
==== Front
Environ Sci Ecotechnol
Environ Sci Ecotechnol
Environmental Science and Ecotechnology
2096-9643
2666-4984
Elsevier

S2666-4984(24)00073-5
10.1016/j.ese.2024.100459
100459
Original Research
The convergence of lactic acid microbiomes and metabolites in long-term electrofermentation
Leininger Aaron ab
Lu Sidan ab
Jiang Jinyue ab
Bian Yanhong ab
May Harold D. b
Ren Zhiyong Jason zjren@princeton.edu
ab⁎
a Department of Civil and Environmental Engineering, Princeton University, USA
b Andlinger Center for Energy and the Environment, Princeton University, USA
⁎ Corresponding author. Department of Civil and Environmental Engineering, Princeton University, USA. zjren@princeton.edu
27 7 2024
11 2024
27 7 2024
22 10045916 10 2023
20 7 2024
20 7 2024
© 2024 The Authors
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Regulating electron transfer in predominantly fermentative microbiomes has broad implications in environmental, chemical, food, and medical fields. Here we demonstrate electrochemical control in fermenting food waste, digestate, and wastewater to improve lactic acid production. We hypothesize that applying anodic potential will expedite and direct fermentation towards lactic acid. Continued operation that introduced epi/endophytic communities (Lactococcus, Lactobacillus, Weissella) to pure culture Lactiplantibacillus plantarum reactors with static electrodes was associated with the loss of anode-induced process intensification despite 80% L. plantarum retention. Employing fluidized electrodes discouraged biofilm formation and extended electrode influence to planktonic gram-positive fermenters using mediated extracellular electron transfer. While short-term experiments differentially enriched Lactococcus and Klebsiella spp., longer-term operations indicated convergent microbiomes and product spectra. These results highlight a functional resilience of environmental fermentative microbiomes to perturbations in redox potential, underscoring the need to better understand electrode induced polymicrobial interactions and physiological impacts to engineer tunable open-culture or synthetic consortia.

Graphical abstract

Image 1

Highlights

• We explore flavin, humic, and heme-based approaches to electrically control microbiomes.

• Microbiomes under different electrode potentials converge over time.

• The anodic tunability of L. plantarum is blocked with continued operation.

• This is the first application of fluidized anodes to fermentative microbiomes.

Keywords

Fermentation
Food waste
Lactic acid
Microbiome
Electrofermentation
Resource recovery
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pmc1 Introduction

In recent years, knowledge of the distribution of extracellular electron transport (EET) processes has extended from metal-reducing, methanogenic, and acetogenic bacteria to primarily fermentative microbes [1], such as lactic acid bacteria [2,3]. In the waste-to-chemicals biorefinery context, control of the assembly and metabolism of fermentative microbiomes is a major barrier to the viability of the carboxylate platform [4]. Generally, longer-chain fermentation products (e.g., butyrate rather than acetate) are more desirable as they are more energy-dense and have orders-of-magnitude higher acid-water separation factors in membrane-based extraction processes [5]. Electrochemical regulation using potentiostatically poised electrodes has been proposed as an engineering control to enable tunability and/or process intensification, but single-batch reports to date, mostly employing cathodic regulation, produced modest effects on the product spectrum [[6], [7], [8], [9], [10], [11], [12], [13]].

There have been mixed findings on how potentiostatically poised electrodes, or engineered electron transfer in general, can regulate the microbiome and products of waste fermentations. Providing an anodic sink for electrons may grant a fitness benefit to fermenters able to regenerate oxidized nicotinamide adenine dinucleotide (NAD+) through outward EET. Some works [6,7,14] suggested that reducing power from a cathode could drive longer-chain products from open-culture fermentation. However, it has also been suggested that negative fitness effects may arise from electron transfer to a fermenter, particularly in the case of forced/coerced transfer (e.g., parasitism) [15]. Suppose the cathode directly, or soluble mediators reduced by the cathode, perform similarly. In that case, long-term cathodic electrofermentation (EF) operation may deselect the fermenters capable of inward-EET over the continued operation. The overwhelming majority of open-culture EF studies have focused on cathodic EF [13], based on the reasoning that additional reducing power from the cathode could increase the generation of more reduced products. However, only two primarily fermentative bacteria have been demonstrated to be capable of endogenous inward EET (Clostridium pastereurianum [1] and Lactiplantibacillus plantarum [16]). EET to a fermenter may have negative fitness effects, while EET from a fermenter to regenerate NAD + may allow for faster metabolic throughput and growth. Our literature review of similarly conducted EF experiments [[6], [7], [8], [9], [10], [11], [12]] showed no clear pattern between applied working electrode potential and change in product length versus the open-circuit control experiment. Reported EF studies have been very short in duration (1–20 days), limited to a single batch fermentation starting with a fresh static planar electrode, and therefore may be too limited to understand the effects of electrode polarization on the assembly and function of the microbiome.

