PCR-amplified 16S rDNA Fragments Research Articles

Non-crop sources of Rapeseed Phyllody phytoplasma (‘Candidatus Phytoplasma asteris’: 16SrI-B and 16SrI-(B/L)L), and closely related strains

In Poland, a phytoplasma belonging to the subgroup 16SrI-B of ‘Ca. Phytoplasma asteris’ species causes disease in winter oilseed rape (Brassica napus), named rapeseed phyllody (RP). Affected plants exhibit malformations of flowers – phyllody, leading to losses in seed production. However, the potential sources of RP phytoplasma (RPp) outside oilseed rape are poorly understood. A screening survey of non-crop wild plants associated with winter oilseed rape was performed in order to identify alternative plant hosts of RPp. Nested polymerase chain reaction (PCR) and sequencing of PCR-amplified 16S rDNA fragments were employed for the detection and identification of phytoplasma strains affecting oilseed rape crops and adjacent wild plants. Phytoplasmas belonging to subgroups 16SrI-B, 16SrI-L, 16SrI-(B/L)L and 16SrI-C, as well as subgroup 16SrXII-H, were detected in non-crop plants. All investigated oilseed rapes manifesting RP disease were positive to ‘Ca. Phytoplasma asteris’ ribosomal subgroup 16SrI-B or heterogenic variant 16SrI-(B/L)L. Wild plants species were predominantly infected with the same phytoplasma subgroups as oilseed rapes. However, comparative sequence analysis revealed single nucleotide polymorphisms that indicate divergence of 16SrI-B and 16SrI-(B/L)L phytoplasma strains in non-crop species. Surprisingly, most of the weeds which tested positive for phytoplasma did not exhibit any characteristic symptoms of infection. In this study we identified for the first time perennial (Elymus repens, Lolium perenne) and annual (Echinochloa crus-galli, Polygonum aviculare, Viola arvensis, Matricaria perforata, Stelaria media) plants as asymptomatic reservoirs of RPp related strains in the winter oilseed rape agroecosystem. We also confirmed RPp related infections in diseased Apera spica-venti, Sisymbrium officinale, Matricaria discoidea, and Papaver rhoeas. These plants potentially play a significant role in RPp evolution and disease aetiology and epidemiology.

Continuous volatile fatty acid production from lignocellulosic biomass by a novel rumen-mimetic bioprocess

Lignocellulosic biomass is an attractive source of biofuels and biochemicals, being abundant in various plant sources. However, processing this type of biomass requires hydrolysis of cellulose. The proposed rumen-mimetic bioprocess consists of dry-pulverization of lignocellulosic biomass and pH-controlled continuous cultivation of ruminal bacteria using ammonium as a nitrogen source. In this study, ruminal bacteria were continuously cultivated for over 60 days and used to digest microcrystalline cellulose, rice straw, and Japanese cedar to produce volatile fatty acids (VFAs). The ruminal bacteria grew well in the chemically defined medium. The amounts of VFAs produced from 20g of cellulose, rice straw, and Japanese cedar were 183±29.7, 69.6±12.2, and 21.8±12.9mmol, respectively. Each digestion completed within 24h. The carbon yield was 60.6% when 180mmol of VFAs was produced from 20g of cellulose. During the cultivation, the bacteria were observed to form flocs that enfolded the feed particles. These flocs likely contain all of the bacterial species necessary to convert lignocellulosic biomass to VFAs and microbial protein symbiotically. Denaturing gradient gel electrophoresis (DGGE) analysis of PCR-amplified 16S rDNA fragments revealed that the bacterial community was relatively stable after 1 week in cultivation, though it was different from the original community structure. Furthermore, sequence analysis of the DGGE bands indicates that the microbial community includes a cellulolytic bacterium, a bacterium acting synergistically with cellulolytic bacteria, and a propionate-producing bacterium, as well as other anaerobic bacteria.

