Nested PCR-denaturing Gradient Gel Electrophoresis Research Articles

Comparison of fungal communities among ten macrophyte rhizospheres

The fungal community composition, size and several physico-chemical properties were individually investigated in ten macrophyte rhizospheric substrates using nested PCR-denaturing gradient gel electrophoresis and soil chemical methods. Results indicated that both Dothideomycetes and Sordariomycetes were dominant fungi in macrophyte rhizospheric substrates, and denitrifying fungi (Fusarium graminearum) was found in nine of ten macrophyte rhizospheres. Fungal Shannon-Wiener diversity index (H) and richness (S) in Thalia dealbata, Typha latifolia, Iris hexagona and Hemerocallis aurantiaca rhizospheres were higher than those in other six rhizospheres. Fungal number and biomass were 1.91 × 103 CFUs g−1 dw and 1.53 μg ergosterol g−1 dw in Iris pseudacor rhizosphere, and were greater than in other nine rhizospheres. The correlation analysis showed that fungal number and biomass significantly and positively correlated to total soil phosphorus, while fungal H and S were significantly and negatively correlated to total organic carbon. The principal components analysis (PCA) showed that the fungal community significantly divided ten macrophyte rhizospheres into four groups, showing the significant difference of fungal communities among ten rhizospheric substrates. The current study revealed for the first time the importance of rhizospheric fungal community in distinguishing macrophyte rhizospheres, thus will undoubtedly widen our insight into fungal communities in aquatic rhizospheres.

Microbial Community Composition Associated with Maotai Liquor Fermentation.

The solid-state fermentation state of Chinese Maotai liquor involves the interaction of several complex microbial communities leading to the generation of the most complex liquor fermentation system in the world and contributes to the unique flavor and aroma of the liquor. In this study, total DNA was extracted from 3 fermented grain samples (FG1, FG2, and FG3) and 12 environmental samples, including Daqu (DA1, DA2, DA3, and DA4), cellar mud (CS1, CS2, and CS3), soil (SL1 and SL2), air (A1 and A2), and sorghum (SH), and the 16S and 18S rRNA genes were amplified. The distribution of typical microorganisms in the samples was analyzed using nested PCR-denaturing gradient gel electrophoresis, while quantitative PCR amplification of 16S rRNA and internal transcribed spacer genes was performed to estimate the microbial abundance present in each sample. The results indicated that Daqu was the primary source of bacteria, followed by the air, soil, and sorghum samples, while the majority of the fungi responsible for Maotai liquor fermentation were from Daqu and sorghum. Highest bacterial concentrations were found in fermented grains, followed by Daqu and sorghum, while the highest fungal concentrations were found in Daqu, followed by sorghum and an air sample from outside the liquor production area. The findings of this study may provide information regarding the mechanisms responsible for flavor development in Maotai liquor, and may be used to further optimize the traditional art of making liquor.

Difference of microbial community stressed in artificial pit muds for Luzhou-flavour liquor brewing revealed by multiphase culture-independent technology.

Artificial pit muds (APMs) is produced by peats, aged pit muds, yellow and black clays etc. and is one of essential factors for Luzhou-flavour liquor production. The microbial community of APMs significantly influence the quality of Luzhou-flavour liquor. The aim of this study was to investigate the differences in bacterial, archaeal and fungal community of APMs, starters and materials. Multiphase culture-independent technology were employed in this study, including nested PCR-denaturing gradient gel electrophoresis (nested PCR-DGGE), phospholipid fatty acid (PLFA), phospholipid ether lipids (PLEL) and fluorescence in situ hybridization (FISH) analysis. Results suggested that the microbial diversity significantly changed under environmental stress and different culture patterns during APMs cultivation. The dominant bacteria in APMs mainly fell into Clostridiales, Lactobacillales, Bacteroidales and Rhizobiales, Archaea affiliated with Methanomicrobiales and Methanosarcinales, and fungi belonged to Saccharomycetales and Eurotiales. Furthermore, the microbial community structures of APMs cultured by ground pile pattern were more similar with that of aged pit muds, meanwhile, the relative bands intensities of microbes, which are the main contributors for liquor brewing, increased with the culture times. Not only the niche selection and biogeochemical properties of APMs, but also the mutual collaboration and constraint between different microbes may result in enriching different liquor-brewing microbes into APMs. APM cultivation technology was necessary to promote enriching functional liquor-brewing microbes into APMs. These results may facilitate understanding the microbial succession during APMs manufacture.

