Ultrasonic pretreatment for an enhancement of biohydrogen production from complex food waste

The efficacy of ultrasonication pretreatment method for complex food waste prior to anaerobic digestion is evaluated for enhancement of H₂ yield (HY) and rate (R). The RSM results showed that the ultimate H₂ production increased with increasing TS content and ultrasonication time (UT). Desirability function integrated with RSM predicted an optimum condition of TS and UT as: 8% TS and 12 min, for maximization of HY and R. The highest HY, 149 mL/g VS_added, and R, 5.23 mL/h, were achieved during the verification test at optimized conditions. Furthermore, a significant decreased lag phase followed by highest molar HBu/HAc ratio (2.2) was also achieved at optimized conditions with lowest specific energy input (13,500 kJ/kg TS). The significant relative enhancement of HY, 75%, and R, 104%, implies that ultrasonically pretreated complex food waste with higher TS loading is about 1.7–2.1 times more effective for enhanced bioH₂ production compared to unsonicated food waste.     

Optimization of conditions for hydrogen production from complex dairy wastewater by anaerobic sludge using desirability function approach

Present study deals with the multiple-response optimization for biohydrogen production using anaerobic sludge and outstanding approach to overcome the drawbacks of conventional response surface methodology (RSM). Dairy wastewater was used as source in batch fermentation was followed for this study. Response surface methodology (RSM), based on a three level, four variable Box–Behnken design, was employed to obtain the best possible combination of substrate concentration, pH, COD/N ratio and COD/P ratio for maximum H₂ yield (HY) and specific hydrogen production rate (SHPR). Experimental data were evaluated by applying RSM integrating a desirability function approach. The optimum H₂ yield and SHPR conditions were: substrate concentration 15.3 g COD/L, pH 5.5, COD/N ratio 100.5 and COD/P ratio 120 with maximum overall desirability D of 0.94. The confirmation experiment under these optimal condition showed a HY and SHPR of 13.54 mmol H₂/g COD and 29.91 mmol H₂/g-VSS.d, respectively. This was only 0.22% and 0.20%, respectively, different from the predicted values, suggesting that the desirability function approach with RSM was a useful technique to get the maximum H₂ yield and SHPR simultaneously.     

Effect of microwave pretreatment of mixed culture on biohydrogen production from waste of sweet produced from Benincasa hispida

To enhance the production of biohydrogen from biomass, various pretreatment methods play important role. In this study, effect of microwave irradiation on the culture was studied on biohydrogen production from Benincasa hispida (Petha) solid waste at different powers for a fixed interval of time. The highest power studied was 800 W with a frequency of 2450 MHz. The amount of soluble sugars found in the waste was 13.9 mg/L having the chemical oxygen demand (COD) of 3000 mg/L. Studies have been performed in batch reactors using mixed consortia and results were also compared with the reactor operated at the normal conditions i.e. without any inoculum pretreatment. Maximum hydrogen produced was 14 mmol H₂ per mol of soluble sugar consumed in the reactor in which the inoculum was exposed to 320 W of microwave for 5 min. SEM analysis of this microwave pretreated culture was done.     

Optimizing biohydrogen production from mushroom cultivation waste using anaerobic mixed cultures

The mushroom bag is a polypropylene bag stuffed with wood flour and bacterial nutrients. After being used for growing mushroom for one to two weeks this bag becomes mushroom cultivation waste (MCW). About 150 million bags (80,000 tons) of MCW are produced annually in Taiwan and are usually burned or discarded. The cellulosic materials and nutrients in MCW could be used as the feedstock and nutrients for anaerobic biohydrogen fermentation. This study aims to select the inoculum from various waste sludges (sewage sludge I, sewage sludge II, cow dung and pig slurry) with or without adding any extra nutrients. A batch test was operated at a MCW concentration of 20 g COD/L, temperature 55 °C and an initial cultivation pH of 8. The results show that extra nutrient addition inhibited hydrogen production rate (HPR) and hydrogen production yield (HY) when using cow dung and pig slurry seeds. However, nutrient addition enhanced the HPR and HY in case of using sewage sludge inoculum and without inoculum. This related to the inhibition caused by high nutrient concentration (such as nitrogen) in cow dung and pig slurry. Peak HY of 0.73 mmol H₂/g TVS was obtained with no inoculum and nutrient addition. However, peak HPR and specific hydrogen production rate (SHPR) of 10.11 mmol H₂/L/d and 2.02 mmol H₂/g VSS/d, respectively, were obtained by using cow dung inoculum without any extra nutrient addition.     

