Enhanced bio-hydrogen production by anaerobic fermentation of apple pomace with enzyme hydrolysis

A series of batch experiments were conducted to investigate the effects of the HCl-pretreated concentrations, enzyme hydrolysis time and temperature, the cellulase dosage, the ultrasonic time and the fermentation substrate concentration on hydrogen (H₂) production from the anaerobic fermentation of apple pomace (AP). The natural mixed microorganisms from river sludge was used as the seed after being boiled for 15 min. A maximum cumulative H₂ yield (CHYm) of 134.04 ml/g total solid (TS) and an average H₂ production rate (AHPR) of 12.00 ml/g TS/h were obtained from the fermentation of the enzyme-hydrolyzed AAP (a AP soaked with 6% ammonia liquor for 24 h) at the substrate concentration of 15 g/L. The optimal enzyme hydrolysis conditions were proposed as follows: AAP with a cellulase dosage of 12.5 mg/g TS at the hydrolysis substrate concentration of 20 g/L after the ultrasonic irradiation for 20 min was hydrolyzed by the enzyme catalysis at 45 °C and initial pH 5.0 for 48 h.     

Hydrogen production from sludge of poultry slaughterhouse wastewater treatment plant pretreated with microwave

This study aims to produce hydrogen from sludge of poultry slaughterhouse wastewater treatment plant (5% total solid) by anaerobic batch fermentation with Enterobactor aerogenes or mixed cultures from hot spring sediment as the inoculums. Sludge was heated in microwave at 850 W for 3 min. Results indicated that a soluble chemical oxygen demand (sCOD) of pretreated sludge was higher than that of raw sludge. Pretreated sludge inoculated with E. aerogenes and supplemented with the Endo nutrient had a higher hydrogen yield (12.77 mL H₂/g tCOD) than the raw sludge (0.18 mL H₂/g tCOD). When considered the hydrogen yield, the optimum initial pH for hydrogen production from microwave pretreated sludge was 5.5 giving the maximum value of 12.77 mL H₂/g tCOD. However, when considered the hydrogen production rate (R_m), the optimum pH for hydrogen production would be 9.0 with the maximum R_m of 22.80 mL H₂/L sludge·h.     

Biohydrogen and biogas production from mashed and powdered vegetable residues by an enriched microflora in dark fermentation

This study conducted the utilization of vegetable residues by an enriched microflora inoculum to produce biohydrogen via anaerobic batch reactor. Dark fermentation processes were carried out with 3 kinds of vegetable residue substrates including broccoli (Brassica oleracea var. italica.), onion (Alium cepa Linn.), and sweet potato (Ipomoea batatas (L.) Lam). Vegetable wastes were pretreated into 2 forms, i.e. mashed and powdered vegetable, prior to the fermentation. The substrate used for the biohydrogen production were vegetable residues and inoculum at the vegetable residues/inoculum ratio of 1:1 (based on TS). The digestion processes were performed under 120 rpm speed of shaking bottle in the incubator with control temperature of 35^?C. In this work, the maximum hydrogen production was achieved by anaerobic digestion at mashed onion with bioreactor inoculum that produced total hydrogen of 424.1 mL H₂ with hydrogen yield and hydrogen concentration of 151.67 mL H₂/g VS_added and 43.54%, respectively. In addition, the hydrogen production continues took only 7 days for the vegetables blended with the bioreactor inoculum. Finally, it was found that the high potential of degradation of vegetable wastes an enriched microflora in dark fermentation also showed alternative solution to eliminate agricultural wastes to produce green energy.     

Effect of acid,heat and combined acid-heat pretreatments of anaerobic sludge on hydrogen production by anaerobic mixed cultures

Activated sludge (AS) from wastewater treatment plant of brewery industry was used as substrate for hydrogen production by anaerobic mixed cultures in batch fermentation process. The AS (10% TS) was pretreated by acid, heat and combined acid and heat. Combined acid- heat treatment (0.5% (w/v) HCl, 110 °C, 60 min) gave the highest soluble COD (sCOD) of 1785.6 ± 27.1 mg/L with the highest soluble protein and carbohydrate of 8.1 ± 0.1 and 38.5 ± 0.8 mg/L, respectively. After the pretreatment, the pretreated sludge was used to produce hydrogen by heat treated upflow anaerobic sludge blanket (UASB) granules. A maximum hydrogen production potential of 481 mL H₂/L was achieved from the AS pretreated with acid (0.5% (w/v) HCl) for 6 h.     

