High-throughput 16S rRNA Gene Research Articles

Soil fungi are more sensitive than bacteria to short-term plant interactions of Picea asperata and Abies faxoniana

Despite increasing knowledge of plant interactions on the aboveground parts, little is known about the belowground mechanisms underlying such interactions. Here, we selected Picea asperata and Abies faxoniana, two dominant species of subalpine forests, to investigate the effects of planting patterns (isolation and intra- and interspecific interactions) on soil microbial communities. High-throughput 16S rRNA and ITS gene sequencing were performed to explore the bacterial and fungal community structures of the soils. The fungal community changed more under interactions of two species than isolation. Intra- and interspecific interactions had different effects on the fungal community. However, neither intra- nor interspecific interactions had effects on bacterial community composition. Compared with bacteria, fungi were more strongly affected by soil properties, such as the nitrate nitrogen concentration, total nitrogen concentration, pH, and β-1,4-N-acetyl-glucosaminidase activity. These results indicated that fungi were more susceptible than bacteria to plant interactions. Fungal community composition and structure, especially those of ectomycorrhizal fungi, were changed by intra- and interspecific interactions. In inter- and intraspecific interactions with P. asperata, the relative abundance of the ectomycorrhizal fungal Inocybe and Trichophaea increased, which may be one of important factors directly or indirectly affecting belowground interactions.

Sinus Microbiota in Patients With Eosinophilic and Non-Eosinophilic Chronic Rhinosinusitis With Nasal Polyps.

Chronic rhinosinusitis with nasal polyps (CRSwNP) is characterized by Th2-skewed inflammation and increased colonization by Staphylococcus aureus. CRSwNP can be distinguished as eosinophilic (ECRSwNP) and non-eosinophilic (NECRSwNP) by the infiltration of eosinophils. The local microbiota plays an important role in the persistent inflammation of CRSwNP. To evaluate the bacterial community composition on the distinct types of CRSwNP patients, we collected nasal swabs from 16 ECRSwNP patients, 18 NECRSwNP patients, and 39 healthy control subjects. The microbiome structure for all the samples were analyzed by high-throughput 16S rRNA gene sequencing. Concentration of S. aureus was determined using TaqMan quantitative polymerase chain reaction (qPCR) targeting the nuclease (nuc) gene. The result showed significant differences in the sinus microbiome among healthy control subjects and CRSwNP patients. Microbiota community diversity was significantly lower in NECRSwNP samples compared to that of healthy control subjects. Interestingly, the abundance of several pathogenic bacteria was diverse between ECRSwNP and NECRSwNP patients. Although Staphylococcus prevailed in all groups, the abundance of Staphylococcus was significantly higher in the healthy control group than the ECRSwNP group. More importantly, the abundance of S. aureus was much higher in NECRSwNP patients. This study highlights that microbiota composition may contribute to the different clinical types of CRSwNP, inspiring new therapeutic strategies to resolve this chronic inflammation process.

Beyond Taxonomic Identification: Integration of Ecological Responses to a Soil Bacterial 16S rRNA Gene Database.