Fermentative hydrogen production from wastewaters: A review and prognosis

Biohydrogen is a promising candidate which can replace a part of our fossil fuels need in day-to-day life due its perceived environmental benefits and availability through dark fermentation of organic substrates. Moreover, advances in biohydrogen production technologies based on organic wastewater conversion could solve the issues related to food security, climate change, energy security and clean development in the future. An evaluation of studies reported on biohydrogen production from different wastewaters will be of immense importance in economizing production technologies. Here we have reviewed biohydrogen production yields and rates from different wastewaters using sludges and microbial consortiums and evaluated the feasibility of biohydrogen production from unexplored wastewaters and development of integrated bioenergy process. Biohydrogen production has been observed in the range of substrate concentration 0.25–160 g COD/L, pH 4–8, temperature 23–60 °C, HRT 0.5–72 h with various types of reactor configuration. The most efficient hydrogen production has been obtained at an organic loading rate (OLR) 320 g COD/L/d, substrate concentration 40 g COD/L, HRT 3 h, pH 5.5–6.0, temperature 35 °C in a continuously-stirred tank reactor system using mixed cultures and fed with condensed molasses fermentation soluble wastewater. The net energy efficiency analysis showed vinasse wastewater has the highest positive net energy gain followed by glycerin wastewater and domestic sewage as 140.39, 68.65, 51.84 kJ/g COD feedstock with the hydrogen yield (HY) of 10 mmol/g COD respectively.     

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.     

The effect of pH on the production of biohydrogen by clostridia: Thermodynamic and metabolic considerations

This study evaluates the effect of pH (4-7) on fermentative biohydrogen production by utilizing three isolated Clostridium species. Fermentative batch experiments show that the maximum hydrogen yield for Clostridium butyricum CGS2 (1.77 mmol/mmol glucose) is achieved at pH 6, whereas a high hydrogen production with Clostridium beijerinckii L9 (1.72 mmol/mmol glucose) and Clostridium tyrobutyricum FYa102 (1.83 mmol/mmol glucose) could be achieved under uncontrolled pH conditions (initial pH of 6.4-6.6 and final pH of 4-4.2). Low hydrogen yields (0-0.6 mmol/mmol glucose) observed at pH 4 are due likely to inhibitory effects on the microbial growth, although a low pH can be thermodynamically favorable for hydrogen production. The low hydrogen yields (0.12-0.64 mmol/mmol glucose) observed at pH 7 are attributed not only to thermodynamically unfavorable, but also metabolically unfavorable for hydrogen production. The relatively high levels of lactate, propionate, or formate observed at pH 7 reflect presumably the high enzymatic activities responsible for their production, together with the low hydrogenase activity, resulting in a low hydrogen production. A correlation analysis of the data from present and previous studies on biohydrogen production with pure Clostridium cultures and mixed microflora indicates a close relation between the hydrogen yield (YH₂) and the (YH₂)/(2(YHAc+YHBu)) ratio, with the observed correlation coefficient (0.787) higher than that (0.175) between YH₂ and the molar ratio of butyrate to acetate (B/A). Based on the (YH₂)/(2(YHAc+YHBu)) ratios observed at different pHs, a control of pH at 5.5-6.8 would seem to be an effective means to enhance the fermentative biohydrogen production.     

Effects of biomass,COD and bicarbonate concentrations on fermentative hydrogen production from POME by granulated sludge in a batch culture

Effects of three selected variables viz. biomass concentration, initial chemical oxygen demand (COD) concentration and initial bicarbonate alkalinity (BA) on biological hydrogen production from palm oil mill effluent (POME) using the granulated sludge in batch culture were investigated. The experimental results were analyzed and modeled using a central composite design (CCD) of response surface methodology (RSM). In order to carry out a comprehensive analysis of the biohydrogen production process, indicative parameters namely hydrogen yield (Y_H), specific hydrogen production rate (SHPR), and COD removal efficiency were studied as the process responses. Maximum hydrogen yield (124.5 mmol H₂/g COD_removed) and specific hydrogen production rate (55.42 mmol H₂/g VSS.d) were achieved at COD_in 3000 and 6500 mg/l, MLVSS 4000 and 2000 mg/l, and initial BA 1100 mg CaCO₃/l, respectively.     

