Dark fermentation of starch factory wastewater with acid- and base-treated mixed microorganisms for biohydrogen production

Biohydrogen (Bio-H₂) can be produced from starch factory wastewater and mixed microorganisms using dark fermentation. Acidic and basic chemicals were used to treat the microorganisms to select the hydrogen (H₂)-producing culture. The experiment used a 120 mL bioreactor at 35 °C and the operation commenced with the initial pH level of wastewater in the pH range 4–7 in batch mode. The bacteria:chemical oxygen demand (COD) ratio was 0.2. The initial pH level of the wastewater in the fermentation process affected the H₂ yield and the specific hydrogen production rate (SHPR). For acid-treated bacteria, the maximum H₂ yield and SHPR were produced at an initial pH of 6.5. The maximum H₂ yield and SHPR were 138 mL/g COD degraded and 7.42 mL/g cells?h, respectively. For the base-treated bacteria, the maximum H₂ yield and SHPR were produced at initial pH of 6.5 and pH 7, respectively. The maximum H₂ yield and SHPR were 182 mL/g COD degraded and 25.60 mL/g cells?h, respectively. The COD degradation efficiency levels were 16 and 20% for acid- and base-treated bacteria, respectively. The digested wastewater remained acidic at pH 4.79–4.83. Throughout the study, no methane gas was observed in the gas mixture produced.     

Modeling of biohydrogen production using generalized multi-scale kinetic model: Impacts of fermentation conditions

This paper presents a new multi-scale kinetic model built upon the multi-stage growth Hypothesis for predicting biohydrogen production. The proposed model represents the significant factors affecting biohydrogen production using a sum of first-order kinetic terms with varying dynamics from slow to fast one. The current work investigates 52 case studies of biohydrogen production that show the double first-order kinetic model provides the best modeling fitness (R² > 0.99). This result suggests two prevalent pathways or microbial groups with distinct dynamics (i.e., fast and slow modes) in biohydrogen production. An increase in temperature (30 °C–43 °C) or substrate concentration (10 g/L to 40 g/L) and the use of simple substrates or mixed cultures can increase the fast-mode dominance up to 100% contribution. Model analysis suggests that the fast mode corresponds to the butyrate production pathway, the growth-associated hydrogen-producing activity, the easily-biodegradable substrates, or the quick hydrogen-producing groups.     

Effects of various pretreatment methods of anaerobic mixed microflora on biohydrogen production and the fermentation pathway of glucose

The influence of different pretreatment methods on anaerobic mixed inoculum was evaluated for selectively enriching the hydrogen (H₂) producing mixed culture using glucose as the substrate. The efficiency of H₂ yield and the glucose fermentation pathway were found to be dependent on the type of pretreatment procedure adopted on the parent inoculum. The H₂ yield could be increased by appropriate pretreatment methods including the use of heat, alkaline or acidic conditions. Heat pretreatment of the inoculum for 30 min at 80 °C increased the H₂ yield to 53.20% more than the control.When the inoculum was heat-pretreated at 80 °C and 90 °C, the glucose degraded via ethanol (HEt) and butric acid (HBu) fermentation pathways. The degradation pathways shifted to HEt and propionate (HPr) types as the heat pretreatment temperature increased to 100 °C. When the inoculum was alkali- or acid-pretreated, the fermentation pathway shifted from glucose to a combination of the HPr and HBu types. This trend became obvious as the acidity increased. As the fermentation pathway shift from the HEt type to the HPr and HBu types, the H₂ yield decreased.     

Simultaneous biohydrogen production and wastewater treatment based on the selective enrichment of the fermentation ecosystem

Biohydrogen production from synthetic wastewater as substrate was studied in anaerobic small scale batch reactors. Enriched anaerobic mixed consortia sampled from various environments were used as parent inocula to start the bioreactors. Selective enrichments were achieved by various physical and chemical pretreatments and changes in the microbial communities were monitored by metagenomic and molecular diagnostics approaches. Experimental data showed the feasibility of biohydrogen production using synthetic wastewater as substrate. The hydrogen generation capability of the different mixed consortia is clearly dependent on the pretreatment methods. The described approach opens the possibility for an alternative way towards simultaneous wastewater treatment and renewable energy generation.     

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.     

