D- and L-lactate consumers are taxonomically, biochemically, and energetically different.

D- and L-lactate are routinely produced as intermediates in fermentative ecosystems. However, the microbial fate of these stereoisomers remains poorly understood. Given that D-lactate is an unavoidable byproduct of digestion and a neurotoxin, understanding its microbial turnover not only holds ecological pertinence but also the potential to uncover new links between gut microbiota metabolism and host health. Here, we used chemostat bioreactors (pH 7.0, 37°C, and a solids retention time of 4 days) to enrich for lactate-consuming communities. DL-lactate-consuming consortia were enriched, characterized, and used as inoculum for duplicate bioreactors fed exclusively with D- or L-lactate. After steady-state was reached, the fed lactate stereoisomers were switched to assess community resilience. Regardless of the fed stereoisomer, the fermentation product spectra were consistent and dominated by acetate, propionate, and CO2. However, microbial communities and biomass yields diverged sharply, with a high relative abundance of Anaerotignum in D-lactate enrichments and Acidipropionibacterium and Propionibacterium in L-lactate enrichments. Notably, the biomass yield for D-lactate feeding was less than half that for L-lactate feeding, suggesting that the two isomers are metabolized through distinct biochemical pathways despite similar product spectra. Metagenomic and metaproteomic analyses confirmed divergence in D- and L-lactate conversion at both the phylogenetic and pathway levels. Our findings reveal how the stereoisomer identity of microbes shapes their niche specialization, with implications for understanding the ecology and clinical impact of lactate metabolism.