Microbial Ecology Studies Research Articles

Alternative agar substitutes for culturing unculturable microorganisms.

Gelling agents are necessary for the preparation of solid or semisolid media. For more than a hundred years, agar has been the primary gelling agent. However, a substantial body of evidence has accumulated suggesting that agar-based media inhibit the growth of many microbial species through the generation of reactive oxygen species (ROS), toxic organic contaminants, or competitive exclusion effects. In this review we have compiled the largest amount of data to date on the use of various gelling agents in microbial isolation and cultivation, with the particular emphasis on rare microbe isolation cases. Our analysis suggested that microbial-derived compounds (especially gellan gum), as gelling agents, are superior to agar in their ability to isolate and maintain either new or known microbial species. We analyzed the reasons behind this success and concluded that there are phylum-level differences in microbial responses to the changes in conditions from natural to the laboratory conditions (with respect to gelling agent usage). Consequently, we hypothesize that at least partial success of microbial-derived gelling agents lies in the recreation of the natural microenvironment conditions (which we address as the "familiarity of conditions" hypothesis). Finally, we present a list of recommendations and suggestions for further microbial ecology studies.

Comparative analysis of size-fractional eukaryotic microbes in subtropical riverine systems inferred from 18S rRNA gene V4 and V9 regions

Eukaryotic microbes play key ecological roles in riverine ecosystems. Amplicon sequencing has greatly facilitated the identification and characterization of eukaryotic microbial communities. Currently, 18S rRNA gene V4 and V9 hypervariable regions are widely used for sequencing eukaryotic microbes. Identifying optimal regions for the profiling of size-fractional eukaryotic microbial communities is critical for microbial ecological studies. In this study, we spanned three rivers with typical natural-human influenced transition gradients to evaluate the performance of the 18S rRNA gene V4 and V9 hypervariable regions for sequencing size-fractional eukaryotic microbes (>180 μm, 20-180 μm, 5-20 μm, 3-5 μm, 0.8-3 μm). Our comparative analysis revealed that amplicon results depend on the specific species and microbial size. The V9 region was most effective for detecting a broad taxonomic range of species. The V4 region was superior to the V9 region for the identification of microbes in the minor 3 μm and at the family and genus levels, especially for specific microbial groups, such as Labyrinthulomycetes. However, the V9 region was more effective for studies of diverse eukaryotic groups, including Archamoebae, Heterolobosea, and Microsporidia, and various algae, such as Haptophyta, Florideophycidae, and Bangiales. Our results highlight the importance of accounting for potential misclassifications when employing both V4 and V9 regions for the identification of microbial sequences. The use of optimal regions for amplification could enhance the utility of amplicon sequencing in environmental studies. The insights gained from this work will aid future studies that employ amplicon-based identification approaches for the characterization of eukaryotic microbial communities and contribute to our understanding of microbial ecology within aquatic systems.

Bacterial Communities across Multiple Ecological Niches (Water, Sediment, Plastic, and Snail Gut) in Mangrove Habitats.

Microbial composition across substrates in mangroves, particularly in the Middle East, remains unclear. This study characterized bacterial communities in sediment, water, Terebralia palustris snail guts, and plastic associated with Avicennia marina mangrove forests in two coastal lagoons in the Sea of Oman using 16S rDNA gene MiSeq sequencing. The genus Vibrio dominated all substrates except water. In the gut of snails, Vibrio is composed of 80-99% of all bacterial genera. The water samples showed a different pattern, with the genus Sunxiuqinia being dominant in both Sawadi (50.80%) and Qurum (49.29%) lagoons. There were significant differences in bacterial communities on different substrata, in particular plastic. Snail guts harbored the highest number of unique Operational Taxonomic Units (OTUs) in both lagoons, accounting for 30.97% OTUs in Sawadi and 28.91% OTUs in Qurum, compared to other substrates. Plastic in the polluted Sawadi lagoon with low salinity harbored distinct genera such as Vibrio, Aestuariibacter, Zunongwangia, and Jeotgalibacillus, which were absent in the Qurum lagoon with higher salinity and lower pollution. Sawadi lagoon exhibited higher species diversity in sediment and plastic substrates, while Qurum lagoon demonstrated lower species diversity. The principal component analysis (PCA) indicates that environmental factors such as salinity, pH, and nutrient levels significantly influence bacterial community composition across substrates. Variations in organic matter and potential anthropogenic influences, particularly from plastics, further shape bacterial communities. This study highlights the complex microbial communities in mangrove ecosystems, emphasizing the importance of considering multiple substrates in mangrove microbial ecology studies. The understanding of microbial dynamics and anthropogenic impacts is crucial for shaping effective conservation and management strategies in mangrove ecosystems, particularly in the face of environmental changes.

