Growth of sedimentary Bathyarchaeota on lignin as an energy source

Tiantian Yua,b,1, Weichao Wuc,d,1, Wenyue Lianga,b, Mark Alexander Levere, Kai-Uwe Hinrichsc,d, and Fengping Wanga,b,2

aState Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, 200240 Shanghai, China; bState Key Laboratory of Ocean Engineering, Shanghai Jiao Tong University, 200240 Shanghai, China; cOrganic Geochemistry Group, MARUM-Center for Marine Environmental Sciences, University of Bremen, 28359 Bremen, Germany; dDepartment of Geosciences, University of Bremen, 28359 Bremen, Germany; and eInstitute of Biogeochemistry and Pollutant Dynamics, Department of Environmental Systems Science, Swiss Federal Institute of Technology Zurich, 8092 Zurich, Switzerland

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

Members of the archaeal phylum Bathyarchaeota are among the other hand, acetogenesis from H2/CO2 has been proposed for most abundant microorganisms on Earth. Although versatile met- some lineages of Bathyarchaeota (3). The reductive acetyl-CoA abolic capabilities such as acetogenesis, methanogenesis, and fer- pathway for carbon fixation has been identified in most of the mentation have been suggested for bathyarchaeotal members, no obtained bathyarchaeotal genomes (3, 12, 14). Among these, direct confirmation of these metabolic functions has been achieved members of the bathyarchaeotal subgroups Bathy-3 and Bathy- through growth of Bathyarchaeota in the laboratory. Here we dem- 8 have been suggested to be capable of methanogenesis (12). All onstrate, on the basis of gene-copy numbers and probing of ar- the above studies indicate great versatility in the metabolic po- chaeal lipids, the growth of Bathyarchaeota subgroup Bathy-8 in tentials of Bathyarchaeota, with some lineages possibly being enrichments of estuarine sediments with the biopolymer lignin. capable of utilizing both organic and inorganic carbon com- Other organic substrates (casein, oleic acid, cellulose, and phenol) pounds for biosynthesis and energy production. However, no did not significantly stimulate growth of Bathyarchaeota.Mean- direct proof of carbon or energy sources has so far been obtained while, putative bathyarchaeotal tetraether lipids incorporated 13C Bathyarchaeota for based on laboratory experiments. SCIENCES 13 from C-bicarbonate only when added in concert with lignin. Our Here we report our efforts to enrich Bathyarchaeota from ENVIRONMENTAL results are consistent with organoautotrophic growth of a bathy- marine sediment in the laboratory by setting up a series of en- archaeotal group with lignin as an energy source and bicarbonate richments with diverse organic compound classes, including lip- ’ as a carbon source and shed light into the cycling of one of Earth s ids (oleic acid), proteins (casein), aromatic monomers (phenol), most abundant biopolymers in anoxic marine sediment. aromatic polymers (lignin), and structural carbohydrates (cellu- lose). The addition of lignin stimulated the growth of Bathy- lignin degradation | Bathyarchaeota | sedimentary carbon cycling | lipid | archaeota affiliated with the Bathy-8 subgroup. During growth on carbon fixation lignin, incorporation of inorganic carbon (IC) into archaeal lipids Bathyarchaeota he members of (formerly referred to as the Significance T“Miscellaneous Crenarchaeotal Group”) (1, 2) are estimated to be among the most abundant microorganisms on the planet Marine sediment holds the largest organic carbon pool on (3) and are particularly common in marine sediments (1, 4–7). earth, where microbial transformation of carbon is considered The phylum Bathyarchaeota contains more than 19 subgroups/ a key process of carbon cycling. Bathyarchaeota are among the lineages with low intragroup similarities (3, 5, 8, 9), and its most abundant and active groups of microorganisms in marine members have been suggested to play a globally important role sediment. It has been suggested that Bathyarchaeota may play in the breakdown of organic matter (10) through fermentation a globally important role in the carbon cycling in the marine (11), acetogenesis (3), and methanogenesis (12). However, due environment through fermentation of complex organic sub- to the lack of pure culture isolates and difficulty of obtaining stances, acetogenesis, and methanogenesis based on meta- enrichments of Bathyarchaeota (13), the metabolic properties genome analysis. Here we provide several lines of converging and capabilities of these abundant and widespread have evidence suggesting the bathyarchaeotal group Bathy-8 is able so far been inferred mostly from single-cell genomic and meta- to grow with lignin as an energy source and bicarbonate as a genomic analyses (2, 3, 11, 12, 14). carbon source. Consequently, members of the Bathyarchaeota The first metabolic insights into Bathyarchaeota came from are probably important, previously unrecognized degraders organic-rich sub-seafloor sediments of the Peru Margin, where of lignin. these Archaea were inferred to be assimilating sedimentary organic carbon based on the δ13C-isotopic compositions of intact polar Bathyarchaeota Author contributions: T.Y. and F.W. designed research; T.Y., W.W., and W.L. performed lipids (10). The organoheterotrophic physiology of research; T.Y., W.W., M.A.L., K.-U.H., and F.W. analyzed data; and T.Y., W.W., M.A.L., was further supported through analyses of bathyarchaeotal ge- K.-U.H., and F.W. wrote the paper. nomes by single-cell genome and metagenome analyses, and genes The authors declare no conflict of interest. encoding enzymes for the degradation, transport, and utilization This article is a PNAS Direct Submission. of detrital proteins, aromatic compounds, and plant-derived car- Published under the PNAS license. bohydrates were identified (2, 11, 14). Peptidase activity in the Data deposition: The sequences reported in this paper have been deposited in the GenBank sediments was measured, and the gene of an extracellular pepti- database (accession nos. PRJNA398600 and PRJNA398689). The metagenome and bathy- dase from Bathyarchaeota was expressed in vitro and character- archaeotal genomes reported in this paper have been deposited in the GenBank database ized, supporting the inferred capacity of Bathyarchaeota to (accession no. PRJNA418892). degrade proteins (11, 15). Meanwhile, stable-isotope probing ex- 1T.Y. and W.W. contributed equally to this work. periments indicated that members of two subgroups assimilated 2To whom correspondence should be addressed. Email: [email protected]. several organic substrates, including acetate, glycine, urea, lipids, This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. and complex mixtures of organic growth substrates, while showing 1073/pnas.1718854115/-/DCSupplemental. no significant incorporation of carbon from proteins (16). On the