Enhanced bio-hydrogen production from sugarcane juice by immobilized Clostridium butyricum on sugarcane bagasse

Immobilized Clostridium butyricum TISTR 1032 on sugarcane bagasse improved hydrogen production rate (HPR) approximately 1.2 times in comparison to free cells. The optimum conditions for hydrogen production by immobilized C. butyricum were initial pH 6.5 and initial sucrose concentration of 25 g COD/L. The maximum HPR and hydrogen yield (HY) of 3.11 L H₂/L substrate·d and 1.34 mol H₂/mol hexose consumed, respectively, were obtained. Results from repeated batch fermentation indicated that the highest HPR of 3.5 L H₂/L substrate·d and the highest HY of 1.52 mol H₂/mol hexose consumed were obtained at the medium replacement ratio of 75% and 50% respectively. The major soluble metabolites in both batch and repeated batch fermentation were butyric and acetic acids.     

Studies on evaluation of pretreatment efficiency of surfactant mediated ultrasonic pretreatment of Sargassum tennerimum (marine macroalgae) for achieving profitable biohydrogen production

Growing energy demand is inevitable in the future, due to increased commercialization and industrialization. Biohydrogen production from renewable biomass is considered as clean energy technology and, in this study, the feasibility of using Sargassum tennerimum as a renewable energy source has been attempted. Pretreatment using sonication pretreatment (ST) and surfactant Dioctyl sodium sulphosuccinate (DOSS) mediated ultrasonic pretreatment (DOSSSP) were investigated and the optimized pretreatment time and sonication power, respectively, were 30 and 200W for ST. DOSS dosage of 0.005 g/g TS was found to be optimum for DOSSSP with the corresponding maximum of 2600 mg/L and 27.36% respectively for sCOD (Soluble chemical oxygen demand) release and COD (Chemical oxygen demand) solubilization percentage. Biohydrogen assay analysis of the pretreated algal biomass was carried out and 86 mL/g COD of maximum biohydrogen production was achieved for DOSSSP pretreated sample than ST and control owing to higher COD solubilization percentage, carbohydrate and protein release. Energy analysis was performed to test the economic viability of the pretreatment process. This analysis showed that the maximum net positive energy of 0.0407 and energy ratio of 2.43 was obtained for DOSSSP revealing its economic viability. This study also reveals that macroalgae Sargassum tennerimum could be a sustainable renewable energy source for biohydrogen production as it is abundant throughout the year.     

Analysis of the three-phase state in biological hydrogen production from coal

The production of biohydrogen from coal is a new research direction in the bioengineering of coalbed methane. In order to study the transformation process and mechanism of the gas-liquid-solid tripe phase in biohydrogen production from coal, a biohydrogen production experiment from low-rank coal is carried out under laboratory conditions. The results show that: The daily gas production of hydrogen reaches a peak value of 1.23 mL/g on the fifth day. The cumulative hydrogen production is 6.24 mL/g. The pH of the liquid products gradually decreases to 5, and the Eh gradually increased from −180 to −50 mV during the experiment. The peak value of lignin degradation rate is 0.42% on the 7th day. The chemical oxygen demand (COD) first increases and then decreases. The highest COD is 4068 mg/L, and the final COD degradation rate is 46.3%. The peak values of cellulase are 0.021 mg/(mL h) and 0.223 mmol H₂/(min mg) at 3 d later than that of hydrogenase. The absorbance of bacterial turbidity first increases and then decreases, with the community structure proving that the hydrolytic bacteria are dominated by Acinetobacter, Comamonas, Intestinimonas, and some fermentation bacteria, including Macellibacteroides. The changes of carbon, oxygen, nitrogen, sulfur and functional groups of carbon and oxygen in coal are obvious, with aliphatic carbon (methoxy, carbonyl, and so on) representing the main part of the biochemical reaction in the macromolecular structure of coal. The analysis of the three-phase state in the process of coal hydrogen production is helpful to study the mechanism of hydrogen production by coal fermentation from different perspectives, and also provides a reference for the promotion of coal hydrogen production by fermentation in the next step.     

