16S rDNA Denaturing Gradient Gel Electrophoresis Research Articles

Progressive change from nitrate to nitrite as the electron acceptor for the oxidation of H2S under feedback control in an anoxic biotrickling filter

Nitrate has been progressively replaced by nitrite as the electron acceptor in the oxidation of H2S from biogas mimic by an anoxic biotrickling filter. Perturbations in the inlet H2S concentration with and without the implementation of feedback controllers have been evaluated. Nitrite was successfully used without decreasing the H2S removal efficiency (94.74 ± 0.01%). Moreover, a PID controller allowed the sulfate selectivity to be maintained and reduced the maximum outlet H2S concentration when compared to the system without control (54.4–64.4%), although elemental sulfur was the main oxidation product. On using ORP control the main disadvantage was the production of peaks in the outlet H2S concentration. The stability and resilience of the biological system were assessed by analyzing the microbial community profiles by 16S rDNA-denaturing gradient gel electrophoresis (DDGE). The modification of the electron acceptor led to a reduction in the diversity and increased the dominance of the microbial populations.

Effects of polyhydroxyalkanoate degradation on soil microbial community

Polyhydroxyalkanoate (PHA) serves as an alternative to some petroleum-based materials due to its biodegradability nature. Nevertheless, no study has been conducted before to evaluate the impact of PHA degradation to the environment. The purpose of this research was to investigate the degradation of PHA and its effects towards the soil microbial community. Four types of PHAs with three different thickness were used in this study; poly(3-hydroxybutyrate) [P(3HB)], poly(3HB-co-8 mol% 3-hydroxyhexanoate) [P(3HB-co-8 mol% 3HHx)], P(3HB-co-12 mol% 3HHx) and P(3HB-co-21 mol% 3HHx). These films were buried at a secondary forest for a duration of 8 weeks. P(3HB-co-21 mol% 3HHx) showed the highest degradation than the rest of the films. 16S rDNA metagenomic analysis revealed that some of the major phyla that were found at the sampling sites included Actinobacteria, Firmicutes and Proteobacteria which had the ability to degrade PHA. 16S rDNA-Denaturing Gradient Gel Electrophoresis (DGGE) profiling successfully showed that the diversity and population of the soil microbial community were correlated with the degradation of PHA. There was a significant change in the soil microbial abundancy before and after the PHA was degraded.

Advanced treatment of refractory organic pollutants in petrochemical industrial wastewater by bioactive enhanced ponds and wetland system

A large-scale combined ponds-wetland system was applied for advanced treatment of refractory pollutants in petrochemical industrial wastewater. The system was designed to enhance bioactivity and biological diversity, which consisted of anaerobic ponds (APs), facultative ponds (FPs), aerobic pond and wetland. The refractory pollutants in the petrochemical wastewater to be treated were identified as alkanes, chloroalkanes, aromatic hydrocarbons, and olefins, which were significantly degraded and transformed along with the influent flowing through the enhanced bioactive ponds-wetland system. 8 years of recent operational data revealed that the average removal rate of stable chemical oxygen demand (COD) was 42.7 % and that influent COD varied from 92.3 to 195.6 mg/L. Final effluent COD could reach 65.8 mg/L (average). COD removal rates were high in the APs and FPs and accounted for 75 % of the total amount removed. This result indicated that the APs and FPs degraded refractory pollutants through the facilitation of bacteria growth. The changes in the community structures of major microbes were assessed by 16SrDNA-denaturing gradient gel electrophoresis. The same analysis was used to identify the main bacterial function for the removal of refractory pollutants in the APs and FPs. The APs and FPs displayed similar microbial diversities, and some of the identified bacteria degraded and removed refractory pollutants. The overall results proved the applicability, stability, and high efficiency of the ponds-wetland system with enhanced bioactivity in the advanced removal of refractory pollutants from petrochemical industrial wastewater.

Do dietary betaine and the antibiotic florfenicol influence the intestinal autochthonous bacterial community in hybrid tilapia (Oreochromis niloticus ♀ × O. aureus ♂)?

The attractant betaine and the antibiotic growth promoter florfenicol are commonly used together in Chinese fresh water aquaculture, but there is no information about the effect of these two feed additive on the intestinal autochthonous bacterial community in hybrid tilapia (Oreochromis nilotica ♀ × O. aureas ♂). Hybrid tilapia (240 fish in total; 20 fish per net cage; three cages per group) were divided into four dietary groups: control group, no betaine or florfenical addition (CK); betaine group, 0.1% betaine added (B); florfenicol group, 0.002% florfenicol added (F); and combination group, 0.1% betaine and 0.002% florfenicol added together (BF). After 8 weeks of feeding, six fish from each cage were chosen randomly, the guts were sampled and pooled, and their intestinal autochthonous bacterial communities were analyzed by 16S rDNA-denaturing gradient gel electrophoresis. Enumeration of total gut autochthonous bacteria was analyzed by quantitative PCR with rpoB as the endogenous control. The results showed that the fish intestinal bacteria of group B were more diverse than that of CK, and that of F and BF groups was reduced in the total numbers and limited to certain bacterial species or genera (P < 0.05). This study revealed that betaine can promote some intestinal autochthonous bacteria, and florfenicol play a depressor role. When combined together, florfenicol may overshadow the effect of betaine on the predominant intestinal bacteria of tilapia.

