Interactions between Soil Bacterial Diversity and Plant-Parasitic Nematodes in Soybean Plants.

Plant-parasitic nematodes are an important group of pests causing economic losses in agriculture worldwide. Among the plant-parasitic nematodes, the root-knot (Meloidogyne spp.) and root-lesion nematodes (Pratylenchus spp.) are considered the two most important ones affecting soybeans. In general, they damage soybean roots, causing a reduction of about one-third in productivity. The soil microbial community can exert a suppressive effect on the parasitism of plant-parasitic nematodes. Here, we investigated the effects of soil bacterial diversity on Meloidogyne javanica (Meloidogyne-assay) and Pratylenchus brachyurus (Pratylenchus-assay) suppression by manipulating microbial diversity using the dilution-to-extinction approach in two independent experiments under controlled conditions. Furthermore, we recorded the changes in the soil microbial community induced by plant-parasitic nematode infection. In Meloidogyne-assay, microbial diversity reduced the population density of M. javanica and improved plant performance. In Pratylenchus-assay, microbial diversity sustained the performance of soybean plants even at high levels of P. brachyurus parasitism. Each nematode population affected the relative abundance of different bacterial genera and altered the core microbiome of key groups within the bacterial community. Our findings provide fundamental insights into the interactions between soil bacterial diversity and plant-parasitic nematodes in soybean plants. IMPORTANCE Root-knot and root-lesion nematodes cause losses of billions of dollars every year to agriculture worldwide. Traditionally, they are controlled by using chemical nematicides, which in general have a negative impact on the environment and human health. Fortunately, the soil microbial community may suppress these pests, acting as an environmentally friendly alternative to control nematodes. However, the effects of soil microbial diversity on the parasitism of plant-parasitic nematodes still poorly understood. In this study, we provide fundamental insight into the interactions between soil bacterial diversity and plant-parasitic nematodes in soybean plants, which may be useful for the development of new strategies to control these phytopathogens.

Isolation of Hermetia illucens larvae core gut microbiota by two different cultivation strategies.

Hermetia illucens larvae (black soldier fly larvae, BSFL) convert efficiently organic waste to high quality biomass. To gain knowledge on the specific functions of gut microbes in this process it is a prerequisite to culture members of the core gut microbiota. Two different cultivation strategies were applied here for this purpose, a dilution-to-extinction cultivation and direct plating using six different media to culture aerobic heterotrophic bacteria. A total of 341 isolates were obtained by the dilution-to-extinction cultivation and 138 isolates by direct plating from guts of BSFL reared on chicken feed. Bacterial isolates were phylogenetically identified at the genus level by 16S rRNA gene sequencing (phylotyping) and differentiated at the strain level by genomic fingerprinting (genotyping). The main proportion of isolates was assigned to Proteobacteria, Firmicutes (Bacilli), and Actinobacteria. Predominant genera discussed in literature as member of a potential BSFL core gut microbiota, Providencia, Proteus, Morganella, Enterococcus, Bacillus, and members of the family Enterobacteriaceae, were isolated. A high intra-phylotype diversity was obtained by genomic fingerprinting which was especially enhanced by the dilution-to-extinction cultivation. This study showed that the application of different cultivation strategies including a dilution-to-extinction cultivation helps to culture a higher diversity of the BSFL gut microbiota and that genomic fingerprinting gives a better picture on the genetic diversity of cultured bacteria which cannot be covered by a 16S rRNA gene sequence based identification alone.

Plant and soil biodiversity have non-substitutable stabilising effects on biomass production.

The stability of plant biomass production in the face of environmental change is fundamental for maintaining terrestrial ecosystem functioning, as plant biomass is the ultimate source of energy for nearly all life forms. However, most studies have focused on the stabilising effect of plant diversity, neglecting the effect of soil biodiversity, the largest reservoir of biodiversity on Earth. Here we investigated the effects of plant and soil biodiversity on the temporal stability of biomass production under varying simulated precipitation in grassland microcosms. Soil biodiversity loss reduced temporal stability by suppressing asynchronous responses of plant functional groups. Greater plant diversity, especially in terms of functional diversity, promoted temporal stability, but this effect was independent of soil biodiversity loss. Moreover, multitrophic biodiversity, plant and soil biodiversity combined, was positively associated with temporal stability. Our study highlights the importance of maintaining both plant and soil biodiversity for sustainable biomass production.

