Ammonia biofiltration and community analysis of ammonia-oxidizing bacteria in biofilters

Biological removal of ammonia was investigated using compost and sludge as packing materials in laboratory-scale biofilters. The aim of this study is to characterize the composition of ammonia-oxidizing bacteria (AOB) in two biofilters designed to remove ammonia. Experimental tests and measurements included analysis of removal efficiency and metabolic products. The inlet concentration of ammonia applied was 20–100 mg m⁻³. Removal efficiencies of BFC and BFS were in the range of 97–99% and 95–99%, respectively. Periodic analysis of the biofilter packing materials showed ammonia was removed from air stream by nitrification and by the improved absorption of NH₃ in the resultant acidity. Nitrate was the dominant product of NH₃ transformation. Changes in the composition of AOB were examined by using nested PCR, denaturing gradient gel electrophoresis (DGGE) and sequencing of DGGE bands. DGGE analysis of biofilter samples revealed that shifts in the community structure of AOB were observed in the experiment; however, the idle phase did not cause the structural shift of AOB. Phylogenetic analysis revealed the population of AOB showed Nitrosospira sp. remains the predominant population in BFC, while Nitrosomonas sp. is the predominant population in BFS.     

应用变性梯度凝胶电泳和16S rDNA序列分析对kefir粒中细菌多样性的研究

以开菲尔(Kefir)粒为材料,经过DNA抽提和16S rDNA V3区PCR扩增,扩增产物经变性梯度凝胶电泳(DGGE)分离并切割电泳条带进行序列测定,并与现有的数据库进行了比较,对Kefir粒的细菌多样性进行分析。结果表明,DGGE图谱中可检测到的8条带的16S rDNA基因序列中有7个基因序列与GenBank数据库登录的相关序列的相似性大于98%,余下的1个基因序列的相似性也大于96%。相似性大于98%的7个克隆中,有3个属于鞘氨醇杆菌属(Sphingobacterium),2个属于乳杆菌属(Lactobacillus),其它2个分别属于肠杆菌属(Enterobacter)和不动杆菌属(Acinetobacter)。首次报道了鞘氨醇杆菌作为优势菌群存在开菲尔Kefir粒中。     

应用变性梯度凝胶电泳和16SrDNA序列分析对kefir粒中细菌多样性的研究

以开菲尔（Kefir）粒为材料,经过DNA抽提和16SrDNA V3区PCR扩增,扩增产物经变性梯度凝胶电泳（DGGE）分离并切割电泳条带进行序列测定,并与现有的数据库进行了比较,对Kefir粒的细菌多样性进行分析。结果表明,DGGE图谱中可检测到的8条带的16SrDNA基因序列中有7个基因序列与GenBank数据库登录的相关序列的相似性大于98％,余下的1个基因序列的相似性也大于96％。相似性大于98％的7个克隆中,有3个属于鞘氨醇杆菌属（Sphingobacterium）,2个属于乳杆菌属（Lactobacillus）,其它2个分别属于肠杆菌属（Errterobacter）和不动杆菌属（Acinetobacter）。首次报道了鞘氨醇杆菌作为优势菌群存在开菲尔Kefir粒中。     

Ethylene removal efficiency and bacterial community diversity of a natural zeolite biofilter

To establish an economical and environmentally friendly technology for ethylene removal from horticultural facilities and industrial point sources, a bench-scale natural zeolite biofiltration system was developed in this study. The system was evaluated for its performance in removing ethylene from an artificially contaminated air stream and characterized for its bacterial diversity under varied ethylene concentrations, and in different spatial stages of the filter. The biofilter enabled to approximately 100% remove ethylene at loading rates of 0.26-3.76 g m⁻³ h⁻¹ when operated with inoculum containing enriched ethylene-degrading bacteria. The bacterial diversity and abundance varied with the height of the biofilter. Moreover, the occurrence and predominance of specific bacterial species varied with the concentrations of ethylene introduced into the biofilter, as observed by PCR-DGGE methods. Phylogenetic analysis indicated that the biofilter system supported a diverse community of ethylene-degrading bacteria, with high similarity to species in the classes Betaproteobacteria, Gammaproteobacteria, Bacilli, and Actinobacteria.     

