Mixed Microbial Communities Research Articles

Conversion of tomato waste into a poly(3-hydroxybutyrate-co-3-hydroxyvalerate-co-3-hydroxyhexanoate) by a mixed microbial community

Tomato waste (TW) originating from tomato processing is produced in large quantities and has a low cost, presenting as an interesting feedstock for biotechnological valorization into value-added bioproducts. This study explored the utilization of TW as feedstock to produce polyhydroxyalkanoates (PHA) by a mixed microbial community. The bioprocess encompassed acidogenic fermentation of TW with a maximum yield of 0.22 ± 0.05 gFP/gVS. A maximum PHA content of 66.8 ± 11.0wt.% was reached in the accumulation assays, with a yield of 0.75 ± 0.10 CmmolPHA/CmmolFP. The biopolymer was composed of 3-hydroxybutyrate (3HB), hydroxyvalerate (3HV) and hydroxyhexanoate (3HHx) with an average composition of 82:11:7 (3HB:3HV:3HHx, molar basis) and similar mechanical and thermal properties to other 3HB:3HV:3HHx terpolymers and comparable to high density polyethylene and polypropylene. These findings demonstrated that TW can be used to efficiently produce a PHA terpolymer with properties that render it suitable for replacing petrochemical-derived plastics.

Mixed and membrane-separated culturing of synthetic cyanobacteria-yeast consortia reveals metabolic cross-talk mimicking natural cyanolichens

Metabolite exchange mediates crucial interactions in microbial communities, significantly impacting global carbon and nitrogen cycling. Understanding these chemically-mediated interactions is essential for elucidating natural community functions and developing engineered synthetic communities. This study investigated membrane-separated bioreactors (mBRs) as a novel tool to identify transient metabolites and their producers/consumers in mixed microbial communities. We compared three co-culture methods (direct mixed, 2-chamber mBR, and 3-chamber mBR) to grow a synthetic binary community of the cyanobacterium Synechococcus elongatus PCC 7942 and the fungus Rhodotorula toruloides NBRC 0880, as well as axenic S. elongatus. Despite not being natural lichen constituents, these organisms exhibited interactions resembling those in cyanolichens. S. elongatus fixed CO2 into sugars as the primary shared metabolite, while R. toruloides secreted various biochemicals, predominantly sugar alcohols, mirroring the metabolite exchange observed in natural lichens. The mBR systems successfully captured metabolite gradients and revealed rapidly consumed compounds, including TCA cycle intermediates and amino acids. Our approach demonstrated that the 2-chamber mBR optimally balanced metabolite exchange and growth dynamics. This study provides insights into cross-species metabolic interactions and presents a valuable tool for investigating and engineering synthetic microbial communities with potential applications in biotechnology and environmental science.

Report on the Swiss-Colombian workshop 2024: “Metagenomics data analysis of mixed microbial communities.”

This is a report (no abstract needed).

Metagenome from ACT-3/CF: an anaerobic chloroform-degrading microbial community.

Here, we present the metagenome of an anaerobic chloroform respiring mixed microbial community used for bioremediation. Our objective was to obtain draft genomes of key microorganisms in the culture.

The bio-cathode of microbial fuel cells systems: Performance analysis under different microbial community conditions

This study aims to investigate the conversion of different microbial community compositions in the cathode chamber in a dual-chamber bioelectrochemical system. A denitrifying bacterial community, or sludge, was enriched for denitrification by microbial consortia and subsequently acclimatized within the cathodic chamber of microbial fuel cells, either within a mixed microbial community sludge microbial fuel cell (M-MFC) or a pure culture of microorganisms in a microbial fuel cell (P-MFC). The bioelectrochemical systems were treated with different nitrate concentrations in the cathodic chamber: the MFC with low concentration nitrate (MFC1, 25 mg/L nitrate; MFC2 50 mg/L nitrate), the MFC with medium concentration nitrate(MFC3, 75 mg/L nitrate; MFC4 100 mg/L nitrate), and MFC with high concentration nitrate (MFC5, 125 mg/L nitrate; MFC6 150 mg/L nitrate; MFC7 175 mg/L nitrate), and the initial COD in the anodic chamber was based on demand(C to N ratio is 5). M-MFC exhibited better-electrogenerated capability than P-MFC. However, P-MFC exhibited better nitrogen removal performance compared to M-MFC. The best performance was attained by both types of MFCs when the nitrate content was 100 mg/L. The maximum output voltages in M-MFC4 and P-MFC4 were 449 mV and 171 mV, respectively. The average conversion rate of M-MFC4 and P-MFC4 were 2.9 mg*L−1 *h−1 and 10.5 mg*L−1 *h−1, respectively. The analysis of the microbial community of two treated MFCs in the cathode chamber in the optimal state indicated that non-electrode denitrification predominantly occurs in P-MFC. The cathode chambers were mostly populated by denitrifying microorganisms. The dominant species in M-MFC were Azonexus, Zoogloea, Ignavibacterium, and Pirellula, and the dominant species in P-MFC were Comamonas, Aeromonas, and Acidovorax. The species was assessed by qPCR analysis, and the results validated the use of a bacterial consortium for domestication as a more suitable method to improve the stability and performance of the MFCs.