Distinct ecological niches and community dynamics: understanding free-living and particle-attached bacterial communities in an oligotrophic deep lake.

Oligotrophic deep-water lakes are unique and sensitive ecosystems with limited nutrient availability. Understanding bacterial communities within these lakes is crucial for assessing ecosystem health, biogeochemical cycling, and responses to environmental changes. In this study, we investigated the seasonal and vertical dynamics of both free-living (FL) and particle-attached (PA) bacteria in Lake Fuxian, a typical oligotrophic deep freshwater lake in southeast China. Our findings revealed distinct seasonal and vertical dynamics of FL and PA bacterial communities, driven by similar physiochemical environmental factors. PA bacteria exhibited higher α- and β-diversity and were enriched with Proteobacteria, Cyanobacteria, Firmicutes, Patescibacteria, Planctomycetota, and Verrucomicrobiota, while FL bacteria were enriched with Actinobacteria and Bacteroidota. FL bacteria showed enrichment in putative functions related to chemoheterotrophy and aerobic anoxygenic photosynthesis, whereas the PA fraction was enriched with intracellular parasites (mainly contributed by Rickettsiales, Chlamydiales, and Legionellales) and nitrogen metabolism functions. Deterministic processes predominantly shaped the assembly of both FL and PA bacterial communities, with stochastic processes playing a greater role in the FL fraction. Network analysis revealed extensive species interactions, with a higher proportion of positively correlated edges in the PA network, indicating mutualistic or cooperative interactions. Cyanobium, Comamonadaceae, and Roseomonas were identified as keystone taxa in the PA network, underscoring potential cooperation between autotrophic and heterotrophic bacteria in organic particle microhabitats. Overall, the disparities in bacterial diversity, community composition, putative function, and network characteristics between FL and PA fractions highlight their adaptation to distinct ecological niches within these unique lake ecosystems.IMPORTANCEUnderstanding the diversity of microbial communities, their assembly mechanisms, and their responses to environmental changes is fundamental to the study of aquatic microbial ecology. Oligotrophic deep-water lakes are fragile ecosystems with limited nutrient resources, rendering them highly susceptible to environmental fluctuations. Examining different bacterial types within these lakes offers valuable insights into the intricate mechanisms governing community dynamics and adaptation strategies across various scales. In our investigation of oligotrophic deep freshwater Lake Fuxian in China, we explored the seasonal and vertical dynamics of two bacterial types: free-living (FL) and particle-attached (PA). Our findings unveiled distinct patterns in the diversity, composition, and putative functions of these bacteria, all shaped by environmental factors. Understanding these subtleties provides insight into bacterial interactions, thereby influencing the overall ecosystem functioning. Ultimately, our research illuminates the adaptation and roles of FL and PA bacteria within these unique lake environments, contributing significantly to our broader comprehension of ecosystem stability and health.

Dynamics of the viral community on the surface of a French smear-ripened cheese during maturation and persistence across production years.

