Growth of sedimentary Bathyarchaeota on lignin as an energy source

Tiantian Yu; Weichao Wu; Wenyue Liang; Mark Alexander Lever; Kai-Uwe Hinrichs; Fengping Wang

doi:10.1073/pnas.1718854115

Edited by Edward F. DeLong, University of Hawaii at Manoa, Honolulu, HI, and approved April 16, 2018 (received for review October 30, 2017)

Significance

Marine sediment holds the largest organic carbon pool on earth, where microbial transformation of carbon is considered a key process of carbon cycling. Bathyarchaeota are among the most abundant and active groups of microorganisms in marine sediment. It has been suggested that Bathyarchaeota may play a globally important role in the carbon cycling in the marine environment through fermentation of complex organic substances, acetogenesis, and methanogenesis based on metagenome analysis. Here we provide several lines of converging evidence suggesting the bathyarchaeotal group Bathy-8 is able to grow with lignin as an energy source and bicarbonate as a carbon source. Consequently, members of the Bathyarchaeota are probably important, previously unrecognized degraders of lignin.

Abstract

Members of the archaeal phylum Bathyarchaeota are among the most abundant microorganisms on Earth. Although versatile metabolic capabilities such as acetogenesis, methanogenesis, and fermentation have been suggested for bathyarchaeotal members, no direct confirmation of these metabolic functions has been achieved through growth of Bathyarchaeota in the laboratory. Here we demonstrate, on the basis of gene-copy numbers and probing of archaeal lipids, the growth of Bathyarchaeota subgroup Bathy-8 in enrichments of estuarine sediments with the biopolymer lignin. Other organic substrates (casein, oleic acid, cellulose, and phenol) did not significantly stimulate growth of Bathyarchaeota. Meanwhile, putative bathyarchaeotal tetraether lipids incorporated ¹³C from ¹³C-bicarbonate only when added in concert with lignin. Our results are consistent with organoautotrophic growth of a bathyarchaeotal group with lignin as an energy source and bicarbonate as a carbon source and shed light into the cycling of one of Earth’s most abundant biopolymers in anoxic marine sediment.

The members of Bathyarchaeota (formerly referred to as the “Miscellaneous Crenarchaeotal Group”) (1, 2) are estimated to be among the most abundant microorganisms on the planet (3) and are particularly common in marine sediments (1, 4 ⇓⇓–7). The phylum Bathyarchaeota contains more than 19 subgroups/lineages with low intragroup similarities (3, 5, 8, 9), and its members have been suggested to play a globally important role in the breakdown of organic matter (10) through fermentation (11), acetogenesis (3), and methanogenesis (12). However, due to the lack of pure culture isolates and difficulty of obtaining enrichments of Bathyarchaeota (13), the metabolic properties and capabilities of these abundant and widespread Archaea have so far been inferred mostly from single-cell genomic and metagenomic analyses (2, 3, 11, 12, 14).

The first metabolic insights into Bathyarchaeota came from organic-rich sub-seafloor sediments of the Peru Margin, where these Archaea were inferred to be assimilating sedimentary organic carbon based on the δ¹³C-isotopic compositions of intact polar lipids (10). The organoheterotrophic physiology of Bathyarchaeota was further supported through analyses of bathyarchaeotal genomes by single-cell genome and metagenome analyses, and genes encoding enzymes for the degradation, transport, and utilization of detrital proteins, aromatic compounds, and plant-derived carbohydrates were identified (2, 11, 14). Peptidase activity in the sediments was measured, and the gene of an extracellular peptidase from Bathyarchaeota was expressed in vitro and characterized, supporting the inferred capacity of Bathyarchaeota to degrade proteins (11, 15). Meanwhile, stable-isotope probing experiments indicated that members of two subgroups assimilated several organic substrates, including acetate, glycine, urea, lipids, and complex mixtures of organic growth substrates, while showing no significant incorporation of carbon from proteins (16). On the other hand, acetogenesis from H₂/CO₂ has been proposed for some lineages of Bathyarchaeota (3). The reductive acetyl-CoA pathway for carbon fixation has been identified in most of the obtained bathyarchaeotal genomes (3, 12, 14). Among these, members of the bathyarchaeotal subgroups Bathy-3 and Bathy-8 have been suggested to be capable of methanogenesis (12). All the above studies indicate great versatility in the metabolic potentials of Bathyarchaeota, with some lineages possibly being capable of utilizing both organic and inorganic carbon compounds for biosynthesis and energy production. However, no direct proof of carbon or energy sources has so far been obtained for Bathyarchaeota based on laboratory experiments.

Here we report our efforts to enrich Bathyarchaeota from marine sediment in the laboratory by setting up a series of enrichments with diverse organic compound classes, including lipids (oleic acid), proteins (casein), aromatic monomers (phenol), aromatic polymers (lignin), and structural carbohydrates (cellulose). The addition of lignin stimulated the growth of Bathyarchaeota affiliated with the Bathy-8 subgroup. During growth on lignin, incorporation of inorganic carbon (IC) into archaeal lipids was demonstrated. This study presents an in vitro enrichment of Bathyarchaeota and suggests the organoautotrophic metabolism of members of this phylum, which likely plays an important role in the carbon cycle in marine sediments.

Results

Enrichments Set-Up.

Numerous enrichment cultures from Dayangshan estuarine sediments of the northern East China Sea (Fig. S1) were set up by adding diverse organic compound classes, i.e., the long-chain fatty acid oleic acid, the protein casein, the aromatic monomer phenol, the phenolic polymer lignin, and the polymeric carbohydrate cellulose (Materials and Methods). Sediment slurries without the addition of organic compounds were used as controls. Changes in the abundances of Bacteria, Archaea, and Bathyarchaeota were then monitored by qPCR using universal bacterial and archaeal 16S rRNA gene primers and Bathyarchaeota-specific 16S rRNA gene primers. The organic compounds were initially added at concentrations of 50 mg/L and were increased to 500 mg/L after 3.5- and 6-mo incubation.

Treatment responses to substrate additions and controls are shown in Fig. 1. In the control samples, the abundance of Bacteria, Archaea, and Bathyarchaeota showed only small fluctuations over time. Casein initiated the strongest growth stimulation in total Bacteria and Archaea, with an increase in bacterial and archaeal 16S rRNA gene copies of about three and nine times at 6-mo incubation (t₆), respectively; bathyarchaeotal gene copies increased only slightly in response to casein. Cellulose stimulated growth of Bacteria after 3.5 mo incubation (t_3.5) but had no influence on archaeal or bathyarchaeotal gene copies. Phenol and oleic acid had no obvious influence on the gene copies of Bacteria, Archaea, or Bathyarchaeota. Lignin showed little influence on the growth of Bacteria but significantly stimulated the growth of Archaea, particularly Bathyarchaeota. Total archaeal gene-copy numbers increased by about two and three times at t₆ and 11-mo incubation (t₁₁), while those of Bathyarchaeota climbed to more than 10 times at t₁₁ compared with the original sample. Thus, strong growth of Bathyarchaeota was achieved only in response to lignin addition. Lignin degradation was monitored by the decrease in the concentrations of total dissolved phenolic compounds after incubation. There were ∼102 mg/L phenolic compounds in the culture when 500 mg/L lignin was added, which decreased to ∼25 mg/L after 2.5-mo incubation.

Fig. 1.

