Impact of whole grain highland hull-less barley on the denaturing gradient gel electrophoresis profiles of gut microbial communities in rats fed high-fat diets.

Polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) is a traditional non-culture technique that can provide a fingerprint of the microbial community. In the field of gut microbiota analysis, PCR-DGGE still holds potential for development. In the present study, we utilized an improved nested PCR-DGGE approach targeting the V3 region of 16S ribosomal DNA to investigate the impact of whole grain highland hull-less barley (WHLB), a cereal known for its significant hypocholesterolemic effect, on the gut microbiota profiles of high-fat diet rats. Seventy-two male Sprague-Dawley rats were divided into four groups and fed a normal control diet, a high-fat diet, or a high-fat diet supplemented with a low or high dose of WHLB for 4 or 8 weeks. The results revealed that the dominant bands varied among different dose groups and further changed with different treatment times. The compositions of bacterial communities in feces and cecal content were similar, but the dominant bacterial bands differed. After performing double DGGE, extracting the bands, sequencing the DNA, and aligning the sequences, a total of 19 bands were classified under the Firmicutes and Bacteroidetes phyla, while two bands were identified as unclassified uncultured bacteria. The relative abundance of Lactobacillus gasseri, Uncultured Prevotella sp., and Clostridium sp. increased following the administration of WHLB. Illumina-based sequencing was employed to assess the reliability of DGGE, demonstrating its reliability in analyzing the dominant taxonomic composition, although it may have limitations in accurately detecting the alpha diversity of bacterial species. IMPORTANCE: While next-generation sequencing has overshadowed polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE), the latter still holds promise for advancing gut microbiota analysis due to its unique advantages. In this study, we used optimized nested PCR-DGGE to investigate the gut microbiota profile of high-fat diet rats after administering whole grain highland hull-less barley. High-throughput sequencing was employed to validate the DGGE results. Our results proved the reliability of PCR-DGGE for analyzing the dominant taxonomic composition while also providing visual evidence of a notable relationship between the composition of cecal and fecal microbial communities, highlighting substantial differences in both richness and abundance.

Use of PCR-DGGE-Based Molecular Methods to Analyze Nematode Community Diversity.

DGGE (denaturing gradient gel electrophoresis) is a nucleic acid separation technique applied to the evaluation of microbial biodiversity. This technique is quite rapid and cheap compared to other types of analysis. Here we describe the comparison of nematode communities inhabiting different ecosystems. After an ecologically representative sampling collection and the nematode extraction from soil, nematodes are centrifuged in Eppendorf tubes to facilitate DNA extraction. DNA from the whole community of each type of soil is extracted, amplified with primers for 18 S rDNA and used in DGGE analysis. The profiles of DGGE can be analyzed with appropriate software, and biodiversity indices can be estimated.

Influence of nutrient, toxic metal and herbicide contents on the soil bacterial communities in tropical vegetable growing areas.

The relationship between bacterial diversity and the bioavailability of nutrients, toxic metals and the herbicide oxyfluorfen in a tropical vegetable growing area was evaluated. The study was conducted in a vegetable growing area located in the mountainous region of Rio de Janeiro (Brazil), and samples were collected in areas of vegetable cultivation and areas of environmental reserve. Fertility analyses and determination of the pseudototal levels of toxic metals in the soil samples were performed. The profile of the soil bacterial community was determined by amplification of the 16S rRNA gene and separation by DGGE. The results showed that the levels of toxic metals and elements associated with soil fertility were higher in vegetable production areas. These differences in the physical and chemical characteristics of the soil favored the presence of a greater number of OTUs in the cultivation areas (17.3-27 OTUs) than in the areas of environmental reserve (13-22 OTUs). Therefore, this study demonstrates that the presence of toxic metals and the herbicide oxyfluorfen and the increase in fertility in soils in areas with intensive vegetable cultivation resulting from the intensive management adopted in these areas promotes a differentiation of the bacterial profiles in soils in tropical vegetable growing areas.

Comparison of culture and PCR-DGGE methods to evaluate the airways of cystic fibrosis patients and determination of their antibiotic resistance profile.

