Continuous Fermentation Research Articles

Inhibition Control by Continuous Extractive Fermentation Enhances De Novo 2-Phenylethanol Production by Yeast.

Current microbial cell factory methods for producing chemicals from renewable resources primarily rely on (fed-)batch production systems, leading to the accumulation of the desired product. Industrially relevant chemicals like 2-phenylethanol (2PE), a flavor and fragrance compound, can exhibit toxicity at low concentrations, inhibit the host activity, and negatively impact titer, rate, and yield. Batch liquid-liquid (L-L) In Situ Product Removal (ISPR) was employed to mitigate inhibition effects, but was not found sufficient for industrial-scale application. Here, we demonstrated that continuous selective L-L ISPR provides the solution for maintaining the productivity of de novo produced 2PE at an industrial pilot scale. A unique bioreactor concept called "Fermentation Accelerated by Separation Technology" (FAST) utilizes hydrostatic pressure differences to separate aqueous- and extractant streams within one unit operation, where both production and product extraction take place - allowing for the control of the concentration of the inhibiting compound. Controlled aqueous 2PE levels (0.43 ± 0.02 g kg-1) and extended production times (>100 h) were obtained and co-inhibiting by-product formation was reduced, resulting in a twofold increase of the final product output of batch L-L ISPR approaches. This study establishes that continuous selective L-L ISPR, enabled by FAST, can be applied for more economically viable production of inhibiting products.

Iron (Magnetite) Nanoparticle-Assisted Dark Fermentation Process for Continuous Hydrogen Production from Rice Straw Hydrolysate

The use of metal nanoparticles (NPs) to enhance hydrogen production in dark fermentation (DF) has become a pioneering field of interest. In particular, iron-based nanoparticles (FeNPs) play a pivotal role in enhancing the activity of metalloenzymes and optimizing feedstock utilization, resulting in improved hydrogen production. This study investigated the effect of FeNPs (magnetite) supplementation at three different concentrations of 50, 100, and 200 ppm in a continuous dark fermenter for the production of hydrogen from rice straw acid hydrolysate. The highest hydrogen production rate of 2.6 ± 0.3 NL H2/L-d was achieved with the addition of 100 ppm of nanoparticles, representing a 53% increase compared to the condition without FeNPs addition. This improvement was driven by a microbial community in which Clostridium was the major dominant genus. In addition, increasing the nanoparticle concentration to 100 ppm resulted in an increase in butyrate concentration to 2.0 ± 0.1 g/L, which is 43% higher than the butyrate concentration without FeNPs. However, when the NP concentration was increased to 200 ppm, the hydrogen production rate decreased to 1.6 ± 0.2 NL H2/L-d. This study can serve as a guideline for future research aimed at evaluating the effects of FeNPs in continuous dark fermentation systems. This work highlights the potential benefits and challenges associated with the use of FeNPs, paving the way for future studies to optimize their application and improve the efficiency of dark fermentation processes.

Control of an integrated first and second-generation continuous alcoholic fermentation process with cell recycling using model predictive control

This study explores controlling a first- and second-generation alcoholic fermentation process with cell recycling, modeled through algebraic differential equations (DAE) with constraints. It evaluates seven controller types: PI, PID, Model Predictive Control (MPC), Neural Network Model Predictive Control (NNMPC), Mixed Model Predictive Control (MMPC), Internal Model Control (IMC), and Linear Quadratic Regulator (LQR). Results show that, while PI and PID controllers can track setpoints, they exhibit slow responses and oscillations. In contrast, MPC controllers respond faster, with both MPC and MMPC demonstrating more robust dynamics, achieving a significant reduction in Integral of the Absolute Error (IAE) across various disturbances, notably an 87% reduction in regulatory scenarios. NNMPC outperforms PI and PID but exhibits overshoot and oscillations, and lacks robustness for servo-type problems. However, MMPC showcases comparable or superior performance to MPC, surpassing other controllers in robustness, especially the IMC and LQR in the regulatory problems. NNMPC maintains a simulation time of under 4 seconds, whereas MPC incurs a computational cost 1,000 times higher. Integrating NNMPC’s optimal increment as an initial estimate in MPC reduces computational time by up to 79.7%. These findings highlight the ANN’s effectiveness in addressing complex control challenges, especially when integrated with MPC. MMPC offers a superior balance between accuracy, robustness, and computational efficiency, serving as a promising solution for reducing computational costs in MPC-type controllers.

