Use of rpoB and 16S rRNA genes to analyse bacterial diversity of a tropical soil using PCR and DGGE

AIM: To evaluate the rpoB gene as a biomarker for PCR-DGGE microbial analyses using soil DNA from the Cerrado, Brazil. METHODS: DNA extraction from soil was followed by Polymerase Chain Reaction (PCR) amplification of rpoB and 16S rRNA genes. PCR products were compared by Denaturing Gradient Gel Electrophoresis (DGGE) to compare gene/community profiles. RESULTS: The rpoB DGGE profiles comprised fewer bands than the 16S rDNA profiles and were easier to delineate and therefore to analyse. Comparison of the community profiles revealed that the methods were complementary. CONCLUSIONS, SIGNIFICANCE AND IMPACT OF THE STUDY: The gene for the beta subunit of the RNA polymerase, rpoB, is a single copy gene unlike 16S rDNA. Multiple copies of 16S rRNA genes in bacterial genomes complicate diversity assessments made from DGGE profiles. Using the rpoB gene offers a better alternative to the commonly used 16S rRNA gene for microbial community analyses based on DGGE.     

Coamplification of Eukaryotic DNA with 16S rRNA Gene-Based PCR Primers: Possible Consequences for Population Fingerprinting of Complex Microbial Communities

The main aim of this study was to evaluate the specificity of three commonly used 16S rRNA gene-based polymerase chain reaction (PCR) primer sets for bacterial community analysis of samples contaminated with eukaryotic DNA. The specificity of primer sets targeting the V₃, V₃-V₅, and V₆-V₈ hypervariable regions of the 16S rRNA gene was investigated in silico and by community fingerprinting of human and fish intestinal samples. Both in silico and PCR-based analysis revealed cross-reactivity of the V₃ and V₃-V₅ primers with the 18S rRNA gene of human and sturgeon. The consequences of this primer anomaly were illustrated by denaturing gradient gel electrophoresis (DGGE) profiling of microbial communities in human feces and mixed gut of Siberian sturgeon. DGGE profiling indicated that the cross-reactivity of 16S rRNA gene primers with nontarget eukaryotic DNA might lead to an overestimation of bacterial biodiversity. This study has confirmed previous sporadic indications in literature indicating that several commonly applied 16S rRNA gene primer sets lack specificity toward bacteria in the presence of eukaryotic DNA. The phenomenon of cross-reactivity is a potential source of systematic error in all biodiversity studies where no subsequent analysis of individual community amplicons by cloning and sequencing is performed.     

Characterization and comparison of microbial community of different typical Chinese liquor Daqus by PCR-DGGE

Aims: To identify and compare microbiota in Chinese liquor Daqu, which were produced in the different regions using different production process. Methods and Results: The DNA exacted from Daqu samples was used as a template for PCR with universal primers of 16S rRNA, 26S rRNA and 18S rRNA, respectively. The amplicons were analysed using denaturing gradient gel electrophoresis (DGGE). It was observed that the bacterial DGGE profile indicated high diversity and predominance of lactic acid bacteria. The results showed that Saccharomycopsis fibuligera and Pichia anomal were dominant yeast species and that several non‐Saccharomyces yeasts including Hanseniaspora guilliermondii, Debaryomyces hansenii, Issatchenkia orientalis and Trichosporon asahii were also detected. As for fungal DGGE, Aspergillus oryzae and Absidia blakesleeana were the most common species amongst different samples. Based on the DGGE analysis, a few differences in community structure were found between Daqu samples. Conclusions: A variety of bacteria, yeast and moulds were identified in Daqu samples, in addition to the present knowledge obtained mainly through the traditional culture‐dependent methods. Moreover, production temperature played a more decisive role on the formation of micro‐organism composition in Daqu than geographical region. Significance and Impact of the Study: PCR–DGGE technique was used in this study to fully observe and asses all microbial community (including bacteria, yeast and mould) in Chinese liquor Daqu for the first time and proved to be effective in profiling Daqu microbial diversity.     

