constructions by removal of protecting groups. Synthesis of the key intermediate 1-C-(2'-oxoalkyl)-5-N-alkylated glycoribo- furanoside involved the introduction of 5-substituted amino and 1-C-2′-oxoalkyl groups from D-ribose. The 5-alkylated amino was introduced through two methods: (a) the 5-aliphatic series amino synthesized by the nucleophilic substitution of 5-mesylate using neat ethylamine, propylamine, butylamine, and hexylamine, (b) the 5-aromatic series amino synthesized by various aromatic aldehydes with C-5 amino under NaBH4 reduction. The 1-C-2′-oxoalkyl groups were introduced through oxidation of the ally group: the 1-C-allyl group was oxidized with Hg(OAc)2 and Jones reagent to the 2-ketonyl C-glycoside; the 1-C-allyl group was oxidized with KMnO4 and CH3I/NaHCO3 to 1-C-methyl acetate glycoside.

一株放线菌发酵产物抗肿瘤活性的研究

本学位论文主要研究一株放线菌发酵产物的抗肿瘤活性。先对该株放线菌进行活化培养，然后进行大批量发酵，发酵液经过冷冻离心，对离心得到的沉淀和上清液用不同极性的有机溶剂进行萃取，得到六个浸膏样品。对六个样品进行初步抗肿瘤活性检测。 然后对活性浸膏进行分离纯化和活性跟踪。本论文主要进行了如下的工作： 1、对菌种进行活化培养，利用该菌株在280C，200r.min-1条件下进行发酵实验，发酵时间为72h,发酵总量为15L。发酵液经过离心得到上清液和沉淀两部分。 2、分别用石油醚、乙酸乙酯、正丁醇萃取沉淀和上清液，得到编号为1—6的六个浸膏样品，对六个浸膏样品进行初步的细胞毒性和抗HepG2肿瘤活性实验，得出结论为5号样品活性最高。在没有分离纯化的情况下GI50达到0.76µg/mL。 3、对5号样品进行TLC实验，找出能够较好分离5号样品中各组分的溶剂组合，最后得出在氯仿：甲醇=8：1时分离效果较好。然后利用氯仿：甲醇=8：1的溶剂组合作为洗脱剂对5号样品进行过硅胶柱分离纯化并进行活性跟踪分离。 4、对分离纯化后得到的样品进行活性跟踪和结构分析。分离后得到样品A，在其浓度为10µg/ml时，抗肿瘤实验细胞的生长率为73.5%。在浓度为1.0mg/ml时，抗单纯疱疹病毒率（HSVⅡ）为74.5%。结构分析得知其分子式最可能为C41H43N8O4. This dissertation studied about the anti-tumor activity of an actinomycete fermentation product. First, we cultured the actinomycete. Second, we fermented it in large quantities, and then centrifuged the fermentation fluid; the next step is that we extract sedimentation and supernatant in different polar organic solvents, in turn to obtained six samples, which were detected about anti-tumor activity. Last, we purified active sample and tracked activity of it. We carried out the following research work: 1. Activation, culture and screening of the actinomyces was carried on. We used the screening strain to carry on the fermentation when the conditions are 280C，200r.min-1,the fermentation time is 72h. Fermentation fluid volume is 15L.And we obtained sedimentation and supernatant after fermentation fluid was centrifuged. 2. We used Petroleum ether, ethyl acetate, n-butanol separately to extract sedimentation and supernatant, and obtained six samples that were numbered 1-6. From the preliminary cell toxicity and the anti-tumor（HepG2） bioactivity experiment, we found that No.5 sample has the highest activity in the samples; the GI50 was 0.76µg/ml which has not been purified. 3. We Carried on TLC experiment on the No.5 sample, found the solvent composition that can separate each component of the No.5 sample. At last, we found that when the proportions are tri-chloromethane: methyl alcohol = 8:1, the Separation result was the best, and then we used the Solvent composition which proportion are tri-chloromethane: methyl alcohol = 8:1 as eluant to Purify No.5 sample by silica gel column. 4. We tracked the activity of pure sample obtained from Purification and analyzed structure of these substances. We got a compound A after separation, and the cell growth rate was 73.5% when its concentration was 10µg/ml. The anti-virus（HSVⅡ） rate was 74.5% when its concentration was 1.0mg/ml. We analyzed the Structure of A, and informed its molecular formula that was the most likely for C41H43N8O4.