High-throughput sequencing 16S rRNA gene surveys have enabled new insights into the diversity of soil bacteria, and furthered understanding of the ecological drivers of abundances across landscapes. However, current analytical approaches are of limited use in formalizing syntheses of the ecological attributes of taxa discovered, because derived taxonomic units are typically unique to individual studies and sequence identification databases only characterize taxonomy. To address this, we used sequences obtained from a large nationwide soil survey (GB Countryside Survey, henceforth CS) to create a comprehensive soil specific 16S reference database, with coupled ecological information derived from survey metadata. Specifically, we modeled taxon responses to soil pH at the OTU level using hierarchical logistic regression (HOF) models, to provide information on both the shape of landscape scale pH-abundance responses, and pH optima (pH at which OTU abundance is maximal). We identify that most of the soil OTUs examined exhibited a non-flat relationship with soil pH. Further, the pH optima could not be generalized by broad taxonomy, highlighting the need for tools and databases synthesizing ecological traits at finer taxonomic resolution. We further demonstrate the utility of the database by testing against geographically dispersed query 16S datasets; evaluating efficacy by quantifying matches, and accuracy in predicting pH responses of query sequences from a separate large soil survey. We found that the CS database provided good coverage of dominant taxa; and that the taxa indicating soil pH in a query dataset corresponded with the pH classifications of top matches in the CS database. Furthermore we were able to predict query dataset community structure, using predicted abundances of dominant taxa based on query soil pH data and the HOF models of matched CS database taxa. The database with associated HOF model outputs is released as an online portal for querying single sequences of interest (https://shiny-apps.ceh.ac.uk/ID-TaxER/), and flat files are made available for use in bioinformatic pipelines. The further development of advanced informatics infrastructures incorporating modeled ecological attributes along with new functional genomic information will likely facilitate large scale exploration and prediction of soil microbial functional biodiversity under current and future environmental change scenarios.

Congruent changes in microbial community dynamics and ecosystem methane fluxes following natural drought in two restored fens

Both the frequency and intensity of drought events are expected to increase, with unresolved alterations to peatland methane cycling and the involved microbial communities. While existing studies have assessed drought effects via experimental approaches under controlled conditions, to our knowledge, no studies have examined the in-situ effects of natural drought in restored temperate fens. In this study, we used quantitative polymerase chain reaction (qPCR) and high throughput 16S rRNA gene amplicon sequencing of DNA and complementary DNA (cDNA) to determine the abundances and community structure of total and putatively active microbial communities following the 2018 European summer drought. Together with geochemical and methane flux data, we compared these results to a non-drought reference dataset. During drought, water level and methane flux rates decreased to a new recent minimum in both fens. This corresponded with pronounced shifts in porewater geochemistry. Microbial community composition in the drought year differed markedly, and was characterized by a greater relative and total abundance of aerobic methanotrophs, and, in one of the two sites, by a decrease in total methanogen abundance. In contrast to the non-drought reference years, type I methanotrophs were clearly more dominant than type II methanotrophs in both fens. cDNA sequencing confirmed the activity of type I methanotrophs during drought, with Methylomonaceae having the highest average relative abundance of bacterial cDNA transcripts. We show that changes in microbial community dynamics, porewater geochemistry, and ecosystem methane fluxes can be substantial following natural drought in restored fens, and provide the first in-situ evidence from a natural drought which suggests type I methanotroph populations are more active than type II methanotrophs in response to drought effects. Type I methanotrophs may represent a key microbial control over methane emissions in restored temperate fens subject to natural drought. • Drought decreased water level and ecosystem methane fluxes substantially. • Microbial community dynamics reflected drought conditions at the sites. • Methanogen gene abundances decreased in one of the two fens. • Proportion of type II methanotrophs was low and similar to a non-drought year. • Type I methanotrophs dominated the active bacterial community profile at both sites.

Handling of spurious sequences affects the outcome of high-throughput 16S rRNA gene amplicon profiling

16S rRNA gene amplicon sequencing is a popular approach for studying microbiomes. However, some basic concepts have still not been investigated comprehensively. We studied the occurrence of spurious sequences using defined microbial communities based on data either from the literature or generated in three sequencing facilities and analyzed via both operational taxonomic units (OTUs) and amplicon sequence variants (ASVs) approaches. OTU clustering and singleton removal, a commonly used approach, delivered approximately 50% (mock communities) to 80% (gnotobiotic mice) spurious taxa. The fraction of spurious taxa was generally lower based on ASV analysis, but varied depending on the gene region targeted and the barcoding system used. A relative abundance of 0.25% was found as an effective threshold below which the analysis of spurious taxa can be prevented to a large extent in both OTU- and ASV-based analysis approaches. Using this cutoff improved the reproducibility of analysis, i.e., variation in richness estimates was reduced by 38% compared with singleton filtering using six human fecal samples across seven sequencing runs. Beta-diversity analysis of human fecal communities was markedly affected by both the filtering strategy and the type of phylogenetic distances used for comparison, highlighting the importance of carefully analyzing data before drawing conclusions on microbiome changes. In summary, handling of artifact sequences during bioinformatic processing of 16S rRNA gene amplicon data requires careful attention to avoid the generation of misleading findings. We propose the concept of effective richness to facilitate the comparison of alpha-diversity across studies.