Bacterial community along a historic lake sediment core of Ardley Island, west Antarctica

The bacterial community in a historic lake sediment core of Ardley Island, Antarctica, spanning approximately 1,600 years, was investigated by molecular approaches targeting the 16S rRNA gene fragments. The cell number in each 1 cm layer of the sediment core was deduced through semi-quantification of the 16S rRNA gene copies by quantitative competitive PCR (QC-PCR). It was found that the total bacterial numbers remained relatively stable along the entire 59 cm sediment core. Denaturing Gradient Gel Electrophoresis (DGGE) analysis and sequencing of PCR-amplified 16S rRNA gene fragments were performed to analyze the bacterial diversity over the entire column. Principle coordinates analysis suggested that the bacterial communities along the sediment core could be separated into three groups. There were obvious bacterial community shift among groups of 1–20 cm, 21–46 cm and 46–59 cm. Diversity indices indicated that the bacterial community in the 21–46 cm depth showed the highest species diversity and uniformity. The main bacterial groups in the sediments fell into 4 major lineages of the gram-negative bacteria: the α, γ and δ subdivision of Proteobacteria, the Cytophaga-Flavobacteria-Bacteroides, and some unknown sequences. The gram-positive bacteria Gemmatimonadetes, Firmicutes and Actinobacteria were also detected. The results demonstrated the presence of highly diverse bacterial community population in the Antarctic lake sediment core. And the possible influence of climate and penguin population change on the bacterial community shift along the sediment core was discussed.Shengkang Li and Xiang Xiao contributed equally to this paper     

Phylogenetic diversity of nitrogen-fixing bacteria in mangrove sediments assessed by PCR-denaturing gradient gel electrophoresis

Culture-independent PCR–denaturing gradient gel electrophoresis (DGGE) was employed to assess the composition of diazotroph species from the sediments of three mangrove ecosystem sites in Sanya, Hainan Island, China. A strategy of removing humic acids prior to DNA extraction was conducted, then total community DNA was extracted using the soil DNA kit successfully for nifH PCR amplification, which simplified the current procedure and resulted in good DGGE profiles. The results revealed a novel nitrogen-fixing bacterial profile and fundamental diazotrophic biodiversity in mangrove sediments, as reflected by the numerous bands present DGGE patterns. Canonical correspondence analysis (CCA) revealed that the sediments organic carbon concentration and available soil potassium accounted for a significant amount of the variability in the nitrogen-fixing bacterial community composition. The predominant DGGE bands were sequenced, yielding 31 different nifH sequences, which were used in phylogenetic reconstructions. Most sequences were from Proteobacteria, e.g. α, γ, β, δ-subdivisions, and characterized by sequences of members of genera Azotobacter, Desulfuromonas, Sphingomonas, Geobacter, Pseudomonas, Bradyrhizobium and Derxia. These results significantly expand our knowledge of the nitrogen-fixing bacterial diversity of the mangrove environment.     

Diversity of gut microbiota increases with aging and starvation in the desert locust

Here we report the effects of starvation and insect age on the diversity of gut microbiota of adult desert locusts, Schistocerca gregaria, using denaturing gradient gel electrophoretic (DGGE) analysis of bacterial 16S rRNA genes. Sequencing of excised DGGE bands revealed the presence of only one potentially novel uncultured member of the Gammaproteobacteria in the guts of fed, starved, young or old locusts. Most of the 16S rRNA gene sequences were closely related to known cultured bacterial species. DGGE profiles suggested that bacterial diversity increased with insect age and did not provide evidence for a characteristic locust gut bacterial community. Starved insects are often more prone to disease, probably because they compromise on immune defence. However, the increased diversity of Gammaproteobacteria in starved locusts shown here may improve defence against enteric threats because of the role of gut bacteria in colonization resistance.     

