DNA-SIP based genome-centric metagenomics identifies key long-chain fatty acid-degrading populations in anaerobic digesters with different feeding frequencies

Abstract

Fats, oils and greases (FOG) are energy-dense wastes that can be added to anaerobic digesters to substantially increase biomethane recovery via their conversion through long-chain fatty acids (LCFAs). However, a better understanding of the ecophysiology of syntrophic LCFA-degrading microbial communities in anaerobic digesters is needed to develop operating strategies that mitigate inhibitory LCFA accumulation from FOG. In this research, DNA stable isotope probing (SIP) was coupled with metagenomic sequencing for a genome-centric comparison of oleate (C18:1)-degrading populations in two anaerobic codigesters operated with either a pulse feeding or continuous-feeding strategy. The pulse-fed codigester microcosms converted oleate into methane at over 20% higher rates than the continuous-fed codigester microcosms. Differential coverage binning was demonstrated for the first time to recover population genome bins (GBs) from DNA-SIP metagenomes. About 70% of the 13C-enriched GBs were taxonomically assigned to the Syntrophomonas genus, thus substantiating the importance of Syntrophomonas species to LCFA degradation in anaerobic digesters. Phylogenetic comparisons of 13C-enriched GBs showed that phylogenetically distinct Syntrophomonas GBs were unique to each codigester. Overall, these results suggest that syntrophic populations in anaerobic digesters can have different adaptive capacities, and that selection for divergent populations may be achieved by adjusting reactor operating conditions to maximize biomethane recovery.

Microbial rRNA gene expression and co‐occurrence profiles associate with biokinetics and elemental composition in full‐scale anaerobic digesters

“…The high representation of syntrophs and methanogens in the co‐occurrence network may also be attributed to their metabolic specialization (Sieber et al ., ), suggesting that their ecological niches are not easily filled by other taxa (Werner et al ., ). Yet, recent work has shown high strain variability within digester syntrophic communities under different feeding strategies (Ziels et al ., , ), indicating that selective forces may still govern the syntrophic community structure in different digester environments.…”

Elucidating syntrophic butyrate-degrading populations in anaerobic digesters using stable isotope-informed genome-resolved metagenomics

“…Additionally, two 13 C-enriched OTUs in both digesters were assigned to Methanothrix (formerly Methanosaeta ), which likely scavenge the 13 C-acetate generated by Syntrophomonas during 13 C-butyrate degradation. While one previous study observed that Syntrophaceae was predominantly enriched in anaerobic digester granular sludge incubated with 13 C-labeled butyrate (10), various other studies also detected Syntrophomonadaceae populations as active syntrophic butyrate degraders in anaerobic digester sludge using 14 C-labeled butyrate and MAR-FISH (15), in anaerobic digester sludge through SIP using 13 C-labeled oleate (12), and in rice paddy soil with SIP using 13 C-labeled butyrate (16). In the latter two studies, acetate-scavenging partners ( Methanothrix and Methanosarcinaceae ) were also enriched.…”

“…Recently, we demonstrated the potential of combining DNA-stable isotope probing (SIP) with genome-resolved metagenomics to identify syntrophic populations degrading the long-chain fatty acid, oleate (C 18:1 ), within anaerobic digesters (12). Stable-isotope informed metagenomic sequencing can enrich metagenomic libraries with genomic sequences of actively-growing microbes that incorporate 13 C into their biomass from an added labeled substrate (13), and thus allows for a ‘zoomed in’ genomic view of low-abundance populations such as syntrophs.…”

“…Stable-isotope informed metagenomic sequencing can enrich metagenomic libraries with genomic sequences of actively-growing microbes that incorporate 13 C into their biomass from an added labeled substrate (13), and thus allows for a ‘zoomed in’ genomic view of low-abundance populations such as syntrophs. We also demonstrated that this approach was amenable for recovering high-quality microbial genomes using a differential-coverage based binning approach, as genomes from active microbes have low abundance in DNA from 12 C controls but are enriched in 13 C-ammended treatments (12). Here, we applied stable-isotope informed metagenomics to resolve the genomic makeup of active syntrophic butyrate-degrading populations within an anaerobic digester.…”

“…Here, we applied stable-isotope informed metagenomics to resolve the genomic makeup of active syntrophic butyrate-degrading populations within an anaerobic digester. We utilized biomass collected from the same anaerobic digesters as were previously used for DNA-SIP with oleate (12) at a similar time point, thus allowing for genomic comparisons using a multi-substrate SIP dataset. This approach identified potential metabolic flexibility in syntrophic populations processing multiple fatty acids within the anaerobic digesters, and elucidated the genomic identity of syntrophic partnerships between active methanogens and bacteria.…”