A metagenome-level analysis of a microbial community fermenting ultra-filtered milk permeate

Abstract

Fermentative microbial communities have the potential to serve as biocatalysts for the conversion of low-value dairy coproducts into renewable chemicals, contributing to a more sustainable global economy. To develop predictive tools for the design and operation of industrially relevant strategies that utilize fermentative microbial communities, there is a need to determine the genomic features of community members that are characteristic to the accumulation of different products. To address this knowledge gap, we performed a 282-day bioreactor experiment with a microbial community that was fed ultra-filtered milk permeate, a low-value coproduct from the dairy industry. The bioreactor was inoculated with a microbial community from an acid-phase digester. A metagenomic analysis was used to assess microbial community dynamics, construct metagenome-assembled genomes (MAGs), and evaluate the potential for lactose utilization and fermentation product synthesis of community members represented by the assembled MAGs. This analysis led us to propose that, in this reactor, members of the Actinobacteriota phylum are important in the degradation of lactose, via the Leloir pathway and the bifid shunt, and the production of acetic, lactic, and succinic acids. In addition, members of the Firmicutes phylum contribute to the chain-elongation-mediated production of butyric, hexanoic, and octanoic acids, with different microbes using either lactose, ethanol, or lactic acid as the growth substrate. We conclude that genes encoding carbohydrate utilization pathways, and genes encoding lactic acid transport into the cell, electron confurcating lactate dehydrogenase, and its associated electron transfer flavoproteins, are genomic features whose presence in Firmicutes needs to be established to infer the growth substrate used for chain elongation.

Wetlands harbor lactic acid-driven chain elongators

“…However, close relatives and even different strains within a single species may exhibit divergent properties. Moreover, predicting chain elongation potential from full genomes remains a challenge, which is further complicated by some isolates producing MCCA only under certain conditions ( 15 , 19 , 20 ). Given these limitations, we conclude that close relatives of known chain elongators, potentially capable of lactic acid-driven MCCA production themselves, were found in wetland soils.…”

Comparison of metagenomes from fermentation of various agroindustrial residues suggests a common model of community organization

“…The operational conditions of the bioreactors are summarized in Table 1 and additional information on sample collection can be found in the respective publications. MAGs were obtained from the inoculum source (two samples, 10 MAGs) (Ingle et al, 2022) and bioreactors fed cellulosic ethanol thin stillage (six samples, 10 MAGs) (Scarborough et al, 2018a;Scarborough et al, 2020), synthetic medium containing xylose as the primary carbon source (three samples, 8 MAGs) (Scarborough et al, 2022), hydrolysate from dairy manure (four samples, 38 MAGs) (Ingle et al, 2021;Ingle et al, 2022), ultra-filtered milk permeate (34 samples, 123 MAGs) (Walters et al, 2022;Walters et al, 2023), and starch ethanol thin stillage (31 samples, 51 MAGs) (Fortney et al, 2021;Fortney et al, 2022). In all cases, only the best-quality representative MAGs determined in each study were used.…”

“…An additional analysis was performed to compare homologs of subunit B of the electron transfer flavoprotein (EtfB). For this, EtfB homologs were identified using known protein sequences (Walters et al, 2023) and tBLASTn (v2.8.1, default parameters) (Camacho et al, 2009) with "pident" (percent identity to the query sequence) > 25% and "qcovhsp" (coverage of the query sequence) > 70%. EtfB homologs were aligned using MUSCLE (v3.8.31, default parameters) and a phylogenetic tree was constructed using RAxML-NG using 500 boostraps.…”

“…MAGs were classified into predicted functional groups using hierarchical clustering based on the detected genes in metabolic pathways important in MCFA production (Walters et al, 2023). Hierarchical clustering was performed in R (v4.1.0) (Core Team, 2018) using the gplots R package (v3.1.3, heatmap.2 command with default parameters, https://github.com/talgalili/gplots/).…”

“…In other cases, it is proposed that ethanol can be used as an electron donor and act as an intermediate during MCFA production (Agler et al, 2012;Kucek et al, 2016a). To evaluate whether this microbial ecology model can be generalized to conceptually explain MCFA production from a variety of carbohydrate-rich organic residues, we evaluated the microbial communities that were enriched when the same inoculum was used in bioreactor experiments that fermented several agroindustrial residues, including thin stillage from starch ethanol production (Fortney et al, 2021;Fortney et al, 2022), thin stillage from cellulosic ethanol production (Scarborough et al, 2018a;Scarborough et al, 2020), xylose (Scarborough et al, 2022), dairy manure hydrolysate (Ingle et al, 2021;Ingle et al, 2022), and ultrafiltered milk permeate (Walters et al, 2022;Walters et al, 2023). In all cases, the inoculum was from an acid-phase anaerobic digester at the local wastewater treatment plant (Madison, WI, United States).…”