Enhancement of fermentative biohydrogen production from textile desizing wastewater via coagulation-pretreatment

A real textile desizing wastewater (TDW) was coagulation-pretreated to enhance its potential of biohydrogen production. Batch fermentation showed that the hydrogen production was efficiently enhanced (550 and 120% increments for hydrogen production rate and hydrogen yield, respectively) and the production performance was substrate-concentration dependent. A peak hydrogen production rate of 3.9 L/L-d and hydrogen yield of 1.52 mol/mol hexose were obtained while using coagulant GGEFloc-653 at a dosage of 1 g/L to pretreat TDW with the concentration of 15 g total sugar/L. The coagulation-pretreatment could have butyrate-type fermentation with high biohydrogen production and the removed some toxic materials that might drive the metabolic pathways to those not favoring biohydrogen production. Based on the data obtained, strategies to operate the coagulation and biohydogen fermentation are suggested. Moreover, fermentation effluent utilization such as for two-stage biogas production and further biohythane (a mixture of H₂ and CH₄) generation are also elucidated.     

Kinetic analysis of biohydrogen production from complex dairy wastewater under optimized condition

Present work describes a kinetic analysis of various aspects of biohydrogen production in batch test using optimized conditions obtained previously. Monod model and Logistic equation have been used to find growth kinetic parameters in batch test under uncontrolled pH. The values of μ_m, K_s, and X_m were 0.64 h⁻¹, 15.89 g-COD L⁻¹, and 7.26 g-VSS L⁻¹, respectively. Modified Leudeking-Piret and Michaelis–Menten equation corroborates a flux of energy to hydrogen production pathway and energy sufficiency in the system. Modified Gompertz equation illustrates that the overall rate and hydrogen yield at 15 g-COD L⁻¹ was higher compared to a dark fermentation of other wastewaters. Besides, Andrew's equation also suggests that since the higher value of K_I (19.95 g-COD L⁻¹), k (255 mL h⁻¹ L⁻¹) was not inhibited at high S. The experimental results implied that the entire products during the fermentation process were growth and substrate degradation associated. The result also confirms that the acetate and butyrate were substantially used for hydrogen production in acidogenic metabolism under uncontrolled pH.     

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.     

Dark fermentative biohydrogen production from confectionery wastewater in continuous-flow reactors

The production of biohydrogen from industrial wastewater through the dark fermentation (DF) process has attracted increased interest in recent years. To implement a DF process on a large scale, a thorough knowledge of laboratory scale process control is required. The operating parameters and design features of the reactors have a great influence on the efficiency of the process. In this work, the possibility of continuous production of biohydrogen from confectionery wastewater was evaluated. The DF process was carried out at 37 ± 1 °C in two different reactors: an upflow anaerobic filter (AF) and a fluidized bed reactor (AFB). Polyurethane foam (PU) was used to immobilize the biomass. The DF process was studied at four hydraulic retention times (HRT) (1.5, 2.5, 7.5 and 15 days) and the corresponding organic loading rates (OLR) (9.21, 6.12, 2.04 and 1.02 g COD_init/(L day)). The highest hydrogen yield (HY) (44.73 ml/g COD_init) and hydrogen production rate (HPR) (92.5 ml/(L day)) was observed in AFB at HRT of 7.5 days and 2.5 days, respectively. The highest concentration of hydrogen in biogas was 34% in AF and 36% in AFB at HRT of 7.5 days. In contrast to AF, the COD removal efficiency in AFB increased with increasing HRT. The pH of the effluent was low (3.95–4.38). However, due to the use of PU for biomass immobilization, it is possible that there were local zones in the reactor that were optimal for the functioning of not only acidogens, but also methanogens. This was evidenced by a rather high content of methane in biogas (2.5% in AF and 9.6% in AFB at HRT of 15 days). These results provide valuable data for optimizing the continuous DF of wastewater from confectionery and other food industries to produce biohydrogen or biohythane.     

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.     

Characteristics of biohydrogen fermentation from various substrates

The characteristics of biohydrogen production from sucrose, slurry-type piggery waste and food waste under the effects of the reactor configurations and operational pHs (6 and 9) were examined by using heat-treated anaerobic sludge as a seed biomass. When sucrose was used in the batch test, the maximum hydrogen yield was 0.12–0.13 g COD (as H₂)/g COD (1.41–1.43 mol/mol hexose) at pH 6. In contrast, 0.10–0.11 g COD (as H₂)/g COD (1.12–1.21 mol/mol hexose) hydrogen yield was achieved from the reactor at pH 9. On the other hand, hydrogen production was not observed in the continuous sequencing batch mode fermenters fed with sucrose. Profile analysis at each cycle revealed hydrogen production at the initial operation periods but eventually only methane at 36 days. When slurry-type piggery waste was used as the substrate, the upflow elutriation-type fermenters produced methane but not hydrogen after 30 days operation. The fermentation intermediate profile showed that the hydrogen produced might have been consumed by homoacetogenic or propionate producing reactions, and eventually converted into methane by acetoclastic methanogens. The downflow leaching bed fermenters using food waste produced 0.013 L H₂/g volatile solids (VS) (0.0061 g COD (as H₂)/g COD) at pH 6 with 54% VS reduction whereas 0.0041 L H₂/g VS (0.0020 g COD (as H₂)/g COD) was produced at pH 9 with 86% VS reduction. The results show that the hydrogen produced should be released rapidly from the reactor before it can be consumed in other biochemical reactions, and substrates with high pH level (>9.0) can be used directly to produce hydrogen without needing to adjust the pH.     