The surface of smear-ripened cheeses constitutes a dynamic microbial ecosystem resulting from the successive development of different microbial groups such as lactic acid bacteria, fungi, and ripening bacteria. Recent studies indicate that a viral community, mainly composed of bacteriophages, also represents a common and substantial part of the cheese microbiome. However, the composition of this community, its temporal variations, and associations between bacteriophages and their hosts remain poorly characterized. Here, we studied a French smear-ripened cheese by both viral metagenomics and 16S metabarcoding approaches to assess both the succession of phages and bacterial communities on the cheese surface during cheese ripening and their temporal variations in ready-to-eat cheeses over the years of production. We observed a clear transition of the phage community structure during ripening with a decreased relative abundance of viral species (vOTUs) associated with Lactococcus phages, which were replaced by vOTUs associated with phages infecting ripening bacteria such as Brevibacterium, Glutamicibacter, Pseudoalteromonas, and Vibrio. The dynamics of the phage community was strongly associated with bacterial successions observed on the cheese surface. Finally, while some variations in the distribution of phages were observed in ready-to-eat cheeses produced at different dates spanning more than 4 years of production, the most abundant phages were detected throughout. This result revealed the long-term persistence of the dominant phages in the cheese production environment. Together, these findings offer novel perspectives on the ecology of bacteriophages in smear-ripened cheese and emphasize the significance of incorporating bacteriophages in the microbial ecology studies of fermented foods.IMPORTANCEThe succession of diverse microbial populations is critical for ensuring the production of high-quality cheese. We observed a temporal succession of phages on the surface of a smear-ripened cheese, with new phage communities showing up when ripening bacteria start covering this surface. Interestingly, the final phage community of this cheese is also consistent over large periods of time, as the same bacteriophages were found in cheese products from the same manufacturer made over 4 years. This research highlights the importance of considering these bacteriophages when studying the microbial life of fermented foods like cheese.

Novel misos shape distinct microbial ecologies: opportunities for flavourful sustainable food innovation

Fermentation is resurgent around the world as people seek healthier, more sustainable, and tasty food options. This study explores the microbial ecology of miso, a traditional Japanese fermented paste, made with novel regional substrates to develop new plant-based foods. Eight novel miso varieties were developed using different protein-rich substrates: yellow peas, Gotland lentils, and fava beans (each with two treatments: standard and nixtamalisation), as well as rye bread and soybeans. The misos were produced at Noma, a restaurant in Copenhagen, Denmark. Samples were analysed with biological and technical triplicates at the beginning and end of fermentation. We also incorporated in this study six samples of novel misos produced following the same recipe at Inua, a former affiliate restaurant of Noma in Tokyo, Japan. To analyse microbial community structure and diversity, metabarcoding (16S and ITS) and shotgun metagenomic analyses were performed. The misos contain a greater range of microbes than is currently described for miso in the literature. The composition of the novel yellow pea misos was notably similar to the traditional soybean ones, suggesting they are a good alternative, which supports our culinary collaborators’ sensory conclusions. For bacteria, we found that overall substrate had the strongest effect, followed by time, treatment (nixtamalisation), and geography. For fungi, there was a slightly stronger effect of geography and a mild effect of substrate, and no significant effects for treatment or time. Based on an analysis of metagenome-assembled genomes (MAGs), strains of Staphylococccus epidermidis differentiated according to substrate. Carotenoid biosynthesis genes in these MAGs appeared in strains from Japan but not from Denmark, suggesting a possible gene-level geographical effect. The benign and possibly functional presence of S. epidermidis in these misos, a species typically associated with the human skin microbiome, suggests possible adaptation to the miso niche, and the flow of microbes between bodies and foods in certain fermentation as more common than is currently recognised. This study improves our understanding of miso ecology, highlights the potential for developing novel misos using diverse local ingredients, and suggests how fermentation innovation can contribute to studies of microbial ecology and evolution.

Effect of target gene sequence evenness and dominance on real-time PCR quantification of artificial sulfate-reducing microbial communities.

Quantitative real-time PCR of phylogenetic and functional marker genes is among the most commonly used techniques to quantify the abundance of microbial taxa in environmental samples. However, in most environmental applications, the approach is a rough assessment of population abundance rather than an exact absolute quantification method because of PCR-based estimation biases caused by multiple factors. Previous studies on these technical issues have focused on primer or template sequence features or PCR reaction conditions. However, how target gene sequence characteristics (e.g., evenness and dominance) in environmental samples affect qPCR quantifications has not been well studied. Here, we compared three primer sets targeting the beta subunit of the dissimilatory sulfite reductase (dsrB) to investigate qPCR quantification performance under different target gene sequence evenness and dominance conditions using artificial gBlock template mixtures designed accordingly. Our results suggested that the qPCR quantification performance of all tested primer sets was determined by the comprehensive effect of the target gene sequence evenness and dominance in environmental samples. Generally, highly degenerate primer sets have equivalent or better qPCR quantification results than a more target-specific primer set. Low template concentration in this study (~105 copies/L) will exaggerate the qPCR quantification results difference among tested primer sets. Improvements to the accuracy and reproducibility of qPCR assays for gene copy number quantification in environmental microbiology and microbial ecology studies should be based on prior knowledge of target gene sequence information acquired by metagenomic analysis or other approaches, careful selection of primer sets, and proper reaction conditions optimization.

