Caproic acid production through vinasse degradation in anaerobic biological processes
This study investigated the microbial diversity, metabolic genetic potential, and functional organization of microbial communities in a two-stage anaerobic system for the conversion of sugarcane vinasse into medium-chain fatty acids (MCFA), particularly caproic acid. Shotgun metagenomics revealed metabolic complementarity and partial overlap between reactor stages. The thermophilic fermentative reactor (FERM) harbored a low-diversity community dominated by Thermoanaerobacterium thermosaccharolyticum, Weizmannia coagulans, Lentilactobacillus hilgardii, and Acidipropionibacterium acidipropionici, with an extensive Carbohydrate-Active enZyme (CAZyme) repertoire and metabolic pathways linked to carbohydrate hydrolysis, acidogenesis, and production of key intermediates, predominantly lactate, acetate, propionate, and butyrate. Lactic acid bacteria in FERM contributed to sustained lactate production, a key electron donor for downstream MCFA synthesis. In the mesophilic Upflow Anaerobic Sludge Blanket (UASB) reactor, a more diverse and specialized microbiome was detected, enriched with Caproicibacterium, Caproicibacter, Clostridium spp., Prevotella cerevisiae, and W. coagulans, carrying gene sets encoding enzymes of reverse ß-oxidation (butyrate biosynthesis), ACP-dependent fatty acid biosynthesis, and utilization of residual polysaccharides. Several MAGs exhibited functional redundancy and the capacity to metabolize fermentative intermediates such as lactate, acetate, and ethanol, supporting continuous fatty acid chain elongation even under fluctuations in FERM effluent composition. Reconstruction of high-quality metagenome-assembled genomes (MAGs) suggests syntrophic interactions among fermentative, acetogenic, and chain-elongating guilds, highlighting a hierarchical hydrolytic system in which T. thermosaccharolyticum, Caproicibacterium amylolyticum, and P. cerevisiae were predicted to degrade polysaccharides and produce organic acids. Overall, these findings demonstrate that primary fermentation and precursor generation occurred in the FERM reactor, followed by efficient carbon chain elongation to MCFA in the UASB reactor. The observed functional genetic redundancy, metabolic complementarity, and putative syntrophic interactions reinforce the applicability of two-stage anaerobic systems as scalable biorefinery platforms for high-value chemical production from agro-industrial residues.  The system consisted of two reactors operating in series, comprising a thermophilic fermentative reactor (FERM) followed by a mesophilic upflow anaerobic sludge blanket (UASB) reactor. The first reactor was continuously fed with raw vinasse from Pernambuco, Brazil, while the second reactor received the digestate produced in the fermentative stage.The first stage (FERM) consisted of a thermophilic fermentative bioreactor constructed from glass (total volume: 4 L; working volume: 2.5 L) and operated at 50°C. The pH was maintained at 5.5 by automated addition of 2 M NaOH. Raw sugarcane vinasse was supplied at a flow rate of 1.44 L/day, corresponding to a hydraulic retention time (HRT) of 1.25 days. Continuous mixing was provided by a magnetic stirrer operating at 120 rpm. No external inoculum was added, and the reactor was self-inoculated by the indigenous microbial community present in the vinasse, which exhibited initial total solids (TS) of 19.5 ± 0.075 g/L and volatile solids (VS) of 14.2 ± 0.55 g/L. The second stage consisted of a UASB reactor constructed from acrylic (total volume: 4 L; working volume: 3.2 L), operated at 37°C with internal liquid recirculation to promote biomass homogeneity. This reactor was fed at 0.58 L/day, resulting in an HRT of 5.5 days, and the pH was adjusted to 5.0 using a 9% (v/v) hydrochloric acid solution. The UASB reactor was inoculated with anaerobic sludge obtained from a brewery wastewater treatment system operating under internal recirculation in P
This study investigated the microbial diversity, metabolic genetic potential, and functional organization of microbial communities in a two-stage anaerobic system for the conversion of sugarcane vinasse into medium-chain fatty acids (MCFA), particularly caproic acid. Shotgun metagenomics revealed metabolic complementarity and partial overlap between reactor stages. The thermophilic fermentative reactor (FERM) harbored a low-diversity community dominated by Thermoanaerobacterium thermosaccharolyticum, Weizmannia coagulans, Lentilactobacillus hilgardii, and Acidipropionibacterium acidipropionici, with an extensive Carbohydrate-Active enZyme (CAZyme) repertoire and metabolic pathways linked to carbohydrate hydrolysis, acidogenesis, and production of key intermediates, predominantly lactate, acetate, propionate, and butyrate. Lactic acid bacteria in FERM contributed to sustained lactate production, a key electron donor for downstream MCFA synthesis. In the mesophilic Upflow Anaerobic Sludge Blanket (UASB) reactor, a more diverse and specialized microbiome was detected, enriched with Caproicibacterium, Caproicibacter, Clostridium spp., Prevotella cerevisiae, and W. coagulans, carrying gene sets encoding enzymes of reverse ß-oxidation (butyrate biosynthesis), ACP-dependent fatty acid biosynthesis, and utilization of residual polysaccharides. Several MAGs exhibited functional redundancy and the capacity to metabolize fermentative intermediates such as lactate, acetate, and ethanol, supporting continuous fatty acid chain elongation even under fluctuations in FERM effluent composition. Reconstruction of high-quality metagenome-assembled genomes (MAGs) suggests syntrophic interactions among fermentative, acetogenic, and chain-elongating guilds, highlighting a hierarchical hydrolytic system in which T. thermosaccharolyticum, Caproicibacterium amylolyticum, and P. cerevisiae were predicted to degrade polysaccharides and produce organic acids. Overall, these findings demonstrate that primary fermentation and precursor generation occurred in the FERM reactor, followed by efficient carbon chain elongation to MCFA in the UASB reactor. The observed functional genetic redundancy, metabolic complementarity, and putative syntrophic interactions reinforce the applicability of two-stage anaerobic systems as scalable biorefinery platforms for high-value chemical production from agro-industrial residues.  The system consisted of two reactors operating in series, comprising a thermophilic fermentative reactor (FERM) followed by a mesophilic upflow anaerobic sludge blanket (UASB) reactor. The first reactor was continuously fed with raw vinasse from Pernambuco, Brazil, while the second reactor received the digestate produced in the fermentative stage.The first stage (FERM) consisted of a thermophilic fermentative bioreactor constructed from glass (total volume: 4 L; working volume: 2.5 L) and operated at 50°C. The pH was maintained at 5.5 by automated addition of 2 M NaOH. Raw sugarcane vinasse was supplied at a flow rate of 1.44 L/day, corresponding to a hydraulic retention time (HRT) of 1.25 days. Continuous mixing was provided by a magnetic stirrer operating at 120 rpm. No external inoculum was added, and the reactor was self-inoculated by the indigenous microbial community present in the vinasse, which exhibited initial total solids (TS) of 19.5 ± 0.075 g/L and volatile solids (VS) of 14.2 ± 0.55 g/L. The second stage consisted of a UASB reactor constructed from acrylic (total volume: 4 L; working volume: 3.2 L), operated at 37°C with internal liquid recirculation to promote biomass homogeneity. This reactor was fed at 0.58 L/day, resulting in an HRT of 5.5 days, and the pH was adjusted to 5.0 using a 9% (v/v) hydrochloric acid solution. The UASB reactor was inoculated with anaerobic sludge obtained from a brewery wastewater treatment system operating under internal recirculation in P