Improved reactor design enables productivity of microbial electrosynthesis on par with classical biotechnology.

Microbial electrosynthesis (MES) converts (renewable) electrical energy into CO2-derived chemicals including fuels. To achieve commercial viability of this process, improvements in production rate, energy efficiency, and product titer are imperative. Employing a compact plate reactor with zero gap anode configuration and NiMo-plated reticulated vitreous carbon cathodes substantially improved electrosynthesis rates of methane and acetic acid. Electromethanogenesis rates exceeded 10 L Lcatholyte-1 d-1 using an undefined mixed culture. Continuous thermophilic MES by Thermoanaerobacter kivui produced acetic acid at a rate of up to 3.5 g Lcatholyte-1 h-1 at a titer of 14 g/L, surpassing continuous gas fermentation without biomass retention and on par with glucose fermentation by T. kivui in chemostats. Coulombic efficiencies reached 80 %-90 % and energy efficiencies up to 30 % for acetate and methane production. The performance of this plate-reactor demonstrates that MES can deliver production rates that are competitive with those of established biotechnologies.

Digital model of biochemical reactions in lactic acid bacterial fermentation of simple glucose and biowaste substrates

As concerns about the environmental impacts of biowaste disposal increase, lactic acid bacterial fermentation is becoming increasingly popular. Current academic research is aimed at the process optimization by developing digital bioreactors. The primary focus is to develop a digital model mimicking the biochemical reactions. In the light of this, this paper intended to build a digital model of biochemical reactions during the fermentation process of both glucose and biowaste substrates, including white pasta and organic municipal waste. For this purpose, near-infrared (NIR) and mid-infrared (MIR) spectroscopy techniques were used to collect spectral information during the fermentation process. Next, the samples were analyzed by High Pressure Liquid Chromatography (HPLC) to measure their glucose, fructose, arabinose, xylose, disaccharide, lactic acid, and acetic acid contents. The results showed that learning algorithms trained on MIR spectra accurately estimated the biochemical reactions for both glucose and biowaste substrates. For the glucose substrate, the results showed R-squared of 0.97 and RMSE of 4.69 g/L for glucose, and R-squared of 0.98 and RMSE of 2.74 g/L for lactic acid. In the case of biowaste substrate, estimations included glucose (R-squared = 0.97, RMSE = 4.69 g/L), fructose (R-squared = 0.88, RMSE = 1.47 g/L), arabinose (R-squared = 0.98, RMSE = 0.55 g/L), xylose (R-squared = 0.93, RMSE = 1.11 g/L), disaccharide (R-squared = 0.90, RMSE = 0.55 g/L), total sugar (R-squared = 0.98, RMSE = 3.79 g/L), lactic acid (R-squared = 0.98, RMSE = 2.74 g/L), and acetic acid (R-squared = 0.97, RMSE = 0.36 g/L). Regarding NIR spectral data, the predictive models were accurate when the substrate was glucose, however, they failed to accurately estimate the chemical reactions in the case of biowaste substrate. The findings of this study can be used to fulfill the requirements for a continuous fermentation process with the objective of maximizing lactic acid production.

Dopamine-assisted surface functionalization of saccharide-responsive fibers for the controlled harvesting and continuous fermentation of Saccharomyces cerevisiae

Continuous fermentation processes increasingly emphasized cell recycling, utilization, and renewal. In this study, to improve the sustainability of the immobilized Saccharomyces cerevisiae, the cells were recovered on the surface of the glucose-responsive supports through manipulating the competitive interactions of phenylboric acid groups between glycoproteins on the cells and glucose. Through a dopamine (DA)-assisted deposition approach, 3-acrylamidophenylboronic acid (APBA) was integrated to design the saccharide-sensitive cotton fibers (APBA@PDA-CF). The optimal co-deposition time (5 h) and ratio (1:1) resulted in an impressive immobilization efficiency of 69.64%. Meanwhile, 93.23% of Saccharomyces cerevisiae was captured and harvested on the surface of APBA@PDA-CF with the fermentation course through regulating the competitive interactions of phenylboric acid groups between glycoproteins on the cells and glucose regardless of pH. Notably, a strong interaction between the yeast cells and APBA@PDA-CF was observed at a low glucose concentration (0.1～2 g/L), with reduced sensitivity at high glucose concentrations (>5 g/L). Moreover, the ethanol production and yield could be increased to 25.37 g/L and 42.4% in the fifth-batch fermentation, respectively. Therefore, based on the feasible and versatile co-deposition method, this study not only broadened the application scope of APBA, but also explored the broad prospects of smart materials in cell immobilization, recovery and continuous fermentation.

