Effects of glyphosate on soil fungal communities: A field study / Efecto del glifosato en las comunidades fúngicas del suelo: estudio a campo

ABSTRACT The driving forces behind many soil processes are microorganisms and they are able to respond immediately to environmental changes. The soil microbial community impacts on many soil properties. More than one-third of the terrestrial ecosystems are semiarid. However, a limited number of studies have been conducted to characterize soil fungal communities in semiarid grasslands, in particular those of agricultural fields. The aim of this study was to explore changes in the diversity and structure of soil fungal communities in semiarid grasslands, after different doses of glyphosate were applied under field conditions. Changes in soil fungal communities were examined using different approaches including culturing, calcofluor white stain and denaturing gradient gel electrophoresis (DGGE). The different approaches complement each other, revealing different aspects of the effect of glyphosate on soil fungal communities. We demonstrated a negative effect of glyphosate on soil fungal biomass at high doses and an early and transitory stimulatory effect on soil fungal biomass. We also found a negative effect of glyphosate on the species richness of cultivable fungi and changes in the molecular structure of soil fungal communities after double doses or long-term glyphosate application. In summary, our findings demonstrate an overall negative effect of glyphosate on soil fungal communities.

RESUMEN Los microorganismos del suelo son los responsables de llevar a cabo la mayoría de los procesos biológicos que ocurren en el suelo, y son capaces de reaccionar ante el estrés ambiental. Más de un tercio de los ecosistemas terrestres son semiáridos. Sin embargo, son escasos los estudios realizados para caracterizar las comunidades fúngicas en suelos agrícolas en ecosistemas semiáridos. El objetivo del presente trabajo fue estudiar los cambios que se producen en la biomasa, la diversidad y la estructura de las comunidades fúngicas del suelo, luego de la aplicación de distintas dosis de glifosato en condiciones de campo. Se emplearon diferentes técnicas incluidas el cultivo, la tinción directa con blanco de calcoflúor y PCR acoplada a electroforesis en geles de gradiente desnaturalizante (DGGE). Las distintas metodologías empleadas se complementan entre sí al detectar cada una distintos aspectos del efecto del glifosato en las comunidades fúngicas del suelo. Se encontró que el glifosato produce un efecto negativo sobre la biomasa fúngica, también se encontró un efecto transitorio estimulante inmediatamente posterior a la aplicación del herbicida. Además, se vio un efecto negativo sobre la riqueza de hongos cultivables, así como también cambios en la estructura molecular de las comunidades luego de aplicaciones repetidas. En conclusión, se demostró un efecto negativo generalizado sobre las comunidades fúngicas del suelo.

A robust method for nucleic acid extraction from fabrics to study bacterial diversity

The importance of studying microbial load on fabrics has been recently realized with reports on fabrics being a source ofspread of infection in medical and hospitality sectors. However, methodological limitations have restricted the analysis ofmicrobial diversity on fabrics. Hence, the study aimed to develop a robust method for extraction of DNA from differenttypes of fabrics. Bacterial community profiles could be successfully generated with DNA extracted from real life samples,together with identification of different bacterial genera on fabrics. The study opens up venues to study effect of environmental factors on microbial load on fabrics. Also, such a technique will aid correlation between microbial load and typesof fabric so as to come up with recommendation for fabrics bearing minimal microbial load for medical and hospitalitysectors.

Bacterial community structure in soils contaminated with electronic waste pollutants from Delhi NCR, India

Background: Microbial community analysis of electronic waste (e-waste)-polluted environments is of interest to understand the effect of toxic e-waste pollutants on the soil microbial community and to evaluate novel microorganisms resisting the toxic environment. The present study aims to investigate the bacterial community structure in soils contaminated with e-waste from various sites of Loni and Mandoli (National Capital Region (NCR), India) where e-waste dumping and recycling activities are being carried out for many years. Results: Interferences to soil metagenomic DNA extraction and PCR amplification were observed because of the presence of inhibiting components derived from circuit boards. Whole-metagenome sequencing on the Illumina MiSeq platform showed that the most abundant phyla were Proteobacteria and Firmicutes. Deltaproteobacteria and Betaproteobacteria were the most common classes under Proteobacteria. Denaturing gradient gel electrophoresis (DGGE) analysis of the bacterial 16S rRNA gene showed that e-waste contamination altered the soil bacterial composition and diversity. There was a decrease in the number of predominant bacterial groups like Proteobacteria and Firmicutes but emergence of Actinobacteria in the contaminated soil samples. Conclusions: This is the first report describing the bacterial community structure of composite soil samples of ewaste-contaminated sites of Loni and Mandoli, Delhi NCR, India. The findings indicate that novel bacteria with potential bioremediating properties may be present in the e-waste-contaminated sites and hence need to be evaluated further.

