Influence of the maize silage to grass silage ratio and feed particle size of rations for ruminants on the community structure of ruminal Firmicutes in vitro

Aims: To investigate the effect of the forage source and feed particle size (FPS) in ruminant rations on the composition of the ruminal Firmicutes community in vitro. Methods and Results: Three diets, varying in maize silage to grass silage ratio and FPS, were incubated in a rumen simulation system. Microbial samples were taken from the liquid fermenter effluents. Microbial community analysis was performed by 16S rRNA‐based techniques. Clostridia‐specific single‐strand conformation polymorphism profiles revealed changes of the community structure in dependence on both factors tested. The coarse grass silage–containing diets seemed to enhance the occurrence of different Roseburia species. As detected by real‐time quantitative PCR, Ruminococcus albus showed a higher abundance with decreasing FPS. A slightly lower proportion of Bacilli was found with increasing grass silage to maize silage ratio by fluorescence in situ hybridization (FISH). In contrast, a slightly higher proportion of bacterial species belonging to the Clostridium‐clusters XIV a and b was detected by FISH with increasing grass silage contents in the diet. Conclusions: The ruminal Firmicutes community is affected by the choice of the forage source and FPS. Significance and Impact of the Study: This study supplies fundamental knowledge about the response of ruminal microbial communities to changing diets. Moreover, the data suggest a standardization of grinding of feeds for in vitro studies to facilitate the comparison of results of different laboratories.     

Effect of Feeding Cows Genetically Modified Maize on the Bacterial Community in the Bovine Rumen

Rumen-cannulated cows (n = 4) were fed successively silage made from either conventional or genetically modified (GM) maize. Results revealed no effects of GM maize on the dynamics of six ruminal bacterial strains (investigated by real-time PCR) compared to the conventional maize silage.     

Bacterial community associated with ensilage process of wilted guinea grass

Aims: To determine the effects of wilting, storage period and bacterial inoculant on the bacterial community and ensiling fermentation of guinea grass silage. Methods and Results: Fermentation products, colony counts and denaturing gradient gel electrophoresis (DGGE) profiles were determined. There was more lactic acid than acetic acid in all silages, but the lactic acid to acetic acid ratio decreased with storage time. This shift from lactic to acetic acid was not prevented even with a combination of wilting and bacterial inoculant. The DGGE analyses suggest that facultatively heterofermentative lactic acid bacteria (Lactobacillus plantarum, Lactobacillus brevis and Lactobacillus pentosus) were involved in the shift to acetic acid fermentation. Conclusions: Lactic acid can dominate the fermentation in tropical grass silage with sufficient wilting prior to ensiling. Prolonged storage may lead to high levels of acetic acid without distinctive changes in the bacterial community. Significance and Impact of the Study: The bacterial community looks stable compared to fermentation products over the course of long storage periods in tropical grass silage. Acetic acid fermentation in tropical grass silage can be a result of the changes in bacterial metabolism rather than community structure.     

Correlations between molecular and operational parameters in continuous lab-scale anaerobic reactors

In this study, the microbial community characteristics in continuous lab-scale anaerobic reactors were correlated to reactor functionality using the microbial resource management (MRM) approach. Two molecular techniques, denaturing gradient gel electrophoresis (DGGE) and terminal-restriction fragment length polymorphism (T-RFLP), were applied to analyze the bacterial and archaeal communities, and the results obtained have been compared. Clustering analyses showed a similar discrimination of samples with DGGE and T-RFLP data, with a clear separation between the meso- and thermophilic communities. Both techniques indicate that bacterial and mesophilic communities were richer and more even than archaeal and thermophilic communities, respectively. Remarkably, the community composition was highly dynamic for both Bacteria and Archaea, with a rate of change between 30% and 75% per 18 days, also in stable performing periods. A hypothesis to explain the latter in the context of the converging metabolism in anaerobic processes is proposed. Finally, a more even and diverse bacterial community was found to be statistically representative for a well-functioning reactor as evidenced by a low Ripley index and high biogas production.     

Microbial community structure in anaerobic co-digestion of grass silage and cow manure in a laboratory continuously stirred tank reactor

The impacts of feeding ratio and loading rate on the microbial community during co-digestion of grass silage with cow manure in an anaerobic laboratory continuously stirred tank reactor were investigated by 16S rRNA gene-based fingerprints. The microbial community remained stable when the reactor was fed with cow manure alone and with up to 20% of grass silage in feedstock at an organic loading rate (OLR) of 2 kg VS m⁻³ day⁻¹. Large changes in the bacterial community were observed when the loading ratio of grass was increased to 40%, while there was little change in the archaeal community. During the increase in OLR from 2 to 4 kg VS m⁻³ day⁻¹ the bacterial community structure showed few differences, whereas Archaea was undetectable. Sequencing of the major DGGE bands indicated that the phylum Bacteriodetes predominated in the bacterial community. Two unclassified bacteria with high abundance survived throughout the operation of the reactor.     