www.pnas.org/cgi/doi/10.1073/pnas.1718854115 PNAS Latest Articles | 1of6 Downloaded by guest on September 27, 2021 was demonstrated. This study presents an in vitro enrichment of on the growth of Bacteria but significantly stimulated the growth Bathyarchaeota and suggests the organoautotrophic metabolism of Archaea, particularly Bathyarchaeota. Total archaeal gene- of members of this phylum, which likely plays an important role copy numbers increased by about two and three times at t6 and in the carbon cycle in marine sediments. 11-mo incubation (t11), while those of Bathyarchaeota climbed to more than 10 times at t11 compared with the original sample. Results Thus, strong growth of Bathyarchaeota was achieved only in re- Enrichments Set-Up. Numerous enrichment cultures from Day- sponse to lignin addition. Lignin degradation was monitored by angshan estuarine sediments of the northern East China Sea the decrease in the concentrations of total dissolved phenolic (Fig. S1) were set up by adding diverse organic compound compounds after incubation. There were ∼102 mg/L phenolic classes, i.e., the long-chain fatty acid oleic acid, the protein ca- compounds in the culture when 500 mg/L lignin was added, sein, the aromatic monomer phenol, the phenolic polymer lignin, which decreased to ∼25 mg/L after 2.5-mo incubation. and the polymeric carbohydrate cellulose (Materials and Meth- ods). Sediment slurries without the addition of organic com- Archaeal and Bathyarchaeotal Community Composition. Since the pounds were used as controls. Changes in the abundances of enrichment of Bathyarchaeota was the aim of this study, and only Bacteria, Archaea, and Bathyarchaeota were then monitored by lignin showed significant stimulation of bathyarchaeotal growth, qPCR using universal bacterial and archaeal 16S rRNA gene further detailed experiments were undertaken with the original primers and Bathyarchaeota-specific 16S rRNA gene primers. sample, lignin enrichments, and controls. Archaeal 16S rRNA gene Bathyarchaeota The organic compounds were initially added at concentrations of analyses show that (40%) and A 50 mg/L and were increased to 500 mg/L after 3.5- and 6-mo (33%) (Fig. 2 ) were the dominant archaeal groups in the Parvarchaea incubation. original sample followed by (8%) and Treatment responses to substrate additions and controls are (5%). After amendment with lignin, the relative abundance of Bathyarchaeota shown in Fig. 1. In the control samples, the abundance of Bac- within the archaeal community increased to over t A teria, Archaea, and Bathyarchaeota showed only small fluctua- 65% at 11 (Fig. 2 ), compared with a strong decrease in control – A tions over time. Casein initiated the strongest growth stimulation samples (8 23% of total Archaea) (Fig. 2 ). The percentage of in total Bacteria and Archaea, with an increase in bacterial and methanogenic reads increased in both lignin cul- archaeal 16S rRNA gene copies of about three and nine times at tures and no-substrate controls, whereas the relative abundance of Thaumarchaeota decreased across lignin treatments and no-substrate 6-mo incubation (t6), respectively; bathyarchaeotal gene copies increased only slightly in response to casein. Cellulose stimulated controls, showing a stronger percent decrease in the former. growth of Bacteria after 3.5 mo incubation (t ) but had no in- The bathyarchaeotal community in the original sample was 3.5 dominated by the subgroups Bathy-8 (49%), Bathy-6 (24%), and fluence on archaeal or bathyarchaeotal gene copies. Phenol and B oleic acid had no obvious influence on the gene copies of Bac- Bathy-12 (10%) (Fig. 2 ) based on a phylogenetic tree of teria, Archaea, or Bathyarchaeota. Lignin showed little influence 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 opera- tional 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 Bath- yarchaeota 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 13 change significantly at t6 and t11. However, the δ C values of phytane, BP-0, and BP-2 showed a different pattern. In the 13C-IC–amended control samples, the δ13C value of phy- tane 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 Fig. 1. The change in abundance of Bacteria, Archaea, and Bathyarchaeota in sediment slurries after incubation with different organic compounds for showed no clear increase relative to concentrations after 6 mo P > 13 3.5, 6, and 11 mo. The abundance (gene copies per gram wet weight of ( 0.05). Production of C-acetate, indicative of acetogenesis −1 13 13 – sediment; gene copies·gww ) is quantified based on the respective 16s rRNA from C-IC, was evident in all C-IC amended controls and genes. The error bars are obtained from replicate incubations. lignin treatments after 6 mo, as indicated by strong increases in