Histamine development and bacterial diversity in microbially-challenged tonggol (Thunnus tonggol) under temperature abuse during canning manufacture.

Histamine formation and bacteriological changes caused by temperature abuse commonly occurring in the manufacturing process of standard canned tuna was assessed in microbiologically challenged tonggol (Thunnus tonggol). The in situ challenge was performed by water-soaking at 26-28°C for 7h to ensure the multiplication and active phase of fish microflora. Right after pre-cooking to back-bone temperature (BBT) of 50-52°C, histamine dropped to 5.17 ± 2.71ppm, and slowly reached 6.84 ± 1.69ppm at 16h abuse. On the contrary, histamine was reduced to 2.87 ± 1.23ppm and eventually reached 5.01 ± 1.32ppm at 24h abuse in the pre-cooked fish previously frozen. The numbers of total aerobic bacteria, Enterobactericeae, psychrotroph, histamine forming bacteria (HFB) and diversity of fish microflora were revealed by cultural and nested PCR-Denaturing Gradient Gel Electrophoresis (PCR-DGGE) techniques. Interestingly, frozen storage effectively halted histamine formation in raw fish throughout 16h abuse despite the presence of HFB. These included the prolific strains of Morganella morganii, Proteus penneri, Proteus mirabilin, Citrobacter spp. The nested PCR-DGGE profile confirmed the presence of M. morganii and Citrobacter spp. in raw fish. These prolific strains were hardly observed in the precooked fish previously frozen. Frozen storage did not only promote even histamine distribution throughout fish muscle but also enhanced histamine loss during thawing and pre-cooking. Therefore, pre-cooking and frozen storage were proven to be the effective combined hurdles not only to reduce but also prolong histamine formation of the challenged toggol throughout 24h of temperature abuse during canning process.

Characterization of fungal community and dynamics during the traditional brewing of Wuyi Hong Qu glutinous rice wine by means of multiple culture-independent methods

The objective of this study was to investigate the fungal community and dynamics during the traditional fermentation of Wuyi Hong Qu (black-skin-red-koji) glutinous rice wine using culture-independent approaches based on nested PCR-denaturing gradient gel electrophoresis (PCR-DGGE), 18S ribosomal RNA (rRNA) gene clone libraries and species-specific quantitative PCR (qPCR). Both of nested PCR-DGGE and 18S rRNA gene clone libraries revealed that the predominant microorganisms varied in different brewing phases. The relative proportions of some fungal species (such as Monascus purpureus, Rhizopus oryzae, Pichia guilliermondii and Saccharomycopsis fibuligera) detected at the early brewing stage decreased as the fermentation progressed, while Saccharomyces cerevisiae became the dominant species at the later stage. The present study revealed that PCR-DGGE fingerprint and 18S rRNA gene clone libraries presented the similar results. Nevertheless, the differences of the fungal species detected by the two different molecular biological methods demonstrated that the combined approach of nested PCR-DGGE and 18S rRNA gene clone libraries would give a more comprehensive profile of the fungal dynamics than either alone. Finally, species-specific qPCR was also performed to quantify some dominant fungal species detected by nested PCR-DGGE and 18S rRNA gene clone libraries. Results showed that R. oryzae increased sharply and dominated during the early brewing period, but decreased continuously from day 10 to the end of the traditional brewing. S. fibuligera and P. guilliermondii were the dominant yeast species at early stage, while S. cerevisiae increased sharply during the early phase and became the primary species at the end of the traditional brewing process. This study provides the first detailed evaluation of fungal community and dynamics in Wuyi Hong Qu glutinous rice wine traditional brewing process using multiplex culture-independent methods, which would show great value in scientifically understanding of the brewing mechanism and be useful for the selection of the suitable and beneficial fungal strains to improve the wine quality.