Background and Objectives: Respiratory infections are the most serious condition in cystic fibrosis (CF) patients; therefore, a thorough comprehension of the diversity and dominant microbial species in CF airways has a crucial role in treatment. Our objective was to determine the antibiotic resistance profile of CF airways microbiota and compare culture methods and PCR-DGGE to evaluate bacterial diversity. Materials and Methods: Pharyngeal swabs from 121 CF patients were collected. The samples were then cultured, identified and antibiotic resistance testing was performed. Thirty samples were subjected to further molecular surveys. DNA contents of these samples were extracted and amplified using nested-PCR technique and their bacterial diversity was assessed by DGGE. The DGGE patterns were visualized and certain bands were excised and purified. Next, the DNA was amplified by another round of PCR and sent out for sequencing. Results: Staphylococcus aureus, Pseudomonas aeruginosa, and Klebsiella pneumoniae were the most prevalent species isolated using culture methods. S. aureus was the most common bacteria among 6 years and younger patients; while, P. aeruginosa had more prevalence among older ones. The PCR-DGGE results showed more diversity than culture methods, particularly in younger patients who exhibited more bacterial diversity than the older groups. Sequencing results unveiled the presence of certain bacterial species including Haemophilus parainfluenzae and Stenotrophomonas maltophilia which were completely missed in culture. Conclusion: Even though culture-dependent methods are cost-effective, PCR-DGGE appeared to be more efficient to determine bacterial diversity. PCR-DGGE detects less abundant species, though their viability could not be determined using this method.

Microbial Community Profiling Using Terminal Restriction Fragment Length Polymorphism (T-RFLP) and Denaturing Gradient Gel Electrophoresis (DGGE).

In their natural environments, microorganisms usually live in organized communities. Profiling analysis of microbial communities has recently assumed special relevance as it allows a thorough understanding of the diversity of the microbiota, its behavior over time, and the establishment of patterns associated with health and disease. The application of molecular biology approaches holds the advantage of including culture-difficult and as-yet-uncultivated phylotypes in the profiles, providing a more comprehensive picture of the microbial community. This chapter focuses on two particular techniques, namely terminal restriction fragment length polymorphism (T-RFLP) and denaturing gradient gel electrophoresis (DGGE), both of which have been widely used in environmental studies and have been recently successfully used by the authors in the study of the oral microbial communities associated with conditions of health and disease.

Homing in on Endogenous Badnaviral Elements: Development of Multiplex PCR-DGGE for Detection and Rapid Identification of Badnavirus Sequences in Yam Germplasm.

Viruses of the genus Badnavirus (family Caulimoviridae) are double-stranded DNA-reverse transcribing (dsDNA-RT) plant viruses and have emerged as serious pathogens of tropical and temperate crops globally. Endogenous badnaviral sequences are found integrated in the genomes of several economically important plant species. Infection due to activation of replication-competent integrated copies of the genera Badnavirus, Petuvirus and Cavemovirus has been described. Such endogenous badnaviral elements pose challenges to the development of nucleic acid-based diagnostic methods for episomal virus infections and decisions on health certification for international movement of germplasm and seed. One major food security crop affected is yam (Dioscorea spp.). A diverse range of Dioscorea bacilliform viruses (DBVs), and endogenous DBV (eDBV) sequences have been found to be widespread in yams cultivated in West Africa and other parts of the world. This study outlines the development of multiplex PCR-dependent denaturing gradient gel electrophoresis (PCR-DGGE) to assist in the detection and analysis of eDBVs, through the example of analysing yam germplasm from Nigeria and Ghana. Primers targeting the three most prevalent DBV monophyletic species groups in West Africa were designed to improve DGGE resolution of complex eDBV sequence fingerprints. Multiplex PCR-DGGE with the addition of a tailor-made DGGE sequence marker enables rapid comparison of endogenous badnaviral sequence diversity across germplasm, as illustrated in this study for eDBV diversity in yam.

Assessment of Bacterial Communities Within the Biofilm of Bladder Calculi in the Neurogenic Bladder Rat Model Following Spinal Cord Injury.

