Start-up of autotrophic nitrogen removal reactors via sequential biocatalyst addition

A procedure for start-up of oxygen-limited autotrophic nitrification-denitrification (OLAND) in a lab-scale rotating biological contactor (RBC) is presented. In this one-step process, NH4+ is directly converted to N2 without the need for an organic carbon source. The approach is based on a sequential addition of two types of easily available biocatalyst to the reactor during start-up: aerobic nitrifying and anaerobic, granular methanogenic sludge. The first is added as a source of aerobic ammonia-oxidizing bacteria (AAOB), the second as a possible source of planctomycetes including anaerobic ammonia-oxidizing bacteria (AnAOB). The initial nitrifying biofilm serves as a matrix for anaerobic cell incorporation. By subsequently imposing oxygen limitation, one can create an optimal environment for autotrophic N removal. In this way, N removal of about 250 mg of N L(-1) d(-1) was achieved after 100 d treating a synthetic NH4+-rich wastewater. By gradually imposing higher loads on the reactor, the N elimination could be increased to about 1.8 g of N L(-1) d(-1) at 250 d. The resulting microbial community was compared with that of the inocula using general bacterial and AAOB- and planctomycete-specific PCR primers. Subsequently, the RBC reactor was shown to treat a sludge digestor effluent under suboptimal and strongly varying conditions. The RBC biocatalyst was also submitted to complete absence of oxygen in a fixed-film bioreactor (FFBR) and proved able to remove NH4+ with NO2- as electron acceptor (maximal 434 mg of NH4+-N (g of VSS)(-1) d(-1) on day 136). DGGE and real-time PCR analysis demonstrated that the RBC biofilm was dominated by members of the genus Nitrosomonas and close relatives of Kuenenia stuttgartiensis, a known AnAOB. The latter was enriched during FFBR operation, but AAOB were still present and the ratio planctomycetes/AAOB rRNA gene copies was about 4.3 after 136 d of reactor operation. Whether this relates to an active role of AAOB in the anoxic N removal process remains to be solved.     

Prevalence of anaerobic ammonium-oxidizing bacteria in contaminated groundwater

Anaerobic ammonium-oxidizing (anammox) bacteria perform an important step in the global nitrogen cycle: anaerobic oxidation of ammonium and reduction of nitrite to form dinitrogen gas (N(2)). Anammox organisms appear to be widely distributed in natural and artificial environments. However, their roles in groundwater ammonium attenuation remain unclear and only limited biomarker-based data confirmed their presence prior to this study. We used complementary molecular and isotope-based methods to assess anammox diversity and activity occurring at three ammonium-contaminated groundwater sites: quantitative PCR, denaturing gradient gel electrophoresis, sequencing of 16S rRNA genes, and (15)N-tracer incubations. Here we show that anammox performing organisms were abundant bacterial community members. Although all sites were dominated by Candidatus Brocadia-like sequences, the community at one site was particularly diverse, possessing four of five known genera of anammox bacteria. Isotope data showed that anammox produced up to 18 and 36% of N(2) at these sites. By combining molecular and isotopic results we have demonstrated the diversity, abundance, and activity of these autotrophic bacteria. Our results provide strong evidence for their important biogeochemical role in attenuating groundwater ammonium contamination.     

PCR-DGGE技术对中华开菲尔微生物菌群的分析

为了解中华开菲尔微生物菌群的结构特征,本论文运用聚合酶链式反应-变性梯度凝胶电泳(PCR-DGGE)技术对开菲尔菌株发酵过程中微生物菌群的结构变化进行了实验分析,结果表明:细菌菌群DGGE图谱上出现有三种不同迁移位置的斑带,而酵母菌群DGGE图谱上只有一条斑带;经过DNA序列的对比分析可知:细菌菌群分别为肠膜明串珠菌(Leuconostoc mesenteroides)、马乳样乳杆菌(Lactobacillus kefiranofaciens)和开菲尔乳杆菌(Lactobacillus kefir),它们的序列同源性都达到100%;酵母菌群为德尔布有孢圆酵母(Torulaspora delbrueckii),其序列同源性为99%。本论文首次报道了德尔布有孢圆酵母在开菲尔菌落中的存在。     