Assessment of bacterial community structure in the deep sub-seafloor biosphere by 16S rDNA-based techniques: a cautionary tale

Investigations into the deep marine environment have demonstrated the presence of a significant microbial biomass buried deep within sediments on a global scale. It is now believed that this deep biosphere plays a major role in the global cycling of elements and contains a large reservoir of organic carbon. This paper reports the development of a DNA extraction protocol that addresses the particular problems faced in applying molecular ecological techniques to samples containing very low biomass. Sediment samples were collected from different geographical locations within the Pacific Ocean and include the Ocean Drilling Program (ODP) Leg 190, Nankai Trough Accretionary Prism. Seven DNA extraction protocols were tested and a commercially available DNA extraction kit with modifications was shown to produce higher yields of polymerase chain reaction (PCR)-amplifiable DNA than standard laboratory methods. Denaturing gradient gel electrophoresis (DGGE) analysis of 16S rRNA gene diversity revealed that template DNA from these extremely low biomass sediment samples was susceptible to PCR bias and random amplification. We propose that it is essential to screen 16S rRNA gene products for bacterial diversity by DGGE or other rapid fingerprinting methods, prior to their use in establishing a representative clone library of deep sub-seafloor bacteria. This represents a cautionary approach to analysis of microbial diversity in such sub-seafloor ecosystems.     

Bacterial community structure and bamA gene diversity in anaerobic degradation of toluene and benzoate under denitrifying conditions

Aim: To characterize the microbial community structure and bamA gene diversity involved in anaerobic degradation of toluene and benzoate under denitrifying conditions. Methods and Results: Nitrate‐reducing enrichment cultures were established on either toluene, benzoate or without additional substrate. Bacterial community structures were characterized by 16S rRNA gene–based PCR‐DGGE analysis. bamA gene diversity was analysed using DGGE and cloning methods. The results showed that bamA gene related to bamA of Thauera chlorobenzoica was dominant in toluene and benzoate cultures. However, a greater diversity of sequences was obtained in benzoate cultures. Low diversity of bamA gene was observed, and some similarities of bamA were also found between active cultures and backgrounds, suggesting that potential natural attenuation of aromatic compounds might occur. Conclusions: The combined analysis of 16S rRNA and bamA genes suggests that the species related to genera Thauera dominated toluene‐ and benzoate‐degrading cultures. The combination of multiple methods (DGGE and cloning) provides a more complete picture of bamA gene diversity. Significance and Impact of the Study: To our knowledge, this is the first report of bamA gene in denitrifying enrichments using DGGE and cloning analysis.     

Differences in microbial communities between healthy and bleached coral Acropora solitaryensis from Xisha Islands,South China Sea

Serious bleaching events have been observed in Acropora solitaryensis, one of the main species of reef-building coral in the area of the Xisha islands in the South China Sea, during 2008–2011. The microbial communities of healthy and bleached coral samples were compared to explore the difference in the bacterial composition using 16S rRNA gene sequencing and denaturing gradient gel electrophoresis (DGGE). Analysis of 16S rRNA gene sequences showed that the diversity of bacteria from the corals was different between the healthy and the bleached. Albeit both the healthy coral and bleached coral displayed similar dominant bacterial species (α- and γ-proteobacteria), the ratio of Vibrio spp. increased sharply in the latter. As the bleaching developed, the diversity of the microbial community was dramatically decreased and the dominant species were replaced by γ-proteobacteria where Vibrio spp. and Escherichia spp. overwhelmed other genera. Similar results were gained by the DGGE technique though the abundance was lower. Furthermore, in the bleached tissues, Vibrio coralliilyticus was identified using both methods. These results indicated that pathogenic Vibrio spp. might be one of the factors causing A. solitaryensis bleaching in the coral reef of the Xisha islands.     