Our study seeks to fill this gap through the first report of long-term operation and community assembly in non-axenic EF. Since the discovery of a flavin-involved extracellular electron transport (FLEET) locus [2] in diverse environmentally relevant gram-positive bacteria, growth in the presence of an anode and exogenous quinone was demonstrated to enhance metabolic throughput in lactic acid bacteria by a factor of around two by enabling a metabolic strategy that blends aspects of fermentation and respiration [3]. Microbiomes can produce lactic acid in high titer (70 g L−1) [17] and hold significant potential in biorefinery applications; examples include bioplastics and substrate for carboxylate upgrading through reverse beta-oxidation. Lower pH increasingly favors the elongation pathway thermodynamics over competing routes such as acetogenesis and the acrylate pathway [18]. We hypothesized that this FLEET metabolism could be leveraged in open-culture fermentations to improve atom-economy towards lactic acid fermentation or chain-elongation products and/or intensify fermentations. First, fluidized anodes were applied in synthetic food waste (kale juice), a plant containing FLEET-encoding Lactococcus spp. as a dominant endo/epiphyte. A spent-media enrichment culture study was then conducted using the effluent from these experiments in a glucose mineral medium, and the performance and microbiome dynamics were observed over 18 semi-batch fill cycles, comparing anodically poised and open-circuit performance for over 1900 h. Anodic, cathodic, and open-circuit fermentations were performed for 24 cycles with glucose mineral media amended with real food waste digestate and the model humic substance, anthraquinone-2,6-disulfonate (AQDS). Finally, domestic wastewater was examined for electrode tunability with and without a heme-based respiratory preacclimation strategy.

2 Materials and methods

2.1 H-cell fermentations

In H-cell experiments, 4 × 4 × 0.635 cm graphite felts connected with Ti wire (28 gauge TEMCo) served as the working electrode. Thick Ti wire (ϕ = 0.2 cm) served as the counter electrode with cation exchange membrane (CMI7000, Membranes International, Ringwood NJ) soaked overnight in M9 salts separating the chambers (300 mL) and an Ag/AgCl reference electrode (1 M KCl, BASi, Inc.) inserted next to the carbon felt. Working electrode potentials were controlled using a multichannel potentiostat (VMP3, BioLogic), and open-circuit potentials were measured with a digital multimeter. Reactor headspaces were constantly purged with nitrogen at 142 cm3 min−1.

Reactors were assembled, filled with deionized water, and autoclaved. Once cool, autoclaved kale juice or mannitol chemically defined media (mCDM) [3] were added to the working electrode chamber, and M9 salts were added to the counter electrode chamber. Reactors were polarized and inoculated with L. plantarum cell preparations. In the kale juice H-cell fermentations, three subsequent half-batch media replacements were performed with raw kale juice, including one group of reactors with 1,4-dihydroxy-2-naphthoic acid (DHNA) (20 μg mL−1) and another without DHNA.

2.2 Fluidized bed electrode fermentations

Fluidized bed reactors (FBRs) were machined from transparent schedule 40 PVC (L = 20.32 cm, ϕ = 5.08 cm) (Supplementary Material Fig. S10). The working electrode consisted of 150 mL granular activated carbon (Alfa Aesar, −20 + 40 mesh) suspended by recirculating flow around a graphite current collector (7.94 × 1.91 × 1.27 cm, McMaster-Carr). Recirculating flow at the minimum fluidization velocity (0.28 cm s−1) was provided by a peristaltic pump and routed through a 42-mesh flow distribution plate mounted within a reducing coupling to suspend the bed. PVC caps with gas bags (Calibrated Instruments, Inc.) were mounted on top of the columns to allow for media replacements without atmospheric intrusion. Counter electrodes consisting of a graphite block (4 × 1.5 × 0.6 cm) within a tubular sleeve of cation exchange membrane (ϕ = 2.5 cm, Membranes International, Ringwood NJ) and filled with 200 mM PBS were installed 3 cm above the top of the current collector. Titanium wire (McMaster-Carr) was used to connect working and counter electrodes. The total working volume of reactors was 360 ml. Reference electrodes were inserted between working and counter electrodes near the top of current collectors. Activated carbon beds were omitted in wastewater experiments, and the graphite current collector served as the working electrode.

