Hydraulic Retention Time and Electric Stimuli as Key Levers for Tailoring Mixotrophic Consortia Toward Enhanced Volatile Fatty Acid Production and Carbon Capture

Hydraulic Retention Time and Electric Stimuli as Key Levers for Tailoring Mixotrophic Consortia Toward Enhanced Volatile Fatty Acid Production and Carbon Capture | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Hydraulic Retention Time and Electric Stimuli as Key Levers for Tailoring Mixotrophic Consortia Toward Enhanced Volatile Fatty Acid Production and Carbon Capture Ana Clara Bonizol Zani, Adalgisa Rodrigues de Andrade, Valeria Reginatto This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9518331/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 4 You are reading this latest preprint version Abstract Background Continuous bioelectrochemical systems represent a promising platform for converting CO 2 into value-added bioproducts, yet operational strategies to enhance carbon capture and product selectivity remain limited. While mixotrophic electrofermentation improves process stability and performance, the role of hydraulic retention time (HRT) in shaping microbial community specialization and inorganic carbon utilization in continuous systems remains poorly understood. Results A continuous electrostimulated bioreactor was operated at a constant potential of 300 mV for approximately 1200 h under three HRTs (33, 19, and 14 h). Decreasing HRT promoted the enrichment of a specialized mixotrophic consortium, with Enterococcus and Clostridium dominating the planktonic phase and Desulfovibrio and Enterococcus enriched in the electrode biofilm. This syntrophic organization enhanced biomass-specific IC removal by 2.6-fold (from 4.92 to 12.88 mg g − 1 h − 1 ) and redirected carbon flux toward acetate, a key product of acetogenic pathways. Acetate reached 1470 mg L − 1 with a volumetric productivity of 107 mg L − 1 h − 1 at HRT = 14 h. Batch assays revealed that ~ 38% of the acetate exceeded heterotrophic stoichiometry, indicating additional CO 2 /HCO 3 − fixation. Functional predictions further revealed the enrichment of carbon fixation pathways, particularly in the electrode-associated biofilm, suggesting that electrode-associated taxa contributed to the generation of reducing equivalents and supported autotrophic metabolism. Conclusion Reducing HRT enhanced inorganic carbon utilization and selectively increased acetate production in a continuous electrostimulated bioreactor. This effect was associated with the enrichment of a functionally specialized microbial consortium and the activation of carbon fixation pathways, particularly in the electrode-associated biofilm, highlighting the role of spatial organization in driving carbon flux. These findings demonstrate that HRT can be used as a practical and energy-efficient engineering strategy to control microbial function and improve CO 2 /HCO 3 − valorization, advancing the development of selective and scalable electrobiotechnological processes. Electrofermentation Microbial electrosynthesis Inorganic carbon fixation Acetogenesis Syntrophic microbial community Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Background Human dependence on fossil fuels has severely disrupted the global carbon cycle, driving atmospheric CO 2 concentrations to unprecedented levels. Annual CO 2 emissions currently exceed 38 billion tons, yet only a negligible fraction, approximately 45 million tons, is converted into value-added products such as acids and biofuels [ 1 ]. In response, 195 nations committed to developing CO 2 mitigation strategies at the 21st United Nations Climate Change Conference in 2015 [ 2 ], spurring interest in technologies that couple carbon capture with the production of useful chemicals. Microbial electrosynthesis (MES) are particularly promising because they exploit microorganisms as biocatalysts to convert CO 2 into value-added organic compounds using electrical energy as the thermodynamic driving force [ 3 ]. A broad range of chemicals has been produced through MES, with volatile fatty acids (VFAs), especially acetate, emerging as the most relevant products [ 4 , 5 ]. Global demand for acetic acid is projected to reach approximately 19.6 million tons per year by 2027 [ 6 ], highlighting the industrial relevance of electrosynthetic acetate production. Acetate can be produced autotrophically by acetogens, which fix CO 2 via the Wood-Ljungdahl pathway (WLP) using H 2 or CO as electron donors [ 7 , 8 ]. The reduction of two CO 2 molecules to acetate via H 2 is exergonic under physiological conditions (approximately − 95 kJ mol − 1 ) [ 9 ]. However, autotrophic growth operates close to thermodynamic limits, making it sensitive to environmental fluctuations and difficult to sustain over long periods [ 10 , 11 ]. A practical strategy to overcome these thermodynamic constraints is to operate under mixotrophic conditions, where microorganisms simultaneously assimilate both inorganic (CO 2 /HCO 3 ⁻) and organic carbon sources. This dual-substrate approach broadens metabolic flexibility, improves thermodynamic favourability, and supports more stable and productive microbial communities [ 12 ]. The co-supply of organic carbon also enables the enrichment of syntrophic consortia in which fermentative organisms provide reducing equivalents — such as H 2 and formate — that acetogens then channel into CO 2 fixation. The application of a low electric potential further enhances this process by promoting extracellular electron transfer, stimulating carbon-fixing pathways, and selectively enriching electroactive genera at electrode surfaces [ 10 , 13 , 14 ]. Under electrofermentation conditions, cathode-associated microorganisms can generate H 2 in situ , supplying a continuous stream of electron donors to planktonic acetogens and thereby amplifying CO 2 conversion rates [ 15 ]. In continuous bioprocesses, hydraulic retention time (HRT) is a key operational parameter that governs dilution rate, imposes selective pressure on microbial growth, and ultimately shapes community structure and metabolic output. Although both electrical stimulation and HRT are recognized as important process levers, their combined impact on microbial specialization and inorganic carbon (IC) utilization in continuous electrobioreactors remains poorly understood. This work demonstrates that HRT can be used as a practical engineering tool to control carbon flux, enhance CO 2 utilization, and selectively drive acetate production in continuous electrobioreactors. Here, we address this gap by operating a continuous electrostimulated bioreactor (E_BR) at a constant applied potential of 300 mV for approximately 1200 h under three successive HRT conditions (33, 19, and 14 h). We monitored biomass concentration, pH, product distribution, and inorganic carbon removal rates across all conditions, and characterized the suspended and electrode-associated microbial communities by 16S rRNA sequencing and metagenomic functional prediction. We demonstrate that decreasing HRT progressively enriches a specialized mixotrophic consortium, enhances IC uptake, and redirects carbon flux toward acetate production, revealing HRT as an effective and simple lever for steering electrofermentation toward enhanced carbon capture. 2. Materials and Methods 2.1 Inoculum Sludge obtained from an anaerobic reactor employed to treat vinasse at a sugar and ethanol plant located in Pradópolis, state of São Paulo, Brazil (Latitude: -21.3189° S; Longitude: -48.1170° W), was applied to inoculate E_BR. The initial volatile suspended solids (VSS) concentration was 2650 mg L − 1 . 2.2 Electrobioreactor (E_BR) operation E_BR with a working volume of 1100 mL was operated in the continuous mode for approximately 1200 h at HRT of 33, 19, and 14 h over time. Two graphite plate electrodes (1.5 × 13.0 × 0.5 cm), which acted as the anode and cathode, were introduced inside E_BR, while a constant potential difference of 300 mV was applied (power supply, Hikari HF-3203S). E_BR was fed with synthetic medium and stirred at 150 rpm with a magnetic stirrer. Anaerobic operation conditions in the reactor were guaranteed by daily flushing the feeding medium and the reactor with CO 2 for 6 min. The temperature was maintained at 30°C by using a heat jacket, and the pH of the feeding medium was adjusted to 6.3. The system was fed with M9 medium supplemented with 3.6 g L − 1 fructose, as the organic carbon source, and 4.0 g L − 1 NaHCO 3 , as the IC source. In addition, the medium contained (in g L − 1 ) 6.78 Na 2 HPO 4 , 3.0 KH 2 PO 4 , 5.0 NaCl, 1.0 NH 4 Cl, 0.49 MgSO 4 .7H 2 O, 0.01 CaCl 2 , and.1.0 yeast extract. Fructose addition helps develop more stable mixotrophic microbial communities. The VSS concentration, which corresponds to the biomass, was measured daily according to APHA (2017) [ 16 ]. During the biomass washout periods, the maximum specific growth rate (µ max ) was determined by using Eq. ( 1 ) [ 17 ]. $$\:\frac{dx}{dt}={{X}_{i}\:e}^{(\mu\:max-D)t}$$ 1 where \(\:{X}_{i}\) is the initial microorganism cell concentration (mg L − 1 ), \(\:x\) is the microorganism cell concentration at time t (mg L − 1 ), D is the dilution rate (h − 1 ) \(\:\left(D=\:\frac{1}{HRT}\right)\) , and µ max is the maximum specific growth rate (h − 1 ). 2.3 Batch assays: fructose consumption and product kinetics During operations at HRTs of 19 h and 14a h, continuous feeding of E_BR was temporarily switched to batch mode to assess fructose consumption and to determine different product formation rates. Kinetic tests were conducted at 456 and 744 h and were labeled batch I (BI) and batch II (BII), respectively. For the batch kinetic tests, 650 mL of the supernatant was replaced with fresh M9 medium in one single step, and CO 2 was purged for 6 min. The fructose consumption and product formation rates (in mg L-1 h-1) were estimated from the slope of a linear regression fitted to the concentration-time profiles. 2.4 Analytical determinations and calculations VSS concentrations during E_BR operation were determined according to Standard Methods (APHA, 2017). Fructose and the products, including acetic (HAc), lactic (HLac), propionic (HPr), butyric (HBut), and formic (HFor) acids, were analyzed on a high-performance liquid chromatograph (Shimadzu LC-20 AT, Japan) equipped with an Aminex HPX-87H column. The column temperature was maintained at 60°C, and the mobile phase consisted of 5 mmol L − 1 H 2 SO 4 at 0.6 mL min − 1 (60 kgf cm − 2 ). A photodiode array detector (PDA) and refractive index detectors (RID) were used to quantify the carboxylic acids and fructose, respectively. The LabSolutions software was employed. IC was quantified from the three final sampling points for each HRT applied. Analyses were carried out on a Shimadzu TOC analyzer (model TOC-VCPN). Samples were pretreated by automatically adding acid, which was followed by gas (oxygen carrier) injection at 230 mL min-1. The IC removal rate and IC specific removal rate were calculated by using Eqs. ( 2 ) and ( 3 ), respectively [ 18 ]. $$\:IC\:removal\:rate\:\left(mg\:{L}^{-1}{h}^{-1}\right)={(IC}_{i}-{IC}_{f})\:x\:D$$ 2 $$\:IC\:Specific\:removal\:\:rate\:\left(mg\:{g}^{-1}{h}^{-1}\right)=\frac{IC\:removal\:rate}{X}$$ 3 where \(\:{IC}_{i}\) and \(\:{IC}_{f\:}\) are the initial and final \(\:IC\) concentrations (mg L − 1 ), respectively, \(\:X\) is the biomass concentration average of three last points (g L − 1 ) at the end of each HRT, and D is dilution rate (h − 1 ) \(\:\left(D=\:\frac{1}{HRT}\right)\) . To evaluate biomass specialization for carboxylic acid production, specific productivity was calculated from the three final sampling points for each applied HRT, using Eq. 4 . $$\:P\:\left(mg\:{g}^{-1}{h}^{-1}\right)=\frac{D\:Cp}{X}$$ 4 where \(\:P\) is the specific productivity (mg g − 1 h − 1 ), D is the dilution rate (h − 1 ), \(\:{C}_{p}\) is the concentration of the respective carboxylic acid (mg L − 1 ), and \(\:X\) is the biomass concentration (g L − 1 ). 2.5 16S rRNA sequencing and metagenomic functional predictions Inoculum and biomass samples were collected from E_BR at the end of each HRT, along with biofilm cells harvested from the graphite electrode, to evaluate how the microbial community adapted to the different HRT conditions. For this purpose, DNA was extracted from the samples by using the ZymoBIOMICS™ DNA Miniprep kit (Zymo Research) and quantified to measure both quality and quantity. The DNA samples were submitted to complete amplification of the 16S rRNA gene by using primers 27F and 1492R (~ 1.6-kb fragment). The amplicons were analyzed on agarose gel; quantified by using the SQK-LSK114 kit (Oxford Nanopore Technologies), to construct a library; and sequenced on a Flongles flow cell (FLO-FLG114) on the MinION platform (Oxford Nanopore Technologies). After the amplicons were sequenced, the resulting reads were base-called by using Guppy Basecaller (v 6.0.7), GPU version. The reads were filtered for quality control in Q10 by employing NanoFilt (v2.3.0) and demultiplexed with Porechop (v0.2.4). The reads were mapped to a 16S reference database by using the KMA tool (v1.4.3). Data were analyzed by applying Python 3.7. The metabolic pathway was predicted with PICRUSt2 v2.3.0. The cluster map displays the 30 most abundant pathways, while percentile filtering (60–90%) generated a refined set of the top 30 pathways [ 19 ]. Sequencing reads were base called by using Guppy v6.4.8 (high-accuracy, GPU) and aligned to the RefSeq 16S database with KMA v1.3.23. 