Microfluidic approaches in microbial ecology.

Microbial life is at the heart of many diverse environments and regulates most natural processes, from the functioning of animal organs to the cycling of global carbon. Yet, the study of microbial ecology is often limited by challenges in visualizing microbial processes and replicating the environmental conditions under which they unfold. Microfluidics operates at the characteristic scale at which microorganisms live and perform their functions, thus allowing for the observation and quantification of behaviors such as growth, motility, and responses to external cues, often with greater detail than classical techniques. By enabling a high degree of control in space and time of environmental conditions such as nutrient gradients, pH levels, and fluid flow patterns, microfluidics further provides the opportunity to study microbial processes in conditions that mimic the natural settings harboring microbial life. In this review, we describe how recent applications of microfluidic systems to microbial ecology have enriched our understanding of microbial life and microbial communities. We highlight discoveries enabled by microfluidic approaches ranging from single-cell behaviors to the functioning of multi-cellular communities, and we indicate potential future opportunities to use microfluidics to further advance our understanding of microbial processes and their implications.

Testate amoebae (Arcellinida, Amoebozoa) community diversity in New England bogs and fens assessed through lineage-specific amplicon sequencing

Testate amoebae (order Arcellinida) are abundant in freshwater ecosystems, including low pH bogs and fens. Within these environments, Arcellinida are considered top predators in microbial food webs and their tests are useful bioindicators of paleoclimatic changes and anthropogenic pollutants. Accurate species identifications and characterizations of diversity are important for studies of paleoclimate, microbial ecology, and environmental change; however, morphological species definitions mask cryptic diversity, which is a common phenomenon among microbial eukaryotes. Lineage-specific primers recently designed to target Arcellinida for amplicon sequencing successfully captured a poorly-described yet diverse fraction of the microbial eukaryotic community. Here, we leveraged the application of these newly-designed primers to survey the diversity of Arcellinida in four low-pH New England bogs and fens, investigating variation among bogs (2018) and then across seasons and habitats within two bogs (2019). Three OTUs represented 66% of Arcellinida reads obtained across all habitats surveyed. 103 additional OTUs were present in lower abundance with some OTUs detected in only one sampling location, suggesting habitat specificity. By establishing a baseline for Arcellinida diversity, we provide a foundation to monitor key taxa in habitats that are predicted to change with increasing anthropogenic pressure and rapid climate change.

Agromyces chromiiresistens sp. nov., Novosphingobium album sp. nov., Sphingobium arseniciresistens sp. nov., Sphingomonas pollutisoli sp. nov., and Salinibacterium metalliresistens sp. nov.: five new members of Microbacteriaceae and Sphingomonadaceae from polluted soil