Fluidized bed electrodes were used to affect the fermentations of raw kale juice, a kale juice enrichment culture, and glucose media with and without adding food waste digestate. Half-media replacements of fluidized bed reactors were performed by filling the gas bag with nitrogen, pumping out half the reactor media, and replacing it with fresh media.

2.3 Media and inoculum

Real food waste, wastewater, and glucose PBS media (200 mM) [19] were used. Preparation of kale (Brassica oleracea var. sabellica) juice media followed the method of Tejedor-Sanz et al. [3] but, in some cases, was not autoclaved in experiments where the epi/endophytic community served as inoculum. 1,4-dihydroxy-2-naphthoic acid (DHNA) was added (20 μg mL−1) as a demethylmenaquinone (DMK) precursor. In the first fill cycles of the fluidized bed fermentations with glucose PBS media, glucose media was augmented with 10% (v/v) digestate (8.76 ± 0.13 gVS L−1) from a nearby food waste digester serving the central New Jersey region (Trenton Renewables, LLC). Later in the experiment, AQDS (206 mg L−1 (500 μM)) was added to the glucose PBS media as a model humic substance. Semi-batch cycles were conducted by half media replacement, using an N2-filled gas bag during media draw-down to ensure anaerobic conditions. Glucose media was degassed for 30 min with N2 using a commercial air stone. In kale juice enrichment culture fluidized bed EF, 2% (v/v) of spent media (anodically fermented kale juice) from a previous experiment was used to inoculate the reactors in the initial fill cycle.

Domestic wastewater was collected from the grit chamber at the headworks from a nearby utility (Stony Brook Regional Sewerage Authority, Princeton, NJ). Before fermentation, respiratory preacclimation was conducted by porcine heme addition (Sigma, 3 or 30 μg mL−1), glucose addition in some cases (1 g L−1), and aeration with a commercial airstone for 3 h, while the control group was N2-purged for the same duration.

2.4 Strains and culture conditions

Some experiments used L. plantarum strain NCIMB 8826 (ATCC) as a model electroactive fermenter. Lyophilized pellets were rehydrated with filter-sterilized glucose De Man, Rogosa, and Sharpe media [20] (gMRS, Oxoid) and incubated with shaking at 37 °C for 24–36 h. Cells were harvested by centrifugation (5200 g, 12 min, 4 °C) and washed 3 × in Dulbecco's PBS. The cell preparation in PBS was used to inoculate autoclaved kale juice to an optical density at 600 nm (OD600) of 0.07.

2.5 Metabolite analysis

1500 μL samples of reactor media were collected during and at the end of semi-batch fill cycles and filtered through 0.22 μm cellulose acetate spin filters. Organic acids and alcohols were analyzed with HPLC (Agilent 1260 Infinity II) using a 4 mM sulfuric acid mobile phase and refractive index detector [21]. Standards ran included glucose, mannitol, formate, succinate, acetate, propionate, butyrate, isovalerate, valerate, and ethanol. The chemical oxygen demand of filtered samples (0.22 μm) was determined with a commercially available assay (Hach HR COD) [22,23].

2.6 Microbiome analysis

Liquid media samples were collected at the end of fill cycles and frozen at −20 °C until extraction. Genomic DNA was extracted with Qiagen PowerSoil Pro kit from 0.2 g of cell pellets resulting from centrifugation of sample aliquot. Extracted DNA was quality checked with Nanodrop, quantified with Qubit HS dsDNA assay, and normalized to 10 ng μL−1 before library preparation. 16S rRNA gene (V4 region) libraries were prepared and sequenced on Illumina Nanoseq (2 × 150 bp). Sequencing data was processed in R through the dada2 pipeline [24] (maxEE = (2,2)), and taxonomy was assigned to amplicon sequence variants using SILVA v132.x reference database. Downstream analyses, including alpha and beta diversity, were conducted in R using phyloseq [25] and visualized with ggplot2. Rarefaction curves were prepared with vegan [26] to confirm that sequencing depth captured microbial diversity [27].