The change in abundance of Bacteria, Archaea, and Bathyarchaeota in sediment slurries after incubation with different organic compounds for 3.5, 6, and 11 mo. The abundance (gene copies per gram wet weight of sediment; gene copies⋅g_ww⁻¹) is quantified based on the respective 16s rRNA genes. The error bars are obtained from replicate incubations.

Archaeal and Bathyarchaeotal Community Composition.

Since the enrichment of Bathyarchaeota was the aim of this study, and only lignin showed significant stimulation of bathyarchaeotal growth, further detailed experiments were undertaken with the original sample, lignin enrichments, and controls. Archaeal 16S rRNA gene analyses show that Thaumarchaeota (40%) and Bathyarchaeota (33%) (Fig. 2A) were the dominant archaeal groups in the original sample followed by Thermoplasmata (8%) and Parvarchaea (5%). After amendment with lignin, the relative abundance of Bathyarchaeota within the archaeal community increased to over 65% at t₁₁ (Fig. 2A), compared with a strong decrease in control samples (8–23% of total Archaea) (Fig. 2A). The percentage of methanogenic Methanococci reads increased in both lignin cultures and no-substrate controls, whereas the relative abundance of Thaumarchaeota decreased across lignin treatments and no-substrate controls, showing a stronger percent decrease in the former.

Fig. 2.

Comparison of archaeal communities at the phylum level (A) and of bathyarchaeotal communities at the subgroup level (B) in response to lignin addition over time. t₀, t_3.5, t₆, and t₁₁ indicate samples that were taken after 0, 3.5, 6, and 11 mo, respectively. Only one sample from t₀ and one sample from t_3.5 were analyzed, compared with four samples from both t₆ and t₁₁. Details of the phylogenetic identifications are in Materials and Methods. AAG, Ancient Archaeal Group; DSEG, Deep Sea Euryarcheotic Group; MBGA, Marine Benthic Group A; MBGB, Marine Benthic Group B; MHVG, Marine Hydrothermal Vent Group; Other, unclassified archaea.

The bathyarchaeotal community in the original sample was dominated by the subgroups Bathy-8 (49%), Bathy-6 (24%), and Bathy-12 (10%) (Fig. 2B) based on a phylogenetic tree of bathyarchaeotal 16S rRNA genes (Fig. S2). Incubation with lignin created a strong selection pressure, resulting in Bathy-8 accounting for 80–90% of the bathyarchaeotal sequences at t₆ and t₁₁ (Fig. 2B). Within the Bathy-8 subgroup, a single operational taxonomic unit (OTU), OTU3326, became dominant, accounting for 63–73% of all bathyarchaeotal 16S rRNA gene reads (Table S1).

IC Assimilation by Lipid Carbon Isotope Analysis.

¹³C-labeled sodium bicarbonate (¹³C-IC) was provided to enrichments with and without lignin to test the hypothesis that members of the Bathyarchaeota assimilate IC via the reductive acetyl-CoA pathway. In the original sediment, the δ¹³C values of the archaeal polar lipid derivatives phytane, biphytane (BP)-0, BP-2, and BP-3 were −35.1‰, −26.5‰, −25.8‰, and −21.9‰, respectively (Fig. 3 and Table S2). No matter whether lignin was added or not, with or without ¹³C-IC, the δ¹³C value of BP-3 did not change significantly at t₆ and t₁₁. However, the δ¹³C values of phytane, BP-0, and BP-2 showed a different pattern.

Fig. 3.

The carbon isotopic composition of archaeal polar lipid derivatives (i.e., phytane, BP-0, BP-2, and BP-3) in the lignin treatment (A) and in the control samples without lignin (B) with/without ¹³C-IC at t₆ and t₁₁.

In the ¹³C-IC–amended control samples, the δ¹³C value of phytane increased by 698‰ at t₆ and by 1,100‰ at t₁₁, whereas the δ¹³C values of BP-0, BP-2, and BP-3 did not change (Fig. 3 and Table S2). When grown on lignin with ¹³C-IC, the δ¹³C values of phytane, BP-0, and BP-2 increased by 885‰, 137‰, and 18‰, respectively, at t₆ (Fig. 3 and Table S2) and by 1,260‰, 234‰, and 81‰, respectively at t₁₁.

Acetate concentration and isotopic data are shown in Table 1. Acetate concentrations show significant fluctuations between treatments and replicates at the two time points sampled after 6 mo and 11 mo. After 6 mo, acetate concentrations were in a similar range in lignin treatments and controls. By comparison, after 11 mo, acetate concentrations had increased in all lignin treatments (P < 0.05, Wilcoxon rank sum test), whereas controls showed no clear increase relative to concentrations after 6 mo (P > 0.05). Production of ¹³C-acetate, indicative of acetogenesis from ¹³C-IC, was evident in all ¹³C-IC–amended controls and lignin treatments after 6 mo, as indicated by strong increases in δ¹³C-acetate. However, after 11 mo, high δ¹³C-acetate values were detected only in lignin treatments, consistent with sustained acetogenesis in the presence of lignin.

View this table:

Table 1.

Carbon isotope composition and concentration of acetate in water medium after incubation with/without lignin and¹³C-bicarbonate

Discussion

Marine sediments are one of Earth’s largest organic carbon sinks. Microbial transformation of carbon is considered a key process influencing the carbon flow in sediment and ultimately atmospheric oxygen and carbon dioxide concentrations (17). Nevertheless, the controls on microbial organic carbon cycling are not well understood, including the biogeochemical role of Archaea, which are often abundant in marine sediments (18). In this study, we demonstrate that lignin addition significantly stimulates the growth of Bathyarchaeota (Fig. 1), which are a dominant group of Archaea in marine sediments (11). The relative abundance of Bathyarchaeota reached up to 65% of the total archaeal community after 11 mo of incubation, with the subgroup Bathy-8 accounting for most of this increase (Fig. 2). Our work suggests that members of the Bathyarchaeota are potentially important, previously unrecognized degraders of lignin in marine sediments.

Lignin is a class of complex cross-linked phenolic polymers and, after cellulose, constitutes the second-most abundant biopolymer on Earth. Lignin is particularly important in the formation of cell walls in vascular plants, comprising up to 25% of plant biomass (19). Structural polymers, such as cellulose, hemicellulose, and lignin, constitute the bulk of terrestrial organic matter. Terrestrial organic matter is estimated to contribute one-third of the total buried organic carbon in marine sediments (20). This contribution decreases from nearshore to offshore but remains high, up to 15%, even in basin sediments (21). In terms of global organic carbon burial, nearshore sediments in estuaries and shallow shelf environments play a very important role, accounting for ∼45% of total organic carbon burial in marine sediments (17 and 22). Consequently, anaerobic lignin degradation in marine sediments, in particular in nearshore environments, is probably a globally important microbial process. The anaerobic biodegradation of lignin in sediments has been demonstrated previously (23), but the responsible microbes have remained elusive.

Metagenomic sequence analyses have suggested that Bathyarchaeota are capable of utilizing a variety of organic compounds including proteins (11), aromatic compounds (2), and carbohydrates (3, 14). However, bathyarchaeotal 16S rRNA gene abundances increased 10-fold in response to lignin addition in this study and did not grow in response to proteins, cellulose, phenol, or oleic acid. This indicates that, at least at the study site, members of this phylum play an important role in the degradation of lignin.

In the enrichment with lignin, the doubling (or generation) time of Bathyarchaeota was estimated to be about 2–3 mo, which is similar to that of other marine-sediment archaea enriched in the laboratory (24 ⇓–26). Considering the typically lower carbon and energy availability in natural sediments, the in situ generation times of bathyarchaeotal cells in marine sediments are likely even longer than in our enrichment cultures. These long generation times may contribute to the difficulty of enriching Bathyarchaeota in the laboratory and explain why no pure isolates exist.