无介体双室微生物燃料电池产电性能初步研究

本文对无介体双室微生物燃料电池的产电性能进行了初步研究，并根据不同运行阶段产电性能的优劣，对其中微生物的差异性进行了比较分析。全文分为两个部分： 第一部分：以乙酸钠为阳极原料构建双室微生物燃料电池（MFC），研究不同阴极受体、外接电阻、乙酸钠浓度和pH等因素对电池产电性能的影响，研究结果表明：在500mL的阴阳极反应体系中，选用乙酸钠作为阳极底物，质量浓度为6.46 g/L， pH 7.0，接入500Ω外电阻，阴极电子受体选择高锰酸钾的情况下，微生物燃料电池产电性能最好，最大电功率密度达到294.72 mW/m2，库伦效率能达到25.87%。在确定最适外接电阻阻值的同时对MFC内阻进行测定，阻值为871.87Ω。 第二部分：微生物燃料电池运行中，比较以厌氧污泥作为接种源的第一阶段和只接入附着有大量微生物电极的第二阶段的产电性能，得出第二阶段产电性能优于第一阶段，最大电功率密度达到353.57mW/m2，比第一阶段提高58.85 mW/m2；库伦效率为39.35%，增幅达52％左右；针对微生物燃料电池运行过程中，底物CH3COONa可能存在其它的代谢途径，本实验进行了第二阶段产电性能与CH3COONa消耗率关系以及阳极液面上方气体成分和含量的研究，发现第二阶段50h前CH3COONa的大量消耗主要用于微生物的生长，在整个运行过程中，阳极液面上方含有CH4和CO2；对气体测定的同时还发现，振荡能增强电功率密度的输出；通过对电极上和污泥中微生物差异性分析得出，δ-变形菌纲、β-变形菌纲和拟杆菌门的菌种更适应微生物燃料电池的运行环境，能在电极上大量富集，提高电池的产电性能，只接入附着有大量微生物的电极能有效降低热袍菌纲的菌种数量，降低了CH3COONa的无为消耗，有效提高了电池的库伦效率。 Electricity production in the mediator-less two-chambered microbial fuel cell(MFC) was researched. Based on the result in the different operation phase in the MFC, the microbial diversity was analysed. The paper involved two parts: Part 1: A two-chambered microbial fuel cell (MFC) was constructed with high-concentration sodium acetate as fuel in the anode. The influence of different electron acceptors in the cathode, external resistance value, pH value and concentration of sodium acetate on electricity generation in MFC was investigated. The result showed that the maximum power density of 294.72 mW/m2 and the coulombic efficiency of 25.87% was achieved at sodium acetate concentration of 6.46 g/L, pH 7.0, external resistance 500Ωin the anode and when using potassium permanganate as electron acceptor in the cathode. While decided the value of resistor, we found that shaking has effect on electricity production in the MFC. Part 2: Comparing the electricity production in different operation phases when using anaerobic sludge as inoculum in the first phase and microbes in the anodic electrode as inoculum in the second phase, the result showed that electricity production in the second phase was more than that in the first phase, the maximum power density of 353.57 mW/m2 and the coulombic efficiency of 39.35% was achieved, 58.85 mW/m2 and 52％ more than that in the first phase, respectively. According to the fact that CH3COONa might be metabolized in other pathway in the running process in the MFC, we determining the relationship between electricity production and CH3COONa consumption, and the gas content in the anode, we found that CH3COONa was mainly used for microbe growth before 50h, and the anode contained CH4 and CO2. At the same time, we found that shaking could improve power density. The analysis on diversity of microbe in the anodic electrode and anaerobic sludge showed that δ-proteobacterium, β-proteobacterium and Bacteroidetes adapted themselves to the running environment of MFC. The anode could enrich them to improve the electricity production while reduced the quantity of Thermotogales, which were obligately anaerobic organotrophs with a fermentative metabolism, to increase the coulombic efficiency effectively.

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.

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.

泥生绿菌的分离鉴定及其在净化废水中作用的初步探讨

本文介绍了从厌氧间歇膨胀光合反应器内的活性污泥中分离并鉴定的泥生绿菌(Chlorobium limicola Nadson)S1,它属严格厌氧光能自养型细菌，在有硫化物和少量碳酸氢盐存在下，有广泛利用有机物的能力，它的最适生长温度为28-30℃,最适生长PH为6.5-7.0，且含有氢化酶。因此，它能与甲烷发酵菌共存而共同作用，达到废水净化之目的。通过光照(2#反应器)和黑暗(1#反应器)对比实验，表明了在光照条件下即有泥生绿菌S1存在下，反应系统能更好地降低CODcr、BOD5 和提高CH4 含量，在四个负荷段的运行中，2#反应器在后三个负荷段的甲烷含量能稳定在91.6%而1#反应器为87%，2#反应器的二氧化碳含量为4.5%而1#反应器为8.8%，于28.35g/l.d的负荷下，2#反应器CODcr去除率达83.4%，BOD去除率达74.53%，分别较1#反应器高10.8%,6.4%。COD去除率提高了14%，BOD去除率提高了9.3%。本试验的试验条件为：白天自然光照，晚上电源光照，光照强度为1000-2500lux，通过连续动态运转，并以恒定的流速将废液注入反应器中，进水PH控制在6.5-7.2，反应器厌氧，恒温室温度控制在30±1℃。为使整个试验中同一水质条件下进行，进水采用化学合成培养基。This paper reports a Chlorobium Liwicola S1's isolation and identification. It is a strictly anaerobic and photosynthetic autotrophic bacterium. Along with sulfidedepondent CO2 assiwilaton,a few simple organic compounds can be photoassimilated. Acetate is most effectively used. Its best conditons of growth are 28-30℃,PH 6.5-7.0, and it contains hydrogenase. So it can live with methanefermentative bacteria in order to treat wastewater. At the same time, the treatment of wastwater using Chlorobium Limicola S1 with methane-fermontative bacteria under dark anaerobic and light anaerobic conditions is studied. In contrast with 1# reactor-darken, 2# reactor-illuminated can lossen wastewater's CODcr, BOD5 and on hance CH4 content better. In the test, 2# reactor's CH4 content is stable at 91.6%, but 1# reactor's is 87%. The CO2 content of 2# reactor is 4.5%, but 1# reactor's is 8.8%. When the load of teatment is 28.35g/l.d, the COD removal effficiency is 83.4% and the BOD removal efficiency is 74.53% in 2# reactor. They are separately 10.8%, 6.4% higher than 1# reactor's.