Raw sequencing reads are publicly available via NCBI SRA (PRJNA1016412 (L. plantarum/kale microbiome succession, Fig. 1) and PRJNA1017815 (fluidized bed kale juice enrichment, Fig. 2)).Fig. 1 a, Anodic electrofermentation of L. plantarum inoculated kale juice followed by three subsequent half-media replacements with raw kale juice in H-cell reactors. b, Distribution of fermentation metabolites with dashed lines indicating half-media replacement. c, Chronoamperometric response of DHNA-amended (n = 3) and non-amended (n = 2) reactors poised at +200 mV vs. Ag/AgCl. All reactors received DHNA during the first fill cycle with autoclaved kale juice. d, Nonmetric dimensional scaling based on Bray-Curtis dissimilarity of suspension microbiomes. e, Top twenty most abundant ASVs across the final three fill cycles. f, The relative abundance of the top twenty ASVs among reads not assigned to genus Lactiplantibacillus. OC: Open-circuit control.

Fig. 1

Fig. 2 Operation of fluidized bed reactors inoculated with fermented kale juice (glucose mineral media) yielded similar fermentation products in anodic (+200 mV) electrofermentation versus open-circuit control across eighteen cycles of serial semi-batch operation. Vertical dashed lines indicate half-media replacement. Glucose (a), lactate (b), acetate (c), propionate (d), butyrate (e), valerate (f), total alcohols and acids (g), and the average carbon length of fermentation products (h).

Fig. 2

2.7 Transmission and environmental scanning electron microscopy

Cell suspensions and associated suspended carbon particles were examined using transmission electron microscopy at 50 kV (Talos F200). Negative-stained specimens were prepared on formvar/carbon-covered 400 mesh Cu grids (Ted Pella) by adding 10 μL of reactor media to the grid and treating with UranyLess (Electron Microscopy Sciences, Hatfield PA) according to the manufacturer protocol.

Biofilm coverage on activated carbon particles in wetted conditions was examined using Quanta 200 FEG E-SEM.

3 Results and discussion

3.1 The presence of alternate charge acceptors and mixed culture of lactic-acid bacteria native to food waste confound anodic tunability

Initial H-cell reactor experiments were carried out using mannitol substrate in chemically defined mineral media (mCDM), and we found that the flavin-based pathway boosted the metabolic throughput of the lactic acid bacterium L. plantarum, leading to faster fermentation and environmental acidification (Supplementary Material Fig. S1). These results support the findings by Tejedor-Sanz et al., 2022 [3], but we did not observe a similar difference in fermentation between anodic and open-circuit when L. plantarum was cultured in gMRS media in FBRs (Supplementary Material Fig. S2), likely attributed to the charge-acceptor rich environment and/or the sorption of electron shuttles to the activated carbon electrode bed. FLEET-style metabolism in L. plantarum depends on a pool of oxidized flavins to reduce but not necessarily an anodically-poised electrode. In the FBR experiment, a greater abundance of charge acceptors was present in the rich media (1 mg L−1 riboflavin in mCDM vs. 4 g L−1 yeast extract in gMRS), likely rendering the polarization of the electrode by potentiostat redundant. This was reflected by the greatly reduced change in OCP in FBRs - the open circuit potential in mCDM H-cells reached as low as −408 mV vs. Ag/AgCl compared with a low point of 138 ± 2 mV in the gMRS FBR during fermentations of comparable length (160 and 103 h, respectively). FLEET-style metabolism in a quinone auxotroph like L. plantarum is reliant on the uptake of quinone from the environment or neighbor to an intra-membrane pool that serves as an intermediate for electrons from NADH dehydrogenase to extracellular flavoproteins, and eventually, through reduced flavins, to the anode [2]. While some FLEET-encoding LAB, such as Lactococcus lactis [28], and secondary lactate fermenters like Propionibacterium freudenreichii [29], can produce their own quinone and leak it to the environment and crossfeeding community members, L. plantarum may have difficulty acquiring relatively hydrophobic quinones from the environment in the presence of strong sorbents like a fresh bed of granular activated carbon in a single-batch experiment.