Response of soil bacterial communities to organic carbon input under soil freeze-thaw in forest ecosystems

The soil microbial community controls the biogeochemical cycle of elements critical to plant growth. Different carbon input modes have different effects on soil carbon storage and the microbial community, but the effects of carbon input control on soil microbial diversity and community structure in temperate forests remain unclear. We used high-throughput 16SrRNA gene sequencing and ecological network analysis methods to analyze the soil bacterial community, symbiotic network, and key species of three different carbon input treatments (litter removal, root removal, litter and root removal) in temperate natural secondary forests during the freeze-thaw season and growing season. The results showed that the change in the carbon source input led to a change in the way in which soil bacteria used carbon, which was mainly manifested as follows: (1) The relative abundance of Acidobacteria, which was oligotrophic bacteria, significantly decreased in the sample plots where the root system was removed. (2) In the litter removal treatment, the abundance of bacteria with functions related to the absorption of CH4 decreased. (3) The correlation between the module characteristic genes in the bacterial network analysis and the physical and chemical properties of soil, CK modules were grouped separately, and the change in the correlation may also indicate the change in the way soil bacteria use carbon. Second, the influence of belowground roots on the soil bacterial community structure was more significant than that of aboveground litter. The root system had a greater influence on the soil bacterial community. Third, the decrease of organic carbon input reduced the stability of the soil bacterial network, and the proportion of positive connections in the bacterial network for litter removal and root treatment increased significantly, indicating that these bacterial communities had the same change trend as the fluctuation of the environment. Finally, the freeze-thaw season had a greater impact than the growing season on bacterial diversity, community structure, and function. The freeze-thaw process allowed the bacterial community to respond to root removal more significantly, and the structural changes were mainly due to changes in temperature, water content, dissolved organic carbon, and nitrate nitrogen.

High-throughput 16S rRNA gene sequencing of the microbial community associated with palm oil mill effluents of two oil processing systems

Palm Oil Mill Effluents (POME) are complex fermentative substrates which habour diverse native microbial contaminants. However, knowledge on the microbiota community shift caused by the anthropogenic effects of POME in the environment is up to date still to be extensively documented. In this study, the bacterial and archaeal communities of POME from two palm oil processing systems (artisanal and industrial) were investigated by Illumina MiSeq Platform. Despite the common characteristics of these wastewaters, we found that their microbial communities were significantly different with regard to their diversity and relative abundance of their different Amplicon Sequence Variants (ASV). Indeed, POME from industrial plants harboured as dominant phyla Firmicutes (46.24%), Bacteroidetes (34.19%), Proteobacteria (15.11%), with the particular presence of Spirochaetes, verrucomicrobia and Synergistetes, while those from artisanal production were colonized by Firmicutes (92.06%), Proteobacteria (4.21%) and Actinobacteria (2.09%). Furthermore, 43 AVSs of archaea were detected only in POME from industrial plants and assigned to Crenarchaeota, Diapherotrites, Euryarchaeota and Nanoarchaeaeota phyla, populated mainly by many methane-forming archaea. Definitively, the microbial community composition of POME from both type of processing was markedly different, showing that the history of these ecosystems and various processing conditions have a great impact on each microbial community structure and diversity. By improving knowledge about this microbiome, the results also provide insight into the potential microbial contaminants of soils and rivers receiving these wastewaters.