肉鸭养殖过程中发酵床垫料菌群结构变化的研究

【目的】探究发酵床使用时间和肉鸭粪便微生物对发酵床垫料菌群结构、总菌和大肠杆菌数量的影响。【方法】采集江苏某肉鸭发酵床养殖场内刚制作完成的发酵床垫料样品,及其饲养4批次、8批次后的垫料样品,同时采集各批次34日龄肉鸭粪便样品,采用变性梯度凝胶电泳技术(Denaturing gradient gel electrophoresis,DGGE)、16S rRNA基因序列分析和实时荧光定量PCR (Quantitative real-time PCR)技术对发酵床使用过程中垫料菌群结构进行定性和定量研究。【结果】0批次(D0)与4批次(D4)、8批次(D8)垫料菌群相似性分别为68.81%、70.82%,而4批次与8批次间垫料菌群的相似性则达81.93%,显著高于D4、D8与D0间相似性(P<0.05)。条带6、8 (最相似菌分别为Leqionella tunisiensis、Pedobacte bauzanensis)在3个时间点垫料菌群中均表现优势,且含量较为稳定;条带10 (最相似菌为Rummeliibacillus suwonesis)仅在2个重复使用垫料菌群中表现优势;条带12、13 (最相似菌分别是Psychrobacter sp. PRwf-1、Iamia majanohamensis)共同存在于垫料样和粪便样。肉鸭粪便中大肠杆菌的数量显著高于4批次、8批次垫料中的数量(P<0.05),与0批次垫料间差异不显著(P>0.05)。【结论】使用时间和肉鸭粪便微生物共同影响了发酵床垫料菌群结构和数量,菌群结构随使用时间的延长而趋于稳定。     

73例中国人血友病甲基因突变的分析

我们用Southern blotting、PCR、变性梯度凝胶电泳(DGGE)和DNA测序等方法对73例血友病甲患者(经上海瑞金医院测定血浆FVⅢ:C和vWF:Ag诊断,其中无亲缘关系患者65例,按FVⅢ:C水平分为轻、中、重三型。FVⅢ:C< 2%为重型,共47例;FVⅢ:C 2%-5%为中型,共9例;FVⅢ:C5%-25%为轻型,共1 7例)进行FVⅢ基因突变检测。共检出内含子22倒位23例,均为重型,约占重型的49%,与国外报道相似。余下50例(其中无亲缘关系者45)用PCR-DGG E分析所有外显子及其侧翼内含子序列,发现异常条带则进行DNA测序。在17例患者中检出突变13种,其中无义突变5种,均为重型;错义突变6种,除1例外都是轻中型;小缺失2例,都是重型;其中,AA466Lys(AAG)-Thr(ACG)、719Tyr(TAC)-Stop(TAG)、AA826 Asp(GAC)-Glu(GAA)、312Ile(ATC)-xxC及AA1551-1552del(AGAA)为新发现的突变。有亲缘关系的患者都有相同的基因突变,而在无亲缘关系患者未发现相同突变。基因突变与临床表现基本相符。 Abstract:We use Southern blotting,PCR,denaturing gradient gel electrophoresis(DGGE)and DNA sequencing to detect gene mutations of haemophilia A in Chinese population.73 cases(47 severe)(FVIII:C<2%),9 moderate(FVIII:C 2%~5%),17 mild(FVIII:C 5%~25%)of haemophilia A were first screened with Southern blotting,23 were found to be the intron 22 inversion type,all being severe cases.The remaining 50 cases without intron 22 in version were examined with PCR-DGGE.Genomic DNA were amplified using GC-clamped primers covering all the exons and all flanking intron regions.Abnormal bands were sequenced.13 different mutations were identified,including 5 nonsense mutations,6 missense mutations and 2 small deletions.5 mutations,AA466Lys(AAG)-Thr(ACG),AA719Tyr(TAC)-Stop(TAG),AA826Asp(GAC)-Glu(GAA),AA312Ile(ATC)-xxC and AA1551-1552del(AGAA)have not been reported before.Generally the genetic defects correspond to the clinical conditions.     