Anodic tunability of L. plantarum pure culture was reported in kale juice substrate supplemented with DHNA (demethylmenaquinone-precursor), and faster fermentation in the anodic condition was observed than in the open-circuit [3]. We hypothesized that this effect could intensify and/or tune mixed culture food waste fermentations with FLEET-encoding autochthonous microbes in a biorefinery concept. We selected kale juice as a model food waste because it was shown to support electroactive fermentation in L. plantarum and contains Lactococcus spp. as a natural endophyte. The short-term experiment comparing the fermentation of kale juice supplemented with DHNA (10 μg mL−1) using fluidized electrodes poised at (−800 to +200 mV vs. Ag/AgCl) did indeed show that the anodically-poised fermentation yielded more than twice the maximum lactic acid production than the cathodic reactor, apparently at the expense of acetic acid, as it reached higher concentration (>800 mg L−1 in cathodic versus 410 mg L−1 in anodic EF) (Supplementary Material Fig. S3a). Anodic/cathodic microbiomes showed similar taxonomic profiles with Lactococcus sp., Klebsiella sp., Citribacter spp., and Clostridum sensu stricto 1, 12, 3 among the ten most abundant amplicon-sequence-variants (ASVs), but a much higher relative abundance of Lactococcus sp., most similar to Lactococcus raffinolactis, was observed in anodically-poised reactors (Supplementary Material Fig. S3b). However, five more repeated trials of single-batch kale juice fermentation under different electrode polarization conditions (+200 mV, −800 mV, open-circuit, and no-electrode) did not show a pattern between electrode polarization and the lactic acid fraction of metabolites (Supplementary Material Fig. S4). This variety of electrode conditions included a no-electrode control that removed the sorptive capacity of the granular activated carbon bed from the environment. These results suggest that factors other than electrode control, for instance, stochastic community assembly, are greater drivers of the product distribution of single-batch MMC fermentations and that any advantage/niche provided by the fluidized activated carbon anode is insignificant in the short term despite the presence of 10.13039/100026099 LAB predicted to encode the EET-locus that granted L. plantarum enhanced metabolic throughput.

To better understand the result differences and the role that microbiome assembly and endo/epiphytes other than the tunable L. plantarum have on anodic tunability, anodic and open-circuit fermentation of kale juice was further conducted in H-cell reactors, starting with a batch of autoclaved kale juice inoculated with L. plantarum and supplemented with DHNA (20 μg mL−1) and then three subsequent half-media replacements with raw kale juice, introducing autochthonous microbes (Fig. 1a). One group of H-cells received raw kale juice that continued to be supplemented with DHNA, while another group was unamended in order to understand the effect of exogenous quinone on the assembly of the electrically influenced microbiome. After five days of fermentation, anodically poised reactors (n = 5) acidified faster and reached a higher lactate titer (6603 ± 341 vs. 5366 ± 288 mg L−1) than open-circuit reactors (n = 3) (Fig. 1b). However, this tunability was not preserved upon the introduction of raw kale juice, with similar product distributions of lactate, acetate, and propionate accumulating in anodic and open-circuit reactors after microbes native to kale were introduced.

Within the anodic group, the continued addition of DHNA (20 μg mL−1) approximately doubled the current density relative to the group without continued DHNA (Fig. 1c) but had no discernible impact on metabolites. Upon introducing the kale endo/epiphytes, current density increased in the DHNA-amended to roughly 2 A m−3 but diminished in the non-amended group. DHNA and flavins have distinct but somewhat overlapping roles in L. plantarum EET, with DHNA taken up by L. plantarum serving as a precursor to intramembrane demethylmenaquinone, allowing the use of flavins as an outward electron shuttle but also able to serve as a diffusible redox mediator on its own from L. plantarum to an anode [30]. DHNA can also exert oxidative stress on L. plantarum, which can be ameliorated by access to an insoluble electron acceptor [31]. DHNA supplementation did not appear to have a discernible impact on microbiome assembly in anodic or open-circuit reactors (Fig. 1e and f).

Microbiomes from all treatments clustered closely in the first and second cycles with raw kale juice (Fig. 1d). L. plantarum-assigned ASVs comprised over 80% of all reads even after three half-media replacements with raw kale juice. Among the introduced (non-Lactiplantibacillus assigned) taxa, LAB assigned to the genera Lactobacillus, Lactococcus, Leuconostoc, Paucilactobacillus, and Weisella were most abundant in all treatment conditions, comprising over 90% of non-Lactiplantibacillus reads (Fig. 1f). The dominance of LAB within these reactors could at least partially be attributed to lactic acid accumulation maintaining pH < 6 (Fig. 1b), selecting for acid-tolerant endo/epiphytes upon their introduction in the second fill cycle. Although metatranscriptomic profiling was not conducted to understand the expression of EET genes by these introduced LAB, it is predicted that some of these introduced taxa (e.g., Lactococcus spp.) encode FLEET loci [3], and current density increased, rather than decreased upon their introduction in the DHNA + group (Fig. 1c).