Comparative analysis of rumen fermentation parameters and bacterial profiles during adaption to different fattening stages in beef cattle fed TMR with various forage silage

We investigated the long-term effects of total mixed rations (TMR) containing various forage silages on the rumen community structure in beef cattle. Fifteen Simmental bulls (437.73 ± 4.01 kg) were fattened for 180 d, split into three trial stages (P1: 1–60 d, P2: 61–120 d, P3: 121–180 d). The diets were composed of corn silage, oat silage, alfalfa silage, wheat straw, and concentrates, formulated into a TMR. Rumen fluid was collected separately on the 60th, 120th, and 180th days among the three fattening stages. High-throughput 16S rRNA gene sequencing was used to analyse rumen bacterial composition and function, and the correlation between rumen bacteria and fermentation parameters was determined using redundant analysis. The results showed that rumen fermentation parameters were stable during long-term fattening. The alpha diversity index indicated that bacterial diversity was higher during the late fattening phase than in the early phase. The most abundant bacteria were Bacteroidetes, Firmicutes, and Prevotella_1 at the three fattening stages. The bacterial communities were clearly separated in the different stages, as indicated by the Box diagram. The thirteen bacterial taxa identified by LEfSe were significantly enriched in the rumen during the three stages. KEGG pathway fractions indicated that carbohydrate metabolism, amino acid metabolism, and energy metabolism pathways were enriched in the rumen during the three stages. In conclusion, we found that long-term feeding of beef cattle with high-quality forage + low levels of concentrate was a healthy and efficient feeding method, promoting a healthy and suitable long-term change in the rumen microbial environment.

Non-Specific Immunity Associated Gut Microbiome in Aristichthys nobilis under Different Rearing Strategies.

To understand the intestinal microbial diversity and community structure of bighead carp (Aristichthys nobilis) under different feeding strategies, 39 fish from three groups (A: 9 fish, natural live food only; B: 15 fish, natural live food + fish formulated feeds; C: 15 fish, natural live food + fish formulated feed + lactic acid bacteria) were obtained for the high throughput 16S rRNA gene sequencing. We first examined five non-specific immunity indications of the carp—lysozyme (LZM), catalase (CAT), glutathione reductase (GR), glutathione peroxidase (GSH-PX), and superoxide dismutase (SOD). Interestingly, the composition of gut microbiota and related non-specific immune indices were affected by the feeding treatment of the bighead carp. Notably, all enzyme activity indexes were significantly different (p < 0.01) in the spleen and three enzyme activity indexes (LZM, GSH-PX, and SOD) had significant differences in the hepatopancreas (p < 0.001) of the carp from the three groups. The 16S rRNA gene sequencing showed higher diversity in groups B and C. Compared to group A, the relative abundance of Actinobacteria increased significantly and the relative abundance of Proteobacteria and Firmicutes decreased significantly in groups B and C at the phylum level. Functional analysis revealed the association between non-specific immune indicators and import genera in the hepatopancreas and spleen of bighead carp. This study provides new insights into the gut microbiomes and non-specific immune of bighead carp.

Unraveling the bacterial community composition across aquatic sediments in the Southwestern coast of India by employing high-throughput 16S rRNA gene sequencing

In this study, the basic bacterial community composition associated with aquatic sediments of the freshwater, mangrove and marine ecosystems of a coastal region in the Southwest coast of India (Mangalore, Karnataka) was explored using high-throughput 16S rRNA gene sequencing. The V3–V4 hypervariable regions of 16S rRNA gene analysis revealed that freshwater and marine ecosystems had a higher abundance of phyla Planctomyces and Proteobacteria which are known to play a major role in the nitrogen cycle, improving the global nitrogen bioavailability in aquatic ecosystems. Functional prediction of xenobiotic degradation pathways using KEGG database from all three samples suggest the resident microbial tolerance to organic contaminants, xenobiotics and pesticides that play a major role in balancing ecological systems by metabolizing the xenobiotics or contaminants to maintain the resilience of the ecosystem. The study highlights the importance of investigating the bacterial community composition across the aquatic ecosystems as it can serve as an indicator of environmental health as well as provide a valuable gene pool for biotechnological applications.

