skip to main content


Title: Characterization of microbial associations with methanotrophic archaea and sulfate-reducing bacteria through statistical comparison of nested Magneto-FISH enrichments

Methane seep systems along continental margins host diverse and dynamic microbial assemblages, sustained in large part through the microbially mediated process of sulfate-coupled Anaerobic Oxidation of Methane (AOM). This methanotrophic metabolism has been linked to consortia of anaerobic methane-oxidizing archaea (ANME) and sulfate-reducing bacteria (SRB). These two groups are the focus of numerous studies; however, less is known about the wide diversity of other seep associated microorganisms. We selected a hierarchical set of FISH probes targeting a range ofDeltaproteobacteriadiversity. Using the Magneto-FISH enrichment technique, we then magnetically captured CARD-FISH hybridized cells and their physically associated microorganisms from a methane seep sediment incubation. DNA from nested Magneto-FISH experiments was analyzed using Illumina tag 16S rRNA gene sequencing (iTag). Enrichment success and potential bias with iTag was evaluated in the context of full-length 16S rRNA gene clone libraries, CARD-FISH, functional gene clone libraries, and iTag mock communities. We determined commonly used Earth Microbiome Project (EMP) iTAG primers introduced bias in some common methane seep microbial taxa that reduced the ability to directly compare OTU relative abundances within a sample, but comparison of relative abundances between samples (in nearly all cases) and whole community-based analyses were robust. The iTag dataset was subjected to statistical co-occurrence measures of the most abundant OTUs to determine which taxa in this dataset were most correlated across all samples. Many non-canonical microbial partnerships were statistically significant in our co-occurrence network analysis, most of which were not recovered with conventional clone library sequencing, demonstrating the utility of combining Magneto-FISH and iTag sequencing methods for hypothesis generation of associations within complex microbial communities. Network analysis pointed to many co-occurrences containing putatively heterotrophic, candidate phyla such as OD1,Atribacteria, MBG-B, and Hyd24-12 and the potential for complex sulfur cycling involvingEpsilon-,Delta-, andGammaproteobacteriain methane seep ecosystems.

 
more » « less
NSF-PAR ID:
10016042
Author(s) / Creator(s):
 ;  ;  
Publisher / Repository:
PeerJ
Date Published:
Journal Name:
PeerJ
Volume:
4
ISSN:
2167-8359
Page Range / eLocation ID:
e1913
Format(s):
Medium: X
Sponsoring Org:
National Science Foundation
More Like this
  1. Abstract

    Documenting anaerobic microbial metabolisms in hypersaline perennially ice‐covered lakes in Antarctica further refines the environmental limits to life and may reveal rare biogeochemical mechanisms and/or novel microbial catalysts of elemental cycling. We assessed rates of sulfate reduction, methanogenesis, and anaerobic oxidation of methane using radiotracers and generated 16S rRNA gene libraries from the microbial communities inhabiting the deep calcium‐chloride‐rich brine and sediments of Lake Vanda, McMurdo Dry Valleys, Antarctica. Sulfate reduction rates were observed in surface sediments but not in the brine overlying the sediments. Methane formation through the methylotrophic, acetoclastic, and hydrogenotrophic pathways was quantified using14C‐labeled methylamine, acetate, and CO2, respectively, and methanogenesis was detected in both the brine and the sediments. Hydrogenotrophic methanogenesis rates were the highest of all substrates tested in the sediments, while methylotrophic methanogenesis was highest in the brines. Anaerobic oxidation of methane was below the limit of detection in both the brines and sediments. The major taxa ofBacteriaandArchaeadetected were most similar to organisms previously observed in hypersaline environments and included examples related to known sulfate‐reducing bacteria other thanDeltaproteobacteria(surprisingly, sulfate‐reducingDeltaproteobacteriawere not observed in this study), and both methanogenic and methanotrophicArchaea. These data indicate an active microbial community in the anoxic brine of Lake Vanda that while similar in terms of community structure and metabolism to other brine habitats, is uniquely evolved to survive in this extreme environment.

