Activity and viability of methanogens in anaerobic digestion of unsaturated and saturated long-chain fatty acids

Abstract

Lipids can be anaerobically digested to methane, but methanogens are often considered to be highly sensitive to the long-chain fatty acids (LCFA) deriving from lipids hydrolysis. In this study, the effect of unsaturated (oleate [C18:1]) and saturated (stearate [C18:0] and palmitate [C16:0]) LCFA toward methanogenic archaea was studied in batch enrichments and in pure cultures. Overall, oleate had a more stringent effect on methanogens than saturated LCFA, and the degree of tolerance to LCFA was different among distinct species of methanogens. Methanobacterium formicicum was able to grow in both oleate- and palmitate-degrading enrichments (OM and PM cultures, respectively), whereas Methanospirillum hungatei only survived in a PM culture. The two acetoclastic methanogens tested, Methanosarcina mazei and Methanosaeta concilii, could be detected in both enrichment cultures, with better survival in PM cultures than in OM cultures. Viability tests using live/dead staining further confirmed that exponential growth-phase cultures of M. hungatei are more sensitive to oleate than are M. formicicum cultures; exposure to 0.5 mM oleate damaged 99% ± 1% of the cell membranes of M. hungatei and 53% ± 10% of the cell membranes of M. formicicum. In terms of methanogenic activity, M. hungatei was inhibited for 50% by 0.3, 0.4, and 1 mM oleate, stearate, and palmitate, respectively. M. formicicum was more resilient, since 1 mM oleate and >4 mM stearate or palmitate was needed to cause 50% inhibition on methanogenic activity.

Fig 1

DGGE pattern of archaeal 16S rRNA gene fragments present in OM and PM enrichment cultures during successive transfers with addition of Methanospirillum hungatei (Mh) or Methanobacterium formicicum (Mf) as hydrogen consumers. Arrows indicate the presence of added methanogens in the enrichment cultures. Numbers 1 to 8 indicate the DGGE bands identified by cloning and sequencing (see the phylogenetic affiliations in Table 1); clones were retrieved from the inoculum sludge used to start up the enrichment series OM and PM.

Fig 2

DGGE pattern of archaeal 16S rRNA gene fragments present in OM and PM enrichment cultures during successive transfers with addition of Methanosaeta concilii (Mc) or Methanosarcina mazei (Mm) as acetate consumers. Methanospirillum hungatei (Mh) and Methanobacterium formicicum (Mf) were added to PM and OM cultures, respectively, to ensure low hydrogen concentration during LCFA degradation. Arrows indicate the presence of added methanogens in the enrichment cultures.

Fig 3

Percentage of membrane damage cells of M. hungatei and M. formicicum in pure cultures when grown on H₂/CO₂, without LCFA addition and in the presence of 0.5 and 1 mM oleate. Live/dead staining images of M. hungatei and M. formicicum are shown in subpanels a to d: the letters correlate the images with the assay and the sampling time indicated by the corresponding letter in the graph. Scale bar, 10 μm.