变性梯度凝胶电泳法研究断奶仔猪粪样细菌区系变化

利用PCR和DGGE技术分析了12头仔猪在断奶后其粪样细菌区系的变化。粪样细菌16S rDNA的V6～V8可变区经PCR扩增,扩增产物经DGGE电泳后再进行相似性分析。结果表明,仔猪断奶当天粪样 DGGE谱带少,同窝仔猪间图谱相似。断奶后,随着断奶时间的推移,每头仔猪的DGGE图谱带逐渐增多,变得复杂和多样,仔猪个体间DGGE图谱差异逐渐增大。仔猪是否同窝以及所采食日粮类型对DGGE图谱没有明显影响。相似性分析还表明,日粮中添加寡果糖的仔猪在断奶后第1周,其粪样微生物区系变化迅速,而后缓慢。     

不同DGGE谱带信息提取方法对分析结果的影响

自1993年Muyzer,et al.[1]将变性梯度凝胶电泳技术(Denaturing gradient gel electrophoresis,DGGE)引入到微生物生态学研究以来,DGGE已被广泛应用于各种生态系统(如淡水、海洋、土壤、动物消化道等)的微生物群落结构分析[2—6]。但对于DGGE凝胶的分析至今仍没有统一的方法,     

Successional development of sulfate-reducing bacterial populations and their activities in an activated sludge immobilized agar gel film

A combination of fluorescence in situ hybridization (FISH), microprofiles, and denaturing gradient gel electrophoresis (DGGE) analysis of PCR-amplified 16S rDNA fragments followed by hybridization analysis with specific probes was applied to investigate successional development of sulfate-reducing bacteria (SRB) community structure and in situ sulfide production activity within an activated sludge immobilized agar gel film. In this model biofilm system, since biases arising from biofilm heterogeneity can be ignored, the population dynamics of SRB in the agar gel is directly related to physiological capability and in situ activity of SRB. Microelectrode measurements showed that an anoxic zone was already developed at the beginning (0 day), a first sulfide production of 0.054 mumol H2S m(-2) x s(-1) was detected during the first week, and the rate increased gradually to 0.221 mumol H2S m(-2) x s(-1) in the fifth week. The most active sulfide production zone moved upward to the chemocline and intensified with time to form a narrow zone with high volumetric sulfide production rates. This result coincided with the shift of the spatial distributions of SRB populations determined by FISH. In situ hybridization with probe SRB385 for mainly general SRB of the delta Proteobacteria plus some gram-positive bacteria and probe 660 for Desulfobulbus indicated that the most abundant populations of SRB were primarily restricted to near the oxic/anoxic interface (chemocline). A close observation of the development of the vertical distributions of SRB populations revealed that the cell numbers of Desulfobulbus tripled (from 0.5 x 10(8) to 1.5 x 10(8) cells cm(-3)) near the oxic/anoxic interface. Similar growth (from 1.0 x10(8) to 4.5 x 10(8) cells cm(-3)) of Desulfovibrio-like SRB that hybridized with probe SRB385 was observed. PCR-DGGE followed by hybridization analysis revealed that one Desulfobulbus strain was detected from the beginning, and another strain appeared after 1 week, coinciding with the first detected sulfide production. In addition, three strains hybridizing with probe 687 (possibly Desulfovibrio) were also dominant SRB in the agar gel.     

凝胶条带内DNA回收测序评估DGGE可靠性

为了评估DGGE的可靠性,对DGGE条带中回收的DNA片段进行了测序比较分析,并引入了DGGE可靠性指数的概念评价其可靠性。结果显示同一条DGGE条带回收的DNA来自同一属的概率为64.7%,相同位置的DGGE条带可以被认为是同一OTU;不同的DGGE条带回收到类似的DNA序列（16S rDNA V3区差异小于4 bp）的概率为10.5%;DGGE可靠性指数为74.8%。以上结果表明尽管DGGE技术与理论预期存在一定的差距,但是DGGE技术基本能够反映微生物群落的多样性。       

贡嘎蝠蛾幼虫肠道细菌多样性分析

[目的]对实验室养殖条件下的重要经济昆虫冬虫夏草寄主-贡嘎蝠蛾(Hepialus gonggaensis,Hg)幼虫肠道微生物群落的多样性进行了研究.[方法]采用常规分离培养与分子鉴定的方法和基于16S rRNA作为分子标记的变性梯度凝胶电泳(denaturing gradient gel electrophoresis,DGGE)的方法.[结果]用常规分离与分子鉴定方法获得8个属的细菌类群,其中肠杆菌属(Enterobacter)是优势菌群,肉食杆菌属(Carnobacterium)是次优势菌群.对通过DGGE方法得到的11条16S rRNA优势条带序列进行了比对和系统进化树分析,结果表明肉食杆菌属(Carnobacterium)的丰度最高,是肠道细菌中主要的优势菌群,芽孢杆菌属(Bacillus)是次优势菌群.DGGE图谱还显示Hg幼虫不同虫龄肠道细菌菌群的结构存在差异,推测可能与其发育生理状态的差异有关系.[结论]结合常规分离法与DGGE法能够更有效的分析肠道微生物的多样性,获得更多更全面的微生物多样性信息.     