Characterization of activity and microbial diversity of typical types of Daqu for traditional Chinese vinegar

Daqu is a fermentation starter used in traditional Chinese vinegar production and determines the quality of the final vinegar products. In this study, the activity and microbial diversity of three typical types of Daqu were investigated using a nested PCR–denaturing gradient gel electrophoresis (DGGE) and plating technique. The bacterial DGGE profile and plate analysis indicated that the bacterial composition of Daqu was dominated by Bacillus sp., Pantoea sp., Saccharopolyspora sp., Enterobacter cloaceae and Cronobacter sakazakii, and lactic acid bacteria including Weissella sp., Pediococcus pentosaceus. DGGE analysis of fungal plates identified Aspergillus niger, Absidia corymbifera, Rhizopus sp. and Penicillium sp. as the predominant fungi involved in Daqu. However, DGGE results indicated that the bacterial microbiota of QC (Qing-Cha) and HX (Hong-Xin) were more complex than that of HR (Huai-Rang) Daqu. The total populations of microorganisms in QC were about 1 log CFU g−1 higher than in HR and HX Daqu. Moreover, the saccharifying power of QC was significantly higher than in HR and HX Daqu, and the liquefying power of QC was significantly higher than in HR, but slightly lower than in HX Daqu. Our results indicate that microbial diversity plays a crucial role in determining the activity of typical types of Daqu.

Nested PCR-denaturing gradient gel electrophoresis analysis of human skin microbial diversity with age

To determine whether the composition and structure of skin microbiota differ with age, cutaneous bacteria were isolated from the axillary fossa of 37 healthy human adults in two age groups (old people and young adults). Bacterial genomic DNA was extracted and characterized by nested PCR-denaturing gradient gel electrophoresis (PCR-DGGE) with primers specifically targeting V3 region of the 16S rRNA gene. The excised gel bands were sequenced to identify bacterial categories. The total bacteria, Staphylococcus spp., Staphylococcus epidermidis and Corynebacterium spp. were further enumerated by quantitative PCR. There were no significant differences in the species diversity profiles between age groups. The similarity index was lower across age groups than that it was intra-group. This indicates that the composition of skin flora is more similar to others of the same age than across age groups. While Staphylococcus spp. and Corynebacterium spp. were the dominant bacteria in both groups, sequencing and quantitative PCR revealed that skin bacterial composition differed by age. The copy number of total bacteria and Corynebacterium spp. were significantly lower in younger subjects, whereas there were no statistical differences in the quantity of Staphylococcus spp. and Staphylococcus epidermidis. These results suggest that the skin flora undergo both quantitative and qualitative changes related to aging.

Characterization of eubacterial and archaeal community diversity in the pit mud of Chinese Luzhou-flavor liquor by nested PCR–DGGE

The aim of this study was to investigate and compare the microbial community structures of eubacteria and archaea in the pit mud of Chinese Luzhou-flavor liquor from the wall (C(w)) and bottom (C(b)) of cellar through nested PCR-denaturing gradient gel electrophoresis (DGGE). The Shannon-Wiener index (H) calculated from the DGGE profiles showed that the community diversities of eubacteria and archaea in samples from C(b) were almost higher than that from C(w). In addition, cluster analysis of the DGGE profiles revealed that some differences were found in the microbial community structure in samples from different locations. The closely relative microorganisms of all eubacterial 16S rRNA gene sequences fell into four phyla (Firmicutes, Proteobacteria, Bacteroidetes and Actinobacteria), including 12 genera and 2 uncultured eubacteria. Moreover, 37.1% eubacteria were affiliated with Clostridium. Particularly, genus Acinetobacter was absent in all samples from C(b) but present in all samples from C(w). The closely relative microorganisms of all archaeal 16S rRNA gene sequences fell into four genera, which included Methanobrevibacter, Methanoculleus, Methanobacterium and Methanosaeta, while the dominant archaea in samples from C(w) and C(b) were similar. Results presented in this study provide further understanding of the spatial differences in microbial community structure in the pit mud, and is of great importance for the production and quality improvement of Luzhou-flavor liquor.