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1718854115 Yu et al. Downloaded by guest on September 27, 2021 Fig. 2. Comparison of archaeal communities at the phylum level (A) and of bathyarchaeotal communities at the subgroup level (B) in response to lignin SCIENCES addition over time. t0, t3.5, 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 ENVIRONMENTAL t3.5 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.

δ13C-acetate. However, after 11 mo, high δ13C-acetate values community after 11 mo of incubation, with the subgroup Bathy- were detected only in lignin treatments, consistent with sustained 8 accounting for most of this increase (Fig. 2). Our work suggests acetogenesis in the presence of lignin. that members of the Bathyarchaeota are potentially important, previously unrecognized degraders of lignin in marine sediments. Discussion Lignin is a class of complex cross-linked phenolic polymers Marine sediments are one of Earth’s largest organic carbon sinks. and, after cellulose, constitutes the second-most abundant bio- Microbial transformation of carbon is considered a key process polymer on Earth. Lignin is particularly important in the for- influencing the carbon flow in sediment and ultimately atmo- mation of cell walls in vascular plants, comprising up to 25% spheric oxygen and carbon dioxide concentrations (17). Never- of plant biomass (19). Structural polymers, such as cellulose, theless, the controls on microbial organic carbon cycling are not hemicellulose, and lignin, constitute the bulk of terrestrial or- well understood, including the biogeochemical role of Archaea, ganic matter. Terrestrial organic matter is estimated to contrib- which are often abundant in marine sediments (18). In this study, ute one-third of the total buried organic carbon in marine we demonstrate that lignin addition significantly stimulates the sediments (20). This contribution decreases from nearshore to growth of Bathyarchaeota (Fig. 1), which are a dominant group offshore but remains high, up to 15%, even in basin sediments of Archaea in marine sediments (11). The relative abundance (21). In terms of global organic carbon burial, nearshore sedi- of Bathyarchaeota reached up to 65% of the total archaeal ments in estuaries and shallow shelf environments play a very

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 13 samples without lignin (B) with/without C-IC at t6 and t11.