Investigation of Bacteria and Fish Pathogenic Bacteria Found in Freshwater Aquaponic System

Bacteria are very vital organisms in aquaponic system decomposing and converting the toxic components of the fish waste into usable vegetable nutrients; however, some unwanted bacteria could cause fish diseases. The purposes of this research were to compare the amount of bacteria in water from aquaponic catfish cultured in different stocking densities and Denaturing Gradient Gel Electrophoresis (DGGE) of Polymerase Chain Reaction (PCR)-amplified 16S rDNA was applied to characterise the bacterial populations. Three different stocking densities; 80, 100 and 120 fish/m2 of hybrid catfish were cultured in aquaponic system. Each treatment contained 3 replications; Treatment 1, 360 catfish (80 fish/m2, without lettuce); Treatment 2, 360 catfish (80 fish/m2, without lettuce); Treatment 3, 450 catfish (100 fish/m2); and treatment 4, 540 catfish (120 fish/m2). This research was conducted for 4 months. Water was sampled two times before fish stocking, it was found that the number of bacteria in the first week were higher than the third week. The average number of bacteria in water from treatment 1, 2, 3 and 4 were (2.49 ± 0.40) ×107, (2.43 ± 0.23) ×107, (2.48 ± 2.22) ×107 and (2.29 ± 2.38) ×107 Colonies forming unit (CFU)/ml respectively. According to four-time samplings after catfish were stocked, the average number of bacteria in water from treatment 1, 2, 3 and 4 were (1.85 ± 0.53) ×107, (2.73 ± 0.15) ×107, (2.45 ± 0.30) ×107 and (2.24 ± 0.19) ×107 CFU/ml respectively. The average number of bacteria in water after lettuce harvest from treatment 1, 2, 3 and 4 were (0.185 ± 0.04) ×107, (0.26 ± 0.04) ×107, (0.30 ± 0.01) ×107 and (0.29 ± 0.05) ×107 CFU/ml respectively. There were no differences in bacterial counts among treatments (p > 0.05). In addition, no significant difference was observed in the number of bacteria in filter tanks (p > 0.05) and the number were quite similar to the ones in catfish tanks. Aeromonas hydrophila and Flavobacterium columnare infected catfish at the beginning of the experiment; however, it got better and no more death found after acclimation. The DGGE analysis of PCR-amplified 16S rDNA fragments revealed that bacterial populations in water were dominated by Aeromonas spp., Pseudomonas spp. and Staphylococcus spp. Antibiotic sensitivity test indicated that enrofloxacin might be the best option for bacterial infection treatment. The results indicate that aquaponic system could reduce the wastewater from catfish culture; however, maintaining a good water quality environment for fish culture should take into account.

Performance of a haloalkaliphilic bioreactor under different ratios

Effects of NO3(-)/SO4(2-) ratio on denitrification and sulfate removal efficiency were investigated in model experiments applying haloalkaliphilic bioreactor. The reduction of both substrates performed well at different NO3(-)/SO4(2-) ratios ranging from 17.6 to l.5. The removal rates of nitrate and sulfate were 6 and 1.39kgm(-3)d(-1), respectively, at NO3(-)/SO4(2-) ratio 3.0, while sulfide concentration reached up to 703gm(-3). The major sulfate-reducing and denitrifying bacteria were Desulfonatronovibrio sp. and Halomonas campisalis, respectively. Decrease in NO3(-)/SO4(2-) ratio led to obvious changes in bacterial community. Although the sulfate reducers became dominant, the population of denitrifying ones also increased as it was demonstrated by analysis of PCR-amplified 16S rDNA fragments, which suggested that SRB and DB coexisted well in bioreactor.

CHARACTERISATION OF SOYBEAN RHIZOBIAL STRAINS FROM JAVA AND SUMATRA

To get insight in the structure of soybean rhizobial population native to Indonesian soils, a thorough survey of the occurrence of the soybean rhizobia were conducted in several locations in Java and Sumatra. A total of 51 different isolates of rhizobial strains were characterised phenotypically based on their symbiotic properties, and genetically using amplified ribosomal DNA restriction analysis (ARDRA). Based on their nodulation capacity on both soybean and the native legume mungbean, these rhizobial strains could be divided into a group of 16 strains specific for soybean only and another group of 35 promiscuous strains that nodulated both leguminous plants. Based on ARDRA of PCRamplified 16S rDNA and 16S-23S rDNA spacer fragments, the rhizobial strains isolated from Java differed with those from Sumatra. Six Java isolates and only one Sumatra isolate were classified as Bradyrhizobium japonicum and these similar to that of B. japonicum strain USDA 110. All these B. japonicum strains were highly specific for soybean. One isolate from Java showed a rather unique position. The remaining strains from Java (20), which were symbiotically promiscuous strains, were clustered in another group. This group and another group containing most Sumatra isolates were distinct from B. japonicum USDA 110 and therefore it is tempting to speculate that these represent indigenous soybean rhizobial bacteria. Application of agricultural practices, such as enhancement of rhizobial population, to increase soybean production is still essential and noteworthy in Sumatra.