Unexpected methane oxidation acceleration by two species of bacteria from the rainwater promoting Methylomonas sp. in the soil

Altering the function of a mixed microbial community through specific microorganisms is a challenge. In this study, we constructed different co-culture systems to not only elucidate the mechanisms of microbial interactions but also explain the significance to the environment. Metagenomic analysis indicated that Methylomonas sp. was the main contributor to aerobic methane oxidation in the soils. Co-culture experiments showed that the introduction of Methylophilus sp. and Rhizobium sp., isolated from rainwater, significantly enhanced aerobic methane oxidation rates, on an hourly scale, representing maximum increases of 50.49 % and 24.62 %, respectively. Furthermore, metatranscriptomic analysis revealed that Methylophilus sp. and Rhizobium sp. could recolonize in the soil with abundance increasing to 1.23 % and 0.02 %. The genes of hxlA (methanol metabolism gene) and cobC (cobalamin synthesis gene) were upregulated, leading to the upregulation of methane oxidation genes such as pmoA and mmoX. The findings uncover the mechanisms of microbial interactions that improve aerobic methane oxidation and highlight the importance of these interactions in maintaining carbon cycle equilibrium in wetlands.

First evidence for temperature’s influence on the enrichment, assembly, and activity of polyhydroxyalkanoate-synthesizing mixed microbial communities

Polyhydroxyalkanoates (PHA) are popular biopolymers due to their potential use as biodegradable thermoplastics. In this study, three aerobic sequencing batch reactors were operated identically except for their temperatures, which were set at 15 °C, 35 °C, and 48 °C. The reactors were subjected to a feast–famine feeding regime, where carbon sources are supplied intermittently, to enrich PHA-accumulating microbial consortia. The biomass was sampled for 16S rRNA gene amplicon sequencing of both DNA (during the enrichment phase) and cDNA (during the enrichment and accumulation phases). All temperatures yielded highly enriched PHA-accumulating consortia. Thermophilic communities were significantly less diverse than those at low or mesophilic temperatures. In particular, Thauera was highly adaptable, abundant, and active at all temperatures. Low temperatures resulted in reduced PHA production rates and yields. Analysis of the microbial community revealed a collapse of community diversity during low-temperature PHA accumulation, suggesting that the substrate dosing strategy was unsuccessful at low temperatures. This points to future possibilities for optimizing low-temperature PHA accumulation.

Anaerobic valorization of sewage sludge pretreated through hydrothermal carbonization: Volatile fatty acids and biomethane production

Hydrothermal carbonization (HTC) emerged as an effective technology for the treatment of various types of wet biomass and organic residues, including sewage sludge, offering the potential for sludge reduction and resource recovery. HTC pretreatment impact on downstream sludge fermentation is investigated. Results obtained at optimal conditions for HTC pretreatment (170 °C for 30 min) indicated that soluble carbon was significantly increased in the liquid fraction, enhancing feedstock availability for fermentation. Semi-continuous fermentation of HTC-treated sludge resulted in a stable process in which a mixed microbial community produced volatile fatty acids (VFAs) with longer chain acids content, acidification yield of 0.59 ± 0.05 g COD-VFA g−1 CODin and volumetric productivity of 1.6 ± 0.5 g COD-VFA L−1 d−1. Biomethane Potential tests evidenced high values for hydrochar. Overall, the HTC pretreatment enables improved conversion efficiencies, in the view of valorizing the liquid for VFA synthesis and the hydrochar for biomethane production.