Within the Bathyarchaeota, the subgroup Bathy-8 became predominant during growth on lignin, comprising ∼90% of the bathyarchaeotal community at the end of experiments (Fig. 2B). Within the Bathy-8, OTU3326 became dominant (Table S1), suggesting that this member of Bathy-8 was the main lignin degrader in the community. Bathy-8 members are widely distributed in various marine and terrestrial habitats, including seeps, hydrothermal vents, shallow marine sediments, sediments of hypersaline, saline, and freshwater lakes, salt marsh sediments, hot springs, and Arctic peat soils (5). BLAST analyses furthermore show that highly similar 16S rRNA gene sequences (≥97% identical with OTU3326) occur in diverse environments, including deep-sea fan sediments (KX952596), estuarine sediments (JQ245924), submarine springs (JF971130), and terrestrial habitats.

The metagenome was obtained from the lignin enrichment and was analyzed (for details see SI Materials and Methods and SI Text). Two genomic bins belonging to Bathy-8 (Bin-L-1 and Bin-L-2) were assembled from the lignin enrichment (Fig. S3 and Table S3). A complete Wood–Ljungdahl pathway (WL; also called the “reductive acetyl-CoA pathway”) was found in both genomes, indicating the ability of these Bathy-8 members to fix IC (Tables S4 and S5). The WL differs from other carbon-fixation pathways in being linear rather than cyclic, involving fewer reaction steps and a smaller set of enzymes, and being used for both energy conservation and biosynthesis by the same organisms; these traits likely confer energetic advantages under low-energy conditions because less metabolic energy is required for the synthesis of enzymes and energetically costly intermediates (27 ⇓–29). High energy efficiency may explain why the WL is widespread among anaerobic microorganisms performing low-energy catabolic reactions, such as methanogenesis and acetogenesis, and in dominant subsurface sedimentary microorganisms. Members of the Bathy-8 group were recently suggested to be capable of methanogenesis because a metagenomic bin contained the entire methyl–coenzyme M reductase (MCR) gene cluster (12). No MCR genes were found in Bin-L-1 and Bin-L-2 (Tables S4 and S5). No known genes for lignin degradation were found in Bin-L-1, while genes of catalase-peroxidase and 4-oxalocrotonate tautomerase for putative lignin and aromatic compound degradation, respectively, were found in Bin-L-2 (Tables S4 and S5). No canonical genes for respiration such as dissimilatory sulfate, nitrate, or iron reduction were found in the genomes (3, 14).

IC was incorporated into phytane as well as biphytanes with 0–2 cycloalkyl rings (Fig. 3). The pattern of label incorporation into these compounds suggests that at least two different functional archaeal groups were assimilating IC for lipid biosynthesis in our experiments. The lignin-unrelated incorporation of ¹³C-IC into phytane is presumably related to the methanogenic class Methanococci, which were the second-most abundant group of Archaea throughout controls and lignin enrichments and which grow using H₂ as an energy source and IC as a carbon source (30). Since the incorporation of ¹³C label into biphytanes was observed only in lignin treatments, which exhibited strong increases only in bathyarchaeotal populations, we infer that the dominant Bathyarchaeota in lignin treatments were responsible for this incorporation. In the White Oak River estuary, which receives high input of terrestrial organic matter, Bathyarchaeota were suggested to be the producers of isoprenoidal tetraether lipids bearing a carbon isotopic signature of autotrophy (31). Bathyarchaeota are therefore a plausible source of ¹³C-labeled BP-0 and BP-2 in our study. The fact that the majority of recovered bathyarchaeotal genomes from various subgroups contain genes of the WL (3, 14) is in line with the observed ¹³C-IC assimilation in lignin treatments. Although the enrichment of Bathyarchaeota in lignin amendments strongly suggests an involvement of Bathyarchaeota in lignin degradation, we cannot entirely rule out the possibility that the IC-derived ¹³C-labeled incorporation into archaeal lipids is due to the selective growth of other archaeal community members whose growth and lipid biosynthesis were also stimulated selectively by the presence of amended lignin. However, no archaeal group other than the Bathy-8 subgroup shows clear growth based on 16S genes and lipid biosynthesis in response to enrichment with lignin (Figs. 2 and 3 and Table S2).

The enrichment of Bathyarchaeota on lignin suggests that members of this phylum could play an important role in the degradation of lignin in anoxic marine sediments. We hypothesize that bathyarchaeotal community members affiliated with Bathy-8 metabolize methoxy-groups of lignin and combine the resulting methyl groups with CO₂ to acetyl-CoA via the carbon monoxide dehydrogenase/acetyl-CoA synthase complex (Codh/Acs). Acetyl CoA is then used as a key intermediate for biosynthesis or is converted to acetate via cleavage of the cofactor to produce energy (3, 28). This mode of lignin metabolism is equivalent to that described for acetogenic bacteria (27), of which 88% of tested cultivars can grow by combining methyl groups from lignin phenols with CO₂ (32). Acetate production was indeed measured in the cultures (Table 1). Although both controls and lignin treatments show clear signs of acetogenic incorporation of CO₂ into acetate, suggesting that acetogenesis is also an important process in unamended samples and perhaps in native sediments, both the higher acetate concentrations and higher ¹³C-enrichment of acetate in lignin treatments after 11 mo indicate that prolonged stimulation of acetogenic activity and CO₂ reduction occurs only in lignin-amended treatments.

In conclusion, the enrichment of the Bathy-8 subgroup and the incorporation of IC into putative bathyarchaeotal lipids in lignin amendments, combined with genomic and geochemical evidence, strongly suggest an organoautotrophic lifestyle for these Bathyarchaeota. Members of this ubiquitous subgroup may play an important, previously unrecognized role in the degradation of terrestrially derived lignin in marine sediment. Such an affinity for lignin is consistent with high abundances of Bathyarchaeota in terrestrially influenced continental margin settings (5, 8, 31). In addition to accounting for a major fraction of organic carbon burial in marine sediments, lignocellulose material is the most abundant renewable energy resource on our planet. Our finding that Bathyarchaeota mediate lignin degradation may thus result in new strategies for the use of plant material during bioenergy production.

Materials and Methods

Sample Collection.

The uppermost 10 cm of intertidal sediments were collected from Dayangshan Island (30.592817 N, 122.083493 E) in Hangzhou Bay, northern East China Sea, China (Fig. S1). Samples were stored anoxically on ice in gas-tight bags and were transported to the laboratory within 3 h, where they were kept at 4 °C until further treatment. The samples were dominated by silt sediment, which is mainly supplied by the Yangtze River (33) and is considered to be rich in terrigenous organic matter (34).

Cultivation Conditions.