Cross-Hemisphere Study Reveals Geographically Ubiquitous, Plastic-Specific Bacteria Emerging from the Rare and Unexplored Biosphere.

ABSTRACTWhile it is now appreciated that the millions of tons of plastic pollution travelling through marine systems carry complex communities of microorganisms, it is still unknown to what extent these biofilm communities are specific to the plastic or selected by the surrounding ecosystem. To address this, we characterized and compared the microbial communities of microplastic particles, nonplastic (natural and wax) particles, and the surrounding waters from three marine ecosystems (the Baltic, Sargasso and Mediterranean seas) using high-throughput 16S rRNA gene sequencing. We found that biofilm communities on microplastic and nonplastic particles were highly similar to one another across this broad geographical range. The similar temperature and salinity profiles of the Sargasso and Mediterranean seas, compared to the Baltic Sea, were reflected in the biofilm communities. We identified plastic-specific operational taxonomic units (OTUs) that were not detected on nonplastic particles or in the surrounding waters. Twenty-six of the plastic-specific OTUs were geographically ubiquitous across all sampled locations. These geographically ubiquitous plastic-specific OTUs were mostly low-abundance members of their biofilm communities and often represented uncultured members of marine ecosystems. These results demonstrate the potential for plastics to be a reservoir of rare and understudied microbes, thus warranting further investigations into the dynamics and role of these microbes in marine ecosystems.IMPORTANCE This study represents one of the largest comparisons of biofilms from environmentally sampled plastic and nonplastic particles from aquatic environments. By including particles sampled through three separate campaigns in the Baltic, Sargasso, and Mediterranean seas, we were able to make cross-geographical comparisons and discovered common taxonomical signatures that define the plastic biofilm. For the first time, we identified plastic-specific bacteria that reoccur across marine regions. Our data reveal that plastics have selective properties that repeatedly enrich for similar bacteria regardless of location, potentially shifting aquatic microbial communities in areas with high levels of plastic pollution. Furthermore, we show that bacterial communities on plastic do not appear to be strongly influenced by polymer type, suggesting that other properties, such as the absorption and/or leaching of chemicals from the surface, are likely to be more important in the selection and enrichment of specific microorganisms.

Amplicon-sequencing of raw milk microbiota: impact of DNA extraction and library-PCR

The highly complex raw milk matrix challenges the sample preparation for amplicon-sequencing due to low bacterial counts and high amounts of eukaryotic DNA originating from the cow. In this study, we optimized the extraction of bacterial DNA from raw milk for microbiome analysis and evaluated the impact of cycle numbers in the library-PCR. The selective lysis of eukaryotic cells by proteinase K and digestion of released DNA before bacterial lysis resulted in a high reduction of mostly eukaryotic DNA and increased the proportion of bacterial DNA. Comparative microbiome analysis showed that a combined enzymatic and mechanical lysis procedure using the DNeasy® PowerFood® Microbial Kit with a modified protocol was best suitable to achieve high DNA quantities after library-PCR and broad coverage of detected bacterial biodiversity. Increasing cycle numbers during library-PCR systematically altered results for species and beta-diversity with a tendency to overrepresentation or underrepresentation of particular taxa. To limit PCR bias, high cycle numbers should thus be avoided. An optimized DNA extraction yielding sufficient bacterial DNA and enabling higher PCR efficiency is fundamental for successful library preparation. We suggest that a protocol using ethylenediaminetetraacetic acid (EDTA) to resolve casein micelles, selective lysis of somatic cells, extraction of bacterial DNA with a combination of mechanical and enzymatic lysis, and restriction of PCR cycles for analysis of raw milk microbiomes is optimal even for samples with low bacterial numbers.Key points• Sample preparation for high-throughput 16S rRNA gene sequencing of raw milk microbiota.• Reduction of eukaryotic DNA by enzymatic digestion.• Shift of detected microbiome caused by high cycle numbers in library-PCR.

