Parallel bioreactor system for accessible and reproducible anaerobic culture.
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ABSTRACT:
SUBMITTER: Monaghan TI
PROVIDER: S-EPMC8208757 | biostudies-literature |
REPOSITORIES: biostudies-literature
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| S-EPMC3878063 | biostudies-literature
Project description:Hydraulic sealing is a crucial condition for the maintenance of sterility during long term operation of microfluidic bioreactors. We developed a miniaturized optically accessible bioreactor (MOAB) allowing perfused culture of 3D cellularised constructs. In the MOAB, the culture chambers are sealed by magnets that generate a weak static magnetic field (SMF). Here, we predicted computationally the exact level of SMF to which cells are subjected during culture in the MOAB and we assessed its influence on the viability, metabolic activity and gene expression of neuroblastoma-derived cells cultured up to seven days. The predicted SMF ranged from 0.32 to 0.57 T using an axial-symmetric model of a single chamber, whereas it ranged from 0.35 to 0.62 T using a 3D model of the complete device. Cell function was evaluated in SH-SY5Y neuroblastoma cells at 2 and 7 days of culture in the MOAB, compared to 2D monolayer, 3D non-perfused constructs, and 3D perfused constructs cultured in a modified MOAB with magnet-free sealing. We measured the cell metabolic activity normalized by the DNA content and the expression levels of heat-shock protein 70 (Hsp-70), Bcl-2 and Bax. We found that the level of SMF applied to cells in the MOAB did not influence their metabolic activity and exerted a stressful effect in 2D monolayer, not confirmed in 3D conditions, neither static not perfused. Instead, the magnets provided a significantly greater hydraulic sealing in long-term culture, thus the MOAB might be potentially exploitable for the development of reliable in vitro models of neurodegeneration.
| S-EPMC6451746 | biostudies-literature
Project description:With its capacity for anaerobic methane oxidation and denitrification, the bacterium Methylomirabilis oxyfera plays an important role in natural ecosystems. Its unique physiology can be exploited for more sustainable wastewater treatment technologies. However, operational stability of full-scale bioreactors can experience setbacks due to, for example, bacteriophage blooms. By shaping microbial communities through mortality, horizontal gene transfer, and metabolic reprogramming, bacteriophages are important players in most ecosystems. Here, we analyzed an infected Methylomirabilis sp. bioreactor enrichment culture using (advanced) electron microscopy, viral metagenomics and bioinformatics. Electron micrographs revealed four different viral morphotypes, one of which was observed to infect Methylomirabilis cells. The infected cells contained densely packed ~55 nm icosahedral bacteriophage particles with a putative internal membrane. Various stages of virion assembly were observed. Moreover, during the bacteriophage replication, the host cytoplasmic membrane appeared extremely patchy, which suggests that the bacteriophages may use host bacterial lipids to build their own putative internal membrane. The viral metagenome contained 1.87 million base pairs of assembled viral sequences, from which five putative complete viral genomes were assembled and manually annotated. Using bioinformatics analyses, we could not identify which viral genome belonged to the Methylomirabilis- infecting bacteriophage, in part because the obtained viral genome sequences were novel and unique to this reactor system. Taken together these results show that new bacteriophages can be detected in anaerobic cultivation systems and that the effect of bacteriophages on the microbial community in these systems is a topic for further study.
| S-EPMC5099504 | biostudies-literature
Project description:BackgroundChain elongation forms a new platform technology for the circular production of biobased chemicals from renewable carbon and energy sources. This study aimed to develop a continuous methanol-based chain elongation process for the open-culture production of a new-generation biofuel precursor and potential platform chemical: n-valerate. Propionate was used as a substrate for chain elongation to n-valerate in an anaerobic open-culture bioreactor. In addition, the co-production of n- and iso-butyrate in addition to n-valerate via, respectively, acetate and propionate elongation was investigated.Resultsn-Valerate was produced during batch and continuous experiments with a pH in the range 5.5-5.8 and a hydraulic retention time of 95 h. Decreasing the pH from 5.8 to 5.5 caused an increase of the selectivity for n-valerate formation (from 58 up to 70 wt%) during methanol-based propionate elongation. n-Valerate and both n- and iso-butyrate were produced during simultaneous methanol-based elongation of propionate and acetate. Propionate was within the open-culture preferred over acetate as a substrate with 10-30% more consumption. Increasing the methanol concentration in the influent (from 250 to 400 mM) resulted in a higher productivity (from 45 to 58 mmol C/L/day), but a lower relative product selectivity (from 49 to 43 wt%) of n-valerate. The addition of acetate as a substrate did not change the average n-valerate productivities. Within the continuous bioreactor experiments, 6 to 17 wt% of formed products was methane. The microbial community during all steady-states in both methanol-based elongation bioreactors was dominated by species related to Clostridium luticellarii and Candidatus Methanogranum. C. luticellarii is the main candidate for n-valerate formation from methanol and propionate.Conclusionsn-Valerate was for the first time proven to be produced from propionate and methanol by an open-culture bioreactor. Methanogenic activity can be inhibited by decreasing the pH, and the n-valerate productivity can be improved by increasing the methanol concentration. The developed process can be integrated with various biorefinery processes from thermochemical, (bio)electrochemical, photovoltaic and microbial technologies. The findings from this study form a useful tool to steer the process of biological production of chemicals from biomass and other carbon and energy sources.
