Methane formation from complex organic compounds is often a team effort. One classic example dates back to 1967, when Methanobacillus omelianskii, initially thought to be a single microorganism capable of converting ethanol to methane, was shown to consist of two metabolically interdependent organisms. One oxidized ethanol to acetate and hydrogen, while the other consumed hydrogen and produced methane1. This type of cooperation is now known as syntrophy.
Syntrophy is particularly important in the anaerobic conversion of organic compounds to methane. In these processes, substrate degradation by one microorganism is often energetically dependent on the activity of another. By removing metabolic products, the partner helps keep the overall reaction thermodynamically favorable, allowing both organisms to grow through close metabolic cooperation. Yet, when considered as a whole, the conversion to methane can be energetically favorable. If the complete reaction can provide energy for microbial growth, why must substrate degradation and methanogenesis be carried out by different microorganisms? Could a single microorganism couple these reactions and conserve energy within one cell? This became a long-standing question for our group.
From alkane degradation to a broader question
An important clue came from genome-based analyses. In 2019, a study combining genomic analysis with environmental observations suggested that a single microorganism “Candidatus Methanoliparia” might possess both the machinery for long-chain alkane degradation and the pathways for methanogenesis2. This raised the possibility that alkane degradation and methane formation could be integrated within one cell, although at that stage the idea remained largely a genomic prediction.
Around the same time, we had obtained an oil reservoir sample containing Candidatus Methanoliparia, giving us an opportunity to test this idea experimentally. Through cultivation, metabolite analyses, and isotope-tracing experiments, we demonstrated that a single methanogen could directly convert long-chain alkanes to methane, a process we term alkylotrophic methanogenesis3.
This naturally led us to ask a broader question: if methanogens can directly utilize long-chain alkanes, what other complex organic compounds might they be able to convert to methane without a syntrophic partner?
One of the first possibilities we pursued was amino acid metabolism. Genome analyses identified members of Archaeoglobi with pathways potentially linked to amino acid utilization and methane formation, and assistant researcher Yue Yi began exploring this possibility together with master's student Zuorong Cao. They collected the samples from Shengli Oil field and began enriching Archaeoglobi cultures. The initial experiments used enrichment cultures, but convincing amino acid degradation to methane was not observed. Because many organisms were present, however, the negative result could not tell us whether the target microorganism itself lacked this capability. To answer this directly, we needed a pure culture.
Shengli Oil field
Yue Yi and Zuorong Cao therefore continued the cultivation and isolation work for roughly one to two years and eventually obtained the strain that we later named Methanoglobus dolicholipophagus AA-75. At last, we could test the original hypothesis directly. The answer was disappointing: even in pure culture, AA-75 did not exhibit the amino-acid-dependent methanogenesis that had motivated its isolation. Still, we now had something valuable in hand: a pure culture of a previously uncultivated methanogen. So, we asked a different question: If AA-75 did not do what we had expected, what could it actually do?
Scanning microscopy photographs of strain AA-75
A second hypothesis
Yue Yi and Hengwu Ding returned to the genome and re-examined the metabolic potential of AA-75 more broadly. One feature attracted their attention: AA-75 encoded pathways with the potential to link fatty-acid degradation to methanogenesis. This immediately brought us back to our original interest in syntrophy. Long-chain fatty-acid conversion to methane is a classic example of syntrophic metabolism. Could AA-75 integrate these functions within a single cell?
This time, the result was different: AA-75 produced methane from both saturated and unsaturated fatty acids. We then used isotopically labeled stearate to confirm that carbon from the fatty acid was transferred into methane. Because AA-75 was a pure culture, we could attribute fatty-acid degradation and methanogenesis to a single microorganism without invoking a syntrophic partner. For us, this was the central conceptual advance: a process classically associated with syntrophic cooperation could be integrated within one cell.
How broad is the metabolic range of methanogens?
Methanogens have long been considered metabolic specialists, restricted to a relatively small range of simple substrates. Our work on long-chain alkanes and now fatty acids suggests that this view may capture only part of their metabolic potential. We therefore see AA-75 not as the end of this story, but as another step towards defining the metabolic boundaries of methanogens. Genomes suggest that pathways associated with the utilization of amino acids, sugars, and other organic compounds occur in different methanogenic lineages4567, so how many other organic compounds can be converted directly to methane? How often can processes currently interpreted as syntrophic be integrated within a single cell?
Syntrophy remains a fundamental principle of anaerobic methane formation. But as more unconventional methanogens are brought into culture, the boundary between what requires a microbial partnership and what one cell can accomplish may prove more flexible than we once assumed.