Spatial and Structural Factors Shape Seagrass-Associated Bacterial Communities in Singapore and Peninsular Malaysia

Plant-microbe relationships play critical roles in the functioning and health of terrestrial plants, but little is known about this relationship in marine angiosperms such as seagrasses. Here, we investigated the microbial communities associated with the seagrass Enhalus acoroides throughout Singapore and Peninsular Malaysia. At each sampling location we collected 10 individual and unconnected plants. Each plant was subsequently broken down into leaves, roots, and rhizomes. In addition to living plant parts a sediment sample was taken in close proximity to each. Using high throughput 16S rRNA gene amplicon sequencing we characterised the bacterial communities associated with each plant part and the associated sediment sample. Results indicate geographic structuring of bacterial communities, with a significant pattern of distance decay suggesting dispersal limitation is a contributing factor to the differences we see in bacterial community structure. Bacterial communities can be further differentiated by the function of the collected sample (leaf, root, and rhizome), and we identified a number of microbial indicator species that are associated with each plant part. Further analysis revealed the presence of several microbial taxa that have previously been identified as indicators of “unhealthy” or “stressed” seagrass meadows. This study addresses a current scientific gap related to the characterisation of seagrass microbiomes, and provides a foundation on which future studies can build, particularly those in the Southeast Asian seagrass biodiversity hotspot.

Compositional and Functional Microbiome Variation Between Tubes of an Intertidal Polychaete and Surrounding Marine Sediment

The decorator worm Diopatra cuprea, a tube-forming marine polychaete common to intertidal and shallow subtidal waters, modifies habitats it occupies through microreef construction and algal gardening. While several studies have demonstrated that decorator worm tubes are hotspots of biogeochemical activity (i.e., nitrogen and sulfur cycling), it is still largely unclear whether the tube microbiome differs compositionally from the surrounding sediment and what distinct functional processes tube microbiomes may have. To address these unknowns, this study analyzed the bacterial communities of D. cuprea tubes and surrounding sediments using high-throughput 16S rRNA gene amplicon sequencing. Tubes and sediments were sampled at three sites along an anthropogenic stress gradient within the Newport River Estuary to also assess geographic variation of tube microbiomes and the possible influence of human disturbance. We found a clear distinction in the microbial community composition and diversity between tubes and surrounding sediment. Tube microbiomes were significantly enriched for the phyla Bacteriodetes, Actinobacteria, Verrucomicrobia, Deferribacteres, Latescibacteria, and Lentisphaerae. Chloroplast sequences of macroalgae and grass species were consistently abundant in tubes and nearly absent in surrounding sediment. Functional annotation of prokaryotic taxa (FAPROTAX)-based functional predictions suggested that tube microbiomes have higher potentials for aerobic chemoheterotrophy, sulfur compound respiration, nitrate reduction, methylotrophy, and hydrocarbon degradation than surrounding sediments. Tube microbiomes vary across sites, though dissimilarity is comparatively low compared to tube-to-sediment differences. Contrary to our hypothesis, the tubes at the most highly impacted site had the highest microbial diversity [i.e., amplicon sequence variant (ASV) richness and Shannon’s diversity], yet tubes from the medium impacted site actually had the lowest microbial diversity. Our findings show that D. cuprea tubes support a microbiome that is significantly distinct in composition and function from the surrounding sediment. Diopatra cuprea tubes appear to create unique microhabitats that facilitate numerous microbially-mediated biogeochemical processes in the marine benthic environment.

