ARPHA Conference Abstracts : Conference Abstract
Conference Abstract
Non-syntrophic Methanogenic Hydrocarbon Degradation by an Archaeal Species
expand article infoZhuo Zhou, Cuijing Zhang§, Pengfei Liu‡,|, Lin Fu, Rafael Laso-Pérez¶,#,¤, Lu Yang, Liping Bai, Jiang Li, Min Yang, Junzhang Lin«, Weidong Wang«, Gunter Wegener¶,#, Meng Li§, Lei Cheng
‡ Biogas Institute of Ministry of Agriculture and Rural Affairs, Chengdu, China
§ Shenzhen University, Shenzhen, China
| Lanzhou University, Lanzhou, China
¶ Max Planck Institute for Marine Microbiology, Bremen, Germany
# University Bremen, Bremen, Germany
¤ Centro Nacional de Biotecnología, Madrid, Spain
« SINOPEC, Dongying, China
Open Access


Methanogenic hydrocarbon biodegradation alters the composition of many subsurface oil reservoirs (Jones et al. 2007). This process reduced the crude oil quality by removing alkanes and thus increasing the oil viscosity. The process has been described for syntrophic associations of hydrocarbon-degrading bacteria and methanogenic archaea (Zengler et al. 1999, Dolfing et al. 2007). However, recent culture-independent studies suggest that the archaeon ‘Candidatus Methanoliparum’ may combine alkane degradation and methanogenesis (Laso-Pérez et al. 2019, Borrel et al. 2019). Here we cultured Ca. Methanoliparum from a subsurface oil reservoir. To study this culture, situ hybridization, metagenomics and metatranscriptomics were combined with stable isotope probing and metabolite analyses for describing its functioning and assessing its potential role in reservoir chemistry.

Incubated an anoxic oily sludge of the Shengli oilfield with sulfate-free medium, we established a methanogenic culture. This culture consumed various different long-chain alkanes, but also alkyl-benzenes and alkyl-cycloalkanes, and produced methane and CO2 as products (Fig. 1a-b). Our analyses revealed that our culture is dominated by a single archaeon, Ca. Methanoliparia (green).

Figure 1.  

Methanogenesis in the oily sludge and visualization of microorganisms.

To study the specific turnover of n-alkanes, the cultures were supplemented with 1,2-13C-labelled or unlabelled n-hexadecane (Fig. 2). Within 100 days of incubation, both compounds were quantitatively converted into methane and carbon dioxide. In the 13C-labelling experiment, around 0.46 mmol of 13CH4 and around 0.15 mmol of 13CO2 were produced, which was equal to 85% to 92% of the stoichiometric conversion of the supplemented labelled hexadecane according to 4C16H34 + 30H2O -- 49CH4 + 15CO2 (Fig. 2a-d).

Figure 2.  

Methanogenic hexadecane degradation by Ca. Methanoliparum.

We examined the functioning of Ca. Methanoliparum in the hexadecane-degrading culture using amplicon sequencing, metagenomics and metatranscriptomics. In the archaeal domain, the relative abundance of Ca. Methanoliparum in the hexadecane-degrading cultures comprised up to 75% of the total abundance according to analysis of archaeal 16S rRNA genes. Furthermore, Ca. Methanoliparum accounted for approximately 34–40% of the total microbial community as determined by metagenomic read recruitment estimation (Fig. 2e-f).

We analysed the gene expression patterns of Ca. Methanoliparum during methanogenic hexadecane degradation (Fig. 3). The genes encoding the methanogenic hexadecane degradation pathway ranked among the top 10% to 25% of all Ca. M. thermophilum transcribed genes. Moreover, genes of Ca. M. thermophilum encoding ACR and MCR ranked among the top 2% of all transcribed genes within the whole community (Fig. 3b). The MAGs of Ca. M. thermophilum also showed the highest transcription among all described MAGs (Fig. 3c). These analyses indicate that Ca. M. thermophilum performs both the degradation of hexadecane and the formation of methane.

Figure 3.  

Hexadecane degradation pathway of Ca. Methanoliparum.