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.     

Biohydrogen production from apple pomace by anaerobic fermentation with river sludge

The biological hydrogen (bio-H₂) production from apple pomace (AP) by fermentation using natural mixed microorganisms in batch process was studied under various experimental conditions. The river sludge was used as a seed after being boiled for 15 min. The results show that the optimal pretreatment for AP was to soak it in the ammonia liquor of 6% for 24 h at room temperature. An optimal fermentation condition for bio-H₂ production was proposed that the pretreated AP at 37 °C, the initial pH of 7.0 and the fermentation concentration of 15 g/l could produce a maximum cumulative H₂ yield (CHYm) of 101.08 ml/g total solid (TS) with an average H₂ production rate (AHPR) of 8.08 ml/g TS/h. During the conversion of AP into H₂, acetic acid, ethanol, propionic acid and butyric acid were main liquid end-products.     

Thermophilic dark fermentation of untreated rice straw using mixed cultures for hydrogen production

Biohydrogen production from untreated rice straw using different heat-treated sludge, initial cultivation pH, substrate concentration and particle size was evaluated at 55 °C. The peak hydrogen production yield of 24.8 mL/g TS was obtained with rice straw concentration 90 g TS/L, particle size <0.297 mm and heat-treated sludge S1 at pH 6.5 and 55 °C in batch test. Hydrogen production using sludge S1 resulted from acetate-type fermentation and was pH dependent. The maximum hydrogen production (P), production rate (R_m) and lag (λ) were 733 mL, 18 mL/h and 45 h respectively. Repeated-batch operation showed decreasing trend in hydrogen production probably due to overloading of substrate and its non-utilization. PCR-DGGE showed both hydrolytic and fermentative bacteria (Clostridium pasteurianum, Clostridium stercorarium and Thermoanaerobacterium saccharolyticum) in the repeated-batch reactor, which perhaps in association led to the microbial hydrolysis and fermentation of raw rice straw avoiding the pretreatment step.     

Cell growth and hydrogen production on the mixture of xylose and glucose using a novel strain of Clostridium sp. HR-1 isolated from cow dung compost

A novel mesophilic hydrogen-producing bacterium was isolated from cow dung compost and designated as Clostridium sp. HR-1 by 16S rRNA gene sequence. The optimum condition for hydrogen production by strain HR-1 was pH of 6.5, temperature of 37 °C and yeast extract as nitrogen sources. The strain HR-1 has the ability to utilize kinds of hexose and pentose as carbon sources for growth and H₂ production. Cell growth and hydrogen productivity were investigated for batch fermentation on media containing different ratios of xylose and glucose. Glucose was the preferred substrate in the glucose and xylose mixtures. The high glucose fraction had higher cell biomass production rate. The rate of glucose consumption was higher than xylose consumption, and remained essentially constant independent of xylose content of the mixture. The rate of xylose utilization was decreased with increasing of the glucose fraction. The average H₂ yield and specific H₂ production rates with xylose and glucose are 1.63 mol-H₂/mol xylose and 11.14-H₂ mmol/h g-cdw, and 2.02 mol-H₂/mol-glucose and 9.37 mmol-H₂/h g-cdw, respectively. Using the same initial substrate concentration, the maximum average H₂ yield and specific H₂ production rates with the mixtures of 9 g/l xylose and 3 g/l glucose was 2.01 mol-H₂/mol-mixed sugar and 12.56 mmol-H₂/h g-cdw, respectively. During the fermentation, the main soluble microbial products were ethanol and acetate which showed trends with the different ratios of xylose and glucose.     

