Converting waste molasses liquor into biohydrogen via dark fermentation using a continuous bioreactor

This study investigated the effects of substrate concentration, HRT (hydraulic retention time), and pre-treatment of the substrate molasses on biohydrogen production from waste molasses (condensed molasses fermentation solubles, CMS) with a CSTR (continuously-stirred tank reactor). First, the hydrogen production was performed with various CMS concentrations (40–90 g COD/L, total sugar 8.7–22.6 g/L) with 6 h HRT. The results show that the maximal hydrogen production rate (HPR) occurred at 80 g COD/L substrate (19.8 g ToSu/L, ToSu: Total Sugar), obtaining an HPR of 0.417 mol/L/d. However, maximum hydrogen yield (HY) of 1.44 mol H₂/mol hexose and overall hydrogen production efficiency (HPE) of 25.6% were achieved with a CMS concentration of 70 g COD/L (17.3 g ToSu/L). The substrate inhibition occurred when CMS concentration was increased to 90 g COD/L (22.6 g ToSu/L). Furthermore, it was observed that the optimal HPR, HY, and HPE all occurred at HRT 6 h. Operating at a lower HRT of 4 h decreased the hydrogen production performance because of lower substrate utilization efficiency. The employment of pre-heating treatment (60 °C for 1 h) of the substrate could markedly enhance the fermentation performance. With 6 h HRT and substrate pre-heating treatment, the HPE raised to 29.9%, which is 18% higher than that obtained without thermal pretreatment.     

Biohydrogen production from acid hydrolyzed wastewater treatment sludge by dark fermentation

Waste generation, waste management, sustainable energy production, and global warming are interrelated environmental issues to be considered together. Wastewater treatment sludge is an organic substance rich waste which causes significant environmental problems. However, these wastes can be used as raw material in biofuel generation. This study was designed to investigate the possible utilization of waste sludge in biohydrogen production by taking these facts into consideration. For this purpose, the sludge was first pre-treated with acid and then, the solid (sludge) and liquid (filtrate) phases of acid pre-treated sludge were used as the substrates for biohydrogen generation dark fermentation. Two-factor factorial experimental design method was used in acid hydrolysis of sludge to determine the effect of pH (pH = 2–6) and reaction period (time, min) elution of chemical oxygen demand (COD), total organic carbon (TOC) and total sugar (TS), NH₄N and PO₄P. Statistical evaluation of the results indicated that pH significantly affects the elution of organic carbon and nutrient content of sludge while the reaction time is significant for only organic carbon content. The optimum pretreatment conditions for maximum organic and nutrient elution were determined as pH = 2 and t = 1440 min. The pretreated products, named as filtrate sludge and sludge, conducted to dark fermentation under mesophilic conditions for biohydrogen generation showed that pretreatment of waste sludge at pH = 6 is the best condition giving the maximum yields (Y_H2) as Y_H2 = 24 mmol g⁻¹ Total Sugar _consumed and Y_H2 = 41 mmol g⁻¹ Total sugar consumed, for filtrate and sludge, respectively.     

Sequential dark and photo-fermentative hydrogen gas production from agar embedded molasses

In this study, molasses and dark fermentation effluent were solidified using agar and used for H₂ production by dark and photo-fermentation. During dark fermentation, the solid jelly form of molasses enabled a slow release of the substrate to the liquid broth hindering fast pH decreases. The initial total sugar concentration, H₂ yield, H₂ rate and lag phase in dark fermentation were 36.2 g/L, 226.24 mL H₂/g TS, 29.85 mL H₂/h and 4.37 h, respectively. Photo-fermentation of 5.77 g TVFA/L embedded dark fermentation effluent did not lead to efficient H₂ production. The best performance in photo-fermentation was obtained with 1.55 g TVFA/L containing diluted dark fermentation effluent. The H₂ yield, H₂ rate and lag phase in photo-fermentation were 870.26 mL H₂/g TVFA, 0.913 mL H₂/h and 54.07 h, respectively. Embedding concentrated substrate using agar can enhance H₂ production performance but only if the release of the substrate does not exceed inhibitory levels and if the rate of diffusion is tolerable for microbial activity.     

