Butyrate is a common fatty acid produced in important fermentative systems, such as the human/animal gut and other H 2 production systems. Despite its importance, there is little information on the partnerships between butyrate producers and other bacteria. The objective of this work was to uncover butyrateproducing microbial communities and possible metabolic routes in a controlled fermentation system aimed at butyrate production. The butyrogenic reactor was operated at 37°C and pH 5.5 with a hydraulic retention time of 31 h and a low hydrogen partial pressure (PH 2). High-throughput sequencing and metagenome functional prediction from 16S rRNA data showed that butyrate production pathways and microbial communities were different during batch (closed) and continuousmode operation. Lactobacillaceae, Lachnospiraceae, and Enterococcaceae were the most abundant phylotypes in the closed system without PH 2 control, whereas Prevotellaceae, Ruminococcaceae, and Actinomycetaceae were the most abundant phylotypes under continuous operation at low PH 2. Putative butyrate producers identified in our system were from Prevotellaceae, Clostridiaceae, Ruminococcaceae, and Lactobacillaceae. Metagenome prediction analysis suggests that nonbutyrogenic microorganisms influenced butyrate production by generating butyrate precursors such as acetate, lactate, and succinate. 16S rRNA gene analysis suggested that, in the reactor, a partnership between identified butyrogenic microorganisms and succinate (i.e., Actinomycetaceae), acetate (i.e., Ruminococcaceae and Actinomycetaceae), and lactate producers (i.e., Ruminococcaceae and Lactobacillaceae) took place under continuousflow operation at low PH 2. IMPORTANCE This study demonstrates how bioinformatics tools, such as metagenome functional prediction from 16S rRNA genes, can help understand biological systems and reveal microbial interactions in controlled systems (e.g., bioreactors). Results obtained from controlled systems are easier to interpret than those from human/animal studies because observed changes may be specifically attributed to the design conditions imposed on the system. Bioinformatics analysis allowed us to identify potential butyrogenic phylotypes and associated butyrate metabolism pathways when we systematically varied the PH 2 in a carefully controlled fermentation system. Our insights may be adapted to butyrate production studies in biohydrogen systems and gut models, since butyrate is a main product and a crucial fatty acid in human/animal colon health. KEYWORDS butyrate production pathways, PICRUSt, Prevotellaceae, hydrogen partial pressure, interconversion reactions, predicted metagenome functional content F ermentation involves the degradation of organic material by anaerobic microorganisms in an environment with low dissolved oxygen and produces short-chain fatty acids (SCFA) (e.g., butyrate and acetate) and gases, including methane (CH 4),