Yu et al. PNAS Latest Articles | 3of6 Downloaded by guest on September 27, 2021 Table 1. Carbon isotope composition and concentration of Text). Two genomic bins belonging to Bathy-8 (Bin-L-1 and Bin- acetate in water medium after incubation with/without lignin L-2) were assembled from the lignin enrichment (Fig. S3 and and13C-bicarbonate Table S3). A complete Wood–Ljungdahl pathway (WL; also “ ” Conditions Time 13C-IC δ13C, ‰ Concentration, μM called the reductive acetyl-CoA pathway )wasfoundinboth genomes, indicating the ability of these Bathy-8 members to fix IC − ± ± Lignin t6 N 14.5 6.5* 4.7 1.9 (Tables S4 and S5). The WL differs from other carbon-fixation −19.9 ± 8.2* 5.6 ± 1.5 pathways in being linear rather than cyclic, involving fewer re- Y530± 10 33.6 ± 1.1 action steps and a smaller set of enzymes, and being used for both 1,990 ± 230 5.4 ± 1.7 energy conservation and biosynthesis by the same organisms; these − t11 N 15.9 26.2 traits likely confer energetic advantages under low-energy condi- −17.2 44.3 tions because less metabolic energy is required for the synthesis of Y510± 80 89 ± 10 enzymes and energetically costly intermediates (27–29). High 1,040 ± 40 35.8 ± 5.4 energy efficiency may explain why the WL is widespread among − Control t6 N 8.2* 6.4 anaerobic microorganisms performing low-energy catabolic reac- −10.8* 5.0 tions, such as methanogenesis and acetogenesis, and in dominant Y 1,730 6.5 subsurface sedimentary microorganisms. Members of the Bathy-8 1,530 ± 400 5.7 ± 3.7 group were recently suggested to be capable of methanogenesis − t11 N 28.1 19.9 because a metagenomic bin contained the entire methyl–coenzyme −21.6 11.0 M reductase (MCR) gene cluster (12). No MCR genes were found Y 130 3.9 in Bin-L-1 and Bin-L-2 (Tables S4 and S5). No known genes for n.d. n.d. lignin degradation were found in Bin-L-1, while genes of catalase- peroxidase and 4-oxalocrotonate tautomerase for putative lignin Data are mean values with SDs from duplicate analyses; those without SD 13 and aromatic compound degradation, respectively, were found in were analyzed once. N, no use of labeled substrate of C-NaHCO3; n.d., not 13 Bin-L-2 (Tables S4 and S5). No canonical genes for respiration detected; Y, use of labeled substrate of C-NaHCO3. *Values are not reliable because the amount was not sufficient (<10 μM) for such as dissimilatory sulfate, nitrate, or iron reduction were found δ13C measurement and are provided for reference only. 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 important role, accounting for ∼45% of total organic carbon these compounds suggests that at least two different functional burial in marine sediments (17 and 22). Consequently, anaerobic archaeal groups were assimilating IC for lipid biosynthesis in our lignin degradation in marine sediments, in particular in near- experiments. The lignin-unrelated incorporation of 13C-IC into shore environments, is probably a globally important microbial phytane is presumably related to the methanogenic class Meth- process. The anaerobic biodegradation of lignin in sediments has anococci, which were the second-most abundant group of Archaea been demonstrated previously (23), but the responsible microbes throughout controls and lignin enrichments and which grow using have remained elusive. H2 as an energy source and IC as a carbon source (30). Since the Metagenomic sequence analyses have suggested that Bathyarchaeota incorporation of 13C label into biphytanes was observed only in are capable of utilizing a variety of organic compounds including lignin treatments, which exhibited strong increases only in bathy- proteins (11), aromatic compounds (2), and carbohydrates (3, archaeotal populations, we infer that the dominant