Evaluation of Bacterial Diversity in Palm Wine by 16S rDNA Analysis of Community DNA

Bacterial diversity and fermentation dynamics in palm wine, a traditional alcoholic fermented beverage, collected from upright palm trees from Idiaba community, Abeokuta, Ogun State, Nigeria were evaluated by DNA based method using the 16S rDNA of the microbial community to verify and complement previous reports, improve our understanding and document yet unreported, uncultured microbial diversity associated with palm wine. The 16S rRNA gene fragments were amplified from microbial community and genomic DNA of isolates, by Polymerase Chain Reaction (PCR) using universal primers; and sequenced. The partial sequences were identified by comparison with sequences deposited in the non-redundant nucleotide database of National Center for Biotechnology Information (NCBI). This analysis revealed that 32 community clones were identified as Lactobacillus sp, Lactobacillus casei strain zhang, Lactobacillusplantarum, Leuconostoc mesenteriodes ssp dextranicum, Leuconcostoc lactis, Pediococcusparvulus strain Bpe-299, Acetobacter pomorum, Acetobacter pasteurianus, Gluconobacter oxydans, Acinetobacter calcoaceticus, Enterobacterium bacterium, Acidovorax sp, Comamonas sp, Bacillus subtilis, Staphylococcuspiscifermentans and uncultured bacteria clone D1-78. The results showed that bacterial diversity in the palm wine sample is dominated by Lactobacillus and Leuconostoc species as reported by previous workers and uncultured bacteria clone D1-78 (1 clone) was detected for the first time in palm wine.     

芝麻香型白酒高温大曲嗜热细菌群落研究

利用高温筛选和传统培养方法,借助于核糖体DNA扩增片段限制性内切酶(ARDRA)分型、分子鉴定以及系统发育分析技术,研究了芝麻香型白酒高温大曲嗜热细菌群落结构。结果显示,55℃高温筛选获得的85株嗜热细菌分属于4个菌属,分别为Thermoactinomyces sp.,Bacillus sp.,Schlegelella sp.以及Streptomyces sp.,其中第1优势菌为Thermoactinomyces vulgaris,丰度为69.41%。该研究首次报道了芝麻香型白酒高温大曲中的嗜热细菌类群,为进一步探索其功能性以及建立其与芝麻香型白酒风味的相关性奠定了基础。     

鉴定乳制品中厌氧芽胞杆菌的3种方法比较

鉴定乳制品中的革兰氏阳性厌氧芽胞杆菌。方法 采用生化鉴定、基质辅助激光解析电离飞行时间质谱(MALDI-TOF MS)鉴定和16S rDNA测序三种方法,对乳制品中5株革兰氏阳性厌氧芽胞杆菌进行鉴定。结果 生化方法鉴定出3株菌,而飞行时间质谱鉴定和16S rDNA测序法对5株菌都给出了鉴定结果。3种方法仅对其中1株菌的鉴定结果一致;飞行时间质谱鉴定和16S rDNA测序的鉴定结果一致性程度高,共有4株菌的鉴定结果一致;而生化方法的鉴定结果与这两种方法差异较大。结论 飞行时间质谱鉴定和16S rDNA测序可作为生化方法的补充,用于革兰氏阳性厌氧芽胞杆菌的快速鉴定。     

Phylogenetic analysis of TCE-dechlorinating consortia enriched on a variety of electron donors