Dilution-to-Stimulation/Extinction Method: a Combination Enrichment Strategy To Develop a Minimal and Versatile Lignocellulolytic Bacterial Consortium.

The engineering of complex communities can be a successful path to understand the ecology of microbial systems and improve biotechnological processes. Here, we developed a strategy to assemble a minimal and effective lignocellulolytic microbial consortium (MELMC) using a sequential combination of dilution-to-stimulation and dilution-to-extinction approaches. The consortium was retrieved from Andean forest soil and selected through incubation in liquid medium with a mixture of three types of agricultural plant residues. After the dilution-to-stimulation phase, approximately 50 bacterial sequence types, mostly belonging to the Sphingobacteriaceae, Enterobacteriaceae, Pseudomonadaceae, and Paenibacillaceae, were significantly enriched. The dilution-to-extinction method demonstrated that only eight of the bacterial sequence types were necessary to maintain microbial growth and plant biomass consumption. After subsequent stabilization, only two bacterial species (Pseudomonas sp. and Paenibacillus sp.) became highly abundant (>99%) within the MELMC, indicating that these are the key players in degradation. Differences in the composition of bacterial communities between biological replicates indicated that selection, sampling, and/or priority effects could shape the consortium structure. The MELMC can degrade up to ∼13% of corn stover, consuming mostly its (hemi)cellulosic fraction. Tests with chromogenic substrates showed that the MELMC secretes an array of endoenzymes able to degrade xylan, arabinoxylan, carboxymethyl cellulose, and wheat straw. Additionally, the metagenomic profile inferred from the phylogenetic composition along with an analysis of carbohydrate-active enzymes of 20 bacterial genomes support the potential of the MELMC to deconstruct plant polysaccharides. This capacity was mainly attributed to the presence of Paenibacillus sp.IMPORTANCE The significance of our study mainly lies in the development of a combined top-down enrichment strategy (i.e., dilution to stimulation coupled to dilution to extinction) to build a minimal and versatile lignocellulolytic microbial consortium. We demonstrated that mainly two selectively enriched bacterial species (Pseudomonas sp. and Paenibacillus sp.) are required to drive the effective degradation of plant polymers. Our findings can guide the design of a synthetic bacterial consortium that could improve saccharification (i.e., the release of sugars from agricultural plant residues) processes in biorefineries. In addition, they can help to expand our ecological understanding of plant biomass degradation in enriched bacterial systems.

Soil biodiversity enhances the persistence of legumes under climate change.

Global environmental change poses threats to plant and soil biodiversity. Yet, whether soil biodiversity loss can further influence plant community's response to global change is still poorly understood. We created a gradient of soil biodiversity using the dilution-to-extinction approach, and investigated the effects of soil biodiversity loss on plant communities during and following manipulations simulating global change disturbances in experimental grassland microcosms. Grass and herb biomass was decreased by drought and promoted by nitrogen deposition, and a fast recovery was observed following disturbances, independently of soil biodiversity loss. Warming promoted herb biomass during and following disturbance only when soil biodiversity was not reduced. However, legumes biomass was suppressed by these disturbances, and there were more detrimental effects with reduced soil biodiversity. Moreover, soil biodiversity loss suppressed the recovery of legumes following these disturbances. Similar patterns were found for the response of plant diversity. The changes in legumes might be partly attributed to the loss of mycorrhizal soil mutualists. Our study shows that soil biodiversity is crucial for legume persistence and plant diversity maintenance when faced with environmental change, highlighting the importance of soil biodiversity as a potential buffering mechanism for plant diversity and community composition in grasslands.

Soil Microbial Diversity Affects the Plant-Root Colonization by Arbuscular Mycorrhizal Fungi.

Terrestrial plants establish symbiosis with arbuscular mycorrhizal fungi (AMF) to exchange water and nutrients. However, the extent to which soil biodiversity influences such association remains still unclear. Here, we manipulated the soil microbial diversity using a "dilution-to-extinction" approach in a controlled pot microcosm system and quantified the root length colonization of maize plants by the AMF Rhizophagus clarus. The experiment was performed by manipulating the soil microbiome within a native and foreign soil having distinct physicochemical properties. Overall, our data revealed significant positive correlations between the soil microbial diversity and AMF colonization. Most importantly, this finding opposes the diversity-invasibility hypothesis and highlights for a potential overall helper effect of the soil biodiversity on plant-AMF symbiosis.