Seed inocula for biohydrogen production from biodiesel solid residues

This study aimed to optimize the hydrogen production from various seed sludges (two kinds of sewage sludges (S1, S2), cow dung (S3), granular sludge (S4) and effluent from condensed soluble molasses H₂ fermenter (S5)) and enhancement of hydrogen production via heat treatment for substrate and seed sludge by using the solid residues of biodiesel production (BDSR). Two batch assay tests were operated at a biodiesel solid residue concentration of 10 g/L, temperature of 55 °C and an initial cultivation pH of 8. The results showed that the peak hydrogen yield (HY) of 94.6 mL H₂/g volatile solid (VS) (4.1 mmolH₂/g VS) was obtained from S1 when substrate and seed sludge were both heat treated at 100 °C for 1 h. However, the peak hydrogen production rate (HPR) and specific hydrogen production rate (SHPR) of 1.48 L H₂/L-d and 0.30 L H₂/g VSS-d were obtained from S2 without any treatment. The heat treatment was found to increase the HY in both the cases of sewage sludges S1 and S2.The HY of 89.5 mL H₂/g VS (without treatment) was increased to 94.6 mL H₂/g VS and 82.6 mL H₂/g VS (without treatment) was increased to 85.7 mL H₂/g VS for S1 and S2. The soluble metabolic product (SMP) analysis showed that the fermentation followed mainly acetate–butyrate pathway with considerable production of ethanol. The total bioenergy production was calculated as 2.8 and 2.9 kJ/g VS for favorable hydrogen and ethanol production, respectively. The BDSR could be used as feedstock for dark fermentative hydrogen production.     

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.     

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.     

Sequential hydrogen and methane production using the residual biocatalyst of biodiesel synthesis as raw material

Residual Fermented Solid (RFS) is the used biocatalyst obtained after enzymatic biodiesel production carried out applying the fermented solid (FS) with lipase activity. Approximately 350 g of RFS are generated for each liter of biodiesel produced from palm residues fermented solid. In this study, this residue was used for the first time as a raw material for biological hydrogen production through dark fermentation and sequential application of the hydrogen production liquid waste (HPLW) for methane obtainment via anaerobic digestion. The RFS was composed mostly of oils and fats (60% wt.%), and carbohydrates, such as mannose, glucose, and xylose. Hydrogen yield reached 239 ± 44 mL H₂/L after 24 h of fermentation using 31 g_RFS/L at the beginning of the process. Additionally, 204 ± 13 mL CH₄/g COD were produced through the anaerobic digestion of HPLW, which represented 61% of efficiency.     

Improving hydrogen production from cassava starch by combination of dark and photo fermentation

The combination of dark and photo fermentation was studied with cassava starch as the substrate to increase the hydrogen yield and alleviate the environmental pollution. The different raw cassava starch concentrations of 10–25 g/l give different hydrogen yields in the dark fermentation inoculated with the mixed hydrogen-producing bacteria derived from the preheated activated sludge. The maximum hydrogen yield (HY) of 240.4 ml H₂/g starch is obtained at the starch concentration of 10 g/l and the maximum hydrogen production rate (HPR) of 84.4 ml H₂/l/h is obtained at the starch concentration of 25 g/l. When the cassava starch, which is gelatinized by heating or hydrolyzed with α-amylase and glucoamylase, is used as the substrate to produce hydrogen, the maximum HY respectively increases to 258.5 and 276.1 ml H₂/g starch, and the maximum HPR respectively increases to 172 and 262.4 ml H₂/l/h. Meanwhile, the lag time (λ) for hydrogen production decreases from 11 h to 8 h and 5 h respectively, and the fermentation duration decreases from 75–110 h to 44–68 h. The metabolite byproducts in the dark fermentation, which are mainly acetate and butyrate, are reused as the substrates in the photo fermentation inoculated with the Rhodopseudomonas palustris bacteria. The maximum HY and HPR are respectively 131.9 ml H₂/g starch and 16.4 ml H₂/l/h in the photo fermentation, and the highest utilization ratios of acetate and butyrate are respectively 89.3% and 98.5%. The maximum HY dramatically increases from 240.4 ml H₂/g starch only in the dark fermentation to 402.3 ml H₂/g starch in the combined dark and photo fermentation, while the energy conversion efficiency increases from 17.5–18.6% to 26.4–27.1% if only the heat value of cassava starch is considered as the input energy. When the input light energy in the photo fermentation is also taken into account, the whole energy conversion efficiency is 4.46–6.04%.     