变性梯度凝胶电泳(DGGE)在微生物生态学中的应用

由于从环境样品中分离和培养细菌的困难,分子生物学方法已发展用来描述和鉴定微生物群落。近年来基于DNA方法的群落分析得到了迅速的发展,如PCR扩增技术,克隆文库法,荧光原位杂交法,限制性酶切片段长度多态性法,变性和温度梯度凝胶电泳法。DGGE已广泛用于分析自然环境中细菌、蓝细菌,古菌、微微型真核生物、真核生物和病毒群落的生物多样性。这一技术能够提供群落中优势种类信息和同时分析多个样品。具有可重复和容易操作等特点,适合于调查种群的时空变化,并且可通过对切下的带进行序列分析或与特异性探针杂交分析鉴定群落成员。DGGE分析微生物群落的一般步骤如下：一是核酸的提取,二是16S rRNA,18S rRNA或功能基因如可容性甲烷加单氧酶羟化酶基因(mmoX)和氨加单氧酶a一亚单位基因(amoA)片段的扩增,三是通过DGGE分析PCR产物。DGGE使用具有化学变性剂梯度的聚丙烯酰胺凝胶,该凝胶能够有区别的解链PCR扩增产物。由PCR产生的不同的DNA片段长度相同但核苷酸序列不同。因此不同的双链DNA片段由于沿着化学梯度的不同解链行为将在凝胶的不同位置上停止迁移。DNA解链行为的不同导致一个凝胶带图案,该图案是微生物群落中主要种类的一个轮廓。DGGE使用所有生物中保守的基因片段如细菌中的16S rRNA基因片段和真菌中的18S rRNA基因片段。然而同其他分子生物学方法一样,DGGE也有缺陷,其中之一是只能分离较小的片段,使用于系统发育分析比较和探针设计的序列信息量受到了限制。在某些情况下,由于所用基因的多拷贝导致一个种类多于一条带,因此不易鉴定群落结构到种的水平。此外,该技术具有内在的如单一细菌种类16S rDNA拷贝之间的异质性问题,可导致自然群落中微生物数量的过多估计。DGGE是分析微生物群落的一种有力的工具。不过为了减少DGGE和其它技术的缺陷,建议研究者结合DGGE和其它分子及微生物学方法以便更详细的观察微生物的群落结构和功能。     

Changes of Soil Bacterial Diversity as a Consequence of Agricultural Land Use in a Semi-Arid Ecosystem

Natural scrublands in semi-arid deserts are increasingly being converted into fields. This results in losses of characteristic flora and fauna, and may also affect microbial diversity. In the present study, the long-term effect (50 years) of such a transition on soil bacterial communities was explored at two sites typical of semi-arid deserts. Comparisons were made between soil samples from alfalfa fields and the adjacent scrublands by two complementary methods based on 16S rRNA gene fragments amplified from total community DNA. Denaturing gradient gel electrophoresis (DGGE) analyses revealed significant effects of the transition on community composition of Bacteria, Actinobacteria, Alpha- and Betaproteobacteria at both sites. PhyloChip hybridization analysis uncovered that the transition negatively affected taxa such as Acidobacteria, Chloroflexi, Acidimicrobiales, Rubrobacterales, Deltaproteobacteria and Clostridia, while Alpha-, Beta- and Gammaproteobacteria, Bacteroidetes and Actinobacteria increased in abundance. Redundancy analysis suggested that the community composition of phyla responding to agricultural use (except for Spirochaetes) correlated with soil parameters that were significantly different between the agricultural and scrubland soil. The arable soils were lower in organic matter and phosphate concentration, and higher in salinity. The variation in the bacterial community composition was higher in soils from scrubland than from agriculture, as revealed by DGGE and PhyloChip analyses, suggesting reduced beta diversity due to agricultural practices. The long-term use for agriculture resulted in profound changes in the bacterial community and physicochemical characteristics of former scrublands, which may irreversibly affect the natural soil ecosystem.     