Expanding the Diversity of Bacterioplankton Isolates and Modeling Isolation Efficacy with Large-Scale Dilution-to-Extinction Cultivation.

Cultivated bacterioplankton representatives from diverse lineages and locations are essential for microbiology, but the large majority of taxa either remain uncultivated or lack isolates from diverse geographic locales. We paired large-scale dilution-to-extinction (DTE) cultivation with microbial community analysis and modeling to expand the phylogenetic and geographic diversity of cultivated bacterioplankton and to evaluate DTE cultivation success. Here, we report results from 17 DTE experiments totaling 7,820 individual incubations over 3 years, yielding 328 repeatably transferable isolates. Comparison of isolates to microbial community data for source waters indicated that we successfully isolated 5% of the observed bacterioplankton community throughout the study; 43% and 26% of our isolates matched operational taxonomic units and amplicon single-nucleotide variants, respectively, within the top 50 most abundant taxa. Isolates included those from previously uncultivated clades such as SAR11 LD12 and Actinobacteria acIV, as well as geographically novel members from other ecologically important groups like SAR11 subclade IIIa, SAR116, and others, providing isolates in eight putatively new genera and seven putatively new species. Using a newly developed DTE cultivation model, we evaluated taxon viability by comparing relative abundance with cultivation success. The model (i) revealed the minimum attempts required for successful isolation of taxa amenable to growth on our media and (ii) identified possible subpopulation viability variation in abundant taxa such as SAR11 that likely impacts cultivation success. By incorporating viability in experimental design, we can now statistically constrain the effort necessary for successful cultivation of specific taxa on a defined medium.IMPORTANCE Even before the coining of the term "great plate count anomaly" in the 1980s, scientists had noted the discrepancy between the number of microorganisms observed under the microscope and the number of colonies that grew on traditional agar media. New cultivation approaches have reduced this disparity, resulting in the isolation of some of the "most wanted" bacterial lineages. Nevertheless, the vast majority of microorganisms remain uncultured, hampering progress toward answering fundamental biological questions about many important microorganisms. Furthermore, few studies have evaluated the underlying factors influencing cultivation success, limiting our ability to improve cultivation efficacy. Our work details the use of dilution-to-extinction (DTE) cultivation to expand the phylogenetic and geographic diversity of available axenic cultures. We also provide a new model of the DTE approach that uses cultivation results and natural abundance information to predict taxon-specific viability and iteratively constrain DTE experimental design to improve cultivation success.

Expansion of Cultured Bacterial Diversity by Large-Scale Dilution-to-Extinction Culturing from a Single Seawater Sample.

High-throughput cultivation (HTC) based on a dilution-to-extinction method has been applied broadly to the cultivation of marine bacterial groups, which has often led to the repeated isolation of abundant lineages such as SAR11 and oligotrophic marine gammaproteobacteria (OMG). In this study, to expand the phylogenetic diversity of HTC isolates, we performed a large-scale HTC with a single surface seawater sample collected from the East Sea, the Western Pacific Ocean. Phylogenetic analyses of the 16S rRNA genes from 847 putative pure cultures demonstrated that some isolates were affiliated with not-yet-cultured clades, including the OPB35 and Puniceicoccaceae marine group of Verrucomicrobia and PS1 of Alphaproteobacteria. In addition, numerous strains were obtained from abundant clades, such as SAR11, marine Roseobacter clade, OMG (e.g., SAR92 and OM60), OM43, and SAR116, thereby increasing the size of available culture resources for representative marine bacterial groups. Comparison between the composition of HTC isolates and the bacterial community structure of the seawater sample used for HTC showed that diverse marine bacterial groups exhibited various growth capabilities under our HTC conditions. The growth response of many bacterial groups, however, was clearly different from that observed with conventional plating methods, as exemplified by numerous isolates of the SAR11 clade and Verrucomicrobia. This study showed that a large number of novel bacterial strains could be obtained by an extensive HTC from even a small number of samples.

Exploration of the key functional proteins from an efficient cellulolytic microbial consortium using dilution-to-extinction approach.