Dark fermentation of ground wheat starch for bio-hydrogen production by fed-batch operation

Ground wheat solution was used for bio-hydrogen production by dark fermentation using heat-treated anaerobic sludge in a completely mixed fermenter operating in fed-batch mode. The feed wheat powder (WP) solution was fed to the anaerobic fermenter with a constant flow rate of 8.33 mL h⁻¹ (200 mL d⁻¹). Cumulative hydrogen production, starch utilization and hydrogen yields were determined at three different WP loading rates corresponding to the feed WP concentrations of 10, 20 and 30 g L⁻¹. The residual starch (substrate) concentration in the fermenter decreased with operation time while starch consumption was increasing. The highest cumulative hydrogen production (3600 mL), hydrogen yield (465 mL H₂ g⁻¹ starch or 3.1 mol H₂ mol⁻¹ glucose) and hydrogen production rate (864 mL H₂ d⁻¹) were obtained after 4 days of fed-batch operation with the 20 g L⁻¹ feed WP concentration corresponding to a WP loading rate of 4 g WP d⁻¹. Low feed WP concentrations (10 g L⁻¹) resulted in low hydrogen yields and rates due to substrate limitations. High feed WP concentrations (30 g L⁻¹) resulted in the formation of volatile fatty acids (VFAs) in high concentrations causing inhibition on the rate and yield of hydrogen production.     

Enhanced hydrogen production from sewage sludge by cofermentation with wine vinasse

The cofermentation of sewage sludge and wine vinasse at different mixing ratios to enhance hydrogen production was investigated. Batch experiments were carried out under thermophilic conditions with thermophilic sludge inoculum obtained from an acidogenic anaerobic reactor. The results showed that the addition of wine vinasse enhances the hydrogen production of sewage sludge fermentation. The highest hydrogen yields, 41.16 ± 3.57 and 43.25 ± 1.52 mL H₂/g VS_added, were obtained at sludge:vinasse ratios of 50:50 and 25:75, respectively. These yields were 13 and 14 times higher than that obtained in the monofermentation of sludge (3.17 ± 1.28 mL H₂/g VS_added). The highest VS removal (37%) was obtained at a mixing ratio of 25:75. Cofermentation had a synergistic effect the hydrogen yield obtained at a sludge:vinasse ratio of 50:50 was 40% higher, comparing to the sum of each waste. Furthermore, kinetic analysis showed that Cone and first-order kinetic models fitted hydrogen production better than the modified Gompertz model.     

Effects of sludge pre-treatment method on bio-hydrogen production by dark fermentation of waste ground wheat

Three different pre-treatment methods were applied on two different anaerobic sludge cultures and their mixtures in order to investigate the effects of pre-treatment methods on bio-hydrogen production from dark fermentation of waste ground wheat solution. Repeated heat, chloroform and combinations of heat and chloroform pre-treatment methods were applied to anaerobic sludges from different sources. Repeated heat treatment (2 × 5 h) was found to be more effective in selecting hydrogen producing bacteria compared to the other treatment methods tested on the basis of cumulative hydrogen production. The highest hydrogen formation (652 ml) and specific hydrogen production rate (SHPR = 25.7 ml H₂ g⁻¹ cells h⁻¹) were obtained with the anaerobic sludge pre-treated by repeated boiling. Both the type of anaerobic sludge and the pre-treatment method had considerable effects on bio-hydrogen production from wheat powder solution (WPS) by dark fermentation.     

Effects of substrate concentration and hydraulic retention time on hydrogen production from common reed by enriched mixed culture in continuous anaerobic bioreactor

This study aims to investigate the effect of substrate concentration and hydraulic retention time (HRT) on hydrogen production in a continuous anaerobic bioreactor from unhydrolyzed common reed (Phragmites australis) an invasive wetland and perennial grass. The bioreactor has capacity of 1 L and working volume of 600 mL. It was operated at pH 5.5, temperature at 37 °C, hydraulic retention time (HRT) 12 h, and variation of substrate concentration from 40, 50, and 60 g COD/L, respectively. Afterward, the HRT was then varied from 12, 8, to 4 h for checking the optimal biohydrogen production. Each condition was run until reach steady state on hydrogen production rate (HPR) which based on hydrogen percentage and daily volume. The results were obtained the peak of substrate concentration was at the 50 g COD/L with HRT 12 h, average HPR and H₂ concentration were 28.71 mL/L/h and 36.29%, respectively. The hydrogen yield was achieved at 106.23 mL H₂/g CODre. The substrate concentration was controlled at 50 g COD/L for the optimal HRT experiments. It was found that the maximum of average HPR and H₂ concentration were 43.28 mL/L/h and 36.96%, respectively peak at HRT 8 h with the corresponding hydrogen yield of 144.35 mL H₂/g CODre. Finally, this study successful produce hydrogen from unhydrolyzed common reed by enriched mixed culture in continuous anaerobic bioreactor.     

