Biohydrogen production from dark fermentation of cheese whey: Influence of pH

Hydrogen production from cheese whey through dark fermentation was investigated in this study in order to systematically analyse the effects of the operating pH. The effluents from pecorino cheese and mozzarella cheese production were the substrates used for the fermentation tests. Either CW only or a mixture of CW and heat-shocked activated sludge were used in mesophilic pH-controlled batch fermentation experiments. The results indicated that hydrogen production was strongly affected by multiple factors including the substrate characteristics, the addition of an inoculum as well as the pH. The process variables were found to affect to a varying extent numerous interrelated aspects of the fermentation process, including the hydrogen production potential, the type of fermentation pathways, as well as the process kinetics. The fermentation products varied largely with the operating conditions and mirrored the H₂ yield. Significant fermentative biohydrogen production was attained at pHs of 6.5–7.5, with the best performance in terms of H₂ generation potential (171.3 NL H₂/kg TOC) being observed for CW from mozzarella cheese production, at a pH value of 6.0 with the heat-shocked inoculum.     

Catalytic biohydrogen production from organic waste materials: A literature review and bibliometric analysis

Global population growth and accelerated urbanisation have resulted in massive amounts of fossil fuel use and waste production. Because of its high energy content, pure nature, and fuel quality, hydrogen fuel is a viable option to fossil fuels. Biohydrogen from agricultural waste, in particular, piques concern because it generates hydrogen while still disposing of waste. This review conducted a bibliometric analysis of biohydrogen production from organic waste to trace the research trends and hotspots based on the literature in the Web of Science (WOS) database from 1970 to 2020. The present review article also focuses on highlighting various processes for converting organic waste into hydrogen, raw materials for biohydrogen production, and catalysts that could distil the latest perceptions that could shed light on a route advancing for successful catalyst design. It also seems that some intentions have been paid on studying waste materials such as pure polysaccharides, disaccharides, and monosaccharides. Among all the catalysts used, non-noble and low-cost active metals over reduced graphene oxide (rGO) support can significantly affect the activity of fermentative hydrogen production from organic waste materials. However, researches focusing on developing anaerobic membrane bioreactors for these technologies are still needed.     

Photofermentative production of hydrogen from organic acids by the purple sulfur bacterium Thiocapsa roseopersicina

A mutant strain of the anaerobic purple sulfur bacterium Thiocapsa roseopersicina, containing only nitrogenase as a functionally active enzyme for H₂ generation was utilized to study the production of H₂ from organic acids (acetate, pyruvate and succinate). Two types of potential substrates for H₂ production, thiosulfate and salts of various organic acids, were compared under photoheterotrophic growth conditions. Thiosulfate proved to be the preferred electron donor for T. roseopersicina; the consumption of organic acids became pronounced only following depletion of the thiosulfate supply. The system is suitable for the generation of H₂ from effluents of heterotrophic dark fermentation processes or waste streams rich in inorganic reduced sulfur compounds and/or simple organic acids.     

Optimization of biohydrogen production by the novel psychrophilic strain N92 collected from the Antarctica

In this study, the response surface methodology (RSM) with central composite design (CCD) was employed to improve the hydrogen production by the psychrophilic N92 strain (EU636058) isolated from Antarctica, which is closely related to Pseudorhodobacter sp. (KT163920). The influence of operational conditions such as temperature (4.7–55.2 °C), initial pH (3.44–10.16), and initial glucose concentration (4.7–55.23 g/dm³), as well as the initial concentrations of (NH₄)₂SO₄ (0.05–3.98 g/dm³), FeSO₄ (0.02–1.33 g/dm³) and NaHCO₃ (0.02–3.95 g/dm³) was evaluated. The linear effect of glucose concentration, along with the quadratic effect of all the six factors were the most significant terms affecting the biohydrogen yield by N92 strain. The optimum conditions for the maximum hydrogen yield of 1.7 mol H₂/mol glucose were initial pH of 6.86, glucose concentration of 28.4 g/dm³, temperature 29 °C and initial concentration of (NH₄)₂SO₄, FeSO₄ and NaHCO₃ of 0.53, 1.55 and 1.64 g/dm³ respectively. Analysis of the metabolites produced under the optimum conditions showed that the most abundant were acetic acid (0.8 g/dm³), butyric acid (0.7 g/dm³) and ethanol (2.1 g/dm³). We suggest that the bioprocess established in this study using the strain N92 could be an alternative for hydrogen production with the advantages of constituting low energy costs in fermentation.     