In the present study, the cellulose binding proteins (CBPs) secreted by a putative cellulolytic microbial consortium were isolated and purified by affinity digestion. The purified CBPs were subsequently separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Using mass spectrometric analyses, eight CBPs were identified and annotated to be similar to known proteins secreted by Clostridium clariflavum DSM 19732 and Paenibacillus sp. W-61. In addition, in combination with dilution-to-extinction approach and zymogram analysis technique, CBPs 6 (97kDa) and 12 (52kDa) were confirmed to be the key functional proteins that influence cellulolytic activities. Moreover, structural domain analyses and enzymatic activity detection indicated that CBPs 6 and 12 contained glycoside hydrolase families (GH) 9 and 48 catalytic modules, which both revealed endoglucandase and xylanase activities. It was suggested that the coexistence of GH9 and GH48 catalytic domains present in these two proteins could synergistically promote the efficient degradation of cellulose.

Two novel Pleosporales species isolated from the bark of Acer saccharum.

During a survey of culturable microfungi from the bark of sugar maple (Acer saccharum), Atrocalyx glutinosus and Nigrograna rubescens, two novel species of Pleosporales (Dothideomycetes) were isolated from several locations in eastern Ontario, Canada. Formal species descriptions are presented based on unique colony phenotypes and micromorphological characteristics and supported using multi-locus molecular phylogenetic comparisons with similar species. Both A. glutinosus and N. rubescens produce pycnidial asexual morphs in culture. As their names imply, under specific culture conditions, A. glutinosus excretes large amounts of the glutinous polysaccharide pullulan and N. rubescens produces a dark red naphthoquinone pigment that diffuses in the culture medium. Citation: Mack JN, Sproule A, Shields SW, Seifert KA, Smith M, Overy DP (2024). Two novel Pleosporales species isolated from the bark of Acer saccharum . Fungal Systematics and Evolution 13: 1-14. doi: 10.3114/fuse.2024.13.01.

Role of Bacillus subtilis exopolymeric genes in modulating rhizosphere microbiome assembly.

BACKGROUND: Bacillus subtilis is well known for promoting plant growth and reducing abiotic and biotic stresses. Mutant gene-defective models can be created to understand important traits associated with rhizosphere fitness. This study aimed to analyze the role of exopolymeric genes in modulating tomato rhizosphere microbiome assembly under a gradient of soil microbiome diversities using the B. subtilis wild-type strain UD1022 and its corresponding mutant strain UD1022eps-TasA, which is defective in exopolysaccharide (EPS) and TasA protein production. RESULTS: qPCR revealed that the B. subtilis UD1022eps-TasA- strain has a diminished capacity to colonize tomato roots in soils with diluted microbial diversity. The analysis of bacterial ß-diversity revealed significant differences in bacterial and fungal community structures following inoculation with either the wild-type or mutant B. subtilis strains. The Verrucomicrobiota, Patescibacteria, and Nitrospirota phyla were more enriched with the wild-type strain inoculation than with the mutant inoculation. Co-occurrence analysis revealed that when the mutant was inoculated in tomato, the rhizosphere microbial community exhibited a lower level of modularity, fewer nodes, and fewer communities compared to communities inoculated with wild-type B. subtilis. CONCLUSION: This study advances our understanding of the EPS and TasA genes, which are not only important for root colonization but also play a significant role in shaping rhizosphere microbiome assembly. Future research should concentrate on specific microbiome genetic traits and their implications for rhizosphere colonization, coupled with rhizosphere microbiome modulation. These efforts will be crucial for optimizing PGPR-based approaches in agriculture.

The selection of copiotrophs may complicate biodiversity-ecosystem functioning relationships in microbial dilution-to-extinction experiments.

The relationships between biodiversity-ecosystem functioning (BEF) for microbial communities are poorly understood despite the important roles of microbes acting in natural ecosystems. Dilution-to-extinction (DTE), a method to manipulate microbial diversity, helps to fill the knowledge gap of microbial BEF relationships and has recently become more popular with the development of high-throughput sequencing techniques. However, the pattern of community assembly processes in DTE experiments is less explored and blocks our further understanding of BEF relationships in DTE studies. Here, a microcosm study and a meta-analysis of DTE studies were carried out to explore the dominant community assembly processes and their potential effect on exploring BEF relationships. While stochastic processes were dominant at low dilution levels due to the high number of rare species, the deterministic processes became stronger at a higher dilution level because the microbial copiotrophs were selected during the regrowth phase and rare species were lost. From the view of microbial functional performances, specialized functions, commonly carried by rare species, are more likely to be impaired in DTE experiments while the broad functions seem to be less impacted due to the good performance of copiotrophs. Our study indicated that shifts in the prokaryotic community and its assembly processes induced by dilutions result in more complex BEF relationships in DTE experiments. Specialized microbial functions could be better used for defining BEF. Our findings may be helpful for future studies to design, explore, and interpret microbial BEF relationships using DTE.

