Reactor microbiome enriches vegetable oil with n-caproate and n-caprylate for potential functionalized feed additive production via extractive lactate-based chain elongation.

BACKGROUND: Biotechnological processes for efficient resource recovery from residual materials rely on complex conversions carried out by reactor microbiomes. Chain elongation microbiomes produce valuable medium-chain carboxylates (MCC) that can be used as biobased starting materials in the chemical, agriculture and food industry. In this study, sunflower oil is used as an application-compatible solvent to accumulate microbially produced MCC during extractive lactate-based chain elongation. The MCC-enriched solvent is harvested as a potential novel product for direct application without further MCC purification, e.g., direct use for animal nutrition. Sunflower oil biocompatibility, in situ extraction performance and effects on chain elongation were evaluated in batch and continuous experiments. Microbial community composition and dynamics of continuous experiments were analyzed based on 16S rRNA gene sequencing data. Potential applications of MCC-enriched solvents along with future research directions are discussed. RESULTS: Sunflower oil showed high MCC extraction specificity and similar biocompatibility to oleyl alcohol in batch extractive fermentation of lactate and food waste. Continuous chain elongation microbiomes produced the MCC n-caproate (nC6) and n-caprylate (nC8) from L-lactate and acetate at pH 5.0 standing high undissociated n-caproic acid concentrations (3 g L-1). Extractive chain elongation with sunflower oil relieved apparent toxicity of MCC and production rates and selectivities reached maximum values of 5.16 ± 0.41 g nC6 L-1 d-1 (MCC: 11.5 g COD L-1 d-1) and 84 ± 5% (e- eq MCC per e- eq products), respectively. MCC were selectively enriched in sunflower oil to concentrations up to 72 g nC6 L-1 and 3 g nC8 L-1, equivalent to 8.3 wt% in MCC-enriched sunflower oil. Fermentation at pH 7.0 produced propionate and n-butyrate instead of MCC. Sunflower oil showed stable linoleic and oleic acids composition during extractive chain elongation regardless of pH conditions. Reactor microbiomes showed reduced diversity at pH 5.0 with MCC production linked to Caproiciproducens co-occurring with Clostridium tyrobutyricum, Clostridium luticellarii and Lactobacillus species. Abundant taxa at pH 7.0 were Anaerotignum, Lachnospiraceae and Sporoanaerobacter. CONCLUSIONS: Sunflower oil is a suitable biobased solvent to selectively concentrate MCC. Extractive reactor microbiomes produced MCC with improved selectivity and production rate, while downstream processing complexity was reduced. Potential applications of MCC-enriched solvents may include feed, food and biofuels purposes.

nZVI Impacts Substrate Conversion and Microbiome Composition in Chain Elongation From D- and L-Lactate Substrates.

Medium-chain carboxylates (MCC) derived from biomass biorefining are attractive biochemicals to uncouple the production of a wide array of products from the use of non-renewable sources. Biological conversion of biomass-derived lactate during secondary fermentation can be steered to produce a variety of MCC through chain elongation. We explored the effects of zero-valent iron nanoparticles (nZVI) and lactate enantiomers on substrate consumption, product formation and microbiome composition in batch lactate-based chain elongation. In abiotic tests, nZVI supported chemical hydrolysis of lactate oligomers present in concentrated lactic acid. In fermentation experiments, nZVI created favorable conditions for either chain-elongating or propionate-producing microbiomes in a dose-dependent manner. Improved lactate conversion rates and n-caproate production were promoted at 0.5-2 g nZVI⋅L-1 while propionate formation became relevant at ≥ 3.5 g nZVI⋅L-1. Even-chain carboxylates (n-butyrate) were produced when using enantiopure and racemic lactate with lactate conversion rates increased in nZVI presence (1 g⋅L-1). Consumption of hydrogen and carbon dioxide was observed late in the incubations and correlated with acetate formation or substrate conversion to elongated products in the presence of nZVI. Lactate racemization was observed during chain elongation while isomerization to D-lactate was detected during propionate formation. Clostridium luticellarii, Caproiciproducens, and Ruminococcaceae related species were associated with n-valerate and n-caproate production while propionate was likely produced through the acrylate pathway by Clostridium novyi. The enrichment of different potential n-butyrate producers (Clostridium tyrobutyricum, Lachnospiraceae, Oscillibacter, Sedimentibacter) was affected by nZVI presence and concentrations. Possible theories and mechanisms underlying the effects of nZVI on substrate conversion and microbiome composition are discussed. An outlook is provided to integrate (bio)electrochemical systems to recycle (n)ZVI and provide an alternative reducing power agent as durable control method.

Consecutive lactate formation and chain elongation to reduce exogenous chemicals input in repeated-batch food waste fermentation.

The production of biochemicals from renewables through biorefinery processes is important to reduce the anthropogenic impact on the environment. Chain elongation processes based on microbiomes have been successfully developed to produce medium-chain fatty acids (MCFA) from organic waste streams. Yet, the sustainability of chain elongation can still be improved by reducing the use of electron donors and additional chemicals. This work aimed to couple lactate production and subsequent chain elongation to decrease chemicals input such as electron donors and hydroxide for pH control in repeated-batch food waste fermentation. Food waste with adjusted pH was used as substrate and fermentation proceeded without pH control. During fermentation, lactate was first formed through the homolactic pathway and then converted to fatty acids (FA), mainly n-butyrate and n-caproate. The highest n-caproate carbon selectivities (mmol C/mmol CFA) and production rates were 38% and 4.2 g COD/L-d, respectively. Hydroxide input was reduced over time to a minimum of 0.47 mol OH-/mol MCFA or 0.79 mol OH-/kg CODFA. Lactate was a key electron donor for chain elongation and its conversion was observed at pH as low as 4.3. The microbiome enriched in this work was dominated by Lactobacillus spp. and Caproiciproducens spp. The high abundance of Caproiciproducens spp. and their co-occurrence with Lactobacillus spp. suggest Caproiciproducens spp. used lactate as electron donor for chain elongation. This work shows the production of n-caproate from food waste with decreased use of hydroxide and no use of exogenous electron donors.