About 600 g of sediment was thoroughly mixed with 2 L of anaerobic artificial seawater medium (26) without Na₂SO₄ in an anaerobic chamber (Type B vinyl; Coy). After centrifugation at 7,610 × g in 500-mL centrifuge bottles, the supernatant was removed, and this process was repeated again to largely eliminate the original porewater. Afterward, the sediment was mixed again with 3 L of anaerobic artificial seawater medium and was dispensed into serum bottles as 100-mL sediment slurries. The following five organic growth substrates were added to different experiments at a dose of 50 mg/L: oleic acid (Sinopharm 30138518), phenol (Sinopharm 100153008), casein (Sinopharm 69006227), alkali lignin (Sigma 45-471003), and cellulose (Sinopharm 68005761). In addition, ¹³C-NaHCO₃ was added to a final concentration of 0.25 µM [the percentage of ¹³C-NaHCO₃ (mol/mol) to total NaHCO₃ was 5%] to test for the assimilation of IC in archaeal lipids. The bottles were sealed with butyl rubber stoppers and aluminum crimp seals, and then the headspace was evacuated by gas pump and refilled with 99.99% N₂ gas in the anaerobic chamber three times, after which a pressure of 200 kPa of N₂ was applied to each vial. Two replicates of each treatment were incubated; meanwhile two slurries were also incubated without the addition of any organic substrates as controls. Slurries were incubated horizontally without shaking in the dark at 20 °C for 3.5 mo (t_3.5), after which 20 mL of slurries were sampled using a syringe with needle. The collected samples were centrifuged at 13,800 × g to separate the supernatant and sediments and were stored at −80 °C for DNA and lipid isolation and measurement of chemical parameters.

Afterward, higher concentrations of organic substrates (500 mg/L of each substrate) and ¹³C-NaHCO₃ [final concentration is 0.5 µM; the percentage of ¹³C-NaHCO₃ (mol/mol) to total NaHCO₃ was 10%] were added to the remaining cultures, which were then incubated for another 2.5 mo (t₆). Then 20 mL of the slurries were collected, more organic substrates (500 mg/L of each substrate) were added again, and these samples were incubated for additional 5 mo. Thus, slurries were obtained at three time points after incubation, i.e., at t_3.5, t₆, and t₁₁ mo.

DNA Extraction.

Total DNA was extracted from 0.2–0.3 g of wet sediment using a PowerSoil DNA Isolation Kit (Mo Bio) according to the manufacturer’s protocol. The concentration of DNA was measured by using a NanoDrop 2000 spectrophotometer (Thermo Scientific), followed by freezing at −80 °C until processing.

qPCR.

Bacterial, archaeal, and bathyarchaeotal 16S rRNA genes were quantified by qPCR and were amplified using the primers Uni519F/Arch908R, Bac341F/prokaryotic519R, and Bathy-442F/Bathy-644R, respectively. The reaction solution included 10 μL of SYBR Premix Ex Taq (TaKaRa), 0.4 μL of ROX reference dye (50×; TaKaRa), 0.8 μM of each primer, and 1 μL of template DNA. Further details, such as primer sequences and PCR conditions, are shown in Table S6. Clones with archaeal, bacterial, and bathyarchaeotal 16S rRNA genes (7) were used for standard curve construction. The R² and efficiency of each individual qPCR assay are shown in Table S6. The abundance of each targeted gene in the DNA assemblage was determined in triplicate analyses.

The mean doubling times (T) of Bathyarchaeota were calculated according to the formula: T = (t₂ − t₁)/(log₂X₂ − log₂X₁) or T = (t₂ − t₁)/[3.322 (logX₂ − logX₁)], which is generated from the formula X₁ × 2^(t2 ⁻ ^t1)/T = X₂. X₁ and X₂ are the bathyarchaeotal abundance at incubation time t₁ and t₂, respectively.

Illumina Sequencing and Data Analysis.

Since bathyarchaeotal growth was observed only in treatments to which lignin had been added as a growth substrate, only the original sample (t₀), the lignin cultures IC+lignin (t_3.5), IC+lignin (t₆) × 2, ¹³C-IC+lignin (t₆) × 2, IC+lignin (t₁₁) × 2, and ¹³C-IC+lignin (t₁₁) × 2, and the control cultures IC (t_3.5), IC (t₆) × 2, ¹³C-IC (t₆) × 2, IC (t₁₁) × 2, and ¹³C-IC (t₁₁) × 2 (a total of 19 samples) were processed for archaeal 16S rRNA gene sequencing. The hypervariable V4 regions of archaeal 16S rRNA genes were amplified from original and enrichment samples using primer sets U519F/Arch806R (Table S6) (35). Each 50-μL reaction solution contained 10× PCR buffer, dNTP (100 μM each), 0.25 μM of each primer, 2.5 U of DNA polymerase (Ex-Taq; TaKaRa), and ∼10 ng of total DNA (measured by a NanoDrop 2000). PCR products were purified using an E.Z.N.A. Gel Extraction Kit (Omega Bio-tek) according to the manufacturer’s instructions.

The 16S rRNA gene amplicons containing unique 8-mer barcodes used for each sample were pooled at equal concentrations and were sequenced on the Illumina MiSeq platform using 2× 250-bp cycles and the 500-cycle MiSeq Reagent Kit v2 (Illumina) according to the manufacturer’s instructions. Raw reads were removed if they contained a 50-bp continuous fragment with an average quality score of less than 30 and/or any ambiguities. Filtered reads were merged together using FLASH version 1.2.6 (36). Further analysis was performed using the QIIME standard pipeline (37). In particular, sequence reads were first filtered to remove low-quality or ambiguous reads, and chimeric sequences were removed using the UCHIME program version 4.2 (38). The sequences were clustered into OTUs using UCLUST (38) with a 97% sequence identity threshold. Taxonomy was assigned using the Greengenes database (version gg_13_5, greengenes.secondgenome.com/). The obtained archaeal sequences from each sample were randomly subsampled to generate a uniform depth of 10,372 sequences. Species richness, species diversity, and coverage were computed using QIIME’s alpha diversity pipeline. Five metrics were calculated: the observed number of species, coverage (through rarefaction analyses), the Chao1 index, the Shannon index, and the Simpson index. The number of archaeal sequences in the above-mentioned 19 samples ranged from 10,425 to 33,848 (Table S7). The number of archaeal OTUs (cutoff, 97%) in these 19 sediment samples ranged from 423 to 869. The coverage calculation indicated that the analyzed sequences covered the diversity of archaeal populations in the investigated samples (Table S7).

Phylogenetic Analyses.

From the Illumina MiSeq dataset, OTUs affiliated with Bathyarchaeota which contained more than 100 sequences were selected for phylogenetic analyses, and the closest phylogenetic affiliations were determined by constructing a bathyarchaeotal 16S rRNA gene tree using reference sequences of the 17 bathyarchaeotal subgroups (5) as phylogenetic anchors. For the construction of the phylogenic tree, the sequences were aligned using CLUSTALX 1.83, and the reference sequences were trimmed, retaining 900 bp after alignment. Neighbor-joining phylogenetic trees were constructed from pairwise comparisons with the Kimura 2-parameter distance model using the molecular evolutionary genetics analysis (MEGA) program, version 6 (39). Bootstrap values are based on 1,000 replicates for testing the robustness of the inferred topology. Interactive Tree of Life (iTOL) (itol.embl.de/) was used to modify the phylogenetic trees.

Data Deposition.

Sequences of Illumina sequencing raw data were submitted to GenBank under accession numbers PRJNA398600 and PRJNA398689.

Lipid Extraction and Analyses.

The total lipid extracts (TLEs) were extracted from lyophilized samples (∼1 g) by a modified Bligh and Dyer method (40). For the samples at t₆, a TLE aliquot of 90% was chemically treated to convert di- and tetraether lipids into their hydrocarbon derivatives phytane and several biphytanes with 0-3 rings via ether-cleavage methods (41). For samples that had been incubated for 11 mo, intact polar archaeal lipid fractions were first purified by preparative HPLC with a fraction collector (42) and then were converted to hydrocarbon derivatives according to the above method (41). The δ¹³C values of archaeal intact polar lipid derivatives were determined on a Finnigan Trace gas chromatograph (Thermo Scientific) connected to a DELTA Plus XP isotope ratio mass spectrometer via a GC combustion III interface (both from Thermo Fisher) according to the method described by Kellermann et al. (43).

