DGGE Gels Research Articles

Rumen Methanogens Diversity Analysis in Indian Buffaloes Using PCR-Denaturing Gradient Gel Electrophoresis

A study aiming to investigate the rumen methanogens diversity using PCR- DGGE in Indian buffaloes (N= 10) was undertaken. Rumen fluid samples were collected from the male fistulated buffaloes. DNA from the rumen fluid samples was isolated using RBB+C method. DGGE gel band pattern was analysed and bands with differential expression were excised, cloned and sequenced for exploring the methanogens diversity in buffaloes. Sequences were aligned using Codon Code Aligner. The phylogeny was constructed based on neighbour joining method with Jukes Cantor nucleotide substitution model. Based on the differential patterns, total 31 bands were analyzed for the methanogen diversity through phylogenetic approach. Results from the study established that Methanobrevibacter is the most prominent genus of methanogens in Indian buffaloes. However, a considerable diversity exists within this genus at species level. Methanobrevibacter woesi represented the largest cluster of methanogens; while Methanobrevibacter millerae constituted the second largest fraction of archaeal community. Methanogens affiliated to Methanomicrobiales and Thermoplasmatales were not detected through DGGE in present study. To identify the methanogens from other minor groups such as Methanomicrobiales, Thermoplasmatales, Methanosarcinales further studies using high throughput techniques are warranted in buffaloes.

Development of a DGGE method to explore Legionella communities

Legionella risk assessment is nowadays based on the presence and concentration of either Legionella pneumophila or Legionella spp. Many species of Legionella can cause Legionnaires’ disease, indeed about half of the known species have been associated with infection. The aim of this work was to develop a method to assess the composition of the Legionella species community in an environmental sample in order to have a better understanding of the contamination of the ecosystem by pathogenic strains.The method is based on the comparison of PCR-DGGE profile of DNA sample with a database consisting in DGGE profiles of Legionella species. Such a database includes all pathogenic Legionella strains. In order to homogenize and normalize the different DGGE fingerprint, a reference marker has been built and added during DGGE gel analysis. This study gives a valuable advance in the methods available for the understanding of Legionella contamination of water environments.

Disease-Specific Enteric Microbiome Dysbiosis in Inflammatory Bowel Disease.

Inflammatory Bowel disease (IBD) is traditionally divided into Crohn's disease (CD) and ulcerative colitis (UC). UC is a relapsing non-transmural inflammatory disease that is restricted to the colon and is characterized by flare-ups of bloody diarrhea. CD is a chronic, segmental localized granulomatous disease that can affect any part of the entire gastrointestinal tract. Ileo-anal pouch is a procedure restoring functionality of the rectum after a colectomy. IBD is a multifactorial disease and flares of IBD are probably triggered by changes in the intestinal microbiota followed by an abnormal immune response. In this study, we aim to analyze the intestinal bacterial diversity in IBD patients during various stages of disease compared with healthy controls. Permission for human experiments and recruitment of participants was obtained from the Ethic Committee for Copenhagen County hospitals (Permission no. KA-03019, Permission no. KA-20060159). Stools from 26 healthy controls, 42 CD, 38 UC and 18 pouch patients were collected. Stool DNA extraction was performed using Qiagen, DNA mini stool kit Denmark. DGGE-PCR amplifying the V2-V3 region of 16S-rDNA gene of the bacteria was amplified by universal primers HDA1 and HDA2. Analysis of DGGE was performed blinded using BioNumerics version 7.5. After normalization, a DGGE gel band matching was performed. The similarities between profiles were calculated with a ranked Pearson correlation coefficient based on the band matching results using band intensities. Simpson's index of diversity and Pielou's species evenness were calculated. Based on the Shannon Diversity Index, UC patients had lower species diversity and bacterial evenness in comparison to healthy persons, p < 0.05. However, only CD and disease pouch patients had lower species diversity compared to those with inactive disease and healthy controls. Well-functioning pouch patients had decreased species evenness in comparison to diseased pouch patients and control group. During the active disease stage in CD and pouch, the patients have a low bacterial diversity in their gut when compared to the inactive stage. In UC patients, a generally low diversity was observed at all stages of the disease compared to healthy controls.