There are many unidentified microbes in polluted soil needing to be explored and nominated to benefit the study of microbial ecology. In this study, a taxonomic research was carried out on five bacterial strains which were isolated and cultivated from polycyclic aromatic hydrocarbons, and heavy metals polluted soil of an abandoned coking plant. Phylogenetical analysis showed that they belonged to the phyla Proteobacteria and Actinobacteria, and their 16S rRNA gene sequence identities were lower than 98.5% to any known and validly nominated bacterial species, suggesting that they were potentially representing new species. Using polyphasic taxonomic approaches, the five strains were classified as new species of the families Microbacteriaceae and Sphingomonadaceae. Genome sizes of the five strains ranged from 3.07 to 6.60 Mb, with overall DNA G+C contents of 63.57–71.22 mol%. The five strains had average nucleotide identity of 72.38–87.38% and digital DNA-DNA hybridization of 14.0–34.2% comparing with their closely related type strains, which were all below the thresholds for species delineation, supporting these five strains as novel species. Based on the phylogenetic, phylogenomic, and phenotypic characterizations, the five novel species are proposed as Agromyces chromiiresistens (type strain H3Y2-19aT = CGMCC 1.61332T), Salinibacterium metalliresistens (type strain H3M29-4T = CGMCC 1.61335T), Novosphingobium album (type strain H3SJ31-1T = CGMCC 1.61329T), Sphingomonas pollutisoli (type strain H39-1-10T = CGMCC 1.61325T), and Sphingobium arseniciresistens (type strain H39-3-25T = CGMCC 1.61326T). Comparative genome analysis revealed that the species of the family Sphingomonadaceae represented by H39-1-10T, H39-3-25T, and H3SJ31-1T possessed more functional protein-coding genes for the degradation of aromatic pollutants than the species of the family Microbacteriaceae represented by H3Y2-19aT and H3M29-4T. Furthermore, their capacities of resisting heavy metals and metabolizing aromatic compounds were investigated. The results indicated that strains H3Y2-19aT and H39-3-25T were robustly resistant to chromate (VI) and/or arsenite (III). Strains H39-1-10T and H39-3-25T grew on aromatic compounds, including naphthalene, as carbon sources even in the presence of chromate (VI) and arsenite (III). These features reflected their adaptation to the polluted soil environment.

Multiscale models driving hypothesis and theory-based research in microbial ecology.

Hypothesis and theory-based studies in microbial ecology have been neglected in favour of those that are descriptive and aim for data-gathering of uncultured microbial species. This tendency limits our capacity to create new mechanistic explanations of microbial community dynamics, hampering the improvement of current environmental biotechnologies. We propose that a multiscale modelling bottom-up approach (piecing together sub-systems to give rise to more complex systems) can be used as a framework to generate mechanistic hypotheses and theories (in-silico bottom-up methodology). To accomplish this, formal comprehension of the mathematical model design is required together with a systematic procedure for the application of the in-silico bottom-up methodology. Ruling out the belief that experimentation before modelling is indispensable, we propose that mathematical modelling can be used as a tool to direct experimentation by validating theoretical principles of microbial ecology. Our goal is to develop methodologies that effectively integrate experimentation and modelling efforts to achieve superior levels of predictive capacity.

Evaluation of Proteins Released to Medium in Yeast-Bacteria Co-culture System

The natural ecosystem contains an astonishing variety of microbial species. Populations of microorga-nisms exist in a common habitat in terms of access to nutrients and other resources in the environment. Cultivation of microorganisms in ideal laboratory conditions seperates them from their natural ecosystem conditions and isolates them from their microbial world, especially from their competitors. With traditional pure culture-oriented cultuvation techniques used in laboratories, interactions mediated by small molecules are not taken into account, resulting in the precise nature of the interactions being largely unknown. Coculture systems are systems in which two or more different cell populations are grown together. In this way, studies on natural interactions between populations can be made and synthetic interactions that are not observed in nature can be provided. With these systems, natural product discovery, microbial ecology, evolution and pathogenesis studies are carried out. In addition, coculture systems are also used in industrial, environmental and medical studies. In this study, the wild strain of Schizosaccharomyces pombe and the DH5α strain of Escherichia coli were grown in their own specific media, then cultured for 48 hours and 72 hours by cultivating in media containing 0,1% glucose with different cell number, and finally the differentiation in the proteins released by the cells into the medium was observed in SDS polyacrylamide gels. Different from the control conditions, new protein bands that emerged under the co-culture conditions were detected and two of these bands were analyzed by mass spectrometry (MS). While 6 of differentaited proteins were released by S.pombe, 257 proteins matched with E.coli proteom. These proteins are; Various carbohydrate-binding proteins, membrane proteins involved in the identification of various signaling molecules and antibiotics, and other proteins involved in various cellular processes.

Sediment Microbiota as a Proxy of Environmental Health: Discovering Inter- and Intrakingdom Dynamics along the Eastern Mediterranean Continental Shelf.