Impact of water quality on bacterioplankton assemblage along Cértima River Basin (central western Portugal) assessed by PCR–DGGE and multivariate analysis

The information on bacterial community composition (BCC) in Portuguese water bodies is very scarce. Cértima River (central western Portugal) is known to have high levels of pollution, namely organic. In the present work, the BCC from a set of 16 water samples collected from Cértima River Basin and its main tributaries was characterized using 16S rDNA-denaturing gradient gel electrophoresis, a culture-independent molecular approach. Molecular data were related to environmental parameters through multivariate analysis to investigate potential impact of water pollution along the river. Principal component analysis using environmental data showed a water quality gradient from more pristine waters (at the mountain tributaries) to waters with increasingly eutrophic potential (such as Fermentelos Lake). This gradient was mainly defined by factors such as organic and inorganic nutrient sources, electrical conductivity, hydrogen carbonate concentration, and pH. Molecular results showed variations in BCC along Cértima River Basin but in the main river section, a Bacteroidetes phylotype (Flavobacterium sp.) proved to be dominant throughout the river course. Multivariate analysis suggests that spatial variation of BCC along the Cértima River Basin depended mainly on parameters such as Chl a, total suspended solid (TSS), total organic carbon, electrical conductivity, and HCO[Formula: see text] levels. Bacteroidetes phylotypes were all related to higher electrical conductivity and HCO[Formula: see text] levels although some of these were also correlated with high SO[Formula: see text] and others with high soluble reactive phosphorus, nitrate, TN, and Kjeld-N levels. The Gammaproteobacteria occurrence was correlated with high SO[Formula: see text] levels. One of the Betaproteobacteria phylotypes showed to correlate with low redox potential (E(h)) and high temperature, pH, TSS, and Chl a levels while another one showed a negative correlation with Chl a values.

Quantitative improvement of 16S rDNA DGGE analysis for soil bacterial community using real-time PCR

Denaturing gradient gel electrophoresis (DGGE) of 16S rDNA fragments has been frequently used to profile a structure of the bacterial community in a given soil. However, this procedure has various types of intrinsic error and bias, thus often misleads the relative abundance of bacterial populations. In order to establish a reliability for the current 16S rDNA DGGE method, we investigated various parameters and potential sources of errors in the DGGE procedures, such as primer mismatch, dNTP concentration, DNA polymerase, PCR cycles, uneven amplification of templates, secondary structure of PCR product, melting domain profiles, and acrylamide/bis concentration. Our result showed that the relative band intensities of the corresponding 16S rDNA templates were closely correlated with the differences of the melting temperature between the higher and lower melting domains of the PCR products. In addition, application of i) real-time PCR, ii) combination of PCR primers and iii) optimization of both dNTP and acrylamide/bis concentrations significantly improved the quantitative representation of bacterial 16S rDNA levels in the mixed samples. Especially, identification of the inflection points of DNA samples through the real-time PCR was crucial for the accurate representation of soil bacterial populations. Beyond these points DNA templates can be over-amplified to a saturated level independently of their initial amounts. Therefore for the accurate analysis of soil bacterial community, a quantitative 16S rDNA DGGE analysis needs to be performed in combination with a real-time PCR.

Comparisons of different hypervariable regions of rrs genes for fingerprinting of microbial communities in paddy soils