| S-EPMC6535856 | biostudies-literature
Project description:BackgroundHuman mesenchymal stem cells (hMSCs) are multipotent by nature and are originally isolated from bone marrow. In light of a future application of hMSCs in the oral cavity, a body compartment with varying oxygen partial pressures and an omnipresence of different bacterial species i.e. periodontitis pathogens, we performed this study to gain information about the behavior of hMSC in an anaerobic system and the response in interaction with oral bacterial pathogens.Methodology/principal findingsWe established a model system with oral pathogenic bacterial species and eukaryotic cells cultured in anaerobic conditions. The facultative anaerobe bacteria Fusobacterium nucleatum, Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans were studied. Their effects on hMSCs and primary as well as permanent gingival epithelial cells (Ca9-22, HGPEC) were comparatively analyzed. We show that hMSCs cope with anoxic conditions, since 40% vital cells remain after 72 h of anaerobic culture. The Ca9-22 and HGPEC cells are significantly more sensitive to lack of oxygen. All bacterial species reveal a comparatively low adherence to and internalization into hMSCs (0.2% and 0.01% of the initial inoculum, respectively). In comparison, the Ca9-22 and HGPEC cells present better targets for bacterial adherence and internalization. The production of the pro-inflammatory chemokine IL-8 is higher in both gingival epithelial cell lines compared to hMSCs and Fusobacterium nucleatum induce a time-dependent cytokine secretion in both cell lines. Porphyromonas gingivalis is less effective in stimulating secretion of IL-8 in the co-cultivation experiments.Conclusions/significanceHMSCs are suitable for use in anoxic regions of the oral cavity. The interaction with local pathogenic bacteria does not result in massive pro-inflammatory cytokine responses. The test system established in this study allowed further investigation of parameters prior to set up of oral hMSC in vivo studies.
| S-EPMC3817215 | biostudies-literature
Project description:BackgroundNowadays, the vast majority of chemicals are either synthesised from fossil fuels or are extracted from agricultural commodities. However, these production approaches are not environmentally and economically sustainable, as they result in the emission of greenhouse gases and they may also compete with food production. Because of the global agreement to reduce greenhouse gas emissions, there is an urgent interest in developing alternative sustainable sources of chemicals. In recent years, organic waste streams have been investigated as attractive and sustainable feedstock alternatives. In particular, attention has recently focused on the production of caproate from mixed culture fermentation of low-grade organic residues. The current approaches for caproate synthesis from organic waste are not economically attractive, as they involve the use of two-stage anaerobic digestion systems and the supplementation of external electron donors, both of which increase its production costs. This study investigates the feasibility of producing caproate from food waste (FW) without the supplementation of external electron donors using a single-phase reactor system.ResultsReplicate leach-bed reactors were operated on a semi-continuous mode at organic loading of 80 g VS FW l-1 and at solid retention times of 14 and 7 days. Fermentation, rather than hydrolysis, was the limiting step for caproate production. A higher caproate production yield 21.86 ± 0.57 g COD l-1 was achieved by diluting the inoculating leachate at the beginning of each run and by applying a leachate recirculation regime. The mixed culture batch fermentation of the FW leachate was able to generate 23 g caproate COD l-1 (10 g caproate l-1), at a maximum rate of 3 g caproate l-1 day-1 under high H2 pressure. Lactate served as the electron donor and carbon source for the synthesis of caproate. Microbial community analysis suggested that neither Clostridium kluyveri nor Megasphaera elsdenii, which are well-characterised caproate producers in bioreactors systems, were strongly implicated in the synthesis of caproate, but that rather Clostridium sp. with 99% similarity to Ruminococcaceae bacterium CPB6 and Clostridium sp. MT1 likely played key roles in the synthesis of caproate. This finding indicates that the microbial community capable of caproate synthesis could be diverse and may therefore help in maintaining a stable and robust process.ConclusionsThese results indicate that future, full-scale, high-rate caproate production from carbohydrate-rich wastes, associated with biogas recovery, could be envisaged.
| S-EPMC5894149 | biostudies-literature
Project description:Mass spectrometry imaging is increasingly used in biological and translational research because it has the ability to determine the spatial distribution of hundreds of analytes in a sample. Being at the interface of proteomics/metabolomics and imaging, the acquired datasets are large and complex and often analyzed with proprietary software or in-house scripts, which hinders reproducibility. Open source software solutions that enable reproducible data analysis often require programming skills and are therefore not accessible to many mass spectrometry imaging (MSI) researchers. We have integrated 18 dedicated mass spectrometry imaging tools into the Galaxy framework to allow accessible, reproducible, and transparent data analysis. Our tools are based on Cardinal, MALDIquant, and scikit-image and enable all major MSI analysis steps such as quality control, visualization, preprocessing, statistical analysis, and image co-registration. Furthermore, we created hands-on training material for use cases in proteomics and metabolomics. To demonstrate the utility of our tools, we re-analyzed a publicly available N-linked glycan imaging dataset. By providing the entire analysis history online, we highlight how the Galaxy framework fosters transparent and reproducible research. The Galaxy framework has emerged as a powerful analysis platform for the analysis of MSI data with ease of use and access, together with high levels of reproducibility and transparency.