PURPOSE: To develop a rat model of bladder calculi in the neurogenic bladder following spinal cord injury (SCI) and assess bacterial communities within the biofilm of bladder calculi using denaturing gradient gel electrophoresis (DGGE). METHODS: The silk tied to a small segment of the Teflon IV catheter was implanted through the urethra into the bladder of rats with SCI induced by T9 laminectomy. After 6 months, the rats were sacrificed and their bladder calculi were collected by opening the bladders through the low-midline incision. Genomic DNA was extracted from the biofilm of bladder calculi followed by DGGE to obtain bacterial DNA. The DNA sequences were compared and analyzed using BLAST (Basic Local Alignment Search Tool) to identify bacteria. RESULTS: After placing silk nidus in the bladder for 6 months, all 6 rats developed bladder calculi. According to DGGE analysis, Pseudomonas aeruginosa was the most dominant strain, while Clostridium sp. and Lactobacillus sp. were relatively dominant strains within the biofilm of bladder calculi in the rats with SCI. CONCLUSION: DGGE analysis showed various microorganisms in the biofilm of calculi arising from a neurogenic bladder rat model. This research design can be the basis for clinical studies and may be applied to calculi in patients with neurogenic bladder following SCI.

Why the importance of geo-origin tracing of edible bird nests is arising?

Edible bird's nest (EBN) swiftlet existed naturally 48,000 years ago in caves as their natural dwellings. Nowadays, edible bird's nest has become a very important industry due to its high nutritional, medicinal and economic value. Additionally, edible bird's nest has a long quality guarantee period. Obviously, the nutritional components and medicinal functions vary depending on geographical origins. Recently, the global demand for edible bird's nest has markedly increased, accompanied by the increasing attention of all key players of the global food trade system, i.e., producers, consumers, traders and the authorities to obtain safe and high-quality edible bird's nest. Hence, this target can be accomplished via the enforcement of an efficient and universal geo-tracing technique. Current methods of the geo-tracking of edible bird's nest, i.e., automation, physical and analytical techniques have several limitations and all of them fail to discriminate different quality grades of edible bird's nest. Meanwhile, in many studies and applications, polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) has proven to be a "cutting edge" technique for greatly enhance food traceability from field to fork through its ability in distinguishing the food products in terms of their quality and safety. This article provides an overview of (1) edible bird's nest as a multiuse strategic food product, (2) quality issues associated with edible bird's nest including implications that the site of acquisition of the edible bird's nest has food safety implications, (3) current regulations and geo-tracking approaches to ensure the safety and quality of edible bird's nest with the special focus on polymerase chain reaction-denaturing gradient gel electrophoresis technique as a vigorous and universal geo-tracing tool to be suggested for edible bird's nest geo-traceability.

Computational analysis of successional changes in the microbial population and community diversity of the immobilized marine nitrifying bacterial consortium in a nitrifying packed bed bioreactor.

Nitrifying bioreactor (NBR) connected to the recirculating aquaculture system (RAS), has a greater emphasis on the biological treatment of wastewater. Nitrifying bacterial consortium (NBC) formed bio-film on the substratum activating the NBR, and it was observed with high nitrification potential in shrimp maturation systems. Scanning Electron Microscopy (SEM) revealed the integrity of the biofilm substantiated with biomineralization. The fate of the matured bio-film population on subsequent operation under RAS, and the aggregated population at different points of RAS, including the rearing water were determined using fingerprints of Denaturing Gradient Gel Electrophoresis (DGGE). Altogether, 38 OTUs of biofilm sample and 35 OTUs of water samples represented the bacterial communities; the shared and unique OTUs indicated the diversity of the population at different time intervals in the operation of the NBR. The mathematical (range-weighted richness) and statistical (diversity indices) interpretation unveiled the OTUs based high bacterial diversity in the biofilm supporting the compositional changes and determined the distance between the community cluster. Ordination analyses indicated the population shift and stability of the activated bio-film till the matured biofilm community got established in the RAS. The DGGE with mathematical and statistical analysis revealed microbial diversity (high Shannon index, species richness and evenness), abundance (relative intensity), consecutive change in the population composition (OTUs, Rr index), and the dynamics (Δt) in the system during the operation.

Impact of Ag2S NPs on soil bacterial community - A terrestrial mesocosm approach.