RDNA-based DGGE analysis and electron microscopic observation of cocoa beans to monitor microbial diversity and distribution during the fermentation process

This study describes the use of a nested PCR-denaturing gradient gel electrophoresis (DGGE) approach and scanning electron microscopy (SEM) to monitor the microbial diversity and distribution involved in spontaneous box fermentation of cocoa beans in Brazil. The nearly complete 16S rRNA and 18S rRNA genes were amplified using universal primers, and the resulting products were subsequently used as a template in a nested PCR to obtain suitable fragments for DGGE. Only two bands corresponding to the yeast species Saccharomyces cerevisiae and Hanseniaspora sp. were detected throughout the fermentation process. Sequencing of purified 16S rDNA amplicons indicated Lactobacillus plantarum, Lactobacillus fermentum and Acetobacter sp. to be the major bacterial groups during the fermentation course. Other partial 16S rDNA sequences corresponded to close relatives of Lactobacillus sp., Bacillus sp., Bacillus licheniformis, Bacillus firmus, Frateuria aurantia, Tatumella ptyseos, Xanthomonas sp. and an uncultured bacterium. SEM showed a well-defined microbial succession that was initially dominated by lemon-shaped yeast cells growing in close association with Lactobacillus (long and curved), and subsequently surpassed by short bacilli-shaped cells. This is the first report of SEM being used for following the microbial distribution during cocoa fermentation. Therefore, the nested PCR-DGGE and SEM techniques seem to offer a relatively fast and reliable method in monitoring microbial diversity and distribution during cocoa fermentation, providing real-time information and theory basis for the development of the starter.

Analysis of Yeast-Like Symbiote Diversity in the Brown Planthopper (BPH), Nilaparvata lugens Stål, Using a Novel Nested PCR-DGGE Protocol

Yeast-like symbiotes (YLS) are endosymbionts that are intimately associated with the growth, development, reproduction of their host, the brown planthopper, Nilaparvata lugens (Hemiptera: Delphacidae). However, it is unclear how many species of YLS are found within N. lugens, and how they are related to each other. Traditional methods or simple amplification based on 18S rDNA sequence does not reliably identify new species quickly and efficiently. Therefore, a novel nested PCR-denaturing gradient gel electrophoresis (DGGE) strategy was developed in this article to analyze the YLS of brown planthopper using a nested PCR protocol that involved the 18S rDNA gene and the 5.8S-ITS gene using fungal universal primers. The nested PCR protocol was developed as follows: firstly, the 18S rDNA gene, and 5.8S-ITS gene were amplified using fungal universal primers. Subsequently, these products were used as a template in a second PCR with primers ITS1GC-ITS2, ITS1FGC-ITS2, and NFGC-NR, which was suitable for DGGE. Using this highly specific molecular approach, we found several previously detected fungi: Noda, Pichia guilliermondii, Candida sp., and some previously undetected fungi, such as Saccharomycetales sp., Debaryomyces hansenii, and some uncultured fungi. In conclusion, the nested PCR system developed in this study, coupled with DGGE fingerprinting, offers a new tool for uncovering fungal endosymbiont diversity within planthoppers.

Analysis of bacterial diversity during the fermentation of inyu, a high-temperature fermented soy sauce, using nested PCR-denaturing gradient gel electrophoresis and the plate count method

The diversity of bacteria associated with the fermentation of inyu, also known as black soy sauce, was studied through the nested PCR-denaturing gradient gel electrophoresis (DGGE) of samples collected from the fermentation stages of the inyu production process. The DGGE profiles targeted the bacterial 16S rDNA and revealed the presence of Citrobacter farmeri, Enterobacter cloacae, Enterobacter hormaechei, Enterococcus faecium, Klebsiella pneumoniae, Pantoea agglomerans, Salmonella enterica, Serratia marcescens, Staphylococcus sciuri and Weissella confusa. The bacterial compositions of 4 fermented samples were further elucidated using the plate count method. The bacteria isolated from the koji-making stage exhibited the highest diversity; Brachybacterium rhamnosum, E. hormaechei, K. pneumoniae, Kurthia gibsonii, Pantoea dispersa, Staphylococcus gallinarum, Staphylococcus kloosii and S. sciuri were identified. Koji collected during the preincubation stage presented the largest cell counts, and E. hormaechei, K. pneumoniae, E. cloacae and Enterobacter pulveris were identified. In brine samples aged for 7 and 31 days, the majority of the bacteria isolated belonged to 4 Bacillus species, but 4 Staphylococcus species and Delftia tsuruhatensis were also detected. This study demonstrates the benefits of using a combined approach to obtain a more complete picture of microbial populations and provides useful information for the control or development of bacterial flora during inyu fermentation.

