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:Transcriptional profiling of the Donna II mixed community containing Dehalococcoides mccartyi strain 195 comparing a batch starved control to the mixed community being fed 1,2,3,4-tetrachlorobenzene as an electron acceptor. The goal was to determine which transcripts are regulated in response to a shift in a different electron acceptor rather than the consistent tetrachloroethene (PCE) that the parent reactor was maintained on.
Project description:This study investigates the role of carbon-to-phosphorus (C/P) ratios in shaping microbial community dynamics and polyhydroxyalkanoates (PHA) production in sequencing batch reactors (SBR) fed with volatile fatty acids. Three conditions, characterized by fixed organic loading rates but varying C/P ratios (Run 1 = 170 Cmol Pmol-1; Run 2 = 235 Cmol Pmol-1; Run 3 = 400 Cmol Pmol-1;), were tested to explore their impact on PHA accumulation, biomass growth, and reactor stability. Results indicate that the moderate phosphorus limitation of Run 2 achieves the best overall performance, with a PHA volumetric productivity of 2.02 g PHA L⁻¹ d⁻¹ and process stability. Under these conditions, PHA-accumulating bacteria, primarily Sphaerotilus and Leadbetterella, dominated the microbial community, with a notable contribution from eukaryotic organisms, particularly Rhogostoma, Vorticella and Tokophrya, which appeared to regulate bacterial populations through selective predation. Conversely, higher C/P ratio increased the storage yield but lowered the productivity (1.55 gPHA L-1 h-1), compromising sludge settleability and reactor stability, indicating impaired microbial functionality. Transmission electron microscopy further confirmed the presence of intracellular PHA granules and polyphosphate reserves, reinforcing the connection between nutrient limitation and adaptive microbial strategies. Overall, these findings highlight the critical role of the C/P ratio in shaping the performance of mixed microbial cultures, demonstrating that a well-balanced nutrient supply can enhance PHA production while maintaining microbial community stability. The results contribute to optimizing the selection process for mixed microbial cultures, offering valuable insights into the impact of carbon-to-nutrient ratios in the feeding strategy.
Project description:This study investigates the role of carbon-to-phosphorus (C/P) ratios in shaping microbial community dynamics and polyhydroxyalkanoates (PHA) production in sequencing batch reactors (SBR) fed with volatile fatty acids. Three conditions, characterized by fixed organic loading rates but varying C/P ratios (Run 1 = 170 Cmol Pmol-1; Run 2 = 235 Cmol Pmol-1; Run 3 = 400 Cmol Pmol-1;), were tested to explore their impact on PHA accumulation, biomass growth, and reactor stability. Results indicate that the moderate phosphorus limitation of Run 2 achieves the best overall performance, with a PHA volumetric productivity of 2.02 g PHA L⁻¹ d⁻¹ and process stability. Under these conditions, PHA-accumulating bacteria, primarily Sphaerotilus and Leadbetterella, dominated the microbial community, with a notable contribution from eukaryotic organisms, particularly Rhogostoma, Vorticella and Tokophrya, which appeared to regulate bacterial populations through selective predation. Conversely, higher C/P ratio increased the storage yield but lowered the productivity (1.55 gPHA L-1 h-1), compromising sludge settleability and reactor stability, indicating impaired microbial functionality. Transmission electron microscopy further confirmed the presence of intracellular PHA granules and polyphosphate reserves, reinforcing the connection between nutrient limitation and adaptive microbial strategies. Overall, these findings highlight the critical role of the C/P ratio in shaping the performance of mixed microbial cultures, demonstrating that a well-balanced nutrient supply can enhance PHA production while maintaining microbial community stability. The results contribute to optimizing the selection process for mixed microbial cultures, offering valuable insights into the impact of carbon-to-nutrient ratios in the feeding strategy.
Project description:This study investigates the role of carbon-to-phosphorus (C/P) ratios in shaping microbial community dynamics and polyhydroxyalkanoates (PHA) production in sequencing batch reactors (SBR) fed with volatile fatty acids. Three conditions, characterized by fixed organic loading rates but varying C/P ratios (Run 1 = 170 Cmol Pmol-1; Run 2 = 235 Cmol Pmol-1; Run 3 = 400 Cmol Pmol-1;), were tested to explore their impact on PHA accumulation, biomass growth, and reactor stability. Results indicate that the moderate phosphorus limitation of Run 2 achieves the best overall performance, with a PHA volumetric productivity of 2.02 g PHA L⁻¹ d⁻¹ and process stability. Under these conditions, PHA-accumulating bacteria, primarily Sphaerotilus and Leadbetterella, dominated the microbial community, with a notable contribution from eukaryotic organisms, particularly Rhogostoma, Vorticella and Tokophrya, which appeared to regulate bacterial populations through selective predation. Conversely, higher C/P ratio increased the storage yield but lowered the productivity (1.55 gPHA L-1 h-1), compromising sludge settleability and reactor stability, indicating impaired microbial functionality. Transmission electron microscopy further confirmed the presence of intracellular PHA granules and polyphosphate reserves, reinforcing the connection between nutrient limitation and adaptive microbial strategies. Overall, these findings highlight the critical role of the C/P ratio in shaping the performance of mixed microbial cultures, demonstrating that a well-balanced nutrient supply can enhance PHA production while maintaining microbial community stability. The results contribute to optimizing the selection process for mixed microbial cultures, offering valuable insights into the impact of carbon-to-nutrient ratios in the feeding strategy.