Differentiation and quantification of the ochratoxin A producers Aspergillus ochraceus and Aspergillus westerdijkiae using PCR‐DGGE

Ochratoxin A (OTA) is a nephrotoxic, teratogenic, immunotoxic, and carcinogenic mycotoxin which is produced in tropical zones mainly by Aspergillus carbonarius, A. niger, A. ochraceus, and A. westerdijkiae. A. ochraceus and A. westerdijkiae species are phenotypically and genomically very close but A. westerdijkiae produce OTA at a very higher level than A. ochraceus. These species have been differentiated recently. The DNA primer pairs which were drawn so far are not specific and a genomic region of the same size is amplified for both species or they are too specific, and in this case, the DNA of a single species is amplified. To help preventing OTA contamination of foodstuffs, the PCR-DGGE (Denaturing Gradient Gel Electrophoresis) method was used to discriminate between A. ochraceus and A. westerdijkiae DNA fragments of the same size but with different sequences and thus faster access to a diagnosis of the toxigenic potential of the fungal microflora. The proposed methodology was able to differentiate A. westerdijkiae from A. ochraceus with only one primer pairs in a single run. A calibration based on initial DNA content was obtained from image analysis of the DGGE gels and a method of quantification of the two strains was proposed.

Effects of stocking density of gray mullet Mugil cephalus on water quality, growth performance, nutrient conversion rate, and microbial community structure in the white shrimp Litopenaeus vannamei integrated system

An experiment (75 days) was conducted in twelve fiberglass tanks (5 m3) to study the effects of stocking density of gray mullet (Mugil cephalus) on water quality, growth performance, nutrient conversion rate, and microbial community structure in the white shrimp (Litopenaeus vannamei) integrated system. Gray mullet (body weight of 1.50 g) were added into nine shrimp tanks with three different densities, namely at 10, 20, and 30% of the initial shrimp biomass as poly-culture treatments. White shrimp (size of 0.50 g) were stocked at a density of 300 shrimp.tank−1. Three tanks stocked only with shrimp served as monoculture treatment (control treatment). Shrimp were fed four times a day using 38%, 35% protein (feed #1, #2, respectively) commercial pellet (Grobest Co. Ltd. Vietnam) with feeding rate was 3 to 10% of shrimp biomass. No external feed was given to the fish during the experiment. The results showed that the mean values of water quality parameters were not significantly different (p > 0.05) between treatments, except TSS, TAN, NO2-N concentration. The best shrimp growth performance, total biomass, and FCR (0.14 g.day−, 2928.68 g.tank−, 1.06 respectively) were found in the low gray mullet density treatment (10% of the initial shrimp biomass), which were significantly better than those in control and other poly-culture treatments. The nutrient conversion rate in poly-culture, which retained 30.41–40.32% of total nitrogen feed input and 25.26–33.54% of total phosphorous feed input, was significantly different between treatments. DGGE bacterial profile from the cultured water illustrated a dynamic microbial community structure in the water column of the experimental tanks, and a considerable difference in community structure between treatments. The phylogeny of the selected band sequences obtained from the DGGE gel was affiliated within the ˠ- Proteobacteria, α- Proteobacteria, Bacterioidetes, Planctomycetes, and Terrabacteria group. The results indicate that gray mullet can be co-cultured with white shrimp at the fish stocking density of 10% shrimp biomass to improve water quality, overall productivity and nutrient utilization efficiency.

Molecular Phylogeny and Diversity of the Salt-Tolerant Alkaliphilic Actinobacteria Inhabiting Coastal Gujarat, India