Sedimentary marine habitats are the largest ecosystem on our planet in terms of area. Marine sediment microbiota govern most of the benthic biological processes and therefore are responsible for much of the global biogeochemical activity. Sediment microbiota respond, even rapidly, to natural change in environmental conditions as well as disturbances of anthropogenic sources. The latter greatly impact the continental shelf. Characterization and monitoring of the sediment microbiota may serve as an important tool for assessing environmental health and indicate changes in the marine ecosystem. This study examined the suitability of marine sediment microbiota as a bioindicator for environmental health in the eastern Mediterranean Sea. Integration of information from Bacteria, Archaea, and Eukaryota enabled robust assessment of environmental factors controlling sediment microbiota composition: seafloor-depth (here representing sediment grain size and total organic carbon), core depth, and season (11%, 4.2%, and 2.5% of the variance, respectively). Furthermore, inter- and intrakingdom cooccurrence patterns indicate that ecological filtration as well as stochastic processes may control sediment microbiota assembly. The results show that the sediment microbiota was robust over 3 years of sampling, in terms of both representation of region (outside the model sites) and robustness of microbial markers. Furthermore, anthropogenic disturbance was reflected by significant transformations in sediment microbiota. We therefore propose sediment microbiota analysis as a sensitive approach to detect disturbances, which is applicable for long-term monitoring of marine environmental health. IMPORTANCE Analysis of data, curated over 3 years of sediment sampling, improves our understanding of microbiota assembly in marine sediment. Furthermore, we demonstrate the importance of cross-kingdom integration of information in the study of microbial community ecology. Finally, the urgent need to propose an applicable approach for environmental health monitoring is addressed here by establishment of sediment microbiota as a robust and sensitive model.

Biogeographic distributions of microbial communities associated with anaerobic methane oxidation in the surface sediments of deep-sea cold seeps in the South China Sea.

Cold seeps are oasis for the microbes in the deep-sea ecosystems, and various cold seeps are located along the northern slope of the South China Sea (SCS). However, by far most microbial ecological studies were limited to specific cold seep in the SCS, and lack of comparison between different regions. Here, the surface sediments (0-4 cm) from the Site F/Haima cold seeps and the Xisha trough in the SCS were used to elucidate the biogeography of microbial communities, with particular interest in the typical functional groups involved in the anaerobic oxidation of methane (AOM) process. Distinct microbial clusters corresponding to the three sampling regions were formed, and significantly higher gene abundance of functional groups were present in the cold seeps than the trough. This biogeographical distribution could be explained by the geochemical characteristics of sediments, such as total nitrogen (TN), total phosphorus (TP), nitrate (NO3 -), total sulfur (TS) and carbon to nitrogen ratios (C/N). Phylogenetic analysis demonstrated that mcrA and pmoA genotypes were closely affiliated with those from wetland and mangroves, where denitrifying anaerobic methane oxidation (DAMO) process frequently occurred; and highly diversified dsrB genotypes were revealed as well. In addition, significantly higher relative abundance of NC10 group was found in the Xisha trough, suggesting that nitrite-dependent DAMO (N-DAMO) process was more important in the hydrate-bearing trough, although its potential ecological contribution to AOM deserves further investigation. Our study also further demonstrated the necessity of combining functional genes and 16S rRNA gene to obtain a comprehensive picture of the population shifts of natural microbial communities among different oceanic regions.

A comparative analysis employing a gene- and genome-centric metagenomic approach reveals changes in composition, function, and activity in waterworks with different treatment processes and source water in Finland

The emergence and development of next-generation sequencing technologies (NGS) has made the analysis of the water microbiome in drinking water distribution systems (DWDSs) more accessible and opened new perspectives in microbial ecology studies. The current study focused on the characterization of the water microbiome employing a gene- and genome-centric metagenomic approach to five waterworks in Finland with different raw water sources, treatment methods, and disinfectant. The microbial communities exhibit a distribution pattern of a few dominant taxa and a large representation of low-abundance bacterial species. Changes in the community structure may correspond to the presence or absence and type of disinfectant residual which indicates that these conditions exert selective pressure on the microbial community. The Archaea domain represented a small fraction (up to 2.5%) and seemed to be effectively controlled by the disinfection of water. Their role particularly in non-disinfected DWDS may be more important than previously considered. In general, non-disinfected DWDSs harbor higher microbial richness and maintaining disinfectant residual is significantly important for ensuring low microbial numbers and diversity. Metagenomic binning recovered 139 (138 bacterial and 1 archaeal) metagenome-assembled genomes (MAGs) that had a >50% completeness and <10% contamination consisting of 20 class representatives in 12 phyla. The presence and occurrence of nitrite-oxidizing bacteria (NOB)-like microorganisms have significant implications for nitrogen biotransformation in drinking water systems. The metabolic and functional complexity of the microbiome is evident in DWDSs ecosystems. A comparative analysis found a set of differentially abundant taxonomic groups and functional traits in the active community. The broader set of transcribed genes may indicate an active and diverse community regardless of the treatment methods applied to water. The results indicate a highly dynamic and diverse microbial community and confirm that every DWDS is unique, and the community reflects the selection pressures exerted at the community structure, but also at the levels of functional properties and metabolic potential.

