The processes of methane formation and oxidation in the soils of the Russian arctic tundra

Methane emission from the following types of tundra soils was studied: coarse humic gleyey loamy cryo soil, peaty gley soil, and peaty gleyey midloamy cryo soil of the arctic tundra. All the soils studied were found to be potential sources of atmospheric methane. The highest values of methane emission were recorded in August at a soil temperature of 8-10 degrees C. Flooded parcels were the sources of atmospheric methane throughout the observation period. The rates of methane production and oxidation in tundra soils of various types at 5 and 15 degrees C were studied by the radioisotope method. Methane oxidation was found to occur in bog water, in the green part of peat moss, and in all the soil horizons studied. Methane formation was recorded in the horizons of peat, in clay with plant roots, and in peaty moss dust of the bogey parcels. At both temperatures, the methane oxidation rate exceeded the rate of methane formation in all the horizons of the mossy-lichen tundra and of the bumpy sinkhole complex. Methanogenesis prevailed only in a sedge-peat moss bog at 15 degrees C. Enrichment bacterial cultures oxidizing methane at 5 and 15 degrees C were obtained. Different types of methanotrophic bacteria were shown to be responsible for methane oxidation under these conditions. A representative of type I methylotrophs oxidized methane at 5 degrees C, and Methylocella tundrae, a psychroactive representative of an acidophilic methanotrophic genus Methylocella, at 15 degrees C.     

Diversity of methanotrophic bacteria in tropical upland soils under different land uses

Three upland soils from Thailand, a natural forest, a 16-year-old reforested site, and an agricultural field, were studied with regard to methane uptake and the community composition of methanotrophic bacteria (MB). The methane uptake rates were similar to rates described previously for forest and farmland soils of the temperate zone. The rates were lower at the agricultural site than at the native forest and reforested sites. The sites also differed in the MB community composition, which was characterized by denaturing gradient gel electrophoresis (DGGE) of pmoA gene fragments (coding for a subunit of particulate methane monooxygenase) that were PCR amplified from total soil DNA extracts. Cluster analysis based on the DGGE banding patterns indicated that the MB communities at the forested and reforested sites were similar to each other but different from that at the farmland site. Sequence analysis of excised DGGE bands indicated that Methylobacter spp. and Methylocystis spp. were present. Sequences of the "forest soil cluster" or "upland soil cluster alpha," which is postulated to represent organisms involved in atmospheric methane consumption in diverse soils, were detected only in samples from the native forest and reforested sites. Additional sequences that may represent uncultivated groups of MB in the Gammaproteobacteria were also detected.     

Soil microbial community structure across a thermal gradient following a geothermal heating event

In this study microbial species diversity was assessed across a landscape in Yellowstone National Park, where an abrupt increase in soil temperature had occurred due to recent geothermal activity. Soil temperatures were measured, and samples were taken across a temperature gradient (35 to 65 degrees C at a 15-cm depth) that spanned geothermally disturbed and unimpacted soils; thermally perturbed soils were visually apparent by the occurrence of dead or dying lodgepole pine trees. Changes in soil microbial diversity across the temperature gradient were qualitatively assessed based on 16S rRNA sequence variation as detected by denaturing gradient gel electrophoresis (DGGE) using both ribosomal DNA (rDNA) and rRNA as PCR templates and primers specific for the Bacteria or Archaea domain. The impact of the major heating disturbance was apparent in that DGGE profiles from heated soils appeared less complex than those from the unaffected soils. Phylogenetic analysis of a bacterial 16S rDNA PCR clone library from a recently heated soil showed that a majority of the clones belonged to the Acidobacterium (51%) and Planctomyces (18%) divisions. Agar plate counts of soil suspensions cultured on dilute yeast extract and R2A agar media incubated at 25 or 50 degrees C revealed that thermophile populations were two to three orders of magnitude greater in the recently heated soil. A soil microcosm laboratory experiment simulated the geothermal heating event. As determined by both RNA- and DNA-based PCR coupled with DGGE, changes in community structure (marked change in the DGGE profile) of soils incubated at 50 degrees C occurred within 1 week and appeared to stabilize after 3 weeks. The results of our molecular and culture data suggest that thermophiles or thermotolerant species are randomly distributed in this area within Yellowstone National Park and that localized thermal activity selects for them.     