Application of a novel Paenibacillus-specific PCR-DGGE method and sequence analysis to assess the diversity of Paenibacillus spp. in the maize rhizosphere

In this study, a Paenibacillus-specific PCR system, based on the specific primer PAEN515F in combination with bacterial primer R1401, was tested and used to amplify specific fragments of the 16S rRNA gene from rhizosphere DNA. The amplicons were used in a second (semi-nested) PCR for DGGE, in which bacterial primers F968GC and R1401 were used. The resulting products were separated into community fingerprints by DGGE. To assess the reliability of the method, the diversity of Paenibacillus species was evaluated on the basis of DNA extracted directly from the rhizospheres of four different cultivars of maize (Zea mays), i.e. CMS04, CMS11, CMS22 and CMS36, sown in two Brazilian field soils (Cerrado and Várzea). In addition, a clone library was generated from the PCR-generated 16S rDNA fragments, and selected clones were sequenced.The results of the bacterial community analyses showed, at the level of clone libraries, that considerable diversity among Paenibacillus spp. was present. The most dominantly found sequences clustered into 12 groups, each one potentially representing a species complex. Sequences closely affiliated with the P. macerans and P. azotofixans complexes were found in all samples, whereas other sequences were scarcer. Clones affiliated with the latter species complex were most abundant, representing 19% of all clones analysed.The Paenibacillus fingerprints generated via semi-nested PCR followed by DGGE showed a clear distinction between the maize plants grown in Cerrado versus Várzea soils. Thus, soil type, instead of maize cultivar type, was the overriding determinative factor that influenced the community structures of the Paenibacillus communities in the rhizospheres investigated. At a lower level (subcluster), there was a trend for maize cultivars CMS11 and CMS22 on the one hand, and CMS36 and CMS04 on the other hand, to cluster together, indicating that these respective pair of cultivars were similar in their Paenibacillus species composition. This trend was tentatively linked to the growth characteristics of these maize cultivars. These results clearly demonstrated the efficacy of the Paenibacillus-specific PCR-DGGE method in describing Paenibacillus species diversity in rhizosphere soils.     

Quantitative and qualitative transitions of methanogen community structure during the batch anaerobic digestion of cheese-processing wastewater

Qualitative and quantitative shifts in methanogen community structure, associated with process performance data, were investigated during the batch anaerobic digestion of a cheese-processing wastewater, whey permeate. Denaturing gradient gel electrophoresis (DGGE) and real-time PCR techniques were applied to obtain qualitative and quantitative microbial data sets, respectively, based on methanogen 16S rRNA genes. Throughout the operation, dynamic variations in both qualitative and quantitative community structures were observed, with repeated shifts in dominance between the aceticlastic Methanosarcinaceae (suggested mainly by the detection of a Methanosarcina-like population) and the hydrogenotrophic Methanomicrobiales (suggested mainly by the detection of a Methanofollis-like population). This trend corresponded well to the diauxic utilization of acetate and longer-chain fatty acids (C₃–C₆), mainly propionate. Joint-plot non-metric multidimensional scaling (NMS) analysis demonstrated that the qualitative and quantitative community shifts had significant correlations with the composition of residual organic acids and the methane production rate, respectively. This suggests the potential use of microbial community shift analysis as an indicative tool for diagnosing anaerobic digestion processes. The results suggest that more attention should be directed to quantitative, as well as qualitative, approaches for a better understanding of anaerobic digestion, particularly in terms of biogas production efficiency, under dynamic and transitional conditions.     

Methanogenic population dynamics during semi‐continuous biogas fermentation and acidification by overloading