Identification of bacterial communities in conventional wastewater treatment sludge to inform inoculation of the anammox process

This study uses 16 S rRNA gene pyrosequencing for the identification of a vast number of wastewater bacterial communities to investigate the evolution of bacterial communities in the Anammox process. Four lab-scale Anammox reactors inoculated with different conventional wastewater treatment sludge (activated sludge, livestock wastewater treatment sludge, denitrification sludge, and anaerobic digestion sludge) were operated under identical operating conditions for more than 400 days. The phylum Planctomycetes was present in all seeds of conventional sludge with a relative abundance of 1–3%. In particular, the known Anammox bacteria Candidatus Brocadia was found in the seed of the denitrification sludge. The reactor inoculated with denitrification sludge demonstrated the most effective nitrogen removal of ∼80% with successful cultivation of Anammox bacteria. This study found that the performance of the Anammox process is related to the presence of Nitrospira genus (nitrite-oxidizing bacteria) and that symbiotic association with other functional groups can lead to nitrogen removal. The outcomes of this study can provide vital insight into the study of microbial ecology for the cultivation of Anammox bacteria.

DNA-, RNA-, and Protein-Based Stable-Isotope Probing for High-Throughput Biomarker Analysis of Active Microorganisms.

Stable-isotope probing (SIP) enables researchers to target active populations within complex microbial communities, which is achieved by providing growth substrates enriched in heavy isotopes, usually in the form of 13C, 18O, or 15N. After growth on the substrate and subsequent extraction of microbial biomarkers, typically nucleic acids or proteins, the SIP technique is used for the recovery and analysis of isotope-labelled biomarkers from active microbial populations. In the years following the initial development of DNA- and RNA-based SIP, it was common practice to characterize labelled populations by targeted gene analysis. Such approaches usually involved fingerprint-based analyses or sequencing clone libraries containing 16S rRNA genes or functional marker gene amplicons. Although molecular fingerprinting remains a valuable approach for rapid confirmation of isotope labelling, recent advances in sequencing technology mean that it is possible to obtain affordable and comprehensive amplicon profiles, or even metagenomes and metatranscriptomes from SIP experiments. Not only can the abundance of microbial groups be inferred from metagenomes, but researchers can bin, assemble, and explore individual genomes to build hypotheses about the metabolic capabilities of labelled microorganisms. Analysis of labelled mRNA is a more recent advance that can provide independent metatranscriptome-based analysis of active microorganisms. The power of metatranscriptomics is that mRNA abundance often correlates closely with the corresponding activity of encoded enzymes, thus providing insight into microbial metabolism at the time of sampling. Together, these advances have improved the sensitivity of SIP methods and allowed using labelled substrates at environmentally relevant concentrations. Particularly as methods improve and costs continue to drop, we expect that the integration of SIP with multiple omics-based methods will become prevalent components of microbial ecology studies, leading to further breakthroughs in our understanding of novel microbial populations and elucidation of the metabolic function of complex microbial communities. In this chapter, we provide protocols for obtaining labelled DNA, RNA, and proteins that can be used for downstream omics-based analyses.