Two rapidly fermented electron donors, lactate and methanol, and two slowly fermented electron donors, propionate and butyrate, were selected for enrichment studies to evaluate the characteristics of anaerobic microbial consortia that reductively dechlorinate TCE to ethene. Each electron donor enrichment subculture demonstrated the ability to dechlorinate TCE to ethene through several serial transfers. Microbial community analyses based upon 16S rDNA, including terminal restriction fragment length polymorphism (T-RFLP) and clone library/sequencing, were performed to assess major changes in microbial community structure associated with electron donors capable of stimulating reductive dechlorination. Results demonstrated that five phylogenic subgroups or genera of bacteria were present in all consortia, including Dehalococcoides sp., low G+C Gram-positives (mostly Clostridium and Eubacterium sp.), Bacteroides sp., Citrobacter sp., and delta Proteobacteria (mostly Desulfovibrio sp.). Phylogenetic association indicates that only minor shifts in the microbial community structure occurred between the four alternate electron donor enrichments and the parent consortium. Inconsistent detection of Dehalococcoides spp. in clone libraries and T-RFLP of enrichment subcultures was resolved using quantitative polymerase chain reaction (Q-PCR). Q-PCR with primers specific to Dehalococcoides 16S rDNA resulted in positive detection of this species in all enrichments. Our results suggest that TCE-dechlorinating consortia can be stably maintained on a variety of electron donors and that quantities of Dehalococcoides cells detected with Dehalococcoides specific 16S rDNA primer/probe sets do not necessarily correlate well with solvent degradation rates.     

酱香型窖泥古菌群落结构的研究

采用ARDRA免培养手段研究酱香型窖泥中的古菌群落结构,通过对窖泥总DNA的提取,扩增古菌16SrDNA序列,构建古菌16S rDNA文库。随机挑选39个阳性克隆子,通过HhaI限制性内切酶酶切,选择酶切图谱不同的25个克隆子用于后续测序,测序结果与NCBI数据库比对分析,获得其分类信息。克隆文库分析结果表明,酱香型窖泥中古菌主要分布于广古菌门中的甲烷袋状菌属（Methanoculleus）、甲烷八叠球菌属（Methanosarcina）、甲烷鬃毛菌（Methanosaeta）和甲烷杆菌属（Methanobacterium）,分别占44%、41%、3%、9%。     

The microbial community in a 2,4-dinitrophenol-digesting reactor as revealed by 16S rDNA gene analysis

The microbial community of a 2,4-dinitrophenol-digesting reactor was investigated using different molecular biological techniques based on 16S rDNA gene sequences. A PCR-denaturing gradient gel electrophoresis (DGGE) analysis of the bacterial community in the reactor showed that one strong and five minor bands were observed in the DGGE profile. The results of excising and sequencing DGGE bands suggested that members of Rhodococcus, Nocardioides, and Nitrospira species were present in the reactor. Partial sequencing of cloned 16S rDNAs revealed diversity among the six main divisions--the alpha, delta subclasses of Proteobacteria, Nitrospira, Cytophagal Flexibacter/Bacteroides, Verrucomicrobia, and Actinobacteria--in the reactor. Two cloned sequence types were not closely affiliated with any described bacterial divisions. The isolation and phylogenetic analysis of 2,4-DNP-degrading bacteria from the reactor revealed that isolated strains were classified into two types of bacteria having different 16S rDNA sequences. One of these strain types was identified as a relative of Rhodococcus koreensis, and the other was identified as a relative of Nocardioides simplex FJ21-A.     

酸马奶中乳杆菌Lb.casei.Zhang和ZL12-1的16S rDNA基因序列及聚类分析

利用遗传学方法对分离自内蒙古锡林郭勒盟牧区采集的酸马奶样品中的乳杆菌Lb．casei．Zhang和ZL12-1的16S rDNA扩增与测序．并将结果与同属的乳杆菌作同源性分析，建立系统发育树，以求更准确地确定其归属，为益生菌的筛选奠定基础。结果表明，菌株Lb.casei.Zhang标准菌株Lb．casei ATCC334^T同源性为100％，菌株zLl2—1与标准Lb.gallinanum ATCC 33199^T的同源性为98％。结合系统发育树及16S rDNA的部分序列分析结果，将Lb.casei.Zhang判定为Lb．caseissubsp.casei.ZL12-1鉴定为Lb.gallinanum，这与传统分类方法所得鉴定结果一致。     