ABSTRACTActinobacteria is a dominant phylum in saline soil and play important roles in the process of organic matter decomposition and biogeochemical cycling. In this study, we investigated the diversity and phylogeny of the haloalkaliphilic actinobacteria that inhabited the saline soil of Coastal Gujarat (India) using conventional and molecular approaches. The actinobacteria were diversified on the basis of their growth patterns, morphology, spore color and sugar utilization. The cultivated actinobacteria were genetically diverse, with the ability to grow at high salt concentrations. The salt resistance feature was widely distributed among the isolates and not confined to any particular phylogenetic cluster. The PCR -DGGE approach was used to assess molecular diversity and to mitigate the limitation of the 16S rRNA sequence approach. Reproducible band profiles confirmed that the PCR-DGGE provided an excellent tool for the 16S rDNA heterogeneity analysis. The migration behavior of the 16S rRNA genes on the DGGE gel suggested lack of correlation between the band numbers and α-diversity. The findings highlighted the trends associated with the microbial community and signify the role of the DGGE in distinguishing a group of species that exhibit 16S rRNA based phylogenetic relatedness with distinct phenotypic characters. Based on the 16S rRNA genes, the actinobacteria were identified as belong to Nocardiopsis, Brachybacterium, Streptomyces and Prauseria. Nocardiopsis was the most predominant actinobacterial genera. The study indicated that a combination of the conventional and molecular approaches could be highly significant in analyzing the diversity of the actinobacteria from the saline habitat.

Effects of storage temperature on bacterial growth rates and community structure in fresh retail sushi

This study was conducted to assess the effects of different storage temperatures (4-20°C), on bacterial concentrations, growth rates and community structure in fresh retail sushi, a popular retail product with a claimed shelf life of 2-3days. The maximum specific growth rate based on aerobic plate count (APC) at 4°C was 0·06h-1 and displayed a sixfold increase (0·37h-1 ) at 20°C. Refrigeration resulted in no growth of hydrogen sulphide (H2 S)-producing bacteria, but this group had the strongest temperature response. The bacterial community structure was determined by PCR/DGGE (denaturing gradient gel electrophoresis). Multivariate analysis based on Bray-Curtis similarities demonstrated that temperature alone was not the major determinant for the bacterial community structure. The total concentration of aerobic bacteria was the variable that most successfully explained the differences between the communities. The dominating organisms, detected by sequencing of DNA bands excised from the DGGE gel, were Brochothrix thermosphacta and genera of lactic acid bacteria (LAB). The relationship between growth rates and storage temperatures clearly demonstrates that these products are sensitive to deviations from optimal storage temperature, possibly resulting in loss of quality during shelf life. Regardless of the storage temperature, the bacterial communities converged towards a similar structure and density, but the storage temperature determined how fast the community reached its carrying capacity. Little information is available on the microbial composition of ready-to-eat food that are prepared with raw fish, subjected to contamination during handling, and susceptible to microbial growth during cold storage. Moreover, the data are a good first possibility to simulate growth of APC, H2 S-producing bacteria and LAB under different temperature scenarios that might occur during production, distribution or storage.

Suitability of Different PCR-DGGE Primer Sets for the Monitoring of Lactic Acid Bacteria in Wine

Lactic acid bacteria (LAB) play a dual role in winemaking as they are the main effectors of malolactic fermentation, but some members can also cause wine spoilage. PCR-DGGE has proved to be a quick tool to study the LAB community and their fluctuation in wine. For detecting wine-associated LAB by PCR-DGGE, the primer sets WLAB1/WLAB2GC, WBAC1/WBAC2GC, Lac1/Lac1o/Lac2GC, 341fGC/518r and rpoB1/rpoB1o/rpoB2GC were tested and evaluated in this study. The primer systems were assessed by the separation of LAB reference strains on DGGE gels and by attributing the resulting amplicons to defined species. Subsequently, the detection of LAB in wine samples and enrichments thereof was compared. While the primer systems WBAC1/WBAC2GC and 341fGC/518r were not appropriate, the Lac1/Lac1o/Lac2GC primer set performed well. However, multiple bands complicated the evaluation. The rpoB1/rpoB1o/rpoB2GC set seemed to be promising for the detection of LAB in wine, although further improvements in terms of the detection limit need to be done. Due to the pronounced sensitivity and the sufficient discrimination of LAB at species level, the WLAB1/WLAB2GC primer system was found to be most suitable for studying the occurrence of LAB in wine.

Association of diverse bacterial communities in human bile samples with biliary tract disorders: a survey using culture and polymerase chain reaction-denaturing gradient gel electrophoresis methods.