多年冻土退化对湿地甲烷排放的影响研究进展

全球气候变暖导致北半球大部分多年冻土区的冻土已经开始退化。多年冻土退化对冻土区湿地CH₄排放产生重要影响,可能直接决定冻土区湿地对全球气候变暖的反馈方式。综述了近年来多年冻土退化对湿地CH₄排放影响的研究。多年冻土退化导致的土壤活动层深度增加和植被类型由中生向湿生的转变都可能会大大增加冻土区湿地CH₄排放量,从而可能对全球气候变暖产生正反馈作用。但多年冻土退化导致的水文条件变化、土壤温度变化和微生物组成及活性变化对湿地CH₄排放的影响却存在一定的不确定性。多年冻土退化除了影响湿地CH₄排放量之外,还可能通过改变土壤冻融过程而影响湿地CH₄排放的季节分配模式。最后提出目前研究中存在的问题,并对未来研究方向进行了展望。     

Bacterial populations occuring in a trichloroethylene-contaminated aquifer during methane injection

Soil core samples were obtained from a trichloroethylene (TCE)-contaminated aquifer before and after the start of methane biostimulation. DNA was extracted directly from the soil samples, and denaturing gradient gel electrophoresis (DGGE) was used to analyse bacterial 16S ribosomal DNA fragments that were PCR amplified from these DNA samples. This analysis consistently detected two phylotypes in the methane-injected samples. These phylotypes were closely related to Methylobacter and Methylomonas , both belonging to type I methanotrophs. A competitive DGGE analysis using Methylosinus trichosporium OB3b cells as an internal quantitative standard showed that these populations accounted for 10⁸−10⁹ cells g⁻¹ soil. These results showed that type I methanotrophs formed a significant proportion of the bacterial community during methane biostimulation. The implications of this finding for TCE bioremediation were discussed.     

Fungi Benefit from Two Decades of Increased Nutrient Availability in Tundra Heath Soil

If microbial degradation of carbon substrates in arctic soil is stimulated by climatic warming, this would be a significant positive feedback on global change. With data from a climate change experiment in Northern Sweden we show that warming and enhanced soil nutrient availability, which is a predicted long-term consequence of climatic warming and mimicked by fertilization, both increase soil microbial biomass. However, while fertilization increased the relative abundance of fungi, warming caused only a minimal shift in the microbial community composition based on the phospholipid fatty acid (PLFA) and neutral lipid fatty acid (NLFA) profiles. The function of the microbial community was also differently affected, as indicated by stable isotope probing of PLFA and NLFA. We demonstrate that two decades of fertilization have favored fungi relative to bacteria, and increased the turnover of complex organic compounds such as vanillin, while warming has had no such effects. Furthermore, the NLFA-to-PLFA ratio for ¹³C-incorporation from acetate increased in warmed plots but not in fertilized ones. Thus, fertilization cannot be used as a proxy for effects on warming in arctic tundra soils. Furthermore, the different functional responses suggest that the biomass increase found in both fertilized and warmed plots was mediated via different mechanisms.     

Diversity of Methanotrophic Bacteria in Tropical Upland Soils under Different Land Uses

Three upland soils from Thailand, a natural forest, a 16-year-old reforested site, and an agricultural field, were studied with regard to methane uptake and the community composition of methanotrophic bacteria (MB). The methane uptake rates were similar to rates described previously for forest and farmland soils of the temperate zone. The rates were lower at the agricultural site than at the native forest and reforested sites. The sites also differed in the MB community composition, which was characterized by denaturing gradient gel electrophoresis (DGGE) of pmoA gene fragments (coding for a subunit of particulate methane monooxygenase) that were PCR amplified from total soil DNA extracts. Cluster analysis based on the DGGE banding patterns indicated that the MB communities at the forested and reforested sites were similar to each other but different from that at the farmland site. Sequence analysis of excised DGGE bands indicated that Methylobacter spp. and Methylocystis spp. were present. Sequences of the “forest soil cluster” or “upland soil cluster α,” which is postulated to represent organisms involved in atmospheric methane consumption in diverse soils, were detected only in samples from the native forest and reforested sites. Additional sequences that may represent uncultivated groups of MB in the Gammaproteobacteria were also detected.     