Population Changes of Eubacteria, Sulfate-Reducing Bacteria and Methanogenic Archaea in an Anaerobic Reactor Processing Ethanol Distillery Vinasse

Abstract The microbiological characterization by molecular techniques (DGGE and quantitative PCR) of Archaea, Bacteria domain and sulfate-reducing bacteria (SRB) from a laboratory scale Up-flow Anaerobic Sludge Blanket (UASB) processing sugar cane vinasse was performed during the operational phase with increasing organic loads. The organic load removal efficiency was between 97% and 75% for volumetric organic loads (VOL) in the range of 0.6 to 15.4 kgCOD.m-3.d−1 and for higher VOL (until 27.0 kgCOD.m-3 .d−1) the removal efficiency decreased to 48%. Archaea represented the majority of the estimated population (107 copies of 16S RNA ribosomal gene. mL-1) followed by bacteria (106 copies of 16S RNA ribosomal gene. mL-1) and sulfate-reducing bacteria (SRB) (105 copies of dsrB gene. mL-1). There was a significant difference between the populations of Archaea and SRB with increasing VOL. DGGE profiles show differences among populations with increasing VOL, especially with respect to sulfate-reducing bacteria. The presence of SRB together with a high sulfate removal percentage (97%) of about 15 kgCOD.m-3d-1h, indicates the establishment of a sulfidogenic process.

Arbuscular mycorrhizal fungal communities in the rhizosphere of a continuous cropping soybean system at the seedling stage

Abstract Arbuscular mycorrhizae (AM) fungi play a crucial role in the growth of soybean; however, the planting system employed is thought to have an effect on AM fungal communities in the rhizosphere. This study was performed to explore the influence of continuous soybean cropping on the diversity of Arbuscular mycorrhizal (AM) fungi, and to identify the dominant AM fungus during the seedling stage. Three soybean cultivars were planted under two and three years continuous cropping, respectively. The diversity of AM fungi in the rhizosphere soil at the seedling stage was subsequently analyzed using polymerase chain reaction (PCR)-denaturing gradient gel electrophoresis (DGGE). The results showed that an increase in cropping years improved the colonization rate of AM in all three soybean cultivars. Moreover, the dominant species were found to be Funneliformis mosseae and Glomus species. The results of cluster analysis further confirmed that the number of years of continuous cropping significantly affected the composition of rhizospheric AM fungal communities in different soybean cultivars.

Bacterial degradation of dissolved organic matter released by Planktothrix agardhii (Cyanobacteria) / Degradação bacteriana da matéria orgânica dissolvida liberada por Planktothrix agardhii (Cyanobacteria)

Abstract Although Planktothrix agardhii often produces toxic blooms in eutrophic water bodies around the world, little is known about the fate of the organic matter released by these abundant Cyanobacteria. Thus, this study focused in estimating the bacterial consumption of the DOC and DON (dissolved organic carbon and dissolved organic nitrogen, respectively) produced by axenic P. agardhii cultures and identifying some of the bacterial OTUs (operational taxonomic units) involved in the process. Both P. agardhii and bacterial inocula were sampled from the eutrophic Barra Bonita Reservoir (SP, Brazil). Two distinct carbon degradation phases were observed: during the first three days, higher degradation coefficients were calculated, which were followed by a slower degradation phase. The maximum value observed for particulate bacterial carbon (POC) was 11.9 mg L-1, which consisted of 62.5% of the total available DOC, and its mineralization coefficient was 0.477 day-1 (t½ = 1.45 days). A similar pattern of degradation was observed for DON, although the coefficients were slightly different. Changes in the OTUs patterns were observed during the different steps of the degradation. The main OTUs were related to the classes Alphaproteobacteria (8 OTUs), Betaproteobacteria (2 OTUs) and Gammaproteobacteria (3 OTUs). The genus Acinetobacter was the only identified organism that occurred during the whole process. Bacterial richness was higher at the slower degradation phase, which could be related to the small amounts of DOM (dissolved organic matter) available, particularly carbon. The kinetics of the bacterial degradation of P. agardhii-originated DOM suggests minimal loss of DOM from the Barra Bonita reservoir.