Optimization of key factors affecting hydrogen production from food waste by anaerobic mixed cultures

Key factors (inoculums concentration, substrate concentration and citrate buffer concentration) affecting hydrogen yield (HY) and specific hydrogen production rate (SHPR) from food waste in batch fermentation by anaerobic mixed cultures were optimized using Response Surface Methodology with Central Composite Design. The experiments were conducted in 120 ml serum bottles with a working volume of 70 mL. Under the optimal condition of 2.30 g-VSS/L of inoculums concentration, 2.54 g-VS/L of substrate concentration, and 0.11 M of citrate buffer concentration, the predicted maximum HY and SHPR of 104.79 mL H₂/g-VS_added and 16.90 mL H₂/g-VSS.h, respectively, were obtained. Concentrations of inoculums, substrate and citrate buffer all had an individual effect on HY and SHPR (P < 0.05). The substrate concentration and citrate buffer concentration had the greatest interactive effect on SHPR (P = 0.0075) while their effects on HY (P = 0.0131) were profound. These results were reproduced in confirmation experiments under optimal conditions and generated an HY of 104.58 mL H₂/g-VS_added and an SHPR of 16.86 mL H₂/g-VSS.h. This was only 0.20% and 0.24%, respectively, different from the predicted values. Microbial community analysis by PCR-DGGE indicated that Clostridium was the pre-dominant hydrogen producer at the optimum and worst conditions. The presence of Lactobacillus sp. and Enterococcus sp. might be responsible for the low HY and SHPR at the worst condition.     

Simultaneous analysis of carbohydrates and volatile fatty acids by HPLC for monitoring fermentative biohydrogen production

The production of biohydrogen through anaerobic fermentation has received increasingly attention and has great potential as an alternative process for clean fuel production in the future. The monitoring of the stages of anaerobic fermentation provides relevant information about the bioprocess. The objective of this study is to propose a novel methodology for simultaneous analysis of sucrose, glucose, fructose and volatile fatty acids (VFAs), such as, acetic, propionic, isobutyric and butyric during anaerobic fermentation by using high-performance liquid chromatography (HPLC). The following chromatographic conditions were optimized: column Aminex HPX-87H, mobile phase consisting of H₂SO₄ 0.005 mol/L, flow rate of 1.0 mL/min and temperature of 55 °C. Sucrose, glucose and fructose were analyzed by refractive index detector (RI) while acetic, propionic, isobutyric and butyric acids were analyzed by ultraviolet (UV) detection at 210 nm. Some analytical parameters of validation, such as, linearity, selectivity, repeatability, intermediate precision, limit of detection and quantification, accuracy and robustness were evaluated. The proposed methodology was successfully applied in the determination of substrates and metabolites during different stages of biohydrogen production.     

Inhibition by the ionic strength of hydrogen production from the organic fraction of municipal solid waste

Composition of the Organic Fraction of Municipal Solid Waste (OFMSW) in organic compounds and inorganic ions is highly variable and might impact the microbial activity in dark fermentation processes. In this study, the effect of the total amount of inorganic ions on fermentative hydrogen production was investigated. Batch experiments were carried out at pH 6 and under a temperature of 37 °C. A freshly reconstituted organic fraction of municipal solid waste (OFMSW) was used as model substrate. At low concentrations in ammonium or chloride ions (2.9–5.1 g L⁻¹, respectively), the hydrogen yield reached a maximum of 40.8 ± 0.5. mLH₂.gVS⁻¹ and 25.1 ± 5.6 mLH₂.gVS⁻¹. In contrast, at high total ionic concentrations of ammonium and chloride (11.1–35.5 g L⁻¹ respectively), a strong inhibition of the fermentative microbial activity and more particularly hydrogen production, was observed. When considering the ionic strength of each ion, the effects of ammonia, chloride or a mixture of different ions (Na⁺, K⁺, H⁺, Li⁺, NH₄⁺, Mn²⁺, NH₄⁺, Mg²⁺, Cl⁻, PO₄³⁻, Br⁻, I⁻, SO₄²⁻) showed very similar inhibitory trends regardless the type of ion or the composition of the ionic mixture. A threshold inhibitory value of the ionic strength was estimated at 0.75 ± 0.13 M with a substantial impact on the fermentative activity from 0.81 ± 0.12 M, with hydrogen yields of 18.1 ± 3.3 and 6.2 ± 4.1 mLH₂.gVS⁻¹, respectively. Microbial community composition was also significantly impacted with a specific decrease in relative abundance of hydrogen-producing bacteria from the genus Clostridium sp. at high ionic strength.     