Bathyarchaeota 14). However, bathyarchaeotal 16S rRNA gene abundances in- in lignin treatments were responsible for this incorporation. In the creased 10-fold in response to lignin addition in this study and White Oak River estuary, which receives high input of terrestrial did not grow in response to proteins, cellulose, phenol, or oleic organic matter, Bathyarchaeota were suggested to be the pro- acid. This indicates that, at least at the study site, members of ducers of isoprenoidal tetraether lipids bearing a carbon isotopic this phylum play an important role in the degradation of lignin. signature of autotrophy (31). Bathyarchaeota are therefore a In the enrichment with lignin, the doubling (or generation) plausible source of 13C-labeled BP-0 and BP-2 in our study. The time of Bathyarchaeota was estimated to be about 2–3 mo, which fact that the majority of recovered bathyarchaeotal genomes from is similar to that of other marine-sediment archaea enriched in various subgroups contain genes of the WL (3, 14) is in line with the laboratory (24–26). Considering the typically lower carbon the observed 13C-IC assimilation in lignin treatments. Although and energy availability in natural sediments, the in situ genera- the enrichment of Bathyarchaeota in lignin amendments strongly tion times of bathyarchaeotal cells in marine sediments are likely suggests an involvement of Bathyarchaeota in lignin degradation, even longer than in our enrichment cultures. These long generation we cannot entirely rule out the possibility that the IC-derived 13C- times may contribute to the difficulty of enriching Bathyarchaeota labeled incorporation into archaeal lipids is due to the selective in the laboratory and explain why no pure isolates exist. growth of other archaeal community members whose growth and Within the Bathyarchaeota, the subgroup Bathy-8 became lipid biosynthesis were also stimulated selectively by the presence predominant during growth on lignin, comprising ∼90% of the of amended lignin. However, no archaeal group other than the bathyarchaeotal community at the end of experiments (Fig. 2B). Bathy-8 subgroup shows clear growth based on 16S genes and Within the Bathy-8, OTU3326 became dominant (Table S1), lipid biosynthesis in response to enrichment with lignin (Figs. 2 suggesting that this member of Bathy-8 was the main lignin de- and 3 and Table S2). grader in the community. Bathy-8 members are widely distrib- The enrichment of Bathyarchaeota on lignin suggests that uted in various marine and terrestrial habitats, including seeps, members of this phylum could play an important role in the hydrothermal vents, shallow marine sediments, sediments of degradation of lignin in anoxic marine sediments. We hypothe- hypersaline, saline, and freshwater lakes, salt marsh sediments, size that bathyarchaeotal community members affiliated with hot springs, and Arctic peat soils (5). BLAST analyses further- Bathy-8 metabolize methoxy-groups of lignin and combine the ≥ more show that highly similar 16S rRNA gene sequences ( 97% resulting methyl groups with CO2 to acetyl-CoA via the carbon identical with OTU3326) occur in diverse environments, including monoxide dehydrogenase/acetyl-CoA synthase complex (Codh/ deep-sea fan sediments (KX952596), estuarine sediments (JQ245924), Acs). Acetyl CoA is then used as a key intermediate for bio- submarine springs (JF971130), and terrestrial habitats. synthesis or is converted to acetate via cleavage of the cofactor to The metagenome was obtained from the lignin enrichment produce energy (3, 28). This mode of lignin metabolism is and was analyzed (for details see SI Materials and Methods and SI equivalent to that described for acetogenic bacteria (27), of