Polyaniline-modified anode with mixed microbial community improved microbial fuel cell performance for power generation treating complex leather tannery wastewater

Polyaniline-modified anode with mixed microbial community improved microbial fuel cell performance for power generation treating complex leather tannery wastewater

Revealing the microbiome diversity and biocontrol potential of field Aedes ssp.: Implications for disease vector management.

The mosquito Aedes spp. holds important relevance for human and animal health, as it serves as a vector for transmitting multiple diseases, including dengue and Zika virus. The microbiome's impact on its host's health and fitness is well known. However, most studies on mosquito microbiomes have been conducted in laboratory settings. We explored the mixed microbial communities within Aedes spp., utilizing the 16S rRNA gene for diversity analysis and shotgun metagenomics for functional genomics. Our samples, which included Ae. aegypti and Ae. albopictus, spanned various developmental stages-eggs, larvae, and adults-gathered from five semiurban areas in Mexico. Our findings revealed a substantial diversity of 8,346 operational taxonomic units (OTUs), representing 967 bacterial genera and 126,366 annotated proteins. The host developmental stage was identified as the primary factor associated with variations in the microbiome composition. Subsequently, we searched for genes and species involved in mosquito biocontrol. Wolbachia accounted for 9.6% of the 16S gene sequences. We observed a high diversity (203 OTUs) of Wolbachia strains commonly associated with mosquitoes, such as wAlb, with a noticeable increase in abundance during the adult stages. Notably, we detected the presence of the cifA and cifB genes, which are associated with Wolbachia's cytoplasmic incompatibility, a biocontrol mechanism. Additionally, we identified 221 OTUs related to Bacillus, including strains linked to B. thuringiensis. Furthermore, we discovered multiple genes encoding insecticidal toxins, such as Cry, Mcf, Vip, and Vpp. Overall, our study contributes to the understanding of mosquito microbiome biodiversity and metabolic capabilities, which are essential for developing effective biocontrol strategies against this disease vector.

Identification of acetaminophen degrading microorganisms in mixed microbial communities using 13C-DNA stable isotope probing

The high frequency of acetaminophen (APAP) detection in water bodies around the world increases the risk to the global environment. A lot of work has concentrated on isolating and identifying the culturable APAP degraders for understanding of the microbiota and their ecophysiology, which greatly underrated the diversity of organisms. Here, 13C-DNA stable isotope probing (DNA-SIP) was applied for the first time to identify microorganisms involved in APAP metabolism under conditions close to in situ from three mixed microbial communities collected in lab-scale nitrification systems. Based on DNA-SIP, Achromobacter (oligotype ATAA), Alicycliphilus (oligotype GTTCG) and Thauera (oligotype GAAACTTTCA) were identified as the key APAP degrading bacteria in the floc sludge, granular sludge and biofilm reactors, respectively, with the relative abundance of 43.20%, 10.12% and 12.60%. Moreover, Leucobacter, Achromobacter, Alicycliphilus, Thauera, Microbacterium and Lactobacillus were found, for the first time, to be directly responsible for APAP biodegradation. However, Achromobacter, Lactobacillus and Alicycliphilus were linked to spread resistance genes (intI1, sul2, qacEdta1-02 and qacH-01). The results highlight that Acinetobacter, Leucobacter, Thauera and Microbacterium could be recommended as bioaugmentation strains for the treatment of wastewater contaminated with APAP. Ultimately, the information acquired in this study could add to the current knowledge on microorganisms degrading APAP and accelerate the implementation of biofortification processes to achieve APAP removal.

IMPACT OF STAPHYLOCOCCUS SPP. ON OCCURRENCE OF INFECTIOUS INFLAMMATORY PROCESSES IN SURGICAL PATIENTS: A BACTERIOLOGICAL STUDY

Introduction. Staphylococci are well-known pathogens associated with purulent-inflammatory processes at various body sites. However, the specific contributions of different microorganisms within microbial communities remain poorly understood. Notably, these diverse microbes can exhibit distinct antibiotic susceptibility profiles due to their varying taxonomic classifications.
 The purpose of this study is to analyze the results of bacteriological examination of pathological material taken from patients in surgical department and to assess the contribution of S. aureus and coagulase-negative staphylococci in the form of mono- and mixed infection in the cases of infectious pathology.
 Results and discussion. 123 samples were obtained from patients in the surgical department; cultures of Staphylococcus bacteria were isolated in 37 cases, which accounted for 30.1% of all studies. Staphylococcus aureus was identified in 18 cultures (14.6%), with 11 isolates (8.9%) found as single infections (monoculture) and 7 isolates (5.7%) identified within mixed microbial communities. Additionally, 19 isolates (15.4%) were identified as coagulase-negative staphylococci. These microbial communities comprised 2 to 4 different types of microorganisms.
 Conclusion. Staphylococcus spp. were isolated from 30.1% of patients in the surgical department. Among these isolates, Staphylococcus aureus was present in 38.9% of mixed microbial cultures, while coagulase-negative staphylococci were found in 33.3% of Staphylococcus isolations. Notably, S. aureus and coagulase-negative staphylococci were never co-isolated within the same mixed culture.