The use of molecular approaches based on 16S rDNA-PCR in microbial ecology has revealed a tremendous prokaryotic diversity in environmental samples. However, there is little or no systematic evaluation of the impacts of hypervariable (V) regions of rrs genes choice on microbial community analysis in soil samples, especially the detailed information about the dominant groups preferentially amplified by different primer pairs. In the present study, eight primer pairs were detected to compare the different V regions for fingerprinting microbial communities in a paddy soil irrigated with petroleum-wastewater, using denaturing gradient gel electrophoresis (DGGE) and amplified ribosomal DNA restriction analysis (ARDRA) techniques. Results reveal the obvious PCR bias produced by different V regions. Both ARDRA analysis of 16S rDNA clone library and DGGE suggest that V 1–V 3 region amplified with primer pair 8f–519r produced the most informative fingerprinting profiles. Additionally, V 3–V 5 region amplified with 341f–907r was another preferable choice for microbial diversity in petroleum-contaminated soil. The V 4–V 5 region and single V region (V 1, V 3, and V 8) were not recommended for the future study of microbial diversity in soil samples. Phylogenetic analysis of 123 sequences from libraries constructed by amplicons generated from six different V regions suggests that different dominant groups were amplified with distinct primer sets. In detail, V 1–V 3 library (amplified with 8f–519r) and V 3–V 5 library were dominated by Actinobacteria (20.4%) (particularly in genus Arthrobacter), V 1–V 3 library (amplified with 63f–518r) was dominated by γ-Proteobacteria (25.0%) and α-Proteobacteria (22.0%) (particularly in genus Brevundimonas), V 3 library was dominated by β-Proteobacteria (22.3%) (particularly in genus Gallionella) and α-Proteobacteria (20.0%), V 6–V 8 library was dominated by Chlamydiae (20.4%) and β-Proteobacteria (20.4%), V 8 library was dominated by γ-Proteobacteria (27.2%) (particularly in genus Acinetobacter) and β-Proteobacteria (14.0%). The present work strongly recommends that primer pairs should be chosen cautiously in community diversity analysis based on PCR amplification of 16S rDNA, and involving at least two different 16S rDNA universal primer pairs would perform better.

Extraction of soil bacteria from a Ferralsol

Extraction of intact bacteria from soil by dispersion and density gradient centrifugation may facilitate analyses of soil bacterial communities which are otherwise hampered by soil particles. Reasonable cell yield and representative fractions with sufficient purity can be extracted from most soils, but highly weathered, clayey and acidic tropical soils like Ferralsols represent a challenge to the method due to low cell yields. At an early stage of our studies with Ferralsols we also found substantial contamination of the extracted bacterial fractions by soil material, measured as total Al and Fe. We have adapted the method to a Ferralsol (pH 4.9, 64% clay, 21% silt, 15% sand) by factorial combinations of pH modification and salt (NaCl) concentration during soil dispersion, and evaluated the yield (microscopic cell counts), purity (optical density and Al + Fe content), and compared the composition of the extracted bacteria (phospholipid fatty acid analysis and 16S rDNA-denaturing gradient gel electrophoresis) versus that of the intact soil community. The cell yield was increased with a factor 2–3 by increasing the pH to 7.5, while it was decreased to a similar extent by adding NaCl (8 g l −1). However, NaCl removed more than 99% of the Al + Fe contamination of the bacterial extracts, and the combination of modified pH and NaCl addition secured reasonable cell yield (4.6% of total number) and low contamination. The observed effects of pH and NaCl are probably due to changes in variable charge (by pH) and ion distribution (NaCl) around interacting particles (soil and bacteria), thus affecting their flocculation. Phospholipid fatty acid and denaturing gradient gel electrophoresis analysis indicate that the bias of the bacterial extracts compared to direct soil extracts increases with the addition of NaCl as well as by pH manipulation. Nevertheless, representativeness was acceptable as indicated by a Bray–Curtis similarity index (bacterial extracts versus soil) of 70% and 87% for phospholipid fatty acid profiles and denaturing gradient gel electrophoresis, respectively. Overall, the results reveal a trade-off among yield, purity and representativeness. Thus, depending on application and analyses, future users can choose the right treatment according to their specific purpose.

Diversity of bacterial communities in container habitats of mosquitoes.

We investigated the bacterial diversity of microbial communities in water-filled, human-made and natural container habitats of the mosquitoes Aedes aegypti and Aedes albopictus in suburban landscapes of New Orleans, Louisiana in 2003. We collected water samples from three classes of containers, including tires (n = 12), cemetery urns (n = 23), and miscellaneous containers that included two tree holes (n = 19). Total genomic DNA was extracted from water samples, and 16S ribosomal DNA fragments (operational taxonomic units, OTUs) were amplified by PCR and separated by denaturing gradient gel electrophoresis (DGGE). The bacterial communities in containers represented diverse DGGE-DNA banding patterns that were not related to the class of container or to the local spatial distribution of containers. Mean richness and evenness of OTUs were highest in water samples from tires. Bacterial phylotypes were identified by comparative sequence analysis of 90 16S rDNA DGGE band amplicons. The majority of sequences were placed in five major taxa: Alpha-, Beta- and Gammaproteobacteria, Actinobacteria, Bacteroidetes, Cyanobacteria, Firmicutes, and an unclassified group; Proteobacteria and Bacteroidetes were the predominant heterotrophic bacteria in containers. The bacterial communities in human-made containers consisted mainly of undescribed species, and a phylogenetic analysis based on 16S rRNA sequences suggested that species composition was independent of both container type and the spatial distribution of containers. Comparative PCR-based, cultivation-independent rRNA surveys of microbial communities associated with mosquito habitats can provide significant insight into community organization and dynamics of bacterial species.