Measurement of Total Phenolic Content.

The lignin degradation was initially surveyed by measuring the concentrations of phenolic compounds in the supernatant of slurries using the Folin–Ciocalteu method (Sinopharm 73104861) as described previously (44).

Acetate Concentration and Carbon Isotope Measurement.

Concentrations and carbon isotope composition of acetate in the supernatants of slurries were analyzed by on LC linked to isotope ratio mass spectrometry (LC-IRMS) according to the method described by Heuer et al. (45).

Acknowledgments

We thank Karl O. Stetter (University of Regensburg) and Hongbo Yu (Huazhong University of Science and Technology) for kind suggestions to improve the manuscript and Jiahua Wang (Shanghai Jiao Tong University) for help with the metagenome analysis. This work was supported financially by State Key Research and Development Project of China Grant 2016YFA0601102, Natural Science Foundation of China Grants 41525011, 91751205, and 91428308, and the Deutsche Forschungsgemeinschaft through the Gottfried Wilhelm Leibniz Program Award Hi 616-14-1 (to K.-U.H.). This study is also a contribution to the Integrated Marine Biosphere Research International Project and the Deep Carbon Observatory.

Footnotes

↵¹T.Y. and W.W. contributed equally to this work.
↵²To whom correspondence should be addressed. Email: fengpingw{at}sjtu.edu.cn.

Author contributions: T.Y. and F.W. designed research; T.Y., W.W., and W.L. performed research; T.Y., W.W., M.A.L., K.-U.H., and F.W. analyzed data; and T.Y., W.W., M.A.L., K.-U.H., and F.W. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
Data deposition: The sequences reported in this paper have been deposited in the GenBank database (accession nos. PRJNA398600 and PRJNA398689). The metagenome and bathyarchaeotal genomes reported in this paper have been deposited in the GenBank database (accession no. PRJNA418892).
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1718854115/-/DCSupplemental.

Published under the PNAS license.