Hydrogen production by sequential dark and photofermentation using wet biomass hydrolysate of Spirulina platensis: Response surface methodological approach

Microalgae and cyanobacteria can be used as a potential biomass to produce hydrogen from stored glycogen and starch through fermentation and photofermentation. In this study, the potential of algal biomass i.e. Spirulina platensis hydrolysate as a substrate for sequential fermentative (I-stage) and photo-fermentative (II-stage) biohydrogen production was evaluated. Response Surface Methodology (RSM) was employed to find the optimum photofermentation conditions. From the preliminary optimization experiments, it was found that the significantly affecting factors for H₂ production were pH, dilution fold (D.F.) of fermentate and Fe(II) sulfate concentration during photofermentation (second stage). In the present study, 1% (w/v) Spirulina platensis hydrolyzate produced 23.06 ± 3.63 mmol of H₂ with yield of 1.92 ± 0.20 mmol H₂/g COD reduced. In the second stage experiment 1510 ± 35 mL/l hydrogen was produced using inoculum volume-20.0% (v/v) and inoculum age-48 h of co-culture of Rhodobacter sphaeroides NMBL-01 and Bacillus firmus NMBL-03 under conditions pH-5.95, D.F. of dark fermentate-20.30 folds, Fe(II) sulfate concentration-0.412 μM, temperature-32±2 °C and light intensity-2.5 klux.     

Biohydrogen production by extracted fermentation from sugar beet

In the study, the production of biohydrogen by extracted fermentation from sugar beet was evaluated. Effects of initial amount of sugar beet, biomass and particle size of sugar beet on biohydrogen formation were investigated. The hydrogen (H₂) gas was predicted to be 78.6 mL at initial dry weight of sugar beet 24.6 g L^?1 and H₂ yield was calculated as 81.9 mLH₂ g^?1TOC while biomass concentration (1 g L^?1) and particle size (0.3 cm) were constant. The peak H₂ gas volume was predicted to be 139.9 mL at the low particle size of 0.1 cm. Hydrogen gas production potential was predicted as 143.6 mL h^?1. The peak value of 197.9 mLH₂ g^?1TOC was obtained with particle size of 0.1 cm when dry weight of sugar beet and initial amount of biomass was kept constant at 24.6 g L^?1 and 1 g L^?1, respectively.     

Biohydrogen production by dark fermentation: Experiences of continuous operation in large lab scale

Hydrogen is a clean energy carrier which can be used as fuel in fuel cells. Today, hydrogen is produced mainly by steam reforming of fossil fuels like natural gas or oil. But only hydrogen produced by renewable sources can be called clean energy production. One possibility for hydrogen production is the biological fermentation of biogenous wastes by hydrogen producing bacteria. For the experimental setup four 30-L-working-volume reactors were constructed for continuous biohydrogen production. As inoculum, heat-treated sludge of a wastewater treatment plant was used. Different hydraulic retention times (HRT) were tested and an organic loading rate (OLR) of 2–14 kg VS/m³*d. As starting substrate, waste sugar medium was used. The pH and other parameters were observed to find boundary conditions for a stable continuous process with a minimum of online-control measurements. The high concentration of organic acids in the reactor led to a very low pH, which was controlled manually and online > 4 up to 5.5, otherwise the biohydrogen production decreased rapidly. The gas amount varied with the different OLRs, but could be stabilised on a high level as well as the hydrogen concentration in the gas with 44–52%. No methane was detected in the gas. It turned out, that continuous biohydrogen production with stable gas amounts and qualities could be achieved at different operation conditions. The results showed, that the operation of a continuous biohydrogen reactor has to be observed very carefully to ensure a constant gas production, and that pH-control is necessary to ensure stable operation conditions.     

Prevention of substrate and product inhibitions by using a dilution strategy during dark fermentative hydrogen production from molasses

Substrate and product inhibitions have a significant effect on dark fermentative hydrogen gas production. Particularly, rapid formation of volatile fatty acids leads to fast pH decreases shifting the metabolic pathway. Therefore, controlling volatile fatty acid accumulation has great importance in maintaining effective hydrogen production. In this context, a dilution strategy was applied to regulate volatile fatty acids levels within the desired concentration range. A three-factor Box-Behnken statistical experiment design was established to assess the effects of dilution time, dilution percentage and initial COD concentration on hydrogen formation yield and rate. Highest hydrogen yield (7.7 mL H₂/mL_reactor) and rate (21. 47 mL H₂/h or 9.38 mmol/L_reactor.h) were achieved when 85 gCOD/L containing fermentation media was diluted with a percentage of 130 of the initial working volume at the 3rd hour of the fermentation period. Moreover, this strategy enabled to start fermentation with 55 g glucose/L.     