Bacterial infection is considered a predisposing factor for disorders of the biliary tract. This study aimed to determine the diversity of bacterial communities in bile samples and their involvement in the occurrence of biliary tract diseases. A total of 102 bile samples were collected during endoscopic retrograde cholangiopancreatography (ERCP). Characterization of bacteria was done using culture and polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) methods. Antimicrobial susceptibility of the isolates was determined based on the Clinical and Laboratory Standards Institute (CLSI) guidelines and identity of the nucleotide sequences of differentiated bands from the DGGE gels was determined based on GenBank data. In total, 41.2% (42/102) of the patients showed bacterial infection in their bile samples. This infection was detected in 21% (4/19), 45.4% (5/11), 53.5% (15/28), and 54.5% (24/44) of patients with common bile duct stone, microlithiasis, malignancy, and gallbladder stone, respectively. Escherichia coli showed a significant association with gallstones. Polymicrobial infection was detected in 48% of the patients. While results of the culture method established coexistence of biofilm-forming bacteria (Pseudomonas aeruginosa, E. coli, Klebsiella pneumoniae, Enterococcus spp., and Acinetobacter spp.) in different combinations, the presence of Capnocytophaga spp., Lactococcus spp., Bacillus spp., Staphylococcus haemolyticus, Enterobacter or Citrobacter spp., Morganella spp., Salmonella spp., and Helicobacter pylori was also characterized in these samples by the PCR-DGGE method. Multidrug resistance phenotypes (87.5%) and resistance to third- and fourth-generation cephalosporins and quinolones were common in these strains, which could evolve through their selection by bile components. Ability for biofilm formation seems to be a need for polymicrobial infection in this organ.

The effect of crop rotation on the structure of the fungal community colonising rice straw residues in paddy rice cultured soil in the Mekong Delta of Vietnam

In this study, we compared a rice monoculture and alternating crop rotation on fungal population . The studied crop rotation systems were rice-rice-rice (CRS1), rice-rice-baby corn (CRS2), rice-rice-mungbean (CRS3) and baby corn-rice-mungbean (CRS4). A fungal primer pair (ITS1–ITS2) was used to amplify internal transcribed spacer (ITS) sequences from total DNA extracts from rice straw residues. Amplified DNA was analyzed by DGGE. Individual bands from DGGE gels were cloned and sequenced. DGGE gels representing the fungal communities of the continuous rice and the rotation site were compared and related to fungal identified from the field.The results demonstrate that the colonizing rice straw residues fungal communities were affected differently depending on crop rotation. Significant differences between the fungal communities from rice straw residues of rice in monoculture and alternating crop rotation of mungbean and baby corn were found. Cloning and sequencing of bands from the DGGE gels revealed the presence of Ascomycota and Basidiomycota that are common in agricultural soils. It was shown that the colonizing rice straw residues of crop rotation seemed to select the phylum Ascomycetes, while different members of the phylum Ascomycota and Basidiomycota were detected in the of colonizing rice straw residues rice monoculture.

Potential Activity, Size, and Structure of Sulfate-Reducing Microbial Communities in an Exposed, Grazed and a Sheltered, Non-Grazed Mangrove Stand at the Red Sea Coast.

After oxygen, sulfate is the most important oxidant for the oxidation of organic matter in mangrove forest soils. As sulfate reducers are poor competitors for common electron donors, their relative success depends mostly on the surplus of carbon that is left by aerobic organisms due to oxygen depletion. We therefore hypothesized that sulfate-cycling in mangrove soils is influenced by the size of net primary production, and hence negatively affected by mangrove degradation and exploitation, as well as by carbon-exporting waves. To test this, we compared quantitative and qualitative traits of sulfate-reducing communities in two Saudi-Arabian mangrove stands near Jeddah, where co-occurring differences in camel-grazing pressure and tidal exposure led to a markedly different stand height and hence primary production. Potential sulfate reduction rates measured in anoxic flow-through reactors in the absence and presence of additional carbon sources were significantly higher in the samples from the non-grazed site. Near the surface (0–2 cm depth), numbers of dsrB gene copies and culturable cells also tended to be higher in the non-grazed sites, while these differences were not detected in the sub-surface (4–6 cm depth). It was concluded that sulfate-reducing microbes at the surface were indeed repressed at the low-productive site as could be expected from our hypothesis. At both sites, sulfate reduction rates as well as numbers of the dsrB gene copies and viable cells increased with depth suggesting repression of sulfate reduction near the surface in both irrespective of production level. Additionally, sequence analysis of DNA bands obtained from DGGE gels based on the dsrB gene, showed a clear difference in dominance of sulfate-reducing genera belonging to the Deltaproteobacteria and the Firmicutes between sampling sites and depths.