CHARACTERISATION OF SOYBEAN RHIZOBIAL STRAINS FROM JAVA AND SUMATRA

To get insight in the structure of soybean rhizobial population native to Indonesian soils, a thorough survey of the occurrence of the soybean rhizobia were conducted in several locations in Java and Sumatra. A total of 51 different isolates of rhizobial strains were characterised phenotypically based on their symbiotic properties, and genetically using amplified ribosomal DNA restriction analysis (ARDRA). Based on their nodulation capacity on both soybean and the native legume mungbean, these rhizobial strains could be divided into a group of 16 strains specific for soybean only and another group of 35 promiscuous strains that nodulated both leguminous plants. Based on ARDRA of PCRamplified 16S rDNA and 16S-23S rDNA spacer fragments, the rhizobial strains isolated from Java differed with those from Sumatra. Six Java isolates and only one Sumatra isolate were classified as Bradyrhizobium japonicum and these similar to that of B. japonicum strain USDA 110. All these B. japonicum strains were highly specific for soybean. One isolate from Java showed a rather unique position. The remaining strains from Java (20), which were symbiotically promiscuous strains, were clustered in another group. This group and another group containing most Sumatra isolates were distinct from B. japonicum USDA 110 and therefore it is tempting to speculate that these represent indigenous soybean rhizobial bacteria. Application of agricultural practices, such as enhancement of rhizobial population, to increase soybean production is still essential and noteworthy in Sumatra.

Microbial community changes during the process of pyrite bioleaching

An analysis of the community composition of predefined mixed cultures (Acidithiobacillus ferrooxidans, Acidithiobacillus thiooxidans, Acidithiobacillus caldus, Leptospirillum ferrooxidans and Sulfobacillus thermosulfidooxidans) under different temperatures and initial pH values in pyrite bioleaching process was carried out. Denaturing gradient gel electrophoresis (DGGE) analysis of PCR-amplified 16S rDNA fragments from bacteria was used to evaluate the changes of the bacterial community in the process of pyrite bioleaching. Microbial community changes were observed in response to temperature and initial pH value changes. The species of predominant bacteria changed with the change of temperature at the same pH value. A. ferrooxidans and A. thiooxidans were the dominant species at 30°C and 35°C; L. ferrooxidans was also found at 35°C, while at 40°C it became the dominant species along with A. caldus and S. thermosulfidooxidans which was the dominant species both at 40°C and 45°C. The microbial community composition on the surface of pyrite was found to be similar with that in the bioleaching solution. It can be concluded that temperature had an important effect on the microbial community structure and composition in pyrite bioleaching.

Changes in the bacterial community structure and diversity during bamboo retting

Microbial retting is a critical step in obtaining fiber bundles from bamboo culm using indigenous microorganisms. A cultivation-independent technique for monitoring the changes in bacteria community during bamboo retting was applied in this work. This technique involves genetic profiling of PCR-amplified small-subunit rRNA and the single-strand conformation polymorphism (SSCP) gel analysis of the PCR-amplified 16S rDNA fragments. The study revealed that both the structure and the diversity of investigated communities varied with the incubation periods and sample locations. The bacteria bands from SCCP gel profiles related to Bacillus sp. decreased in intensity, and Phaeospirillum sp. and Azospirillum brasilense completely disappeared during the 4(th) and 5(th) month of incubation, while the bands related to the Sphingomonas japonica, Alphaproteobacterium Ellin335 and Microbacterium sp. increased. The bands closely related to Sphingomonads, Brevundimonas brasilense, Pseudoclavibacter sp., Agrococcus jenensis and Oxalophagus oxalicus remained dominant during the whole incubation period. This study showed that the use of PCR assay targeting 16S rRNA and SCCP profiling provided valuable information on monitoring the bacteria dynamic changes occurring in the bacteria community during bamboo retting, which is crucial for controlling the quality of the retting process and improving the retting efficiency, and thus benefits for fiber recovery.