Resumo Embora Planktothrix agardhii frequentemente forme florações tóxicas em corpos d'água pelo mundo, pouco ainda se sabe sobre o destino da matéria orgânica liberada por essa abundante Cyanobacteria. Assim, este estudo foi focado na estimativa do consumo bacteriano do carbono orgânico dissolvido (DOC) e nitrogênio orgânico dissolvido (DON) produzido por culturas axênicas de P. agardhii e identificação de algumas das unidades taxonômicas operacionais (OTUs) bacterianas envolvidas no processo. Ambos a linhagem de P. agardhii e o inóculo bacteriano foram amostrados do reservatório eutrófico de Barra Bonita (SP, Brasil). Foram observadas duas fases distintas da degradação do DOC: durante os três primeiros dias, coeficientes mais altos de degradação foram calculados, que foram então seguidos por uma fase mais lenta da degradação do carbono. O valor máximo calculado para o carbono bacteriano particulado (POC) foi de 11,9 mgL-1, o que equivale a aproximadamente 62,5% do DOC disponível para consumo, e o seu coeficiente de mineralização foi de 0,477 dia-1 (t1/2 = 1,45 dias). Um padrão similar de degradação foi observado para DON, embora os coeficientes sejam ligeiramente diferentes. Foram observadas mudanças nos padrões de OTUs durante os diferentes passos da degradação. As principais OTUs foram relacionadas às classes Alphaproteobacteria (8 OTUs), Betaproteobacteria (2 OTUs) e Gammaproteobacteria (3 OTUs). O gênero Acinetobacter foi o único organismo identificado que ocorreu durante todo o processo. A maior riqueza bacteriana foi observada durante a fase lenta de degradação, o que pode estar relacionado às pequenas quantidades de matéria orgânica dissovida (DOM) disponíveis, particularmente o carbono. A cinética da degradação bacteriana da MOD de P. agardhii, quando comparada ao tempo de retenção do reservatório, sugere que existe uma perda mínima após sua liberação em Barra Bonita.

Development and diversity of lactic acid producing bacteria and bifidobacteria in healthy full term Indian infants from Himachal Pradesh

BACKGROUND/AIMS: The initial microbial colonization is a crucial step for the healthy development of an infant. Previous studies from India reported the dominance of target microbial species among Indian infants without any analysis on the diversity of target groups. This is the first study from India with an objective to investigate the establishment and diversity of lactic acid producing bacteria (LAB) and bifidobacteria in vaginally delivered, full term, breastfed infants for the first 4 months after birth. METHODS: Present study used polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) based sequence analysis of LAB and bifidobacteria in healthy infants. The results were used to compare the development and early colonization by LAB and bifidobacteria using diversity indices during the initial months of development of gut microbiota in infants. RESULTS: During the first 4 months, the Shannon diversity index (H) of LAB increased from 1.16 to 1.318 and for bifidobacteria the H increased from 0.975 to 1.293 (P < 0.05). Higher Sorenson’s pair wise similarity coefficient was observed for LAB and bifidobacteria during 2nd and the 3rd month. The species of the genera Enterococcus, Streptococcus, and Lactobacillus were dominant among the LAB group whereas Bifidobacterium breve was dominant species among Bifidobacterium group. CONCLUSIONS: Our results indicate that in breast fed infants, the microbial diversity of LAB and bifidobacteria increased during the period of study.

Ecology and biotechnological potential of bacterial community from three marine sponges of the coast of Rio de Janeiro, Brazil

ABSTRACT Marine sponges has been a large reservoir of microbial diversity, with the presence of many species specific populations as well as producing biologically active compounds, which has attracted great biotechnological interest. In order to verify the influence of the environment in the composition of the bacterial community present in marine sponges and biotechnological potential of bacteria isolated from these organisms, three species of sponges and the waters surrounding them were collected in different beaches of Rio de Janeiro, Brazil. The profile of the bacterial community present in sponges and water was obtained by PCR-DGGE technique and the biotechnological potential of the strains isolated by producing amylase, cellulase, protease and biosurfactants. The results showed that despite the influence of the environment in the composition of the microbial community, studied marine sponges shown to have specific bacterial populations, with some, showing potential in the production of substances of biotechnological applications.