Molecular analyses of the methane-oxidizing microbial community in rice field soil by targeting the genes of the 16S rRNA, particulate methane monooxygenase, and methanol dehydrogenase

Rice field soil with a nonsaturated water content induced CH4 consumption activity when it was supplemented with 5% CH4. After a lag phase of 3 days, CH4 was consumed rapidly until the concentration was less than 1.8 parts per million by volume (ppmv). However, the soil was not able to maintain the oxidation activity at near-atmospheric CH4 mixing ratios (i.e., 5 ppmv). The soil microbial community was monitored by performing denaturing gradient gel electrophoresis (DGGE) during the oxidation process with different PCR primer sets based on the 16S rRNA gene and on functional genes. A universal small-subunit (SSU) ribosomal DNA (rDNA) primer set and 16S rDNA primer sets specifically targeting type I methylotrophs (members of the gamma subdivision of the class Proteobacteria [gamma-Proteobacteria]) and type II methylotrophs (members of the alpha-Proteobacteria) were used. Functional PCR primers targeted the genes for particulate methane monooxygenase (pmoA) and methanol dehydrogenase (mxaF), which code for key enzymes in the catabolism of all methanotrophs. The yield of PCR products amplified from DNA in soil that oxidized CH4 was the same as the yield of PCR products amplified from control soil when the universal SSU rDNA primer set was used but was significantly greater when primer sets specific for methanotrophs were used. The DGGE patterns and the sequences of major DGGE bands obtained with the universal SSU rDNA primer set showed that the community structure was dominated by nonmethanotrophic populations related to the genera Flavobacterium and Bacillus and was not influenced by CH4. The structure of the methylotroph community as determined with the specific primer sets was less complex; this community consisted of both type I and type II methanotrophs related to the genera Methylobacter, Methylococcus, and Methylocystis. DGGE profiles of PCR products amplified with functional gene primer sets that targeted the mxaF and pmoA genes revealed that there were pronounced community shifts when CH4 oxidation began. High CH4 concentrations stimulated both type I and II methanotrophs in rice field soil with a nonsaturated water content, as determined with both ribosomal and functional gene markers.     

用于分析土壤微生物结构变化规律的变性梯度凝胶电泳条件研究

研究确定土壤微生物基因组DNA提取方法、PCR扩增条件、DGGE电泳条件,为进一步研究分析土壤中微生物结构变化规律提供理论依据。土壤微生物基因组DNA提取采用直接法和间接法进行比较; PCR扩增条件调整扩增体系、DGGE电泳条件调整变性剂范围,并对其结果进行比较分析。通过对DGGE电泳相关条件的研究,结果显示,土壤中粗基因组DNA采用直接法提取,然后进行纯化; PCR扩增体系中加入BSA,DGGE电泳系统组成中变性剂浓度范围为35%～55%。确定了土壤微生物基因组DNA提取方法、PCR扩增条件、DGGE电泳条件,为后续的相关研究提供理论依据。     

Diversity in methane enrichments from agricultural soil revealed by DGGE separation of PCR amplified 16S rDNA fragments

Denaturing gradient gel electrophoresis (DGGE) profiles of PCR amplified V3 regions of 16S rRNA genes were used to assess the diversity in enrichment cultures with methane as the only carbon and energy source. The enrichments originated from two agricultural soils. One was a sandy soil with low (10%) organic content, the other an organic soil with approximately 50% organic content. DGGE provided a fast evaluation of the distribution of amplifiable sequence types indicating that specific bacterial populations had been enriched from each soil. The DGGE profiles revealed a broader range of amplified V3 fragments in the community derived from organic soil than from sandy soil. Fragments from 19 individual DGGE bands were sequenced and compared with 27 previously published 16S rRNA gene sequences. The sequences confirmed the high diversity with the presence of different methylotrophic populations in each enrichment. No affiliation was found with type I methanotrophs, instead type II methanotroph sequences were found in the enrichments from both soil types. Some of the fragments from the organic soil enrichment were not affiliated with methylotrophs. Most of the sequences clustered distantly on a branch within the α-Proteobacteria. These facts suggested that previously undescribed methylotrophs are abundant in methane enrichments from agricultural soil.     