Denaturing gradient gel electrophoresis analyses of the vertical distribution and diversity of Vibrio spp. populations in the Cariaco Basin

The Cariaco system is the second largest permanently anoxic marine water body in the world. Its water column is characterized by a pronounced vertical layering of microbial communities. The goal of our study was to investigate the vertical distribution and diversity of Vibrio spp. present in the Cariaco Basin waters using denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rDNA fragments. Representatives of the Vibrio genus were detected by nested and direct PCR in seawater at 10 depths. Sequence analyses of 55 DGGE bands revealed that only 11 different operational taxonomic units (OTU) are identified as Vibrio species. Between one and five OTUs were detected at each depth and the most common OTUs were OTU 1 and OTU 2, which phylogenetically clustered with Vibrio chagasii and Vibrio fortis, respectively. OTUs 3 and 4 were only found in the anoxic zone and were identified as Vibrio orientalis and Vibrio neptunius, respectively. Several Vibrio species detected are potentially pathogenic to human, prawns and corals such as Vibrio parahaemolyticus, Vibrio fischeri and Vibrio shilonii. In the Cariaco Basin, different Vibrio species were found to be specific to specific depths strata, suggesting that this genus is a natural component of the microbial communities in this marine redox environment.

PCR-DGGE analysis of bacterial communities in funazushi, fermented crucian carp with rice, during fermentation

Funazushi (fermented Crucian Carp with rice) is a fermented fish product found only around Lake Biwa in Shiga Prefecture, Japan. It is characterized by a unique cheese-like flavor and characteristic sour taste. We analyzed the changes in the microbial community during funazushi fermentation by denaturing gradient gel electrophoresis of PCR-amplified 16S rDNA fragments (PCR-DGGE) and by plate counts. The plate counts showed that lactic acid bacteria reached 8.0 log10 CFU/g within 7 days of fermentation initiation before decreasing slowly to 4.0 log10 CFU/g during the remainder of 1-year study period. PCR-DGGE revealed that the dominant bacteria in the initial (days 14 and 30) and latter (days 90, 180, and 360) periods of fermentation were Lactobacillus plantarum and L. acetotolerans. This is the first identification of L. acetotolerans in funazushi as traditional cultivation techniques have not been sufficiently sensitive. This is the first report of PCR-DGGE being used to assess the microbial community in funazushi. This technique was also found to be effective in profiling microbial diversity.

Different colonization and residence time of Listonella anguillarum and Vibrio splendidus in the rotifer Brachionus plicatilis determined by real-time PCR and DGGE

Listonella anguillarum 90-11-287 and Vibrio splendidus DMC-1 were incorporated in the rotifer Brachionus plicatilis, which was subsequently maintained under larval rearing conditions to determine the residence time of both pathogens in rotifers. Real-time PCR was applied to specifically detect and quantify both pathogens. L. anguillarum colonized rotifers more efficiently than V. splendidus and both pathogenic strains were released from rotifers to seawater, after infected rotifers were transferred to rearing tanks. V. splendidus grew and became predominant in the seawater of tanks. Both pathogens remained in rotifer or seawater enough time to infect fish larvae, but their different behaviour could determine different infection patterns, preferentially by ingestion of prey or by active intake or contact with surrounding seawater. The effect of L. anguillarum and V. splendidus on the bacterial community associated with rotifers and seawater of rearing tanks was analysed by DGGE of PCR-amplified 16S rDNA fragments. The bacterial community of rotifers did not present a marked species dominance. The incorporation of L. anguillarum or V. splendidus did not reduce bacterial diversity and shifts could be explained by bacterial exchange between rotifers and seawater. Main bacterial groups were identified by sequencing the DNA extracted from Marine Agar (MA) bacterial isolates and DGGE excised bands. Only 2 DGGE bands corresponded to bacteria isolated from MA plates, suggesting that bacterial groups present in rotifers may not be easily cultivable. The bacterial community of rotifers was composed by Gram negative bacteria belonging to α-Proteobacteria ( Ruegeria spp), γ-Proteobacteria ( Alteromonas alvinellae, Marinobacter sp, an Oceanospirillaceae bacterium and Pseudoalteromonas sp), Cytophaga–Flexibacter–Bacteroides group ( Polaribacter dokdonensis, Roseivirga spongicola and Tenacibaculum soleae) and a Gram positive bacterium ( Microbacterium sp).