Predictive and explicative models of fermentative hydrogen production from solid organic waste: Role of butyrate and lactate pathways

Solid organic waste represents an abundant, cheap, and available source of biodegradable substrates not yet exploited to produce biohydrogen by dark fermentation. The impact of the composition of solid organic waste on microbial metabolic pathways and subsequently on biohydrogen production, has not been clearly elucidated. The aim of this study is to determine the compositional features of different substrates that influence bioH2 production. For this, we measured Biological hydrogen potentials (BHP) on 26 different substrates and performed a multivariate statistical analysis of the experimental data using a partial least square regression. The results showed that the BHP values correlated well with the initial carbohydrate content measured after mild hydrolysis. A predictive model explaining more than 89% of the experimental variability was then built to predict the maximal biohydrogen yield with a high accuracy and for a large spectrum of organic waste. An explicative model showed that only carbohydrates, butyrate and lactate concentrations were significant variables explaining more than 98% of biohydrogen yield variability. Interestingly, an interaction term between carbohydrates and lactate concentrations was required to explain microbial pathways producing hydrogen.     

Influence of pH, temperature and volatile fatty acids on hydrogen production by acidogenic fermentation

The aim of this work was to study the influence of pH and temperature on acidogenic fermentation and bio-hydrogen production. A centered factorial design was generated with respect to pH (4-6 units) and temperature (26-40 °C), and these conditions were used in batch experiments. Biomass cultivation was conducted in a sequential batch reactor (SBR). A mixed-acidogenic culture enriched from activated sludge and fed with a 9 g/l glucose solution was used in the experiments. At low pH values, hydrogen production was favored when the temperatures were low, a result contrary to those described in literature. Working at higher temperatures reduced the length of the lag phase. Additionally, the hydrogen production rate was increased at these temperatures. These opposite trends indicated that an inhibition effect occurred during the experiment. Hydrogen production was studied by using a response surface methodology, being the highest hydrogen production occurred at pH 5.4 and 26 °C. Regarding to the relationship between the hydrogen and acid production, the hydrogen produced per unit of acetate produced increased as the pH increased. On the other hand, hydrogen produced from other acids was constant and similar to theoretical yields. These values of hydrogen produced per unit of acid produced allowed to estimate the experimental hydrogen production. This result indicated that pH was the most important factor in acidogenic fermentation.     

Biohydrogen production from organic solid waste in a sequencing batch reactor: An optimization of the hydraulic and solids retention time

Hydrogen production from organic solids waste was evaluated using a sequencing batch reactor (SBR) under mesophilic conditions, to investigate the effect of the hydraulic retention time (HRT) and solids retention time (SRT) on hydrogen production. The examined HRT and SRT values were from 4.6 to 27 h and 17–102 h, respectively. The results showed high hydrogen production rates (1.86 LH₂/L·d) and a yield of 127.26 mLH₂/gCOD_removed for an SRT of 60 h and an HRT of 16 h. The highest chemical oxygen demand (COD) removal (38.6 ± 6.9%) was also obtained under those conditions. The highest substrate hydrolysis percentage (73.0 ± 11.4%) was obtained at an HRT of 16 h and an SRT of 102 h. A short SRT of 20 h affected hydrogen production, which decreased up to 90%. With an SRT of 20 h and an HRT of 16 h, acetic acid-like fatty acids were mainly obtained. In experiments with a long SRT (60 h), the obtained fatty acid was butyrate. The conversion efficiencies for converting particulate material into fatty acids were 51–47% using a long SRT; a short HRT resulted in percentages of 37–40%. A 3D surface analysis was performed using the maximum hydrogen yield conditions as the central point, showing that the optimal hydrogen production can be obtained with an HRT of 16 h and an SRT of 55 h. Microbial analysis showed the predominance of the Olsenella genus at an HRT< 8 h and the presence of Clostridium at an HRT of 16 h. The HRT is the main parameter leading the community composition in the process.     

