【作者】 周盛；

【导师】 韦朝海；

【作者基本信息】 华南理工大学 ， 环境工程， 2009， 博士

【摘要】 为了快速、准确地从焦化废水活性污泥中筛选一些功能微生物和一些能激活活性污泥中的功能微生物的激活剂;同时为了探索焦化废水活性污泥中功能微生物对废水生物处理的强化作用。研究通过以下两种种新途径即PCR-DGGE作辅助筛选手段、改进后BIOLOG微平板分别对功能微生物和一些能激活活性污泥中的功能微生物的激活剂进行筛选和研究:1.通过苯酚富集后平板分离常规筛选法和通过牛肉膏蛋白胨固体培养基、高氏1号固体培养基、完全固体培养基（TYG culture medium）TYG直接平板分离并通过PCR-DGGE从理论上鉴定。结果筛选到两株A1、C1为原生境中主要功能微生物;两株B1、D1为原生境中非主要功能微生物;5株A、B、C、D、F1为原生境中非功能微生物。后经检测结果表明:在活性污泥中分别投加目标功能菌A1、B1、C1、D1、E1进行强化时,系统内COD为600 mg/L的焦化废水分别经40 h、48 h、32 h、40 h、48 h的曝气,COD分别降至128 mg/L、160 mg/L、118 mg/L、156 mg/L、170 mg/L后稳定。当在活性污泥中分别投加目标非功能菌F1、A、B、C、D进行强化时,焦化废水分别经56 h曝气,COD分别降至218 mg/L、216 mg/L、215 mg/L、219 mg/L、217 mg/L后稳定,该结果与对照组降解实验结果基本一致。结果表明:活性污泥中主要功能菌的投加,对活性污泥生物处理的强化效果最为明显;非主要功能菌的投加对废水生物处理起强化效果不佳,不如主要功能菌对活性污泥的强化效果明显。非功能菌的投加对废水生物处理基本无任何强化作用。因此,通过PCR-DGGE作筛选功能微生物辅助手段是一种新的有效的筛选方法,该方法可从PCR-DGGE的图谱上直接预先判断出从原活性污泥生境中筛选到的微生物是否为原活性污泥生境中的功能微生物,避免常规方法筛选到的非主要功能微生物甚至非功能微生物。2.研究通过改进后的BIOLOG微平板技术从微生物生长繁殖所须的生长因子（氨基酸,碱基）、维生素、大量元素、微量元素中进行筛选。结果筛选到6种焦化废水活性污泥激活剂。后经PCR-DGGE和分子进化树对被激活剂激活后的微生物功能菌进行分析得知γ-Ami,VB12,L-Pro,DL-Ala激活剂极可能分别对活性污泥中Rhodococcus erythropolis,Alcaligenes faecalis,Uncultured Pseudomonas sp.,Pseudomonas sp.苯酚降解功能菌有激活作用;而激活剂A、L-His激活剂极可能分别对活性污泥中Micrococcus Sp.,Uncultured Bacillus sp.非苯酚降解功能菌有激活作用。结果表明:Biolog微平板筛选法简单、方便、占用空间小、耗材少,是多种待筛选的目标物有效的高通量筛选方法。稀土盐是焦化废水活性污泥一特殊激活剂。研究通过PCR-DGGE,构建系统发育树,对不同剂量的稀土盐氯化镧（LaCl₃·3H₂O）对焦化废水活性污泥在TYG培养基中生长进行分子生态学研究以及其生物多样性分析。结果表明低剂量浓度5 mg/L和10 mg/L LaCl₃·3H₂O对焦化废水活性污泥在TYG培养基中生长有一定刺激作用,生物多样性比对照组多;高剂量浓度200 mg/L和400 mg/L LaCl₃·3H₂O对焦化废水活性污泥在TYG培养基中生长有一定抑制作用,生物多样性比对照组少。低剂量浓度5 mg/L和10 mg/L LaCl₃·3H₂O极可能刺激较有代表的微生物类群为未培养微生物Uncultured Pseudomonas sp.（AB076873）、Uncultured Bacillus sp.（EF072899）、Uncultured bacterium clone SR40（DQ298294）及可培养微生物类群bacterium rj11。更多还原