Spatial and seasonal variations in bacterial communities of the Yellow Sea by T-RFLP analysis

Four typical coastal sites (rocky shore, sandy shore, mud flat shore, and artificial harbor) at the Yellow Sea were chosen to investigate the spatial and seasonal variations in bacterial communities. This was accomplished by using terminal restriction fragment length polymorphism (T-RFLP) analysis of PCR amplified 16S rDNA fragments. Two kinds of tetrameric restriction enzymes, HhaI and MspI, were used in the experiment to depict the bacterial community diversity in different marine environments. It was found that the community compositions digested by the two enzymes separately were different. However, the results of bacterial community diversity derived from them were similar. The MDA analysis results of T-RFLP profiles coming from HhaI and MspI both exhibited a significant seasonal community shift for bacteria and a relatively low spatial variation among the four locations. With HhaI as the sample, the pair wise T-tests also revealed that variations were minor between each pair of marine environments, with R ranging from 0.198 to 0.349. However, the bacterial community structure in the mud flat site depicted a larger difference than each of the other three sites (R ranging from 0.282 to 0.349).

DNA Microarrays for Identifying Fishes

In many cases marine organisms and especially their diverse developmental stages are difficult to identify by morphological characters. DNA-based identification methods offer an analytically powerful addition or even an alternative. In this study, a DNA microarray has been developed to be able to investigate its potential as a tool for the identification of fish species from European seas based on mitochondrial 16S rDNA sequences. Eleven commercially important fish species were selected for a first prototype. Oligonucleotide probes were designed based on the 16S rDNA sequences obtained from 230 individuals of 27 fish species. In addition, more than 1200 sequences of 380 species served as sequence background against which the specificity of the probes was tested in silico. Single target hybridisations with Cy5-labelled, PCR-amplified 16S rDNA fragments from each of the 11 species on microarrays containing the complete set of probes confirmed their suitability. True-positive, fluorescence signals obtained were at least one order of magnitude stronger than false-positive cross-hybridisations. Single nontarget hybridisations resulted in cross-hybridisation signals at approximately 27% of the cases tested, but all of them were at least one order of magnitude lower than true-positive signals. This study demonstrates that the 16S rDNA gene is suitable for designing oligonucleotide probes, which can be used to differentiate 11 fish species. These data are a solid basis for the second step to create a “Fish Chip” for approximately 50 fish species relevant in marine environmental and fisheries research, as well as control of fisheries products.

Polymerase chain reaction-based denaturing gradient gel electrophoresis in the evaluation of oral microbiota

Clinical evaluation of oral microbial reduction after a standard prophylactic treatment has traditionally been based on bacterial cultivation methods. However, not all microbes in saliva or dental plaque can be cultivated. Polymerase chain reaction-based denaturing gradient gel electrophoresis (PCR-DGGE) is a cultivation-independent molecular fingerprinting technique that allows the assessment of the predominant bacterial species present in the oral cavity. This study sought to evaluate the oral microbial changes that occurred after a standard prophylactic treatment with a conventional oral care product using PCR-DGGE. Twelve healthy adults participated in the study. Pooled plaque samples were collected at baseline, 24 h after prophylaxis (T1), and 4 days after toothbrushing with fluoride toothpaste (T4). The total microbial genomic DNA of the plaque was isolated. PCR was performed with a set of universal bacterial 16S rDNA primers. The PCR-amplified 16S rDNA fragments were separated by DGGE. The effects of the treatment and of dental brushing were assessed by comparing the PCR-DGGE fingerprinting profiles. The mean numbers of detected PCR amplicons were 22.3 +/- 6.1 for the baseline group, 13.0 +/- 3.1 for the T1 group, and 13.5 +/- 4.3 for the T4 group; the differences among the three groups were statistically significant (P < 0.01). The study also found a significant difference in the mean similarities of microbial profiles between the baseline and the treatment groups (P < 0.001). PCR-based DGGE has been shown to be an excellent means of rapidly and accurately assessing oral microbial changes in this clinical study.