Establecimiento y evaluación de dos métodos de pre tratamiento de muestras de suelo para la extracción de ADN para el estudio de la diversidad bacteriana / Establishment and evaluation of two methods of pre-treatment of soil samples for the extraction of DNA for the study of bacterial diversity

La necesidad de ampliar los conocimientos respecto a los mecanismos bioquímicos y fisiológicos desarrollados por los microorganismos presentes en suelos requiere de una descripción completa de la diversidad microbiana, para lo cual en las últimas décadas se han desarrollado diferentes técnicas moleculares (qPCR, DGGE, T-RFLP, RAPD.) las mismas que requieren una adecuada técnica de extracción de ADN que aseguren el éxito de la descripción de la diversidad microbiana, considerando las características de las muestras de suelos a ser estudiadas. Los protocolos de extracción de ADN generalmente utilizados están basados en la separación de los microorganismos de la matriz antes de la extracción de ADN mediante lisis física o química y por otro lado, la extracción directa del ADN microbiano a partir de muestras de suelo, sin embargo la presencia de sustancias húmicas y fenólicas afectan la calidad del ADN extraído, lo que repercuten en el desarrollo de posteriores estudios moleculares. La finalidad de este estudio fue la de establecer procedimientos de pre tratamientos de 3 tipos de nuestras de suelo (arenoso, arcilloso y francos) para posteriormente describir la riqueza y diversidad bacteriana de las muestras en estudio mediante PCR DGGE. De esta manera se determinó que la adición de CaCO3 en muestras de suelos francos permite la identificación de una mayor diversidad y riqueza bacteriana (10 bandas). Asimismo, la adición de PVPP a suelos arenosos (8 bandas) y arcillosos (3 bandas) también permite obtener las características descritas anteriormente utilizando el método PCR-DGGE. Lo cual indica que los procedimientos de pre tratamiento con CaCO3 y PVPP son específicos para la extracción de ciertas comunidades microbianas.

The knowledge about biochemical and physiological mechanisms by microorganisms in soils are required for a complete description of microbial diversity, lately different molecular techniques have been developed to study this feature (qPCR, DGGE, T- RFLP, RAPD). DNA extraction techniques ensure the description of the microbial diversity success, according the soil samples characteristics. Generally, DNA extraction protocols used for separation of microorganism of matrix soil before DNA extraction by physical and chemical lysis. Other protocol is direct extraction of microbial DNA from soil samples, humic acids and phenolic substances affect the quality of DNA, which affect the development of subsequent molecular studies. The purpose of this study was to establish pretreatment procedures for different kind of soil samples (frank, sandy and clayey) in order to describe richness and bacterial diversity by PCR DGGE. In this sense, we determined the addition of CaCO3 in frank soils samples allows the identification of greater diversity and bacterial richness (10 bands) than the other method. Besides, PVPP pretreatment is no only useful to obtain bacterial diversity in sandy soil (8 bands), but also in clayey soils (3 bands) soils by PCR-DGGE method. This indicates that the pretreatment procedures with CaCO3 and PVPP are specific for soil microbial community's isolation.

Analysis of microbial diversity in Shenqu with different fermentation times by PCR-DGGE

Abstract Shenqu is a fermented product that is widely used in traditional Chinese medicine (TCM) to treat indigestion; however, the microbial strains in the fermentation process are still unknown. The aim of this study was to investigate microbial diversity in Shenqu using different fermentation time periods. DGGE (polymerase chain reaction-denaturing gradient gel electrophoresis) profiles indicated that a strain of Pediococcus acidilactici (band 9) is the predominant bacteria during fermentation and that the predominant fungi were uncultured Rhizopus, Aspergillus oryzae, and Rhizopus oryzae. In addition, pathogenic bacteria, such as Enterobacter cloacae, Klebsiella oxytoca, Erwinia billingiae, and Pantoea vagan were detected in Shenqu. DGGE analysis showed that bacterial and fungal diversity declined over the course of fermentation. This determination of the predominant bacterial and fungal strains responsible for fermentation may contribute to further Shenqu research, such as optimization of the fermentation process.

The effect of heavy metal contamination on the bacterial community structure at Jiaozhou Bay, China

Abstract In this study, determination of heavy metal parameters and microbiological characterization of marine sediments obtained from two heavily polluted sites and one low-grade contaminated reference station at Jiaozhou Bay in China were carried out. The microbial communities found in the sampled marine sediments were studied using PCR-DGGE (denaturing gradient gel electrophoresis) fingerprinting profiles in combination with multivariate analysis. Clustering analysis of DGGE and matrix of heavy metals displayed similar occurrence patterns. On this basis, 17 samples were classified into two clusters depending on the presence or absence of the high level contamination. Moreover, the cluster of highly contaminated samples was further classified into two sub-groups based on the stations of their origin. These results showed that the composition of the bacterial community is strongly influenced by heavy metal variables present in the sediments found in the Jiaozhou Bay. This study also suggested that metagenomic techniques such as PCR-DGGE fingerprinting in combination with multivariate analysis is an efficient method to examine the effect of metal contamination on the bacterial community structure.