Impact of Zinc Orthophosphate on Simulated Drinking Water Biofilms Influenced by Lead and Copper

AbstractPhosphate, a key nutrient for bacterial growth, is also a key component of many corrosion-control programs to manage lead and copper corrosion in premise plumbing. Bench-scale stagnant water galvanic macrocells with lead and copper components were fed with drinking water containing three levels of zinc orthophosphate [0 (control), 1, and 3 mg l−1-PO4]. Suspended polycarbonate coupons, representing benign downstream fixtures, were placed in the macrocells, thus enabling biofilm formation on this material. Community profiling using denaturing gradient gel electrophoresis (16S rDNA PCR-DGGE) revealed that phosphate dose (primarily) and metal type (to a lesser extent) influenced biofilm community diversity. Generally, community diversity increased with increasing heterotrophic plate counts that in turn rose in response to elevated phosphate. Partial 16s rDNA sequences obtained from DGGE gel bands identified the dominant bacterial taxa as the phyla Verrumicrobia, Firmicutes, Bacteroidetes, and α-Prote...

Molecular Analysis of Methanogen Richness in Landfill and Marshland Targeting 16S rDNA Sequences.

Methanogens, a key contributor in global carbon cycling, methane emission, and alternative energy production, generate methane gas via anaerobic digestion of organic matter. The methane emission potential depends upon methanogenic diversity and activity. Since they are anaerobes and difficult to isolate and culture, their diversity present in the landfill sites of Delhi and marshlands of Southern Assam, India, was analyzed using molecular techniques like 16S rDNA sequencing, DGGE, and qPCR. The sequencing results indicated the presence of methanogens belonging to the seventh order and also the order Methanomicrobiales in the Ghazipur and Bhalsawa landfill sites of Delhi. Sequences, related to the phyla Crenarchaeota (thermophilic) and Thaumarchaeota (mesophilic), were detected from marshland sites of Southern Assam, India. Jaccard analysis of DGGE gel using Gel2K showed three main clusters depending on the number and similarity of band patterns. The copy number analysis of hydrogenotrophic methanogens using qPCR indicates higher abundance in landfill sites of Delhi as compared to the marshlands of Southern Assam. The knowledge about “methanogenic archaea composition” and “abundance” in the contrasting ecosystems like “landfill” and “marshland” may reorient our understanding of the Archaea inhabitants. This study could shed light on the relationship between methane-dynamics and the global warming process.

Microencapsulated chlorpyrifos: Degradation in soil and influence on soil microbial community structures

Degradation kinetics of microencapsulated chlorpyrifos (CPF-MC) in soil and its influence on soil microbial community structures were investigated by comparing with emulsifiable concentration of chlorpyrifos (CPF-EC) in laboratory. The residual periods of CPF-MC with fortification levels of 5 and 20mg/kg reached 120days in soil, both of the degradation curves did not fit the first-order model, and out-capsule residues of chlorpyrifos in soil were maintained at 1.76 (±0.33) and 5.92 (±1.20) mg/kg in the period between 15 and 60days, respectively. The degradation kinetics of CPF-EC fit the first-order model, and the residual periods of 5 and 20mg/kg treatments were 60days. Bacterial community structures in soil treated with two concentrations of CPF-MC showed similarity to those of the control during the test period, as seen in the band number and relative intensities of the individual band on DGGE gels (p>0.05). Fungal community structures were slightly affected in the 5mg/kg treatments and returned to the control levels after 30days, but initially differed significantly from control in the 20mg/kg treatments (p<0.05) and did not recover to control levels until 90days later. The CPF-EC significantly altered microbial community structures (p<0.05) and effects did not disappear until 240days later. The results indicated that the microcapsule technology prolonged the residue periods of chlorpyrifos in soil whereas it decreased its side-effects on soil microbes as compared with the emulsifiable concentration formulation.