一种新奇的甲烷氧化细菌——能兼性营养和厌氧生长的沼气甲基弯菌

经过细心的条件选择实现了无甲烷条件下甲烷氧化菌沼气甲基弯菌81Z(methylosinus methanica 81Z)利用C2化合物的生长，同时 发现二碳代谢中间产物甘氨酸的胞外积累及对生长的抑制作用。又在此基础上从81Z原种中富集得到一株菌81Z-A，兼性生长能力大 幅度提高，而且除乙酸外又能利用丙酮酸、苹果酸、柠檬酸、葡萄糖而生长。对细胞氧化各种有机底物时氧吸收的测定及酶分析结 果发现了在其它甲烷氧化细菌中未曾发现的异柠檬酸裂解酶和苹果酸酶的存在，表明81Z除了具有通常II型菌的碳代谢途径外，具 有特殊的补偿代谢途径——乙醛酸支路以及从乙酸生糖的回补途径。因此推证其兼性生长的能力是固有的，从而说明了甲烷氧化菌 的专一性概念没有普遍意义。说明了81Z还能在含有二碳的培养基中厌氧生长，包括细胞的分裂和增值行为。虽然这种厌氧生长还 很弱，但至少可以说明它不是严格好氧的，这对于传统的关于甲烷氧化菌的严格好氧的概念是一个冲击。81Z正常条件下是利用甲 烷而生长的，当供给它乙酸、乙醛酸和丝氨酸时能促进含C-C键有机物氧化的活性，而对甲烷单加氧酶和其它C2化合物的氧化有抑 制或阻遏作用，对碳同化的丝氨酸途径的关键酶羟基丙酮酸还原酶有阻抑作用。同时也证明了81Z的甲烷单加氧酶和甲醇氧化活性 可被甲烷、甲醇所诱导，而因甲酸而降低。The growth of Methylosinus methanica 81Z on C2-compounds without methane was realized by selecting suitable conditions. The intermediate product Gly from its C2 metabolism was found to accumulate out cells and inhibit its growth. 81Z-A, which was obtained from 81Z by richening, could grow on C2- compounds rapidly. It can even grow on pyruvate, malate, citrate and glucose. The results of measurements of oxygen consumption by cell suspensions in the presence of various organic compounds and the results of enzyme assays of detected activities of isocitrate lyaze and malic enzyme in cell extracts which were not found in other methantrophs showed that 81Z possesses not only the regular carbon metabalic pathways but also some peculiar anaplerotic pathways--the glyoxylate cycle and the gluconeogenic pathway from acetate. As a consequence of these studies, its ability of facultative growth is inherent. Therefore, the concept of obligate dependence on C2- compounds of methanotrophs is not of universal significance. The ability of 81Z's growth(including desintegration and proliferation behaviour) on C2-compounds anaerobically was also demonstrated. Despite of the weakness of this growth, at least it could be said that 81Z is not strictly aerobic. This is a strike to the traditonal concept about the strictly aerobic action of methanotrophs. Regularly, 81Z grows on methane. The presence of acetate, glyoxylate and serine could increaze its ability of oxidizing the organic coumpounds containing C-C ponds. In contrast, they could inhibit the activities of MMO and other C2-compounds oxidation, they also repressed the key enzyme hydroxypyruvate reductase of the serine-pathway for carbon assimilation. At the some time, it was testified that the activities of MMO and methanol oxidation were inducible by methane or methanol and were lower in the presence of formate.