An advanced molecular strategy to identify bacterial communities on art objects

The application of culture-independent techniques based on molecular biological methods, especially on the PCR amplification of 16S rRNA genes, attempts to overcome some shortcomings of conventional cultivation methods and reveals far more complex bacterial communities on art objects than can be shown by cultivation methods. One of the major challenges of investigating microbial growth on art objects by molecular means is the extraction of DNA, due to small sample amounts and PCR inhibitors. In the present study, we introduce a DNA extraction protocol, which allowed the extraction of PCR-amplifiable DNA from samples derived from lime wall paintings and loamy soil underground. The DNA extracts were used to amplify 16S ribosomal fragments, which were subsequently analyzed by denaturing gradient gel electrophoresis (DGGE). In parallel with the DGGE analysis, clone libraries containing PCR fragments of the ribosomal gene were constructed and clones were screened by DGGE. Clone libraries allow the inclusion of the entire 16S rDNA sequence in the phylogenetic analyses of microorganisms, providing a more reliable phylogenetic identification of microorganisms than is obtained from sequence analyses of excised and directly sequenced DGGE bands.     

Detection of methanotrophs with highly divergent pmoA genes from Arctic soils

Tundra soil samples from the Canadian Arctic community, Kuujjuaq, were analyzed for the presence of the soluble (sMMO) and particulate (pMMO) methane monooxygenase genes. Total genomic DNA extracted from these soils was used as template for PCR using sMMO- and pMMO-specific primers, mmoX1-mmoX2 and A189-A682, respectively. pMMO and sMMO genes were detected in the Kuujjuaq soil samples. Isolation of sMMO-possessing methanotrophic microorganisms from the three soils, as determined by the colony naphthalene oxidation assay, was carried out using direct plating (5 degrees C) and methane enrichment studies (5 degrees C and 25 degrees C). Direct plating did not yield sMMO-possessing methanotrophic bacteria, whereas methane enrichments yielded isolates possessing and expressing sMMO activity. Analysis of derived amino acid sequences of pmoA genes and partial 16S rRNA genes obtained by PCR, using DNA isolated directly from this environment and from isolates, revealed the presence of highly divergent PmoA/AmoA sequences and 16S rRNA sequences that cluster closely with but are distinct from the genes from the genera Methylosinus and Methylocystis.     

Methane emission from Siberian arctic polygonal tundra: eddy covariance measurements and modeling

Eddy covariance measurements of methane flux were carried out in an arctic tundra landscape in the central Lena River Delta at 72°N. The measurements covered the seasonal course of mid‐summer to early winter in 2003 and early spring to mid‐summer in 2004, including the periods of spring thaw and autumnal freeze back. The study site is characterized by very cold and deep permafrost and a continental climate with a mean annual air temperature of ?14.7 °C. The surface is characterized by wet polygonal tundra, with a micro‐relief consisting of raised moderately dry sites, depressed wet sites, polygonal ponds, and lakes. We found relatively low fluxes of typically 30 mg CH₄ m^?2 day^?1 during mid‐summer and identified soil temperature and near‐surface atmospheric turbulence as the factors controlling methane emission. The influence of atmospheric turbulence was attributed to the high coverage of open water surfaces in the tundra. The soil thaw depth and water table position were found to have no clear effect on methane fluxes. The excess emission during spring thaw was estimated to be about 3% of the total flux measured during June–October. Winter emissions were modeled based on the functional relationships found in the measured data. The annual methane emission was estimated to be 3.15 g m^?2. This is low compared with values reported for similar ecosystems. Reason for this were thought to be the very low permafrost temperature in the study region, the sandy soil texture and low bio‐availability of nutrients in the soils, and the high surface coverage of moist to dry micro‐sites. The methane emission accounted for about 14% of the annual ecosystem carbon balance. Considering the global warming potential of methane, the methane emission turned the tundra into an effective greenhouse gas source.     