3. Results and Discussion 3.1 Monitoring the electrobioreactor at different HRT: cell biomass and pH We operated the electrostimulated bioreactor (E_BR) continuously for approximately 1200 h under three successive hydraulic retention times (33, 19, and 14 h), monitoring volatile suspended solids (VSS) as a proxy for biomass concentration and pH throughout the entire operation (Fig. 1 ). At the initial HRT of 33 h, VSS decreased from 2650 to 1993 mg L − 1 within the first 144 h, indicating partial biomass washout under the imposed dilution rate. However, continued operation at this HRT allowed the surviving community to recover, and VSS subsequently rose to 2666 mg L − 1 , suggesting that microbial growth rates surpassed the washout threshold. To impose stronger selective pressure and drive community specialization, HRT was reduced to 19 h, which caused a 14.5% decrease in VSS — from 2910 to 2490 mg L − 1 — at 456 h (Fig. 1 ). Immediately, following this transition, continuous feeding was momentarily switched to batch mode (Batch I, BI) to characterize substrate consumption and product formation kinetics under the microbial community established at HRT = 19 h (Section 3.3 ). After Bath I (BI), continuous operation was resumed, marking the beginning of the 14 a h phase. At this stage, the HRT was further reduced to 14 h, resulting in a 26.7% decrease in VSS, from 3039 to 2396 mg L − 1 (Fig. 1 ). The E_BR was maintained at this HRT until 744 h. At this point, a second batch assay (Batch II, BII) was initiated to probe substrate and product dynamics under the more stringently selected community (Section 3.3 ). Following BII, continuous operation resumed — beginning the 14 b h phase — at HRT = 14 h, and was sustained until the experiment’s end at 1156 h. The 14 b h phase began with a VSS of 4193 mg L − 1 , reflecting biomass accumulation during BII, and VSS declined to 2360 mg L − 1 , a 43.7% reduction, underscoring the strong washout pressure imposed at the shortest HRT. Each HRT transition was accompanied by a biomass washout period, which enabled estimation of the maximum specific growth rate (µ max ) of the consortium using Eq. 1 and the corresponding washout profiles (Fig. S1 ). The estimated µ max values were 0.025, 0.050, 0.068, and 0.069 h − 1 for HRT = 33, 19, 14 a , and 14 b h, respectively. These values fall within the range reported for mesophilic acetogens (0.03–0.12 h − 1 ) [ 20 ]. For reference, µ max values of 0.069, 0.078, and 0.018 h − 1 have been reported for Acetobacterium wieringae (DSM 1911), Blautia hydrogenotrophica (DSM 10507), and Sporomusa acidovorans (DSM 3132), respectively [ 21 ]. Clostridium aceticum , one of the genera that became dominant in this study (Section 3.2 ), achieves µ max = 0.052 h − 1 under pH-controlled conditions (pH 8.0), and only 0.006 h − 1 without pH control [ 22 ]. The near-identical µ max values at HRT = 14 a and 14 b h (0.068 vs. 0.069 h − 1 ) suggest that the community had reached a stable growth phenotype by the lowest HRT, with BII causing only a transient perturbation in biomass concentration rather than a shift in intrinsic growth capacity. Throughout the entire operation, pH fluctuated within a narrow range of 6.3 to 7.0 (Fig. 1 ). This range is mechanistically significant for two reasons. First, at pH ≈ 6.5, the dissolved inorganic carbon system approaches the first acidity constant of the carbonate equilibrium (pKa1 = 6.35), at which point aqueous CO 2 and bicarbonate (HCO 3 − ) coexist at comparable concentrations. This coexistence makes inorganic carbon bioavailable via two complementary routes: gaseous CO 2 is directly accessible to microorganisms that employ the WLP, while HCO 3 − serves as an ionic reservoir accessible via carbonic anhydrase activity [ 23 , 24 ]. Second, the slightly acidic to near-neutral pH maintained throughout operation (6.3–7.0) thermodynamically favors acidogenesis over solventogenesis in Clostridium -containing communities, as discussed in Section 3.2 . Consistent with this, no solvents were detected at any point during operation — only carboxylic acids accumulated as metabolic products. 3.2 Product distribution and microbial community dynamics Changes in HRT produced marked shifts in both product distribution and microbial community composition (Fig. 2 A and 2 B). The soluble products detected throughout E_BR operation were exclusively carboxylic acids; no corresponding alcohols were observed under any condition. As discussed in Section 3.1 , the pH range of 6.3–7.0 thermodynamically favors acidogenesis over solventogenesis in Clostridium -containing communities [ 25 ]. Moreover, with the main VFAs (pKa ≈ 4.7–4.8) persisting in their dissociated anionic forms at the operating pH, undissociated acid concentrations remained low, preventing intracellular acidification and eliminating the redox imbalance that would otherwise trigger solvent formation as a reductant-relief mechanism [ 26 ]. 3.2.1 Suspended Microbial Community and Product Profile At HRT = 33 h, acetate (HAc) and propionate (HPr) co-dominated the product profile, with HAc and HPr reaching 846.8 and 782.0 mg L − 1 , respectively, and butyrate (HBut) present at low concentrations (100.3 mg L − 1 ). The microbial community at this stage was dominated by Trichococcus (44.8%), a well-characterized acidogenic lactic acid bacterium that ferments sugars to lactate, formate, and acetate [ 27 , 28 ], and Klebsiella (27.5%), which increased from 1.3% in the inoculum. Neither genus is associated with propionate production, indicating that HPr likely originated from less abundant or taxonomically unassigned community members (∼9% of sequences), possibly through the acrylate or succinate-propionate pathways. At HRT of 19 h, the community underwent a substantial restructuring: Trichococcus declined sharply from 44.8% to 1.8%, while Enterococcus (25.2%), Arcobacter (22.3%), and Robinsoniella (18.9%) emerged as the dominant genera, together accounting for 66.4% of the total relative abundance (Fig. 2 B). Concomitantly, low concentrations of formate (HFor; 50–70 mg L − 1 ) appeared for the first time, pointing to activation of the methyl branch of the WLP, where formate serves as a key intermediate [ 29 ]. HAc, HPr, and HBut concentrations increased modestly to 1171.3, 790.6, and 114.6 mg L − 1 , respectively. Arcobacter , known for its fermentative and acidogenic activity and exoelectrogenic behavior in anodic microbial fuel cell (MFC) biofilms [ 30 , 31 ], likely benefited from the applied potential difference of 300 mV. Enterococcus , a lactic acid bacterium capable of H 2 and VFA production, and with demonstrated capacity for extracellular electron transfer (EET) both via direct contact and soluble mediators [ 32 ], was consistent with the stable VFA formation observed at this stage. At HRT of 14 a h, the community retained Enterococcus (31.7%) and Arcobacter (13.3%), while Clostridium increased 3.5-fold relative to HRT = 19 h, reaching 9.5% of the total abundance. Ilyobacter (16.1%) and Robinsoniella (8.5%) were also present; however, both Arcobacter and Robinsoniella showed relative declines of 9.0% and 10.4%, respectively. The enrichment of Clostridium at the lowest HRT coincided with a significant increase in HAc concentration, consistent with the genus's known capacity to fix CO 2 into acetate via the WLP in the presence of H 2 [ 33 ]. The enrichment of Clostridia under H 2 /CO 2 -fed conditions has been consistently associated with acetate-dominated VFA production in homoacetogenic systems [ 34 ]. At the final continuous stage (HRT = 14 b h), HAc reached its maximum concentration of 1474.6 mg L − 1 and remained stable, while HPr decreased to 450.7 mg L − 1 — a 54.3% reduction relative to HRT = 14 a h — confirming that the carbon flux was progressively redirected toward acetate as the community became more specialized. HBut increased by 149.6% relative to HRT = 33 h, reaching 250.3 mg L − 1 , while HFor concentrations remained low across all HRTs. Enterococcus (26.1%) and Clostridium (15.8%) persisted as the dominant genera, accounting for 41.8% of the community, while Robinsoniella and Arcobacter declined to 3.5% and 3.2%, respectively. The persistence of Enterococcus and Clostridium , which have been reported to interact synergistically in continuous H 2 -producing reactors [ 35 ], likely underpinned the stable and elevated acetate accumulation observed at the lowest HRT. 3.2.2 Electrode Biofilm Community At the end of the experiment (1156 h), cells harvested from the graphite electrode surface revealed a community composition distinctly different from the planktonic phase. Enterococcus was the most abundant genus in electrode, representing 39.2% of the biofilm community — a higher proportion than in the suspended phase (26.1%). Desulfovibrio (9.1%) and Geobacte r (3.0%) were substantially enriched in the biofilm relative to the bulk liquid, while Clostridium , which dominated the planktonic community (15.8%), accounted for only 1.4% of the biofilm. Additionally, 18.5% of biofilm sequences remained taxonomically unassigned, suggesting the presence of poorly characterized electroactive taxa. The prominence of Enterococcus in the electrode biofilm is noteworthy. Beyond its fermentative role, Enterococcus faecalis has been shown to transfer electrons generated during fermentative metabolism to electrode surfaces both directly and via soluble redox mediators, establishing it as a functionally electroactive genus in bioelectrochemical systems [ 32 ]. Its H 2 -producing capability also positions it as an indirect electron donor for acetogens in the planktonic phase. Desulfovibrio is a key electrotrophic genus in bioelectrochemical systems. It accepts electrons from the cathode and produces H 2 or formate through periplasmic hydrogenases and outer-membrane cytochromes, generating a continuous supply of reducing equivalents [ 36 ]. This function is particularly critical for syntrophic interactions with planktonic Clostridium , which utilizes H 2 to reduce CO 2 via the WLP [ 15 ]. Recent studies have highlighted that the coupling of H 2 -producing electrotrophs such as Desulfovibrio with acetogens such as Clostridium synergistically drives CO 2 reduction through WLP [ 37 ], which is entirely consistent with the community architecture observed here. Geobacter , while well-established as the dominant genus in anodic MFC biofilms [ 38 ], appeared at low abundance (3.0%) in the E_BR electrode biofilm. Its role in electrofermentation systems is less well characterized. Overall, the electrode biofilm and planktonic community exhibited a clear functional division of labor: the biofilm was enriched in electrotrophic and H 2 -producing genera ( Desulfovibrio , Enterococcus ), which supplied reducing equivalents to the planktonic acetogens ( Clostridium ), enabling CO 2 fixation and acetate accumulation in the bulk liquid. This syntrophic architecture, shaped and sustained by the combined effects of HRT and electrical stimulation, is discussed further in Section 3.4 in terms of metabolic pathway enrichment. 