Bacterial degradation of dissolved organic matter released by Planktothrix agardhii (Cyanobacteria)

Abstract Although Planktothrix agardhii often produces toxic blooms in eutrophic water bodies around the world, little is known about the fate of the organic matter released by these abundant Cyanobacteria. Thus, this study focused in estimating the bacterial consumption of the DOC and DON (dissolved organic carbon and dissolved organic nitrogen, respectively) produced by axenic P. agardhii cultures and identifying some of the bacterial OTUs (operational taxonomic units) involved in the process. Both P. agardhii and bacterial inocula were sampled from the eutrophic Barra Bonita Reservoir (SP, Brazil). Two distinct carbon degradation phases were observed: during the first three days, higher degradation coefficients were calculated, which were followed by a slower degradation phase. The maximum value observed for particulate bacterial carbon (POC) was 11.9 mg L-1, which consisted of 62.5% of the total available DOC, and its mineralization coefficient was 0.477 day-1 (t½ = 1.45 days). A similar pattern of degradation was observed for DON, although the coefficients were slightly different. Changes in the OTUs patterns were observed during the different steps of the degradation. The main OTUs were related to the classes Alphaproteobacteria (8 OTUs), Betaproteobacteria (2 OTUs) and Gammaproteobacteria (3 OTUs). The genus Acinetobacter was the only identified organism that occurred during the whole process. Bacterial richness was higher at the slower degradation phase, which could be related to the small amounts of DOM (dissolved organic matter) available, particularly carbon. The kinetics of the bacterial degradation of P. agardhii-originated DOM suggests minimal loss of DOM from the Barra Bonita reservoir.

Resumo Embora Planktothrix agardhii frequentemente forme florações tóxicas em corpos dágua pelo mundo, pouco ainda se sabe sobre o destino da matéria orgânica liberada por essa abundante Cyanobacteria. Assim, este estudo foi focado na estimativa do consumo bacteriano do carbono orgânico dissolvido (DOC) e nitrogênio orgânico dissolvido (DON) produzido por culturas axênicas de P. agardhii e identificação de algumas das unidades taxonômicas operacionais (OTUs) bacterianas envolvidas no processo. Ambos a linhagem de P. agardhii e o inóculo bacteriano foram amostrados do reservatório eutrófico de Barra Bonita (SP, Brasil). Foram observadas duas fases distintas da degradação do DOC: durante os três primeiros dias, coeficientes mais altos de degradação foram calculados, que foram então seguidos por uma fase mais lenta da degradação do carbono. O valor máximo calculado para o carbono bacteriano particulado (POC) foi de 11,9 mgL-1, o que equivale a aproximadamente 62,5% do DOC disponível para consumo, e o seu coeficiente de mineralização foi de 0,477 dia-1 (t1/2 = 1,45 dias). Um padrão similar de degradação foi observado para DON, embora os coeficientes sejam ligeiramente diferentes. Foram observadas mudanças nos padrões de OTUs durante os diferentes passos da degradação. As principais OTUs foram relacionadas às classes Alphaproteobacteria (8 OTUs), Betaproteobacteria (2 OTUs) e Gammaproteobacteria (3 OTUs). O gênero Acinetobacter foi o único organismo identificado que ocorreu durante todo o processo. A maior riqueza bacteriana foi observada durante a fase lenta de degradação, o que pode estar relacionado às pequenas quantidades de matéria orgânica dissovida (DOM) disponíveis, particularmente o carbono. A cinética da degradação bacteriana da MOD de P. agardhii, quando comparada ao tempo de retenção do reservatório, sugere que existe uma perda mínima após sua liberação em Barra Bonita.

Effect of Gegen Qinlian Decoction on LPS, TNF-α, IL-6, and intestinal flora in diabetic KK-Ay mice / 中草药

