Nitrite-driven anaerobic ethane oxidation

Abstract

Ethane, the second most abundant gaseous hydrocarbon in vast anoxic environments, is an overlooked greenhouse gas. Microbial anaerobic oxidation of ethane can be driven by available electron acceptors such as sulfate and nitrate. However, despite nitrite being a more thermodynamically feasible electron acceptor than sulfate or nitrate, little is known about nitrite-driven anaerobic ethane oxidation. In this study, a microbial culture capable of nitrite-driven anaerobic ethane oxidation was enriched through the long-term operation of a nitrite-and-ethane-fed bioreactor. During continuous operation, the nitrite removal rate and the theoretical ethane oxidation rate remained stable at approximately 25.0 mg NO₂ ^-N L^-1 d^-1 and 11.48 mg C₂H₆ L^-1 d^-1, respectively. Batch tests demonstrated that ethane is essential for nitrite removal in this microbial culture. Metabolic function analysis revealed that a species affiliated with a novel genus within the family Rhodocyclaceae, designated as 'Candidatus Alkanivoras nitrosoreducens', may perform the nitrite-driven anaerobic ethane oxidation. In the proposed metabolic model, despite the absence of known genes for ethane conversion to ethyl-succinate and succinate-CoA ligase, 'Ca. A. nitrosoreducens' encodes a prospective fumarate addition pathway for anaerobic ethane oxidation and a complete denitrification pathway for nitrite reduction to nitrogen. These findings advance our understanding of nitrite-driven anaerobic ethane oxidation, highlighting the previously overlooked impact of anaerobic ethane oxidation in natural ecosystems.

Keywords: Anaerobic ethane oxidation; Denitrification; Fumarate addition pathway; Greenhouse gas; Microbial culture.

Graphical abstract

Fig. 1

Reactor performance during 350 days of operation. a, Nitrite loading rate and removal rate. b, HRT, the nitrite concentration in influent, and the concentrations of nitrate, nitrite, and ammonium in effluent. Phase I indicates the start-up stage; Phase II indicates the continuous stage.

Fig. 2

Nitrite removal performance in batch tests on day 209. Circles: 95% C₂H₆ + 5% CO₂ in headspace; squares: 95% Ar + 5% CO₂ in headspace.

Fig. 3

Microbial community changes during long-term operation. Relative order-level abundance of microbial population (>2% in at least one sample) was shown.

Fig. 4

Relative abundance and transcript level of genes involved in nitrogen (a) and carbon (b) metabolism pathways. Relative abundance was calculated by DiTing. The asterisks indicate that the metagenomic reads and trimmed metatranscriptomic reads were mapped onto metagenomic assembly using Bowtie2 (v2.3.2) and StringTie (v2.1.5). The relative gene abundance was determaind as GPM, and relative transcriptional abundance was determaind as TPM. MT, metatranscriptomic profiles on day 250. Nitrate reductase (NAR, narB); nitrate reductase (NAD(P)H) (NR); assimilatory nitrate reductase (NAS, nasAB); nitrite reductase (NAD(P)H) (NIT-6); ferredoxin-nitrite reductase (NiR, nirA); membrane-bound (NAR, narGHI) and periplasmic (NAP, napAB) dissimilatory nitrate reductases; dissimilatory nitrite reductase (ccNIR, nrfAH); haem-containing (cd1-NIR, nirS) and copper-containing (Cu-NIR, nirK) nitrite reductases; nitric oxide reductase (NOR, norBC); nitrous oxide reductase (NOS, nosZ); nifKDH, nitrogenase; ammonia monooxygenase (AMO, amoABC); hydroxylamine dehydrogenase (HAO, hao); nitrite oxidoreductase (NXR, nxrAB); hydrazine synthase (HZS, hzs); hydrazine dehydrogenase (HDH, hdh); rTCA, reductive tricarboxylic acid cycle; TCA cycle, tricarboxylic acid cycle; phosphoribulokinase (PRK, prkB); ATP-citrate lyase (aclAB); citryl-CoA synthetase/ lyase (ccsAB/ccl); acetyl-CoA synthetase (ACS, acsABCDE); acetyl-CoA decarbonylase/synthase (CODH/ACS, cdhCDE); methane/ ammonia monooxygenase, (MMO/AMO, mmoABC/amoABC); alcohol/methanol dehydrogenase (ADH, adh; MDH, mdh); acetaldehyde dehydrogenase (ALDH, ald).

Fig. 5

Phylogenetic affiliation and metabolic potential of prospective functional MAGs. Branch lengths in the species tree were hidden if shorter than 0.01. Five neighbor public genomes and bacterial species 'Candidatus Alkanivorans nitratireducens' were included in the species tree. ETC, electron transfer chain; TCA, tricarboxylic acid cycle; long-chain fatty acid-CoA ligase (fadD, lcfB); methylmalonyl-CoA mutase (mcm); propionyl-CoA carboxylase (pccB, accD); succinate-CoA ligase (sucCD); succinate dehydrogenase (sdhABCD, frdB); alkylsuccinate synthase (assAs).

Fig. 6

Prospective metabolic model of nitrite-driven anaerobic ethane oxidation by bacteria 'Ca. A. nitrosoreducens'. The black mark of the gene indicates that the gene has been identified in the genome, while the gray mark indicates that the gene has not been identified. TCA, tricarboxylic acid cycle; acetyl-CoA decarbonylase/synthase (CODH/ACS); alkylsuccinate synthase (assAs); long-chain fatty acid-CoA ligase (fadD, lcfB); methylmalonyl-CoA mutase (mcm); propionyl-CoA carboxylase (pccB, accD); succinate-CoA ligase (sucCD); succinate dehydrogenase (sdhABCD, frdB); 5,10-methylenetetrahydrofolate reductase (Mthfr); Methylenetetrahydrofolate dehydrogenase (Mthfd); Formyltetrahydrofolate deformylase (Fthd); Formate dehydrogenase (fdh); haem-containing (NirS, nirS) and copper-containing (NirK, nirK) nitrite reductases; nitric oxide reductase (NorB, norB); nitrous oxide reductase (NosZ, nosZ); dissimilatory nitrite reductase (nrfAH).