Soil microbial community structures are shaped by agricultural systems revealing little temporal variation

Many studies in soil microbial ecology are undertaken with a single sampling event, with the influence of temporal progression rarely being considered. Under field conditions, soil samples were taken from different agricultural systems; a sown grassland to maize rotation (MC), an intensively managed permanent grassland (INT), as well as extensively managed permanent grasslands with high (EXT_HP), low to sufficient (EXT_LP) and deficient available P (EXT_DP), six times throughout the 2017 growing season. Thus, this study aimed to determine if any differences in soil microbiome structures between both sharply contrasting (MC – INT – EXT), slightly differing (EXT_HP – EXT_DP) and quite similar (EXT_HP – EXT_LP and EXT_LP – EXT_DP) agricultural systems persist through changing growth conditions within the growing season. For both fungal and bacterial community structure, the influence of agricultural system (CV = 0.256, P < 0.001 and CV = 0.145, P < 0.01, respectively) was much greater than that of temporal progression (√CV = 0.065 and 0.042, respectively, both P < 0.001). Importantly, nearly all agricultural systems persistently harbored significantly distinct fungal community structures across each of the six sampling events (all at least P < 0.05). There were not as many pairwise differences in bacterial community structure between the agricultural systems, but some did persist (MC and EXT_HP ∼ EXT_DP, all P < 0.001). Additionally, persistent indicator fungal OTUs (IndVal >0.7, P ≤ 0.05) associated to each agricultural system (except EXT_LP) were found in each of the six sampling events. These results highlight the temporal stability of pairwise differences in soil microbiome structures between established agricultural systems through changing plant growth conditions, even between those with a comparable management regime. This is a highly relevant finding in informing the sampling strategy of studies in soil microbial ecology as well as for designing efficient soil biodiversity monitoring systems.

The Fungal Species and Fungal Numbers Associated with Non-Rhizosphere Soil of Spinacea oleracea (L.) During Kharif and Rabi Seasons

The microbes associated with non-rhizosphere soil, rhizosphere and phylloplane were found to differ quantitatively and qualitatively. The study of such associations are of special interest in the study of microbial ecology. The microbes in turn exert influence on the plants in supplying useful micro and macro nutrients to the above ground community The present study is aimed to study the qualitative and quantitative aspects of fungal species inhabiting the Non-Rhizosphere Soil of Spinacea oleracea (L.). The fungal numbers and species were estimated in two Kharif seasons (Kharif season –I and Kharif season -II) and two Rabi seasons (Rabi season –I and Rabi season – II).A total of 68 fungal species were isolated from non-rhizosphere soils.The number of fungal species isolated during Rabi season-I were more than that of Kharif season – I. The dominant groups were Fusaria followed by Sterile mycelia, Fungi imperfecti and Aspergilli which were dominant throughout the Kharif season II. The dominant species during Rabi Season II were Aspergillus niger. Acrophialophora nainiana, Myrothecium gramineum, Aspergillus nidulans, Aspergillus flavus, Phoma humicola, Cladosporium cladosporioides. Fusarium dimerum, Bahupaathra sp. and Curvularia sp.

The Effects of DNA Extraction Kits and Primers on Prokaryotic and Eukaryotic Microbial Community in Freshwater Sediments.

DNA based sequencing technology has revolutionized the field of microbial ecology and environmental studies. However, biases can be introduced at all experimental steps and, thus, affect the interpretation of microbial community. So far, previous studies on the biases introduced from the key steps of DNA extraction and primer sets mainly focused on the bacterial communities in soil or sediment samples, while little is known about the effect on the eukaryotic microbial communities. Here, we studied the effects of three different DNA extraction kits on both prokaryotic and micro-eukaryotic communities by 16S and 18S rRNA gene amplicon sequencing, and further disentangled the influence of primer choice on the micro-eukaryotic communities. Our results showed that the FastDNA SPIN Kit for Soil and DNeasy PowerSoil Kit produced much higher DNA yield with good reproducibility, and observed more eukaryotic OTUs compared to the MinkaGene DNA extraction kit, but all three kits exhibited comparable ability in recovering bacterial alpha diversity. Of the two primer sets, both targeting the V4 region of the 18S rRNA gene, the TAR primer set detected higher number of unique OTUs than the EK primer set, while the EK primer set resulted in longer amplicons and better reproducibility between replicates. Based on our findings, we recommend using the DNeasy PowerSoil Kit with the EK primer set to capture the abundant micro-eukaryotic taxa from freshwater sediment samples. If a more complete picture of the eukaryotic microbial community is desired, the TAR primer set in combination with the FastDNA SPIN Kit is more efficient in this study.