变性梯度凝胶电泳在实验小鼠细菌检测中的应用

目的 研究变性梯度凝胶电泳(denatured gradient gel electrophoresis, DGGE)在实验小鼠细菌检测中的应用。方法 根据16S rDNA V3区引物,PCR扩增3种实验小鼠(KM小鼠、NIH小鼠和BALB/c小鼠)呼吸道和盲肠段的细菌基因组DNA;扩增产物运用DGGE进行电泳检测,并分析条带数量间差异的统计学意义。结果 KM小鼠盲肠段条带12~18条,呼吸道条带5~10条;NIH小鼠盲肠段条带15~20条,呼吸道条带4~10条;BALB/c小鼠盲肠段条带10~15条,呼吸道条带0~7条。统计分析结果显示,KM小鼠和NIH小鼠在盲肠和呼吸道电泳条带数量上的差异无统计学意义( P >0.05);BALB/c小鼠与KM小鼠、NIH小鼠间的差异均有统计学意义( P <0.05)。结论 DGGE 在实验小鼠盲肠和呼吸道细菌检测中能较好地反映菌群的物种多样性。     

青海两盐湖细菌多样性研究

用DGGE法和纯培养法,对青海柯柯盐湖、茶卡盐湖底泥及周边土壤样品的细菌多样性进行了研究。结果显示,两个盐湖存在大量的未知细菌, 分离到的纯培养仅占实有细菌的小部分。采用多相分类方法,鉴定了12株纯培养细菌,属5个可能的新种,另有一株菌可能成立一个新属。认为提出新思路、设计新程序,从自然极端环境取样,分离未知菌,是微生物资源开发利用的关键之一。     

Methanotrophic diversity in an agricultural soil as evaluated by denaturing gradient gel electrophoresis profiles of pmoA, mxaF and 16S rDNA sequences

Molecular methods were used to characterize the diversity of a methanotrophic population in an agricultural soil. For this purpose we have used DGGE analysis of functional and phylogenetic markers. Functional markers utilised comprised the pmoA-gene coding for the -subunit of the particulate methane monooxygenase (pMMO) present in all known methanotrophs and the mxaF-gene coding for the -subunit of methanol dehydrogenase (MDH) present in all Gram-negative methylotrophs. In addition, we have used 16S rDNA as a phylogenetic marker. DGGE patterns of an enrichment culture, and sequencing of major DGGE bands obtained with the bacterial specific primers showed that the community structure was dominated by methanotrophic populations related to Methylobacter sp. and Methylomicrobium sp. The PCR products amplified with the functional primer sets were related to both type I and type II methanotrophs. We also designed a new pmoA-targeting primer set which could be used in a nested protocol to amplify PCR-products from DNA extracted directly from the soil.     

几株红假单胞菌属细菌的表观特征及其遗传多样性研究

采用变性梯度胶电泳(DGGE)分析方法和传统的表型特征研究方法、化学方法、核酸杂交方法等技术对14株紫色非硫细菌进行了多相研究。它们均具有红假单胞菌属(Rhodopseudomonas)的基本特征：具片层状光合内膜结构,出芽分裂,含细菌叶绿素α和正常的螺菌黄素等。根据形态大小、黑暗条件下能否形成好氧菌落及碳源利用上的差异可将14株菌分为T群和gc群两群。用一对引物341f～906r扩增3株标准菌株Rps.palustris ATCC 17001、Rps.rutila R1、Rps.julia ATCC 51105和14株分离株的16S rRNA基因片断,作DGGE分析,结果发现,17株菌中有5个遗传型：gc型、R1型、T型、pal型、jul型。3个标准菌株分别是R1型、pal型、jul型,而14株分离菌株除包括R1型外,另有2个新的遗传型：T型和gc型。几个代表菌株的总DNA的杂交结果表明,T型和gc型可能代表2个新的种群。     

Effect of disodium fumarate on ruminal metabolism and rumen bacterial communities as revealed by denaturing gradient gel electrophoresis analysis of 16S ribosomal DNA