真菌单宁酶发酵五倍子和有机溶剂中酶法合成没食子酸丙酯的研究

本文报告了丝状真菌单宁酶发酵五倍子及有机溶剂中酶法合成没食子酸丙酯的研究。利用单宁和/或五倍子诱导丝状真菌产生单宁 酶的原理，借助二级发酵程序，对从天然源得到的75株菌进行了生物转化实验研究。选择出既能水解单宁或五倍子成没食子酸，又 能把没食子酸和丙醇合成没食子酸丙酯，而且生物催化活性都较高的1株菌，这株菌经初步鉴定为黑曲霉(Aspergillus niger No.17)。随后对它开展了产酶条件和参数优化实验，得出了最佳培养条件。立足于参数优化实验方案的基础上，经由液体培养发酵 制备单宁酶制剂，并把该酶通过化学手段共价结合到一种新型载体—聚乙烯醇和戊二醛反应生成的缩醛上，制备得到固定化单宁酶 。这种固定化生物催化剂在两种有机介质体系中都具有逆向催化合成没食子酸丙酯的能力。最后建立起来一条有效可行的微生物酶 法制备没食子酸的技术途径，没食子酸产率达到70%。对这种物质进行元素 分析：含C,49.45%;含H,3.63%。它的熔点为237℃～243 ℃，三种溶剂系统的TLC均只给出一个斑点。这些数据都与标准品一致。有机溶剂中酶法合成没食子酸丙酯的技术途径已经建立。 水溶性单宁酶在潜溶剂体系中也能催化上述酯化反应，反应混合物中的PG浓度为16.4mmol/L，制备薄层被用于分离反应混合物所含 的PG，这种产物被红外、质谱及三种溶剂系统的TLC等方法鉴定，确证为目标产物。在这一学位论文的实验研究过程中，还包括一 些生化分析方法的建立和应用，这些方法用于鉴定底物和产物及测定它们的浓度，其内容主要包括TLC定性/半定量分析、元素分析 、质谱、红外等手段的综合运用。本工作为开发我国特有的天然产物资源—五倍子的生物化工加工技术及非水相生物催化技术的开 发，提供了有用的基础数据资料，具有应用基础研究工作的重要性。In this thesis, the studies on the fermentation of Chinese gallotannin by filamentous fungi with tannase activity and enzymatic synthesis of propyl gallate(PG) in organic solvents were described through these biocatalysts. Based on the principles of induction enzyme, the tannase produced from filamentous fungi by adding tannic acid(TA) and/or Chinese gallotannin into media was investigated, and the screening experiments of bioconversion were done with 75 strains by means of a two-stage fermentation procedure. These strains were isolated with the enrichment culture technique from natural sources. Hence we selected one strain (Aspergillus niger No.17) that can not only catalyze the hydrolyses of TA and/or Chinese gallotannin into gallic acid(GA) in the liquid cultures, but also be used to synthesize PG from propanol and GA in the non-aqueous media. At the same time both of its biocatalytical activities were higher. This strain was calssified to be Aspergillus niger by the primary identification. Then optimum conditions for production of the tannase and its parameters were examined. In this way, one set of optimum culture conditions was selected. Making use of the optimum proposal, the tanase was prepared through a liquid fermentation procedure. The enzyme was convalently coupled to a new type of carrier which was made chemically from polyvinyl alcohol(PVA)and glutaraldehyde. The immobilized enzymes were able to synthesize PG reversely in two organic media. Finally, an effective enzymatic technique for production of GA was developed. The yield of GA products was up to 70%。Element analysis for this substance: calce: C, 49.42%; H, 3.56%; found: C, 49.45%, H, 3.63%. Its melting point was 237℃～ 243℃ and TLCs on three solvent systems gave only one spot respectively. These data were identical with theauthentic GA. The enzymatic synthesis of PG in organic solvents was extablished with reverse route of tannase catalytical hydrolysis. Aqueous enzyme perparation also catalyzed above esterification in a buffer system. The PG concentration in the reaction mixture was 16.4mmol/L. The reparative-scale TLC was used to isolate PG from the reaction mixture. This product separated was identified by IR, MS and TLC on three solvent systems. In this study of thesis, some biochemical analytical mehtods were developed and used to identify substrates and products, and to determinate their concentration. These methods, including TLC qualitative/half quantitative analysis, element analysis, MS, IR and so on, were useful, available and performable. This work provided basic data and information for developing the biochemical engineering and bio-processing of Chinese gallotannin-a special natural resource in China and the non-aqueous phase biocatalysis. Thus, this study possesses importance in the applied and basic research work.