Objective: To explore the effect of Gegen Qinlian Decoction (GGQLD) on LPS, TNF-α, IL-6, and intestinal flora in diabetic KK-Ay mice. Methods: C57BL/6J mice with ordinary feed were taken as the normal control group and orally administrated with equal distilled water. The KK-Ay mice fed with high-fat diet were divided into five groups: pioglitazone group, blank group (model group), high, medium, and low dose GGQLD group, and orally administrated with pioglitazone hydrochloride (5 mg/kg), distilled water, and GGQLD (crude drug 40, 13.3, and 4.44 g/kg), respectively. The oral administration for six groups lasted for four weeks. Tumor necrosis factor (TNF-α), interleukin 6 (IL-6), and endotoxin (LPS) levels in the plasma were determined by enzyme-linked immunosorbent assay (ELISA); Gut microbial communities were assayed by polymerase chain reaction (PCR) and PCR-denaturing gradient gel electrophoresis (PCR-DGGE) methods. Results: Compared with the model group, the LPS levels in the plasma of mice were significantly reduced by 15.61% and 14.48% respectively in the Gegenqinlian Decoction of high and medium dose group (P < 0.05), the IL-6 levels in plasma of mice were significantly reduced by 56.86%, 37.12% and 30.21% respectively in high, medium, and low dose GGQLD group (P < 0.05), and the TNF-α levels in plasma of mice were significantly reduced by 28.32%, 30.70%, and 23.42% respectively in high, medium, and low dose GGQLD group (P < 0.05). The number of DGGE bands in high dose group significantly increased, and by cloning, sequencing, and Blast analysis, Lactobacillus johnsonii only existed in the high dose group; The results showed that GGQLD could regulate the structure of intestinal flora in KK-Ay mice. Conclusion: The mechanisms of anti-diabetic effects of GGQLD in type 2 diabetic KK-Ay mice are probably related with the anti-inflammation and regulation of intestinal flora.

Study on dynamic changing regularity of microflora in fermentation process of Sojae Semen Praeparatum by PCR-DGGE / 中草药

Objective: To reveal the dynamic changing regularity of microflora in the fermentation process of Sojae Semen Praeparatum (SSP) and lay the foundation for revealing the mechanism of SSP processing by denaturing gradient gel electrophoresis (DGGE). Methods: The dynamic changes of microflora, both bacteria and fungi in fermentation process were monitored by PCR-denaturing gradient gel electrophoresis. According to the unweighted pair group method using arithmetic average clustering, the samples of SSP in various stages were analyzed. Results: Bacterial flora had diversity, and Aspergillus was the major fungus in the first stage called "yellow cladding". The major bacteria was Lactobacillus, while the major fungus was Cryptococcus at the "secondary fermentation" stage. The major microorganism was Bacillus subtillis and Pseudomonas putida on day 1, and Stenotrophomonas maltophilia, Sphingobacterium sp, and A. oryzae on day 3. Then on day 6, B. amyloliquefaciens, Aspergillus, and Trichosporon ovoides became the primary microorganisms. B. subtillis, T. ovoides, and A. niger were the major microorganism on day 3 of "secondary fermentation". On day 9 of this stage, the major strains were B. subtilis, L. concavus, L. nasuensis, and Cryptococcus randhawi. On day 15 of "secondary fermentation", they were B. subtilis, L. concavus, C. randhawi, Trichosporon, and two fungi cannot be cultured. Klebsiella oxytoca, B. subtilis, and L. concavus were dominant strains in the whole fermentation process. The composition of microflora in "yellow cladding" stage was different to that of the "secondary fermentation". The microbial community on day 3 and 6 was similar to 76.4%. While the lowest similarity between the samples on day 3 and 9, it was similar to 24.5% during samples on day 6 and 9 in "secondary fermentation" stage. The highest similarity of fungal composition was between day 3 and 6 samples, and the lowest one was between day 3 and 15 of "secondary fermentation", which was similar to 11.2% only. Conclusion: The results show that the unique flavor and function of SSP may be determined by the dynamic microbial communities and microbial flora in the fermentation process, and the secondary fermentation is proved to be irreplaceable from the microbiological point of view.

Functional Genes of Microorganisms, Comprehending the Dynamics of Agricultural Ecosystems

ABSTRACT The microbial composition of different types,in ecosystems (including agro-ecosystems), has been investigated in a rapidly growing number of studies in the past few years. The importance of microorganisms, regarding the maintenance and stability of nutrients in agroecosystems, is a key to maintain the sustainability of a crop. Molecular tools to study microbial communities are possible through many methods such as RISA, DGGE, TGGE, clone libraries, T-RFLP, RAPD, SSCP and more recently NGS (Next-Generation Sequencing). DGGE is widely employed to characterize the diversity and the community dynamics of microorganisms in the environment, making possible to find out specific groups through functional genes, allowing access to data that cannot be obtained by cultural methods. The aim of this paper is to review the functional groups related to agroecosystems and to indicate the critical choice of DNA primers pairs and targeted DNA regions that may be used in PCR-based methods such as the DGGE technique in order to evaluate the microbial communities in a variety of environments.