The aim of the present study was to examine the effects of feeding diets with addition of disodium fumarate (DF) to goats on ruminal metabolism and changes of rumen bacterial communities. Four cannulated goats were used in a 4 × 4 Latin square design. The results showed that ruminal pH increased linearly (P<0.01)as the amount of DF added increased, while lactate production decreased linearly (P<0.01). DF addition did not affect the production of acetate, propionate, butyrate, TVFA and NH₃-N. The effect of DF on the changes in rumen bacterial-community structure of goats was analyzed using 16S rDNA-based approaches. Amplicons of the V6-V8 variable regions of bacterial 16S rDNA were analyzed by denaturing gradient gel electrophoresis (DGGE), cloning and sequencing. Differences in rumen bacterial community structure were determined based on the Shannon index of diversity for pairwise comparison of the DGGE fingerprints and revealed significant changes in rumen microbiota after DF addition. As compared with those fed with the control diet, goats fed on the diets with DF addition showed a higher bacterial diversity. The sequences of seven amplicons in total 11 clones showed less than 97% similarity with those of previously identified or unidentified bacteria, suggesting that most bacteria in the gastrointestinal tract have not been cultured or identified. Amplicons related to Succinivibrio dextrinosolvens species were found in most DGGE fingerprints derived from goats on the diet containing DF, but not in goats on the control diet. These results demonstrated the ability of DF to improve the metabolism of rumen lactate fermentation and to influence the bacterial composition of the rumen in goats.     

三江源地区高寒草甸土壤氨氧化细菌多样性研究

目的:利用16S rDNA-PCR-DGGE方法首次对青藏高原核心区三江源自然保护区的高寒草甸样地的土壤氨氧化细菌进行了多样性研究。方法:从土壤中抽提微生物总DNA,运用巢式PCR扩增氨氧化细菌16S rRNA基因的V3可变区,结合DGGE(denaturing gradient gel electrophoresis)技术分析样品氨氧化细菌群落组成,并利用相关性分析探讨了群落结构多样性与环境因子的联系。结果:4个样地(玉树S1;囊谦S2;治多S3和S4)共检测到14个多态性条带,多样性指数为1.69～2.00。相关性分析显示多样性指数与海拔、C/N呈负相关,与NO3-呈显著正相关。结论:海拔、C/N及NO3-可能是影响土壤氨氧化细菌多样性的重要因子。     

有机碳源下废水厌氧氨氧化同步脱氮除碳

为明确有机碳源胁迫下,厌氧氨氧化反应器的同步脱氮除碳规律及功能微生物群落结构的动态变化,采用成功启动的厌氧氨氧化UASB反应器,通过逐步提升进水有机负荷,探究有机碳源下废水厌氧氨氧化同步脱氮除碳。研究表明,当进水化学需氧量(Chemical oxygen demand,COD)浓度从172 mg/L升至620 mg/L,反应器维持较高的脱氮效率,氨氮和总氮去除率均在85%以上,并对COD具有平均56.6%的去除率,高浓度COD未对Anammox菌活性构成显著抑制作用。聚合酶链式反应和变性梯度凝胶电泳(PCR-DGGE)图谱和割胶测序结果表明,变形菌门Proteobacteria、浮霉菌门Planctomycetes、绿曲挠菌门Chloroflexi以及绿菌门Chlorobi等微生物共存于同一反应体系中,推测反应器内存在复杂的脱氮除碳途径。而且,代表厌氧氨氧化的部分浮霉菌门微生物能耐受高浓度有机碳源,在高有机负荷下依旧发挥着高效的脱氮作用,为反应器高效脱氮提供了保障。     

16S rDNA技术研究新生腹泻仔猪粪样细菌区系的多样性变化

用PCR/DGGE技术跟踪一窝5头新生腹泻仔猪自然康复、补饲、断奶过程中粪样细菌区系的演变,构建3头仔猪42日龄粪样的16S rDNA克隆库,分析匹配于DGGE优势谱带23个克隆的16S rDNA序列。结果表明,DGGE图谱由简单(2日龄)到复杂(10日龄),再回复简单(16日龄)到复杂(断奶),最后趋于稳定。2、16日龄DGGE图谱最简单、相似,最优势谱带为大肠杆菌;10日龄(补饲后3天)图谱复杂,大肠杆菌存在但不是最优势谱带,补饲前后图谱的相似性低,补饲导致了粪样细菌区系结构的显著变化;断奶前(27日龄)和后(35、42日龄)图谱复杂,优势谱带、图谱相似性均趋向稳定。序列分析表明,23个克隆中除5个与未知细菌最相似外,其余最相似菌分属于肠球菌(Enterococcus),链球菌(Streptococcus),梭菌(Clostridium),消化链球菌(Peptostreptococcus)和乳酸杆菌(Lactobacillus)。