Soils might be a final sink for Ag2S nanoparticles (NPs). Still, there are limited data on their effects on soil bacterial communities (SBC). To bridge this gap, we investigated the effects of Ag2S NPs (10 mg kg-1 soil) on the structure and function of SBC in a terrestrial indoor mesocosm, using a multi-species design. During 28 days of exposure, the SBC function-related parameters were analysed in terms of enzymatic activity, community level physiological profile, culture of functional bacterial groups [phosphorous-solubilizing bacteria (P-SB) and heterotrophic bacteria (HB)], and SBC structure was analysed by 16S rRNA gene-targeted denaturing gradient gel electrophoresis. The SBC exposed to Ag2S NPs showed a significative decrease of functional parameters, such as ß-glucosidase activity and L-arginine consumption, and increase of the acid phosphatase activity. At the structural level, significantly lower richness and diversity were detected, but at later exposure times compared to the AgNO3 treatment, likely because of a low dissolution rate of Ag2S NPs. In fact, stronger effects were observed in soils spiked with AgNO3, in both functional and structural parameters. Changes in SBC structure seem to negatively correlate with parameters related to phosphorous (acid phosphatase activity) and carbon cycling (abundance of HB, P-SB, and ß-glucosidase activity). Our results indicate a significant effect of Ag2S NPs on SBC, specifically on parameters related to carbon and phosphorous cycling, at doses as low as 10 mg kg-1 soil. These effects were only observed after 28 days, highlighting the importance of long-term exposure experiments for slowly dissolving NPs.

Sub-Lethal Effects of Pesticides on the DNA of Soil Organisms as Early Ecotoxicological Biomarkers.

This review describes the researches performed in the last years to assess the impact of pesticide sub-lethal doses on soil microorganisms and non-target organisms in agricultural soil ecosystems. The overview was developed through the careful description and a critical analysis of three methodologies based on culture-independent approaches involving DNA extraction and sequencing (denaturing gradient gel electrophoresis, DGGE; next-generation sequencing, NGS) to characterize the microbial population and DNA damage assessment (comet assay) to determine the effect on soil invertebrates. The examination of the related published articles showed a continuous improvement of the possibility to detect the detrimental effect of the pesticides on soil microorganisms and non-target organisms at sub-lethal doses, i.e., doses which have no lethal effect on the organisms. Considering the overall critical discussion on microbial soil monitoring in the function of pesticide treatments, we can confirm the usefulness of PCR-DGGE as a screening technique to assess the genetic diversity of microbial communities. Nowadays, DGGE remains a preliminary technique to highlight rapidly the main differences in microbial community composition, which is able to give further information if coupled with culture-dependent microbiological approaches, while thorough assessments must be gained by high-throughput techniques such as NGS. The comet assay represents an elective technique for assessing genotoxicity in environmental biomonitoring, being mature after decades of implementation and widely used worldwide for its direct, simple, and affordable implementation. Nonetheless, in order to promote the consistency and reliability of results, regulatory bodies should provide guidelines on the optimal use of this tool, strongly indicating the most reliable indicators of DNA damage. This review may help the European Regulation Authority in deriving new ecotoxicological endpoints to be included in the Registration Procedure of new pesticides.

Botanical microbiomes on the cheap: Inexpensive molecular fingerprinting methods to study plant-associated communities of bacteria and fungi.

High-throughput sequencing technologies have revolutionized the study of plant-associated microbial populations, but they are relatively expensive. Molecular fingerprinting techniques are more affordable, yet yield considerably less information about the microbial community. Does this mean they are no longer useful for plant microbiome research? In this paper, we review the past 10 years of studies on plant-associated microbiomes using molecular fingerprinting methodologies, including single-strand conformation polymorphism (SSCP), denaturing gradient gel electrophoresis (DGGE), amplicon length heterogeneity PCR (LH-PCR), ribosomal intergenic spacer analysis (RISA) and automated ribosomal intergenic spacer analysis (ARISA), and terminal restriction fragment length polymorphism (TRFLP). We also present data juxtaposing results from TRFLP methods with those generated using Illumina sequencing in the comparison of rhizobacterial populations of Brazilian maize and fungal surveys in Canadian tomato roots. In both cases, the TRFLP approach yielded the desired results at a level of resolution comparable to that of the MiSeq method, but at a fraction of the cost. Community fingerprinting methods (especially TRFLP) remain relevant for the identification of dominant microbes in a population, the observation of shifts in plant microbiome community diversity, and for screening samples before their use in more sensitive and expensive approaches.

Characterization of eubacterial communities by Denaturing Gradient Gel Electrophoresis (DGGE) and Next Generation Sequencing (NGS) in a desulfurization biotrickling filter using progressive changes of nitrate and nitrite as final electron acceptors.