GenBank数据库中微生物rDNA序列准确性研究

rDNA序列同源性分析是目前常用的一种微生物分子鉴定方法。在该方法中,待鉴定微生物的rDNA序列需要与GenBank数据库中的相关序列进行同源性比对,从而得出该菌株的具体种属。从GenBank数据库中收集了细菌16S rDNA序列、丝状真菌ITS序列以及酵母26S rDNA D1/D2区域序列共88条,通过BLAST比对和构建系统发育树的方法对所收集rDNA序列的准确性进行了验证。结果发现有17条序列(19.3%)的长度过短,无法提供足够的系统发育信息;5条序列(5.6%)存在错误命名或测序不准确问题;58条序列(65.9%)没有相关文章发表;62条序列(70.4%)无法获取对应的微生物保藏物。从而提示了GenBank数据库的有限参考价值,在微生物分子鉴定过程中需要对相关rDNA序列的准确性进行验证。     

Succession of a microbial community during stable operation of a semi-continuous garbage-decomposing system

The microbial community in a garbage-decomposing system was analyzed using denaturing gradient gel electrophoresis (DGGE) on the basis of 16S rDNA. The system treated 1 kg of garbage everyday for two months at ambient temperature with almost constant decomposition efficiency, although a transient pH increase occurred. Succession of the banding pattern of the DGGE profile suggested that the bacterial community was not directly affected by the continuous addition of non-sterilized garbage into the open system, but changed with the fluctuation of pH. These resistance and resilience characteristics of the community structure may be effective to keep the decomposition efficiency stable. The analyses of the DNA sequences from the DGGE bands suggested the existence of uncultured or novel bacteria as well as Lactobacillus sp., Corynebacterium spp., Enterococcus spp., and Staphylococcus sp. A specific PCR detection was performed to evaluate the existence of Escherichia coli within the community. E. coli 16S rDNAs were not detected from the decomposing system.     

Effect of dilution rate on structure of a mesophilic acetate-degrading methanogenic community during continuous cultivation

The community structures of two mesophilic acetate-degrading methanogenic consortia enriched at dilution rates of 0.025 and 0.6 d(-1) were analyzed by fluorescence in situ hybridization (FISH) and phylogenetic analyses based on 16S rDNA clonal sequences and quantitative real-time polymerase chain reaction (PCR). FISH experiments with archaeal and bacterial domain-specific probes showed that archaeal cells were predominant and only a small number of bacterial cells were detected at both dilution rates. In the domain Archaea, the number of cells closely related to Methanosarcina barkeri was shown to be greater at the high dilution rate using FISH with species-specific probes. Taxonomic analyses based on rDNA clonal sequences obtained at the low and high dilution rates showed that 43% of 100 clones and 72% of 92 clones, respectively, were affiliated with the domain Archaea and the remainders at each dilution rate were affiliated with the domain Bacteria. Within the domain Archaea, all rDNA clones at both dilution rates were affiliated with the genera Methanosaeta or Methanosarcina of the aceticlastic methanogens. Within the domain Bacteria, the rDNA clones obtained at the low dilution rate were affiliated with four phyla, Firmicutes (36%), Bacteroidetes (9%), Chloroflexi (6%) and candidate division OP12 (5%). The rDNA clones obtained at the high dilution rate were affiliated with four phyla, Firmicutes (16%), Bacteroidetes (8%), Proteobacteria (1%) and candidate division OP12 (3%). Real-time quantitative PCR experiments showed that the number of rDNA sequences affiliated with the genus Methanosarcina was greater at the high dilution rate. In addition, a significant number of rDNA sequences affiliated with the genus Methanoculleus were detected only at the low dilution rate. Detection of a hydrogenotrophic methanogen at the low dilution rate suggests that the syntrophic acetate oxidation by hydrogenotrophic methanogens and acetate-oxidizing bacteria could occur at the low dilution rate.     