Unexpectedly high bacterial diversity in arctic tundra relative to boreal forest soils, revealed by serial analysis of ribosomal sequence tags

Arctic tundra and boreal forest soils have globally relevant functions that affect atmospheric chemistry and climate, yet the bacterial composition and diversity of these soils have received little study. Serial analysis of ribosomal sequence tags (SARST) and denaturing gradient gel electrophoresis (DGGE) were used to compare composite soil samples taken from boreal and arctic biomes. This study comprises an extensive comparison of geographically distant soil bacterial communities, involving the analysis of 12,850 ribosomal sequence tags from six composite soil samples. Bacterial diversity estimates were greater for undisturbed arctic tundra soil samples than for boreal forest soil samples, with the highest diversity associated with a sample from an extreme northern location (82(o)N). The lowest diversity estimate was obtained from an arctic soil sample that was disturbed by compaction and sampled from a greater depth. Since samples from the two biomes did not form distinct clusters on the basis of SARST data and DGGE fingerprints, factors other than latitude likely influenced the phylogenetic compositions of these communities. The high number of ribosomal sequences analyzed enabled the identification of possible cosmopolitan and endemic bacterial distributions in particular soils.     

Molecular analyses of novel methanotrophic communities in forest soil that oxidize atmospheric methane

Forest and other upland soils are important sinks for atmospheric CH(4), consuming 20 to 60 Tg of CH(4) per year. Consumption of atmospheric CH(4) by soil is a microbiological process. However, little is known about the methanotrophic bacterial community in forest soils. We measured vertical profiles of atmospheric CH(4) oxidation rates in a German forest soil and characterized the methanotrophic populations by PCR and denaturing gradient gel electrophoresis (DGGE) with primer sets targeting the pmoA gene, coding for the alpha subunit of the particulate methane monooxygenase, and the small-subunit rRNA gene (SSU rDNA) of all life. The forest soil was a sink for atmospheric CH(4) in situ and in vitro at all times. In winter, atmospheric CH(4) was oxidized in a well-defined subsurface soil layer (6 to 14 cm deep), whereas in summer, the complete soil core was active (0 cm to 26 cm deep). The content of total extractable DNA was about 10-fold higher in summer than in winter. It decreased with soil depth (0 to 28 cm deep) from about 40 to 1 microg DNA per g (dry weight) of soil. The PCR product concentration of SSU rDNA of all life was constant both in winter and in summer. However, the PCR product concentration of pmoA changed with depth and season. pmoA was detected only in soil layers with active CH(4) oxidation, i.e., 6 to 16 cm deep in winter and throughout the soil core in summer. The same methanotrophic populations were present in winter and summer. Layers with high CH(4) consumption rates also exhibited more bands of pmoA in DGGE, indicating that high CH(4) oxidation activity was positively correlated with the number of methanotrophic populations present. The pmoA sequences derived from excised DGGE bands were only distantly related to those of known methanotrophs, indicating the existence of unknown methanotrophs involved in atmospheric CH(4) consumption.     

Different influences of DNA purity indices and quantity on PCR-based DGGE and functional gene microarray in soil microbial community study

Based on the comparative study of the DNA extracts from two soil samples obtained by three commercial DNA extraction kits, we evaluated the influence of the DNA quantity and purity indices (the absorbance ratios A260/280 and A260/230, as well as the absorbance value A320 indicating the amount of humic substances) on polymerase chain reaction (PCR)-based denaturing gradient gel electrophoresis (DGGE) and a functional gene microarray used in the study of microbial communities. Numbers and intensities of the DGGE bands are more affected by the A260/280 and A320 values than by the ratio A260/230 and conditionally affected by the DNA yield. Moreover, we demonstrated that the DGGE band pattern was also affected by the preferential extraction due to chemical agents applied in the extraction. Unlike DGGE, microarray is more affected by the A260/230 and A320 values. Until now, the successful PCR performance is the mostly used criterion for soil DNA purity. However, since PCR was more influenced by the A260/280 ratio than by A260/230, it is not accurate enough any more for microbial community assessed by non-PCR-based methods such as microarray. This study provides some useful hints on how to choose effective DNA extraction method for the subsequent assessment of microbial community.     