Integrated process to produce biohydrogen from wheat straw by enzymatic saccharification and dark fermentation

In this study, different pretreatment methods, including lyophilization, hydrothermal pretreatment, and ultrasound combined with dilute alkali post-cooking, were investigated to enhance the efficiency of enzymatic saccharification and biohydrogen production of the wheat straw. All pretreatment methods could effectively remove lignin and hemicellulose while retaining cellulose, further enhancing the biomass accessibility for subsequently enzymatic saccharification and biohydrogen production. A reducing sugar concentration of 13.18 g/L was acquired when wheat straw was treated with ultrasound and dilute alkali cooking (R_U). The sequential fermentative hydrogen yield of the substrate R_U was 133.6 mL/g total solids (TS), which was 5.6-fold larger than that of the raw material (23.9 mL/g TS). The study confirmed that ultrasound combined with dilute alkali cooking was an effective method, which not only provided significant guideline for improving biohydrogen production but also presented helpful direction for the efficient pretreatment of other lignocellulosic biomass.     

Ultrasonic pretreatment of palm oil mill effluent: Impact on biohydrogen production,bioelectricity generation,and underlying microbial communities

Substrate bioavailabity is one of the critical factors that determine the relative biohydrogen (bioH₂) yield in fermentative hydrogen production and bioelectricity output in a microbial fuel cell (MFC). In the present undertaking, batch bioH₂ production and MFC-based biolectricity generation from ultrasonically pretreated palm oil mill effluent (POME) were investigated using heat-pretreated anaerobic sludge as seed inoculum. Maximum bioH₂ production (0.7 mmol H₂/g COD) and COD removal (65%) was achieved at pH 7, for POME which was ultrasonically pretreated at a dose of 195 J/mL. Maximum value for bioH₂ productivity and COD removal at this sonication dose was higher by 38% and 20%, respectively, than unsonicated treatments. In batch MFC experiments, the same ultrasound dose led to reduced lag-time in bioelectricity generation with concomitant 25% increase in bioelectricity output (18.3 W/m³) and an increase of COD removal from 30% to 54%, as compared to controls. Quantitative polymerase chain reaction (qPCR) tests on sludge samples from batch bioH₂ production reflected an abundance of gene fragments coding for both clostridial and thermoanaerobacterial [FeFe]-hydrogenase. Fluorescence in situ hybridization (FISH) tests on sludge from MFC experiments showed Clostridium spp. and Thermoanaerobacterium spp. as the dominant microflora. Results suggest the potential of ultrasonicated POME as sustainable feedstock for dark fermentation-based bioH₂ production and MFC-based bioelectricity generation.     

Impact of regulated pH on proto scale hydrogen production from xylose by an alkaline tolerant novel bacterial strain, Enterobacter cloacae DT-1

A hydrogen producing facultative anaerobic alkaline tolerant novel bacterial strain was isolated from crude oil contaminated soil and identified as Enterobacter cloacae DT-1 based on 16S rRNA gene sequence analysis. DT-1 strain could utilize various carbon sources; glycerol, CMCellulose, glucose and xylose, which demonstrates that DT-1 has potential for hydrogen generation from renewable wastes. Batch fermentative studies were carried out for optimization of pH and Fe²⁺ concentration. DT-1 could generate hydrogen at wide range of pH (5–10) at 37 °C. Optimum pH was; 8, at which maximum hydrogen was obtained from glucose (32 mmol/L), when used as substrate in BSH medium containing 5 mg/L Fe²⁺ ion. Decrease in hydrogen partial pressure by lowering the total pressure in the fermenter head space, enhanced the hydrogen production performance of DT-1 from 32 mmol H₂/L to 42 mmol H₂/L from glucose and from 19 mmol H₂/L to 33 mmol H₂/L from xylose. Hydrogen yield efficiency (HY) of DT-1 from glucose and xylose was 1.4 mol H₂/mol glucose and 2.2 mol H₂/mol xylose, respectively. Scale up of batch fermentative hydrogen production in proto scale (20 L working volume) at regulated pH, enhanced the HY efficiency of DT-1 from 2.2 to 2.8 mol H₂/mol xylose (1.27 fold increase in HY from laboratory scale). 84% of maximum theoretical possible HY efficiency from xylose was achieved by DT-1. Acetate and ethanol were the major metabolites generated during hydrogen production.     