Amelioration of photofermentative hydrogen production from molasses dark fermenter effluent by zeolite-based removal of ammonium ion

One of the challenges in the development of integrated dark and photofermentative biological hydrogen production systems is the presence of ammonium ions in dark fermentation effluent (DFE). Ammonium strongly inhibits the sequential photofermentation process, and so its removal is required for successful process integration. In this study, the removal of ammonium ions from molasses DFE using a natural zeolite (clinoptilolite) was investigated. The samples were treated with batch suspensions of Na-form clinoptilolite. The ammonium ion concentration could be reduced from 7.60 mM to 1.60 mM and from 12.30 mM to 2.40 mM for two different samples. Photofermentative hydrogen production on treated and untreated molasses DFE samples were investigated in batch photobioreactors by an uptake hydrogenase deleted (hup⁻) mutant strain of Rhodobacter capsulatus. Maximum hydrogen productivities of 1.11 mmol H₂/L_c·h and 1.16 mmol H₂/L_c·h and molar yields of 79% and 90% were attained in the treated DFE samples, while the untreated samples resulted in no hydrogen production. The results showed that ammonium ions in molasses DFE could be effectively removed using clinoptilolite by applying a cost-effective, simple batch process.     

Biohydrogen production from soluble condensed molasses fermentation using anaerobic fermentation

Using anaerobic micro-organisms to convert organic waste to produce hydrogen gas gives the benefits of energy recovery and environmental protection. The objective of this study was to develop a biohydrogen production technology from food wastewater focusing on hydrogen production efficiency and micro-flora community at different hydraulic retention times. Soluble condensed molasses fermentation (CMS) was used as the substrate because it is sacchariferous and ideal for hydrogen production. CMS contains nutrient components that are necessary for bacterial growth: microbial protein, amino acids, organic acids, vitamins and coenzymes. The seed sludge was obtained from the waste activated sludge from a municipal sewage treatment plant in Central Taiwan. This seed sludge was rich in Clostridium sp.A CSTR (continuously stirred tank reactor) lab-scale hydrogen fermentor (working volume, 4.0 L) was operated at a hydraulic retention time (HRT) of 3–24 h with an influent CMS concentration of 40 g COD/L. The results showed that the peak hydrogen production rate of 390 mmol H₂/L-d occurred at an organic loading rate (OLR) of 320 g COD/L-d at a HRT of 3 h. The peak hydrogen yield was obtained at an OLR of 80 g COD/L-d at a HRT of 12 h. At HRT 8 h, all hydrogenase mRNA detected were from Clostridium acetobutylicum-like and Clostridium pasteurianum-like hydrogen-producing bacteria by RT-PCR analysis. RNA based hydrogenase gene and 16S rRNA gene analysis suggests that Clostridium exists in the fermentative hydrogen-producing system and might be the dominant hydrogen-producing bacteria at tested HRTs (except 3 h). The hydrogen production feedstock from CMS is lower than that of sucrose and starch because CMS is a waste and has zero cost, requiring no added nutrients. Therefore, producing hydrogen from food wastewater is a more commercially feasible bioprocess.     

Biohydrogen yield efficiency and the benefits of dark,photo and dark-photo fermentative production technology in circular Asian economies

Twenty-six new data envelopment analysis (DEA) models with 55 biohydrogen production experiments categorized into three groups including dark fermentation (DF), photo fermentation (PF), and dark-photo sequential fermentation (DF-PF) technologies, are used to evaluate their biohydrogen yield efficiency. The results reveal the average yield efficiencies of DF, PF and DF-PF are 0.2844, 0.3460 and 0.7040, respectively. The most efficient overall combination of biohydrogen inputs is PhBR1/Rhodobacter capsulatus B10/Rhodobacter capsulatus in DF-PF. Statistical tests demonstrate DF-PF has statistically double the efficiency of PF and DF, and the efficiency of PF significantly exceeds that of DF, supporting some of the literature findings. A flexible DEA model must be carefully chosen when evaluating biohydrogen production. All inputs and outputs of biohydrogen statistically influenced yield efficiency to a significant level. India and Japan are the top two economies benefitting from improved biohydrogen yield efficiency. Improving biohydrogen yield efficiency can improve macroeconomic growth and develop the renewable hydrogen and biohydrogen industry.     