Analysis and comparison of bacterial communities in two types of ‘wheatQu’, the starter culture of Shaoxing rice wine, using nested PCR-DGGE

Shaoxing rice wine, manufactured in Shaoxing region of Zhejiang Province, is the best-known Chinese rice wine. In the current study, the bacterial communities associated with two types of ‘wheat Qu’, the Shaoxing rice wine starter cultures, were identified and compared using nested PCR–denaturing gradient gel electrophoresis analysis. DNA sequencing of the highly variable V3 or V9 regions of the 16S rRNA genes obtained from PCR–DGGE fingerprints identified common species related to Saccharopolyspora hordei, S. rectivirgula, Bacillus subtilis, B. amyloliquefaciens and so on. Some sequences associated with a particular type were also identified. For example, Clavibacter michiganensis and Saccharopolyspora sp. were found in the machine- but not in the human-made type, while Curtobacterium plantarum, Thermoactinomyces intermedius, T. vulqaris, B. licheniformis and Bacillus sp. were found in human- but not in machine-made type of wheat Qu. The information gathered from the present research enriches the present knowledge of Shaoxing rice wine-related bacteria, and will also facilitate the long-term objective of selecting bacterial combinations to be used as starter cultures that will more tightly regulate the fermentation process, as well as improve the quality and safety of wheat Qu. Copyright © 2012 The Institute of Brewing & Distilling

Microbial diversity of traditional fermentation starters for Hong Qu glutinous rice wine as determined by PCR-mediated DGGE

Hong Qu glutinous rice wine, one of the most popular traditional rice wines in China, is brewed from glutinous rice with the addition of two traditional fermentation starters – Hong Qu (hóng qū) and Yao Qu (yào qū). In this study, the microbial compositions of fungi and bacteria associated with these two fermentation starters were investigated by nested PCR-denaturing gradient gel electrophoresis (DGGE).The bacterial DGGE profile targeting the V3 region of the 16S rRNA gene indicated that the bacterial composition of starters was highly variable in species composition and dominated by Bacillus species, including Bacillus ginsengihumi, Bacillus megaterium or Bacillus aryabhattai, Bacillus subtilis, Bacillus methylotrophicus and Bacillus amyloliquefaciens. Lactic acid bacteria (LAB) including Weissella paramesenteroides, Pediococcus pentosaceus and Pediococcus acidilactici were also detected in some fermentation starters. In analysis of fungi, PCR-DGGE fingerprints revealed that Rhizopus oryzae, Rhizopus microsporus and Aspergillus sp. were the most frequent species in Yao Qu, while Monascus sp. dominated in Hong Qu. Three non-Saccharomyces yeasts (Saccharomycopsis fibuligera, Pichia guilliermondii and Pichia farinose) were also detected in some starter samples. Multivariate comparisons of DGGE fingerprints showed the complex bacterial and fungal communities were different between Hong Qu and Yao Qu. The Shannon–Weaver diversity index (H′) and Simpson dominance index (D), calculated from the DGGE profiles also showed considerable qualitative differences in the community structure between Hong Qu and Yao Qu.PCR-DGGE was found to be useful for evaluating the quality and the type of traditional wine starters. The prevalence of potential spoilage microorganism highlights the need for more attention when evaluating the microbiological safety of naturally fermented, traditional food products. Moreover, the above results are relevant for the development of starter cultures and predictability of fermentation process for traditional Hong Qu glutinous rice wine, and to aid their intermediate and large scale production.