Bio-hydrogen production from waste fermentation: Mixing and static conditions

One of the main disadvantages of the dark fermentation process is the cost associated with the stages needed for obtaining H₂ producing microorganisms. Using anaerobic microflora in fermentation systems directly is an alternative which is gaining special interest when considering the implementation of large-scale plants and the use of wastes as substrate material. The performance of two H₂ producing microflora obtained from different anaerobic cultures was studied in this paper. Inoculum obtained from a waste sludge digester and from a laboratory digester treating slaughterhouse wastes were used to start up H₂ fermentation systems. Inoculum acclimatized to slaughterhouse wastes gave better performance in terms of stability. However, due to the limited availability of this seed material, further work was performed to study the behaviour of the inoculum obtained from the municipal wastewater treatment plant. The process was evaluated under static and mixing conditions. It was found that application of a low organic loading rate favoured the performance of the fermentation systems, and that agitation of the reacting mass could alleviate unsteady performance. Specific H₂ production obtained was in the range of 19–26 L/kg SV_fed with maximum peak production of 38–67 L/kg SV_fed. Although the performance of the systems was unsteady, recovery could be achieved by suspending the feeding process and controlling the pH in the range of 5.0–5.5. Testing the recovery capacity of the systems under temperature shocks resulted in total stoppage of H₂ production.     

Hydrogen production from melon and watermelon mixture by dark fermentation

Hydrogen gas production from melon and watermelon mixture by dark fermentation was studied with and without inoculum addition. In this context, hydrogen production performance of natural and external inoculation was compared in batch experiments by varying fruit mixture concentration between 0.74 and 37 g TS/L. Hydrogen production increased by increasing the substrate concentration due to higher initial total sugar content at elevated TS (total solids) concentrations. Hydrogen productivity at 37 g TS/L for natural microflora was 80.62 mLH₂/L_reactor.h. However, this value significantly increased to 351.12 mLH₂/L_reactor.h at same solid concentration when the fruit mixture was externally inoculated with heat treated anaerobic sludge. Most favorable nutrient and inoculum composition for hydrogen gas production were at 37 g TS/L. Moreover, the presence of the natural microflora in the fruit mixture led to less inoculum requirement and contribution for hydrogen formation.     

Preparation of active hydrogen-producing cultures from palm oil mill sludge for biohydrogen production system

Methods are investigated to prepare active hydrogen (H₂)-producing cultures originating from palm oil mill sludge using dark fermentation. The first successful method that produces potent H₂-producing cultures and avoids growing H₂-consuming methanogens involves heat pretreatment of the sludge at 100 °C for 2 h and then the sludge sample is shocked in an ice bath for 15 min. Subsequently, a glucose solution rich in nutrients (glucose-based substrate) of 14.80 g chemical oxygen demand (COD)/L is fed in to enrich the H₂-producing cultures. The H₂ production reaches 78.63% on day 31. The second method involves acid pretreatment of sludge with 10 M hydrochloric acid at pH 3 for 48 h. Glucose-based substrate of 25.47 g COD/L is fed into the system. The H₂ production is 69.41% on day 27. For both methods, the H₂ production is stable after the H₂ content reached its maximum. The operation is performed semi-continuously using a hydraulic retention time of 1 day and at 30 °C. The optimum bacterial cells-to-COD level of substrate is approximately 0.60 in the start-up medium. The fermentation medium has an optimum initial pH of 5 and a final pH of 5.2–5.3. These two methods are recommended to produce active H₂-producing cultures for plant start-up in bio-H₂ production.