Anoxic biotrickling filters (BTFs) represent a technology with high H2S elimination capacity and removal efficiencies widely studied for biogas desulfurization. Three changes in the final electron acceptors were made using nitrate and nitrite during an operating period of 520 days. The stability and performance of the anoxic BTF were maintained when a significant perturbation was applied to the system that involved the progressive change of nitrate to nitrite and vice versa. Here the impact of electron acceptor changes on the microbial community was characterized by denaturing gel gradient electrophoresis (DGGE) and next generation sequencing (NGS). Both platforms revealed that the community underwent changes during the perturbations but was resilient because the removal capacity did not significantly change. Proteobacteria and Bacteroidetes were the main Phyla and Sulfurimonas and Thiobacillus the main nitrate-reducing sulfide-oxidizing bacteria (NR-SOB) genera involved in the biodesulfurization process.

The correlation analyses of bacterial community composition and spatial factors between freshwater and sediment in Poyang Lake wetland by using artificial neural network (ANN) modeling.

As one of the most important components of the lake ecosystem, microorganisms from the freshwater and sediment play an important role in many ecological processes. However, the difference and correlation of bacterial community between these two niches were not clear. This study investigated the diversity of microbial community of freshwater and sediment samples from fifteen locations in Poyang Lake wetland. The correlation between the bacterial community and physicochemical property of Poyang Lake wetland was analyzed by artificial neural network (ANN). Our results demonstrated that the freshwater and sediment bacterial community were dominated by groups of the Bacteroidetes (23.33%) and ß-Proteobacteria (22.54%) separately, whereas, Canalipalpata, Bacillariophyta, Gemmatimonadetes, and Verrucomicrobia were detected in freshwater niches only. Phylogenetic analysis further indicated that bacterial composition in freshwater significantly differed with the sediment niches. There are 34 unique species accounted for 85% in fresh water samples and 28 unique species accounted for 82% in sediment samples. Cluster analysis further proved that all the samples from freshwater niches clustered closely together, far from the rest sediment samples. ANN analysis revealed that the freshwater with high N and P nutrients will greatly increase the diversity of the bacterial communities. In general, both environmental physicochemical properties, not each factor independently, contributed to the shift in the bacterial community structure. The five tributaries (Gan, Fu, Xin, Rao, Xiu Rivers) play a vital role in shaping the bacterial communities of Poyang Lake. This study provides new insights for understanding of microbial community compositions and structures of Poyang Lake wetland.

Short communication: Dynamic changes in bacterial diversity during the production of powdered infant formula by PCR-DGGE and high-throughput sequencing.

Microorganisms such as thermophilic and psychrotrophic bacteria cause spoilage of milk and milk products [e.g., powdered infant formula (PIF)], mainly because they produce heat-stable extracellular enzymes. However, the dynamic changes in microbial diversity during PIF production are still not well understood. We used denaturing gradient gel electrophoresis (DGGE) and high-throughput sequencing (HTS) to investigate bacterial community structure and distribution during the major stages of PIF production: raw milk, pasteurization, mixing, evaporation, and spray-drying. Our PCR-DGGE analysis indicated that Lactobacillus and Pseudomonas spp. were the dominant bacteria at the raw milk and pasteurization stages; Lactococcus, Streptococcus, Enterococcus, and Lactobacillus spp. were abundant during mixing, evaporation, and spray-drying. Our HTS analysis showed that Pseudomonas had an abundance of 96.79% at the raw milk stage. Lactobacillus, Streptococcus, Thermus, Acinetobacter, and Bacteroides spp. were most common after pasteurization. The index of bacterial diversity was highest at the evaporation stage, suggesting a high potential risk of microbial contamination. The results from DGGE and HTS were consistent in reflecting changes in dominant flora, but different in reflecting the richness of bacterial communities present during PIF production: HTS revealed a much higher richness of bacterial species than DGGE. Our findings from DGGE and HTS showed that psychrophilic and thermophilic bacteria were the main flora present during PIF production: psychrophilic bacteria were mainly Pseudomonas spp. and thermophilic bacteria were mainly Lactobacillus, Streptococcus, and Bacillus spp. To our knowledge, this is the first study to report dynamic changes in microbial communities during PIF production. Our results provide insight into bacterial communities and identify potential contamination sources that could serve as a guide for reducing microbial risk.