Optimization of media composition for hydrogen gas production from hydrolyzed wheat starch by dark fermentation

Effects of N/C, P/C and Fe(II)/C ratios in fermentation medium on biohydrogen production by dark fermentation of acid-hydrolyzed wheat starch was investigated. The powdered wheat was autoclaved at pH = 3 and 90 °C for 15 min and the resulting sugar solution was fermented after external addition of N, P and Fe(II) to overcome nutrient limitations. Box–Wilson statistical experiment design was used by considering the N/C (0–0.05, w w⁻¹), P/C (0–0.02) and Fe(II)/C (0–0.03) ratios as the independent variables while the hydrogen yield and specific hydrogen production rate (SHPR) were the objective functions to be optimized. A quadratic response function was used to correlate the response functions with the independent variables. Low levels of the variables (N/C < 0.02, P/C < 0.01, Fe(II)/C < 0.01) resulted in low hydrogen yield and SHPR due to nutrient limitations and high levels of nutrients caused inhibitions. The optimum conditions yielding the maximum hydrogen yield (Y = 2.84 mol H₂ mol⁻¹ glucose) were N/C = 0.02, P/C = 0.008 and Fe(II)/C = 0.015. The maximum SHPR (96 mL H₂ g⁻¹ biomass h⁻¹) was obtained at N/C = 0.025, P/C = 0.008 and Fe(II)/C = 0.015 (w w⁻¹).     

Optimization of particle number,substrate concentration and temperature of batch immobilized reactor system for biohydrogen production by dark fermentation

Immobilized cell bioreactor was operated in batch mode for biohydrogen generation by dark fermentation from acid hydrolyzed waste wheat powder. It was aimed to optimize the fermentation conditions with the purpose of obtaining the highest hydrogen yield (Y_H2) and production rate (HPR) by applying Box–Wilson statistical experimental design method. Particle number (PN = 120–240; X₁), initial total sugar concentration (TS₀ = 10–30 g/l; X₂) and fermentation temperature (T = 35–55 °C; X₃) were selected as independent variables. Polyester fibers with particle diameter “Dp” = 0.5 cm were used as support material to immobilize microorganisms with heat-pretreated sludge. Quadratic equations for production yield and rate were developed by using experimental results. The maximum Y_H2 (3.21 mol H₂/mol glucose) and HPR (73.3 ml H₂/h) were predicted at the optimum conditions of PN = 240, TS₀ = 10 g/l and T = 44.9 °C. Also, analysis of variance, as well as sum of ranking difference test results demonstrated that fitting models were statistically significant.     

Optimization and modelling of dark fermentative hydrogen production from cheese whey by Enterobacter aerogenes 2822

In India, annually about 3.3–5 million tons of cheese whey is produced which may causes serious problems for the environment if left untreated. In this study, pretreated cheese whey was utilized to produce hydrogen via dark fermentation by Enterobacter aerogenes 2822 cells in 2 L double walled cylindrical bioreactor having working volume of 1.5 L. Effect of change in total carbohydrate concentration in cheese whey (CW_TC, 20–45 g L^?1), temperature (T, 25–37 °C) and pH (5.5–7.5) was investigated on volumetric hydrogen production rate (VHPR) using Box Behnken design (BBD). Experimental VHPR of 24.7 mL L^?1 h^?1 was attained at an optimum concentration of 32.5 g L^?1 CW_TC, 31 °C T and 6.5 pH, which was in good correlation with predicted rate of 23.2 mL L^?1 h^?1. Mathematical models based on Monod and logistic equations were developed to describe the kinetics of substrate consumption and growth profile of E. aerogenes 2822 under optimum conditions. While for the modelling of fermentative hydrogen production in batch mode, Modified Gompertz equation and Leudeking-Piret models were used which gave proper simulated fitting. These results will add significant values to cheese whey by converting it into a clean form of bioenergy.     

Dark fermentative hydrogen production from simple sugars and various wastewaters by a newly isolated Thermoanaerobacterium thermosaccharolyticum SP-H2