利用含纤维素原料发酵产氢的研究

本文从新鲜大熊猫粪便和实验室保存的沼气发酵富集物中筛选得到 4 株厌氧纤维素分解菌B5、C3、D3-2、D4-1，利用这4 株菌预处理秸秆，然后将预处理后的秸秆用本实验室保存的厌氧产氢菌来发酵进行生物产氢。同时还比较研究了：○1 用1% H2SO4、25% NH3 · H2O和12% NaOH对秸秆进行化学预处理；○2 用厌氧纤维素分解菌对秸秆进行生物预处理；○3 化学与生物组合预处理对秸秆发酵生物产氢的影响。实验结果表明：12% NaOH和生物组合预处理后的秸秆发酵产氢效果最好，其产氢量为21.04 mL g-1，是未经预处理秸秆的75 倍；最高氢气浓度为57.3%，是未经预处理秸秆的96 倍；其产氢的最适pH 为4.5 ~ 6.0，最佳底物浓度为45 ~ 55 g L-1；其发酵过程中的挥发性脂肪酸（VFAs）以乙酸和丁酸为主。 本实验筛选到的 4 株厌氧纤维素分解菌株中，B5 和D4-1 在降解纤维素的同时还具有直接以纤维素为底物产氢的功能，因此本文分别对菌株B5 和D4-1 以及二者的组合菌株B5+D4-1 直接利用秸秆为基质发酵生物产氢做了初步探索研究。结果发现：组合菌株发酵产氢的效果以及对秸秆纤维素和半纤维素的降解率要比单菌株好。菌株B5+D4-1 发酵，秸秆的产氢量为11.4 mL g-1，分别是B5 和D4-1 单菌株的1.6 倍和3.1 倍；组合菌株B5+D4-1 发酵的最大氢气浓度为31.6%，分别是B5 和D4-1 单菌株的1.3 倍和2.4 倍。在发酵过程中，组合菌株B5+D4-1 对秸秆纤维素和半纤维素的最高降解率分别为35.0%和11.8%，分别是菌株B5 的1.2 倍和1.1 倍，是菌株D4-1的1.5 倍和1.3 倍。菌株B5，D4-1 以及组合菌株B5+D4-1 发酵过程产生的挥发性脂肪酸（VFAs）均以乙酸为主。菌株B5 单独发酵过程中只检测到乙酸和丁酸，菌株D4-1 单独发酵以及组合菌株B5+D4-1 发酵过程检测到有乙醇、乙酸和丁酸。 The fermentative bio-hydrogen production by anaerobic hydrogen bacteria preserved in our laboratory from the straw which had been pretreated by four anaerobic cellulolytic decomposition strains of B5, C3, D3-2, D4-1 which were isolated and screened from giant panda’s excrement and biogas fermentation enrichments conserved in our laboratory was studied. Besides, the impact of chemical(1% H2SO4、25% NH3·H2O and 12% NaOH), biological (cellulolytic strains of B5, C3, D3-2, D4-1) and chemical-biological combination pretreatment on bio-hydrogen production from straw by fermentation was also comparatively studied. The experiments showed that the best results of bio-hydrogen production were obtained from the straw with 12% NaOH-biological combination pretreatment method, its capability of bio-hydrogen production was 21.04 mL g-1, which was 75 times higher than the straw without pretreatment; the maximum concentration of H2 was 57.3%, which was 96 times higher than the straw without pretreatment; its optimum pH range was 4.5 ~ 6.0, and its optimum range of substrate concentration was 45 ~ 55 g L-1; In the process of fermentation, the main composition of VFAs were acetate and butyrate. Among the four strains of B5, C3, D3-2, D4-1, B5 and D4-1 have the function of hydrogen-producing by cellulose used as substrate when it decompose cellulose, so the preliminary exploration and research on fermentative bio-hydrogen production by B5, D4-1 and B5+D4-1 which directly used straw as substrate was carried out. The results showed that the combination strains of B5+D4-1 was strikingly better than either B5 or D4-1 strain in the fermentative hydrogen production. The hydrogen-production capability of B5+D4-1 was 11.4 mL g-1 which was respectively 1.6 times and 3.1times higher than B5 and D4-1; the maximum hydrogen concentration of B5+D4-1 was 31.6% which was respectively 1.3 times and 2.4 times higher than B5 and D4-1. In the process of fermentation, the maximum degradation rate of cellulose and hemicellulose in straw was respectively 35.0% and 11.8% by B5+D4-1, which was 1.2 times and 1.1 times higher than B5, and was 1.5 times and 1.3 times higher than D4-1 respectively. The Volatile Fattty Acids(VFAs) generated in the process of fermentation with strains of B5, D4-1 and B5+D4-1 were all mainly acetate. Acetate and butyrate were detected in the process of fermentation with B5, ethonal, acetate and butyrate were detected in the process of fermentation with D4-1 and B5+D4-1.