Extreme-thermophilic biohydrogen production from lignocellulosic bioethanol distillery wastewater with community analysis of hydrogen-producing microflora

Extreme-thermophilic biohydrogen production from distillery wastewater was investigated in batch and continuous-mode operation. Hydrogen-producing mixed culture was enriched by repeated batch cultivations. Effect of temperature and pH on biohydrogen yield was investigated in batch experiments. The highest hydrogen yield of 196. mL/g-volatile solids_addded (VS_added) was obtained at 70 °C and pH 7.0 in batch culture. Continuous biohydrogen production was performed in CSTR reactor with yield of 172.0 mL/g-VS_added at HRT (hydraulic retention time) of 4 days. The main metabolic products were acetate, lactate, and ethanol. Community structure of hydrogen-producing microflora was investigated by 16S rRNA gene sequence analysis. The microorganisms involved in both batch and continuous-mode operation were similar and hydrogen production was carried out by a group of extreme-thermophilic bacterial species related to Thermotoga, Coprothermobacter, Caldanaerobacter, Thermobrachium, and Caldicellulosiruptor.     

Selective adaptation of an anaerobic microbial community: Biohydrogen production by co-digestion of cheese whey and vegetables fruit waste

The co-digestion process of crude cheese whey (CCW) with fruit vegetable waste (FVW) for biohydrogen production was investigated in this study. Five different C/N ratios (7, 17, 21, 31, and 46) were tested in 2 L batch systems at a pH of 5.5 and 37 °C. The highest specific biohydrogen production rate of 10.68 mmol H₂/Lh and biohydrogen yield of 449.84 mL H₂/g COD were determined at a C/N ratio of 21. A pyrosequencing analysis showed that the main microbial population at the initial stage of the co-digestion consisted of Bifidobacterium, with 85.4% of predominance. Hydrogen producing bacteria such as Klebsiella (9.1%), Lactobacillus (0.97%), Citrobacter (0.21%), Enterobacter (0.27%), and Clostridium (0.18%) were less abundant at this culture period. The microbial population structure was correlated with the lactate, acetate, and butyrate profiles obtained. Results demonstrated that the co-digestion of CCW with FVW improves biohydrogen production due to a better nutrient balance and improvement of the system's buffering capacity.     

Production efficiency and economic benefit evaluation of biohydrogen produced using macroalgae as a biomass feedstock in Asian circular economies

This study uses three data envelopment analysis models to determine the production efficiency of biohydrogen which is produced from macroalgae and other sources by dark fermentation. The efficiency of macroalgae is greatest in batch mode for S. Japonica using a sDFMEC process at pH 5.3, 35 °C, 1 g COD/L and a hydrogen production rate (HPR) of 0.34 L/L/h. The highest efficiency is using an internal circulation batch reactor in continuous mode for beverage waste water. The HPR and substrate concentration are the most important factor of biohydrogen efficiency, and efficiency and temperature are the most important factors of HPR. Malaysia and India are the two economies that most benefit from increased production efficiency due to the use of macroalgae. Increasing biohydrogen yield efficiency will improve macroeconomic growth and establish a renewable hydrogen and biohydrogen industry, which is especially efficient related to the economic recovery during the COVID-19 pandemic.     