Biohydrogen production by immobilized Enterobacter aerogenes on functionalized multi-walled carbon nanotube

Hydrogen production by immobilized Enterobacter aerogenes on functionalized multi-walled carbon nanotube (MWCNT-COOH) in repeated batch mode was studied. Fourier transform infrared (FTIR) spectroscopy and field emission scanning electron microscopy (FESEM) were employed to confirm immobilization of E. aerogenes successfully. The effect of MWCNT-COOH concentrations (0.2, 0.6, and 1.2 mg/mL) on hydrogen production was investigated. The present study showed that immobilized E. aerogenes on 1.2 mg/mL MWCNT-COOH resulted in higher hydrogen yield (2.2 moL/mol glucose), hydrogen production rate (2.72 L/L.h), and glucose degradation efficiency (96.20%) and shorter the lag phase (1 h) compared to the free E. aerogenes. Modified Gompertz and Logistic models were employed to predict the cumulative hydrogen production successfully.     

Pilot-scale outdoor photofermentative hydrogen production from molasses using pH control

The duration of photofermentative hydrogen production from sugar-based nutrients is limited by gradual acidification caused by the production of organic acids, leading to suboptimal pH. To address this issue, a custom pH control system was built and installed on a 20 L tubular photobioreactor, and operated under outdoor conditions. Long-term, single-stage hydrogen production from molasses was achieved using the purple non-sulfur bacterium, Rhodobacter capsulatus. The run lasted for 48 days, the longest duration achieved in a tubular photobioreactor on molasses as the only feed. pH was maintained close to its optimum value. High-purity hydrogen (above 90% by mole, on average) and near-complete conversion of sucrose was observed. The highest hydrogen productivity was 0.69 molH₂/(m³.h). On the other hand, hydrogen production was observed to cease after periods of activity. Production resumed after dilution followed by artificial illumination, indicating that the production activity could be recovered during prolonged runs.     

Biohydrogen production: Current perspectives and the way forward

Global research is moving forward in developing biological production of hydrogen (biohydrogen) as a renewable energy source to alleviate stresses due to carbon dioxide emissions and depleting fossil fuels resource. Biohydrogen has the potential to replace current hydrogen production technologies relying heavily on fossil fuels through electricity generation. While biohydrogen research is still immature, extensive work on laboratory- and pilot-scale systems with promising prospects has been reported. This work presents a review of advances in biohydrogen production focusing on production pathways, microbiology, as well as bioreactor configuration and operation. Challenges and prospects of biohydrogen production are also outlined.     

Cladophora sp. as a sustainable feedstock for dark fermentative biohydrogen production

Macroalgae are rich in carbohydrates which can be used as a promising substrate for fermentative biohydrogen production. In this study, Cladophora sp. biomass was fermented for biohydrogen production at various inoculum/substrate (I/S) ratios against a control of inoculum without substrate in laboratory-scale batch reactors. The biohydrogen production yield ranged from 40.8 to 54.7 ml H₂/g-VS, with the I/S ratio ranging from 0.0625 to 4. The results indicated that low I/S ratios caused the overloaded accumulation of metabolic products and a significant pH decrease, which negatively affected hydrogen production bacteria's metabolic activity, thus leading to the decrease of hydrogen fermentation efficiency. The overall results demonstrated that Cladophora sp. biomass is an efficient fermentation feedstock for biohydrogen production.     

Dark fermentative hydrogen gas production from molasses using hot spring microflora

This study reports hydrogen gas (H₂) production from molasses by hot spring microflora in three stages. During the first two stages most convenient temperature, inoculation percentage (INP) ensuring the highest H₂ yield and rate were determined using suspended culture. Then, H₂ was produced by the same culture immobilized on porous ceramic rings at three different hydraulic retention times. For the suspended culture experiments, the most effective H₂ production resulting 202.32 mL H₂/g COD was obtained at 37 °C with 10 INP. The highest H₂ formation of 534.35 mLH₂/d was realized for the biofilm culture at 0.53-day hydraulic retention time and H₂ production using hot spring microflora in biofilm form was found to be promising. The pH of the experiments remained stable around 5.5–6.5 without a requirement for pH adjustment during the fermentation.     