有机碳对混合菌群亚硝酸盐氮氧化的影响

自养硝化过程在自然界氮素循环和污水处理系统脱氮过程中起着关键作用。因此，了解有机碳对硝化的影响和硝化菌与异养菌之间的竞争对微生物生态学和污水处理系统设计都很重要。目前对氨氧化到硝酸盐氮过程的研究文献很多，但对亚硝酸盐氧化过程在异养菌的存在下如何受到有机碳影响的研究甚少。本文从生理生化指标、基因组学、蛋白组学三方面考察了在实验室条件下有机碳（乙酸钠）对硝化细菌和异养菌组成的混合菌群的硝化性能、菌群结构及代谢功能的变化的影响。 全文分为两大部分： 第一部分为乙酸钠对游离态硝化混合菌群的硝化性能和菌群结构的短期影响。混合菌株先在自养条件下进行连续培养，两个月后硝化速率达到20 mg N/(L·d)；而后离心收集菌体进行批式实验。在批式反应器中，初始亚硝氮均为126mg N/ L，乙酸钠-C 与亚硝酸盐-N 的比分别为0，0.44，0.88，4.41，8.82。结果表明：在低C/N 比（0.44 和0.88）时，亚硝酸盐去除速率比C/N=0 下高，细菌呈现一次生长；而在高C/N 比（4.41 和8.82）时，出现连续的硝化反硝化，亚硝酸盐去除率仍比对照下高，细菌呈现二次生长。不同C/N 比下微生物群落明显不同，优势菌群从自养和寡营养细菌体系（包括亚硝酸盐氧化菌，拟杆菌门,α-变形菌纲,浮霉菌门和绿色非硫细菌下的一些菌株）过渡到异养和反硝化菌体系 （γ-变形菌纲的菌株尤其是反硝化菌Pseudomonas stutzeri 和P. nitroreducens 占主导）。 第二部分为乙酸钠对硝化混合菌群生物膜的硝化性能和菌群结构的长期影响。接种富集的硝化混合菌群于装有组合式填料的三角瓶中，于摇床中自养培养；两个月后填料上形成生物膜的硝化速率达到20 mg N/ (L·d)；而后进行长期实验，每12 小时更换混合营养培养基（亚硝氮约200 mg N/ L，C/N 比同上）。结果显示：相较于C/N 比=0 时的亚硝酸盐氧化反应来说，低C/N 比出现了部分的反硝化，而高C/N 比则是几乎完全的反硝化。与对照比，C/N=0.44 时亚硝酸盐氧化速率并未受乙酸钠的影响，反而上升了，但C/N=0.88 时亚硝酸盐氧化速率有所下降。菌群结构分析表明自养对照与混合营养下微生物群落的不同；PCR-DGGE未检测出混合营养下硝化杆菌的存在，而显示异养菌尤其是反硝化菌的大量存 在。荧光定量PCR 结果表明随C/N 比上升，硝化杆菌数量从2.42 × 104 下降到1.34× 103 16S rRNA gene copies/ ng DNA，反硝化菌由0 增加至2.51 × 104 nosZgene copies/ ng DNA。SDS-PAGE 的结果表明不同C/N 比下的蛋白组较为复杂且呈现一定的差异性。 有机碳对亚硝氮氧化及微生物群落的影响很复杂，本文分别讨论了对游离态和生物膜固定态两种状态的混合菌群相应的短期和长期影响研究。研究发现，有机碳并非一定带来硝化的负影响，如果控制在适当的C/N 比范围，有机碳是有利于亚硝氮氧化的。这些发现阐明了有机碳和硝化反硝化的关系，填补了硝化微生物生态学上的空白，对污水处理系统中减少异养菌的影响并提高氮去除率有一定理论指导意义。 Nitrification plays a key role in the biological removal of nitrogen in both nature and wastewater treatment plant (WWTP). So, understanding of the effect of organic carbon on nitrification and the competition between nitrifying bacteria and heterotrophic bacteria is important for both microbial ecology and WWTP design and operation. Despite the fact that the nitrification process of ammonia to nitrate has been extensively investigated, it is not known how the process of nitrite oxidization is affected by organic carbon when heterotrophic bacteria are present. By measuring different physiological and biochemical parameters, as well as using genomic DNA and proteome analysis, we investigated the influence of organic (acetate) on nitrite oxidizing performance, community structure and metabolic function of nitrite-oxidizing and heterotrophic bacteria under laboratory conditions. The dissertation involves two parts: Part one deals with the effect of organic matter on functional performance and bacterial community shift of nitrite-oxidizing and heterotrophic bacteria under suspended state. The bacteria were prepared in a continuous-flow stirred reactor under autotrophic condition; after two months, the nitrification rate of the culture reached about 20 mg N/ (L·d); then the bacteria were harvested for the next batch experiments. The initial concentrations of nitrite were 126 ± 6 mg N/ L in all flasks, and sodium acetate (C) to nitrite (N) ratios were 0, 0.44, 0.88, 4.41, and 8.82, respectively. The results showed that at low C/N ratios (0.44 or 0.88), the nitrite removal rate was higher than that obtained under autotrophic condition and the