A Clostridium group IV species dominates and suppresses a mixed culture fermentation by tolerance to medium chain fatty acids products

A microbial community is engaged in a complex economy of cooperation and competition for carbon and energy. In engineered systems such as anaerobic digestion and fermentation, these relationships are exploited for conversion of a broad range of substrates into products, such as biogas, ethanol, and carboxylic acids. Medium chain fatty acids (MCFAs), for example, hexanoic acid, are valuable, energy dense microbial fermentation products, however, MCFA tend to exhibit microbial toxicity to a broad range of microorganisms at low concentrations. Here, we operated continuous mixed population MCFA fermentations on biorefinery thin stillage to investigate the community response associated with the production and toxicity of MCFA. In this study, an uncultured species from the Clostridium group IV (related to Clostridium sp. BS-1) became enriched in two independent reactors that produced hexanoic acid (up to 8.1 g L−1), octanoic acid (up to 3.2 g L−1), and trace concentrations of decanoic acid. Decanoic acid is reported here for the first time as a possible product of a Clostridium group IV species. Other significant species in the community, Lactobacillus spp. and Acetobacterium sp., generate intermediates in MCFA production, and their collapse in relative abundance resulted in an overall production decrease. A strong correlation was present between the community composition and both the hexanoic acid concentration (p = 0.026) and total volatile fatty acid concentration (p = 0.003). MCFA suppressed species related to Clostridium sp. CPB-6 and Lactobacillus spp. to a greater extent than others. The proportion of the species related to Clostridium sp. BS-1 over Clostridium sp. CPB-6 had a strong correlation with the concentration of octanoic acid (p = 0.003). The dominance of this species and the increase in MCFA resulted in an overall toxic effect on the mixed community, most significantly on the Lactobacillus spp., which resulted in a decrease in total hexanoic acid concentration to 32 ± 2% below the steady-state average. As opposed to the current view of MCFA toxicity broadly leading to production collapse, this study demonstrates that varied tolerance to MCFA within the community can lead to the dominance of some species and the suppression of others, which can result in a decreased productivity of the fermentation

Adjusting Organic Load as a Strategy to Direct Single-Stage Food Waste Fermentation from Anaerobic Digestion to Chain Elongation

Production of medium chain carboxylic acids (MCCA) as renewable feedstock bio-chemicals, from food waste (FW), requires complicated reactor configurations and supplementation of chemicals to achieve product selectivity. This study evaluated the manipulation of organic loading rate in an un-supplemented, single stage stirred tank reactor to steer an anaerobic digestion (AD) microbiome towards acidogenic fermentation (AF), and thence to chain elongation. Increasing substrate availability by switching to a FW feedstock with a higher COD stimulated chain elongation. The MCCA species n-caproic (10.1 ± 1.7 g L−1) and n-caprylic (2.9 ± 0.8 g L−1) acid were produced at concentrations comparable to more complex reactor set-ups. As a result, of the adjusted operating strategy, a more specialised microbiome developed containing several MCCA-producing bacteria, lactic acid-producing Olsenella spp. and hydrogenotrophic methanogens. By contrast, in an AD reactor that was operated in parallel to produce biogas, the retention times had to be doubled when fed with the high-COD FW to maintain biogas production. The AD microbiome comprised a diverse mixture of hydrolytic and acidogenic bacteria, and acetoclastic methanogens. The results suggest that manipulation of organic loading rate and food-to-microorganism ratio may be used as an operating strategy to direct an AD microbiome towards AF, and to stimulate chain elongation in FW fermentation, using a simple, un-supplemented stirred tank set-up. This outcome provides the opportunity to repurpose existing AD assets operating on food waste for biogas production, to produce potentially higher value MCCA products, via simple manipulation of the feeding strategy.This project has received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No 66599

Selecting fermentation products for food waste valorisation with HRT and OLR as the key operational parameters.