The hydrogen-producing bacterium SP-H2 was isolated from a thermophilic acidogenic reactor inoculated with municipal sewage sludge and processing a carbohydrate-rich simulated food waste. Based on the 16S rRNA gene sequence, the bacterium was identified as Thermoanaerobacterium thermosaccharolyticum. The maximum growth rate was observed at 55–60 °C and pH 7.5. The H₂-producing activity of the bacterium was studied using mono-, di- and tri-saccharides related to both hexoses (maltose, glucose, mannose, fructose, lactose, galactose, sucrose, raffinose, cellobiose) and pentoses (xylose and arabinose), as well as using real wastewaters (cheese whey, confectionery wastewater, sugar-beet processing wastewater). The highest H₂ yield was observed during dark fermentation (DF) of maltose (1.91 mol H₂/mol hexose or 77.8 mmol H₂/L). The maximum H₂ production rate was observed during DF of xylose (13.3 ml H₂/g COD/h) and cellobiose (2.47 mmol H₂/L/h). The main soluble metabolite products were acetate, ethanol and butyrate. The acetate concentration had a statistically significant positive correlation with the H₂ content in biogas and the specific H₂ yield. Based on the results of the correlation analysis, it was tentatively assumed that in the formic acid (mixed-acid) type fermentation, the rate of H₂ production was higher than in the butyric acid type fermentation. With regard to real wastewater, cheese whey and confectionery wastewater were distinguished by a higher H₂ yield (152 ml H₂/g COD) and H₂ production rate (0.57 mmol H₂/L/h), respectively. The highest concentrations of confectionery wastewater and cheese whey, at which the DF process took place, were 5915 and 7311 mg COD/L, respectively. At the same time, SP-H2 dominated in the microbial community, despite the presence of indigenous microorganisms in wastewater. Thus, T. thermosaccharolyticum SP-H2 is a promising strain for DF of carbohydrate-rich unsterile wastewater under thermophilic conditions.     

Effect of pre-treatment and hydraulic retention time on biohydrogen production from organic wastes

This study investigated the effect of pre-treatment and hydraulic retention time (HRT) on biohydrogen production from organic wastes. Various pre-treatments including thermal, base, acid, ultrasonication, and hydrogen peroxide were applied alone or in combination to enhance biohydrogen production from potato and bean wastewater in batch tests. All the pre-treated samples showed higher hydrogen production than the control tests. Hydrogen peroxide pre-treatment achieved the best results of 939.7 and 470 mL for potato and bean wastewater, respectively. Continuous biohydrogen production from sucrose, potato and bean wastewater was significantly influenced by reducing the HRT as 24, 18 and 12 h. Sucrose and potato showed similar behavior, where the hydrogen production rate (HPR) increased with decreasing the HRT. Optimum hydrogen yield results of 320 mL-H₂/g-VS (sucrose) and 150 mL-H₂/g-VS (potato) were achieved at HRT of 18 h. Bean wastewater showed optimum HPR of 0.65 L/L.d with hydrogen yield of 80 mL-H₂/g-VS at 24 h HRT.     

Combined effects of temperature and pH on biohydrogen production by anaerobic digested sludge

A full factorial design was conducted to investigate the combined effects of temperatures and initial pH on fermentative hydrogen production by mixed cultures in batch tests. The experimental results showed that the modified Logistic model can be used to describe the progress of cumulative hydrogen production in the batch tests of this study. The modified Ratkowsky model can be used to describe the combined effects of the temperatures and initial pH on the substrate degradation efficiency, hydrogen yield and average hydrogen production rate. The temperatures and initial pH had interactive impact on fermentative hydrogen production. The maximum substrate degradation efficiency, the maximum hydrogen yield and the maximum average hydrogen production rate was predicted at the temperature of 37.8 °C and the initial pH of 7.1, 37.4 °C and 6.9, and 38.2 °C and 7.2, respectively. In general, the optimal temperature for the fermentative hydrogen production was around 37.8 °C and the optimal initial pH for the fermentative hydrogen production was around 7.1.     

Use of organic waste for biohydrogen production and volatile fatty acids via dark fermentation and further processing to methane

Despite the suitability of organic waste for dark fermentation (DF), anaerobic digestion (AD) counteracts its large-scale use for biohydrogen production. Therefore, 12 types of organic waste obtained from sugar, textile, food, and milk industries are investigated in batch single-stage AD and compared energetically to batch two-stage DF with subsequent AD. From the viewpoint of DF, a parametric study of mesophilic and thermophilic conditions, different substrate concentrations, and mixed cultures, i.e., granular and digested sludge, is conducted. Hydrogen yields of 90–160 L_N/kg_oDM (mean) and maximum yields of 199–291 L_N/kg_oDM are achieved with starchy and sugary wastes. Concentrations of volatile fatty acids of 9.7–14.5 g/L (mean) show the possible material uses. Thermophilic conditions are more suitable than mesophilic ones. Furthermore granular sludge is applicable for DF. The energetic comparison of the procedures demonstrates a method for assessing the applicability of waste and allows preliminary economic estimations.     

