When we first observed the phenomenon of methane-dependent selenate reduction, my supervisor He-Ping Zhao, a professor in Zhejiang University, said he would invite us to an AWESOME buffet (which is generally expensive to students) if we deciphered how this coupled bio-process worked. I hope he remembers those words and look forward to eating together! (^_<)

Joke aside. Methane is not only a potent greenhouse gas able to intensify global warming, but also an excellent carbon source to support microbial growth by coupling the reduction of diverse electron acceptors. Our group is super interested in the microorganisms and underlying mechanism of methane cycling, and has spent almost ten years in applying methane to treat wastewater contaminated by nitrate, perchlorate, and heavy metals (e.g., selenate, chromate, antimonate), to remove water pollutants and decrease greenhouse gas emissions simultaneously.

Selenate is a representative case. Although selenium is known as an essential element for living organisms, high concentrations of selenium -- for example in the agricultural drainage and industrial wastewater -- may result in significant ecological damage, as only one order of magnitude separates essential and toxic levels. Microbial reduction of selenium oxyanions to precipitated elemental selenium, in the presence of carbon source, is a promising remediation for selenate-contaminated water. However, it is really a VERY long journey since we firstly observed the methane-dependent selenate reduction, till this paper is published recently.

Methane-based bio-reduction processes can be performed by methanotrophs either independently (e.g., bio-reduction of nitrate, nitrite, iron, manganese) or in consortia with other microbial partners (e.g., bio-reduction of sulfate). To explore whether and how methane oxidation could couple to selenate reduction, we constructed and ran a bioreactor in 2015, inoculated by wetland sediment and with methane and selenate as the sole electron donor and acceptor, under hypoxic conditions. After around one month of operation, the reactor turned light red, which is a character of the reduction product elemental selenium. We were all excited and continued the operation to enrich the microbial community in the subsequent 300 days. Then we incubated the enrichment culture in batch experiments and verified the observed phenomenon of methane-dependent selenate reduction. A problem arose at that time: 16S rRNA gene sequencing and quantitative PCR, as we always performed in the past, only gave a rough profile for community structure and active genes, which were insufficient to decipher the responsible members or the possibly trophic relationships for the coupled process. So we asked Professor Marc Strous, who is a pioneer in methane oxidation field, for help and very luckily, Marc kindly agreed to provide the established meta-omic platform for us!

Then I flied to University of Calgary in the winter of 2017. Um…it was really cold in Canada, down to -50 ºC sometimes, but I did feel the warmth because Strous’ group was very nice and patient to give me huge help both in the research and in the life. Combining metagenomics and metaproteomics with the help of Xiao-Li Dong and Angela Kouris, we found the interesting results that different lineages of microorganisms performed methane oxidation and selenate reduction, respectively, with methane-derived organic acids serving as interspecies electron shuttles. This amazed us because such trophic relationship using diffusible intermediates of methane oxidation has never been confirmed experimentally.

We then conducted a series of batch experiments and confirmed the meta-omic predicted trophic relationships. Excitedly, we drafted the manuscript, revised for dozens of rounds, submitted with confidence, but finally received the rejection… The reviewers requested the gene knockout experiment and fluorescence in situ hybridization (FISH) images, to directly validate the roles of proposed gene and the distribution of different microorganisms. After trying three months of FISH, we realized it was difficult to obtain an image with high quality by ourselves. So we consulted Professor Gene Tyson, an expert in methane oxidation also accumulating much experience in performing FISH, and again were very luck to receive a positive feedback!

The biological samples were then prepared and sent to Australia, for FISH imaging carried out by Simon McIlroy. Meanwhile, we performed the gene knockout experiment on a purchased isolate which is phylogenetically similar to the predicted key member, because the purification and isolation from a complex community had no guarantee to succeed. These supplementary experiments validated the contribution of the gene initially annotated as periplasmic nitrate reductase in reducing selenate, and of the diffusible organic acids in connecting separated microorganisms. After incorporating the new results and restructuring the paper, we submitted again and feel very fortunate to see the publication of this paper which takes almost six years by scientists from three countries!.

It is absolutely not the end; instead, a new journey starts. This paper reveals the trophic relationship using organic acids produced by the fermentation of methane-derived carbon as interspecies electron shuttles during the methane-dependent selenate reduction. It still remains unknown whether this trophic interaction is specific or universal for methane-based bio-reduction processes. For example, would diffusible intermediates also play a role in methane-dependent nitrate or perchlorate reduction? In the future, our group will continue to investigate the phenomena, microorganisms and mechanisms during methane oxidation, for the sake of the simultaneous removal of water pollutants and greenhouse gas.

Finally, we would like to take the chance to thank all the members from Strous’s group, Tyson’s group, and Zhao’s group that supported us and made this long journey highly enjoyable. Thanks legends. Hope to raise a toast with you all some day!

Please check our paper for the full details at https://doi.org/10.1038/s41396-021-01044-3.