3.3 Batch Assays (BI and BII): Insights into Substrate and Product Dynamics To gain mechanistic insight into substrate consumption and product formation kinetics, detail that is difficult to resolve under continuous flow, continuous feeding of E_BR was temporarily suspended and the system was operated in batch mode twice: at 456 h (Batch I, BI) and at 744 h (Batch II, BII), corresponding to community states established at HRT of 19 h and HRT of 14 a h, respectively (Fig. 3 A and 3 B). In both assays, fructose was consumed rapidly, and a characteristic transient intermediate profile emerged (Fig. 3 A and 3 B). Lactate (HLac), which was not detected during continuous operation, appeared within the first 4 h of each batch assay, concomitant with fructose consumption, and was completely depleted by the end of the batch period. The transient accumulation and subsequent disappearance of HLac under batch conditions, but not under continuous flow, reflects the kinetics of the underlying microbial interactions: In continuous mode, lactate was probably produced by Enterococcus and other lactic acid bacteria and is consumed by lactate-fermenting bacteria (e.g., Clostridium ) as fast as it is generated, precluding its accumulation to detectable levels. In batch mode, the transiently higher substrate concentration and the absence of dilution allow lactate to accumulate before being metabolized downstream. HAc, HPr, and HBut gradually accumulated throughout both batch assays, BI and BII (Fig. 3 A and 3 B), respectively, with concentrations increasing sharply after lactate began to be depleted, consistent with their formation through lactate conversion rather than directly from fructose. By 24 h, HAc, HPr, and HBut concentrations in BII exceeded those in BI by 46.4%, 18.4%, and 24.2%, respectively, demonstrating that the microbial community selected under the lower HRT (14 a h) had developed a greater capacity for carboxylic acid production — particularly acetate — compared with the community present at HRT = 19 h. The dominant lactate-converting metabolism in this system is consistent with the acrylate/succinate-propionate pathway, by which Clostridium spp. convert lactate into propionate and acetate at an expected stoichiometric ratio of 3 lactate : 2 propionate : 1 acetate [ 39 ]. However, the observed final acetate concentration in BII (6.61 mmol L − 1 ) substantially exceeded the stoichiometric maximum predicted by this pathway alone (4.80 mmol L − 1 ), a discrepancy that cannot be attributed to analytical error or differences in initial fructose concentration. This excess acetate — accounting for approximately 38% of the total HAc produced — points to the involvement of additional autotrophic carbon-fixing routes. Specifically, it implicates CO 2 /HCO 3 − assimilation via the WLP, in which H 2 generated by Clostridium fermentation and by Desulfovibrio electrotrophic activity at the electrode surface (Section 3.2.2 ) is used to reduce inorganic carbon to acetyl-CoA and ultimately acetate [ 40 , 41 ]. This interpretation is further supported by the metabolic pathway enrichment data presented in Section 3.4 , which shows a progressive increase in the abundance of prokaryotic carbon fixation pathways across decreasing HRTs, as well as their pronounced enrichment in the electrode biofilm. Taken together, the batch assays reveal that the E_BR community is not purely heterotrophic: fructose drives lactic acid fermentation, lactate is subsequently channelled into propionate and acetate, and the reducing equivalents liberated by these fermentative reactions — supplemented by bioelectrochemical H 2 production — feed the WLP, generating additional acetate from inorganic carbon. 3.4 Inorganic Carbon Capture and Metabolic Pathway Enrichment To quantify how HRT modulated the capacity of the microbial community to assimilate inorganic carbon, we measured IC concentrations at the inlet and outlet of E_BR at the end of each HRT phase and calculated IC removal rates and biomass-specific IC removal rates (Eqs. 2 and 3 ; Fig. 4 ). IC removal increased progressively as HRT decreased. At HRT = 33 h, the IC removal rate was 13.1 mg L − 1 h − 1 , corresponding to a biomass-specific rate of 4.92 mg g − 1 h − 1 . Reducing HRT to 19 h nearly doubled the specific IC removal rate (9.35 mg g − 1 h − 1 ; 1.9-fold increase), and at HRT = 14 h, the specific rate reached 12.88 mg g − 1 h − 1 — a 2.6-fold increase relative to the highest HRT — with a volumetric removal rate of 30.5 mg L − 1 h − 1 . These results demonstrate that shorter residence times did not merely wash out biomass; they selectively enriched a community that was intrinsically more efficient at capturing and assimilating inorganic carbon per unit of cell mass. The progressive enrichment of IC capture capacity across decreasing HRTs is mechanistically consistent with the community shifts described in Section 3.2 . At the lowest HRT, the dominance of Clostridium in the planktonic phase and Desulfovibrio in the electrode biofilm established a syntrophic architecture well-suited for CO 2 fixation: Desulfovibrio electrotroph activity supplied a continuous H 2 flux at the cathode, which Clostridium channelled through the WLP to reduce CO 2 to acetyl-CoA and, ultimately, acetate. This interpretation is corroborated by the stoichiometric excess of acetate observed in the batch assays (Section 3.3 ), which could not be accounted for by heterotrophic lactate conversion alone. To evaluate whether these metabolic shifts were reflected in the functional genomic composition of the community, we predicted metabolic pathway abundances using PICRUSt2 and compared the planktonic and electrode-associated communities across HRT conditions (Fig. 5 ). The heatmap revealed that HRT strongly modulated the enrichment of metabolic pathways and that the electrode biofilm was functionally distinct from the planktonic community. At HRT = 33 h, pathways associated with carbon fixation and energy metabolism showed relatively low abundance, suggesting limited metabolic turnover and reduced carbon flux through autotrophic routes. As HRT decreased to 19 and 14 h, these pathways became progressively more abundant, indicating functional enrichment toward carbon fixation and energy-conserving processes. Notably, prokaryotic carbon fixation pathways were consistently most abundant in the electrode-associated biofilm across all conditions, confirming that the electrode community — dominated by Desulfovibrio and Enterococcus — played a disproportionate role in driving inorganic carbon assimilation relative to its biomass contribution. Amino acid biosynthesis pathways were also prominent in the heatmap, reflecting active biosynthetic metabolism in a community under sustained growth and selective pressure. These findings provide functional genomic support for the syntrophic model proposed above and demonstrate that the combination of electrical stimulation and progressively shorter HRT shaped not only the taxonomic composition but also the metabolic specialization of both the planktonic and biofilm communities toward enhanced carbon capture. 3.5 Specific Productivities and Biomass Specialization To assess how HRT-driven community specialization translated into productive efficiency, we calculated the specific productivities of individual carboxylic acids and the total VFA-specific productivity at each HRT, using the last three steady-state measurements of biomass and product concentrations at each condition (Eq. 4 ; Fig. 6 ). Overall, decreasing HRT was accompanied by a consistent increase in both individual and total VFA-specific productivity, reflecting progressive biomass specialization toward carboxylic acid production. Acetate and propionate were the dominant metabolites throughout all operating conditions. At HRT = 33 h, the total VFA-specific productivity was 24.4 mg g − 1 h − 1 , with acetate and propionate as co-dominant products. Whereas decreasing the HRT to 19 h and 14 a h increased the productivity to 47.76 and 74.68 mg g − 1 h − 1 , respectively. When HRT was reduced to 19 h, acetate-specific productivity increased by 25.5%, while propionate productivity declined modestly. The most pronounced shift occurred between HRT = 14 a and 14 b h. At HRT = 14 b h, acetate-specific productivity reached 42.3 mg g − 1 h − 1 , a 65.9% increase over HRT = 19 h. In contrast, propionate-specific productivity fell by 54.3% between the two 14 h phases. This confirms a progressive and selective redirection of carbon flux toward acetate. The volumetric acetate productivity at the end of HRT = 14 b reached 107 mg L − 1 h − 1 . This value compares favourably with several previous electrofermentation and MES studies. Acetate production rates reported for bioelectrochemical systems remain relatively low, typically ranging from 4.88 to 32.2 mg L − 1 h − 1 depending on operational conditions [ 15 , 42 , 43 ]. The acetate productivity reported here therefore, represents a substantial improvement over these benchmarks. Two aspects of the experimental design are particularly noteworthy. First, the high acetate productivity achieved under mild cathodic polarization suggests that the syntrophic community architecture — rather than purely electrochemical reduction —was the primary driver of acetate accumulation, indicating that energy-efficient electrofermentation can be sustained under near-thermodynamic conditions. Second, the supplementation of the medium with fructose as an organic co-substrate supported metabolic maintenance and community stability, enabling sustained operation over 1200 h without the biomass loss and productivity collapse that have been reported in autotrophic-only MES systems. For instance, acetate production was not restored after repeated batch cycles due to substantial biomass loss during catholyte exchange in mixed-culture systems [ 10 ]. Taken together, these results demonstrate that operating an electrostimulated bioreactor under progressively shorter HRTs is an effective strategy to enhance biomass-specific productivity, redirect carbon flux toward acetate, and maintain stable, long-term performance — outcomes that are difficult to achieve in purely autotrophic or non-electrostimulated systems. 4. Conclusion This study demonstrates that hydraulic retention time (HRT) is a key operational parameter to control carbon flux distribution and enhance CO 2 conversion in continuous electrostimulated bioreactors. Reducing HRT increased specific productivity from 24.4 to 74.68 mg g − 1 h − 1 and promoted the selection of a functionally specialized mixotrophic consortium. This shift enabled efficient coupling between electrochemical processes and microbial metabolism, resulting in acetate production beyond heterotrophic stoichiometric limits and confirming additional CO 2 fixation. The spatial organization of the community, with distinct roles between planktonic and electrode-associated populations, supported this enhanced carbon conversion. Overall, HRT reduction emerges as a simple, energy-efficient, and scalable strategy to intensify CO 2 valorization and steer continuous electrofermentation toward selective acetate production as a platform chemical, reinforcing its potential for sustainable bioprocesses. Abbreviations HRT Hydraulic retention time IC Inorganic carbon VFA Volatile fatty acid WLP Wood-Ljungdahl pathway MES Microbial electrosynthesis E_BR Electrostimulated bioreactor VSS Volatile suspended solids μ max Maximum specific growth rate HAc Acetic acid HPr Propionic acid HBut Butyric acid HFor Formic acid Declarations Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Availability of data and materials The datasets generated and/or analyzed during the current study are available from the corresponding author on reasonable request. Competing interests The authors declare no competing interests. Funding The authors acknowledge financial support from the São Paulo Research Foundation (FAPESP; processes 2024/00725-0 and 2023/07992-0), the Coordination for the Improvement of Higher Education Personnel (CAPES), and Petróleo Brasileiro S.A. (process 2023/00640-1). Authors’ contributions A.C.B.Z.: Conceptualization, Methodology, Investigation, Writing – original draft, Data curation. A.R.A.: Validation, Resources, Writing – review and editing, Supervision, Project administration. V.R.: Conceptualization, Validation, Resources, Writing – review and editing, Supervision, Project administration. Acknowledgements The authors thank Cynthia Maria de Campos Prado Manso for English language revision and the members of LABIORE and LEEA for their support. Ana Clara Bonizol Zani acknowledges her family (Tatiana, Renato, Carol, and Helena) for their continuous support, João Carlos de Souza for valuable academic discussions, and her advisors, Valeria Reginatto and Adalgisa R. de Andrade, for their guidance and encouragement throughout this work. References Chung TH, Dhillon SK, Shin C, Pant D, Dhar BR. Microbial electrosynthesis technology for CO 2 mitigation, biomethane production, and ex-situ biogas upgrading. Biotechnol Adv . 2024; doi: 10.1016/j.biotechadv.2024.108474 . Anderson TR, Hawkins E, Jones PD. CO₂, the greenhouse effect and global warming: from the pioneering work of Arrhenius and Callendar to today’s Earth system models. Endeavour . 2016; doi: 10.1016/j.endeavour.2016.07.002 . Luan L, Ji X, Guo B, Cai J, Dong W, Huang Y, Zhang S. Bioelectrocatalysis for CO 2 reduction: Recent advances and challenges to develop a sustainable system for CO 2 utilization. Biotechnol. Adv . 2023; doi: 10.1016/j.biotechadv.2023.108098 . 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Mater . 2018; doi: 10.1016/j.jhazmat.2018.06.005 . Wang R, Lv N, Li C, Cai G, Pan X, Li Y, Zhu G. Novel strategy for enhancing acetic and formic acids generation in acidogenesis of anaerobic digestion via targeted adjusting environmental niches. Water Res . 2021; doi: 10.1016/j.watres.2021.116896 . Schuchmann K, Müller V. Energetics and application of heterotrophy in acetogenic bacteria. Appl. Environ. Microbiol . 