Influence of different sources of contamination on the microbiological quality of lettuce in the Teresópolis region, RJ, Brazil / Influência de diferentes fontes de contaminação na qualidade microbiológica de alface na região de Teresópolis, RJ

ABSTRACT Irrigation water and cultivated soil have been identified as possible sources of contamination in several crops. In certain vegetables that are eaten raw, such as lettuce, this contamination can lead to public health problems. Aiming to evaluate the influence of these sources on the quality of lettuce grown in the Córrego Sujo Basin, Teresópolis, RJ, an important agricultural pole whose production services the metropolitan region of Rio de Janeiro, water from different sources (spring, weir and river) was collected in this region, as well as samples of soil and lettuce irrigated with these waters, to carry out conventional microbiological analyzes (counts of total heterotrophic bacteria and thermotolerant coliforms) and molecular analyzes (PCR-DGGE). The count of fecal coliforms in lettuce suggests that there is an influence of irrigation water and the cultivated soil on the contamination of these vegetables. The grouping of bacterial communities in the different samples obtained by the PCR-DGGE technique shows that irrigation water has a greater influence on the contamination of these vegetables in relation to the soil where they are grown. These results corroborate the need to monitor water bodies used for irrigation and demonstrate that the PCR-DGGE technique is of great value for the study of microbial communities and, when associated with specific primers, can help in the detection of pathogens in food.

RESUMO A água de irrigação e o solo de cultivo têm sido apontados como possíveis fontes de contaminação em diversas culturas. Em determinadas hortaliças consumidas cruas, como a alface, essa contaminação pode causar problemas de saúde pública. Objetivando avaliar a influência dessas fontes na qualidade das alfaces cultivadas na Bacia do Córrego Sujo, Teresópolis, RJ; importante polo agrícola com produção voltada à região metropolitana do Rio de Janeiro, coletou-se nesta região águas proveniente de diferentes fontes (nascente, açude e rio); solos e alfaces irrigados com essas águas, para realização de análises microbiológicas convencionais (contagens de bactérias heterotróficas totais e coliformes termotolerantes) e moleculares (PCR-DGGE). A contagem de coliformes fecais na alface sugere que existe influência da água de irrigação e do solo na contaminação desses vegetais. O agrupamento das comunidades bacterianas nas diferentes amostras obtido pela técnica de PCR-DGGE mostra que a água de irrigação tem influência maior na contaminação dessas hortaliças em relação ao solo onde são cultivadas. Esses resultados corroboram a necessidade de monitoramento de corpos d'água utilizados para irrigação e demonstram ser a técnica do PCR-DGGE de grande valia para o estudo das comunidades microbianas e, quando associada a iniciadores específicos podem ajudar na detecção de patógenos em alimentos.

Shifts in the bacterial community of saliva give insights on the relationship between obesity and oral microbiota in adolescents.

The current study aimed at the determination of the impact of obesity on the salivary microbiome in adolescents. Sixty subjects ranging 14-17 years old were enrolled (obese: n = 30-50% females, and normal weight: n = 30-50% females). Stimulated saliva was collected for denaturing gradient gel electrophoresis (DGGE) band patterns and massive 16S rRNA gene sequencing using the Ion Torrent platform. Overall, data analysis revealed that male subjects harbored a higher diverse salivary microbiome, defined by a significant higher richness (32.48 versus 26.74) and diversity (3.36 versus 3.20), higher Simpson values (0.96 versus 0.95) and distinct bacterial community structure considering either sex or condition (p < 0.05). Bacterial community fingerprinting analysis in human saliva showed a positive correlation with increased body mass index (BMI) in adolescents. Veillonella, Haemophilus and Prevotella occurrence was found to be affected by BMI, whereas Neisseria and Rothia occurrence was significantly impacted by sex in obese subjects. Our findings suggest that male and female adolescents may harbor a naturally distinct salivary microbiota and that obesity may specifically have an impact on their oral bacterial community. The potential dysbiotic oral microbiome in obese adolescents raises new insights on the etiology and prevention of future conditions in these populations.

PCR-denaturing Gradient Gel Electrophoresis as a Simple Identification Tool of Arbuscular Mycorrhizal Fungal Isolates.