Evolution of urban black and odorous water: The characteristics of microbial community and driving-factors

Urban black blooms that are primarily caused by organic carbon are deleterious environmental problems. However, detailed studies on the microbial characteristics that form urban black blooms are lacking. In this study, we observed the composition, diversity, and function of bacterial community in the overlying water and sediments during the occurrence and remediation of urban black blooms using high-throughput 16S rRNA gene amplicon sequencing analysis. First, we found that pivotal consortia in the overlying water increased significantly during the formation of black blooms, including the genera Acidovorax, Brevundimonas, Pusillimonas, and Burkholderiales involved in the degradation of refractory organics, as well as the genera Desulfovibrio, Dechloromonas, and Rhizobium related to the production of black and odorous substances. An RDA analysis revealed that chemical oxygen demand, dissolved oxygen, and oxidation reduction potential were related to the changes in microbial community composition. Furthermore, aeration was found to accelerate the removal of ammonia nitrogen and enhance the function of microbial community by stimulating the growth of order Planktomycetes during the remediation of black blooms, but aeration substantially damaged the microbial diversity and richness. Therefore, the health of the aquatic ecosystem should be comprehensively considered when aeration is applied to restore polluted waterbodies. Notably, we observed a large number of pathogenic bacteria in urban black blooms, which emphasizes the importance of treating domestic sewage so that it is harmless. Together, these findings provide new insights and a basis to prevent and manage urban black blooms.

Handling of spurious sequences affects the outcome of high-throughput 16S rRNA gene amplicon profiling

Handling of spurious sequences affects the outcome of high-throughput 16S rRNA gene amplicon profiling

Refinement of 16S rRNA gene analysis for low biomass biospecimens

High-throughput phylogenetic 16S rRNA gene analysis has permitted to thoroughly delve into microbial community complexity and to understand host-microbiota interactions in health and disease. The analysis comprises sample collection and storage, genomic DNA extraction, 16S rRNA gene amplification, high-throughput amplicon sequencing and bioinformatic analysis. Low biomass microbiota samples (e.g. biopsies, tissue swabs and lavages) are receiving increasing attention, but optimal standardization for analysis of low biomass samples has yet to be developed. Here we tested the lower bacterial concentration required to perform 16S rRNA gene analysis using three different DNA extraction protocols, three different mechanical lysing series and two different PCR protocols. A mock microbiota community standard and low biomass samples (108, 107, 106, 105 and 104 microbes) from two healthy donor stools were employed to assess optimal sample processing for 16S rRNA gene analysis using paired-end Illumina MiSeq technology. Three DNA extraction protocols tested in our study performed similar with regards to representing microbiota composition, but extraction yield was better for silica columns compared to bead absorption and chemical precipitation. Furthermore, increasing mechanical lysing time and repetition did ameliorate the representation of bacterial composition. The most influential factor enabling appropriate representation of microbiota composition remains sample biomass. Indeed, bacterial densities below 106 cells resulted in loss of sample identity based on cluster analysis for all tested protocols. Finally, we excluded DNA extraction bias using a genomic DNA standard, which revealed that a semi-nested PCR protocol represented microbiota composition better than classical PCR. Based on our results, starting material concentration is an important limiting factor, highlighting the need to adapt protocols for dealing with low biomass samples. Our study suggests that the use of prolonged mechanical lysing, silica membrane DNA isolation and a semi-nested PCR protocol improve the analysis of low biomass samples. Using the improved protocol we report a lower limit of 106 bacteria per sample for robust and reproducible microbiota analysis.