Project description:Chinese Hamster ovary (CHO) cells are the main platform used to produce recombinant proteins in the biopharmaceutical industry and are commonly cultured in either fed-batch or perfusion mode. However, the optimization of the complex biological systems used in such processes is extremely challenging. Omics approaches can reveal otherwise unknown characteristics of these systems and identify culture parameters that can be manipulated to optimize the cultivation process. Here we have applied proteomic profiling to a monoclonal antibody (mAb) production operated in perfusion mode to explore how cell biology and reactor environment change as the cell density reaches ≥ 200 x 106 cells/mL. The proteomics data show an increase of structural proteins as cell density increase, signs of oxidative stress and changes in glutathione metabolism at very high cell densities. Additionally, metabolomic profiling was carried out. See article “High cell density culture has a maintained exoproteome and metabolome” for more information.
Project description:A continuous culture of Bifidobacterium longum NCC2705 was carried out in a 2.5-l reactor (Bioengineering AG, Wald, Switzerland), equipped with a Biospectra control system (Biospectra AG, Schlieren, Switzerland) and containing 2 l of MRS, added of 0.05% cysteine, inoculated with 2 % (v/v) preculture. The temperature was maintained at 37°C and the pH at 6.0 by addition of 5 M NaOH. The culture was stirred constantly at 250 rpm using two rushton type propellers. Anaerobic conditions were maintained by flushing the headspace of the reactor with CO2. After 8 h in batch mode the culture was run in continuous mode at a dilution rate of 0.1 h-1. Fresh medium was added using a peristaltic pump (Alitea, Bioengineering AG, Wald, Switzerland), and fermented broth harvested with a second peristaltic pump (Alitea, Bioengineering AG, Wald, Switzerland) set at a slightly higher flow rate. A stabilization period of 90 h (corresponding to nine reactor volume changes) was operated prior culture monitoring (t=0). Aliquots of 2 ml taken at t=31, 134 and 211 h were centrifuged (4,000 g, 1 min, room temperature) for transcriptomic analysis. Supernatants were discarded and cell pellets snap frozen in liquid nitrogen and stored at -80ºC until RNA-extraction. Keywords: Time course of Bifidobacterium longum in continuous culture
Project description:Two-stage two-phase biogas reactor systems consisting each of one batch downflow hydrolysis reactor (HR, vol. 10 L), one process fluid storage tank (vol. 10 L), and one downstream upflow anaerobic filter reactor (AF, vol. 10 L), were operated at mesophilic (M, 37 °C) and thermophilic (T, 55 °C) temperatures and over a period of > 750 d (Figure 1, Additional file 1). For each reactor system and for each process temperature, two replicates were conducted in parallel, denominated further as biological replicates. Further process details were as previously published. Start-up of all fermenters were performed using liquid fermenter material from a biogas plant converting cattle manure in co-digestion with grass and maize silage and other biomass at varying concentrations and at mesophilic temperatures. Silage of perennial ryegrass (Lolium perenne L.) was digested as sole substrate in batches of varying amounts with retention times of 28 d (storage of bale silage at -20 °C, cutting length 3 cm, volatile substances (VS) 32 % of fresh mass (FM), total Kjeldahl nitrogen 7.6 g kgFM-1, NH4+-N 0.7 g kgFM-1, acetic acid 2.6 g kgFM-1, propionic acid < 0.04 g kgFM-1, lactic acid 2.6 g kgFM-1, ethanol 2.2 g kgFM-1, C/N ratio 19.3, chemical oxygen demand (COD) 357.7 g kgFM-1, analysis of chemical properties according to [6]. No spoilage was observed in the silage. Biogas yields were calculated as liters normalized to 0 °C and 1013 hPa (LN) per kilogram volatile substances (kgVS). For chemical analysis, samples were taken from the effluents of HR and AF. For sequencing of 16S rRNA gene amplicon libraries, microbial metagenomes, and microbial metatranscriptomes, samples were taken from the silage digestate in the HR digested for 2 d. At this time point, high AD rates were detected as indicated by the fast increase of volatile fatty acids (VFA), e.g., acetic acid. Sampling was performed at two different organic loading rates (OLR), i.e., batch-fermentation of 500 g (denominated as “low OLR”, samples MOLR500 and TOLR500) and 1,500 g silage (denominated as “increased OLR”, samples MOLR1500 and TOLR1500).
Project description:A continuous culture of Bifidobacterium longum NCC2705 was carried out in a 2.5-l reactor (Bioengineering AG, Wald, Switzerland), equipped with a Biospectra control system (Biospectra AG, Schlieren, Switzerland) and containing 2 l of MRS, added of 0.05% cysteine, inoculated with 2 % (v/v) preculture. The temperature was maintained at 37°C and the pH at 6.0 by addition of 5 M NaOH. The culture was stirred constantly at 250 rpm using two rushton type propellers. Anaerobic conditions were maintained by flushing the headspace of the reactor with CO2. After 8 h in batch mode the culture was run in continuous mode at a dilution rate of 0.1 h-1. Fresh medium was added using a peristaltic pump (Alitea, Bioengineering AG, Wald, Switzerland), and fermented broth harvested with a second peristaltic pump (Alitea, Bioengineering AG, Wald, Switzerland) set at a slightly higher flow rate. A stabilization period of 90 h (corresponding to nine reactor volume changes) was operated prior culture monitoring (t=0). Aliquots of 2 ml taken at t=31, 134 and 211 h were centrifuged (4,000 g, 1 min, room temperature) for transcriptomic analysis. Supernatants were discarded and cell pellets snap frozen in liquid nitrogen and stored at -80ºC until RNA-extraction. Keywords: Time course of Bifidobacterium longum in continuous culture Bifidobacterium longum NCC2705 at time 31 versus time 134 h and versus time 211 h in continuous culture. Two technical replicares with dyes swaps