Determination of microbial diversity in Daqu, a fermentation starter culture of Maotai liquor, using nested PCR-denaturing gradient gel electrophoresis

This study endeavored to investigate the diversity of microbes present during the shaping, ripening and drying of Daqu, a fermentation starter culture and substrata complex of Maotai alcoholic spirit. A nested PCR-denaturing gradient gel electrophoresis technique was utilized with different combinations of primers. The results showed the presence of bacteria, yeasts and molds. The microflora, which originate from wheat, were readily detectable during every stage of the fermentation process. However, the microbial structure had clear differences in the shaping, ripening and drying processes. In the shaping stage, there was a high level of diversity of the LAB (lactic acid bacteria) and fungi in the shaped samples. In the ripening stage, however, a reduction of diversity of fungi with a high level of diversity of the Bacilli was observed in the ripened samples. In the drying stage, the diversity of Bacilli and fungi, especially acid-producing bacteria, reduced dramatically. Interestingly, uncultured Lactococcus sp., Microbacterium testaceum, Cochliobolus sp., and Thermoascus crustaceus were the first to be detected in the fermentation starters used in liquor production. This study revealed the microbial diversity and distributions during the shaping, ripening and drying of Daqu-making, facilitating evaluation of the hygienic conditions and aiding in the design of specific starter and/or adjunct cultures.

Distribution and Diversity of Gallionella -Like Neutrophilic Iron Oxidizers in a Tidal Freshwater Marsh

Microbial iron oxidation is an integral part of the iron redox cycle in wetlands. Nonetheless, relatively little is known about the composition and ecology of iron-oxidizing communities in the soils and sediments of wetlands. In this study, sediment cores were collected across a freshwater tidal marsh in order to characterize the iron-oxidizing bacteria (FeOB) and to link their distributions to the geochemical properties of the sediments. We applied recently designed 16S rRNA primers targeting Gallionella-related FeOB by using a nested PCR-denaturing gradient gel electrophoresis (DGGE) approach combined with a novel quantitative PCR (qPCR) assay. Gallionella-related FeOB were detected in most of the samples. The diversity and abundance of the putative FeOB were generally higher in the upper 5 to 12 cm of sediment than in deeper sediment and higher in samples collected in April than in those collected in July and October. Oxygen supply by macrofauna appears to be a major force in controlling the spatial and temporal variations in FeOB communities. The higher abundance of Gallionella-related FeOB in April coincided with elevated concentrations of extractable Fe(III) in the sediments. Despite this coincidence, the distributions of FeOB did not exhibit a simple relationship to the redox zonation inferred from the geochemical depth profiles.

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

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

Impact of Silver Nanoparticle Contamination on the Genetic Diversity of Natural Bacterial Assemblages in Estuarine Sediments

Recent advancements in the nanotechnology industry have seen a growing interest in integrating silver nanoparticles (Ag-NPs) into consumer and medical products. To date, there has been little research into the toxicological impact that Ag-NPs will have when they enter the marine realm. Of particular concern are the possible effects of Ag-NPs on natural bacterial assemblages, given the antimicrobial activity of silver. In this study, estuarine sediment samples were dosed in triplicate for 20 days from a stock solution of Ag-NPs, with a final cumulative treatment of either 0 microg L(-1) (control), 25 microg L(-1) or 1000 microg L(-1). The experimental tanks were left for a further 10 days to allow for any recovery. Inductively coupled plasma-optical emission spectrometery (ICP-OES) of water and sediment samples confirmed that the Ag-NPs concentration in the aqueous phase decreased after each dosing and were transported to accumulate in the surface layer of the sediment (approximately top 3 mm). The overall concentration of Ag-NPs in the water column, however, increased steadily during the 20 days of dosing but decreased rapidly during the following 10 days without dosing. Nevertheless, the Ag-NPs did not have any impact on the prokaryotic abundance in the water column over the incubation period (ANOVA, P < 0.05). Environmental DNA was extracted from sediment samples and a two-step nested PCR-denaturing gradient gel electrophoresis (DGGE) approach, using PCR primers specific to the phylum Bacteria, was adopted to assess their diversity. Multivariate statistical analyses of presence/ absence matrices produced from DGGE profiles revealed negligible differences in bacterial diversity between treatments, suggesting that, under the selected experimental regime, Ag-NPs present little or no impact on estuarine sediment bacterial diversity. Possible reasons for this could include environmental factors, in particular the chloride ions in estuary water affecting the chemistry and behavior of Ag-NPs.