Self-assembling a 1,4-dioxane-degrading consortium and identifying the key role of Shinella sp. through dilution-to-extinction and reculturing.

IMPORTANCE: Assembling a functional microbial consortium and identifying key degraders involved in the degradation of 1,4-dioxane are crucial for the design of synergistic consortia used in enhancing the bioremediation of 1,4-dioxane-contaminated sites. However, due to the vast diversity of microbes, assembling a functional consortium and identifying novel degraders through a simple method remain a challenge. In this study, we reassembled 1,4-dioxane-degrading microbial consortia using a simple and easy-to-operate method by combining dilution-to-extinction and reculture techniques. We combined differential analysis of community structure and metabolic function and confirmed that Shinella species have a stronger 1,4-dioxane degradation ability than Xanthobacter species in the enriched consortium. In addition, a new dioxane-degrading bacterium was isolated, Shinella yambaruensis, which verified our findings. These results demonstrate that DTE and reculture techniques can be used beyond diversity reduction to assemble functional microbial communities, particularly to identify key degraders in contaminant-degrading consortia.

A targeted liquid cultivation method for previously uncultured non-colony forming microbes.

A large number of microbes are not able to form colonies using agar-plating methods, which is one of the reasons that cultivation based on solid media leaves the majority of microbial diversity in the environment inaccessible. We developed a new Non-Colony-Forming Liquid Cultivation method (NCFLC) that can selectively isolate non-colony-forming microbes that exclusively grow in liquid culture. The NCFLC method involves physically separating cells using dilution-to-extinction (DTE) cultivation and then selecting those that could not grow on a solid medium. The NCFLC was applied to marine samples from a coastal intertidal zone and soil samples from a forest area, and the results were compared with those from the standard direct plating method (SDP). The NCFLC yielded fastidious bacteria from marine samples such as Acidobacteriota, Epsilonproteobacteria, Oligoflexia, and Verrucomicrobiota. Furthermore, 62% of the isolated strains were potential new species, whereas only 10% were novel species from SDP. From soil samples, isolates belonging to Acidobacteriota and Armatimonadota (which are known as rare species among identified isolates) were exclusively isolated by NCFLC. Colony formation capabilities of isolates cultivated by NCFLC were tested using solid agar plates, among which approximately one-third of the isolates were non-colony-forming, approximately half-formed micro-colonies, and only a minority could form ordinary size colonies. This indicates that the majority of the strains cultivated by NCFLC were previously uncultured microbial species unavailable using the SDP method. The NCFCL method described here can serve as a new approach to accessing the hidden microbial dark matter.

Does a decrease in microbial biomass alter mycorrhizal attributes and soil quality indicators in coal mining areas under revegetation process?

Losses of microbial diversity in degraded ecosystems still have obscure consequences, especially when considering the interaction between arbuscular mycorrhizal fungi (AMF) and soil bacteria. This study investigates the effect of decreasing microbial biomass on mycorrhizal attributes and soil quality indicators. The dilution-to-extinction approach was applied in microcosms to search for associations among bacterial diversity, mycorrhizal attributes, and soil quality indicators. The experiment was conducted with four soil treatments (undiluted control 100 = D0, 10-3 = D3, 10-6 = D6, and 10-9 = D9) from a short-term (two years = 2Y) and a long-term (15 years = 15Y) coal mine revegetation area. Microcosms were inoculated with 300 spores of Acaulospora colombiana, Gigaspora albida, and Claroideoglomus etunicatum with millet as the host plant. Results included the total number of AMF spores, mycorrhizal colonization, soil aggregation, glomalin, fluorescein diacetate hydrolysis (FDA), basal soil respiration, microbial biomass, and soil bacterial microbiome. Larger differences were observed between areas than between dilution treatments within the sampling area. Attributes that presented differences in the dilutions compared to D0 2Y samples were mycorrhizal colonization (D0 = 85% and D9 = 43.3%), FDA (D0 = 77.2% and D9 = 55.5%), extractable glomalin-related soil protein (D0 = 0.09 and D9 = 0.11) and bacterial diversity (D0 = 7.3 and D6 = 5.3). D0 15Y samples presented differences in microbial biomass nitrogen (D0: 232.0) and bacterial diversity (D0: 7.9, D9: 5.6) compared to the dilutions. Bacterial microbiome present in the D0 samples formed distinct clusters as to other samples and correlated with soil aggregation and basal respiration attributes. Results suggest that AMF inoculation and dilution-to-extinction did not affect soil quality indicators preeminently, but the bacterial community is affected and can influence the process of environmental revegetation. A long-term revegetation period is substantial to improve quality indicators and establish the diversity of microorganisms and consequently revegetation in areas impacted by coal mining.