The effect of sample size in studies of soil microbial community structure

Replicate soil samples of 0.01, 0.1, 0.25, 1.0 and 10.0 g were taken from a single, large, homogenized sample from a field maintained as continuous meadow. The samples were processed for direct enumeration of bacterial cells and community structure assays by DGGE analysis of PCR-amplified 16S-rDNA fragments from whole community extracts. The goal was to determine the sample size or size range that produced the most consistent results (i.e., mean values) and the lowest variance. Enumeration data were analyzed by ANOVA, and the community composition fingerprints were analyzed by discriminant analysis (DA). Acceptable results were obtained for sample sizes from 0.1 to 1.0 g for both enumeration and community fingerprinting, but the size that yielded the best results for both measures was 0.25 g. The results suggest that for well homogenized silt loam soils with moderate organic matter concentrations, this sample size should produce high quality consistent results. For soils that differ in organic concentrations or clay content, a reconnaissance survey similar to the present examination is recommended.

The use of wood in practice – a hygienic risk?

The survival of bacteria on wood was investigated in laboratory experiments using naturally occurring and hygienically relevant bacteria and in a trial performed in a meat factory. Different types of wood dust (pine, larch, maple) and polyethylene chips were inoculated with chicken manure to study the interactions of bacteria and wood. The survival of the different bacteria from chicken manure was followed by plating on selective media. In addition, denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rDNA fragments from directly extracted DNA was used to precisely analyze changes in the relative abundance and composition of the manure-derived bacterial community on wood and plastic. Bands of DGGE-community profiles which reflected changes in the composition of the bacterial community on wood and plastic were excised, re-amplified, cloned and analyzed by sequencing. A rapid decrease of CFU of manure-derived bacteria was observed on pine-wood which correlated with the decreased amount of DNA extracted from pine-wood. Bacteria applied on larch, maple and plastic survived longer than on pine. Investigations have shown that some bacterial groups, e.g. enterobacteria, are more affected by wood and plastic than other bacteria. Furthermore, our results indicated that extractives from different wood types influenced the survival of bacterial species differently. Better hygienic characteristics of pine-wood compared to plastic as a material for transportation pallets were also observed in preliminary tests under practice conditions during a 4-week test in a meat processing factory.

Profiling and monitoring of microbial populations by denaturing high-performance liquid chromatography

We describe a new molecular technique for the analysis of microbial species and complex microbial populations based on the separation of PCR-amplified 16S rDNA fragments by denaturing high-performance liquid chromatography (DHPLC). Using marine bacterial samples, we determined the optimum conditions for the analysis of bacterial species and the examination of complex bacterial assemblages obtained from different environments. The incorporation of a 40-bp GC clamp into the amplification primer was essential to effectively discriminate genetic differences in DHPLC—primers with a 20-, 10-, or 0-bp GC clamp length were less efficient. A 64.5 °C column temperature in DHPLC allowed optimal separation of species in a complex bacterial population. PCR–DHPLC analysis of bacterial assemblages demonstrated profiles with distinguishable peaks, which constituted the different populations and their degree of abundance. Fraction collection and DNA sequencing from profile peaks enabled bacterial identification. PCR–DHPLC analysis can also provide opportunities for describing bacterial communities, cloning bacteria, and monitoring bacterial populations in environments of interest.

Optimized sequence retrieval from single bands of temperature gradient gel electrophoresis profiles of the amplified 16S rDNA fragments from an activated sludge system