Acidogenic fermentation is attractive for food waste valorisation. A better understanding is required on how operation affects product selectivity. This study demonstrated that the hydraulic retention time (HRT) and organic loading rate (OLR) selected fermentation pathways in a single-stage, semicontinuous stirred tank reactor. Three combinations of HRT and OLR were tested to distinguish the effect of each parameter. Three fermentation profiles with distinct microbial communities were obtained. Predominantly n-butyric acid (13 ± 2 gCOD L-1, 55 ± 14% of carboxylates) was produced at an HRT of 8.5 days and OLR around 12 gCOD L-1 d-1. Operating at an HRT two days longer, yet with similar OLR, stimulated chain elongation (up to 13.6 gCOD L-1 of n-caproic acid). This was reflected by a microbial community twice as diverse at longer HRT as indicated by first and second order Hill number (1D = 24 ± 4, 2D = 12 ± 3) and by a higher relative abundance of genera related to secondary fermentation, such as the VFA-elongating Caproiciproducens spp., and secondary lactic acid fermenter Secundilactobacillus spp.. Operating at a higher OLR (20 gCOD L-1 d-1) but HRT of 8.5 days, resulted in typical lactic acid fermentation (34 ± 5 gCOD L-1) harbouring a less diverse community (1D = 8.0 ± 0.7, 2D = 5.7 ± 0.9) rich in acid-resistant homofermentative Lactobacillus spp. These findings demonstrate that a flexible product portfolio can be achieved by small adjustments in two key operating conditions. This improves the economic potential of acidogenic fermentation for food waste valorisation

Food waste as substrate to obtain two different enriched microbiomes for model development

This project has received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No 66599

Self-generated ethanol in stillage fermentation for carboxylate upgrade

IntroductionVolatile fatty acids (VFA) are intermediates of the biogas process, thus can be produced from waste streams, and have a higher economic value as they can be used as industrial precursors and bulk chemicals. Several specific organisms present in mixed cultures have the capacity to elongate short VFA to caproate or others with a higher value using ethanol as a reducing agent. In this study we investigated different operational conditions to enhance VFA production from stillage of a bio-ethanol biorefinery. The main goals were to i) increase the titers of VFA; and ii) evaluate the ethanol requirements for carboxylate upgrade. MethodsTwo continuous reactors were fed with distilled stillage and operated at pH 5.5 and 6.5. They run for 300 days at different HRT. Batch tests with inoculum from reactor 5.5 were performed with different proportions of beer and stillage. ResultsA decrease of HRT improved the production rate of VFA from 2.7 to 8.8 g COD L-1d-1, but final concentration and composition remained similar within the same reactor (Fig.1). A decrease of pH inhibited methane production by VFA present in acidic form, but also reduced VFA concentrations in the broth. pH reduction induced ethanol production and therefore, a higher presence of longer carboxylates.Mixing beer (ethanol-rich stream) in a fermentation broth included active yeast in the system and this used VFA to generate more ethanol available for chain elongation (Fig.2). However, high concentrations of ethanol inhibited VFA production.ConclusionsLow pH induces ethanol generation in stillage fermentation and the introduction of small portions of beer can enhance the generation of ethanol and thus elongate carboxylates.<br/

Adjusting Organic Load as a Strategy to Direct Single-Stage Food Waste Fermentation from Anaerobic Digestion to Chain Elongation

Production of medium chain carboxylic acids (MCCA) as renewable feedstock bio-chemicals, from food waste (FW), requires complicated reactor configurations and supplementation of chemicals to achieve product selectivity. This study evaluated the manipulation of organic loading rate in an un-supplemented, single stage stirred tank reactor to steer an anaerobic digestion (AD) microbiome towards acidogenic fermentation (AF), and thence to chain elongation. Increasing substrate availability by switching to a FW feedstock with a higher COD stimulated chain elongation. The MCCA species n-caproic (10.1 ± 1.7 g L−1) and n-caprylic (2.9 ± 0.8 g L−1) acid were produced at concentrations comparable to more complex reactor set-ups. As a result, of the adjusted operating strategy, a more specialised microbiome developed containing several MCCA-producing bacteria, lactic acid-producing Olsenella spp. and hydrogenotrophic methanogens. By contrast, in an AD reactor that was operated in parallel to produce biogas, the retention times had to be doubled when fed with the high-COD FW to maintain biogas production. The AD microbiome comprised a diverse mixture of hydrolytic and acidogenic bacteria, and acetoclastic methanogens. The results suggest that manipulation of organic loading rate and food-to-microorganism ratio may be used as an operating strategy to direct an AD microbiome towards AF, and to stimulate chain elongation in FW fermentation, using a simple, un-supplemented stirred tank set-up. This outcome provides the opportunity to repurpose existing AD assets operating on food waste for biogas production, to produce potentially higher value MCCA products, via simple manipulation of the feeding strategy.This project has received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No 66599