Continuous Primary Beer Fermentation with Yeast Immobilized in Alginate–Chitosan Microcapsules with a Liquid Core

Continuous Primary Beer Fermentation with Yeast Immobilized in Alginate–Chitosan Microcapsules with a Liquid Core

Investigating drug-gut microbiota interactions: reductive and hydrolytic metabolism of oral glucocorticoids by in vitro artificial gut microbiota

Elucidation of the role of gut microbiota in the metabolism of orally administered drugs may improve therapeutic effectiveness and contribute to the development of personalized medicine. In this study, ten different artificial gut microbiota (AGM), obtained by culturing fecal samples in a continuous fermentation system, were challenged for their metabolizing capacity on a panel of six glucocorticoids selected from either prodrugs or drugs. Data from metabolic stability assays highlighted that, while the hydrolysis-mediated conversion of prodrugs to drugs represented only a minor metabolic pathway, significant differences in the stability of parent compounds and in their conversion rates to multiple reductive metabolites were obtained for the selected drugs. In the latter case, a taxonomic composition-dependent ability to convert parent drugs to metabolites was observed. Indeed, the artificial microbial communities dominated by the genus Bacteroides showed the maximal conversion of parent glucocorticoids to several metabolites. Furthermore, the effect of drugs on AGM was also evaluated through shallow shotgun sequencing and flow cytometry-based total bacterial cell count highlighting that these drugs can affect both the taxonomic composition and growth performances of the human gut microbiota.

Brewery spent grain valorization through fermentation: Targeting biohydrogen, carboxylic acids and methane production

This study investigated three different fermentation approaches to explore the potential for producing biohydrogen, carboxylic acids, and methane from hydrolysates of thermally dilute acid pretreated brewer's spent grains (BSG). Initially, the research focused on maximizing the volumetric hydrogen production rate (HPR) in the continuous dark fermentation (DF) of BSG hydrolysates by varying the hydraulic retention time (HRT). The highest HPR reported to date of 5.9 NL/L-d was achieved at 6 h HRT, with a Clostridium-dominated microbial community. The effect of the operational pH (4, 5, 6, and 7) on the continuous acidogenic fermentation was then investigated. A peak carboxylic acid concentration of 17.3 g CODequiv./L was recorded at pH 6, with an associated volumetric productivity of 900.5 ± 13.1 mg CODequiv./L-h and a degree of acidification of 68.3 %. Lactic acid bacteria such as Limosilactobacillus and Lactobacillus were dominant at pH 4–5, while Weissella, Enterococcus, and Lachnoclostridium appeared at pH 6 and 7. Finally, this study evaluated the biochemical methane potential of the DF broth and the unfermented hydrolysates and found high methane yields of 659 and 517 NmL CH4/g-VSadded, respectively, both within one week. Overall, the results showed that pretreated BSG can be a low-cost feedstock for the production of bioenergy and valuable bio-based chemicals in a circular economy.

Effects of Caffeine Doses on Rumen Fermentation Profile and Nutrient Digestibility Using a Lactating Cow Diet under Continuous Cultures Conditions

Caffeine is a plant secondary metabolite, commonly known for its bioactivity properties. This molecule increases microbial activity during anaerobic digestion. The aim of this study was to determine the effects of caffeine doses on the rumen fermentation profile and nutrient digestibility when continuous culture fermenters were fed a lactating cow’s diet. We hypothesize that adding caffeine doses into continuous culture fermenters with a rumen fluid inoculum will not affect anaerobic fermentation or nutrient utilization. Fermenters were fed twice a day (at 0800 and 2000 h) with an experimental diet of high-producing dairy cows (53.55 g/DM day; Forage:Concentrate ratio, F:C of 40:60). Four levels of caffeine (0 ppm, 50 ppm, 100 ppm, and 150 ppm) were added to the diets as a treatment. The experiment was arranged in a randomized complete block design. Two blocks of four fermenters were run in two replicated periods of ten days. Statistical analyses were conducted in SAS version 9.4 for Windows (SAS Institute Inc., Cary, NC) using the GLIMMIX procedure. The addition of caffeine at a 50 ppm dose on continuous culture fermentation decreased the protozoal counts (Diplodinium spp.) (p = 0.03) and ammonia concentrations (p < 0.05). A treatment of 50 ppm of caffeine increased the DM, OM, and starch digestibility (p < 0.05). When caffeine doses increased further than 50 ppm, the OM, DM, and starch digestibility decreased linearly (p = 0.01). The total volatile fatty acids and fatty acid proportions were unaffected. However, the acetate-to-propionate ratio (A:P) tended to decrease linearly among treatments (p = 0.07). The means of pH measurements and maximum pH had a significantly linear decrease effect (p < 0.01). Caffeine may represent a potential rumen fermentation modifier for use in lactating cow diets.