Long-term effects of acetylene on denitrifying N2O production: Biomass performance and microbial community

Maximizing production and use of N2O produced by the process of denitrification has emerged as a promising concept for energy recovery from nitrogen. Acetylene is well known as an inhibitor of N2O reductase activity, however, to date, few studies have utilized long-term cultivation with acetylene to achieve denitrifying N2O recovery. This work assessed the performance of N2O production and shifts in the denitrifying community in response to long-term exposure of acetylene during nitrite denitrification. Batch tests showed that long-term exposure to acetylene at high concentrations (volume fraction >40 %) resulted in high N2O accumulation, up to 98.6 %. High-throughput 16S rRNA gene analysis suggested that Saprospiraceae dominated the microbial community at the end of the cultivation period with its abundance increasing from the initial 3.2% to 42.2%. Metagenomic analysis revealed that after the long-term cultivation under high level acetylene, the relative abundance of nosZ in the community did not change significantly. Instead, the nosZⅡ-harboring bacteria became dominant. Batch tests in series demonstrated that N2O reduction could be restored rapidly upon removal of acetylene. This study describes an effective method for high yield production of N2O during nitrite denitrification that can be used to enhance energy recovery and improves our understanding of the long-term effect of acetylene on the denitrifying community.

Effect of Bacillus spp. on Lettuce Growth and Root Associated Bacterial Community in a Small-Scale Aquaponics System

The integration of probiotics in aquaponics systems is a strategy for mitigating environmental impacts and for promoting sustainable agriculture. In order to understand the role of probiotics, we investigated the effect of a commercial probiotic mixture of Bacillus subtilis and B. licheniformis on the growth of lettuce (Lactuca sativa L.) under deep-water culture integrated with Mozambique tilapia (Oreochromis mossambicus). We determined plant growth, water quality parameters, and leaf mineral analysis, and assessed the influence of a probiotic mixture on the microbiota. Bacterial communities were analyzed by high-throughput 16S rRNA gene sequencing. Compared to the control systems, the addition of the probiotic Bacillus significantly increased the concentration of nitrate and phosphate in deep water culture solution, which contributed to improved lettuce growth. In both the growth trials, the Fv/Fm, the mean shoot dry weight, and the mean fresh weight of the harvested shoots from the Bacillus treatment were significantly higher than those observed for the control plants. Higher concentrations of phosphorus, potassium, and zinc in the lettuce leaves were found in systems that received the Bacillus. Although differences were observed at the phylum level, Proteobacteria and Bacteroidetes were predominant in both the Bacillus-treatment and the control systems. At the genus level, however, the communities present in the two types of systems were heterogeneous with Bacillus-treated systems, containing significantly higher numbers of Chryseobacterium, Bacillus, Nitrospira, Polynucleobacter, and Thermomonas. The results indicate that Bacillus supplementation can effectively alleviate nutrient deficiencies, improve water quality, and modify the composition of bacterial communities in aquaponics systems.

Administration of Bovine Milk Oligosaccharide to Weaning Gnotobiotic Mice Inoculated with a Simplified Infant Type Microbiota.

Bovine milk oligosaccharides (BMO) share structural similarity to selected human milk oligosaccharides, which are natural prebiotics for infants. Thus, there is a potential in including BMOs as a prebiotic in infant formula. To examine the in vivo effect of BMO-supplementation on the infant gut microbiota, a BMO-rich diet (2% w/w) was fed to gnotobiotic mice (n = 11) inoculated with an infant type co-culture and compared with gnotobiotic mice receiving a control diet (n = 9). Nuclear magnetic resonance metabolomics in combination with high-throughput 16S rRNA gene amplicon sequencing was used to compare metabolic activity and microbiota composition in different compartments of the lower gastrointestinal tract. BMO components were detected in cecum and colon contents, revealing that BMO was available for the gut bacteria. The gut microbiota was dominated by Enterobacteriaceae and minor abundance of Lactobacilliaceae, while colonization of Bifidobacteriaceae did not succeed. Apart from a lower E. coli population in cecum content and lower formate (in colon) and succinate (in colon and cecum) concentrations, BMO supplementation did not result in significant changes in microbiota composition nor metabolic activity. The present study corroborates the importance of the presence of bifidobacteria for obtaining microbial-derived effects of milk oligosaccharides in the gastrointestinal tract.