bacteria had single growth phase, while at high C/N ratios (4.41 or 8.82), continuous aerobic nitrification and denitrification occurred besides higher nitrite removal rates, and the bacteria had double growth phases. The community structure of total bacteria strikingly varied with the different C/N ratios; the dominant populations shifted from autotrophic and oligotrophic bacteria (NOB, and some strains of Bacteroidetes, Alphaproteobacteria, Actinobacteria, and green nonsulfur bacteria) to heterotrophic and denitrifying bacteria (strains of Gammaproteobacteria, especially Pseudomonas stutzeri and P. nitroreducens). Part two describes the influence of acetate on nitrite oxidizing performance, community structure and metabolic function of nitrite-oxidizing and heterotrophic bacteria in biofilms. Bacterial enrichments was transferred into flasks with polypropylene carriers and cultured under agitated and autotrophic condition. After two month, the biofilms grown on the carriers had a nitrification rate of about 20 mg N/ (L·h); then the biofilms were refreshed with mixotrophic medium (nitrite were 200 mg N/ L in all flasks, and C/N ratios was the same as above) every 12 h. the results show: normal nitrite oxidization reactions were performed when C/N = 0, but nitrite oxidization and partial denitrification occurred with low C/N ratios (0.44 or 0.88). At high C/N ratios (4.41 or 8.82), we mainly observed denitrification. In contrast to C/N = 0, the nitrite oxidization rate was unaffected when C/N = 0.44, but decreased with C/N = 0.88. The structure of bacterial communities varied significantly between autotrophic and mixotrophic conditions. Nitrobacter was hard to detect by PCR-DGGE while heterotrophs and especially denitrifiers were in the majority under mixotrophic conditions. Real-time PCR indicated that the Nitrobacter population decreased from 2.42 × 104 to 1.34 × 103 16S rRNA gene copies/ ng DNA, while the quantity of denitrifiers obviously increased from 0 to 2.51×104 nosZ gene copies/ ng DNA with an increasing C/N ratio. SDS-PAGE indicated the complexity of and a certain difference between the proteome of nitrite-oxidizing and heterotrophic bacteria at different C/N ratios. We conclude that the influence of organic matter on nitrite oxidation and the community structure of NOB and heterotrophic bacteria is complex. In this dissertation, we focused on how sodium acetate influenced the system both under suspended state and in biofilms. We observed that acetate did not necessarily have a negative impact on nitrification. Instead, an appropriate amount of acetate benefited both nitrite oxidization and denitrification. These findings provide a greater understanding about the relationship between organics and nitrification; they fill the gaps in the field of microbial ecology of nitrifying bacteria; they also provide insight into how to minimize the negative impact of heterotrophic bacteria and maximize the benefit of nitrogen removal in biological treatment systems.