Immobilization of Saccharomyces cerevisiae on polyhydroxyalkanoate/konjac glucan nanofiber membranes: Characterization, immobilization efficiency and cellular activity

Yeast immobilization systems can recoup yeast losses in continuous batch fermentation and relieve substrate or product inhibition. We report the use of solution blow spinning process to efficiently prepare polyhydroxyalkanoate (PHB) /konjac glucomannan (KGM) nanofiber membranes as immobilization carriers for Saccharomyces cerevisiae. The prepared PHB/KGM nanofiber membranes had fiber diameters similar to the scale of yeast cells. Incorporating KGM significantly enhanced the porosity (from 87.21 % to 91.74 %), crystallinity, and hydrophilicity (reducing water contact angle from 135.8° to 110.1°), while increasing the specific surface area (from 10.24 to 17.79 m2/g) of pure PHB nanofiber membranes. Thermal stability was maintained (degradation temperatures above 250 °C). These changes enhanced the force between the nanofiber membranes and the cells and facilitated their autoimmobilization on the nanofiber membranes. The highest yeast immobilization efficiency of 87.93 % could be achieved at a KGM addition ratio of 400:2. Yeast showed no loss of cellular activity on the immobilized carriers of natural materials and maintained or even improved fermentation kinetics during at least three consecutive alcoholic fermentations These findings indicate that PHB/KGM nanofiber membranes can serve as effective carriers for yeast immobilization, promoting the sustainable production of fermented foods.

Treatment of dual-flow continuous culture fermenters with an organic essential oil product minimally influenced prokaryotic microbiome

Treatment of dual-flow continuous culture fermenters with an organic essential oil product minimally influenced prokaryotic microbiome

Optimizing Lactic Acid Production Through Dynamic Simulation in Repeated-Batch Fermentation System

In this study, we explored the lactic acid production from molasses through a repeated-batch fermentation process using dynamic simulation. Our investigation revealed that sugar concentrations ranging from 50 to 150 g/l significantly impacted the dynamic profiles of sugar consumption concentration, microbial Enterococcus faecalis RKY1 growth, and lactic acid production. Through dynamic simulation, we identified the optimal inlet sugar concentration as 68 g/l (equivalent to 130 g/l molasses), resulting in a lactic acid average productivity of 3.9 g/l h (3-repeated batch). The simulation demonstrated the viability of a sustainable lactic acid production process using Enterococcus faecalis RKY1, enabling the repeated-batch fermentation process for multiple cycles. Comparative analysis with continuous fermentation indicated that repeated-batch fermentation offers a superior alternative, characterized by higher substrate conversion to lactic acid. This study contributes valuable insights into optimizing lactic acid production processes, emphasizing the efficiency and sustainability of the repeated-batch fermentation approach.

Enzyme induced polymer degradation as a pathway to create microbial habitats for biomanufacturing intensification

Polymer hydrogels are instrumental in cell immobilization for biomanufacturing, yet traditional hydrogel bioreactors often compromise cell viability, function, and mass transfer. This results in uncontrolled microbial proliferation and significant cell leakage, thereby impeding the continuous production of high-value products. To address these challenges, we introduce an innovative liquid microcavity technique employing a blend of Gelatin-methacryloyl (Gel-MA) and Pluronic F127diacrylate (F127-DA) for precision bioprinting, followed by selective enzymatic digestion of gelatin to create microbial habitats. Through tailoring of the hydrogel’s pore structure and integration of diverse networks, our approach facilitates controlled nutrient transfer, efficient waste removal, and stable mechanical support, all while maintaining a leak-free suspension culture of microorganisms. We validated the effectiveness of our method by fabricating yeast-infused Three-dimensional (3D) lattice constructs, successfully achieving continuous fermentation of glucose into ethanol in a flow reactor with minimal efficiency reduction (≥90 % yield) over five cycles. This research not only confronts the spatial limitations inherent in hydrogel-based systems but also demonstrates a foundational potential for revolutionary change in biomanufacturing practices.