We searched the cell extracts of the hexadecane-degrading cultures for hexadecyl-CoM formation using Q-Exactive Plus Orbitrap masss pectrometry. The unlabelled hexadecane culture contained a prominent mass peak of m/z = 365.21868 that matches the mass produced by synthesized authentic standard of hexadecyl-CoM. Fragmentation of both peaks yielded hexadecyl-thiol (m/z = 257.23080, C16H33S), ethenesulfonate (m/z = 106.98074, C2H3SO3) and bisulfite (m/z = 80.96510, HSO3). Moreover, cultures supplied with 1,2-13C-hexadecane produced a peak at m/z = 367.22524 for 1,2-13C-hexadecyl-CoM and the fragment 259.23721 for 1,2-13C-hexadecyl-thiol, with a mass shift of 2 units compared with the unlabelled group. These analyses confirmed the activation of n-hexadecane as hexadecyl-CoM (Fig. 4).

Figure 4.  

Identification of the intermediate hexadecyl-CoM.

Here we demonstrate the activation of different hydrocarbon classes by ACRs of Ca. Methanoliparum, expanding the substrate range of this enzyme to an unforeseen number of compounds. Ca. Methanoliparum couples the degradation of long-chain alkanes and alkyl-substituted hydrocarbons to methane formation, proposed as alkylotrophy. Its metabolic pathways represent an additional mode of methanogenesis, adding to CO2 reduction, methylotrophy, methyl reduction, acetate fermentation and the recently reported methoxydotrophy. Ca. Methanoliparum grows in a wide temperature range, at least between 35 and 55 °C, covering the temperature range of most biodegraded oil reservoirs. Indeed, sequences of Ca. Methanoliparum are present in various anoxic hydrocarbon-rich environments worldwide. Thus, the demonstration of the unique features of Ca. Methanoliparum in hydrocarbon conversion may fundamentally change our view of crude oil transformation and biogeochemical processes in subsurface oil reservoirs. Future studies with Ca. Methanoliparia cultures will resolve the biochemical mechanisms of methanogenic hydrocarbon degradation in archaea, and will be helpful for the application of microbial-enhanced energyrecovery from depleted oil reservoirs.


Alkane, Biodegradation, Methane

Presenting author

Zhuo Zhou

Presented at



We thank A. Oren (The Hebrew University of Jerusalem) for discussing thenaming of the different Ca. Methanoliparum species; R. Conrad and W. B. Whitman fordiscussing the manuscript; K. Wrighton for providing access to the server Zenith; Q. Yuan,Y. Liu, J. Pan, M.-w. Cai and Y.-n. Tang for assisting in data analysis; L.-r. Dai, D. Zhang and L. Li for assisting in cultivation and experiments; and Z. Zhou for technical support. 

Funding program

National Natural Science Foundation of China (National Science Foundation of China) - 92051108 [Cheng]Agricultural Science and Technology Innovation Project of the Chinese Academy of Agriculture Science (Grant No. CAAS-ASTIP-2016-BIOMA).the Central Public-interest Scientific Institution Basal Research Fund (Y2021PT02, Y2021XK06). [Cheng]National Natural Science Foundation of China (National Science Foundation of China) - 41802179 [Zhang]Deutsche Forschungsgemeinschaft (German Research Foundation) - EXC-2077-390741603 [Laso-Pérez]National Natural Science Foundation of China (National Science Foundation of China) - 41802179 [Yang]National Natural Science Foundation of China (National Science Foundation of China) - 31970066 [Bai]Deutsche Forschungsgemeinschaft (German Research Foundation) - EXC-2077-390741603 [Wegener]National Natural Science Foundation of China (National Science Foundation of China) - 91851105 [Li]

Author contributions

L.C. and M.L. initiated the study. L.C., M.L., G.W. and P.-f.L. designed research. J.-z.L., W.-d.W. and Z.Z. collected the oily sludge samples. Z.Z., J.L., M.Y. and L.C.conducted cultivation experiments. Z.Z. and L.Y. performed oil analysis. C.-j.Z., P.-f.L., Z.Z., R.L.-P. and M.L. performed all bioinformatics analyses. R.L.-P. and L.C. designed CARD-FISH probes, and R.L.-P. performed CARD-FISH and cell visualization. L.F., L.C. and L.-p.B. performed metabolite analyses. P.-f.L., R.L.-P., G.W., M.L. and L.C. analysed data and wrote the manuscriptwith contributions from all of the co-authors. 

Conflicts of interest

The authors have declared that no competing interests exist.


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