Energy analysis of the system of two-stage anaerobic processing of liquid organic waste with production of hydrogen- and methane-containing biogases

In recent years, public attention has been increasingly attracted to solving two inextricably linked problems - preventing the depletion of natural resources and protecting the environment from anthropogenic pollution. The annual consumption of livestock waste for biogas production is about 240 thousand m³ per year, which is 0.17% of the total manure produced at Russian agricultural enterprises. At present, the actual use of organic waste potentially suitable for biogas production is 2–3 orders of magnitude lower than the existing potential for organic waste. Currently, hydrogen energy is gaining immense popularity in the world due to the problem of depletion of non-renewable energy sources - hydrocarbons, and environmental pollution caused by their increasing consumption. Of particular interest is the dark process of producing hydrogen-containing biogas in the processing of organic waste under anaerobic conditions, which allows you to take advantage of both energy production and solving the problem of organic waste disposal. An energy analysis of a two-stage anaerobic liquid organic waste processing system with the production of hydrogen- and methane-containing biogases based on experimental data obtained in a laboratory plant with increased volume reactors was performed. The energy efficiency of the system is in the range of 1.91–2.74. Maximum energy efficiency was observed with a hydraulic retention time of 2.5 days in a dark fermentation reactor. The cost of electricity to produce 1 m³ of hydrogen was 1.093 kW·h with a hydraulic retention time of 2.5 days in the dark fermentation reactor. When the hydraulic retention time in the dark fermentation reactor was 1 day, the specific (in ratio to the processing rate of organic waste) energy costs to produce of 1 m³ of hydrogen were minimal in the considered hrt range, and amounted to 26 (W/m³ of hydrogen)/(m³ of waste/day). Thus, the system of two-stage anaerobic processing of liquid organic waste to produce hydrogen and methane-containing biogases is an energy-efficient way to both produce hydrogen and process organic waste.     

Influence of the initial proportion of carbohydrates,proteins, and lipids on biohydrogen production by dark fermentation: A multi-response optimization approach

The present study investigated the effect of the initial proportions of carbohydrates, proteins and lipids within the substrate on the resulting biohydrogen productivity by dark fermentation. Organic matter removal and the related metabolic by-products generated during the process were also assessed. The results obtained showed that initial substrate composition in terms of carbohydrates, proteins and lipids has a significant effect on maximal potential hydrogen production (H_max), hydrogen production rate (R_max), hydrogen yield (Y_H2) and metabolites distribution. Tests with proteins and lipids as unique substrate did not produce H₂. A simplex-centroid design (SCD) and compositional data analysis of the substrate was used to determine the best condition to convert the substrate into H₂. H_max, R_max and Y_H2 were significantly increased using an initial proportion of 56% carbohydrates (15 g/L), 22% proteins (6 g/L), and 22% lipids (6 g/L), which was concomitant with the generation of acetic and butyric acids. Protein and lipid proportions higher than 29% and lower than 12% led to decreased H_max, R_max and Y_H2 values with a consequent accumulation of propionic acid.     

Lactate wastewater dark fermentation: The effect of temperature and initial pH on biohydrogen production and microbial community

Biohydrogen production using dark fermentation (hydrolysis and acidogenesis) is one of the ways to recover energy from lactate wastewater from the food-processing industry, which has high organic matter. Dark fermentation can be affected by the temperature, pH and the microbial community structure. This study investigated the effects of temperature and initial pH on the biohydrogen production and the microbial community from a lactate wastewater using dark fermentation. Biohydrogen production was successful only at lower temperature levels (35 and 45 °C) and initial pH 6.5, 7.5 and 8.5. The highest hydrogen yield (0.85 mol H₂/mol lactate consumed) was achieved at 45 °C and initial pH 8.5. The COD reduction achieved by fermenting the lactate wastewater at 35 °C ranged between 21 and 30% with the maximum COD reduction at pH 8.5, and at 45 °C, the COD reduction ranged between 12 and 21%, with the maximum at pH 7.5. At 35 °C, the lactate degradation ranged between 54 and 95%, while at 45 °C, it ranged between 77 and 99.8%. 16S rRNA sequencing revealed that at 35 °C, bacteria from the Clostridium genera were the most abundant at the end of the fermentation in the reactors that produced hydrogen, while at 45 °C Sporanaerobacter, Clostridium and Pseudomonas were the most abundant.