Biohydrogen production from hydrolyzed waste wheat by dark fermentation in a continuously operated packed bed reactor: The effect of hydraulic retention time

The aim of the study is biohydrogen production from hydrolyzed waste wheat by dark fermentation in a continuously operated up-flow packed bed reactor. For this purpose, the effect of hydraulic retention time (HRT) on the rate (R_H2) and yield (Y_H2) of hydrogen gas formation were investigated. In order to determine the most suitable hydraulic retention time yielding the highest hydrogen formation, the reactor was operated between HRT = 1 h and 8 h. The substrate was the acid hydrolyzed wheat powder (AHWP). Waste wheat was sieved down to 70 μm size (less than 200 mesh) and acid hydrolyzed at pH = 2 and 90 °C in an autoclave for 15 min. The sugar solution obtained from hydrolysis of waste wheat was used as substrate at the constant concentration of 15 g/L after neutralization and nutrient addition for biohydrogen production by dark fermentation. The microbial growth support particle was aquarium biological sponge (ABS). Heat-treated anaerobic sludge was used as inoculum. Total gas volume and hydrogen percentage in total gas, hydrogen gas volume, total sugar and total volatile fatty acid concentrations in the feed and in the effluent of the system were monitored daily throughout the experiments. The highest yield and rate of productions were obtained as Y_H2 = 645.7 mL/g TS and R_H2 = 2.51 L H₂/L d at HRT = 3 h, respectively.     

Biohydrogen production from biomass and industrial wastes by dark fermentation

Hydrogen is a clean energy carrier which has a great potential to be an alternative fuel. Abundant biomass from various industries could be a source for biohydrogen production where combination of waste treatment and energy production would be an advantage. This article summarizes the dark fermentative biohydrogen production from biomass. Types of potential biomass that could be the source for biohydrogen generation such as food and starch-based wastes, cellulosic materials, dairy wastes, palm oil mill effluent and glycerol are discussed in this article. Moreover, the microorganisms, factors affecting biohydrogen production such as undissociated acid, hydrogen partial pressure and metal ions are also discussed.     

Biohydrogen production by a novel integration of dark fermentation and mixotrophic microalgae cultivation

Biohydrogen is usually produced via dark fermentation, which generates CO₂ emissions and produces soluble metabolites (e.g., volatile fatty acids) with high chemical oxygen demand (COD) as the by-products, which require further treatments. In this study, mixotrophic culture of an isolated microalga (Chlorella vulgaris ESP6) was utilized to simultaneously consume CO₂ and COD by-products from dark fermentation, converting them to valuable microalgae biomass. Light intensity and food to microorganism (F/M) ratio were adjusted to 150 μmol m⁻² s⁻¹ and F/M ratio, 4.5, respectively, to improve the efficiency of assimilating the soluble metabolites. The mixotrophic microalgae culture could reduce the CO₂ content of dark fermentation effluent from 34% to 5% with nearly 100% consumption of soluble metabolites (mainly butyrate and acetate) in 9 days. The obtained microalgal biomass was hydrolyzed with 1.5% HCl and subsequently used as the substrate for bioH₂ production with Clostridium butyricum CGS5, giving a cumulative H₂ production of 1276 ml/L, a H₂ production rate of 240 ml/L/h, and a H₂ yield of 0.94 mol/mol sugar.     

Effect of light intensity and initial pH during hydrogen production by an integrated dark and photofermentation process

Photofermentation was carried out with the spent fermentation broth obtained from the anaerobic dark fermentation in a two-stage process. For the first stage, i.e. dark fermentation Enterobacter cloacae DM 11 was used as hydrogen producing microorganism. For photofermentation Rhodobacter sphaeroides O.U. 001, a photo-heterotrophic purple non-sulfur bacterium, was used. pH study revealed that cumulative hydrogen production was maximum at initial medium pH of 7.0 ± 0.2. Biomass yield was also high at the vicinity of pH 7.0 and it decreased as the pH increased from 7.0 to 8.0. Increased light intensity resulted in an increase in the total volume of hydrogen evolved and also hydrogen production rate. However, light conversion efficiency decreased by increasing light intensity. A four-fold increase in light intensity resulted in a three-fold decrease in light conversion efficiency although the cumulative volume of hydrogen gas production increased. It was observed that only a maximum of 0.51% light conversion efficiency could be achieved but at the expense of very low light intensity of 2500 lux (3.75 W m⁻²).