Charting the evolution of biohydrogen production technology through a patent analysis

This study models the evolution of technologies for hydrogen production from the fermentation of biomass. We used a patent-clustering method to construct a technology network based on the mutual citation relationships between representative technology patents. Subsequently, we established an approximate matrix by analyzing the density of this citation network and identified the core technology cluster. We evaluated 2125 US patents from 2012 related to fermentative hydrogen production from biomass and divided the patents into four clusters according to their main technological areas. The largest cluster featured “the methods and systems that process useful gas by using waste and wastewater as feedstock and by enhancing biological (e.g., aerobic and anaerobic) processes,” indicating that this technology area currently represents the mainstream technology for such hydrogen production.     

Application of nanotechnology in dark fermentation for enhanced biohydrogen production using inorganic nanoparticles

Fermentation is an important innovation by mankind and this process is used for converting organic substrate into useful products. Using natural conditions, specifically, light and dark conditions, photo-fermentation and dark fermentation techniques can be developed and operated under controlled conditions. Generally, products such as biofuels, bioactive compounds and enzymes have been produced using the dark fermentation method. However, the major requirement for today's industralized world is biofuels in its clean and pure forms. Biohydrogen is the most efficient and cleanest form of energy produced using dark fermentation of organic substrates. Nevertheless, the quantity of biohydrogen produced via dark fermentation is low. In order to increase the product quantity and quality, several internal and external stress or alterations are made to conventional fermentation conditions. In recent times, nanotechnology has been introduced to enhance the rate of dark fermentation. Nanoparticles (NPs), specifically, inorganic NPs such as silver, iron, titanium oxide and nickel have increased the production rate of biohydrogen. Therefore, the present review focuses on exploring the potential of nanotechnology in the dark fermentation of biohydrogen production, the mechanisms involved, substrates used and changes to be made to increase the production efficiency of dark fermentation.     

Fungal solid-state fermentation of food waste for biohydrogen production by dark fermentation

The dark fermentation process was evaluated for biohydrogen production from food waste through fungal solid-state fermentation (SSF). Three fungal cultures (one strain of Aspergillus tubingensis and two strains of Meyerozyma caribbica) were compared, being A. tubingensis the best hydrolyser culture for releasing soluble carbohydrates. The biochemical hydrogen potential of food waste hydrolysate (FWH) at different substrate-inoculum ratios obtained a lower hydrogen yield than untreated food waste (RFW). The highest hydrogen yield value corresponded to treatments RFW-20 and RFW-30 with 77.0 ± 2.6 and 76.9 ± 1.4 mL H₂ normalized by per gram volatile solid added (NmL H₂/gVS_added), respectively. The microbial community of food waste was analysed, being detected lactic-acid bacteria genera as Latilactobacillus and Leuconostoc. The presence of actively growing bacteria during the SSF could explain the lowest hydrogen yield (20.1–36.0 NmL H₂/gVS_added) in the FWH treatment due to the substrate competition between lactic-acid bacteria and hydrogen-producing bacteria, where the lactic-acid bacteria were favoured by their faster growth rate.     

Effects of rice husk particle size on biohydrogen production under solid state fermentation

As a renewable energy source bio-hydrogen production from lignocellulosic wastes is a promising approach which can produce clean fuel with no CO₂ emissions. Utilization of agro-industrial residues in solid state fermentation (SSF) is offering a solution to solid wastes disposal and providing an economical process of value-added products such as hydrogen.In this study three different particle size of rice husk (<2000 μm, <300 μm, <74 μm) was subjected to batch SSF with a Clostridium termitidis: Clostridium intestinale ratio of 5:1. C. termitidis is a cellulolytic microorganism that has the ability to hydrolyze cellulosic substances and C. intestinale is able to grow on glucose having a potential of enhancing hydrogen production when used in the co-culture. 5 g dw rice husk with 75% humidity was used as substrate in SSF under mesophilic conditions. The highest HF Volume (29.26 mL) and the highest yield (5.9 mL H₂ g⁻¹ substrate) were obtained with the smallest particle size (<74 μm). The main metabolites obtained from the fermentation media were acetic, butyric, propionic and lactic acids. The second best production yield (3.99 mL H₂ g⁻¹ substrate) was obtained with the middle particle size (<300 μm) rice husk with a HF of 19.71 mL.     