Cultivation of Dominant Freshwater Bacterioplankton Lineages Using a High-Throughput Dilution-to-Extinction Culturing Approach Over a 1-Year Period.

Although many culture-independent molecular analyses have elucidated a great diversity of freshwater bacterioplankton, the ecophysiological characteristics of several abundant freshwater bacterial groups are largely unknown due to the scarcity of cultured representatives. Therefore, a high-throughput dilution-to-extinction culturing (HTC) approach was implemented herein to enable the culture of these bacterioplankton lineages using water samples collected at various seasons and depths from Lake Soyang, an oligotrophic reservoir located in South Korea. Some predominant freshwater bacteria have been isolated from Lake Soyang via HTC (e.g., the acI lineage); however, large-scale HTC studies encompassing different seasons and water depths have not been documented yet. In this HTC approach, bacterial growth was detected in 14% of 5,376 inoculated wells. Further, phylogenetic analyses of 16S rRNA genes from a total of 605 putatively axenic bacterial cultures indicated that the HTC isolates were largely composed of Actinobacteria, Bacteroidetes, Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, and Verrucomicrobia. Importantly, the isolates were distributed across diverse taxa including phylogenetic lineages that are widely known cosmopolitan and representative freshwater bacterial groups such as the acI, acIV, LD28, FukuN57, MNG9, and TRA3-20 lineages. However, some abundant bacterial groups including the LD12 lineage, Chloroflexi, and Acidobacteria could not be domesticated. Among the 71 taxonomic groups in the HTC isolates, representative strains of 47 groups could either form colonies on agar plates or be revived from frozen glycerol stocks. Additionally, season and water depth significantly affected bacterial community structure, as demonstrated by 16S rRNA gene amplicon sequencing analyses. Therefore, our study successfully implemented a dilution-to-extinction cultivation strategy to cultivate previously uncultured or underrepresented freshwater bacterial groups, thus expanding the basis for future multi-omic studies.

Dilution-to-Extinction Platform for the Isolation of Marine Bacteria-Producing Antitumor Compounds.

Gram-negative marine bacteria are an underexplored source of new chemical entities for a wide range of applications. Even though, some have shown a high antitumor activity. This chapter describes an isolation and screening protocol based on the Dilution-to-Extinction approach coupled with an antiproliferative test oriented to the discovery of new cytotoxic compounds synthesized by marine bacteria. In addition to the discovery of new bioactive secondary metabolites, this protocol provides a high-throughput isolation and screening platform for discarding no bioactive strains during the first steps of the drug discovery process.

Assessing the Diversity and Metabolic Potential of Psychrotolerant Arsenic-Metabolizing Microorganisms From a Subarctic Peatland Used for Treatment of Mining-Affected Waters by Culture-Dependent and -Independent Techniques.