Detection and differentiation of Entamoeba histolytica and Entamoeba dispar in clinical samples through PCR-denaturing gradient gel electrophoresis

Amebiasis is one of the twenty major causes of disease in Mexico; however, the diagnosis is difficult due to limitations of conventional microscopy-based techniques. In this study, we analyzed stool samples using polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) to differentiate between Entamoeba histolytica (pathogenic) and E. dispar (non-pathogenic). The target for the PCR amplification was a small region (228 bp) of the adh112 gene selected to increase the sensitivity of the test. The study involved 62 stool samples that were collected from individuals with complaints of gastrointestinal discomfort. Of the 62 samples, 10 (16.1%) were positive for E. histolytica while 52 (83.9%) were negative. No sample was positive for E. dispar. These results were validated by nested PCR-RFLP (restriction fragment length polymorphism) and suggest that PCR-DGGE is a promising tool to differentiate among Entamoeba infections, contributing to determine the specific treatment for patients infected with E. histolytica, and therefore, avoiding unnecessary treatment of patients infected with the non-pathogenic E. dispar.

Comparative analysis of the intestinal microbial flora diversity of adult and young African lions / 中国兽医学报

In this study,the intestinal microbial flora diversity of adult and young African lions in the same breeding environment was detected by PCR-DGGE technique.Total bacterial DNA was extracted and 16S rDNA V3 region was amplified,then conducting PCR-DGGE.Subsequently,the specific bands of DGGE were cloned and sequenced.The bacterial species were identified by comparing the sequence through BLAST.The results indicated that the intestinal microbial flora of adult African lions includes Clostridium,Lachnospiraceae bacterium,Anaerovorax,Lactococcus,Peptostreptococcus and Blautia.While the intestinal microbial flora of young African lions is lesser,most bacteria are common to adult and young lions,such as Bacteroidetes bacterium and rumen bacterium.The UPGMA clustering analysis of the DGGE fingerprint showed the similarities of the bacteria structures between adult and young African lions were only 34％.These results revealed that the intestinal microbial flora has significant difference in different stages of African lions.This study lays a foundation for the development of microecological agents in different growth stages of wild animals.

The oral microbiome community variations associated with normal, potentially malignant disorders and malignant lesions of the oral cavity

The human oral microbiome has been known to show strong association with various oral diseases including oral cancer. This study attempts to characterize the community variations between normal, oral potentially malignant disorders (OPMD) and cancer associated microbiota using 16S rDNA sequencing. Swab samples were collected from three groups (normal, OPMD and oral cancer) with nine subjects from each group. Bacteria genomic DNA was isolated in which full length 16S rDNA were amplified and used for cloned library sequencing. 16S rDNA sequences were processed and analysed with MOTHUR. A core oral microbiome was identified consisting of Firmicutes, Proteobacteria, Fusobacteria, Bacteroidetes and Actinobacteria at the phylum level while Streptococcus, Veillonella, Gemella, Granulicatella, Neisseria, Haemophilus, Selenomonas, Fusobacterium, Leptotrichia, Prevotella, Porphyromonas and Lachnoanaerobaculum were detected at the genus level. Firmicutes and Streptococcus were the predominant phylum and genus respectively. Potential oral microbiome memberships unique to normal, OPMD and oral cancer oral cavities were also identified. Analysis of Molecular Variance (AMOVA) showed a significant difference between the normal and the cancer associated oral microbiota but not between the OPMD and the other two groups. However, 2D NMDS showed an overlapping of the OPMD associated oral microbiome between the normal and cancer groups. These findings indicated that oral microbes could be potential biomarkers to distinguish between normal, OPMD and cancer subjects.

Effect of biofertilizer on the diversity of nitrogen - fixing bacteria and total bacterial community in lowland paddy fields in Sukabumi West Java, Indonesia