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1. Inagaki F, et al.
(2003) Microbial communities associated with geological horizons in coastal subseafloor sediments from the sea of Okhotsk. Appl Environ Microbiol 69:7224–7235.
OpenUrl Abstract/FREE Full Text
↵
1. Meng J, et al.
(2014) Genetic and functional properties of uncultivated MCG Archaea assessed by metagenome and gene expression analyses. ISME J 8:650–659.
OpenUrl CrossRef PubMed
↵
1. He Y, et al.
(2016) Genomic and enzymatic evidence for acetogenesis among multiple lineages of the archaeal phylum Bathyarchaeota widespread in marine sediments. Nat Microbiol 1:16035.
OpenUrl
↵
1. Jiang L,
2. Zheng Y,
3. Chen J,
4. Xiao X,
5. Wang F
(2011) Stratification of achaeal communities in shallow sediments of the Pearl River Estuary, Southern China. Antonie Van Leeuwenhoek 99:739–751.
OpenUrl CrossRef PubMed
↵
1. Kubo K, et al.
(2012) Archaea of the Miscellaneous Crenarchaeotal Group are abundant, diverse and widespread in marine sediments. ISME J 6:1949–1965.
OpenUrl CrossRef PubMed
↵
1. Ruff SE, et al.
(2015) Global dispersion and local diversification of the methane seep microbiome. Proc Natl Acad Sci USA 112:4015–4020.
OpenUrl Abstract/FREE Full Text
↵
1. Yu T,
2. Liang Q,
3. Niu M,
4. Wang F
(2017) High occurrence of Bathyarchaeota (MCG) in the deep-sea sediments of South China Sea quantified using newly designed PCR primers. Environ Microbiol Rep 9:374–382.
OpenUrl
↵
1. Lazar CS, et al.
(2015) Environmental controls on intragroup diversity of the uncultured benthic Archaea of the Miscellaneous Crenarchaeotal Group lineage naturally enriched in anoxic sediments of the White Oak River estuary (North Carolina, USA). Environ Microbiol 17:2228–2238.
OpenUrl CrossRef PubMed
↵
1. Fillol M,
2. Auguet J-C,
3. Casamayor EO,
4. Borrego CM
(2016) Insights in the ecology and evolutionary history of the Miscellaneous Crenarchaeotic Group lineage. ISME J 10:665–677.
OpenUrl CrossRef PubMed
↵
1. Biddle JF, et al.
(2006) Heterotrophic Archaea dominate sedimentary subsurface ecosystems off Peru. Proc Natl Acad Sci USA 103:3846–3851.
OpenUrl Abstract/FREE Full Text
↵
1. Lloyd KG, et al.
(2013) Predominant Archaea in marine sediments degrade detrital proteins. Nature 496:215–218.
OpenUrl CrossRef PubMed
↵
1. Evans PN, et al.
(2015) Methane metabolism in the archaeal phylum Bathyarchaeota revealed by genome-centric metagenomics. Science 350:434–438.
OpenUrl Abstract/FREE Full Text
↵
1. Gagen EJ,
2. Huber H,
3. Meador T,
4. Hinrichs KU,
5. Thomm M
(2013) Novel cultivation-based approach to understanding the Miscellaneous Crenarchaeotic Group (MCG) Archaea from sedimentary ecosystems. Appl Environ Microbiol 79:6400–6406.
OpenUrl Abstract/FREE Full Text
↵
1. Lazar CS, et al.
(2016) Genomic evidence for distinct carbon substrate preferences and ecological niches of Bathyarchaeota in estuarine sediments. Environ Microbiol 18:1200–1211.
OpenUrl CrossRef PubMed
↵
1. Michalska K, et al.
(2015) New aminopeptidase from “microbial dark matter” archaeon. FASEB J 29:4071–4079.
OpenUrl CrossRef PubMed
↵
1. Seyler LM,
2. McGuinness LM,
3. Kerkhof LJ
(2014) Crenarchaeal heterotrophy in salt marsh sediments. ISME J 8:1534–1543.
OpenUrl CrossRef PubMed
↵
1. Berner RA
(1989) Biogeochemical cycles of carbon and sulfur and their effect on atmospheric oxygen over Phanerozoic time. Palaeogeogr Palaeoclimatol Palaeoecol 75:97–122.
OpenUrl CrossRef
↵
1. Lipp JS,
2. Morono Y,
3. Inagaki F,
4. Hinrichs KU
(2008) Significant contribution of Archaea to extant biomass in marine subsurface sediments. Nature 454:991–994.
OpenUrl CrossRef PubMed
↵
1. Wei H, et al.
(2009) Natural paradigms of plant cell wall degradation. Curr Opin Biotechnol 20:330–338.
OpenUrl CrossRef PubMed
↵
1. Burdige DJ
(2005) Burial of terrestrial organic matter in marine sediments: A re-assessment. Glob Biogeochem Cycles 19:GB4011.
OpenUrl CrossRef
↵
1. Prahl FG,
2. Ertel JR,
3. Goni MA,
4. Sparrow MA,
5. Eversmeyer B
(1994) Terrestrial organic carbon contributions to sediments on the Washington margin. Geochim Cosmochim Acta 58:3035–3048.
OpenUrl CrossRef
↵
1. Hedges JI,
2. Keil RG
(1995) Sedimentary organic matter preservation: An assessment and speculative synthesis. Mar Chem 49:81–115.
OpenUrl CrossRef
↵
1. Benner R,
2. Maccubbin AE,
3. Hodson RE
(1984) Anaerobic biodegradation of the lignin and polysaccharide components of lignocellulose and synthetic lignin by sediment microflora. Appl Environ Microbiol 47:998–1004.
OpenUrl Abstract/FREE Full Text
↵
1. Girguis PR,
2. Orphan VJ,
3. Hallam SJ,
4. DeLong EF
(2003) Growth and methane oxidation rates of anaerobic methanotrophic Archaea in a continuous-flow bioreactor. Appl Environ Microbiol 69:5472–5482.
OpenUrl Abstract/FREE Full Text
↵
1. Krüger M,
2. Wolters H,
3. Gehre M,
4. Joye SB,
5. Richnow H-H
(2008) Tracing the slow growth of anaerobic methane-oxidizing communities by (¹⁵)N-labelling techniques. FEMS Microbiol Ecol 63:401–411.
OpenUrl CrossRef PubMed
↵
1. Zhang Y,
2. Henriet J-P,
3. Bursens J,
4. Boon N
(2010) Stimulation of in vitro anaerobic oxidation of methane rate in a continuous high-pressure bioreactor. Bioresour Technol 101:3132–3138.
OpenUrl CrossRef PubMed
↵
1. Drake HL,
2. Gössner AS,
3. Daniel SL
(2008) Old acetogens, new light. Ann N Y Acad Sci 1125:100–128.
OpenUrl CrossRef PubMed
↵
1. Lever MA
(2012) Acetogenesis in the energy-starved deep biosphere–A paradox? Front Microbiol 2:284.
OpenUrl CrossRef PubMed
↵
1. Hügler M,
2. Sievert SM
(2011) Beyond the Calvin cycle: Autotrophic carbon fixation in the ocean. Annu Rev Mar Sci 3:261–289.
OpenUrl CrossRef PubMed
↵
1. Rosenberg E,
2. DeLong EF,
3. Lory S,
4. Stackebrandt E,
5. Thompson F
1. Whitman WB,
2. Bowen TL,
3. Boone DR
(2014) The methanogenic bacteria. The Prokaryotes: Other Major Lineages of Bacteria and the Archaea, eds Rosenberg E, DeLong EF, Lory S, Stackebrandt E, Thompson F (Springer, Heidelberg), pp 123–163.
↵
1. Meador TB, et al.
(2015) The archaeal lipidome in estuarine sediment dominated by members of the Miscellaneous Crenarchaeotal Group. Environ Microbiol 17:2441–2458.
OpenUrl CrossRef
↵
1. Lever MA, et al.
(2010) Acetogenesis in deep subseafloor sediments of the Juan de Fuca Ridge Flank: A synthesis of geochemical, thermodynamic, and gene-based evidence. Geomicrobiol J 27:183–211.
OpenUrl CrossRef
↵
1. Dai Z-J,
2. Liu JT,
3. Xie H-L,
4. Shi W-Y
(2014) Sedimentation in the outer Hangzhou Bay, China: The influence of Changjiang sediment load. J Coast Res 30:1218–1225.
OpenUrl
↵
1. Xu F,
2. Ji Z,
3. Wang K,
4. Jin H,
5. Loh PS
(2016) The distribution of sedimentary organic matter and implication of its transfer from Changjiang Estuary to Hangzhou Bay, China. Open J Mar Sci 6:103–114.
OpenUrl
↵
1. Song Z-Q, et al.
(2013) Bacterial and archaeal diversities in Yunnan and Tibetan hot springs, China. Environ Microbiol 15:1160–1175.
OpenUrl
↵
1. Magoč T,
2. Salzberg SL
(2011) FLASH: Fast length adjustment of short reads to improve genome assemblies. Bioinformatics 27:2957–2963.
OpenUrl CrossRef PubMed
↵
1. Caporaso JG, et al.
(2010) QIIME allows analysis of high-throughput community sequencing data. Nat Methods 7:335–336.
OpenUrl CrossRef PubMed
↵
1. Edgar RC,
2. Haas BJ,
3. Clemente JC,
4. Quince C,
5. Knight R
(2011) UCHIME improves sensitivity and speed of chimera detection. Bioinformatics 27:2194–2200.
OpenUrl CrossRef PubMed
↵
1. Tamura K,
2. Stecher G,
3. Peterson D,
4. Filipski A,
5. Kumar S
(2013) MEGA6: Molecular evolutionary genetics analysis version 6.0. Mol Biol Evol 30:2725–2729.
OpenUrl CrossRef PubMed
↵
1. Sturt HF,
2. Summons RE,
3. Smith K,
4. Elvert M,
5. Hinrichs KU
(2004) Intact polar membrane lipids in prokaryotes and sediments deciphered by high-performance liquid chromatography/electrospray ionization multistage mass spectrometry–New biomarkers for biogeochemistry and microbial ecology. Rapid Commun Mass Spectrom 18:617–628.
OpenUrl CrossRef PubMed
↵
1. Liu X-L, et al.
(2012) Mono- and dihydroxyl glycerol dibiphytanyl glycerol tetraethers in marine sediments: Identification of both core and intact polar lipid forms. Geochim Cosmochim Acta 89:102–115.
OpenUrl CrossRef
↵
1. Zhu C, et al.
(2013) Comprehensive glycerol ether lipid fingerprints through a novel reversed phase liquid chromatography–mass spectrometry protocol. Org Geochem 65:53–62.
OpenUrl CrossRef
↵
1. Kellermann MY, et al.
(2012) Autotrophy as a predominant mode of carbon fixation in anaerobic methane-oxidizing microbial communities. Proc Natl Acad Sci USA 109:19321–19326.
OpenUrl Abstract/FREE Full Text
↵
1. Cicco N,
2. Lanorte MT,
3. Paraggio M,
4. Viggiano M,
5. Lattanzio V
(2009) A reproducible, rapid and inexpensive Folin–Ciocalteu micro-method in determining phenolics of plant methanol extracts. Microchem J 91:107–110.
OpenUrl
↵
1. Heuer V, et al.
(2006) Online δ¹³C analysis of volatile fatty acids in sediment/porewater systems by liquid chromatography‐isotope ratio mass spectrometry. Limnol Oceanogr Methods 4:346–357.
OpenUrl CrossRef

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Biological Sciences
Environmental Sciences

Proceedings of the National Academy of Sciences: 115 (23)

Table of Contents

Submit

[1] ↵
Inagaki F, et al.
(2003) Microbial communities associated with geological horizons in coastal subseafloor sediments from the sea of Okhotsk. Appl Environ Microbiol 69:7224–7235.
OpenUrl Abstract/FREE Full Text

[2] Inagaki F, et al.

[3] ↵
Meng J, et al.
(2014) Genetic and functional properties of uncultivated MCG Archaea assessed by metagenome and gene expression analyses. ISME J 8:650–659.
OpenUrl CrossRef PubMed

[4] Meng J, et al.

[5] ↵
He Y, et al.
(2016) Genomic and enzymatic evidence for acetogenesis among multiple lineages of the archaeal phylum Bathyarchaeota widespread in marine sediments. Nat Microbiol 1:16035.
OpenUrl

[6] He Y, et al.

