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 (C_18:1)-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.

Figure 1

Cumulative methane production (minus blank controls) for the microcosms fed with ¹²C- and ¹³C-labeled oleate over three repeated batch feeding periods. The number above each plot indicates the batch oleate feed round. The black dashed line shows the theoretical methane potential of the added oleate (25.5 ml CH₄; based on 2.9 g COD/g oleate, 8 mm concentration and 35 °C temperature). The black solid line represents the predicted methane production based on non-linear model fitting with a modified Gompertz equation (Supplementary Table 1). Error bars represent the standard deviation of the biological replicates.

Figure 2

Total copies of Syntrophomonas 16S rRNA genes measured by qPCR for each density-gradient fraction recovered from isopycnic separation of DNA from ¹³C-incubated microcosms and ¹²C-controls for both anaerobic codigesters. The filled circles indicate gradient fractions that were pooled for subsequent 16S rRNA gene amplicon sequencing and metagenomic sequencing. Both biological replicate microcosms are shown, and each point represents an average of duplicate technical replicates.

Figure 3

Relative fractions of the 11 most abundant genera in the heavy gradient fractions of ¹³C-incubated samples and ¹²C-controls for sample sets from both codigesters, based on 16S rRNA gene amplicon sequencing. Relative fractions were determined using DESeq2 normalized read counts for all OTUs (Love et al., 2014), and were then aggregated at the genus level. Both biological replicate microcosms are shown.

Figure 4

Phylogenetic overview of differentially abundant genome bins identified in both anaerobic codigester DNA-SIP metagenomes. The tree was constructed based on a concatenated alignment of conserved marker genes within the metagenomic contigs of each bin using PhyloPhlAn v.1.3 (Segata et al., 2013). The color of each genome bin node represents the codigester biomass source, and the height of the outer bars represent the genome bin coverage in the heavy gradient fractions of the ¹³C-incubated samples and ¹²C-controls. The tree was illustrated using GraPhLan v.0.9.7 (Asnicar et al., 2015).

Figure 5

Cumulative coverage values of KEGG ECs potentially involved in LCFA degradation (KEGG map 00071), based on the coverage of all differentially abundant genome bins in the ¹³C-incubated DNA-SIP metagenomes. Values from duplicate biological replicates are shown for both codigesters, and the size of each marker is proportional to the log₁₀ of the EC read coverage.