2016; doi: 10.1128/AEM.00882-16 de Souza JC, Zani ACB, Silva JP, dos Santos A, Umbuzeiro GA, Bueno AV, Lobo FL, Reginatto V, de Andrade AR. High-performance microbial fuel cell for aromatic hydrocarbon bioremediation: Leveraging a unique mangrove-derived electrogenic consortium. ACS Omega 2025; doi: 10.1021/acsomega.5c05703 Ruiz V, Ilhan ZE, Kang D.-W, Krajmalnik-Brown R, Buitrón G. The source of inoculum plays a defining role in the development of MEC microbial consortia fed with acetic and propionic acid mixtures. J. Biotechnol . 2014; doi: 10.1016/j.jbiotec.2014.04.016 Pankratova G, Leech D, Gorton L, Hederstedt L. Extracellular electron transfer by the Gram-positive bacterium Enterococcus faecalis . Biochemistry . 2018; doi: 10.1021/acs.biochem.8b00600 . Zhang L, Zhao R, Jia D, Jiang W, Gu Y. Engineering Clostridium ljungdahlii as the gas-fermenting cell factory for the production of biofuels and biochemicals. Curr Opin Chem Biol. 2020; doi: 10.1016/j.cbpa.2020.04.010 . He Y, Cassarini C, Lens PNL. Enrichment of homoacetogens converting H 2 /CO 2 into acids and ethanol and simultaneous methane production. Eng. Life Sci . 2022; doi: 10.1002/elsc.202200027 . Morra S, Arizzi M, Allegra P, La Licata B, Sagnelli F, Zitella P, Gilardi G, Valetti F. Expression of different types of [FeFe]-hydrogenase genes in bacteria isolated from a population of a bio-hydrogen pilot-scale plant. Int J Hydrogen Energy . 2014; doi: 10.1016/j.ijhydene.2014.04.009 . Mills S, Dessì P, Pant D, Farràs P, Sloan WT, Collins G, Ijaz UZ. A meta-analysis of acetogenic and methanogenic microbiomes in microbial electrosynthesis. npj Biofilms Microbiomes . 2022; doi: 10.1038/s41522-022-00337-5 . Dessì P, Buenaño-Vargas C, Martínez-Sosa S, Mills S, Trego A, Ijaz UZ, Pant D, Puig S, O'Flaherty V, Farràs P. Microbial electrosynthesis of acetate from CO 2 in three-chamber cells with gas diffusion biocathode under moderate saline conditions. Environ. Sci. Ecotechnol . 2023; doi: 10.1016/j.ese.2023.100261 . Xu F, Cao F-Q, Kong Q, Zhou L-L, Yuan Q, Zhu Y-J, Wang Q, Du Y-D, Wang Z-D. Electricity production and evolution of microbial community in the constructed wetland–microbial fuel cell. Chem. Eng. J. 2018; doi: 10.1016/j.cej.2018.02.003 . Seeliger S, Janssen PH, Schink B. Energetics and kinetics of lactate fermentation to acetate and propionate via methylmalonyl-CoA or acrylyl-CoA. FEMS Microbiol. Lett. 2002; doi: 10.1016/S0378-1097(02)00651-1 . Cardeña R, Valencia-Ojeda C, Chazaro-Ruiz LF, Razo-Flores E. Regulation of the dark fermentation products by electro-fermentation in reactors without membrane. Int. J. Hydrogen Energy 2024; doi: 10.1016/j.ijhydene.2023.06.253 . Isipato M, Dessì P, Sánchez C, Mills S, Ijaz UZ, Asunis F, Spiga D, De Gioannis G, Mascia M, Collins G, Muntoni A, Lens PNL. Propionate production by bioelectrochemically-assisted lactate fermentation and simultaneous CO 2 recycling. Front. Microbiol . 2020; doi: 10.3389/fmicb.2020.599438 . Song YE, Mohamed A, Kim C, Kim M, Li S, Sundstrom E, Beyenal H,. Kim JR, Biofilm matrix and artificial mediator for efficient electron transport in CO 2 microbial electrosynthesis. Chem. Eng. J. 2022; doi: 10.1016/j.cej.2021.131885 . Tharak A, Katakojwala R, Kajla S, Mohan SV, Chemolithoautotrophic reduction of CO 2 to acetic acid in gas and gas-electro fermentation systems: Enrichment, microbial dynamics, and sustainability assessment. Chem. Eng. J . 2023; doi: 10.1016/j.cej.2022.140200 . Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Reviewers invited by journal 05 May, 2026 Editor assigned by journal 04 May, 2026 Submission checks completed at journal 04 May, 2026 First submitted to journal 24 Apr, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9518331","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":635107546,"identity":"c8a92e82-31c9-492a-8762-c9aad5740b29","order_by":0,"name":"Ana Clara Bonizol Zani","email":"","orcid":"","institution":"Universidade de São Paulo","correspondingAuthor":false,"prefix":"","firstName":"Ana","middleName":"Clara Bonizol","lastName":"Zani","suffix":""},{"id":635107547,"identity":"9fcc00b1-b619-4369-81ba-0e59b60d063e","order_by":1,"name":"Adalgisa Rodrigues de Andrade","email":"","orcid":"","institution":"Universidade de São Paulo","correspondingAuthor":false,"prefix":"","firstName":"Adalgisa","middleName":"Rodrigues","lastName":"de Andrade","suffix":""},{"id":635107549,"identity":"345648f8-ff3b-4d01-b996-e582e1a7f03a","order_by":2,"name":"Valeria Reginatto","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA+UlEQVRIiWNgGAWjYFAC5gYGBgMgzd744ANDAVSQsQGfFkaoFp7DhjPADOK0gIBEMpFaDI4fbHxcUWCXLz/zMWPDD4N7eQwS6c8eMO64h1vLmcRmwzMGyZYbbiczNvYYFBczSOSYGzCeKcapRbIhsU2ywYDZwEA6//hjBoOExAaJHDYJxrYE3Fr6H7b/bDCoN5CfeZixGaIl/RleLfwSiW2MDQaHDRhuMMO0JJgR0PKwGeiw4wYGZ8B+SShm43ljbpB4BrcWNv7kgx8b/lQbyLcfBoZYRUIePzswxD7uwK0FAySwAc1hIEEDA1gxGykaRsEoGAWjYPgDAP2+UVm/H6lrAAAAAElFTkSuQmCC","orcid":"","institution":"Universidade de São Paulo","correspondingAuthor":true,"prefix":"","firstName":"Valeria","middleName":"","lastName":"Reginatto","suffix":""}],"badges":[],"createdAt":"2026-04-24 14:24:48","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9518331/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9518331/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":109296189,"identity":"a41d7730-d913-42a3-97ac-8d0a24fa38bb","added_by":"auto","created_at":"2026-05-15 08:46:00","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":160511,"visible":true,"origin":"","legend":"\u003cp\u003eVolatile suspended solids concentration (VSS) and pH during electrobioreactor operation at different hydraulic retention time (HRT).\u003c/p\u003e","description":"","filename":"floatimage1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9518331/v1/d55190c7bf3791e1b226fd1e.jpg"},{"id":109255938,"identity":"87cf874f-a371-44be-822d-2be3818feee5","added_by":"auto","created_at":"2026-05-14 10:06:37","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":343671,"visible":true,"origin":"","legend":"\u003cp\u003eProduct distribution (A) and microbial community structure (B) of the E_BR. The microbial biofilm that adhered to the electrode was also analyzed (Electrode cell, Fig. 2B).\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-9518331/v1/83cd9784464b7b6ab96ed39c.png"},{"id":109296568,"identity":"ca99af1b-38d7-47f0-81f8-5daa5e778fee","added_by":"auto","created_at":"2026-05-15 08:48:15","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":264633,"visible":true,"origin":"","legend":"\u003cp\u003eKinetic batch tests BI, at 456 h (A), and BII, at 744 h (B).\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-9518331/v1/952cdf7ae7cb163f36c44694.png"},{"id":109255933,"identity":"53b35d24-1c30-47a1-95c6-18c60b9692f7","added_by":"auto","created_at":"2026-05-14 10:06:37","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":24610,"visible":true,"origin":"","legend":"\u003cp\u003eInorganic carbon (IC) removal rate and IC specific removal rate as a function of the hydraulic retention time (HRT).\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-9518331/v1/3682cacaba83b8ebd9630ffa.png"},{"id":109297798,"identity":"438ec41a-95b5-42c9-bc83-e5e39fb3d2b5","added_by":"auto","created_at":"2026-05-15 09:05:53","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":597581,"visible":true,"origin":"","legend":"\u003cp\u003eCluster map of dominant metabolic functions in each E_BR sample based on microbiota composition and diversity; logarithmic scale normalized values were used.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-9518331/v1/474236810bb7ecc5d1a3b11a.png"},{"id":109297822,"identity":"9286472f-fc86-4f9a-b5db-082359c14191","added_by":"auto","created_at":"2026-05-15 09:06:25","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":117775,"visible":true,"origin":"","legend":"\u003cp\u003eSpecific productivities of carboxylic acids as a function of HRT.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-9518331/v1/fa8e850567ec45d6cc02739d.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"Hydraulic Retention Time and Electric Stimuli as Key Levers for Tailoring Mixotrophic Consortia Toward Enhanced Volatile Fatty Acid Production and Carbon Capture","fulltext":[{"header":"1. Background","content":"\u003cp\u003eHuman dependence on fossil fuels has severely disrupted the global carbon cycle, driving atmospheric CO\u003csub\u003e2\u003c/sub\u003e concentrations to unprecedented levels. Annual CO\u003csub\u003e2\u003c/sub\u003e emissions currently exceed 38\u0026nbsp;billion tons, yet only a negligible fraction, approximately 45\u0026nbsp;million tons, is converted into value-added products such as acids and biofuels [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. In response, 195 nations committed to developing CO\u003csub\u003e2\u003c/sub\u003e mitigation strategies at the 21st United Nations Climate Change Conference in 2015 [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e], spurring interest in technologies that couple carbon capture with the production of useful chemicals.\u003c/p\u003e \u003cp\u003eMicrobial electrosynthesis (MES) are particularly promising because they exploit microorganisms as biocatalysts to convert CO\u003csub\u003e2\u003c/sub\u003e into value-added organic compounds using electrical energy as the thermodynamic driving force [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. A broad range of chemicals has been produced through MES, with volatile fatty acids (VFAs), especially acetate, emerging as the most relevant products [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Global demand for acetic acid is projected to reach approximately 19.6\u0026nbsp;million tons per year by 2027 [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], highlighting the industrial relevance of electrosynthetic acetate production.\u003c/p\u003e \u003cp\u003eAcetate can be produced autotrophically by acetogens, which fix CO\u003csub\u003e2\u003c/sub\u003e via the Wood-Ljungdahl pathway (WLP) using H\u003csub\u003e2\u003c/sub\u003e or CO as electron donors [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. The reduction of two CO\u003csub\u003e2\u003c/sub\u003e molecules to acetate via H\u003csub\u003e2\u003c/sub\u003e is exergonic under physiological conditions (approximately\u0026thinsp;\u0026minus;\u0026thinsp;95 kJ mol\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. However, autotrophic growth operates close to thermodynamic limits, making it sensitive to environmental fluctuations and difficult to sustain over long periods [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eA practical strategy to overcome these thermodynamic constraints is to operate under mixotrophic conditions, where microorganisms simultaneously assimilate both inorganic (CO\u003csub\u003e2\u003c/sub\u003e/HCO\u003csub\u003e3\u003c/sub\u003e⁻) and organic carbon sources. This dual-substrate approach broadens metabolic flexibility, improves thermodynamic favourability, and supports more stable and productive microbial communities [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. The co-supply of organic carbon also enables the enrichment of syntrophic consortia in which fermentative organisms provide reducing equivalents \u0026mdash; such as H\u003csub\u003e2\u003c/sub\u003e and formate \u0026mdash; that acetogens then channel into CO\u003csub\u003e2\u003c/sub\u003e fixation.\u003c/p\u003e \u003cp\u003eThe application of a low electric potential further enhances this process by promoting extracellular electron transfer, stimulating carbon-fixing pathways, and selectively enriching electroactive genera at electrode surfaces [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Under electrofermentation conditions, cathode-associated microorganisms can generate H\u003csub\u003e2\u003c/sub\u003e \u003cem\u003ein situ\u003c/em\u003e, supplying a continuous stream of electron donors to planktonic acetogens and thereby amplifying CO\u003csub\u003e2\u003c/sub\u003e conversion rates [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn continuous bioprocesses, hydraulic retention time (HRT) is a key operational parameter that governs dilution rate, imposes selective pressure on microbial growth, and ultimately shapes community structure and metabolic output. Although both electrical stimulation and HRT are recognized as important process levers, their combined impact on microbial specialization and inorganic carbon (IC) utilization in continuous electrobioreactors remains poorly understood. This work demonstrates that HRT can be used as a practical engineering tool to control carbon flux, enhance CO\u003csub\u003e2\u003c/sub\u003e utilization, and selectively drive acetate production in continuous electrobioreactors.