Arsenic contamination in water by natural causes or industrial activities is a major environmental concern, and treatment of contaminated waters is needed to protect water resources and minimize the risk for human health. In mining environments, treatment peatlands are used in the polishing phase of water treatment to remove arsenic (among other contaminants), and peat microorganisms play a crucial role in arsenic removal. The present study assessed culture-independent diversity obtained through metagenomic and metatranscriptomic sequencing and culture-dependent diversity obtained by isolating psychrotolerant arsenic-tolerant, arsenite-oxidizing, and arsenate-respiring microorganisms from a peatland treating mine effluent waters of a gold mine in Finnish Lapland using a dilution-to-extinction technique. Low diversity enrichments obtained after several transfers were dominated by the genera Pseudomonas, Polaromonas, Aeromonas, Brevundimonas, Ancylobacter, and Rhodoferax. Even though maximal growth and physiological activity (i.e., arsenite oxidation or arsenate reduction) were observed at temperatures between 20 and 28°C, most enrichments also showed substantial growth/activity at 2-5°C, indicating the successful enrichments of psychrotolerant microorganisms. After additional purification, eight arsenic-tolerant, five arsenite-oxidizing, and three arsenate-respiring strains were obtained in pure culture and identified as Pseudomonas, Rhodococcus, Microbacterium, and Cadophora. Some of the enriched and isolated genera are not known to metabolize arsenic, and valuable insights on arsenic turnover pathways may be gained by their further characterization. Comparison with phylogenetic and functional data from the metagenome indicated that the enriched and isolated strains did not belong to the most abundant genera, indicating that culture-dependent and -independent methods capture different fractions of the microbial community involved in arsenic turnover. Rare biosphere microorganisms that are present in low abundance often play an important role in ecosystem functioning, and the enriched/isolated strains might thus contribute substantially to arsenic turnover in the treatment peatland. Psychrotolerant pure cultures of arsenic-metabolizing microorganisms from peatlands are needed to close the knowledge gaps pertaining to microbial arsenic turnover in peatlands located in cold climate regions, and the isolates and enrichments obtained in this study are a good starting point to establish model systems. Improved understanding of their metabolism could moreover lead to their use in biotechnological applications intended for bioremediation of arsenic-contaminated waters.

High-throughput cultivation based on dilution-to-extinction with catalase supplementation and a case study of cultivating acI bacteria from Lake Soyang.

Multi-omics approaches, including metagenomics and single-cell amplified genomics, have revolutionized our understanding of the hidden diversity and function of microbes in nature. Even in the omics age, cultivation is an essential discipline in microbial ecology since microbial cultures are necessary to assess the validity of an in silico prediction about the microbial metabolism and to isolate viruses infecting bacteria and archaea. However, the ecophysiological characteristics of predominant freshwater bacterial lineages remain largely unknown due to the scarcity of cultured representatives. In an ongoing effort to cultivate the uncultured majority of freshwater bacteria, the most abundant freshwater Actinobacteria acI clade has recently been cultivated from Lake Soyang through catalase-supplemented high-throughput cultivation based on dilution-to-extinction. This method involves physical isolation of target microbes from mixed populations, culture media simulating natural habitats, and removal of toxic compounds. In this protocol, we describe detailed procedures for isolating freshwater oligotrophic microbes, as well as the essence of the dilution-to-extinction culturing. As a case study employing the catalase-supplemented dilution-to-extinction protocol, we also report a cultivation trial using a water sample collected from Lake Soyang. Of the 480 cultivation wells inoculated with a single lake-water sample, 75 new acI strains belonging to 8 acI tribes (acI-A1, A2, A4, A5, A6, A7, B1, B4, C1, and C2) were cultivated, and each representative strain per subclade could be revived from glycerol stocks. These cultivation results demonstrate that the protocol described in this study is efficient in isolating freshwater bacterioplankton harboring streamlined genomes.

Construction of Simplified Microbial Consortia to Degrade Recalcitrant Materials Based on Enrichment and Dilution-to-Extinction Cultures.

The capacity of microbes to degrade recalcitrant materials has been extensively explored for environmental remediation and industrial production. Significant achievements have been made with single strains, but focus is now going toward the use of microbial consortia owning to their functional stability and efficiency. However, assembly of simplified microbial consortia (SMC) from complex environmental communities is still far from trivial due to large diversity and the effect of biotic interactions. Here we propose a strategy, based on enrichment and dilution-to-extinction cultures, to construct SMC with reduced diversity for degradation of keratinous materials. Serial dilutions were performed on a keratinolytic microbial consortium pre-enriched from a soil sample, monitoring the dilution effect on community growth and enzymatic activities. An appropriate dilution regime (10-9) was selected to construct a SMC library from the enriched microbial consortium. Further sequencing analysis and keratinolytic activity assays demonstrated that obtained SMC displayed actual reduced microbial diversity, together with various taxonomic composition, and biodegradation capabilities. More importantly, several SMC possessed equivalent levels of keratinolytic efficiency compared to the initial consortium, showing that simplification can be achieved without loss of function and efficiency. This methodology is also applicable to other types of recalcitrant material degradation involving microbial consortia, thus considerably broadening its application scope.