Adaptive laboratory evolution in a novel parallel shaken pH-auxostat.

Adaptive laboratory evolution (ALE) is a widely used microbial strain development and optimization method. ALE experiments, to select for faster-growing strains, are commonly performed as serial batch cultivations in shake flasks, serum bottles, or microtiter plates or as continuous cultivations in bioreactors on a laboratory scale. To combine the advantages of higher throughput in parallel shaken cultures with continuous fermentations for conducting ALE experiments, a new Continuous parallel shaken pH-auxostat (CPA) was developed. The CPA consists of six autonomous parallel shaken cylindrical reactors, equipped with real-time pH control of the culture medium. The noninvasive pH measurement and control are realized by biocompatible pH sensor spots and a programmable pump module, to adjust the dilution rate of fresh medium for each reactor separately. Two different strains of the methylotrophic yeast Ogataea polymorpha were used as microbial model systems for parallel chemostat and pH-auxostat cultivations. During cultivation, the medium is acidified by the microbial activity of the yeast. For pH-auxostat cultivations, the growth-dependent acidification triggers the addition of fresh feed medium into the reactors, leading to a pH increase and thereby to the control of the pH to a predetermined set value. By controlling the pH to a predetermined set value, the dilution rate of the continuous cultivation is adjusted to values close to the washout point, in the range of the maximum specific growth rate of the yeast. The pH control was optimized by conducting a step-response experiment and obtaining tuned PI controller parameters by the Chien-Hrones-Reswick (CHR) PID tuning method. Two pH-auxostat cultivations were performed with two different O. polymorpha strains at high dilution rates for up to 18 days. As a result, up to 4.8-fold faster-growing strains were selected. The increased specific maximum growth rates of the selected strains were confirmed in subsequent batch cultivations.

Resilience towards organic load and activated sludge variations in co-fermentation for carboxylic acid production

Two perturbations were investigated in acidogenic co-fermentation of waste activated sludge (WAS) and food waste in continuous mesophilic fermenters: increasing the organic loading rate (OLR) and changing the WAS. A control reactor maintained an OLR of 11 gVS/(L·d), while a test reactor had a prolonged OLR change to 18 gVS/(L·d). For each OLR, two WAS were studied. The change in OLR led to differentiated fermentation product profile without compromising the fermentation yields (∼300 mgCOD/gVS). At 11 gVS/(L·d), the product profile was dominated by acetic, butyric, and propionic acids while at 18 gVS/(L·d) it shifted to acetic acid, ethanol, and caproic acid. Reverting the OLR also reverted the fermentation profile. The biomass immigration with the WAS changed the fermentation microbial structure and introduced acetic acid-consuming methanogens, which growth was only delayed by the OLR increase. Microbial monitoring and post-fermentation tests can be used for early detection of acetic acid-consuming events.

A comprehensive model for estimating the shelf life of young radish (Raphanus sativus L.) kimchi packaged in partially breathable film pouches

Continuous fermentation by lactic acid bacteria (LAB) causes packaging bursting, presenting a challenge in the commercialization of kimchi by reducing its shelf life. This study explored the use of partially breathable film pouches (PBFP) as a packaging solution for young radish kimchi (YRK). Furthermore, the shelf life of YRK was estimated through multivariate accelerated shelf life testing (MASLT). Packaged YRK samples were stored for 60 d at different temperatures (0, 5, 10, and 15 °C). The gas permeability of the PBFP and LAB metabolism controlled the volume and headspace gas composition of the packaged YRK samples during storage. The PBFP did not negatively impact YRK quality. Principal component (PC) analysis successfully reduced the physicochemical and microbial characteristics of YRK to a single variable. The exponential plateau model better described the variation in the time-related PC score than the zero- and first-order models. The P value within 15% of the comprehensive model, incorporating exponential and Arrhenius models, indicated its adequacy for predicting the shelf life of YRK. This study offers both insights into packaging technology and a predictive model, contributing to the improvement of the distribution management system for fermented vegetables.

Sustainable menaquinone-7 production through continuous fermentation in biofilm bioreactors.