Sequence retrieval from single bands of polymerase chain reaction (PCR)-denaturing gel electrophoresis (DGE) profiles is an important but often difficult step for molecular diversity analysis of complex microbial communities such as activated sludge systems. We analyzed the temperature gradient gel electrophoresis (TGGE) profiles of PCR-amplified 16S rDNA fragments from an activated sludge sample of a coking wastewater treatment plant. Single bands were excised, and a clone library was constructed for each. Sequence heterogeneity in each single band was found to be significantly overestimated due to single-stranded DNA (ssDNA) contamination formed during the PCR amplification, since only 10–60% of library clones of each single TGGE band had identical migration behavior compared with the parent band. Three methods, digestion with mung bean nuclease, optimization of PCR amplification, and purification via denatured polyacrylamide gel electrophoresis (d-PAGE), were compared for their ability to minimize ssDNA contamination, with the last one being the most efficient. After using d-PAGE to minimize ssDNA to a nearly nondetectable level, 70–100% of library clones for each single TGGE band had identical migration compared with the parent band. Several sequences were found in each of six single bands, and this co-migration could be predicted with the Poland software. The predominant bacteria of the activated sludge were assessed via a combination of sequence retrieval from each single TGGE band and band intensity analysis. Only beta and alpha subclasses of the Proteobacteria were detected, 93.8% and 6.2%, respectively. Our work suggests that prior to constructing a clone library to retrieve the actual sequence diversity of a single DGE band, it is advisable to minimize ssDNA contamination to a nondetectable level.

Bacterial population dynamics and community structure in a pharmaceutical manufacturing water supply system determined by real-time PCR and PCR-denaturing gradient gel electrophoresis

To control bacteria in the pharmaceutical water supply system. Bacteria were enumerated by conventional culture method and fluorescent vital staining. Activated carbon treatment and storage in a tank provided favourable environments for bacterial growth. The bacterial population of the water in both the post-activated carbon treatment and the tank was analysed by denaturing gradient gel electrophoresis (DGGE) with PCR-amplified 16S rDNA fragments including V6, -7, and -8 regions. The bacterial community structure in activated carbon treated water was stable throughout the year. Several kinds of bacteria such as genus Aquaspirillum and Methylobacterium were found in the water after activated carbon treatment. The bacterial community structure was changed and other bacteria such as mycobacteria were detected after storage. Mycobacteria were quantified in water samples using real-time PCR targeting the 16S rDNA gene. Mycobacteria were also detected in tap water and their number was increased 10(3)-10(4)-fold higher after storage. These data suggest the importance of culture-independent methods for quality control of water used in pharmaceutical manufacturing. Critical steps and specified bacteria that should be controlled in the water supply system were recognized by culture-independent methods. These data will enable effective control of water used in the pharmaceutical industry.

Microbial composition and structure of a rotating biological contactor biofilm treating ammonium-rich wastewater without organic carbon.

High nitrogen losses were observed in a rotating biological contactor (RBC) treating ammonium-rich (up to 500 mg NH4(+)-N/L) but organic-carbon-poor leachate from a hazardous waste landfill in Kölliken, Switzerland. The composition and spatial structure of the microbial community in the biofilm on the RBC was analyzed with specific attention for the presence of aerobic ammonium and nitrite oxidizing bacteria and anaerobic ammonium oxidizers. Anaerobic ammonium oxidation (anammox) involves the oxidation of ammonium with nitrite to N2. First the diversity of the biofilm community was determined from sequencing cloned PCR-amplified 16S rDNA fragments. This revealed the presence of a number of very unusual 16S rDNA sequences, but very few sequences related to known ammonium or nitrite oxidizing bacteria. From analysis of biofilm samples by fluorescence in situ hybridization with known phylogenetic probes and by dot-blot hybridization of the same probes to total RNA purified from biofilm samples, the main groups of microorganisms constituting the biofilm were found to be ammonium-oxidizing bacteria from the Nitrosomonas europaea/eutropha group, anaerobic ammonium-oxidizing bacteria of the "Candidatus Kuenenia stuttgartiensis" type, filamentous bacteria from the phylum Bacteroidetes, and nitrite-oxidizing bacteria from the genus Nitrospira. Aerobic and anaerobic ammonium-oxidizing bacteria were present in similar amounts of around 20 to 30% of the biomass, whereas members of the CFB phylum were present at around 7%. Nitrite oxidizing bacteria were only present in relatively low amounts (less than 5% determined with fluorescence in situ hybridization). Data from 16S rRNA dot-blot and in situ hybridization were not in all cases congruent. FISH analysis of thin-sliced and fixed biofilm samples clearly showed that the aerobic nitrifiers were located at the top of the biofilm in an extremely high density and in alternating clusters. Anammox bacteria were exclusively present in the lower half of the biofilm, whereas CFB-type filamentous bacteria were present throughout the biofilm. The structure and composition of these biofilms correlated very nicely with the proposed physiological functional separations in ammonium conversion.