Aims: This study was to investigate the methanogenic community in a biogas reactor from start‐up to acidification conditions. Furthermore, reliability and accuracy of the applied quantitative real‐time PCR method (Q‐PCR) was briefly evaluated. Methods and Results: A mesophilic (37°C), maize silage fed, continuously stirred tank reactor was surveyed. It was operated semi‐continuously with increasing daily organic loading rates (OLRs) to reach acidification. Gas production and organic acid composition were measured. Methanogenic community structure was determined by 16S rDNA‐based Q‐PCR to estimate the abundance of key methanogenic micro‐organisms. 16S rDNA of hydrogenotrophic Methanobacteriales was most abundant at OLRs of ≥3·7 g dry organic matter (DOM) l^?1 day^?1. By contrast, that of aceticlastic Methanosaetaceae predominated at lower OLRs but disappeared at OLRs of ≥4·1 g DOM l^?1 day^?1. At the same OLR, the propionate concentration increased dramatically indicating the acidification of the digester. Application of internal standards to examine Q‐PCR’s accuracy revealed that the detected amount of 16S rDNA may vary within one log cycle. Conclusions: These results suggest that the absence of Methanosaetaceae might be taken as biological indicator for process’ instability. Inhibitory effects on Q‐PCR analyses could not be determined based on the spiking experiments. Significance and Impact of the Study: In this study, reactors’ microbiology was observed over time using Q‐PCR. Insights into the abundance of different methanogens might be used to improve the performance of biogas reactors.     

Taxonomic composition and gene content of a methane-producing microbial community isolated from a biogas reactor

A total community DNA sample from an agricultural biogas reactor continuously fed with maize silage, green rye, and small proportions of chicken manure has recently been sequenced using massively parallel pyrosequencing. In this study, the sample was computationally characterized without a prior assembly step, providing quantitative insights into the taxonomic composition and gene content of the underlying microbial community. Clostridiales from the phylum Firmicutes is the most prevalent phylogenetic order, Methanomicrobiales are dominant among methanogenic archaea. An analysis of Operational Taxonomic Units (OTUs) revealed that the entire microbial community is only partially covered by the sequenced sample, despite that estimates suggest only a moderate overall diversity of the community. Furthermore, the results strongly indicate that archaea related to the genus Methanoculleus, using CO(2) as electron acceptor and H(2) as electron donor, are the main producers of methane in the analyzed biogas reactor sample. A phylogenetic analysis of glycosyl hydrolase protein families suggests that Clostridia play an important role in the digestion of polysaccharides and oligosaccharides. Finally, the results unveiled that most of the organisms constituting the sample are still unexplored.     

Dynamics of Vaginal Bacterial Communities in Women Developing Bacterial Vaginosis, Candidiasis, or No Infection, Analyzed by PCR-Denaturing Gradient Gel Electrophoresis and Real-Time PCR

The microbial flora of the vagina plays a major role in preventing genital infections, including bacterial vaginosis (BV) and candidiasis (CA). An integrated approach based on PCR-denaturing gradient gel electrophoresis (PCR-DGGE) and real-time PCR was used to study the structure and dynamics of bacterial communities in vaginal fluids of healthy women and patients developing BV and CA. Universal eubacterial primers and Lactobacillus genus-specific primers, both targeted at 16S rRNA genes, were used in DGGE and real-time PCR analysis, respectively. The DGGE profiles revealed that the vaginal flora was dominated by Lactobacillus species under healthy conditions, whereas several potentially pathogenic bacteria were present in the flora of women with BV. Lactobacilli were the predominant bacterial population in the vagina for patients affected by CA, but changes in the composition of Lactobacillus species were observed. Real-time PCR analysis allowed the quantitative estimation of variations in lactobacilli associated with BV and CA diseases. A statistically significant decrease in the relative abundance of lactobacilli was found in vaginal fluids of patients with BV compared to the relative abundance of lactobacilli in the vaginal fluids of healthy women and patients with CA.     

Elevated Ground-Level O<Subscript>3</Subscript> Changes the Diversity of Anoxygenic Purple Phototrophic Bacteria in Paddy Field

The knowledge of the impact of elevated ground-level O₃ below ground the agro-ecosystem is limited. A field experiment in China Ozone Free-Air Concentration Enrichment (FACE-O₃) facility on a rice–wheat rotation system was carried out to investigate responses of anoxygenic phototrophic purple bacteria (AnPPB) to elevated ground-level O₃. AnPPB community structures and sizes in paddy soil were monitored by molecular approaches including PCR–DGGE and real-time quantitative PCR based upon the pufM gene on three typical rice growth stages. Repetitive sequence-based PCR (rep-PCR) in combination with culture-reliant method was conducted to reveal changes in genotypic diversity. Elevated ground-level O₃ statistically reduce AnPPB abundance and percentage in total bacterial community in flooded rice soil via decreasing their genotypic diversity and metabolic versatility. Concomitantly, their community composition changed after rice anthesis stage under elevated ground-level O₃. Our results from AnPPB potential responses imply that continuously elevated ground-level O₃ in the future would eventually harm the health of paddy ecosystem through negative effect on soil microorganisms.     