\u003c/p\u003e \u003cp\u003eHere, we address this gap by operating a continuous electrostimulated bioreactor (E_BR) at a constant applied potential of 300 mV for approximately 1200 h under three successive HRT conditions (33, 19, and 14 h). We monitored biomass concentration, pH, product distribution, and inorganic carbon removal rates across all conditions, and characterized the suspended and electrode-associated microbial communities by 16S rRNA sequencing and metagenomic functional prediction. We demonstrate that decreasing HRT progressively enriches a specialized mixotrophic consortium, enhances IC uptake, and redirects carbon flux toward acetate production, revealing HRT as an effective and simple lever for steering electrofermentation toward enhanced carbon capture.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Inoculum\u003c/h2\u003e \u003cp\u003eSludge obtained from an anaerobic reactor employed to treat vinasse at a sugar and ethanol plant located in Prad\u0026oacute;polis, state of S\u0026atilde;o Paulo, Brazil (Latitude: -21.3189\u0026deg; S; Longitude: -48.1170\u0026deg; W), was applied to inoculate E_BR. The initial volatile suspended solids (VSS) concentration was 2650 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Electrobioreactor (E_BR) operation\u003c/h2\u003e \u003cp\u003eE_BR with a working volume of 1100 mL was operated in the continuous mode for approximately 1200 h at HRT of 33, 19, and 14 h over time. Two graphite plate electrodes (1.5 \u0026times; 13.0 \u0026times; 0.5 cm), which acted as the anode and cathode, were introduced inside E_BR, while a constant potential difference of 300 mV was applied (power supply, Hikari HF-3203S). E_BR was fed with synthetic medium and stirred at 150 rpm with a magnetic stirrer. Anaerobic operation conditions in the reactor were guaranteed by daily flushing the feeding medium and the reactor with CO\u003csub\u003e2\u003c/sub\u003e for 6 min. The temperature was maintained at 30\u0026deg;C by using a heat jacket, and the pH of the feeding medium was adjusted to 6.3.\u003c/p\u003e \u003cp\u003eThe system was fed with M9 medium supplemented with 3.6 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e fructose, as the organic carbon source, and 4.0 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e NaHCO\u003csub\u003e3\u003c/sub\u003e, as the IC source. In addition, the medium contained (in g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) 6.78 Na\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e, 3.0 KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e, 5.0 NaCl, 1.0 NH\u003csub\u003e4\u003c/sub\u003eCl, 0.49 MgSO\u003csub\u003e4\u003c/sub\u003e.7H\u003csub\u003e2\u003c/sub\u003eO, 0.01 CaCl\u003csub\u003e2\u003c/sub\u003e, and.1.0 yeast extract. Fructose addition helps develop more stable mixotrophic microbial communities.\u003c/p\u003e \u003cp\u003eThe VSS concentration, which corresponds to the biomass, was measured daily according to APHA (2017) [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. During the biomass washout periods, the maximum specific growth rate (\u0026micro;\u003csub\u003emax\u003c/sub\u003e) was determined by using Eq.\u0026nbsp;(\u003cspan refid=\"Equ1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003cdiv id=\"Equ1\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ1\" name=\"EquationSource\"\u003e\n$$\\:\\frac{dx}{dt}={{X}_{i}\\:e}^{(\\mu\\:max-D)t}$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e1\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003ewhere \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{X}_{i}\\)\u003c/span\u003e\u003c/span\u003e is the initial microorganism cell concentration (mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:x\\)\u003c/span\u003e\u003c/span\u003e is the microorganism cell concentration at time t (mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), D is the dilution rate (h\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\left(D=\\:\\frac{1}{HRT}\\right)\\)\u003c/span\u003e\u003c/span\u003e, and \u0026micro;\u003csub\u003emax\u003c/sub\u003e is the maximum specific growth rate (h\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Batch assays: fructose consumption and product kinetics\u003c/h2\u003e \u003cp\u003eDuring operations at HRTs of 19 h and 14a h, continuous feeding of E_BR was temporarily switched to batch mode to assess fructose consumption and to determine different product formation rates. Kinetic tests were conducted at 456 and 744 h and were labeled batch I (BI) and batch II (BII), respectively. For the batch kinetic tests, 650 mL of the supernatant was replaced with fresh M9 medium in one single step, and CO\u003csub\u003e2\u003c/sub\u003e was purged for 6 min. The fructose consumption and product formation rates (in mg L-1 h-1) were estimated from the slope of a linear regression fitted to the concentration-time profiles.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Analytical determinations and calculations\u003c/h2\u003e \u003cp\u003eVSS concentrations during E_BR operation were determined according to Standard Methods (APHA, 2017). Fructose and the products, including acetic (HAc), lactic (HLac), propionic (HPr), butyric (HBut), and formic (HFor) acids, were analyzed on a high-performance liquid chromatograph (Shimadzu LC-20 AT, Japan) equipped with an Aminex HPX-87H column. The column temperature was maintained at 60\u0026deg;C, and the mobile phase consisted of 5 mmol L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e H\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e at 0.6 mL min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (60 kgf cm\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e). A photodiode array detector (PDA) and refractive index detectors (RID) were used to quantify the carboxylic acids and fructose, respectively. The LabSolutions software was employed.\u003c/p\u003e \u003cp\u003eIC was quantified from the three final sampling points for each HRT applied. Analyses were carried out on a Shimadzu TOC analyzer (model TOC-VCPN). Samples were pretreated by automatically adding acid, which was followed by gas (oxygen carrier) injection at 230 mL min-1. The IC removal rate and IC specific removal rate were calculated by using Eqs.\u0026nbsp;(\u003cspan refid=\"Equ2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) and (\u003cspan refid=\"Equ3\" class=\"InternalRef\"\u003e3\u003c/span\u003e), respectively [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003cdiv id=\"Equ2\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ2\" name=\"EquationSource\"\u003e\n$$\\:IC\\:removal\\:rate\\:\\left(mg\\:{L}^{-1}{h}^{-1}\\right)={(IC}_{i}-{IC}_{f})\\:x\\:D$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e2\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Equ3\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ3\" name=\"EquationSource\"\u003e\n$$\\:IC\\:Specific\\:removal\\:\\:rate\\:\\left(mg\\:{g}^{-1}{h}^{-1}\\right)=\\frac{IC\\:removal\\:rate}{X}$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e3\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003ewhere \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{IC}_{i}\\)\u003c/span\u003e\u003c/span\u003e and \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{IC}_{f\\:}\\)\u003c/span\u003e\u003c/span\u003eare the initial and final \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:IC\\)\u003c/span\u003e\u003c/span\u003e concentrations (mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), respectively, \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:X\\)\u003c/span\u003e\u003c/span\u003e is the biomass concentration average of three last points (g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) at the end of each HRT, and D is dilution rate (h\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\left(D=\\:\\frac{1}{HRT}\\right)\\)\u003c/span\u003e\u003c/span\u003e.\u003c/p\u003e \u003cp\u003eTo evaluate biomass specialization for carboxylic acid production, specific productivity was calculated from the three final sampling points for each applied HRT, using Eq.\u0026nbsp;\u003cspan refid=\"Equ4\" class=\"InternalRef\"\u003e4\u003c/span\u003e.\u003cdiv id=\"Equ4\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ4\" name=\"EquationSource\"\u003e\n$$\\:P\\:\\left(mg\\:{g}^{-1}{h}^{-1}\\right)=\\frac{D\\:Cp}{X}$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e4\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003ewhere \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:P\\)\u003c/span\u003e\u003c/span\u003e is the specific productivity (mg g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e h\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), D is the dilution rate (h\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{C}_{p}\\)\u003c/span\u003e\u003c/span\u003e is the concentration of the respective carboxylic acid (mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), and \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:X\\)\u003c/span\u003e\u003c/span\u003e is the biomass concentration (g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 16S rRNA sequencing and metagenomic functional predictions\u003c/h2\u003e \u003cp\u003eInoculum and biomass samples were collected from E_BR at the end of each HRT, along with biofilm cells harvested from the graphite electrode, to evaluate how the microbial community adapted to the different HRT conditions.\u003c/p\u003e \u003cp\u003eFor this purpose, DNA was extracted from the samples by using the ZymoBIOMICS\u0026trade; DNA Miniprep kit (Zymo Research) and quantified to measure both quality and quantity. The DNA samples were submitted to complete amplification of the 16S rRNA gene by using primers 27F and 1492R (~\u0026thinsp;1.6-kb fragment). The amplicons were analyzed on agarose gel; quantified by using the SQK-LSK114 kit (Oxford Nanopore Technologies), to construct a library; and sequenced on a Flongles flow cell (FLO-FLG114) on the MinION platform (Oxford Nanopore Technologies).\u003c/p\u003e \u003cp\u003eAfter the amplicons were sequenced, the resulting reads were base-called by using Guppy Basecaller (v 6.0.7), GPU version. The reads were filtered for quality control in Q10 by employing NanoFilt (v2.3.0) and demultiplexed with Porechop (v0.2.4). The reads were mapped to a 16S reference database by using the KMA tool (v1.4.3). Data were analyzed by applying Python 3.7.\u003c/p\u003e \u003cp\u003eThe metabolic pathway was predicted with PICRUSt2 v2.3.0. The cluster map displays the 30 most abundant pathways, while percentile filtering (60\u0026ndash;90%) generated a refined set of the top 30 pathways [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Sequencing reads were base called by using Guppy v6.4.8 (high-accuracy, GPU) and aligned to the RefSeq 16S database with KMA v1.3.23.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results and Discussion","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Monitoring the electrobioreactor at different HRT: cell biomass and pH\u003c/h2\u003e \u003cp\u003eWe operated the electrostimulated bioreactor (E_BR) continuously for approximately 1200 h under three successive hydraulic retention times (33, 19, and 14 h), monitoring volatile suspended solids (VSS) as a proxy for biomass concentration and pH throughout the entire operation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAt the initial HRT of 33 h, VSS decreased from 2650 to 1993 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e within the first 144 h, indicating partial biomass washout under the imposed dilution rate. However, continued operation at this HRT allowed the surviving community to recover, and VSS subsequently rose to 2666 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, suggesting that microbial growth rates surpassed the washout threshold. To impose stronger selective pressure and drive community specialization, HRT was reduced to 19 h, which caused a 14.5% decrease in VSS \u0026mdash; from 2910 to 2490 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e \u0026mdash; at 456 h (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Immediately, following this transition, continuous feeding was momentarily switched to batch mode (Batch I, BI) to characterize substrate consumption and product formation kinetics under the microbial community established at HRT\u0026thinsp;=\u0026thinsp;19 h (Section \u003cspan refid=\"Sec13\" class=\"InternalRef\"\u003e3.3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAfter Bath I (BI), continuous operation was resumed, marking the beginning of the 14\u003csup\u003ea\u003c/sup\u003e h phase. At this stage, the HRT was further reduced to 14 h, resulting in a 26.7% decrease in VSS, from 3039 to 2396 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The E_BR was maintained at this HRT until 744 h. At this point, a second batch assay (Batch II, BII) was initiated to probe substrate and product dynamics under the more stringently selected community (Section \u003cspan refid=\"Sec13\" class=\"InternalRef\"\u003e3.3\u003c/span\u003e). Following BII, continuous operation resumed \u0026mdash; beginning the 14\u003csup\u003eb\u003c/sup\u003e h phase \u0026mdash; at HRT\u0026thinsp;=\u0026thinsp;14 h, and was sustained until the experiment\u0026rsquo;s end at 1156 h. The 14\u003csup\u003eb\u003c/sup\u003e h phase began with a VSS of 4193 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, reflecting biomass accumulation during BII, and VSS declined to 2360 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, a 43.7% reduction, underscoring the strong washout pressure imposed at the shortest HRT.