腌制苋菜梗的微生物菌群结构及其品质

腌制苋菜梗是浙东传统的特色腌制食品,了解其细菌种群结构并探讨对亚硝酸盐等理化品质的影响,以确保腌制食品的安全性。采用16S rDNA基因克隆文库的方法,对腌制成熟的苋菜梗中细菌组成结构多样性进行了分析,共检测出了乳杆菌、类香菌、弓形杆菌等6个菌属,其中乳杆菌属占优势为总数的83.9%。与此同时,测定了腌制苋菜梗体系亚硝酸盐等一些理化指标,腌制成熟时pH值为4.35,盐度为5.5,亚硝酸盐质量分数为3.99mg/kg(未超标),细菌和乳酸菌浓度分别为8.8×106 CFU/mL和1.6×106 CFU/mL。     

Dynamics of the functional gene copy number and overall bacterial community during microcystin-LR degradation by a biological treatment facility in a drinking water treatment plant

Information is limited on the potential for microcystins (MCs) degradation by carrier-attached biofilms obtained in winter that were not exposed to detectable levels of MCs in the preceding months. Under controlled laboratory conditions, we confirmed that microcystin-LR (MCLR) was effectively biodegraded within 5.5 days in cultures of the biofilm sampled in winter. Quantitative polymerase chain reaction (qPCR) assays revealed that seasonal variations in the MCLR-degradation potential of the biofilm were closely related to the initial MCLR-degrader population in the biofilm. Indigenous MCLR-degraders in the biofilm could accumulate by exposure to natural MCLR in the water column, accelerating MCLR-degradation. The qPCR assay suggested that MCLR may be a primary substrate for the degraders in the presence of another labile organic carbon associated with the biofilm under the present study conditions. qPCR and PCR-denaturing gradient gel electrophoresis (DGGE) for 16S rDNA demonstrated that the overall bacterial population from the winter biofilm rapidly increased with the MCLR-degrader population and remained stable after day 3.5, while the overall bacterial community structure shifted throughout the entire biodegradation period. This study is important to the in-depth understanding of microbial degradation of MCs and could facilitate the bioremediation of MCs in polluted habitats.     

利用PCR-DGGE技术筛选分离海南黄帝椒产品微生物

黄帝椒是海南特产高辣度辣椒。该研究以黄帝椒产品为原料,采用聚合酶链式反应-变性梯度凝胶电泳(PCR-DGGE)技术分析了其微生物种群关系,并结合传统平板筛选法进行菌种分离及鉴定,获得了海南特辣黄帝椒产品优势微生物。研究结果显示,海南黄帝椒产品平板筛选分离到了Pseudomonas stutzeri,Lactobacillus plantarum,Lactobacillus brevis等可培养的菌株;DGGE图谱中检测到了6个条带,其中,乳酸菌占总菌数的65%,处于最优势地位,假单胞菌占总菌数的16%,处于次要地位。该研究也首次提及了不同的微生物对黄帝椒产品的颜色、脆性的影响。     

Bioaugmentation and adsorption treatment of coking wastewater containing pyridine and quinoline using zeolite-biological aerated filters

Bioaugmented zeolite-biological aerated filters (Z-BAFs), i.e. adding isolated degrading bacteria into the BAFs with zeolite as fillings, were designed to treat coking wastewater containing high concentrations of pyridine and quinoline and to explore the bacterial community of biofilm on the zeolite surface. The investigation was carried out for 91 days of column operation and the treatment of pyridine, quinoline, total organic carbon (TOC), and ammonium was shown to be highly efficient by bioaugmentation and adsorption. Biomass determination and bacterial diversity detection based on 16S rDNA and rRNA techniques supported the treatment data and indicated that bioaugmentation could recover the bacterial richness and diversity from pyridine and quinoline loading shocks. However, bioaugmentation accelerated the shift of the bacterial community structure resulting in a more distinct difference from the starting community. Clone library analysis revealed that pyridine and quinoline were more harmful to Bacterodietes among all ingenious bacteria, and bioaugmentation promoted the growth of Planctomycetes in the biofilm. Moreover, the introduced bacteria did not remain dominant in the bioaugmented biofilm, indicating the indigenous degrading bacteria played the most significant role in the treatment. This bioaugmented Z-BAF method was shown to be an alternative technology for the treatment of wastewater containing pyridine and quinoline or other N-heterocyclic aromatic compounds.     