Functionally redundant but dissimilar microbial communities within biogas reactors treating maize silage in co-fermentation with sugar beet silage

Numerous observations indicate a high flexibility of microbial communities in different biogas reactors during anaerobic digestion. Here, we describe the functional redundancy and structural changes of involved microbial communities in four lab-scale continuously stirred tank reactors (CSTRs, 39°C, 12 L volume) supplied with different mixtures of maize silage (MS) and sugar beet silage (SBS) over 80 days. Continuously stirred tank reactors were fed with mixtures of MS and SBS in volatile solid ratios of 1:0 (Continuous Fermenter (CF) 1), 6:1 (CF2), 3:1 (CF3), 1:3 (CF4) with equal organic loading rates (OLR 1.25 kgVS m⁻³ d⁻¹) and showed similar biogas production rates in all reactors. The compositions of bacterial and archaeal communities were analysed by 454 amplicon sequencing approach based on 16S rRNA genes. Both bacterial and archaeal communities shifted with increasing amounts of SBS. Especially pronounced were changes in the archaeal composition towards Methanosarcina with increasing proportion of SBS, while Methanosaeta declined simultaneously. Compositional shifts within the microbial communities did not influence the respective biogas production rates indicating that these communities adapted to environmental conditions induced by different feedstock mixtures. The diverse microbial communities optimized their metabolism in a way that ensured efficient biogas production.     

Denaturing gradient gel electrophoresis (DGGE) analysis of bacterial community composition in deep-sea sediments of the South China Sea

The South China Sea, which is one of the largest marginal seas in the world, is predicted to have suitable accumulation conditions and exporting prospects for natural gas hydrate. The aim of this study was to explore the bacterial community composition of deep-sea sediments from such an ecosystem. DNA was extracted by five different methods and used as templates for PCR amplification of the V3 regions of the 16S rRNA gene. Denaturing gradient gel electrophoresis (DGGE) was used to separate the amplified products and analyse the 16S rRNA gene diversity of sediment samples. The results of DGGE indicated that the bacterial community composition is influenced by DNA extraction methods. Sequencing dominant bands demonstrated that the major phylogenetic groups identified by DGGE belong to Proteobacteria, Bacteroidetes, gram-positive bacteria and Archaea. Integrating different DNA extraction procedures are needed to analyse the actual bacterial diversity from environment when the amplification of 16S rRNA gene and construction of representative clone library were adopted.     

Dynamics of vaginal bacterial communities in women developing bacterial vaginosis, candidiasis, or no infection, analyzed by PCR-denaturing gradient gel electrophoresis and real-time PCR

The microbial flora of the vagina plays a major role in preventing genital infections, including bacterial vaginosis (BV) and candidiasis (CA). An integrated approach based on PCR-denaturing gradient gel electrophoresis (PCR-DGGE) and real-time PCR was used to study the structure and dynamics of bacterial communities in vaginal fluids of healthy women and patients developing BV and CA. Universal eubacterial primers and Lactobacillus genus-specific primers, both targeted at 16S rRNA genes, were used in DGGE and real-time PCR analysis, respectively. The DGGE profiles revealed that the vaginal flora was dominated by Lactobacillus species under healthy conditions, whereas several potentially pathogenic bacteria were present in the flora of women with BV. Lactobacilli were the predominant bacterial population in the vagina for patients affected by CA, but changes in the composition of Lactobacillus species were observed. Real-time PCR analysis allowed the quantitative estimation of variations in lactobacilli associated with BV and CA diseases. A statistically significant decrease in the relative abundance of lactobacilli was found in vaginal fluids of patients with BV compared to the relative abundance of lactobacilli in the vaginal fluids of healthy women and patients with CA.     

Monitoring bacterial and archaeal community shifts in a mesophilic anaerobic batch reactor treating a high-strength organic wastewater

Shifts in bacterial and archaeal communities, associated with changes in chemical profiles, were investigated in an anaerobic batch reactor treating dairy-processing wastewater prepared with whey permeate powder. The dynamics of bacterial and archaeal populations were monitored by quantitative real-time PCR and showed good agreement with the process data. A rapid increase in bacterial populations and a high rate of substrate fermentation were observed during the initial period. Growth and regrowth of archaeal populations occurred with biphasic production of methane, corresponding to the diauxic consumption of acetate and propionate. Bacterial community structure was examined by denaturing gel gradient electrophoresis (DGGE) targeting 16S rRNA genes. An Aeromonas -like organism was suggested to be mainly responsible for the rapid fermentation of carbohydrate during the initial period. Several band sequences closely related to the Clostridium species, capable of carbohydrate fermentation, lactate or ethanol fermentation, and/or homoacetogenesis, were also detected. Statistical analyses of the DGGE profiles showed that the bacterial community structure, as well as the process performance, varied with the incubation time. Our results demonstrated that the bacterial community shifted, reflecting the performance changes and, particularly, that a significant community shift corresponded to a considerable process event. This suggested that the diagnosis of an anaerobic digestion process could be possible by monitoring bacterial community shifts.     