Analysis of microbial communities in doenjang, a Korean fermented soybean paste, using nested PCR-denaturing gradient gel electrophoresis

Doenjang is a traditional Korean fermented soybean paste that provides a major source of protein. The microbial diversity of 10 samples of doenjang (5 commercially manufactured products and 5 homemade products) was investigated using nested PCR-denaturing gradient gel electrophoresis (DGGE). In the first step, the nearly complete 16S rRNA and 18S rRNA genes were amplified using universal primers. Subsequently, these products were used as a template in a nested PCR to obtain fragments suitable for DGGE. The bacterial DGGE profile targeting the V3 region of the 16S rRNA gene indicated that lactic acid bacteria such as Leuconostoc mesenteroide, Tetragenococcus halophilus, and Enterococcus faecium were the predominant species. However, bands corresponding to Bacillus species, known to be the main organisms in doenjang, were not detected under the conditions described above. When selective PCR was conducted using a primer specific for Bacillus species, Bacillus subtilis and B. licheniformis were detected in several doenjang samples. In analysis of fungi, Mucor plumbeus, Aspergillus oryzae, and Debaryomyces hansenii were the most common species in the doenjang samples. On the basis of DGGE, a few differences in community structure were found for different samples. Also, cluster analysis of the DGGE profile revealed that the microbial diversity did not differ clearly between commercially manufactured and homemade products. The nested PCR-DGGE technique was used for the first time in this study to asses a microbial community in doenjang and proved to be effective in profiling microbial diversity.

Development of group-specific PCR-DGGE fingerprinting for monitoring structural changes of Thauera spp. in an industrial wastewater treatment plant responding to operational perturbations

A Thauera-specific nested-PCR denaturing gradient gel electrophoresis (DGGE) method was developed, and its usefulness was demonstrated by monitoring the structural shifts of Thauera spp. in an anaerobic-anoxic-oxic fixed-biofilm coking wastewater treatment plant (WWTP) responding to operational perturbations. The specificity of the PCR method was confirmed by the fact that all 16 S rRNA gene sequences, cloned from the amplicons of a biofilm sample, belonged to Thauera genus. 16 S rRNA gene V3 region was then amplified from the first round Thauera-specific PCR product and applied for DGGE analysis. All Thauera clones, with 13 different V3 regions, migrated into 10 positions on DGGE gel, which demonstrated the high resolution of this DGGE method. When the WWTP experienced a gradual deterioration in chemical oxygen demand (COD) removal function due to a mechanical failure of the recirculation pump, biofilm samples were collected from the reactor and analyzed by this method. Principal component analysis (PCA) of the DGGE fingerprinting data showed that the composition of Thauera group exhibited a time related trajectory when the plant's COD removal rate decreased from 84.1 ± 2.7% in the first 4 weeks to less than 75% at week 5 and 6, suggesting a concomitant shift of Thauera composition and the system's COD removal function. This group-specific PCR DGGE fingerprinting technology has the potential to be a profiling tool for monitoring structural shifts of Thauera spp. in industrial WWTPs.

Nested PCR-Denaturing Gradient Gel Electrophoresis Approach To Determine the Diversity of Sulfate-Reducing Bacteria in Complex Microbial Communities

Here, we describe a three-step nested-PCR-denaturing gradient gel electrophoresis (DGGE) strategy to detect sulfate-reducing bacteria (SRB) in complex microbial communities from industrial bioreactors. In the first step, the nearly complete 16S rRNA gene was amplified using bacterial primers. Subsequently, this product was used as a template in a second PCR with group-specific SRB primers. A third round of amplification was conducted to obtain fragments suitable for DGGE. The largest number of bands was observed in DGGE patterns of products obtained with primers specific for the Desulfovibrio-Desulfomicrobium group, indicating a large diversity of these SRBs. In addition, members of other phylogenetic SRB groups, i.e., Desulfotomaculum, Desulfobulbus, and Desulfococcus-Desulfonema-Desulfosarcina, were detected. Bands corresponding to Desulfobacterium and Desulfobacter were not detected in the bioreactor samples. Comparative sequence analysis of excised DGGE bands revealed the identity of the community members. The developed three-step PCR-DGGE strategy is a welcome tool for studying the diversity of sulfate-reducing bacteria.