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

Abstract

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.

Keywords: carboxylates; lactate isomer metabolism; lactate isomerization; lactate racemization; microbial chain elongation; n-butyrate; n-caproate; zero-valent iron.

FIGURE 1

Experiment I—Increase in monomeric lactate concentrations and pH due to nZVI addition. Data shows the initial and final results after 18 days of reaction. Error bars depict ± one standard deviation.

FIGURE 2

Experiment II—Substrates (A,B), pH (C), and metabolites (D–F) profile of lactate-based chain elongation with different nZVI concentrations. Error bars show duplicates absolute deviation from the average.

FIGURE 3

Experiment II—Net conversion by the end of the experiments at different nZVI concentrations. Oligomers were estimated as a fraction (0.36) of total lactate. The unconverted fraction shows missing electrons (from nZVI and oligomers) between metabolic products and substrates added (acetate, total lactate and Fe⁰). Error bars show duplicates absolute deviation from average.

FIGURE 4

Experiment IV—Substrate (A,B), n-butyrate (C) and pH (D) profiles for D-lactate, L-lactate and racemic lactate conversion. A positive or negative sign between braces indicate absence (–) or presence (+) of nZVI (added at 1 g⋅L^–1). Error bars indicate ± one standard deviation.

FIGURE 5

Microbiome composition in experiment II (A) and experiment IV (B). Experiment IV used D-lactate (D), L-lactate (L) or racemic lactate (R) in the absence (–) or presence (+) of nZVI (1 g⋅L^–1) (B). Blue and red shaded taxa indicate whether relative abundance was found positively or negatively correlated with nZVI presence (*** indicates p < 0.0005; ** indicates p < 0.001; * indicates p < 0.01).

FIGURE 6

Proposed effects of nZVI on lactate-based chain elongation (A), enantiomeric proportions during lactate conversion to elongated carboxylates (B) and to propionate through the acrylate pathway (C). Blue and red boxes depict even- and odd-chained carboxylates, respectively. Abbreviations: Lar, lactate racemase; D-Ldh, D-lactate dehydrogenase; L-Ldh, L-lactate dehydrogenase; PFOR, pyruvate:ferredoxin oxidoreductase. (B,C) Adapted from Schweiger and Buckel (1984); Kuchta and Abeles (1985), Hino and Kuroda (1993), and Angenent et al. (2016).