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eEach HRT transition was accompanied by a biomass washout period, which enabled estimation of the maximum specific growth rate (\u0026micro;\u003csub\u003emax\u003c/sub\u003e) of the consortium using Eq.\u0026nbsp;\u003cspan refid=\"Equ1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and the corresponding washout profiles (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). The estimated \u0026micro;\u003csub\u003emax\u003c/sub\u003e values were 0.025, 0.050, 0.068, and 0.069 h\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e for HRT\u0026thinsp;=\u0026thinsp;33, 19, 14\u003csup\u003ea\u003c/sup\u003e, and 14\u003csup\u003eb\u003c/sup\u003e h, respectively. These values fall within the range reported for mesophilic acetogens (0.03\u0026ndash;0.12 h\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. For reference, \u0026micro;\u003csub\u003emax\u003c/sub\u003e values of 0.069, 0.078, and 0.018 h\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e have been reported for \u003cem\u003eAcetobacterium wieringae\u003c/em\u003e (DSM 1911), \u003cem\u003eBlautia hydrogenotrophica\u003c/em\u003e (DSM 10507), and \u003cem\u003eSporomusa acidovorans\u003c/em\u003e (DSM 3132), respectively [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. \u003cem\u003eClostridium aceticum\u003c/em\u003e, one of the genera that became dominant in this study (Section \u003cspan refid=\"Sec10\" class=\"InternalRef\"\u003e3.2\u003c/span\u003e), achieves \u0026micro;\u003csub\u003emax\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.052 h\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e under pH-controlled conditions (pH 8.0), and only 0.006 h\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e without pH control [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. The near-identical \u0026micro;\u003csub\u003emax\u003c/sub\u003e values at HRT\u0026thinsp;=\u0026thinsp;14\u003csup\u003ea\u003c/sup\u003e and 14\u003csup\u003eb\u003c/sup\u003e h (0.068 vs. 0.069 h\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) suggest that the community had reached a stable growth phenotype by the lowest HRT, with BII causing only a transient perturbation in biomass concentration rather than a shift in intrinsic growth capacity.\u003c/p\u003e \u003cp\u003eThroughout the entire operation, pH fluctuated within a narrow range of 6.3 to 7.0 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). This range is mechanistically significant for two reasons. First, at pH\u0026thinsp;\u0026asymp;\u0026thinsp;6.5, the dissolved inorganic carbon system approaches the first acidity constant of the carbonate equilibrium (pKa1\u0026thinsp;=\u0026thinsp;6.35), at which point aqueous CO\u003csub\u003e2\u003c/sub\u003e and bicarbonate (HCO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e) coexist at comparable concentrations. This coexistence makes inorganic carbon bioavailable via two complementary routes: gaseous CO\u003csub\u003e2\u003c/sub\u003e is directly accessible to microorganisms that employ the WLP, while HCO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e serves as an ionic reservoir accessible via carbonic anhydrase activity [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Second, the slightly acidic to near-neutral pH maintained throughout operation (6.3\u0026ndash;7.0) thermodynamically favors acidogenesis over solventogenesis in \u003cem\u003eClostridium\u003c/em\u003e-containing communities, as discussed in Section \u003cspan refid=\"Sec10\" class=\"InternalRef\"\u003e3.2\u003c/span\u003e. Consistent with this, no solvents were detected at any point during operation \u0026mdash; only carboxylic acids accumulated as metabolic products.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Product distribution and microbial community dynamics\u003c/h2\u003e \u003cp\u003eChanges in HRT produced marked shifts in both product distribution and microbial community composition (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). The soluble products detected throughout E_BR operation were exclusively carboxylic acids; no corresponding alcohols were observed under any condition. As discussed in Section \u003cspan refid=\"Sec9\" class=\"InternalRef\"\u003e3.1\u003c/span\u003e, the pH range of 6.3\u0026ndash;7.0 thermodynamically favors acidogenesis over solventogenesis in \u003cem\u003eClostridium\u003c/em\u003e-containing communities [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Moreover, with the main VFAs (pKa\u0026thinsp;\u0026asymp;\u0026thinsp;4.7\u0026ndash;4.8) persisting in their dissociated anionic forms at the operating pH, undissociated acid concentrations remained low, preventing intracellular acidification and eliminating the redox imbalance that would otherwise trigger solvent formation as a reductant-relief mechanism [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section3\"\u003e \u003ch2\u003e3.2.1 Suspended Microbial Community and Product Profile\u003c/h2\u003e \u003cp\u003eAt HRT\u0026thinsp;=\u0026thinsp;33 h, acetate (HAc) and propionate (HPr) co-dominated the product profile, with HAc and HPr reaching 846.8 and 782.0 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, respectively, and butyrate (HBut) present at low concentrations (100.3 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e). The microbial community at this stage was dominated by \u003cem\u003eTrichococcus\u003c/em\u003e (44.8%), a well-characterized acidogenic lactic acid bacterium that ferments sugars to lactate, formate, and acetate [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], and \u003cem\u003eKlebsiella\u003c/em\u003e (27.5%), which increased from 1.3% in the inoculum. Neither genus is associated with propionate production, indicating that HPr likely originated from less abundant or taxonomically unassigned community members (\u0026sim;9% of sequences), possibly through the acrylate or succinate-propionate pathways.\u003c/p\u003e \u003cp\u003eAt HRT of 19 h, the community underwent a substantial restructuring: \u003cem\u003eTrichococcus\u003c/em\u003e declined sharply from 44.8% to 1.8%, while \u003cem\u003eEnterococcus\u003c/em\u003e (25.2%), \u003cem\u003eArcobacter\u003c/em\u003e (22.3%), and \u003cem\u003eRobinsoniella\u003c/em\u003e (18.9%) emerged as the dominant genera, together accounting for 66.4% of the total relative abundance (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Concomitantly, low concentrations of formate (HFor; 50\u0026ndash;70 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) appeared for the first time, pointing to activation of the methyl branch of the WLP, where formate serves as a key intermediate [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. HAc, HPr, and HBut concentrations increased modestly to 1171.3, 790.6, and 114.6 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, respectively. \u003cem\u003eArcobacter\u003c/em\u003e, known for its fermentative and acidogenic activity and exoelectrogenic behavior in anodic microbial fuel cell (MFC) biofilms [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], likely benefited from the applied potential difference of 300 mV. \u003cem\u003eEnterococcus\u003c/em\u003e, a lactic acid bacterium capable of H\u003csub\u003e2\u003c/sub\u003e and VFA production, and with demonstrated capacity for extracellular electron transfer (EET) both via direct contact and soluble mediators [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e], was consistent with the stable VFA formation observed at this stage.\u003c/p\u003e \u003cp\u003eAt HRT of 14\u003csup\u003ea\u003c/sup\u003e h, the community retained \u003cem\u003eEnterococcus\u003c/em\u003e (31.7%) and \u003cem\u003eArcobacter\u003c/em\u003e (13.3%), while \u003cem\u003eClostridium\u003c/em\u003e increased 3.5-fold relative to HRT\u0026thinsp;=\u0026thinsp;19 h, reaching 9.5% of the total abundance. \u003cem\u003eIlyobacter\u003c/em\u003e (16.1%) and \u003cem\u003eRobinsoniella\u003c/em\u003e (8.5%) were also present; however, both \u003cem\u003eArcobacter\u003c/em\u003e and \u003cem\u003eRobinsoniella\u003c/em\u003e showed relative declines of 9.0% and 10.4%, respectively. The enrichment of \u003cem\u003eClostridium\u003c/em\u003e at the lowest HRT coincided with a significant increase in HAc concentration, consistent with the genus's known capacity to fix CO\u003csub\u003e2\u003c/sub\u003e into acetate via the WLP in the presence of H\u003csub\u003e2\u003c/sub\u003e [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. The enrichment of \u003cem\u003eClostridia\u003c/em\u003e under H\u003csub\u003e2\u003c/sub\u003e/CO\u003csub\u003e2\u003c/sub\u003e-fed conditions has been consistently associated with acetate-dominated VFA production in homoacetogenic systems [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAt the final continuous stage (HRT\u0026thinsp;=\u0026thinsp;14\u003csup\u003eb\u003c/sup\u003e h), HAc reached its maximum concentration of 1474.6 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and remained stable, while HPr decreased to 450.7 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e \u0026mdash; a 54.3% reduction relative to HRT\u0026thinsp;=\u0026thinsp;14\u003csup\u003ea\u003c/sup\u003e h \u0026mdash; confirming that the carbon flux was progressively redirected toward acetate as the community became more specialized.\u003c/p\u003e \u003cp\u003eHBut increased by 149.6% relative to HRT\u0026thinsp;=\u0026thinsp;33 h, reaching 250.3 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, while HFor concentrations remained low across all HRTs. \u003cem\u003eEnterococcus\u003c/em\u003e (26.1%) and \u003cem\u003eClostridium\u003c/em\u003e (15.8%) persisted as the dominant genera, accounting for 41.8% of the community, while \u003cem\u003eRobinsoniella\u003c/em\u003e and \u003cem\u003eArcobacter\u003c/em\u003e declined to 3.5% and 3.2%, respectively. The persistence of \u003cem\u003eEnterococcus\u003c/em\u003e and \u003cem\u003eClostridium\u003c/em\u003e, which have been reported to interact synergistically in continuous H\u003csub\u003e2\u003c/sub\u003e-producing reactors [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e], likely underpinned the stable and elevated acetate accumulation observed at the lowest HRT.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section3\"\u003e \u003ch2\u003e3.2.2 Electrode Biofilm Community\u003c/h2\u003e \u003cp\u003eAt the end of the experiment (1156 h), cells harvested from the graphite electrode surface revealed a community composition distinctly different from the planktonic phase. \u003cem\u003eEnterococcus\u003c/em\u003e was the most abundant genus in electrode, representing 39.2% of the biofilm community \u0026mdash; a higher proportion than in the suspended phase (26.1%). \u003cem\u003eDesulfovibrio\u003c/em\u003e (9.1%) and \u003cem\u003eGeobacte\u003c/em\u003er (3.0%) were substantially enriched in the biofilm relative to the bulk liquid, while \u003cem\u003eClostridium\u003c/em\u003e, which dominated the planktonic community (15.8%), accounted for only 1.4% of the biofilm. Additionally, 18.5% of biofilm sequences remained taxonomically unassigned, suggesting the presence of poorly characterized electroactive taxa.\u003c/p\u003e \u003cp\u003eThe prominence of \u003cem\u003eEnterococcus\u003c/em\u003e in the electrode biofilm is noteworthy. Beyond its fermentative role, \u003cem\u003eEnterococcus faecalis\u003c/em\u003e has been shown to transfer electrons generated during fermentative metabolism to electrode surfaces both directly and via soluble redox mediators, establishing it as a functionally electroactive genus in bioelectrochemical systems [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Its H\u003csub\u003e2\u003c/sub\u003e-producing capability also positions it as an indirect electron donor for acetogens in the planktonic phase.