A procedure to evaluate the efficiency of surface sterilization methods in culture-independent fungal endophyte studies

Extraneous DNA interferes with PCR studies of endophytic fungi. A procedure was developed with which to evaluate the removal of extraneous DNA. Wheat (Triticum aestivum) leaves were sprayed with Saccharomyces cerevisiae and then subjected to physical and chemical surface treatments. The fungal ITS1 products were amplified from whole tissue DNA extractions. ANOVA was performed on the DNA bands representing S. cerevisiae on the agarose gel. Band profile comparisons using permutational multivariate ANOVA (PERMANOVA) and non-metric multidimensional scaling (NMDS) were performed on DGGE gel data, and band numbers were compared between treatments. Leaf surfaces were viewed under variable pressure scanning electron microscopy (VPSEM). Yeast band analysis of the agarose gel showed that there was no significant difference in the mean band DNA quantity after physical and chemical treatments, but they both differed significantly (p < 0.05) from the untreated control. PERMANOVA revealed a significant difference between all treatments (p < 0.05). The mean similarity matrix showed that the physical treatment results were more reproducible than those from the chemical treatment results. The NMDS showed that the physical treatment was the most consistent. VPSEM indicated that the physical treatment was the most effective treatment to remove surface microbes and debris. The use of molecular and microscopy methods for the post-treatment detection of yeast inoculated onto wheat leaf surfaces demonstrated the effectiveness of the surface treatment employed, and this can assist researchers in optimizing their surface sterilization techniques in DNA-based fungal endophyte studies.

Bacterial-biota dynamics of eight bryophyte species from different ecosystems

Despite the importance of bryophyte-associated microorganisms in various ecological aspects including their crucial roles in the soil-enrichment of organic mass and N2 fixation, nonetheless, little is known about the microbial diversity of the bryophyte phyllospheres (epi-/endophytes). To get insights into bacterial community structures and their dynamics on the bryophyte habitats in different ecosystems and their potential biological roles, we utilized the 16S rRNA gene PCR-DGGE and subsequent phylogenetic analyses to investigate the bacterial community of eight bryophyte species collected from three distinct ecosystems from western Japan. Forty-two bacterial species belonging to γ-proteobacteria and Firmicutes with 71.4% and 28.6%, respectively, were identified among 90 DGGE gel band population. These DGGE-bands were assigned to 13 different genera with obvious predomination the genus Clostridium with 21.4% from the total bacterial community. These analyses provide new insights into bryophyte-associated bacteria and their relations to the ecosystems.

Dietary Supplementation of Usnic Acid, an Antimicrobial Compound in Lichens, Does Not Affect Rumen Bacterial Diversity or Density in Reindeer

Reindeer (Rangifer tarandus tarandus) may include large proportions of lichens in their winter diet. These dietary lichens are rich in phenolic secondary compounds, the most well-known being the antimicrobial usnic acid. Previous studies have shown that reindeer host rumen bacteria resistant to usnic acid and that usnic acid is quickly detoxified in their rumen. In the present study, reindeer (n = 3) were sampled before, during, and after usnic acid supplementation to determine the effect on their rumen microbial ecology. Ad libitum intake of usnic acid averaged up to 278 mg/kg body mass. Population densities of rumen bacteria and methanogenic archaea determined by real-time PCR, ranged from 1.36 × 10(9) to 11.8 × 10(9) and 9.0 × 10(5) to 1.35 × 10(8) cells/g wet weight, respectively, and the two populations did not change significantly during usnic acid supplementation (repeated measures ANOVA) or vary significantly between the rumen liquid and particle fraction (paired t test). Rumen bacterial community structure determined by denaturing gradient gel electrophoresis did not change in response to intake of usnic acid. Firmicutes (38.7 %) and Bacteriodetes (27.4 %) were prevalent among the 16S rRNA gene sequences (n = 62) from the DGGE gels, but representatives of the phyla Verrucomicrobia (14.5 %) and Proteobacteria (1.6 %) were also detected. Rapid detoxification of the usnic acid or resistance to usnic acid may explain why the diversity of the dominant bacterial populations and the bacterial density in the reindeer rumen does not change during usnic acid supplementation.