Relationship between rumen methanogens and methane production in dairy cows fed diets supplemented with a feed enzyme additive

Aims: To investigate the relationship between ruminal methanogen community and host enteric methane (CH₄) production in lactating dairy cows fed diets supplemented with an exogenous fibrolytic enzyme additive. Methods and Results: Ecology of ruminal methanogens from dairy cows fed with or without exogenous fibrolytic enzymes was examined using PCR–denaturing gradient gel electrophoresis (PCR–DGGE) analyses and quantitative real‐time PCR (qRT‐PCR). The density of methanogens was not affected by the enzyme additive or sampling times, and no relationship was observed between the total methanogen population and CH₄ yield (as g per head per day or g kg^?1 DMI). The PCR–DGGE profiles consisted of 26 distinctive bands, with two bands similar to Methanogenic archaeon CH1270 negatively correlated, and one band similar to Methanobrevibacter gottschalkii strain HO positively correlated, with CH₄ yield. Three bands similar to Methanogenic archaeon CH1270 or Methanobrevibacter smithii ATCC 35061 appeared after enzyme was added. Conclusions: Supplementing a dairy cow diet with an exogenous fibrolytic enzyme additive increased CH₄ yield and altered the composition of the rumen methanogen community, but not the overall density of methanogens. Significance and Impact of the Study: This is the first study to identify the correlation between methanogen ecology and host CH₄ yield from lactating dairy cows.     

Denaturing gradient gel electrophoresis (DGGE)-based monitoring of intestinal lactobacilli and bifidobacteria of pigs during a feeding trial

The aim of this study was to determine the influence of different feeding strategies on the gut microbiota of organic growing-finishing pigs. A total of 76 pigs were allocated to four different dietary treatments (control, probiotics, maize silage and grass silage). Effects of the applied probiotic preparation on the composition of the intestinal and faecal microbiota were monitored. By using a DGGE (denaturing gradient gel electrophoresis)-based methodology, fingerprints of the intestinal microbiota were obtained. The total microbial DNA was isolated from faecal and colon samples and amplified with PCR using different primer sets to detect bifidobacteria and lactobacilli. PCR products were separated using DGGE and the resulting profiles were compared with the findings of the other dietary treatments. Bands were excised from the gels and sequenced for further identification. Particularly two different DGGE profiles of bifidobacteria were observed, while lactobacilli showed larger variety within the dietary treatments.     

Effect of the corn silage to grass silage ratio and feed particle size of diets for ruminants on the ruminal Bacteroides-Prevotella community in vitro

This study examined whether different corn silage to grass silage ratios in ruminant rations and different grinding levels of the feed affect the composition of the ruminal Bacteroides-Prevotella community in vitro. Three diets, composed of 10% soybean meal as well as of different corn silage and grass silage proportions, were ground through 1 mm or 4 mm screened sieves and incubated in a semi-continuous rumen simulation system. On day 14 of the incubation microbes were harvested by centrifugation from the liquid effluent of fermenter vessels. Microbial DNA was extracted for single strand conformation polymorphism (SSCP) analysis of 16S rRNA genes followed by sequencing of single SSCP bands. Fluorescence in situ hybridization (FISH) and real-time quantitative (q) PCR were used to quantify differences in the relative abundance of Bacteroides-Prevotella and Prevotella bryantii. SSCP profiles revealed a significant influence of the forage source as well as of the feed particle size on the community structure of the Bacteroides-Prevotella group. Different, phylogenetically distinct, so far uncultured Prevotella species were detected by sequence analysis of several treatment-dependent occurring SSCP bands indicating different nutritional requirements of these organisms for growth. No quantitative differences in the occurrence of Bacteroides-Prevotella-related species were detected between diets by FISH with probe BAC303. However, real-time qPCR data revealed a higher abundance of P. bryantii with increasing grass silage to corn silage ratio, thus again indicating changes within the community composition of the Bacteroides-Prevotella group. As P. bryantii possesses high proteolytic activity its higher abundance may have been caused by the higher contents of crude protein in the grass silage containing diets. To conclude, results of this study show an influence of the forage source on the ruminal community of Bacteroides-Prevotella. Furthermore, they suggest an effect of the feed particle size on this bacterial group.