Self-generated ethanol in stillage fermentation for carboxylate upgrade

IntroductionVolatile fatty acids (VFA) are intermediates of the biogas process, thus can be produced from waste streams, and have a higher economic value as they can be used as industrial precursors and bulk chemicals. Several specific organisms present in mixed cultures have the capacity to elongate short VFA to caproate or others with a higher value using ethanol as a reducing agent. In this study we investigated different operational conditions to enhance VFA production from stillage of a bio-ethanol biorefinery. The main goals were to i) increase the titers of VFA; and ii) evaluate the ethanol requirements for carboxylate upgrade. MethodsTwo continuous reactors were fed with distilled stillage and operated at pH 5.5 and 6.5. They run for 300 days at different HRT. Batch tests with inoculum from reactor 5.5 were performed with different proportions of beer and stillage. ResultsA decrease of HRT improved the production rate of VFA from 2.7 to 8.8 g COD L-1d-1, but final concentration and composition remained similar within the same reactor (Fig.1). A decrease of pH inhibited methane production by VFA present in acidic form, but also reduced VFA concentrations in the broth. pH reduction induced ethanol production and therefore, a higher presence of longer carboxylates.Mixing beer (ethanol-rich stream) in a fermentation broth included active yeast in the system and this used VFA to generate more ethanol available for chain elongation (Fig.2). However, high concentrations of ethanol inhibited VFA production.ConclusionsLow pH induces ethanol generation in stillage fermentation and the introduction of small portions of beer can enhance the generation of ethanol and thus elongate carboxylates.<br/

Dataset on experimental data available in the literature on "Medium chain carboxylic acids from complex organic feedstock by mixed culture fermentation"

This dataset was created as a compilation of experimental data in the literature on the production of medium chain carboxylic acids (MCCAs) by microbial mixed cultures (MMC) fermentation. The intention was to provide a dataset as comprehensive as possible that includes the majority of experimental results available in this research area to the best of our knowledge. The focus lied on MMC-based studies processing complex organic feedstock, yet selected studies were included on synthetic substrates. The relevant literature studies were collected and experimental results categorized according to bioreactor operation, i.e. batch, fed-batch and (semi-)continuous. Operational parameters, such as feedstock type, organic loading rate, temperature, etc., were extracted from information reported in studies and placed alongside product outcome in terms of MCCA production for each experiment. This dataset forms the backbone of the discussion and figure generation of the literature review "Medium chain carboxylic acids from complex organic feedstock by mixed culture fermentation" by V. De Groof, M. Coma, T. Arnot, D. Leak, A. Lanham. Published in MDPI Molecules: Special Issue "Chemicals from Food Supply Chain By-Products and Waste Streams" 2019.The experimental data was collected through literature review. Studies were included that specifically target chain elongation in mixed microbial culture fermentation, but the scope was extended to include other studies that have noted chain elongation products as by-products from, for instance, volatile fatty acids (short chain carboxylic acids) or hydrogen production. Studies were selected via various research data bases based on topic relevance and key word combinations (e.g. "medium chain carboxylic acids", "medium chain fatty acids", "chain elongation in mixed cultures", ...) combined with authors' expertise in the research area and data availability.Experimental values were copied from cited studies and converted to uniform units using conversion constants given in "Read me" tab. Where data was not available or not applicable for the particular experiment "NA" is placed. Experiments were ordered according to reactor operation style, and processed for generating Figures provided in the accompanying paper.Comments provided in single spreadsheet cell provide further information regarding calculation or estimation method

Dataset for "Adjusting organic load as a strategy to direct single-stage food waste fermentation from anaerobic digestion to chain elongation"

This dataset includes the results summary from a lab-scale bioreactor experiment as discussed in the research paper with the same name, published at Processes MDPI (De Groof, V.; Coma, M.; Arnot, T.C.; Leak, D.J.; Lanham, A.B. Adjusting Organic Load as a Strategy to Direct Single-Stage Food Waste Fermentation from Anaerobic Digestion to Chain Elongation. Processes 2020, 8, 1487.). The study comprised two operational phases of duplicate reactors fed with food waste, each set to target a different product. The data comprises a summary on feedstock composition, microbial community analysis and operational conditions and product outcome per operational phase. The archaeal and bacterial community data includes the final sequences of the operational taxonomic units found and their relative abundance in each sample as determined by 16s rRNA amplicon sequencing. The raw data files have been submitted in the specialized EMBL-EBI database and are available under the accession number PRJEB39281.AnalyticalThis dataset was prepared and processed in Microsoft Excel from raw analytical data. The bioinformatic processing prior to the microbial community summary in the spreadsheet was done as outlined in the publication, and results were processed via the DNASense data analysis app (applies Rstudio IDE v.3.5.1 with the ampvis v.2.5.8. package)