[7] ↵
Jiang L,
Zheng Y,
Chen J,
Xiao X,
Wang F
(2011) Stratification of achaeal communities in shallow sediments of the Pearl River Estuary, Southern China. Antonie Van Leeuwenhoek 99:739–751.
OpenUrl CrossRef PubMed

[8] Jiang L,

[9] Zheng Y,

[10] Chen J,

[11] Xiao X,

[12] Wang F

[13] ↵
Kubo K, et al.
(2012) Archaea of the Miscellaneous Crenarchaeotal Group are abundant, diverse and widespread in marine sediments. ISME J 6:1949–1965.
OpenUrl CrossRef PubMed

[14] Kubo K, et al.

[15] ↵
Ruff SE, et al.
(2015) Global dispersion and local diversification of the methane seep microbiome. Proc Natl Acad Sci USA 112:4015–4020.
OpenUrl Abstract/FREE Full Text

[16] Ruff SE, et al.

[17] ↵
Yu T,
Liang Q,
Niu M,
Wang F
(2017) High occurrence of Bathyarchaeota (MCG) in the deep-sea sediments of South China Sea quantified using newly designed PCR primers. Environ Microbiol Rep 9:374–382.
OpenUrl

[18] Yu T,

[19] Liang Q,

[20] Niu M,

[21] Wang F

[22] ↵
Lazar CS, et al.
(2015) Environmental controls on intragroup diversity of the uncultured benthic Archaea of the Miscellaneous Crenarchaeotal Group lineage naturally enriched in anoxic sediments of the White Oak River estuary (North Carolina, USA). Environ Microbiol 17:2228–2238.
OpenUrl CrossRef PubMed

[23] Lazar CS, et al.

[24] ↵
Fillol M,
Auguet J-C,
Casamayor EO,
Borrego CM
(2016) Insights in the ecology and evolutionary history of the Miscellaneous Crenarchaeotic Group lineage. ISME J 10:665–677.
OpenUrl CrossRef PubMed

[25] Fillol M,

[26] Auguet J-C,

[27] Casamayor EO,

[28] Borrego CM

[29] ↵
Biddle JF, et al.
(2006) Heterotrophic Archaea dominate sedimentary subsurface ecosystems off Peru. Proc Natl Acad Sci USA 103:3846–3851.
OpenUrl Abstract/FREE Full Text

[30] Biddle JF, et al.

[31] ↵
Lloyd KG, et al.
(2013) Predominant Archaea in marine sediments degrade detrital proteins. Nature 496:215–218.
OpenUrl CrossRef PubMed

[32] Lloyd KG, et al.

[33] ↵
Evans PN, et al.
(2015) Methane metabolism in the archaeal phylum Bathyarchaeota revealed by genome-centric metagenomics. Science 350:434–438.
OpenUrl Abstract/FREE Full Text

[34] Evans PN, et al.

[35] ↵
Gagen EJ,
Huber H,
Meador T,
Hinrichs KU,
Thomm M
(2013) Novel cultivation-based approach to understanding the Miscellaneous Crenarchaeotic Group (MCG) Archaea from sedimentary ecosystems. Appl Environ Microbiol 79:6400–6406.
OpenUrl Abstract/FREE Full Text

[36] Gagen EJ,

[37] Huber H,

[38] Meador T,

[39] Hinrichs KU,

[40] Thomm M

[41] ↵
Lazar CS, et al.
(2016) Genomic evidence for distinct carbon substrate preferences and ecological niches of Bathyarchaeota in estuarine sediments. Environ Microbiol 18:1200–1211.
OpenUrl CrossRef PubMed

[42] Lazar CS, et al.

[43] ↵
Michalska K, et al.
(2015) New aminopeptidase from “microbial dark matter” archaeon. FASEB J 29:4071–4079.
OpenUrl CrossRef PubMed

[44] Michalska K, et al.

[45] ↵
Seyler LM,
McGuinness LM,
Kerkhof LJ
(2014) Crenarchaeal heterotrophy in salt marsh sediments. ISME J 8:1534–1543.
OpenUrl CrossRef PubMed

[46] Seyler LM,

[47] McGuinness LM,

[48] Kerkhof LJ

[49] ↵
Berner RA
(1989) Biogeochemical cycles of carbon and sulfur and their effect on atmospheric oxygen over Phanerozoic time. Palaeogeogr Palaeoclimatol Palaeoecol 75:97–122.
OpenUrl CrossRef

[50] Berner RA

[51] ↵
Lipp JS,
Morono Y,
Inagaki F,
Hinrichs KU
(2008) Significant contribution of Archaea to extant biomass in marine subsurface sediments. Nature 454:991–994.
OpenUrl CrossRef PubMed

[52] Lipp JS,

[53] Morono Y,

[54] Inagaki F,

[55] Hinrichs KU

[56] ↵
Wei H, et al.
(2009) Natural paradigms of plant cell wall degradation. Curr Opin Biotechnol 20:330–338.
OpenUrl CrossRef PubMed

[57] Wei H, et al.

[58] ↵
Burdige DJ
(2005) Burial of terrestrial organic matter in marine sediments: A re-assessment. Glob Biogeochem Cycles 19:GB4011.
OpenUrl CrossRef

[59] Burdige DJ

[60] ↵
Prahl FG,
Ertel JR,
Goni MA,
Sparrow MA,
Eversmeyer B
(1994) Terrestrial organic carbon contributions to sediments on the Washington margin. Geochim Cosmochim Acta 58:3035–3048.
OpenUrl CrossRef

[61] Prahl FG,

[62] Ertel JR,

[63] Goni MA,

[64] Sparrow MA,

[65] Eversmeyer B

[66] ↵
Hedges JI,
Keil RG
(1995) Sedimentary organic matter preservation: An assessment and speculative synthesis. Mar Chem 49:81–115.
OpenUrl CrossRef

[67] Hedges JI,

[68] Keil RG

[69] ↵
Benner R,
Maccubbin AE,
Hodson RE
(1984) Anaerobic biodegradation of the lignin and polysaccharide components of lignocellulose and synthetic lignin by sediment microflora. Appl Environ Microbiol 47:998–1004.
OpenUrl Abstract/FREE Full Text

[70] Benner R,

[71] Maccubbin AE,

[72] Hodson RE

[73] ↵
Girguis PR,
Orphan VJ,
Hallam SJ,
DeLong EF
(2003) Growth and methane oxidation rates of anaerobic methanotrophic Archaea in a continuous-flow bioreactor. Appl Environ Microbiol 69:5472–5482.
OpenUrl Abstract/FREE Full Text

[74] Girguis PR,

[75] Orphan VJ,

[76] Hallam SJ,

[77] DeLong EF

[78] ↵
Krüger M,
Wolters H,
Gehre M,
Joye SB,
Richnow H-H
(2008) Tracing the slow growth of anaerobic methane-oxidizing communities by (¹⁵)N-labelling techniques. FEMS Microbiol Ecol 63:401–411.
OpenUrl CrossRef PubMed

[79] Krüger M,

[80] Wolters H,

[81] Gehre M,

[82] Joye SB,

[83] Richnow H-H

[84] ↵
Zhang Y,
Henriet J-P,
Bursens J,
Boon N
(2010) Stimulation of in vitro anaerobic oxidation of methane rate in a continuous high-pressure bioreactor. Bioresour Technol 101:3132–3138.
OpenUrl CrossRef PubMed

[85] Zhang Y,

[86] Henriet J-P,

[87] Bursens J,

[88] Boon N

[89] ↵
Drake HL,
Gössner AS,
Daniel SL
(2008) Old acetogens, new light. Ann N Y Acad Sci 1125:100–128.
OpenUrl CrossRef PubMed

[90] Drake HL,

[91] Gössner AS,

[92] Daniel SL

[93] ↵
Lever MA
(2012) Acetogenesis in the energy-starved deep biosphere–A paradox? Front Microbiol 2:284.
OpenUrl CrossRef PubMed

[94] Lever MA

[95] ↵
Hügler M,
Sievert SM
(2011) Beyond the Calvin cycle: Autotrophic carbon fixation in the ocean. Annu Rev Mar Sci 3:261–289.
OpenUrl CrossRef PubMed

[96] Hügler M,

[97] Sievert SM

[98] ↵
Rosenberg E,
DeLong EF,
Lory S,
Stackebrandt E,
Thompson F
Whitman WB,
Bowen TL,
Boone DR
(2014) The methanogenic bacteria. The Prokaryotes: Other Major Lineages of Bacteria and the Archaea, eds Rosenberg E, DeLong EF, Lory S, Stackebrandt E, Thompson F (Springer, Heidelberg), pp 123–163.