Due to their obligate symbiotic nature and lack of long-term storage methods, the strain collection of arbuscular mycorrhizal (AM) fungi requires periodic proliferation using a pot culture with host plants. Therefore, a method to evaluate the purity of proliferated AM fungal cultures is critical for the quality control of their collection. In a simple evaluation of the purity and identity of a proliferated AM fungal culture, DNA extracted from the culture was amplified using AM fungi-specific PCR followed by an analysis with denaturing gradient gel electrophoresis (PCR-DGGE). The present results showed that the DGGE band patterns of AM fungal strains differed according to their phylogenetic positions, allowing for the rapid and easy identification of the proliferated AM fungal strains. When a culture was contaminated with another AM fungal strain, the DGGE pattern became a mixture of those strains. A contaminant strain was detectable even when its ratio was 1/9 of the main strain. It was also possible to confirm the purity of the culture by comparing whether the DGGE band pattern of the proliferated culture was identical to that obtained from single spores isolated from the culture. Therefore, PCR-DGGE is useful as a quality control tool for maintaining culture collections of AM fungi.

Diversity and abundance of denitrifiers during cow manure composting / Diversidad y abundancia de desnitrificadores durante el compostaje del estiércol de vaca

Diversity and abundance of the denitrifying genes nirK, nirS and nosZ were investigated in cow manure compost using polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) and real-time quantitative PCR (qPCR), respectively. These three genes were detected in all the stages of the composting process. Phylogenetic analysis showed that the nirK gene was closely related to Rhizobiales, Burkholderiales, the nirS gene was closely related to Pseudomonadales and Burkholderiales, and the nosZ gene was closely related to Rhodospirillales, Rhizobiales, Pseudomonadales, and Alteromonadales. qPCR results showed that the abundance of these three genes (nirK, nirS and nosZ) reached the peak value in the late thermophilic stage of composting and abundance of the nirK gene was higher than that of the nosZ gene and the nirS gene. Redundancy analysis (RDA) showed that the diversity of the nirK and nirS genes was significantly correlated with ammonium (p < 0.05), the diversity of the nosZ gene was significantly correlated with pH (p < 0.05) and the abundance of the nirK nirS and nosZ genes was significantly correlated with temperature (p< 0.05).

La diversidad y la abundancia de los genes desnitrificadores nirK, nirS, nosZ en el compost de estiércol de vaca se investigaron por medio de la reacción en cadena de la polimerasa seguida de electroforesis en gel con gradiente de desnaturalización (PCR-DGGE) y por PCR cuantitativa (qPCR) en tiempo real, respectivamente. Estos 3 genes fueron detectados durante todas las fases del compostaje. El análisis filogenético mostró estrecha relación del gen nirK con Rhizobiales y Burkholderiales, del gen nirS con Pseudomonadales y Burkholderiales y del gen nosZ con Rhodospirillales, Rhizobiales, Pseudomonadales y Alteromonadales. Los resultados de la qPCR mostraron que la abundancia de los genes nirK, nirSy nosZ alcanzó el valor máximo en la fase termofílica tardía del compostaje, y que la abundancia del gen nirK era más elevada que los de los genes nosZ y nirS. El análisis de redundancia (RDA) mostró que la diversidad de los genes nirK y nirS estaba significativamente correlacionada con la concentración de amonio (p<0,05), mientras que la del gen nosZ estaba significativamente correlacionada con el pH (p<0,05). También mostró que la abundancia de los genes nirK, nirS y nosZ estaba significativamente correlacionada con la temperatura (p<0,05).

Bacterial and archeal dynamics of a labscale HYBRID gas fermentation bioreactor fed with CO2 and H2.

16s rDNA-based methods were used in order to identify the dynamics of microbial profiles in a HYBRID gas fermentation bio-methanization reactor. The effects of various H2 and CO2 ratios on microbial community were investigated. The HYBRID gas fermentation reactor was composed of granular anaerobic seed and the system fed with only H2 and CO2 gases. No additional organic material and trace element was fed during the throughout the experiments; thus, the microbial diversity was directly related to production of methane. The dynamics of the microbial communities were investigated with DGGE and real-time PCR analysis. The results showed that Methanobacteriales members were more dominated than Methanosarcinales and Methanomicrobiales members in the system. DGGE results indicated that Methanosaeta concilii, Methanoculleus sp., Methanosphaerula palustris, Methanofollis formosanus, Methanolinea sp., and Methanobacterium palustre were the most prominent methanogens depending on different H2/CO2 ratios. DGGE profiles suggested that hydrogenotrophic and acetoclastic species were responsible for the production of methane. The survival of syntrophic bacteria and acetoclastic methanogens was attributed to their utilization of organic materials provided by lysis. To the best of our knowledge, this is the first microbial profile detection study in a hybrid bioreactor system operated with only pure hydrogen and carbon dioxide.