Microbial community analysis in the denitrification process of saline-wastewater by denaturing gradient gel electrophoresis of PCR-amplified 16S rDNA and the cultivation method

The metallurgic wastewater generated from the processes of recovering precious metals from industrial wastes contains high concentrations of nitrogen compounds and salts. Biological nitrogen removal from this wastewater was attempted using a circulating bioreactor system equipped with an anaerobic packed bed or an anaerobic fluidized bed. The denitrification capability of the system with the anaerobic packed bed was more stable than that of the system with the anaerobic fluidized bed. The NOx removal rate of the anaerobic packed bed was as high as 97%. Microbial community analysis by denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S ribosomal DNA (rDNA) fragments and the cultivation method revealed that the community diversity varied in accordance with wastewater composition such as the level of salinity and so on. Phylogenetic analysis suggested that the taxonomic affiliation of the dominant species in the anaerobic reactors was to the gamma-Proteobacteria including Halomonadaceae species. The PCR-DGGE method as a non-cultivation method was found to be a powerful tool for analysis of the microbial community, because the cultivation method could detect only a fraction of the microbial species present in these systems. The genetic diversity of the isolated bacteria belonging to the gamma-Proteobacteria which reduced both nitrate and nitrite in the anaerobic packed bed was higher than that of the bacteria in the anaerobic fluidized bed. This suggested that a genetically diverse microbial community stabilized the denitrifying performance in the anaerobic packed bed.     

The sequence heterogenicities among 16S rRNA genes of Salmonella serovars and the effects on the specificity of the primers designed

Previously, we have reported a 16S rDNA targeted polymerase chain reaction (PCR) method for the specific detection of Salmonella serovars [J. Appl. Bacteriol. 80 (1996) 659]. The target sites of its primers, i.e. 16SFI and 16SIII, according to the data in GenBank, were found mismatched to the corresponding sequences of some Salmonella serovars, such as those of S. Houten, S. Chingola, S. Bareilly, and S. Weltevreden. Accordingly, a PCR method using a nonspecific primer MINf combined with a primer modified from our 16SFI primer, i.e. the primer MINr, was developed and displayed better detection specificity [Int. J. Food Microbiol. 80 (2003) 67]. In this study, we show the sequence heterogenicity at the primer 16SFI targeting sites for some Salmonella serovars. Thus, the sequence used for designing of PCR primers might be just one of the several possible sequences. Such a situation may lead to the misjudgment on evaluation of the specificity of the primers if this was only based on the data in GenBank. Strains of the above described Salmonella serovars with target sequences from GenBank mismatched to the primer 16SF1 were reidentified and their PCR results were confirmed. Meanwhile, their 16SFI/16SIII primer annealing sites were sequenced and the sequences obtained were found completely and highly homologous to those of 16SFI and complementary to those of 16SIII primer, respectively.     

两株土壤放线菌ZSM-1和ZSM-2的分离与鉴定

对分离自土壤的2 株编号为ZSM-1和ZSM-2的放线菌进行分类鉴定,提取基因组脱氧核糖核酸（deoxyribonucleic acid,DNA）后经聚合酶链式反应（polymerase chain reaction,PCR）扩增菌株16S rDNA,基因测序并进行同源性分析,构建进化树并进行系统发育分析,结合培养特征、生理生化特征,将该2 株菌分别鉴定为纳士维尔链霉菌（Streptomyces nashvillensis）与土地戈登氏菌（Gordonia terrae）。