     
    more » « less
  2. Abstract

    Deep‐sea methane seeps are amongst the most biologically productive environments on Earth and are often characterised by stable, low oxygen concentrations and microbial communities that couple the anaerobic oxidation of methane to sulfate reduction or iron reduction in the underlying sediment. At these sites, ferrous iron (Fe2+) can be produced by organoclastic iron reduction, methanotrophic‐coupled iron reduction, or through the abiotic reduction by sulfide produced by the abundant sulfate‐reducing bacteria at these sites. The prevalence of Fe2+in the anoxic sediments, as well as the availability of oxygen in the overlying water, suggests that seeps could also harbour communities of iron‐oxidising microbes. However, it is unclear to what extent Fe2+remains bioavailable and in solution given that the abiotic reaction between sulfide and ferrous iron is often assumed to scavenge all ferrous iron as insoluble iron sulfides and pyrite. Accordingly, we searched the sea floor at methane seeps along the Cascadia Margin for microaerobic, neutrophilic iron‐oxidising bacteria, operating under the reasoning that if iron‐oxidising bacteria could be isolated from these environments, it could indicate that porewater Fe2+can persist is long enough for biology to outcompete pyritisation. We found that the presence of sulfate in our enrichment media muted any obvious microbially‐driven iron oxidation with most iron being precipitated as iron sulfides. Transfer of enrichment cultures to sulfate‐depleted media led to dynamic iron redox cycling relative to abiotic controls and sulfate‐containing cultures, and demonstrated the capacity for biogenic iron (oxyhydr)oxides from a methane seep‐derived community. 16S rRNA analyses revealed that removing sulfate drastically reduced the diversity of enrichment cultures and caused a general shift from a Gammaproteobacteria‐domainated ecosystem to one dominated byRhodobacteraceae(Alphaproteobacteria). Our data suggest that, in most cases, sulfur cycling may restrict the biological “ferrous wheel” in contemporary environments through a combination of the sulfur‐adapted sediment‐dwelling ecosystems and the abiotic reactions they influence.

     
    more » « less
  3. Abstract

    The processes that lead to the precipitation of authigenic calcium phosphate minerals in certain marine pore waters remain poorly understood. Phosphogenesis occurs in sediments beneath some oceanic upwelling zones that harbor polyphosphate‐accumulating bacteria. These bacteria are believed to concentrate phosphate in sediment pore waters, creating supersaturated conditions with respect to apatite precursors. However, the relationship between microbes and phosphorite formation is not fully resolved. To further study this association, we examined microbial community data generated from two sources: sediment cores recovered from the shelf of the Benguela upwelling region where phosphorites are currently forming, andDNApreserved within phosphoclasts recovered from a phosphorite deposit along the Benguela shelf.iTag and clone library sequencing of the 16SrRNAgene showed that many of our sediment‐hosted communities shared large numbers of phylotypes with one another, and that the same metabolic guilds were represented at localities across the shelf. Sulfate‐reducing bacteria and sulfur‐oxidizing bacteria were particularly abundant in our datasets, as were phylotypes that are known to carry out nitrification and the anaerobic oxidation of ammonium. TheDNAextracted from phosphoclasts contained the signature of a distinct microbial community from those observed in the modern sediments. While some aspects of the modern and phosphoclast communities were similar, we observed both an enrichment of certain common microbial classes found in the modern phosphogenic sediments and a relative depletion of others. The phosphoclast‐associatedDNAcould represent a relict signature of one or more microbial assemblages that were present when the apatite or its precursors precipitated. While these taxa may or may not have contributed to the precipitation of the apatite that now hosts their genetic remains, several groups represented in the phosphoclast extract dataset have the genetic potential to metabolize polyphosphate, and perhaps modulate phosphate concentrations in pore waters where carbonate fluorapatite (or its precursors) are known to be precipitating.