耐酸产甲烷菌的分离及其复合菌剂的研究

本文从成都龙泉垃圾填埋场和宜宾造纸厂分离到耐酸性能优良的高温产甲烷菌RY3和中温产甲烷菌SH4，并将其与实验室现有的利用不同底物的产甲烷菌配伍组合成了复合菌剂。采用活性污泥作为固体附着物，研制出了固体产甲烷菌复合菌剂。 菌株RY3的pH耐受范围为5.5～10.5，最适生长pH 6.0～8.0。菌株RY3为革兰氏阳性，长杆状，多数单生，不运动；菌落浅黄色，形状近圆形；利用H2+CO2或甲酸盐作为唯一碳源生长，不利用乙酸盐，对氯霉素非常敏感。该菌最适生长温度为55℃～65℃，最适NaCl浓度为0～2%。根据形态和生理生化特性及16S rDNA序列分析将其初步定为热自养甲烷热杆菌（Methanothermobacter thermautotrophicus）。添加RY3菌液与仅添加厌氧污泥作为接种物相比一周内可使达到最大产甲烷速率所需时间缩短三分之二，甲烷总产量提高约1.8倍。菌株SH4的生长pH范围5.5～9.5，其对酸碱具有良好的适应性，培养3天后，在初始pH值为6.0～8.0的培养基中甲烷产量相差不大，且基本达到最大产量。SH4革兰氏染色阳性，短杆状，多数单生，不运动；菌落近圆形，微黄；利用H2+CO2或甲酸盐作为唯一碳源生长，不利用乙酸盐，对氯霉素非常敏感。SH4最适生长pH 为7.0，最适生长温度为35℃，最适NaCl浓度为0～1.5%。实验表明，添加SH4菌液与仅添加厌氧污泥作为接种物相比可使产甲烷启动时间缩短三分之一，甲烷总产量亦有大幅提高。从形态和生理生化特征以及16S rDNA序列分析表明SH4为嗜树木甲烷短杆菌（Methanobrevibacter arboriphilus）。 以活性污泥为附着物，与培养基和菌种经搅拌后厌氧发酵可得产甲烷菌固体复合菌剂。固体复合菌剂的pH耐受范围为5.5～9.5，温度耐受范围为15℃～65℃，表明其对环境的适应性较强。以猪粪为底物进行厌氧发酵，接种复合菌剂进行试验，以接种实验室长期富集的产甲烷厌氧污泥作为对照，在20℃时，发酵甲烷浓度与对照基本一致，但每日产气量优于对照，第15天时接种复合菌剂的发酵瓶每日产气量是对照的1.59倍；50℃时达到最大甲烷含量所需时间比对照缩短三分之二，三周内总产气量约为对照的2.7倍，甲烷总产量约为2.8倍。以不加接种物为对照，接种复合菌剂20℃时发酵甲烷含量达到50%约需2周，对照2周内甲烷含量最高仅为4.3%；50℃时接种复合菌剂发酵仅需约1周甲烷含量便可达50%，对照则至少需要2周。 In this paper, high-temperature Methanogen RY3 and middle-temperature SH4 were isolated from Chengdu Longquan refuse landfill and Yibin paper mill. They could be used to make compound inoculum that producing methane with the existing Methanogens utilized different substrate. With using anaerobic activated sludge be solid fixture, the process had been designed to produce solid compound inoculum. Strain RY3 possessed excellent capacity of acid and alkali-tolerant. The pH-tolerant scale of RY3 was 5.5～10.5 and its optimum pH value for growth was 6.0～8.0. RY3 was G+, long-rod shape, monothetic and nonmotile, the colony was pale yellow with suborbicular-shape. Formate or H2+CO2 but not acetate was utilized by RY3 as sole C-source, and it was very sensitive to chloramphenicol. Besides, strain RY3 grew fastest at 55℃～65 and 0℃～2% NaCl. Characteristics of modality and physiology with sequence analysis of the 16s rDNA gene of strain RY3 preliminarily showed that it was Methanothermobacter thermautotrophicus. The experiments indicated that the time which began to produce methane with the highest velocity could be shortened two third by adding RY3 in one week, and the total methane production also was 1.8 times than before. Strain SH4 possessed wide scale of growing pH（5.5～9.5）and excellent ability of acclimatizing itself to acid-alkali. The methane production had no apparent difference among those cultivated in different initial pH（6.0～8.0）after three days and equaled to the maximum production basically. Cells of SH4 were G+, short-rod sharp, monothetic and nonmotile. The colony was pale yellow with suborbicular-shape. Formate or H2+CO2 but not acetate was utilized by SH4 as sole C-source, and it was very sensitive to chloramphenicol. Besides, it grew fastest at pH 7.0，55 ℃～65 and 0℃～2% NaCl concentration. The experiment indicated the time that began to produce methane could be shortening one third by adding SH4. And the total methane production also rose apparently. Characteristic of modality and physiology with sequence analysis of the 16S rDNA gene of strain SH4 demonstrated it was Methanobrevibacter arboriphilus. The activated sludge was utilized as fixture, mixed with culture medium and inocolum, that the solid compound inoculum could be produced by anaerobic fermentation. The compound inoculum could grow between pH 5.5～9.5, 15℃～65. It demonstrated the compound inoculum ha℃ve great ability of adapting to circumstance. In the experiment that making pig manure be substrate and taking the anaerobic sludge producing methane that cultured in long term in laboratory to be comparison, the concentration of methane in fermentation added compound inoculum almost equal to the comparison at 20℃, but the volume of gas production could be a little higher. The gas production everyday inoculated compound inoculum was 1.59 times to comparison. The time that the concentration of methane to maximum could be shortening by two third by adding compound inoculum, and the total gas production was 2.7 times to comprison while the total methane production was 2.8 times. If take the no inoculum be the comprasion, anaerobic fermentation added compound inoculum made the concentration of methane to 50% in 2 weeks but the comparison only to 4.3% at 20℃. The time that the concentration of methane to 50% by adding compound inoculum only need 1 week, but the comparison need 2 weeks at 50℃.

Two-stage photo-biological production of hydrogen by marine green alga Platymonas subcordiformis

A marine green alga, Platymonas subcordiformis, was demonstrated to photobiologically evolve hydrogen (H-2) after the first stage of photosynthesis, when subjected to a two-phase incubation protocol in a second stage of H2 production: anaerobic incubation in the dark followed by the exposure to light illumination. The anaerobic incubation induced hydrogenase activity to catalyse H? evolution in the following phase of light illumination. H,) evolution strongly depended upon the duration of anaerobic incubation, deprivation of sulphur (S) from the medium and the medium pH. An optimal anaerobic incubation period of 32 h gave the maximum H2 evolution in the second phase in the absence of sulphur. Evolution of H,) was greatly enhanced by 13 times when S was deprived from the medium. This result suggests that S plays a critical role in the mediation of H-2 evolution from R subcordiformis. A 14-fold increase in H-2 production was obtained when the medium pH increased from 5 to 8; with a sharp decline at pH above eight. H-2 evolution was enhanced by 30-50% when supplementing the optimal concentrations of 25 mM acetate and 37.5 mM glucose. (C) 2003 Elsevier B.V. All rights reserved.