| S-EPMC6901077 | biostudies-literature
Project description:Improvements in genome sequencing and assembly are enabling high-quality reference genomes for all species. However, the assembly process is still laborious, computationally and technically demanding, lacks standards for reproducibility, and is not readily scalable. Here we present the latest Vertebrate Genomes Project assembly pipeline and demonstrate that it delivers high-quality reference genomes at scale across a set of vertebrate species arising over the last ~500 million years. The pipeline is versatile and combines PacBio HiFi long-reads and Hi-C-based haplotype phasing in a new graph-based paradigm. Standardized quality control is performed automatically to troubleshoot assembly issues and assess biological complexities. We make the pipeline freely accessible through Galaxy, accommodating researchers even without local computational resources and enhanced reproducibility by democratizing the training and assembly process. We demonstrate the flexibility and reliability of the pipeline by assembling reference genomes for 51 vertebrate species from major taxonomic groups (fish, amphibians, reptiles, birds, and mammals).
| S-EPMC10327048 | biostudies-literature
Project description:The rat pancreatic progenitor cell line B-13 is of interest for research on drug metabolism and toxicity since the cells trans-differentiate into functional hepatocyte-like cells (B-13/H) when treated with glucocorticoids. In this study we investigated the trans-differentiation and liver-specific functions of B-13/H cells in a three-dimensional (3D) multi-compartment bioreactor, which has already been successfully used for primary liver cell culture. Undifferentiated B-13 cells were inoculated into the bioreactor system and exposed to dexamethasone to promote hepatic trans-differentiation (B-13/HT). In a second approach, pre-differentiated B-13 cells were cultured in bioreactors for 15 days to evaluate the maintenance of liver-typical functions (B-13/HP). During trans-differentiation of B-13 cells into hepatocyte-like cells in the 3D bioreactor system (approach B-13/HT), an increase in glucose metabolism and in liver-specific functions (urea and albumin synthesis; cytochrome P450 [CYP] enzyme activity) was observed, whereas amylase - characteristic for exocrine pancreas and undifferentiated B-13 cells - decreased over time. In bioreactors with pre-differentiated cells (approach B-13/HP), the above liver-specific functions were maintained over the whole culture period. Results were confirmed by gene expression and protein analysis showing increased expression of carbamoyl-phosphate synthase 1 (CPS-1), albumin, CYP2E1, CYP2C11 and CYP3A1 with simultaneous loss of amylase. Immunohistochemical studies showed the formation of 3D structures with expression of liver-specific markers, including albumin, cytokeratin (CK) 18, CCAAT/enhancer-binding protein beta (CEBP-β), CYP2E1 and multidrug resistance protein 2 (MRP2). In conclusion, successful culture and trans-differentiation of B-13 cells in the 3D bioreactor was demonstrated. The requirement for only one hormone and simple culture conditions to generate liver-like cells makes this cell type useful for in vitro research using 3D high-density culture systems.
| S-EPMC6062372 | biostudies-literature
Project description:Ex vivo colon fermentation systems are highly versatile as models for analyzing gastrointestinal tract microbiota composition and functionality. Ex vivo colon models range in size and functionality from bench-top micro fermenters to large units housed in individualized cabinets. The length of set-up time (including stabilization periods) for each fermentation system can range from hours to weeks to months. The aim of this study was to investigate a single-use cassette mini-fermentation system as a reproducible batch model of the colon. The online data log from the cassettes (triplicate wells across four different cassettes, n = 12) was sensitive enough to identify real-time changes in pH, temperature, dissolved oxygen or liquid addition (sodium hydroxide) during the runs which could be addressed if an alarm set-point was triggered. The alpha diversity indices also showed little variation between cassettes with the samples clustering around the mean. The weighted beta diversity PCoA analysis illustrated that 95% of the variance between the samples was accounted for by the time-point and not the fermentation run/cassette used. The variation in taxonomic diversity between cassettes was limited to less than 20 out of 115 genera. This study provides evidence that micro-bioreactors provide some very attractive advantages as batch models for the human colon. We show for the first time the use of the micro-Matrix a 24-well sophisticated parallel controlled cassette-based bioreactors as a batch colon model. We demonstrated a high level of reproducibility across fermentation cassettes when used in conjunction with a standardized fecal microbiota. The machine can operate 24 individual fermentations simultaneously and are relatively cost effective. Based on next generation sequencing analysis, the micro-bioreactors offer a high degree of reproducibility together with high-throughput capacity. This makes it a potential system for large screening projects that can then be scaled up to large fermenters or human/animal in vivo experiments.
| S-EPMC6096000 | biostudies-literature