Biases in community structures of ammonia/ammonium-oxidizing microorganisms caused by insufficient DNA extractions from Baijiang soil revealed by comparative analysis of coastal wetland sediment and rice paddy soil

Repetitive extraction of DNAs from surface sediments of a coastal wetland in Mai Po Nature Reserve (MP) of Hong Kong and surface Baijiang soils from a rice paddy (RP) in Northeast China was conducted to compare the microbial diversity in this study. Community structures of ammonia/ammonium-oxidizing microorganisms in these samples were analyzed by PCR-DGGE technique. The diversity and abundance of ammonia-oxidizing archaea (AOA), ammonia-oxidizing bacteria (AOB), and anaerobic ammonium-oxidizing (anammox) bacteria were also analyzed based on archaeal and bacterial ammonia monooxygenase subunit A encoding (amoA) and anammox bacterial 16S rRNA genes, respectively. DGGE profiles of archaeal and bacterial amoA and anammox bacterial 16S rRNA genes showed a similar pattern among all five repetitively extracted DNA fractions from both MP and RP, except the anammox bacteria in RP, indicating a more diverse anammox community retrieved in the second to the fifth fractions than the first one. Both soil and marine group AOA were detected while soil and coastal group AOB and Scalindua-anammox bacteria were dominant in MP. Soil group AOA and marine group AOB were dominant in RP, while both Scalindua and Kuenenia species were detected in RP. Pearson correlation analysis showed that the abundance of archaeal and bacterial amoA and anammox bacterial 16S rRNA genes was significantly correlated with the DNA concentrations of the five DNA fractions from MP, but not from RP (except the archaeal amoA gene). Results suggest that anammox bacteria diversity may be biased by insufficient DNA extraction of rice paddy soil samples.     

Endophytic bacteria in long-term in vitro cultivated “axenic” pineapple microplants revealed by PCR–DGGE

In vitro propagated plants are believed to be free of microbes. However, after 5 years of in vitro culture of pineapple plants, without evidence of microbial contamination, the use of culture-independent molecular approach [classifying heterogeneous nucleic acids amplified via universal and specific 16S rRNA gene by polymerase chain reaction (PCR)], and further analysis by denaturing gradient gel electrophoresis (DGGE) revealed endophytic bacteria in roots, young and mature leaves of such plants. The amplification of 16S rRNA gene (Bacteria domain) with the exclusion of the plant chloroplast DNA interference, confirmed the presence of bacterial DNA, from endophytic microorganisms within microplant tissues. PCR–DGGE analysis revealed clear differences on bacterial communities depending on plant organ. Group-specific DGGE analyses also indicated differences in the structures of Actinobacteria, Alphaproteobacteria and Betaproteobacteria communities in each part of plants. The results suggest the occurrence of a succession of bacterial communities colonizing actively the microplants organs. This study is the first report that brings together evidences that pineapple microplants, previously considered axenic, harbor an endophytic bacterial community encompassing members of Actinobacteria, Alphaproteobacteria and Betaproteobacteria group which is responsive to differences in organs due to plant development.     

High abundance of heterotrophic prokaryotes in hydrothermal springs of the Azores as revealed by a network of 16S rRNA gene-based methods

Two hydrothermal springs (AI: 51 °C, pH 3; AIV: 92 °C, pH 8) were analysed to determine prokaryotic community composition. Using pyrosequencing, 93,576 partial 16S rRNA gene sequences amplified with V2/V3-specific primers for Bacteria and Archaea were investigated and compared to 16S rRNA gene sequences from direct metagenome sequencing without prior amplification. The results were evaluated by fluorescence in situ hybridization (FISH). While in site AIV Bacteria and Archaea were detected in similar relative abundances (Bacteria 40 %, Archaea 35 %), the acidic spring AI was dominated by Bacteria (68 %). In spring AIV the combination of 16S rRNA gene sequence analysis and FISH revealed high abundance (>50 %) of heterotrophic bacterial genera like Caldicellulosiruptor, Dictyoglomus, and Fervidobacterium. In addition, chemolithoautotrophic Aquificales were detected in the bacterial community with Sulfurihydrogenibium being the dominant genus. Regarding Archaea, only Crenarchaeota, were detected, dominated by the family Desulfurococcaceae (>50 %). In addition, Thermoproteaceae made up almost 25 %. In the acidic spring (AI) prokaryotic diversity was lower than in the hot, slightly alkaline spring AIV. The bacterial community of site AI was dominated by organisms related to the chemolithoautotrophic genus Acidithiobacillus (43 %), to the heterotrophic Acidicaldus (38 %) and to Anoxybacillus (7.8 %). This study reveals differences in the relative abundance of heterotrophic versus autotrophic microorganisms as compared to other hydrothermal habitats. Furthermore, it shows how different methods to analyse prokaryotic communities in complex ecosystems can complement each other to obtain an in-depth picture of the taxonomic composition and diversity within these hydrothermal springs.     

