Project description:Polyhydroxyalkanoates (PHAs) are bio-based, biodegradable polyesters that can be produced from organic-rich waste streams using mixed microbial cultures. To maximize PHA production, mixed microbial cultures may be enriched for PHA-producing bacteria with a high storage capacity through the imposition of cyclic, aerobic feast-famine conditions in a sequencing batch reactor (SBR). Though enrichment SBRs have been extensively investigated a bulk solutions-level, little evidence at the proteome level is available to describe the observed SBR behavior to guide future SBR optimization strategies. As such, the purpose of this investigation was to characterize proteome dynamics of a mixed microbial culture in an SBR operated under aerobic feast-famine conditions using fermented dairy manure as the feedstock for PHA production. At the beginning of the SBR cycle, excess PHA precursors were provided to the mixed microbial culture (i.e., feast), after which followed a long duration devoid of exogenous substrate (i.e., famine). Two-dimensional electrophoresis was used to separate protein mixtures during a complete SBR cycle, and proteins of interest were identified.
Project description:Characterization of post-translational modification of nitrogenase in Rhodopseudomonas palustris strains that produce hydrogen gas constitutively.
Project description:Background. Transforming waste and non-food materials into bulk biofuels and chemicals represents a major stride in creating a sustainable bioindustry, optimizing the use of resources while reducing environmental footprints. Yet, despite these advancements, the production of high-value natural products often continues to rely on first-generation substrates, underscoring the intricate processes and specific requirements of their biosynthesis. This is also true for Streptomyces lividans, a renowned host organism celebrated for its capacity to produce and uncover a wide array of natural products, attributed to its genetic versatility and potent secondary metabolism. Given this context, it becomes imperative to assess and optimize this microorganism for the synthesis of natural products specifically from waste and non-food substrates. Results. We metabolically engineered S. lividans TK24 to heterologously produce the ribosomally synthesized and post-translationally modified peptide, bottromycin, as well as the polyketide, pamamycin. The modified strains successfully produced these compounds using waste and non-food model substrates like protocatechuate (derived from lignin), 4-hydroxybenzoate (sourced from plastic waste), and mannitol (from seaweed). Comprehensive transcriptomic and metabolomic analyses offered insights into how these substrates influenced the cellular metabolism of S. lividans. When evaluating production efficiency, S. lividans showcased remarkable tolerance, especially in a fed-batch process using a mineral medium containing the toxic aromatic 4-hydroxybenzoate, leading to enhanced and highly selective bottromycin production. Additionally, it generated a unique spectrum of pamamycins when cultured in mannitol-rich seaweed extract without the need for added nutrients. Conclusion. Our study showcases the successful production of high-value natural products using varied waste and non-food raw materials, thereby circumventing the reliance on costly, food-competing resources. S. lividans exhibited remarkable adaptability and resilience across these diverse substrates. When cultured on aromatic compounds, it displayed a distinct array of intracellular CoA esters, presenting promising avenues for polyketide production. Future research could focus on enhancing S. lividans' substrate utilization pathways to more efficiently process the intricate mixtures commonly found in waste and non-food sources.
Project description:Anaerobic digestion is a popular and effective microbial process for waste treatment. The performance of anaerobic digestion processes is contingent on the balance of the microbial food web in utilizing various substrates. Recently, co-digestion, i.e., supplementing the primary substrate with an organic-rich co-substrate has been exploited to improve waste treatment efficiency. Yet the potential effects of elevated organic loading on microbial functional gene community remains elusive. In this study, functional gene array (GeoChip 5.0) was used to assess the response of microbial community to the addition of poultry waste in anaerobic digesters treating dairy manure. Consistent with 16S rRNA gene sequences data, GeoChip data showed that microbial community compositions were significantly shifted in favor of copiotrophic populations by co-digestion, as taxa with higher rRNA gene copy number such as Bacilli were enriched. The acetoclastic methanogen Methanosarcina was also enriched, while Methanosaeta was unaltered but more abundant than Methanosarcina throughout the study period. The microbial functional diversity involved in anaerobic digestion were also increased under co-digestion.
Project description:Caldicellulosiruptor saccharolyticus is an extremely thermophilic, gram-positive anaerobe which ferments a broad range of substrates to mainly acetate, CO2, and hydrogen gas (H2). Its high hydrogen-producing capacity make this bacterium an attractive candidate for microbial biohydrogen production. However, increased H2 levels tend to inhibit hydrogen formation and leads to the formation of other reduced end products like lactate and ethanol. To investigate the organism’s strategy for dealing with elevated H2 levels and to identify alternative pathways involved in the disposal of the reducing equivalents, the effect of the hydrogen partial pressure (PH2) on fermentation performance was studied. For this purpose cultures were grown under high and low PH2 in a glucose limited chemostat setup. Transcriptome analysis revealed the up-regulation of genes involved in the disposal of reducing equivalents under high PH2, like lactate dehydrogenase and alcohol dehydrogenase as well as the NADH-dependent and ferredoxin-dependent hydrogenases. These findings were in line with the observed shift in fermentation profiles from acetate production under low PH2 to a mixed production of acetate, lactate and ethanol under high PH2. In addition, differential transcription was observed for genes involved in carbon metabolism, fatty acid biosynthesis and several transport systems. The presented transcription data provides experimental evidence for the involvement of the redox sensing Rex protein in gene regulation under high PH2 cultivation conditions. Overall, these findings indicate that the PH2 dependent changes in the fermentation pattern of C. saccharolyticus are, in addition to the known regulation at the enzyme/metabolite level, also regulated at the transcription level.
Project description:Mining waste streams of food production for bioactive plant polysaccharides that affect the fitness and expressed activities of targeted human gut microbes