Heterogeneous lineage-specific arginine deiminase expression within dental microbiome species.

Tooth decay is the most common preventable chronic disease, affecting more than two billion people globally. The development of caries on teeth is primarily a consequence of acid production by cariogenic bacteria that inhabit the plaque microbiome. Other bacterial strains in the oral cavity may suppress or prevent tooth decay by producing ammonia as a byproduct of the arginine deiminase metabolic pathway, increasing the pH of the plaque biofilm. While the benefits of arginine metabolism on oral health have been extensively documented in specific bacterial groups, the prevalence and consistency of arginine deiminase system (ADS) activity among oral bacteria in a community context remain an open question. In the current study, we use a multi-omics approach to document the pervasiveness of the expression of the ADS operon in both health and disease to better understand the conditions in which ADS activity may prevent tooth decay.

Occurrence of Rhodococcus sp. RR1 prmA and Rhodococcus jostii RHA1 prmA across microbial communities and their enumeration during 1,4-dioxane biodegradation

1,4-Dioxane, a likely human carcinogen, is a co-contaminant at many chlorinated solvent contaminated sites. Conventional treatment technologies, such as carbon sorption or air stripping, are largely ineffective, and so many researchers have explored bioremediation for site clean-up. An important step towards this involves examining the occurrence of the functional genes associated with 1,4-dioxane biodegradation. The current research explored potential biomarkers for 1,4-dioxane in three mixed microbial communities (wetland sediment, agricultural soil, impacted site sediment) using monooxygenase targeted amplicon sequencing, followed by quantitative PCR (qPCR). A BLAST analysis of the sequencing data detected only two of the genes previously associated with 1,4-dioxane metabolism or co-metabolism, namely propane monooxygenase (prmA) from Rhodococcus jostii RHA1 and Rhodococcus sp. RR1. To investigate this further, qPCR primers and probes were designed, and the assays were used to enumerate prmA gene copies in the three communities. Gene copies of Rhodococcus RR1 prmA were detected in all three, while gene copies of Rhodococcus jostii RHA1 prmA were detected in two of the three sample types (except impacted site sediment). Further, there was a statistically significant increase in RR1 prmA gene copies in the microcosms inoculated with impacted site sediment following 1,4-dioxane biodegradation compared to the control microcosms (no 1,4-dioxane) or to the initial copy numbers before incubation. Overall, the results indicate the importance of Rhodococcus associated prmA, compared to other 1,4-dioxane degrading associated biomarkers, in three different microbial communities. Also, the newly designed qPCR assays provide a platform for others to investigate 1,4-dioxane biodegradation potential in mixed communities and should be of particular interest to those considering bioremediation as a potential 1,4-dioxane remediation approach.

MicroMPN: methods and software for high-throughput screening of microbe suppression in mixed populations.

We created a unified set of software and laboratory protocols for screening microbe libraries to assess the suppression of a pathogen in a mixed microbial community. Existing methods of fluorescent labeling were combined with the most probable number (MPN) assay in a microplate format to enumerate the reduction of a pathogenic soil microbe from complex soil matrices. This work provides a fluorescent expression vector available from Addgene, step-by-step laboratory protocols hosted by protocols.io, and MicroMPN, a command-line software for processing plate reader outputs. MicroMPN simplifies MPN estimation from 96- and 384-well microplates. The microplate screening assay is amenable to robotic automation with standard liquid handling robots, further reducing the hands-on processing time. This tool was designed to evaluate synthetic microbial communities for use as microbial inoculates or probiotics. The fluorMPN method is also useful for screening chemical and antimicrobial libraries for pathogen suppression in complex bacterial communities like soil.

Electrical current disrupts the electron transfer in defined consortia.