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1718854115 Yu et al. Downloaded by guest on September 27, 2021 which 88% of tested cultivars can grow by combining methyl qPCR. Bacterial, archaeal, and bathyarchaeotal 16S rRNA genes were quan- tified by qPCR and were amplified using the primers Uni519F/Arch908R, groups from lignin phenols with CO2 (32). Acetate production Bac341F/prokaryotic519R, and Bathy-442F/Bathy-644R, respectively. The re- was indeed measured in the cultures (Table 1). Although both μ μ controls and lignin treatments show clear signs of acetogenic action solution included 10 L of SYBR Premix Ex Taq (TaKaRa), 0.4 L of ROX reference dye (50×; TaKaRa), 0.8 μM of each primer, and 1 μL of template incorporation of CO2 into acetate, suggesting that acetogenesis is DNA. Further details, such as primer sequences and PCR conditions, are also an important process in unamended samples and perhaps in shown in Table S6. Clones with archaeal, bacterial, and bathyarchaeotal 16S native sediments, both the higher acetate concentrations and rRNA genes (7) were used for standard curve construction. The R2 and effi- 13 higher C-enrichment of acetate in lignin treatments after 11 mo ciency of each individual qPCR assay are shown in Table S6. The abundance indicate that prolonged stimulation of acetogenic activity and CO2 of each targeted gene in the DNA assemblage was determined in triplicate reduction occurs only in lignin-amended treatments. analyses. In conclusion, the enrichment of the Bathy-8 subgroup and the The mean doubling times (T)ofBathyarchaeota were calculated according = − − = − − incorporation of IC into putative bathyarchaeotal lipids in lignin to the formula: T (t2 t1)/(log2X2 log2X1)orT (t2 t1)/[3.322 (logX2 × (t2 − t1)/T = amendments, combined with genomic and geochemical evi- logX1)], which is generated from the formula X1 2 X2. X1 and X2 are the bathyarchaeotal abundance at incubation time t and t , respectively. dence, strongly suggest an organoautotrophic lifestyle for these 1 2 Bathyarchaeota . Members of this ubiquitous subgroup may play Illumina Sequencing and Data Analysis. Since bathyarchaeotal growth was an important, previously unrecognized role in the degradation of observed only in treatments to which lignin had been added as a growth

terrestrially derived lignin in marine sediment. Such an affinity substrate, only the original sample (t0), the lignin cultures IC+lignin (t3.5), IC+ Bathyarchaeota 13 13 for lignin is consistent with high abundances of in lignin (t6) × 2, C-IC+lignin (t6) × 2, IC+lignin (t11) × 2, and C-IC+lignin 13 terrestrially influenced continental margin settings (5, 8, 31). In (t11) × 2, and the control cultures IC (t3.5), IC (t6) × 2, C-IC (t6) × 2, IC (t11) × 2, 13 × addition to accounting for a major fraction of organic carbon and C-IC (t11) 2 (a total of 19 samples) were processed for archaeal 16S burial in marine sediments, lignocellulose material is the most rRNA gene sequencing. The hypervariable V4 regions of archaeal 16S rRNA abundant renewable energy resource on our planet. Our finding genes were amplified from original and enrichment samples using primer μ that Bathyarchaeota mediate lignin degradation may thus result sets U519F/Arch806R (Table S6) (35). Each 50- L reaction solution contained 10× PCR buffer, dNTP (100 μM each), 0.25 μM of each primer, 2.5 U of DNA in new strategies for the use of plant material during bioenergy polymerase (Ex-Taq; TaKaRa), and ∼10 ng of total DNA (measured by a production. NanoDrop 2000). PCR products were purified using an E.Z.N.A. Gel Extrac- tion Kit (Omega Bio-tek) according to the manufacturer’s instructions.