Aims: Some of methanotrophic bacteria and nitrous oxide (N2O) reducing bacteria have been proven able to support the plant growth and increase productivity of paddy. However, the effect of application of the methanotrophics and N2O reducing bacteria as a biofertilizer to indigenous nitrogen-fixing bacteria and total bacterial community are still not well known yet. The aim of the study was to analyze the diversity of nitrogen-fixing bacteria and total bacterial communty in lowland paddy soils. Methodology and results: Soil samples were taken from lowland paddy fields in Pelabuhan Ratu, Sukabumi, West Java, Indonesia. There were two treatments applied to the paddy field i.e biofertilizer-treated field (biofertilizer with 50 kg/ha NPK) and control (250 kg/ha NPK fertilizer). There were nine different nifH bands which were successfully sequenced and most of them were identified as unculturable bacteria and three of them were closely related to Sphingomonas sp., Magnetospirillum sp. and Ideonella dechloratans respectively. In addition, there were 20 different 16S rDNA bands which were successfully sequenced. Phylogenetic analysis of the sequence showed that there were 5 phyla of bacteria, i.e. Proteobacteria (Alphaproteobacteria and Gammaproteobacteria), Chlorofexi, Gemmatimonadetes, Clostridia, and Bacteroidetes respectively. Alphaproteobacteria was the most dominant group in lowland paddy field. Microbial diversities in the biofertilizer-treated field were lower than that of 100% fertilizer-treated field either based on nifH and 16S rDNA genes. Conclusion, significance and impact study: Biofertilizer treatment has lower microbial diversity than control, either based on nifH and 16S rDNA genes.

Effects of different forms of nitrogen on rhizosphere microbial community structure of Eichhornia crassipes (Pontederiaceae)

AbstractRhizosphere microbial communities are important for phytoremediation, plant nutrition, health and metabolism. Many factors, including plant species, pH and nutritional factors influence rhizosphere microbiology. In this study, we analysed the effects of different forms of nitrogen on the structures of rhizosphere microbial communities of E. crassipes. Using a conventional culture method with special media, bacteria, actinobacteria and molds were cultured. We found that the numbers of bacteria were largely similar across the three culture conditions, while the numbers of actinobacteria and molds from the rhizosphere of E. crassipes cultured in NH4Cl solution were two orders of magnitude higher than those from the rhizospheres of plants cultured in distilled water and KNO3 solution. Using a culture-independent method of polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) of 16S rDNA, we found that the form of nitrogen could influence the components of the rhizosphere microbial community. Pseudoxanthomonas, Enterobacter and Citrobacter were present in all of the samples cultured under the three different experimental conditions. The genus Reyranella was found only in samples cultured in KNO3 solution; Acinetobacter and Streptomyces were unique to samples cultured in NH4Cl solution, and Pseudomonas, Pseudacidovorax and Methylosinus were found only in samples cultured in distilled water. Pseudoxanthomonas and Acidovorax were the dominant genera in the rhizosphere microbial community of E. crassipes cultured in KNO3 solution, while Novosphingobium was the dominant genus in the sample cultured in a nitrogen-deficient medium. Our results provide a theoretical foundation for using E. crassipes as a phytoremediation plant and controlling the widespread distribution of E. crassipes around the world using principles of nutrient metabolism.

ResumenComunidades microbianas de la rizósfera son importantes para la fitorremediación, nutrición vegetal, salud y metabolismo. Muchos factores, incluyendo la especie de planta, el pH y los factores nutricionales influyen en la microbiología de la rizósfera. En este estudio, se analizaron los efectos de las diferentes formas del nitrógeno en la estructura de las comunidades microbianas de la rizósfera de E. crassipes. Mediante métodos de cultivo convencional con medios especiales se cultivaron: bacterias, actinobacterias y mohos. Se encontró que el número de bacterias era en gran parte similar a través de las tres condiciones de cultivo, mientras que el número de actinobacterias y mohos de la rizósfera de E. crassipes cultivadas en solución de NH4Cl era dos órdenes de magnitud superior a los de las rizósferas de plantas cultivadas en agua destilada y solución de KNO3. Utilizando un método de cultivo independiente de electroforesis en gel con gradiente de desnaturalización (PCR-DGGE) del ADNr 16S, se encontró que la forma de nitrógeno podría influir en los componentes de la comunidad microbiana de la rizósfera. Pseudoxanthomonas, Enterobacter y Citrobacter estaban presentes en todas las muestras cultivadas en las tres condiciones experimentales. El género Reyranella se encontró sólo en muestras cultivadas en solución de KNO3; Acinetobacter y Streptomyces eran las únicas muestras cultivadas en solución de NH4Cl, y Pseudomonas, Pseudacidovorax y Methylosinus se encontraron sólo en muestras cultivadas en agua destilada. Pseudoxanthomonas y Acidovorax eran los géneros dominantes en la comunidad microbiana de la rizósfera de E. crassipes cultivadas en solución de KNO3, mientras que Novos phingobium fue el género dominante en la muestra cultivada en un medio deficiente de nitrógeno. Nuestros resultados proporcionan una base teórica para el uso de E. crassipes como planta fitorremediadora y para controlar la distribución generalizada de E. crassipes en todo el mundo a través de los principios del metabolismo de nutrientes.