The Differences in Colonic Mucosal Microbiota Between Normal Individual and Colon Cancer Patients by Polymerase Chain Reaction-denaturing Gradient Gel Electrophoresis

The aim of this study was to analyze the differences in the intestinal composition between normal individuals and colon cancer patients. To establish the criteria for screening a normal individual for colon cancer, human colonic biopsies were obtained at routine colonoscopy. For patients with colon cancer, samples were obtained from cancerous regions. For normal individuals, colonic biopsies were taken from 3 sites of large intestine (descending, transverse, and ascending colon). Thereafter, a comparison of the microbiota structure by polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) was carried out. At last, bacterial species were identified by sequencing special bands from DGGE gels and comparing data with sequence databases. With PCR-DGGE, we have discovered that the diversity and richness of the bacterial community from colon cancer patient's colonic mucosa were lower than that of the normal individual's sample. Then, a special DGGE band was found in the colon cancer patients. After sequencing, we confirmed that it had a high level of similarity with bacteroides. Colon cancers are closely related with the alteration of intestinal flora such as the reduction of biodiversity and richness of the bacterial community. Furthermore, the increase in proportion of bacteroides may be directly associated with colon cancer.

Development of microbial properties and enzyme activities in copper mine wasteland during natural restoration

A copper mine wasteland consists of a pedogenetic substrate, and regeneration of ecosystems on mine wasteland undergoes typical primary succession. In this study, microbial biomass, basic respiration, microbial diversity and enzyme activities were investigated during the natural successional process in two copper mine wastelands with different restoration development times. As the plant ecosystem progressed from bare land with cryptogamic crusts to vascular plant communities, organic matter, total nitrogen (N), available phosphorus (P) and water holding capacity increased, whereas pH and toxicity of heavy metals decreased. Along with the natural process of soil development resulting from the amelioration of substrate condition, microbial biomass C and N, and urease and alkaline phosphatase activities in surface layers increased, and the metabolic quotient decreased. Microbial biomass gradually exhibited strong spatial heterogeneity during succession. Microbial biomass and enzyme activities had positive correlations with organic matter and nutrient conditions (N and P), and negative correlations with pH and heavy metals. The concentrations of organic matter, N, P and available Cu significantly influenced microbial biomass and activity. The analysis of 16S rDNA fragments by polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) showed no obvious trend in microbial diversity during succession. Phylogenetic analysis of 16S rDNA sequences generated from DGGE gels indicated that bacteria mainly included Proteobacteria (alpha-, beta- and gamma-Proteobacteria), Firmicutes (Bacillus and Clostridia), Actinobacteria, Nitrospirae, Deinococcus-Thermus and Bacteroidetes. The natural successional series consisted of species adapted to mine wastelands. Soil microbial biomass and enzyme activities tended to be more sensitive to heavy metals.

Bioaugmentation with a pyridine-degrading bacterium in a membrane bioreactor treating pharmaceutical wastewater

The bacterial strain Paracoccus denitrificans W12, which could utilize pyridine as its sole source of carbon and nitrogen, was added into a membrane bioreactor (MBR) to enhance the treatment of a pharmaceutical wastewater. The treatment efficiencies investigated showed that the removal of chemical oxygen demand, total nitrogen, and total phosphorus were similar between bioaugmented and non-bioaugmented MBRs, however, significant removal of pyridine was obtained in the bioaugmented reactor. When the hydraulic retention time was 60 hr and the influent concentration of pyridine was 250–500 mg/L, the mean effluent concentration of pyridine without adding W12 was 57.2 mg/L, while the pyridine was degraded to an average of 10.2 mg/L with addition of W12. The bacterial community structure of activated sludge during the bioaugmented treatment was analyzed using polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE). The results showed that the W12 inoculum reversed the decline of microbial community diversity, however, the similarity between bacterial community structure of the original sludge and that of the sludge after bioaugmentation decreased steadily during the wastewater treatment. Sequencing of the DNA recovered from DGGE gel indicated that Flavobacteriaceae sp., Sphingobium sp., Comamonas sp., and Hyphomicrobium sp. were the dominant organisms in time sequence in the bacterial community in the bioaugmented MBR. This implied that the bioaugmentation was affected by the adjustment of whole bacterial community structure in the inhospitable environment, rather than being due solely to the degradation performance of the bacterium added.