Materials and Methods The 16S rRNA gene amplicons containing unique 8-mer barcodes used for SCIENCES Sample Collection. The uppermost 10 cm of intertidal sediments were col- each sample were pooled at equal concentrations and were sequenced on ENVIRONMENTAL lected from Dayangshan Island (30.592817 N, 122.083493 E) in Hangzhou Bay, the Illumina MiSeq platform using 2× 250-bp cycles and the 500-cycle MiSeq northern East China Sea, China (Fig. S1). Samples were stored anoxically on Reagent Kit v2 (Illumina) according to the manufacturer’s instructions. Raw ice in gas-tight bags and were transported to the laboratory within 3 h, reads were removed if they contained a 50-bp continuous fragment with an where they were kept at 4 °C until further treatment. The samples were average quality score of less than 30 and/or any ambiguities. Filtered reads dominated by silt sediment, which is mainly supplied by the Yangtze River were merged together using FLASH version 1.2.6 (36). Further analysis was (33) and is considered to be rich in terrigenous organic matter (34). performed using the QIIME standard pipeline (37). In particular, sequence reads were first filtered to remove low-quality or ambiguous reads, and Cultivation Conditions. About 600 g of sediment was thoroughly mixed with chimeric sequences were removed using the UCHIME program version 4.2

2 L of anaerobic artificial seawater medium (26) without Na2SO4 in an an- (38). The sequences were clustered into OTUs using UCLUST (38) with a 97% aerobic chamber (Type B vinyl; Coy). After centrifugation at 7,610 × g in sequence identity threshold. was assigned using the Greengenes 500-mL centrifuge bottles, the supernatant was removed, and this process was database (version gg_13_5, greengenes.secondgenome.com/). The obtained repeated again to largely eliminate the original porewater. Afterward, the archaeal sequences from each sample were randomly subsampled to gen- sediment was mixed again with 3 L of anaerobic artificial seawater medium erate a uniform depth of 10,372 sequences. Species richness, species di- and was dispensed into serum bottles as 100-mL sediment slurries. The fol- versity, and coverage were computed using QIIME’s alpha diversity pipeline. lowing five organic growth substrates were added to different experiments Five metrics were calculated: the observed number of species, coverage at a dose of 50 mg/L: oleic acid (Sinopharm 30138518), phenol (Sinopharm (through rarefaction analyses), the Chao1 index, the Shannon index, and 100153008), casein (Sinopharm 69006227), alkali lignin (Sigma 45-471003), the Simpson index. The number of archaeal sequences in the above-men- 13 and cellulose (Sinopharm 68005761). In addition, C-NaHCO3 was added to tioned 19 samples ranged from 10,425 to 33,848 (Table S7). The number of 13 a final concentration of 0.25 μM [the percentage of C-NaHCO3 (mol/mol) to archaeal OTUs (cutoff, 97%) in these 19 sediment samples ranged from total NaHCO3 was 5%] to test for the assimilation of IC in archaeal lipids. The 423 to 869. The coverage calculation indicated that the analyzed sequences bottles were sealed with butyl rubber stoppers and aluminum crimp seals, and covered the diversity of archaeal populations in the investigated samples