Polymerase chain reaction-denaturing gradient gel electrophoresis analysis of bacterial community structure in the food,intestines, and feces of earthworms

The bacterial communities in the food, intestines, and feces of earthworms were investigated by PCR-denaturing Gradient gel electrophoresis (DGGE). In this study, PCR-DGGE was optimized by testing 6 universal primer sets for microbial 16S rRNA in 6 pure culture strains of intestinal microbes in earthworms. One primer set effectively amplified 16S rRNA from bacterial populations that were found in the food, intestines, and feces of earthworms. Compared with the reference markers from the pure culture strains, the resulting DGGE profiles contained 28 unique DNA fragments. The dominant microorganisms in the food, intestines, and feces of earthworms included Rhodobacterales bacterium, Fusobacteria, Ferrimonas marina, Aeromonas popoffii, and soil bacteria. Other straisn, such as Acinetobacter, Clostridium, and Veillonella, as well as rumen bacteria and uncultured bacteria also were present. These results demonstrated that PCR-DGGE analysis can be used to elucidate bacterial diversity and identify unculturable microorganisms.     

Microbial Diversity in High Arsenic Groundwater in Hetao Basin of Inner Mongolia,China

The microbial diversity and community structure in twenty-one groundwater samples from high arsenic shallow aquifers of Hetao Basin, Inner Mongolia, China was investigated with an integrated approach including polymerase chain reaction (PCR)-denaturing gradient gel electrophoresis (DGGE) and 16S rRNA gene phylogenetic analyses. A total of 25 bacterial and 32 archaeal DGGE bands were exercised for sequencing. Phylogenetic analyses showed that the bacterial DGGE bands were dominated by Proteobacteria, and the archaeal bands were dominated by Thaumarchaeota and Euryarchaeota. Based on arsenic concentrations, three samples (corresponding to low, medium, and high level of arsenic, respectively) were selected for construction of 16S rRNA gene clone libraries. A total of 912 (468 and 444 for bacteria and archaea, respectively) 16S rRNA gene clone sequences were obtained and subjected to phylogenetic analyses. The results showed that bacterial communities of these samples were dominated by Acinetobacter, Pseudomonas, Massilia, Dietzia, Planococcus, Brevundimonas, Aquabacterium and Geobacter, and archaeal communities by Nitrosophaera, Thermoprotei and Methanosaeta. The relative abundance of major groups varied as a function of changes in groundwater geochemistry. Acinetobacter, Brevundimonas, Geobacter, Thermoprotei and Methanosaeta dominated in high arsenic samples with high concentrations of methane and Fe(II), and low concentrations of SO^2? ₄ and NO^? ₃, while Pseudomonas and Nitrosophaera were abundant in low arsenic groundwater. These results imply that microbes play an important role in arsenic mobilization in the shallow aquifers of Hetao Basin, Inner Mongolia.     

Bacilli community of saline–alkaline soils from the Ararat Plain (Armenia) assessed by molecular and culture-based methods

The bacterial community composition in the A horizon of a natural saline–alkaline soil located in Ararat Plain (Armenia) was studied using molecular and culture-based methods The sequence analysis of a 16S rRNA gene clone library and denaturing gradient gel electrophoresis (DGGE) profiles indicated dominance of Firmicutes populations. The majority of the sequences of the bacterial 16S rRNA gene library were close relatives of representatives belonging to the genera Halobacillus (41.2%), Piscibacillus (23.5%), Bacillus (23.5%) and Virgibacillus (11.8%). Eight novel moderately halophilic bacilli isolates were successfully obtained from the enriched cultures of the saline–alkaline soil samples. 16S rRNA gene sequence analyses of isolates revealed their affiliation (97.7–99.7% similarity) to representatives of the genera Bacillus, Piscibacillus and Halobacillus. All isolates were able to tolerate high concentrations of NaCl and highly alkaline conditions. This is the first study combining cultivation-independent and -dependent approaches to reveal the bacterial diversity of the saline–alkaline soils of Ararat Plain and it suggested an important role of bacilli as key microbes in biogeochemical cycles of these environments.     