These results collectively point towards a unique scenario where introducing additional putative electroactive fermenters to a system is linked to the loss of electrode-induced tunability. These findings suggest several outstanding questions that need to be addressed before anodic EF can be engineered for non-axenic systems – did L. plantarum's FLEET expression, and associated product tunability, change with continued operation or with other LAB as neighbors; did other LAB outcompete L. plantarum for substrate, quinones, or flavins, or did community members otherwise interfere with L. plantarum's outward EET?

3.2 Convergence of anodic, open-circuit kale juice enrichment cultures through months of fluidized bed glucose fermentations

Inconsistencies in kale juice EF could be attributed to substrate or endophyte community variations. To decipher these uncertainties, we conducted spent-media enrichment culture experiments where a homogenous substrate was fermented over 18 semi-batch cycles after an initial inoculation with anodically fermented kale juice. FBRs were employed with the aim that the ball-milling/scouring effect from the bed fluidization would discourage thick biofilm formation on the working electrode over long-term operation. Continued operation and biofilm formation were previously linked to decreased chain-elongation tunability and the discrepancy between the attached and planktonic microbiome [14], possibly by inhibiting EET to planktonic fermenters. There was no prior report of fluidized anodes to engineer fermentations, although similar FBRs have been demonstrated to support outward EET and growth of the canonical electrogen Geobacter sulfurreducens [32]. In addition, LAB as gram-positive fermenters may generally prefer a planktonic lifestyle with the ability to self-aggregate or adhere to a surface as a stress response [33,34].

Through the first seven semi-batch cycles, anodically-poised reactors performed similarly to the open-circuit control, with no differences observed in glucose consumption (Fig. 2a) or metabolite production. Over the first 1000 h of operation, the average carbon length of metabolites increased from near 2.0 to over 3.0 (Fig. 2h) as the proportion of propionate (Fig. 2d) and butyrate (Fig. 2e) increased. Two operational changes were made in the final eight cycles to favor increased lactic acid accumulation. We reduced the interval between cycles to 24 h and increased the yeast extract concentration 0.1–5 g L−1 to address 10.13039/100026099 LAB auxotrophy concerns and provide additional flavins to support EET activity [35]. These changes resulted in more than doubled lactic acid accumulation in both poised and open circuit conditions (Fig. 2b), while no clear difference was observed. Valerate, which previously had been a minor metabolite (<100 mg L−1), was observed to accumulate close to 1500 mg L−1 (Fig. 2f), consistent with a previous observation of lactate secondary fermentation yielding odd-carbon chain elongation products resulting from elongation of propionate produced via the acrylate pathway [36]. Propionate production also appeared to be impacted by applied potential, with greater yield in the control group during the first seven cycles and greater yield in the anodically-poised group after increasing yeast extract and reducing hydraulic retention time (Fig. 2d).

Throughout the initial 1000 h of the experiment, it became apparent that the anodically-poised and control microbiomes increasingly resembled each other at the end of each semi-batch fill cycle (Fig. 3a). This convergence occurred irrespective of the applied potential, indicating that the electrode did not play a significant role in driving differential microbiome assembly, mirroring the distribution of metabolic processes. Notably, homolactic Lactococcus spp. was observed in comparable proportions in both anodically-poised and open-circuit reactors, and associated lactate was observed as a transient metabolite, likely converted to acetate, propionate, or butyrate by Clostridium spp. and/or Pseudomonas sp. (Fig. 3b). Rather than known DHNA-producers like Propionibacterium spp., Clostridium sensu stricto 12 was observed with increasing relative abundance over operational time (Fig. 3b) coinciding with increased accumulation of butyrate, pointing towards their role as putative lactate consumers as has been observed in other lactate-consuming microbiomes [37,38]. Although the inoculum (fermented kale juice) was unlikely to contain a high abundance of strong electrogens, application of oxidizing potentials did not lead to enrichment of canonical strong electrogens (e.g., Geobacter spp. was not observed) in primarily fermentative microbiomes even over long timescales.Fig. 3 a, Convergence of microbiomes was observed through the first six semi-batch cycles of fluidized bed kale juice enrichment cultures through NMDS on Bray-Curtis distance. Data points are labelled by the corresponding fill cycle of DNA sequencing. b, Top 20 most abundant ASVs from triplicate anodic and duplicate open-circuit fermentation reactors. c–d, ESEM images from the conclusion of the study period show biofilm formation in weathered regions of granular activated particles (c) but qualitatively little attachment across particles weathered smooth through “ball-milling” (d).

