Chain elongators, friends and foes (or the microbial ecology of chain elongation microbiomes)

C

Enrichment and characterisation of ethanol chain elongating communities from natural and engineered environments

Chain elongation is a microbial process in which an electron donor, such as ethanol, is used to elongate short chain carboxylic acids, such as acetic acid, to medium chain carboxylic acids. This metabolism has been extensively investigated, but the spread and differentiation of chain elongators in the environment remains unexplored. Here, chain elongating communities were enriched from several inocula (3 anaerobic digesters, 2 animal faeces and 1 caproic acid producing environment) using ethanol and acetic acid as substrates at pH 7 and 5.5. This approach showed that (i) the inoculum’s origin determines the pH where native chain elongators can grow; (ii) pH affects caproic acid production, with average caproic acid concentrations of 6.4 ± 1.6  g·L−1 at pH 7, versus 2.3 ± 1.8  g·L−1 at pH 5.5; however (iii) pH does not affect growth rates significantly; (iv) all communities contained a close relative of the known chain elongator Clostridium kluyveri; and (v) low pH selects for communities more enriched in this Clostridium kluyveri-relative (57.6 ± 23.2% at pH 7, 96.9 ± 1.2% at pH 5.5). These observations show that ethanol-consuming chain elongators can be found in several natural and engineered environments, but are not the same everywhere, emphasising the need for careful inoculum selection during process development

Electrolytic extraction drives volatile fatty acid chain elongation through lactic acid and replaces chemical pH control in thin stillage fermentation

Background: Volatile fatty acids (VFA) are building blocks for the chemical industry. Sustainable, biological production is constrained by production and recovery costs, including the need for intensive pH correction. Membrane electrolysis has been developed as an in situ extraction technology tailored to the direct recovery of VFA from fermentation while stabilizing acidogenesis without caustic addition. A current applied across an anion exchange membrane reduces the fermentation broth (catholyte, water reduction: H2O + e- → 12 H2 + OH-) and drives carboxylate ions into a clean, concentrated VFA stream (anolyte, water oxidation: H2O → 2e- + 2 H+ + O2). Results: In this study, we fermented thin stillage to generate a mixed VFA extract without chemical pH control. Membrane electrolysis (0.1 A, 3.22 ± 0.60 V) extracted 28 ± 6 % of carboxylates generated per day (on a carbon basis) and completely replaced caustic control of pH, with no impact on the total carboxylate production amount or rate. Hydrogen generated from the applied current shifted the fermentation outcome from predominantly C2 and C3 VFA (64 ± 3 % of the total VFA present in the control) to majority of C4 to C6 (70 ± 12 % in the experiment), with identical proportions in the VFA acid extract. A strain related to Megasphaera elsdenii (maximum abundance of 57 %), a bacteria capable of producing mid-chain VFA at a high rate, was enriched by the applied current, alongside a stable community of Lactobacillus spp. (10 %), enabling chain elongation of VFA through lactic acid. A conversion of 30 ± 5 % VFA produced per sCOD fed (60 ± 10 % of the reactive fraction) was achieved, with a 50 ± 6 % reduction in suspended solids likely by electro-coagulation. Conclusions: VFA can be extracted directly from a fermentation broth by membrane electrolysis. The electrolytic water reduction products are utilized in the fermentation: OH- is used for pH control without added chemicals, and H2 is metabolized by species such as Megasphaera elsdenii to produce greater value, more reduced VFA. Electro-fermentation displays promise for generating added value chemical co-products from biorefinery sidestreams and wastes.</p

Top-down and bottom-up microbiome engineering approaches to enable biomanufacturing from waste biomass.

UNLABELLED: Growing environmental concerns and the need to adopt a circular economy have highlighted the importance of waste valorization for resource recovery. Microbial consortia-enabled biotechnologies have made significant developments in the biomanufacturing of valuable resources from waste biomass that serve as suitable alternatives to petrochemical-derived products. These microbial consortia-based processes are designed following a top-down or bottom-up engineering approach. The top-down approach is a classical method that uses environmental variables to selectively steer an existing microbial consortium to achieve a target function. While high-throughput sequencing has enabled microbial community characterization, the major challenge is to disentangle complex microbial interactions and manipulate the structure and function accordingly. The bottom-up approach uses prior knowledge of the metabolic pathway and possible interactions among consortium partners to design and engineer synthetic microbial consortia. This strategy offers some control over the composition and function of the consortium for targeted bioprocesses, but challenges remain in optimal assembly methods and long-term stability. In this review, we present the recent advancements, challenges, and opportunities for further improvement using top-down and bottom-up approaches for microbiome engineering. As the bottom-up approach is relatively a new concept for waste valorization, this review explores the assembly and design of synthetic microbial consortia, ecological engineering principles to optimize microbial consortia, and metabolic engineering approaches for efficient conversion. Integration of top-down and bottom-up approaches along with developments in metabolic modeling to predict and optimize consortia function are also highlighted. ONE-SENTENCE SUMMARY: This review highlights the microbial consortia-driven waste valorization for biomanufacturing through top-down and bottom-up design approaches and describes strategies, tools, and unexplored opportunities to optimize the design and stability of such consortia

A novel high-throughput method for kinetic characterisation of anaerobic bioproduction strains, applied to Clostridium kluyveri

Hexanoic acid (HA), also called caproic acid, can be used as an antimicrobial agent and as a precursor to various chemicals, such as fuels, solvents and fragrances. HA can be produced from ethanol and acetate by the mesophilic anaerobic bacterium Clostridium kluyveri, via two successive elongation steps over butyrate. A high-throughput anaerobic growth curve technique was coupled to a data analysis framework to assess growth kinetics for a range of substrate and product concentrations. Using this method, growth rates and several kinetic parameters were determined for C. kluyveri. A maximum growth rate (mu(max)) of 0.24 +/- 0.01 h(-1) was found, with a half-saturation index for acetic acid (K-S,K-AA) of 3.8 +/- 0.9 mM. Inhibition by butyric acid occurred at of 124.7 +/- 5.7 mM (K-I,K-BA), while the final product, HA, linearly inhibited growth with complete inhibition above 91.3 +/- 10.8 mM (K-HA of 10.9*10(-3) +/- 1.3*10(-3) mM(-1)) at pH = 7, indicating that the hexanoate anion also exerts toxicity. These parameters were used to create a dynamic mass-balance model for bioproduction of HA. By coupling data collection and analysis to this modelling framework, we have produced a powerful tool to assess the kinetics of anaerobic micro-organisms, demonstrated here with C. kluyveri, in order further explore the potential of micro-organisms for chemicals production