Improving methane production through electrical current application to anaerobic digesters has garnered interest in optimizing such microbial electrochemical technologies, with claims suggesting direct interspecies electron transfer (DIET) at the cathode enhances methane yield. However, previous studies with mixed microbial communities only reported interspecies interactions based on species co-occurrence at the cathode, lacking insight into how a poised cathode influences well-defined DIET-based partnerships. To address this, we investigated the impact of continuous and discontinuous exposure to a poised cathode (-0.7 V vs. standard hydrogen electrode) on a defined consortium of Geobacter metallireducens and Methanosarcina barkeri, known for their DIET capabilities. The physiology of DIET consortia exposed to electrical current was compared to that of unexposed consortia. In current-exposed incubations, overall metabolic activity and cell numbers for both partners declined. The consortium, receiving electrons from the poised cathode, accumulated acetate and hydrogen, with only 32% of the recovered electrons allocated to methane production. Discontinuous exposure intensified these detrimental effects. Conversely, unexposed control reactors efficiently converted ethanol to methane, transiently accumulating acetate and recovering 88% of electrons in methane. Our results demonstrate the overall detrimental effect of electrochemical stimulation on a DIET consortium. Besides, the data indicate that the presence of an alternative electron donor (cathode) hinders efficient electron retrieval by the methanogen from Geobacter, and induces catabolic repression of oxidative metabolism in Geobacter. This study emphasizes understanding specific DIET-based interactions to enhance methane production during electrical stimulation, providing insights for optimizing tailored interspecies partnerships in microbial electrochemical technologies.

Eukaryotic genomes from a global metagenomic data set illuminate trophic modes and biogeography of ocean plankton.

Single-celled eukaryotes play ecologically significant roles in the marine environment, yet fundamental questions about their biodiversity, ecological function, and interactions remain. Environmental sequencing enables researchers to document naturally occurring protistan communities, without culturing bias, yet metagenomic and metatranscriptomic sequencing approaches cannot separate individual species from communities. To more completely capture the genomic content of mixed protistan populations, we can create bins of sequences that represent the same organism (metagenome-assembled genomes [MAGs]). We developed the EukHeist pipeline, which automates the binning of population-level eukaryotic and prokaryotic genomes from metagenomic reads. We show exciting insight into what protistan communities are present and their trophic roles in the ocean. Scalable computational tools, like EukHeist, may accelerate the identification of meaningful genetic signatures from large data sets and complement researchers' efforts to leverage MAG databases for addressing ecological questions, resolving evolutionary relationships, and discovering potentially novel biodiversity.

Geobacter grbiciae—A New Electron Donor in the Formation of Co-Cultures via Direct Interspecies Electron Transfer

Geobacter grbiciae can grow via coupling oxidation of ethanol to the reduction of various forms of soluble Fe(III) and poorly crystalline Fe(III) oxide, suggesting that G. grbiciae can act as an electron-donor microbe for forming co-cultures through direct interspecies electron transfer (DIET). In this report, potential co-cultures through DIET of G. grbiciae and Methanosarcina barkeri 800, G. sulfurreducens Δhyb, or Methanospirillum hungatei, as electron-acceptor microbes, were examined. Co-cultures of G. grbiciae and G. sulfurreducens Δhyb were performed with ethanol as the sole electron-donor substance and fumarate as the electron-acceptor substance in the presence of granular activated carbon (GAC), magnetite, or polyester felt. The conditions for co-culturing G. grbiciae and M. barkeri 800 (or M. hungatei) were the same as those for G. grbiciae and G. sulfurreducens Δhyb, except fumarate was absent and different cultivation temperatures were used. All co-cultures were anaerobically cultivated. Samples were regularly withdrawn from the co-cultures to monitor methane, fumarate, and succinate via gas or high-performance liquid chromatography. G. grbiciae formed functional co-cultures with M. barkeri 800 in the presence of GAC or magnetite. No co-culture of G. grbiciae with the H2/formate-utilizing methanogen M. hungatei was observed. Additionally, G. grbiciae formed functional co-cultures with H2/formate-un-utilizing G. sulfurreducens Δhyb without the GAC or magnetite supplement. These findings indicate electron transfer between G. grbiciae and M. barkeri 800/G. sulfurreducens Δhyb is via DIET rather than H2/formate, confirming that G. grbiciae acts as an electron-donor microbe. Although the co-cultures of G. grbiciae and M. barkeri 800 syntrophically converted ethanol to methane through DIET, the conversion of propionate or butyrate to methane was not observed. These findings expand the range of microbes that can act as electron donors for interaction with other microbes through DIET. However, propionate and butyrate metabolism through DIET in mixed microbial communities with methane as a product requires further analysis. This study provides a framework for finding new electron-donor microbes.