Fig. 3

Environmental SEM allowed examination of the fluidized bed particles in wet conditions after the final cycle of operation and suggested that the bed fluidization was effective in scouring the granular activated carbon particles relatively smooth (Fig. 3c), with qualitatively little attached growth evident, relegated to the crevices of the weathered particles (Fig. 3d). Taken together, these results suggest that despite the metabolite variations under different operational conditions, over long-term microbiome assembly, the presence of an anode did not create an ecological niche in a planktonic system favoring certain primary fermenters or driving differential glucose fermentation patterns.

3.3 Anaerobic sludge enrichment ferments mixed municipal food waste and glucose to similar end products in a fluidized bed regardless of polarization

In a biorefinery context, it may be desirable to tune food waste as an energy and carbon-rich substrate towards more hydrophobic, longer-chain products to facilitate product extraction. To this end, almost all reports of environmental EF have employed reducing working electrode potentials [13]. However, cathodic EF may be hypothesized to select away from electroactive fermenters in a community if the electron transfer is a coerced process, similar to the phenomenon of “EET parasitism” [15]. Providing selective pressure on electroactive LAB as primary fermenters rather than acetogens and mixed-acid fermenters may be an avenue towards tuning products because lactate can serve as a favorable electron donor to elongate other organic acids. To our best knowledge, no report exists comparing performance and microbiome assembly between anodic/cathodic/OC fermentation of food waste. Therefore, we performed long-term, serial semi-batch fermentations with both digestate from a regional food waste digester and glucose as a model substrate over 24 cycles in three FBRs, one poised anodically (0.0 mV vs. Ag/AgCl), one cathodically (−800 mV vs. Ag/AgCl), and one open-circuit (Fig. 4).Fig. 4 Long-term operation of fluidized bed reactors showed similar performance across anodic (0.0 mV vs. Ag/AgCl), cathodic (−800 mV vs. Ag/AgCl), and open-circuit control across operational regimes separated with dashed lines: continuous digestate addition (1), only glucose PBS media (2), and with AQDS addition (3). Fermentation products (a) and the average carbon length of fermentation products (b) across serial semi-batch operation.

Fig. 4

After three cycles where food waste digestate was added at 10% (v/v) to glucose PBS media, subsequent cycles of glucose fermentation resulted in products dominated by acetate and propionate in all conditions (Fig. 4a), with increasing carbon length of metabolites increasing from close to 2.00 to near 2.75 (Fig. 4b). However, no clear difference between polarization conditions was evident. This lack of tunability continued with adding the model humic substance, AQDS (206 mg L−1). Humic substances are often present in waste streams. AQDS was previously shown as a redox mediator to increase butyrate production from a single batch fermentation of a mixed microbial culture fermenting glucose in the presence of a cathode [6].

The initial inoculation phase of this experiment with 10% digestate (v/v) was repeated twice with 16S rRNA gene sequencing of the planktonic microbiome after each of four semi-batch cycles. In these experiments, microbiomes were observed to diverge under polarization conditions. Pluralibacter spp., Enterobacter spp., and Klebsiella spp. were present in the digester inoculum and enriched in cathodic and open-circuit conditions, but ASVs assigned to Lactococcus and unique to the anodic condition became dominant (>50%) in duplicate anodic reactors by the fourth fill cycle (Supplementary Material Figs. S5a–c). These Lactococcus-dominated microbiomes accumulated the highest butyrate, up to 179 vs. 56 ± 33 mg L−1 from cathodic/open-circuit (Supplementary Material Fig. S5d). These results are consistent with the six trials of single-batch kale juice fermentation (Supplementary Material Fig. S4) in that variation in the form and function of EF microbiomes may occur during the startup of EF, but it may not accurately reflect their long-term performance. This highlights the importance of exercising caution when interpreting results from short-term open-culture EF studies.