\u003c/p\u003e \u003cp\u003e \u003cem\u003eDesulfovibrio\u003c/em\u003e is a key electrotrophic genus in bioelectrochemical systems. It accepts electrons from the cathode and produces H\u003csub\u003e2\u003c/sub\u003e or formate through periplasmic hydrogenases and outer-membrane cytochromes, generating a continuous supply of reducing equivalents [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. This function is particularly critical for syntrophic interactions with planktonic \u003cem\u003eClostridium\u003c/em\u003e, which utilizes H\u003csub\u003e2\u003c/sub\u003e to reduce CO\u003csub\u003e2\u003c/sub\u003e via the WLP [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Recent studies have highlighted that the coupling of H\u003csub\u003e2\u003c/sub\u003e-producing electrotrophs such as \u003cem\u003eDesulfovibrio\u003c/em\u003e with acetogens such as \u003cem\u003eClostridium\u003c/em\u003e synergistically drives CO\u003csub\u003e2\u003c/sub\u003e reduction through WLP [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e], which is entirely consistent with the community architecture observed here. \u003cem\u003eGeobacter\u003c/em\u003e, while well-established as the dominant genus in anodic MFC biofilms [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e], appeared at low abundance (3.0%) in the E_BR electrode biofilm. Its role in electrofermentation systems is less well characterized.\u003c/p\u003e \u003cp\u003eOverall, the electrode biofilm and planktonic community exhibited a clear functional division of labor: the biofilm was enriched in electrotrophic and H\u003csub\u003e2\u003c/sub\u003e-producing genera (\u003cem\u003eDesulfovibrio\u003c/em\u003e, \u003cem\u003eEnterococcus\u003c/em\u003e), which supplied reducing equivalents to the planktonic acetogens (\u003cem\u003eClostridium\u003c/em\u003e), enabling CO\u003csub\u003e2\u003c/sub\u003e fixation and acetate accumulation in the bulk liquid. This syntrophic architecture, shaped and sustained by the combined effects of HRT and electrical stimulation, is discussed further in Section \u003cspan refid=\"Sec14\" class=\"InternalRef\"\u003e3.4\u003c/span\u003e in terms of metabolic pathway enrichment.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Batch Assays (BI and BII): Insights into Substrate and Product Dynamics\u003c/h2\u003e \u003cp\u003eTo gain mechanistic insight into substrate consumption and product formation kinetics, detail that is difficult to resolve under continuous flow, continuous feeding of E_BR was temporarily suspended and the system was operated in batch mode twice: at 456 h (Batch I, BI) and at 744 h (Batch II, BII), corresponding to community states established at HRT of 19 h and HRT of 14\u003csup\u003ea\u003c/sup\u003e h, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003eIn both assays, fructose was consumed rapidly, and a characteristic transient intermediate profile emerged (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Lactate (HLac), which was not detected during continuous operation, appeared within the first 4 h of each batch assay, concomitant with fructose consumption, and was completely depleted by the end of the batch period. The transient accumulation and subsequent disappearance of HLac under batch conditions, but not under continuous flow, reflects the kinetics of the underlying microbial interactions: In continuous mode, lactate was probably produced by \u003cem\u003eEnterococcus\u003c/em\u003e and other lactic acid bacteria and is consumed by lactate-fermenting bacteria (e.g., \u003cem\u003eClostridium\u003c/em\u003e) as fast as it is generated, precluding its accumulation to detectable levels. In batch mode, the transiently higher substrate concentration and the absence of dilution allow lactate to accumulate before being metabolized downstream.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eHAc, HPr, and HBut gradually accumulated throughout both batch assays, BI and BII (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB), respectively, with concentrations increasing sharply after lactate began to be depleted, consistent with their formation through lactate conversion rather than directly from fructose. By 24 h, HAc, HPr, and HBut concentrations in BII exceeded those in BI by 46.4%, 18.4%, and 24.2%, respectively, demonstrating that the microbial community selected under the lower HRT (14\u003csup\u003ea\u003c/sup\u003e h) had developed a greater capacity for carboxylic acid production \u0026mdash; particularly acetate \u0026mdash; compared with the community present at HRT\u0026thinsp;=\u0026thinsp;19 h.\u003c/p\u003e \u003cp\u003eThe dominant lactate-converting metabolism in this system is consistent with the acrylate/succinate-propionate pathway, by which \u003cem\u003eClostridium\u003c/em\u003e spp. convert lactate into propionate and acetate at an expected stoichiometric ratio of 3 lactate : 2 propionate : 1 acetate [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. However, the observed final acetate concentration in BII (6.61 mmol L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) substantially exceeded the stoichiometric maximum predicted by this pathway alone (4.80 mmol L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), a discrepancy that cannot be attributed to analytical error or differences in initial fructose concentration. This excess acetate \u0026mdash; accounting for approximately 38% of the total HAc produced \u0026mdash; points to the involvement of additional autotrophic carbon-fixing routes. Specifically, it implicates CO\u003csub\u003e2\u003c/sub\u003e/HCO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e assimilation via the WLP, in which H\u003csub\u003e2\u003c/sub\u003e generated by \u003cem\u003eClostridium\u003c/em\u003e fermentation and by \u003cem\u003eDesulfovibrio\u003c/em\u003e electrotrophic activity at the electrode surface (Section \u003cspan refid=\"Sec12\" class=\"InternalRef\"\u003e3.2.2\u003c/span\u003e) is used to reduce inorganic carbon to acetyl-CoA and ultimately acetate [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThis interpretation is further supported by the metabolic pathway enrichment data presented in Section \u003cspan refid=\"Sec14\" class=\"InternalRef\"\u003e3.4\u003c/span\u003e, which shows a progressive increase in the abundance of prokaryotic carbon fixation pathways across decreasing HRTs, as well as their pronounced enrichment in the electrode biofilm. Taken together, the batch assays reveal that the E_BR community is not purely heterotrophic: fructose drives lactic acid fermentation, lactate is subsequently channelled into propionate and acetate, and the reducing equivalents liberated by these fermentative reactions \u0026mdash; supplemented by bioelectrochemical H\u003csub\u003e2\u003c/sub\u003e production \u0026mdash; feed the WLP, generating additional acetate from inorganic carbon.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Inorganic Carbon Capture and Metabolic Pathway Enrichment\u003c/h2\u003e \u003cp\u003eTo quantify how HRT modulated the capacity of the microbial community to assimilate inorganic carbon, we measured IC concentrations at the inlet and outlet of E_BR at the end of each HRT phase and calculated IC removal rates and biomass-specific IC removal rates (Eqs.\u0026nbsp;\u003cspan refid=\"Equ2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and \u003cspan refid=\"Equ3\" class=\"InternalRef\"\u003e3\u003c/span\u003e; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIC removal increased progressively as HRT decreased. At HRT\u0026thinsp;=\u0026thinsp;33 h, the IC removal rate was 13.1 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e h\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, corresponding to a biomass-specific rate of 4.92 mg g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e h\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. Reducing HRT to 19 h nearly doubled the specific IC removal rate (9.35 mg g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e h\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e; 1.9-fold increase), and at HRT\u0026thinsp;=\u0026thinsp;14 h, the specific rate reached 12.88 mg g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e h\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e \u0026mdash; a 2.6-fold increase relative to the highest HRT \u0026mdash; with a volumetric removal rate of 30.5 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e h\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. These results demonstrate that shorter residence times did not merely wash out biomass; they selectively enriched a community that was intrinsically more efficient at capturing and assimilating inorganic carbon per unit of cell mass.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe progressive enrichment of IC capture capacity across decreasing HRTs is mechanistically consistent with the community shifts described in Section \u003cspan refid=\"Sec10\" class=\"InternalRef\"\u003e3.2\u003c/span\u003e. At the lowest HRT, the dominance of \u003cem\u003eClostridium\u003c/em\u003e in the planktonic phase and \u003cem\u003eDesulfovibrio\u003c/em\u003e in the electrode biofilm established a syntrophic architecture well-suited for CO\u003csub\u003e2\u003c/sub\u003e fixation: \u003cem\u003eDesulfovibrio\u003c/em\u003e electrotroph activity supplied a continuous H\u003csub\u003e2\u003c/sub\u003e flux at the cathode, which \u003cem\u003eClostridium\u003c/em\u003e channelled through the WLP to reduce CO\u003csub\u003e2\u003c/sub\u003e to acetyl-CoA and, ultimately, acetate. This interpretation is corroborated by the stoichiometric excess of acetate observed in the batch assays (Section \u003cspan refid=\"Sec13\" class=\"InternalRef\"\u003e3.3\u003c/span\u003e), which could not be accounted for by heterotrophic lactate conversion alone.\u003c/p\u003e \u003cp\u003eTo evaluate whether these metabolic shifts were reflected in the functional genomic composition of the community, we predicted metabolic pathway abundances using PICRUSt2 and compared the planktonic and electrode-associated communities across HRT conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). The heatmap revealed that HRT strongly modulated the enrichment of metabolic pathways and that the electrode biofilm was functionally distinct from the planktonic community.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAt HRT\u0026thinsp;=\u0026thinsp;33 h, pathways associated with carbon fixation and energy metabolism showed relatively low abundance, suggesting limited metabolic turnover and reduced carbon flux through autotrophic routes. As HRT decreased to 19 and 14 h, these pathways became progressively more abundant, indicating functional enrichment toward carbon fixation and energy-conserving processes. Notably, prokaryotic carbon fixation pathways were consistently most abundant in the electrode-associated biofilm across all conditions, confirming that the electrode community \u0026mdash; dominated by \u003cem\u003eDesulfovibrio\u003c/em\u003e and \u003cem\u003eEnterococcus\u003c/em\u003e \u0026mdash; played a disproportionate role in driving inorganic carbon assimilation relative to its biomass contribution. Amino acid biosynthesis pathways were also prominent in the heatmap, reflecting active biosynthetic metabolism in a community under sustained growth and selective pressure.\u003c/p\u003e \u003cp\u003eThese findings provide functional genomic support for the syntrophic model proposed above and demonstrate that the combination of electrical stimulation and progressively shorter HRT shaped not only the taxonomic composition but also the metabolic specialization of both the planktonic and biofilm communities toward enhanced carbon capture.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.5 Specific Productivities and Biomass Specialization\u003c/h2\u003e \u003cp\u003eTo assess how HRT-driven community specialization translated into productive efficiency, we calculated the specific productivities of individual carboxylic acids and the total VFA-specific productivity at each HRT, using the last three steady-state measurements of biomass and product concentrations at each condition (Eq.\u0026nbsp;\u003cspan refid=\"Equ4\" class=\"InternalRef\"\u003e4\u003c/span\u003e; Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eOverall, decreasing HRT was accompanied by a consistent increase in both individual and total VFA-specific productivity, reflecting progressive biomass specialization toward carboxylic acid production. Acetate and propionate were the dominant metabolites throughout all operating conditions.