     
    more » « less
  4. Abstract

    Archaeal anaerobic methanotrophs (“ANME”) and sulfate-reducing Deltaproteobacteria (“SRB”) form symbiotic multicellular consortia capable of anaerobic methane oxidation (AOM), and in so doing modulate methane flux from marine sediments. The specificity with which ANME associate with particular SRB partners in situ, however, is poorly understood. To characterize partnership specificity in ANME-SRB consortia, we applied the correlation inference technique SparCC to 310 16S rRNA amplicon libraries prepared from Costa Rica seep sediment samples, uncovering a strong positive correlation between ANME-2b and members of a clade of Deltaproteobacteria we termed SEEP-SRB1g. We confirmed this association by examining 16S rRNA diversity in individual ANME-SRB consortia sorted using flow cytometry and by imaging ANME-SRB consortia with fluorescence in situ hybridization (FISH) microscopy using newly-designed probes targeting the SEEP-SRB1g clade. Analysis of genome bins belonging to SEEP-SRB1g revealed the presence of a complete nifHDK operon required for diazotrophy, unusual in published genomes of ANME-associated SRB. Active expression of nifH in SEEP-SRB1g within ANME-2b—SEEP-SRB1g consortia was then demonstrated by microscopy using hybridization chain reaction (HCR-) FISH targeting nifH transcripts and diazotrophic activity was documented by FISH-nanoSIMS experiments. NanoSIMS analysis of ANME-2b—SEEP-SRB1g consortia incubated with a headspace containing CH4 and 15N2 revealed differences in cellular 15N-enrichment between the two partners that varied between individual consortia, with SEEP-SRB1g cells enriched in 15N relative to ANME-2b in one consortium and the opposite pattern observed in others, indicating both ANME-2b and SEEP-SRB1g are capable of nitrogen fixation, but with consortium-specific variation in whether the archaea or bacterial partner is the dominant diazotroph.

     
    more » « less
  5. As marine sediments are buried, microbial communities transition from sulfate-reduction to methane-production after sulfate is depleted. When this biogenic methane diffuses into the overlying sulfate-rich sediments, it forms a sulfate-methane transition zone (SMTZ) because sulfate reducers deplete hydrogen concentrations and make hydrogenotrophic methanogenesis exergonic in the reverse direction, a process called the anaerobic oxidation of methane (AOM). Microbial participation in these processes is often inferred from geochemistry, genes, and gene expression changes with sediment depth, using sedimentation rates to convert depth to time. Less is known about how natural sediments transition through these geochemical states transition in real-time. We examined 16S rRNA gene amplicon libraries and metatranscriptomes in microcosms of anoxic sediment from the White Oak River estuary, NC, with three destructively sampled replicates with methane added (586-day incubations) and three re-sampled un-amended replicates (895-day incubations). Sulfate dropped to a low value (∼0.3 mM) on similar days for both experiments (312 and 320 days, respectively), followed by a peak in hydrogen, intermittent increases in methane-cycling archaea starting on days 375 and 362 (mostly Methanolinea spp. and Methanosaeta spp., and Methanococcoides sp. ANME-3), and a methane peak 1 month later. However, methane δ 13 C values only show net methanogenesis 6 months after methane-cycling archaea increase and 4 months after the methane peak, when sulfate is consistently below 0.1 mM and hydrogen increases to a stable 0.61 ± 0.13 nM (days 553–586, n = 9). Sulfate-reducing bacteria (mostly Desulfatiglans spp. and Desulfosarcina sp. SEEP-SRB1) increase in relative abundance only during this period of net methane production, suggesting syntrophy with methanogens in the absence of sulfate. The transition from sulfate reduction to methane production in marine sediments occurs through a prolonged period of methane-cycling by methanogens at low sulfate concentrations, and steady growth of sulfate reducers along with methanogens after sulfate is depleted. 
    more » « less