【Abstract】 To screen some functional microbes from coking wastewater activated sludge and to screen some activator that the functional microbes in coking wastewater activated sludge could be activated by those activators, at the same time for the sake of researching the functional microorganisms strengthen effect in the coking wastewater activated sludge, three approachs have been used to screen and study as follow:Firstly,Four domainent bacteria strains A, B, C, D has been isolated after enriched from the enrichment environment. And six domainent bacteria strains A1, B1, C1, D1, E1, F1 has been isolated by TYG, Gao1 or Beef-cream peptone medium but not enriched from the enrichment environment. Ten strains A, B, C, D A1, B1, C1, D1, E1, F1 has been identify by PCR-DGGE in theory. Two strains A1and C1 are dominent functional microorganism, three strains B1, D1and E1 are non-dominent functional microorganism and five strains F1, A, B, C, D, is non- functional microorganism. As it shows from experiment result that strengthened with dominent functional microorganism A1 in activated sludge, the COD of coking wastewater decreases to 128 mg/L after 40 hours of aeration and it go stable; In the same way, strengthened with functional microorganism B1, C1, D1, E1 the COD decreases to 160 mg/L, 118 mg/L, 156 mg/L, 170 mg/L after 48 h, 32 h, 40 h , 48 h of aeration and they go stable respectively; strengthened with non-functional microorganism F1, A, B, C, D the COD decreases to 218 mg/L , 216 mg/L ,215 mg/L ,219 mg/L 217 mg/L after 56 hours of aeration and they go stable respectively, basically the same with the COD when no candidates bacteria are present in the activated sludge, which decreases to 220 mg/L after 56 h of aeration and it go stable. Conclusions: The presence of dominant functional microbes in activated sludge is the key factor in strengthening its capability of biological treatment; and the significance of the presence of non-dominant ones is second compared with dominant ones; the presence of non-functional microbes shows no effects. This study suggests that PCR-DGGE is effective as a new supplementary means of screening functional microbes from activated sludge. More importantly, the profile of PCR-DGGE allows the researchers to be conscious of whether the screened microbes are functional ones in the habitat of original activated sludge in advance, so that the non-functional microbes and functional ones can be identified clearly.Seeondly,This study employed the BIOLOG micro-plate technique to screen out the activators from growth factors （amino acids, bases）, vitamins, macroelements, trace elements, which are the necessities of microorganism reproduction. Six sorts of coking wastewater activated sludge activators were screened out. PCR-DGGE and molecular phylogenetic tree were used for the analysis of microorganism functional bacteria that have been already activated by the screened activators. The analysis suggests that it is possibly for activatorsγ-Ami,VB12,L-Pro,DL-Ala to respectively activate phenol degradation functional bacteria Rhodococcus erythropolis,Alcaligenes faecalis,Uncultured Pseudomonas sp.,Pseudomonas sp. in activated sludge; and it is possibly for activators A and L-His to respectively activate non-phenol degradation functional bacteria Micrococcus Sp.,Uncultured Bacillus sp in activated sludge. The research results suggest also that Biolog micro-plate screening program is characterized by simplicity, convenience, small space and less materials. Especially, multiple objects to be screened can be placed into only one micro-plate to perform high throughout screening simultaneously.The rare-earth salt is a special activator of activated sludge. It is not need the BIOLOG micro-plate technique to screen out. In this study,PCR-DGGE, built phylogenetic tree has been used in this study to analyze its biological diversity and to make molecular ecology study on the effects of rare-earth salt lanthanum chloride （LaCl₃·3H₂O） on the growth of some coking wastewater activated sludge microorganisms in TGY culture. It shows that at low dose of 5mg/L and 10mg/L, LaCl₃·3H₂O could stimulate the growth of coking wastewater activated sludge in TYG culture medium to some extent, and its diversity was more than the control group; at high dose of 200mg/L and 400mg/L, LaCl₃·3H₂O could inhibit the growth of coking wastewater activated sludge in TGY culture medium to some extent, and its diversity was less than control group. At the low dose of 5mg/L and 10mg/L, LaCl₃·3H₂O could stimulate the more representative microorganism groups, non-culturable microorganism group Uncultured Pseudomonas sp., Uncultured Bacillus sp. and Uncultured bacterium clone SR40 and culturable microorganism group Bacterium rJ11..更多还原