Menaquinone-7 (MK-7), a vital vitamin with numerous health benefits, is synthesized and secreted extracellularly by the formation of biofilm, dominantly in Bacillus strains. Our team developed an innovative biofilm reactor utilizing Bacillus subtilis natto cells to foster biofilm growth on plastic composite supports to produce MK-7. Continuous fermentation in biofilm reactors offers a promising strategy for achieving sustainable and efficient production of Menaquinone-7 (MK-7). Unlike conventional batch fermentation, continuous biofilm reactors maintain a steady state of operation, which reduces resource consumption and waste generation, contributing to sustainability. By optimizing fermentation conditions, MK-7 production was significantly enhanced in this study, demonstrating the potential for sustainable industrial-scale production. To determine the optimal operational parameters, various dilution rates were tested. These rates were selected based on their potential to enhance nutrient supply and biofilm stability, thereby improving MK-7 production. By carefully considering the fermentation conditions and systematically varying the dilution rates, MK-7 production was significantly enhanced during continuous fermentation. The MK-7 productivity was found to increase from 0.12mg/L/h to 0.33mg/L/h with a dilution rate increment from 0.007 to 0.042h-1). This range was chosen to explore the impact of various nutrient supply rates on MK-7 production and to identify the optimal conditions for maximizing productivity. However, a further increase in the dilution rate to 0.084h-1 led to reduced productivity at approximately 0.16mg/L/h, likely due to insufficient retention time for effective biofilm formation. Consequently, a dilution rate of 0.042h-1 exhibited the highest productivity of 0.33mg/L/h, outperforming all investigated dilution rates and demonstrating the critical balance between nutrient supply and retention time in continuous fermentation. These findings validate the feasibility of operating continuous fermentation at a 0.084h-1 dilution rate, corresponding to a 48h retention time, to achieve the highest MK-7 productivity compared to conventional batch fermentation. The significant advancements achieved in enhancing Menaquinone-7 (MK-7) productivity through continuous fermentation at optimal dilution rates in the present work indicate promising prospects for even greater efficiency and sustainability in MK-7 production through future developments.

Simultaneous design of fermentation and microbe

AbstractConstraint‐based optimization of microbial strains and model‐based bioprocess design have been used extensively to enhance yields in biotechnological processes. However, strain and process optimization are usually carried out in sequential steps, causing underperformance of the biotechnological process when scaling up to industrial fermentation conditions. Herein, we propose the optimization formulation SimulKnock that combines the optimization of a fermentation process with metabolic network design in a bilevel optimization program. The upper level maximizes space‐time yield and includes mass balances of a continuous fermentation, while the lower level is based on flux balance analysis. SimulKnock predicts optimal gene deletions and finds the optimal trade‐off between growth rate and product yield. Results of a case study with a genome‐scale metabolic model of Escherichia coli indicate higher space‐time yields than a sequential approach using OptKnock for almost all target products considered. By leveraging SimulKnock, we reduce the gap between strain and process optimization.

Enhancing Biohydrogen Production: The Role of Iron-Based Nanoparticles in Continuous Lactate-Driven Dark Fermentation of Powdered Cheese Whey

Here, a comprehensive investigation was conducted under various operational strategies aimed at enhancing biohydrogen production via dark fermentation, with a specific focus on the lactate metabolic pathway, using powdered cheese whey as a substrate. Initially, a batch configuration was tested to determine both the maximum hydrogen yield (100.2 ± 4.2 NmL H2/g CODfed) and the substrate (total carbohydrates) consumption efficiency (94.4 ± 0.8%). Subsequently, a transition to continuous operation was made by testing five different operational phases: control (I), incorporation of an inert support medium for biomass fixation (II), addition of carbon-coated, zero-valent iron nanoparticles (CC-nZVI NPs) at 100 mg/L (III), and supplementation of Fe2O3 nanoparticles at concentrations of 100 mg/L (IV) and 300 mg/L (V). The results emphasized the critical role of the support medium in stabilizing the continuous system. On the other hand, a remarkable increase of 10% in hydrogen productivity was observed with the addition of Fe2O3 NPs (300 mg/L). The analysis of the organic acids’ composition unveiled a positive correlation between high butyrate concentrations and improved volumetric hydrogen production rates (25 L H2/L-d). Moreover, the presence of iron-based NPs effectively regulated the lactate concentration, maintaining it at low levels. Further exploration of the bacterial community dynamics revealed a mutually beneficial interaction between lactic acid bacteria (LAB) and hydrogen-producing bacteria (HPB) throughout the experimental process, with Prevotella, Clostridium, and Lactobacillus emerging as the predominant genera. In conclusion, this study highlighted the promising potential of nanoparticle addition as a tool for boosting biohydrogen productivity via lactate-driven dark fermentation.