基于PCR DGGE的小鼠肠道菌群基因组提取方法的建立

通过比较4种小鼠粪便细菌总DNA提取方法对基于PCR-DGGE检测的肠道菌群多样性分析的影响,旨在建立适于PCR—DGGE的小鼠肠道微生物宏基因组提取的稳定、经济、快捷的方法。采用SDS裂解法、某国产市售粪便DNA提取试剂盒、改进的化学裂解法、改进的溶菌酶法4种方法提取小鼠粪便细菌总DNA,通过琼脂糖凝胶电泳、紫外分光光度法、细菌16S rRNAV3区PCR扩增结合DGGE对提取结果进行比较分析。SDS裂解法和国产市售试剂盒2种方法提取粪便细菌总DNA均未得到理想结果,另2种方法均能够检测到粪便中20种左右的细菌。改进的化学裂解法和改进的溶菌酶提取法的建立为基于PCR—DGGE进行肠道菌群结构的定量及定性分析提供了可靠的前提基础和实验保障。     

A diverse bacterial community in an anoxic quinoline-degrading bioreactor determined by using pyrosequencing and clone library analysis

There is a concern of whether the structure and diversity of a microbial community can be effectively revealed by short-length pyrosequencing reads. In this study, we performed a microbial community analysis on a sample from a high-efficiency denitrifying quinoline-degrading bioreactor and compared the results generated by pyrosequencing with those generated by clone library technology. By both technologies, 16S rRNA gene analysis indicated that the bacteria in the sample were closely related to, for example, Proteobacteria, Actinobacteria, and Bacteroidetes. The sequences belonging to Rhodococcus were the most predominant, and Pseudomonas, Sphingomonas, Acidovorax, and Zoogloea were also abundant. Both methods revealed a similar overall bacterial community structure. However, the 622 pyrosequencing reads of the hypervariable V3 region of the 16S rRNA gene revealed much higher bacterial diversity than the 130 sequences from the full-length 16S rRNA gene clone library. The 92 operational taxonomic unit (OTUs) detected using pyrosequencing belonged to 45 families, whereas the 37 OTUs found in the clone library belonged to 25 families. Most sequences obtained from the clone library had equivalents in the pyrosequencing reads. However, 64 OTUs detected by pyrosequencing were not represented in the clone library. Our results demonstrate that pyrosequencing of the V3 region of the 16S rRNA gene is not only a powerful tool for discovering low-abundance bacterial populations but is also reliable for dissecting the bacterial community structure in a wastewater environment.     

Analysis of Bacterial and Fungal Communities in Japanese- and Chinese-Fermented Soybean Pastes Using Nested PCR–DGGE

The microbial diversity of Japanese- and Chinese-fermented soybean pastes was investigated using nested PCR–denaturing gradient gel electrophoresis (DGGE). Five Japanese-fermented soybean paste samples and three Chinese-fermented soybean paste samples were analyzed for bacteria and fungi. Extracted DNA was used as a template for PCR to amplify 16S rRNA and 18S rRNA genes. The nearly complete 16S rRNA and 18S rRNA genes were amplified using universal primers, and the resulting products were subsequently used as a template in a nested PCR to obtain suitable fragments for DGGE. Tetragenococcus halophilus and Staphylococcus gallinarum were found to dominate the bacterial microbiota in Japanese samples, whereas Bacillus sp. was detected as the predominant species in Chinese samples. DGGE analysis of fungi in soybean pastes determined the presence of Aspergillus oryzae and Zygosaccharomyces rouxii in most of the Chinese and Japanese samples. Some differences were observed in the bacterial diversity of Japanese- and Chinese-fermented soybean pastes.