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 Archaea 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 Thaumarchaeota 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 Thermoplasmata 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