Unexpectedly High Bacterial Diversity in Arctic Tundra Relative to Boreal Forest Soils, Revealed by Serial Analysis of Ribosomal Sequence Tags

Arctic tundra and boreal forest soils have globally relevant functions that affect atmospheric chemistry and climate, yet the bacterial composition and diversity of these soils have received little study. Serial analysis of ribosomal sequence tags (SARST) and denaturing gradient gel electrophoresis (DGGE) were used to compare composite soil samples taken from boreal and arctic biomes. This study comprises an extensive comparison of geographically distant soil bacterial communities, involving the analysis of 12,850 ribosomal sequence tags from six composite soil samples. Bacterial diversity estimates were greater for undisturbed arctic tundra soil samples than for boreal forest soil samples, with the highest diversity associated with a sample from an extreme northern location (82^oN). The lowest diversity estimate was obtained from an arctic soil sample that was disturbed by compaction and sampled from a greater depth. Since samples from the two biomes did not form distinct clusters on the basis of SARST data and DGGE fingerprints, factors other than latitude likely influenced the phylogenetic compositions of these communities. The high number of ribosomal sequences analyzed enabled the identification of possible cosmopolitan and endemic bacterial distributions in particular soils.     

Methanogenesis from methanol at low temperatures by a novel psychrophilic methanogen, "Methanolobus psychrophilus" sp. nov., prevalent in Zoige wetland of the Tibetan plateau

The Zoige wetland of the Tibetan plateau is at permanent low temperatures and is a methane emission heartland of the plateau; however, cold-adaptive methanogens in the soil are poorly understood. In this study, a variety of methanogenic enrichments at 15 degrees C and 30 degrees C were obtained from the wetland soil. It was demonstrated that hydrogenotrophic methanogenesis was the most efficient type at 30 degrees C, while methanol supported the highest methanogenesis rate at 15 degrees C. Moreover, methanol was the only substrate to produce methane more efficiently at 15 degrees C than at 30 degrees C. A novel psychrophilic methanogen, strain R15, was isolated from the methanol enrichment at 15 degrees C. Phylogenetic analysis placed strain R15 within the genus Methanolobus, loosely clustered with Methanolobus taylorii (96.7% 16S rRNA similarity). R15 produced methane from methanol, trimethylamine, and methyl sulfide and differed from other Methanolobus species by growing and producing methane optimally at 18 degrees C (specific growth rate of 0.063 +/- 0.001 h(-1)) and even at 0 degrees C. Based on these characteristics, R15 was proposed to be a new species and named "Methanolobus psychrophilus" sp. nov. The K(m) and V(max) of R15 for methanol conversion were determined to be 87.5 +/- 0.4 microM and 0.39 +/- 0.04 mM h(-1) at 18 degrees C, respectively, indicating a high affinity and conversion efficiency for methanol. The proportion of R15 in the soil was determined by quantitative PCR, and it accounted for 17.2% +/- 2.1% of the total archaea, enumerated as 10(7) per gram of soil; the proportion was increased to 42.4% +/- 2.3% in the methanol enrichment at 15 degrees C. This study suggests that the psychrophilic methanogens in the Zoige wetland are likely to be methylotrophic and to play a role in methane emission of the wetland.     

利用DGGE评价不同培养基回收番茄根际细菌类群的能力

用营养肉汤、YG、根系分泌物、土壤浸渍液4种培养基从番茄根际分离培养细菌,并结合变性梯度凝胶电泳(DGGE)技术,对4种培养基回收番茄根际细菌种群的能力进行了比较研究。结果表明,不同培养基和培养温度,回收到的细菌种群有一定差异;低营养浓度的YG培养基在较低的培养温度20℃下进行较长时间的培养,比高营养浓度营养肉汤培养基产生更多、更具代表性的细菌;以根系分泌物为基础的培养基从番茄根际回收到的优势菌群最多。该研究初步建立了用DGGE技术对不同培养基回收分离细菌种群能力进行评价的方法。     

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.