then the headspace was evacuated by gas pump and refilled with 99.99% N2 (Table S7). 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; Phylogenetic Analyses. From the Illumina MiSeq dataset, OTUs affiliated with meanwhile two slurries were also incubated without the addition of any or- Bathyarchaeota which contained more than 100 sequences were selected for ganic substrates as controls. Slurries were incubated horizontally without phylogenetic analyses, and the closest phylogenetic affiliations were de- shaking in the dark at 20 °C for 3.5 mo (t3.5), after which 20 mL of slurries were termined by constructing a bathyarchaeotal 16S rRNA gene tree using ref- sampled using a syringe with needle. The collected samples were centrifuged erence sequences of the 17 bathyarchaeotal subgroups (5) as phylogenetic at 13,800 × g to separate the supernatant and sediments and were stored at anchors. For the construction of the phylogenic tree, the sequences were −80 °C for DNA and lipid isolation and measurement of chemical parameters. aligned using CLUSTALX 1.83, and the reference sequences were trimmed, Afterward, higher concentrations of organic substrates (500 mg/L of each retaining 900 bp after alignment. Neighbor-joining phylogenetic trees were 13 substrate) and C-NaHCO3 [final concentration is 0.5 μM; the percentage of constructed from pairwise comparisons with the Kimura 2-parameter dis- 13 C-NaHCO3 (mol/mol) to total NaHCO3 was 10%] were added to the tance model using the molecular evolutionary genetics analysis (MEGA) remaining cultures, which were then incubated for another 2.5 mo (t6). Then program, version 6 (39). Bootstrap values are based on 1,000 replicates for 20 mL of the slurries were collected, more organic substrates (500 mg/L of testing the robustness of the inferred topology. Interactive Tree of Life each substrate) were added again, and these samples were incubated for (iTOL) (itol.embl.de/) was used to modify the phylogenetic trees. additional 5 mo. Thus, slurries were obtained at three time points after in-

cubation, i.e., at t3.5, t6, and t11 mo. Data Deposition. Sequences of Illumina sequencing raw data were submitted to GenBank under accession numbers PRJNA398600 and PRJNA398689. 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 manufac- Lipid Extraction and Analyses. The total lipid extracts (TLEs) were extracted turer’s protocol. The concentration of DNA was measured by using a from lyophilized samples (∼1 g) by a modified Bligh and Dyer method (40).

NanoDrop 2000 spectrophotometer (Thermo Scientific), followed by freez- For the samples at t6, a TLE aliquot of 90% was chemically treated to convert ing at −80 °C until processing. di- and tetraether lipids into their hydrocarbon derivatives phytane and

Yu et al. PNAS Latest Articles | 5of6 Downloaded by guest on September 27, 2021 several biphytanes with 0-3 rings via ether-cleavage methods (41). For Acetate Concentration and Carbon Isotope Measurement. Concentrations and samples that had been incubated for 11 mo, intact polar archaeal lipid carbon isotope composition of acetate in the supernatants of slurries were fractions were first purified by preparative HPLC with a fraction collector analyzed by on LC linked to isotope ratio mass spectrometry (LC-IRMS) (42) and then were converted to hydrocarbon derivatives according to the according to the method described by Heuer et al. (45). above method (41). The δ13C values of archaeal intact polar lipid derivatives were determined on a Finnigan Trace gas chromatograph (Thermo Scien- ACKNOWLEDGMENTS. We thank Karl O. Stetter (University of Regensburg) tific) connected to a DELTA Plus XP isotope ratio mass spectrometer via a GC and Hongbo Yu (Huazhong University of Science and Technology) for kind combustion III interface (both from Thermo Fisher) according to the method suggestions to improve the manuscript and Jiahua Wang (Shanghai Jiao described by Kellermann et al. (43). 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 Measurement of Total Phenolic Content. The lignin degradation was initially 41525011, 91751205, and 91428308, and the Deutsche Forschungsgemein- surveyed by measuring the concentrations of phenolic compounds in the schaft through the Gottfried Wilhelm Leibniz Program Award Hi 616-14-1 supernatant of slurries using the Folin–Ciocalteu method (Sinopharm 73104861) (to K.-U.H.). This study is also a contribution to the Integrated Marine Bio- as described previously (44). sphere Research International Project and the Deep Carbon Observatory.

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