Bi-directional regulation of electroactive microbial community using Au/antimicrobial peptide nanocomposite

The power generation and pollutants removal efficiencies of mixed electroactive microbial community rely on the effective electron transfer between outer membrane proteins of electroactive electricigens. However, the poor conductivity of protein and various non-electrogenic bacteria greatly hinder the electron transfer and effective contact between electricigens. Herein, we report a novel “bi-directional” microbial community regulation method that can selectively inhibit Gram-positive non-electrogenic bacteria and enhances the conductivity and electron transfer between electrogenic bacteria and the electrode in MFC by taking the advantages of the excellent conductive and bacteriostatic properties of Au/Nisin nanocomposite. The Au/Nisin stimulation endows microbial fuel cell (MFC) a high-efficiency functional community, which increases the power density and decolorization rate by 103 % and 33 %, respectively. The high conductivity, and selectivity and antimicrobial activity of the nanocomposite improve the electrochemical activity of the bioanode, hereby promoting the microbial extracellular electron transfer and increasing the proportion of electrogenic bacteria such as Proteobacteria, Bacteroidetes and Desulfovibrio in the biofilm. Our work successfully demonstrates that the construction of multifunctional metallic peptide nanocomposite can effectively regulate microbial community to improve the electrochemical performance of MFC.

Differences Between Heterotrophic and Nitrate-dependent Iron-oxidizing Microbial Communities in Bioreactor Sediment Treating Mine Wastewater

Nitrate-dependent iron oxidation (NDFO) is a novel mechanism for microbial bioremediation of metal and metalloid contaminants. During NDFO, microbes catalyze a redox reaction wherein nitrate is reduced to nitrite and nitrogen gas while Fe(II) is oxidized to solid Fe(III) hydroxide minerals. Metalloid contaminants such as selenium and arsenic have a propensity for adsorption to iron minerals produced during NDFO; some contaminants may also be concurrently bioreduced. A number of bacterial isolates have been shown to be capable of NDFO (e.g., Kappler et al. (2005), Kiskira et al. (2017)), but little work has been done to date characterizing mixed microbial communities performing NDFO. Some autotrophic communities have been characterized, with high relative abundances for strains of Gallionellaceae in both a freshwater sediment enrichment culture and an activated sludge culture (Blöthe and Roden 2009, Tian et al. 2020). In mixotrophic activated sludge cultures, the concentration of Fe(II) amendment was found to significantly impact microbial community composition; these cultures were fed with methanol in addition to Fe(II), and the dominant community members were Methyloversatilis and other methylotrophic strains (Liu et al. 2018). The work presented here examines microbial communities performing NDFO in the context of remediation, and in particular how differences between NDFO and heterotrophic communities may influence remediation effectiveness. This research characterizes and compares microbial communities performing NDFO versus heterotrophic denitrification during removal of selenium and nickel from mining wastewater. Sediment and influent water from a subsurface bioreactor treating mining wastewater were used to construct batch bioreactors, which were amended with selenium and nickel as well as either Fe(II) or methanol to investigate contaminant removal and microbial community composition in NDFO versus heterotrophic microbial communities. Both Fe(II) and methanol reactors removed total aqueous selenium to below the quantification limit, but Fe(II) reactors removed it more rapidly, likely due to adsorption of selenite. For nickel, removal to below the detection limit was achieved with methanol amendment, while Fe(II) amendment resulted in 42-95% removal. This was likely due to precipitation of nickel sulfide during sulfate reduction in methanol-amended reactors. DNA from the batch bioreactors will be sequenced and the results analyzed for differences among communities. Permutational multivariate analysis of variance and non-metric multidimensional scaling will be used to determine significant correlations of community composition with experimental variables, selenium and nickel removal, and NDFO (Roberts 2023, Kruskal 1964). Indicator species analyses (De Cáceres et al. 2010) will be applied to identify taxa found significantly more often (i.e., at a higher relative abundance) in one group of microbial communities than in any other group. The indicator species analysis may reveal whether there are groups of denitrifiers that predominate in NDFO conditions vs. groups that predominate during heterotrophic denitrification. The results of these microbial community analyses, in combination with the geochemical analyses, will improve our understanding of microbial communities performing NDFO in remediation environments.