Genetic characterisation of commercially important Brachionus sp. strains with 16s rDNA sequencing and DGGE (denaturing gradient gel electrophoresis) analysis

Genetic characterisation of commercially important Brachionus sp. strains with 16s rDNA sequencing and DGGE (denaturing gradient gel electrophoresis) analysis

Analysis of bacterial communities on aging flue-cured tobacco leaves by 16S rDNA PCR–DGGE technology

Many microorganisms, growing on aging flue-cured tobacco leaves, play a part in its fermentation process. These microflora were identified and described by culture-dependent methods earlier. In this study we report the identity of the microflora growing on the tobacco leaf surface by employing culture-independent methods. We have amplified microbial 16S rDNA sequences directly from the leaf surface and used denaturing gradient gel electrophoresis (DGGE) to identify bacterial community on the tobacco leaves. Our culture-independent methods for the study of microbial community on tobacco leaves showed that microbial community structures on leaves of variety Zhongyan 100, NC89 and Zhongyan 101 were similar between 0 and 6 months aging, and between 9 and 12 months aging, while the similarity is low between 0 and 6, and between 9 and 12 months aging, respectively. There were certain similarities of bacterial communities (similarity up to 63%) among the three tobacco varieties for 0 to 6 months aging. Five dominant 16S rDNA DGGE bands A, B, C, D and E were isolated, cloned, and sequenced. They were most similar to two cultured microbial species Bacteriovorax sp. EPC3, Bacillus megaterium, and three uncultured microbial species, respectively.

Microbial diversity along a transect of agronomic zones

The diversity of microbial communities constitutes a critical component of good soil-management practices. To characterize the effects of different management practices, molecular indicators such as phospholipid fatty acid (PLFA), denaturing gradient gel electrophoresis (DGGE) and composition of ammonia-oxidizing bacteria were used to analyze bacterial community structure and diversity from four eastern Washington State soils. Samples from four sites were collected representing a transect of high-precipitation to low-precipitation areas that covered different agronomic zones with different management and cropping practices. Biomass amounts estimated from extractable PLFA were significantly higher in the no-till (NT) soil than in the conventional-till (CT) soil. Similarities among the different 16S rDNA DGGE band profiles were analyzed quantitatively using correspondence analysis and this confirmed that the CT soil was the most dissimilar soil. DGGE analysis of 16S rDNA ammonia-oxidizing bacteria from the four soils revealed two identical bands, indicating little effect of agronomic practices and precipitation on these species. A second set of primers, specific for amoA (ammonia monooxygenase) genes, was used to examine ammonia oxidizers in the samples. Six banding patterns (clusters) from amplified rDNA restriction analysis of 16S rDNA fragments were observed after restriction analysis with HinfI. Sequencing of these clones revealed the presence of only Nitrosospira-like sequences. Analysis of the sequences showed that ammonia oxidizers from the NT soil were more diverse compared to those from the CT and conservation reserve program soils. Our data showed that management and agronomic practices had more impact on bacterial community structure than annual precipitation.

Microbial diversity along a transect of agronomic zones

The diversity of microbial communities constitutes a critical component of good soil-management practices. To characterize the effects of different management practices, molecular indicators such as phospholipid fatty acid (PLFA), denaturing gradient gel electrophoresis (DGGE) and composition of ammonia-oxidizing bacteria were used to analyze bacterial community structure and diversity from four eastern Washington State soils. Samples from four sites were collected representing a transect of high-precipitation to low-precipitation areas that covered different agronomic zones with different management and cropping practices. Biomass amounts estimated from extractable PLFA were significantly higher in the no-till (NT) soil than in the conventional-till (CT) soil. Similarities among the different 16S rDNA DGGE band profiles were analyzed quantitatively using correspondence analysis and this confirmed that the CT soil was the most dissimilar soil. DGGE analysis of 16S rDNA ammonia-oxidizing bacteria from the four soils revealed two identical bands, indicating little effect of agronomic practices and precipitation on these species. A second set of primers, specific for amoA (ammonia monooxygenase) genes, was used to examine ammonia oxidizers in the samples. Six banding patterns (clusters) from amplified rDNA restriction analysis of 16S rDNA fragments were observed after restriction analysis with HinfI. Sequencing of these clones revealed the presence of only Nitrosospira-like sequences. Analysis of the sequences showed that ammonia oxidizers from the NT soil were more diverse compared to those from the CT and conservation reserve program soils. Our data showed that management and agronomic practices had more impact on bacterial community structure than annual precipitation.