[99] Rosenberg E,

[100] DeLong EF,

[101] Lory S,

[102] Stackebrandt E,

[103] Thompson F

[104] Whitman WB,

[105] Bowen TL,

[106] Boone DR

[107] ↵
Meador TB, et al.
(2015) The archaeal lipidome in estuarine sediment dominated by members of the Miscellaneous Crenarchaeotal Group. Environ Microbiol 17:2441–2458.
OpenUrl CrossRef

[108] Meador TB, et al.

[109] ↵
Lever MA, et al.
(2010) Acetogenesis in deep subseafloor sediments of the Juan de Fuca Ridge Flank: A synthesis of geochemical, thermodynamic, and gene-based evidence. Geomicrobiol J 27:183–211.
OpenUrl CrossRef

[110] Lever MA, et al.

[111] ↵
Dai Z-J,
Liu JT,
Xie H-L,
Shi W-Y
(2014) Sedimentation in the outer Hangzhou Bay, China: The influence of Changjiang sediment load. J Coast Res 30:1218–1225.
OpenUrl

[112] Dai Z-J,

[113] Liu JT,

[114] Xie H-L,

[115] Shi W-Y

[116] ↵
Xu F,
Ji Z,
Wang K,
Jin H,
Loh PS
(2016) The distribution of sedimentary organic matter and implication of its transfer from Changjiang Estuary to Hangzhou Bay, China. Open J Mar Sci 6:103–114.
OpenUrl

[117] Xu F,

[118] Ji Z,

[119] Wang K,

[120] Jin H,

[121] Loh PS

[122] ↵
Song Z-Q, et al.
(2013) Bacterial and archaeal diversities in Yunnan and Tibetan hot springs, China. Environ Microbiol 15:1160–1175.
OpenUrl

[123] Song Z-Q, et al.

[124] ↵
Magoč T,
Salzberg SL
(2011) FLASH: Fast length adjustment of short reads to improve genome assemblies. Bioinformatics 27:2957–2963.
OpenUrl CrossRef PubMed

[125] Magoč T,

[126] Salzberg SL

[127] ↵
Caporaso JG, et al.
(2010) QIIME allows analysis of high-throughput community sequencing data. Nat Methods 7:335–336.
OpenUrl CrossRef PubMed

[128] Caporaso JG, et al.

[129] ↵
Edgar RC,
Haas BJ,
Clemente JC,
Quince C,
Knight R
(2011) UCHIME improves sensitivity and speed of chimera detection. Bioinformatics 27:2194–2200.
OpenUrl CrossRef PubMed

[130] Edgar RC,

[131] Haas BJ,

[132] Clemente JC,

[133] Quince C,

[134] Knight R

[135] ↵
Tamura K,
Stecher G,
Peterson D,
Filipski A,
Kumar S
(2013) MEGA6: Molecular evolutionary genetics analysis version 6.0. Mol Biol Evol 30:2725–2729.
OpenUrl CrossRef PubMed

[136] Tamura K,

[137] Stecher G,

[138] Peterson D,

[139] Filipski A,

[140] Kumar S

[141] ↵
Sturt HF,
Summons RE,
Smith K,
Elvert M,
Hinrichs KU
(2004) Intact polar membrane lipids in prokaryotes and sediments deciphered by high-performance liquid chromatography/electrospray ionization multistage mass spectrometry–New biomarkers for biogeochemistry and microbial ecology. Rapid Commun Mass Spectrom 18:617–628.
OpenUrl CrossRef PubMed

[142] Sturt HF,

[143] Summons RE,

[144] Smith K,

[145] Elvert M,

[146] Hinrichs KU

[147] ↵
Liu X-L, et al.
(2012) Mono- and dihydroxyl glycerol dibiphytanyl glycerol tetraethers in marine sediments: Identification of both core and intact polar lipid forms. Geochim Cosmochim Acta 89:102–115.
OpenUrl CrossRef

[148] Liu X-L, et al.

[149] ↵
Zhu C, et al.
(2013) Comprehensive glycerol ether lipid fingerprints through a novel reversed phase liquid chromatography–mass spectrometry protocol. Org Geochem 65:53–62.
OpenUrl CrossRef

[150] Zhu C, et al.

[151] ↵
Kellermann MY, et al.
(2012) Autotrophy as a predominant mode of carbon fixation in anaerobic methane-oxidizing microbial communities. Proc Natl Acad Sci USA 109:19321–19326.
OpenUrl Abstract/FREE Full Text

[152] Kellermann MY, et al.

[153] ↵
Cicco N,
Lanorte MT,
Paraggio M,
Viggiano M,
Lattanzio V
(2009) A reproducible, rapid and inexpensive Folin–Ciocalteu micro-method in determining phenolics of plant methanol extracts. Microchem J 91:107–110.
OpenUrl

[154] Cicco N,

[155] Lanorte MT,

[156] Paraggio M,

[157] Viggiano M,

[158] Lattanzio V

[159] ↵
Heuer V, et al.
(2006) Online δ¹³C analysis of volatile fatty acids in sediment/porewater systems by liquid chromatography‐isotope ratio mass spectrometry. Limnol Oceanogr Methods 4:346–357.
OpenUrl CrossRef

[160] Heuer V, et al.

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Growth of sedimentary Bathyarchaeota on lignin as an energy source

Significance

Abstract

Results

Enrichments Set-Up.

Archaeal and Bathyarchaeotal Community Composition.

IC Assimilation by Lipid Carbon Isotope Analysis.

Discussion

Materials and Methods

Sample Collection.

Cultivation Conditions.

DNA Extraction.

qPCR.

Illumina Sequencing and Data Analysis.

Phylogenetic Analyses.

Data Deposition.

Lipid Extraction and Analyses.

Measurement of Total Phenolic Content.

Acetate Concentration and Carbon Isotope Measurement.

Acknowledgments

Footnotes

References

Citation Manager Formats

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Main menu

User menu

Search

Significance

Abstract

Results

Enrichments Set-Up.

Archaeal and Bathyarchaeotal Community Composition.

IC Assimilation by Lipid Carbon Isotope Analysis.

Discussion

Materials and Methods

Sample Collection.

Cultivation Conditions.

DNA Extraction.

qPCR.

Illumina Sequencing and Data Analysis.

Phylogenetic Analyses.

Data Deposition.

Lipid Extraction and Analyses.

Measurement of Total Phenolic Content.

Acetate Concentration and Carbon Isotope Measurement.

Acknowledgments

Footnotes

References

Citation Manager Formats

Article Classifications

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