3.4 Electrode influence and heme-based preacclimation strategies did not affect domestic wastewater fermentation patterns

The production of organic acids is an attractive alternative to biogas recovery from wastewater, which can decarbonize the sector and increase its return value [39]. Domestic wastewater contains a microbiome reflective of its sewer origin that is diverse temporally and spatially, making process intensification or tunability via EF difficult to engineer due to the preponderance of EET mechanisms that may be present in the wastewater microbiome. However, domestic wastewater commonly contains lactic acid bacteria (Supplementary Material Fig. S6). In addition to FLEET-style energy conservation, LAB can achieve an energetic boost via more classical respiratory pathways enabled by environmental heme uptake [40]. In contrast to quinone, which is leaked by members in crossfeeding microbiomes and produced in situ, heme and humic substances could be sourced directly from urban waste streams. Heme is very bioavailable iron and heme addition to wastewater plus subsequent aeration was proposed to serve as a selective pressure towards LAB as primary fermenters and result in anode-induced tunability towards lactic acid as a product or transient metabolite in wastewater fermentation (Supplementary Material Fig. S6).

Across all heme treatments, VFA production (Fig. 5a) followed similar trends, with products dominated by acetate (Fig. 5a) up to a final titer of 171 ± 46 mg L−1. Lactate was observed at sub-ppm levels in the initial 24 h. Respiratory preacclimation did not impact the chronoamperometric response of heme-amended wastewater poised at +200 mV (Fig. 5c) after the initial 4 h, and the open-circuit evolution of domestic wastewater among all groups followed a similar pattern (Fig. 5d). This suggests that heme addition and respiratory preacclimation did not alter the redox behavior of the wastewater microbiome. Final microbiomes were similar across all treatments with deselection from LAB apparent – Lactococcus spp. present in the initial wastewater were not among the top 20 ASVs in the final community (Fig. S7). These experiments were repeated with the addition of 1 g L−1 glucose, higher heme concentration (30 μg mL−1), and riboflavin/DHNA to provide more labile substrate during the preacclimation period and additional electron mediators. Some lactate (∼25 mg L−1) was produced, but there was again no distinct impact of the electrode on substrate utilization or metabolite distribution.Fig. 5 Impact of a heme (3 μg mL−1) and aeration-based respiratory preacclimation strategy for domestic wastewater fermentation. COD equivalents of analyzed constituents at the end of 140 h of fermentation (a), pH (b), volumetric current density (c), and the evolution of open-circuit potential (d).

Fig. 5

4 Conclusions

While electron transport has been demonstrated to be an important process in some primary fermentative organisms, most studies only reported short-term observations of EF to mixed effect. Here, a long-term comparison between anodic, cathodic, and open-circuit treatments with multiple environmentally relevant substrates and microbial communities suggests that microbiomes associated with leafy greens (kale), food waste digestion, and domestic wastewater are resistant to change in form and function by anodically and cathodically-poised fluidized and static electrodes. Once tunability had been established with static electrodes and the model electroactive fermenter L. plantarum in H-cell reactors, continued operation while introducing LAB native to food waste was demonstrated to interfere with electrode-induced tunability despite high retention (>80%) of the model electroactive fermenter. These results suggest that environmental fermentative microbiomes exhibit robust functionality when subjected to changes in redox potential. This study instead highlights the knowledge gaps in biology that need to be addressed before electrochemical technology can be applied to tune and accelerate mixed culture electrofermentation, including the relevance of FLEET style metabolism in different growth phases and the connection between electroactive primary fermenters and competitors and cross-feeders that secrete quinone. Further work should be directed towards understanding the physiological effects of the electrode on model organisms. For example, defined coculture experiments of naturally co-occurring LAB or LAB and secondary fermenters with/without polarized electrodes and mediators could elucidate best practices for using electricity to intensify non-axenic fermentations within the biorefinery.

CRediT authorship contribution statement

Aaron Leininger: Writing - Original Draft, Methodology, Investigation, Formal Analysis, Data Curation, Conceptualization. Sidan Lu: Methodology, Investigation. Jinyue Jiang: Methodology, Investigation, Data Curation. Yanhong Bian: Methodology, Investigation, Data Curation. Harold D. May: Writing - Review & Editing, Conceptualization. Zhiyong Jason Ren: Writing - Review & Editing, Resources, Project Administration, Investigation, Funding Acquisition, Conceptualization.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix A Supplementary data

The following is the Supplementary data to this article.Multimedia component 1

Multimedia component 1

Acknowledgements

This work is supported by the US Department of Agriculture (INFEWS/T1:AWD1006334 ) and the US Department of Energy (DE-EE0009494 ). We appreciate Dr. Wei Wang at the Princeton Genomics Core Facility and John Schreiber from Princeton Imaging and Analysis Center for their support in library preparation/sequencing and imaging, respectively.

Appendix A Supplementary data to this article can be found online at https://doi.org/10.1016/j.ese.2024.100459.
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