\u003c/p\u003e \u003cp\u003eAt HRT\u0026thinsp;=\u0026thinsp;33 h, the total VFA-specific productivity was 24.4 mg g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e h\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, with acetate and propionate as co-dominant products. Whereas decreasing the HRT to 19 h and 14\u003csup\u003ea\u003c/sup\u003e h increased the productivity to 47.76 and 74.68 mg g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e h\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, respectively. When HRT was reduced to 19 h, acetate-specific productivity increased by 25.5%, while propionate productivity declined modestly. The most pronounced shift occurred between HRT\u0026thinsp;=\u0026thinsp;14\u003csup\u003ea\u003c/sup\u003e and 14\u003csup\u003eb\u003c/sup\u003e h. At HRT\u0026thinsp;=\u0026thinsp;14\u003csup\u003eb\u003c/sup\u003e h, acetate-specific productivity reached 42.3 mg g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e h\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, a 65.9% increase over HRT\u0026thinsp;=\u0026thinsp;19 h. In contrast, propionate-specific productivity fell by 54.3% between the two 14 h phases. This confirms a progressive and selective redirection of carbon flux toward acetate.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe volumetric acetate productivity at the end of HRT\u0026thinsp;=\u0026thinsp;14\u003csup\u003eb\u003c/sup\u003e reached 107 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e h\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. This value compares favourably with several previous electrofermentation and MES studies. Acetate production rates reported for bioelectrochemical systems remain relatively low, typically ranging from 4.88 to 32.2 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e h\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e depending on operational conditions [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. The acetate productivity reported here therefore, represents a substantial improvement over these benchmarks.\u003c/p\u003e \u003cp\u003eTwo aspects of the experimental design are particularly noteworthy. First, the high acetate productivity achieved under mild cathodic polarization suggests that the syntrophic community architecture \u0026mdash; rather than purely electrochemical reduction \u0026mdash;was the primary driver of acetate accumulation, indicating that energy-efficient electrofermentation can be sustained under near-thermodynamic conditions. Second, the supplementation of the medium with fructose as an organic co-substrate supported metabolic maintenance and community stability, enabling sustained operation over 1200 h without the biomass loss and productivity collapse that have been reported in autotrophic-only MES systems. For instance, acetate production was not restored after repeated batch cycles due to substantial biomass loss during catholyte exchange in mixed-culture systems [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTaken together, these results demonstrate that operating an electrostimulated bioreactor under progressively shorter HRTs is an effective strategy to enhance biomass-specific productivity, redirect carbon flux toward acetate, and maintain stable, long-term performance \u0026mdash; outcomes that are difficult to achieve in purely autotrophic or non-electrostimulated systems.\u003c/p\u003e \u003c/div\u003e"},{"header":"4. Conclusion","content":"\u003cp\u003eThis study demonstrates that hydraulic retention time (HRT) is a key operational parameter to control carbon flux distribution and enhance CO\u003csub\u003e2\u003c/sub\u003e conversion in continuous electrostimulated bioreactors. Reducing HRT increased specific productivity from 24.4 to 74.68 mg g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e h\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and promoted the selection of a functionally specialized mixotrophic consortium. This shift enabled efficient coupling between electrochemical processes and microbial metabolism, resulting in acetate production beyond heterotrophic stoichiometric limits and confirming additional CO\u003csub\u003e2\u003c/sub\u003e fixation. The spatial organization of the community, with distinct roles between planktonic and electrode-associated populations, supported this enhanced carbon conversion. Overall, HRT reduction emerges as a simple, energy-efficient, and scalable strategy to intensify CO\u003csub\u003e2\u003c/sub\u003e valorization and steer continuous electrofermentation toward selective acetate production as a platform chemical, reinforcing its potential for sustainable bioprocesses.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eHRT \u0026nbsp; \u0026nbsp;Hydraulic retention time\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIC \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Inorganic carbon\u003c/p\u003e\n\u003cp\u003eVFA \u0026nbsp; \u0026nbsp;\u0026nbsp;Volatile fatty acid\u003c/p\u003e\n\u003cp\u003eWLP \u0026nbsp; \u0026nbsp;Wood-Ljungdahl pathway\u003c/p\u003e\n\u003cp\u003eMES \u0026nbsp; \u0026nbsp;Microbial electrosynthesis\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eE_BR\u0026nbsp;\u0026nbsp; Electrostimulated bioreactor\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eVSS\u0026nbsp; \u0026nbsp; \u0026nbsp; Volatile suspended solids\u003c/p\u003e\n\u003cp\u003e\u0026mu;\u003csub\u003emax\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/sub\u003eMaximum specific growth rate\u003c/p\u003e\n\u003cp\u003eHAc \u0026nbsp; \u0026nbsp;\u0026nbsp;Acetic acid\u003c/p\u003e\n\u003cp\u003eHPr \u0026nbsp; \u0026nbsp; \u0026nbsp;Propionic acid\u003c/p\u003e\n\u003cp\u003eHBut \u0026nbsp;\u0026nbsp;Butyric acid\u003c/p\u003e\n\u003cp\u003eHFor \u0026nbsp; \u0026nbsp;Formic acid\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors acknowledge financial support from the S\u0026atilde;o Paulo Research Foundation (FAPESP; processes 2024/00725-0 and 2023/07992-0), the Coordination for the Improvement of Higher Education Personnel (CAPES), and Petr\u0026oacute;leo Brasileiro S.A. (process 2023/00640-1).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA.C.B.Z.: Conceptualization, Methodology, Investigation, Writing \u0026ndash; original draft, Data curation. A.R.A.: Validation, Resources, Writing \u0026ndash; review and editing, Supervision, Project administration. V.R.: Conceptualization, Validation, Resources, Writing \u0026ndash; review and editing, Supervision, Project administration.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors thank Cynthia Maria de Campos Prado Manso for English language revision and the members of LABIORE and LEEA for their support. Ana Clara Bonizol Zani acknowledges her family (Tatiana, Renato, Carol, and Helena) for their continuous support, Jo\u0026atilde;o Carlos de Souza for valuable academic discussions, and her advisors, Valeria Reginatto and Adalgisa R. de Andrade, for their guidance and encouragement throughout this work.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eChung TH, Dhillon SK, Shin C, Pant D, Dhar BR. Microbial electrosynthesis technology for CO\u003csub\u003e2\u003c/sub\u003e mitigation, biomethane production, and ex-situ biogas upgrading. \u003cem\u003eBiotechnol Adv\u003c/em\u003e. 2024; doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.biotechadv.2024.108474\u003c/span\u003e\u003cspan address=\"10.1016/j.biotechadv.2024.108474\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAnderson TR, Hawkins E, Jones PD. 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J\u003c/em\u003e. 2023; doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.cej.2022.140200\u003c/span\u003e\u003cspan address=\"10.1016/j.cej.2022.140200\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":false,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"biotechnology-for-biofuels-and-bioproducts","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bbio","sideBox":"Learn more about [Biotechnology for Biofuels](http://biotechnologyforbiofuels.biomedcentral.com/)","snPcode":"13068","submissionUrl":"https://submission.nature.com/new-submission/13068/3","title":"Biotechnology for Biofuels and Bioproducts","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Electrofermentation, Microbial electrosynthesis, Inorganic carbon fixation, Acetogenesis, Syntrophic microbial community","lastPublishedDoi":"10.21203/rs.3.rs-9518331/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9518331/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eContinuous bioelectrochemical systems represent a promising platform for converting CO\u003csub\u003e2\u003c/sub\u003e into value-added bioproducts, yet operational strategies to enhance carbon capture and product selectivity remain limited. While mixotrophic electrofermentation improves process stability and performance, the role of hydraulic retention time (HRT) in shaping microbial community specialization and inorganic carbon utilization in continuous systems remains poorly understood.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eA continuous electrostimulated bioreactor was operated at a constant potential of 300 mV for approximately 1200 h under three HRTs (33, 19, and 14 h). Decreasing HRT promoted the enrichment of a specialized mixotrophic consortium, with \u003cem\u003eEnterococcus\u003c/em\u003e and \u003cem\u003eClostridium\u003c/em\u003e dominating the planktonic phase and \u003cem\u003eDesulfovibrio\u003c/em\u003e and \u003cem\u003eEnterococcus\u003c/em\u003e enriched in the electrode biofilm. This syntrophic organization enhanced biomass-specific IC removal by 2.6-fold (from 4.92 to 12.88 mg g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e h\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) and redirected carbon flux toward acetate, a key product of acetogenic pathways. Acetate reached 1470 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e with a volumetric productivity of 107 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e h\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e at HRT\u0026thinsp;=\u0026thinsp;14 h. Batch assays revealed that ~\u0026thinsp;38% of the acetate exceeded heterotrophic stoichiometry, indicating additional CO\u003csub\u003e2\u003c/sub\u003e/HCO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e fixation. Functional predictions further revealed the enrichment of carbon fixation pathways, particularly in the electrode-associated biofilm, suggesting that electrode-associated taxa contributed to the generation of reducing equivalents and supported autotrophic metabolism.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eReducing HRT enhanced inorganic carbon utilization and selectively increased acetate production in a continuous electrostimulated bioreactor. This effect was associated with the enrichment of a functionally specialized microbial consortium and the activation of carbon fixation pathways, particularly in the electrode-associated biofilm, highlighting the role of spatial organization in driving carbon flux. These findings demonstrate that HRT can be used as a practical and energy-efficient engineering strategy to control microbial function and improve CO\u003csub\u003e2\u003c/sub\u003e/HCO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e valorization, advancing the development of selective and scalable electrobiotechnological processes.\u003c/p\u003e","manuscriptTitle":"Hydraulic Retention Time and Electric Stimuli as Key Levers for Tailoring Mixotrophic Consortia Toward Enhanced Volatile Fatty Acid Production and Carbon Capture","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-05-14 10:06:33","doi":"10.21203/rs.3.rs-9518331/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewersInvited","content":"","date":"2026-05-06T01:23:57+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-05-04T08:47:38+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-05-04T07:23:35+00:00","index":"","fulltext":""},{"type":"submitted","content":"Biotechnology for Biofuels and Bioproducts","date":"2026-04-24T14:09:35+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"biotechnology-for-biofuels-and-bioproducts","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bbio","sideBox":"Learn more about [Biotechnology for Biofuels](http://biotechnologyforbiofuels.biomedcentral.com/)","snPcode":"13068","submissionUrl":"https://submission.nature.com/new-submission/13068/3","title":"Biotechnology for Biofuels and Bioproducts","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"4bda19bd-f03f-478a-b8fd-fb71d19a92fa","owner":[],"postedDate":"May 14th, 2026","published":true,"recentEditorialEvents":[{"type":"reviewersInvited","content":"6","date":"2026-05-06T01:23:57+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-05-04T08:47:38+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-05-04T07:23:35+00:00","index":"","fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-14T10:06:33+00:00","versionOfRecord":[],"versionCreatedAt":"2026-05-14 10:06:33","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9518331","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9518331","identity":"rs-9518331","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}