Effect of system optimizing conditions on biohydrogen production from herbal wastewater by slaughterhouse sludge

The study evaluates the biohydrogen production from herbal wastewater as the substrate by the enriched mixed slaughterhouse sludge as the seed source. In the following experiments, batch-fermentations are carried out with the optimum substrate concentrations, fermentation pH and fermentation temperature to observe the effects of H₂ production, hydrogen yield and other fermentation end products at different conditions. The hydrogen production is increased as substrate concentration increased up to 8 g COD/L WW, but drastically decreased at 10 g COD/L WW. When the pH of fermentation is controlled to 6.5, a maximum amount of hydrogen yield could be obtained. The hydrogen production is maximum at 50 °C (930 ± 30 mL/L WW) compared to 30 °C (436 ± 16 mL/L WW). Acid-forming pathway with butyric acid as a major metabolite dominated the metabolic flow during the hydrogen production. The experimental results indicated that effective hydrogen production from the herbal wastewater could be obtained by thermophilic acidogenesis at proper operational conditions.     

Improvement of photofermentative biohydrogen production using pre-treated brewery wastewater with banana peels waste

In this study, the impacts of banana peels pre-treatment stage on photofermentative hydrogen production of Rhodobacter sphaeroides 158 DSM using brewery wastewater (BWW) were investigated in a batch bioreactor. The experimental results indicate that banana peels pre-treatments can significantly enhance the cumulative hydrogen production. The maximum hydrogen production yield (408.33 mL H₂ L⁻¹_wastewater) was achieved from the substrate, which was composed of 50% BWW pretreated with 1 g L⁻¹ of banana peels for 2 h and 50% standard medium. This highest amount of hydrogen production was 2.7-folds higher than those that applied the same percentage of raw BWW as the substrate source.     

Effect of food to microorganism ratio on biohydrogen production from food waste via anaerobic fermentation

The effect of different food to microorganism ratios (F/M) (1–10) on the hydrogen production from the anaerobic batch fermentation of mixed food waste was studied at two temperatures, 35 ± 2 °C and 50 ± 2 °C. Anaerobic sludge taken from anaerobic reactors was used as inoculum. It was found that hydrogen was produced mainly during the first 44 h of fermentation. The F/M between 7 and 10 was found to be appropriate for hydrogen production via thermophilic fermentation with the highest yield of 57 ml-H₂/g VS at an F/M of 7. Under mesophilic conditions, hydrogen was produced at a lower level and in a narrower range of F/Ms, with the highest yield of 39 ml-H₂/g VS at the F/M of 6. A modified Gompertz equation adequately (R² > 0.946) described the cumulative hydrogen production yields. This study provides a novel strategy for controlling the conditions for production of hydrogen from food waste via anaerobic fermentation.     

Techno-economic evaluation of biohydrogen production from wastewater and agricultural waste

The world is facing serious climate change caused in part by human consumption of fossil fuel. Therefore, developing a clean and environmentally friendly energy resource is necessary given the depletion of fossil fuels, the preservation of the earth's ecosystem and self-preservation of human life. Biological hydrogen production, using dark fermentation is being developed as a promising alternative and renewable energy source, using biomass feedstock. In this study, beverage wastewater and agricultural waste were examined as substrates for dark fermentation to produce clean biohydrogen energy.     

Effect of fermentation conditions on biohydrogen production from lipid-rich food material

Among the basic components of organic materials, such as carbohydrate, protein, and lipid, the hydrogen yield of carbohydrate fermentation has been reported to be significantly higher than that of lipid. This study used lard as a model organic matter for lipid and investigated its H₂ production potential in batch anaerobic fermentation experiments under various combinations of stirring and CO₂-scavenging conditions. A significant increase in the hydrogen yield was observed in both CO₂-scavenging and stirring conditions; the CO₂-scavenging condition yield was 2.9 times higher than the stirring condition (116.7 and 40.3 mL H₂/g volatile solid [VS], respectively), which was much greater than reported previously. A maximal hydrogen yield of 185.8 mL H₂/g VS was obtained in the presence of both CO₂-scavenging and stirring, and the H₂ content of the total biogas was as high as 99% (v/v). In addition, there was less H₂ and more CH₄ production in the absence of CO₂-scavenging and/or stirring, which suggests that the consumption of H₂ and CO₂ for methanogenesis was the major mechanism of the poor hydrogen yield from lipid. The volatile fatty acids in all the tests consisted primarily of valeric (47.2–54.9%) and propionic acids (26.6–30.3%), and higher concentrations of these acids remained in the fermentation liquid without CO₂ removal. These results suggest that lipid-rich food waste is a potential source for H₂ production if the fermentation process is optimized to minimize the partial pressure of CO₂ and H₂ and restrain the activities of H₂-consuming bacteria.