Dark fermentative hydrogen production from sucrose and molasses

There are many factors affecting the dark fermentative hydrogen production. The interaction of these factors, that is, their combined effects, should be investigated for better design of the systems with stable and higher hydrogen yields. This study aimed to investigate the combined effects of initial substrate, pH, and biomass (or initial substrate to biomass) values on hydrogen production from sucrose and sugar‐beet molasses. Therefore, optimum initial chemical oxygen demand (COD), pH, and volatile suspended solids (VSS) or initial substrate to biomass (VSS) ratio (S/X_o) values leading to the highest dark fermentative hydrogen production were investigated in batch reactors. An experimental design approach (response surface methodology) was used. Results revealed that when sucrose was the substrate, maximum hydrogen production yield (HY) of 2.3 mol H₂/mol sucrose_added was obtained at initial pH of 7 and COD of 10 g/L. Initial S/X_o values studied (4–20 g COD/g VSS) had no effect on HY, while the initial pH was found as the parameter mostly affecting both HY and hydrogen production rate (HPR). When substrate was molasses, initial COD concentration was the only variable affecting HY and HPR. Maximum of both was achieved at 10 g/L initial COD. Initial VSS values studied (2.5–7.5 g/L) had no effect on HPR and HY. This study also indicated that molasses leads to homoacetogenesis for potentially containing intrinsic microorganism and/or natural constituents; thus, sucrose is more advantageous for hydrogen production via fermentation. Homoacetogenesis should be prevented for effective optimization via response surface methodology, if substrate is a natural carbon source potential to have intrinsic microorganisms. Copyright © 2017 John Wiley & Sons, Ltd.     

Photocatalytic pretreatment for the redox conversion of waste activated sludge to enhance biohydrogen production

Photocatalytic pretreatment of waste activated sludge (WAS) using a flat photocatalytic reactor was undertaken. Photocatalytic pretreatment enhanced the release of soluble substances from WAS, in which the soluble protein and soluble carbohydrate concentration increased by about 50% and 80%, respectively. Significant removal of heavy metal ions from the liquid phase of WAS was also achieved after photocatalytic pretreatment. In addition, the highest hydrogen yield and the highest concentration of volatile fatty acids (VFAs) were achieved from the photocatalysis pretreated WAS by batch anaerobic digestion (55 °C). The cumulative hydrogen yield from photocatalysis pretreated WAS was 211.0 ml/l-sludge, much higher than those from UV pretreated WAS (111.0 ml/l-sludge) and from raw WAS (93.0 ml/l-sludge). The results indicate that photocatalysis is a promising WAS pretreatment method for the enhancement of biohydrogen production, probably due to the photo-oxidation of organics and simultaneous photo-reduction of heavy metal ions in WAS.     

Biohydrogen production from rice straw: Effect of combinative pretreatment,modelling assessment and energy balance consideration

Biohydrogen production from agro waste biomass through combinative pretreatments is an emerging cost effective, alternative energy technology. The present study aimed to ascertain the extent to which the combinative dispersion thermochemical disintegration (DTCD) enhances the cost effective and energy efficient biohydrogen production from rice straw. The efficiency of the combinative pretreatment was evaluated in terms of degree of disintegration and biohydrogen generation. The optimal conditions for combinative pretreatments are pH 10, temperature 80 °C, rpm 12000 and disintegration time 30 mins. A higher degree of disintegration of about 20.9% was achieved through DTCD pretreatment when compared to dispersion thermal disintegration (DTD) (13.2%) and disperser disintegration (DD) (9.5%). The specific energy spent to achieve maximal degree of disintegration for the three pretreatments were in the following order: DD (1469 kJ/kg Rice Straw) > DTD (1044 kJ/kg Rice Straw) > DTCD (742 kJ/kg Rice Straw). Hence, a considerable amount of energy could be saved through this combinative pretreatment. First order kinetic model (exponential rise to maximum) of biohydrogen production is helpful in deriving the two parameters of uncertainty: substrate biodegradability and hydrolysis rate constant. These two parameters evaluate the maximal biohydrogen yield potential of rice straw through combinative pretreatments. As expected, a higher biohydrogen yield of about (129 mL/g COD) was observed in DTCD when compared to DTD (81 mL/g COD) DD (58 mL/g COD) and Control (8 mL/g COD). To gain insights into the feasibility of implementing the pretreatment at large scale, scalable studies are essential in terms of energy balance and cost. A higher positive net energy of about 0.39621 kWh/kg rice straw was achieved for DTCD when compared to others.