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.     

Biohydrogen production from various feedstocks by Bacillus firmus NMBL-03

In present work our objective was to find out the potential substrates for fermentative hydrogen production using microbial culture of Bacillus firmus NMBL-03 isolated from municipal sludge. A wide variety of substrates (glucose, xylose, arabinose, lactose, sucrose, and starch) and carbohydrate rich waste products (bagasse hydrolysate, molasses, potato peel and cyanobacterial mass) have been used for dark fermentative hydrogen production. All studies were done at optimized physico-chemical conditions. Among all substrates glucose, arabinose, lactose, starch, molasses and bagasse hydrolysate were found to be the favorable substrates for hydrogen production. The highest VHPR i.e. 177.5 ± 7.07 ml/L.h (7.95 ± 0.29 mmol H₂/L.h) and maximum H₂ production (22.58 ± 2.65 mmol H₂/L) was achieved using starch as the substrate. The maximum yield 1.29 ± 0.11 mol H₂/mol reducing sugar was obtained from bagasse hydrolysate as substrate. Butyrate and acetate were detected as the end product in all the cases while lactate was also detected from glucose and cyanobacterial hydrolysate. Since considerable amount of H₂ also evolved when cyanobacterial mass was used, therefore this microbe can be exploited for hydrogen production through a three stage integrated system. Residual carbohydrate containing biomass left after cyanobacterial H₂ production can be utilized in dark fermentative H₂ production. Spent media obtained after dark fermentative H₂ production contain considerable amount of volatile fatty acids that can be potential substrate for photo fermentative H₂ production.     

Biohydrogen production from cornstalk wastes by anaerobic fermentation with activated sludge

An anaerobic fermentation process to produce hydrogen from cornstalk wastes was systematically investigated in this work. Batch experiments numbered series I, II and III were designed to investigate the effects of acid pretreatment, enzymatic hydrolysis (enzymatic temperature, enzymatic time and enzymatic pH) on hydrogen production by using the natural sludge as inoculant. A maximum cumulative H₂ yield of 126.22 ml g⁻¹-CS (Cornstalk, or 146.94 ml g⁻¹-TS, Total Solid) and an average H₂ production rate of 9.58 ml g⁻¹-CS h⁻¹ were obtained from fermentation cornstalk with a concentration of 20 g/L and an initial pH of 7.0 at 36 °C through an optimal pretreatment process. The optimal process was that the substrate was soaked with an HCl concentration of 0.6 wt% at 90 °C for 2 h, and subsequently enzymatic hydrolysis for 72 h at 50 °C and pH 4.8 before fermentation. The biogas consisted of only H₂ and CO₂. In addition, the fermentation system was the typical ethanol-type fermentation according to ethanol and acetate as the main liquid by-products.     

Microbial hydrogen production from sewage sludge bioaugmented with a constructed microbial consortium

A constructed microbial consortium was formulated from three facultative H₂-producing anaerobic bacteria, Enterobacter cloacae IIT-BT 08, Citrobacter freundii IIT-BT L139 and Bacillus coagulans IIT-BT S1. This consortium was tested as the seed culture for H₂ production. In the initial studies with defined medium (MYG), E. cloacae produced more H₂ than the other two strains and it also was found to be the dominant member when consortium was used. On the other hand, B. coagulans as a pure culture gave better H₂ yield (37.16 ml H₂/g COD_consumed) than the other two strains using sewage sludge as substrate. The pretreatment of sludge included sterilization (15% v/v), dilution and supplementation with 0.5% w/v glucose, which was found to be essential to screen out the H₂ consuming bacteria and ameliorate the H₂ production. Considering (1:1:1) defined consortium as inoculum, COD reduction was higher and yield of H₂ was recorded to be 41.23 ml H₂/g COD_reduced. Microbial profiling of the spent sludge showed that B. coagulans was the dominant member in the constructed consortium contributing towards H₂ production. Increase in H₂ yield indicated that in consortium, the substrate utilization was significantly higher. The H₂ yield from pretreated sludge (35.54 ml H₂/g sludge) was comparatively higher than that reported in literature (8.1–16.9 ml H₂/g sludge). Employing formulated microbial consortium for biohydrogen production is a successful attempt to augment the H₂ yield from sewage sludge.     

Application of rice rhizosphere microflora for hydrogen production from apple pomace

The combination of substrate materials and bacteria is an important factor affecting conversion technology for biological hydrogen production. We performed anaerobic hydrogen fermentation of apple pomace wastes using rhizosphere bacterial microflora of rice as the parent inoculum. In the vial test, the optimal condition for hydrogen fermentation was initial pH 6.0, 35 °C, and 73.4 g pomace per liter of medium (equivalent to 10 g-hexose/L). In the batch experiment (pH 6.0, temperature 35 °C) the hydrogen yield reached 2.3 mol-H₂/mol-hexose. The time course of biogas production and PCR-DGGE analysis suggest that Clostridium spp. decomposed degradable carbohydrates rapidly and a part of the refractory carbohydrate (e.g. pectin) gradually in the apple pomace slurry. In addition to hydrogen, volatile fatty acids (VFAs) were produced in the anaerobic fermentation of apple pomace, which can be a substrate for methane fermentation. The rice rhizosphere can be a promising source of inoculum bacteria for hydrogen fermentation in combination with plant material waste like apple pomace.     

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 production from poplar leaves pretreated by different methods using anaerobic mixed bacteria

Leaves are one of the main by-products of forestry. In this study, batch experiments were carried out to convert poplar leaves pretreated by different methods into hydrogen using anaerobic mixed bacteria at 35 °C. The effects of acid (HCl), alkaline (NaOH) and enzymatic (Viscozyme L, a mixture of arabanase, cellulase, β-glucanase, hemicellulase and xylanase) pretreatments on the saccharification of poplar leaves were studied. Furthermore, the effects of acid and enzymatic pretreatment on hydrogen production, together with their corresponding degradation efficiencies for the total reducing sugar (TRS) and metabolites were compared. A maximum cumulative hydrogen yield of 44.92 mL/g-dry poplar leaves was achieved from substrate pretreated with 2% Vicozyme L, which was approximately 3-fold greater than that in raw substrate and 1.34-fold greater than that from substrate pretreated with 4% HCl. The results show that enzymatic pretreatment is an effective method for enhancing the hydrogen yield from poplar leaves.     

Biohydrogen production from wastewater-based microalgae: Progresses and challenges

Microalgae originating from wastewater has been exhibiting particularly promising results in terms of biohydrogen production and wastewater treatment. This paper aims to review the factors affecting production, pretreatment techniques to improve synthesis, advanced technologies utilized for enhancing biohydrogen production, and techno-economic feasibility evaluation of the processes at a commercial scale. Microalgae possess metabolic components to synthesize biohydrogen using photobiological and fermentative processes but must undergo pretreatment for efficient biohydrogen production. The efficiency of these processes is influenced by factors such as the microalgae species, light intensity, cell density, pH, temperature, substrates, and the type of bioreactors. Moreover, many limitations, such as oxygen sensitivity, altered thylakoid constitution, low photon conversion efficiency, light capture disruption, and the evolution of harmful by-products hinder the sustainability of biohydrogen production processes. High operational and maintenance costs serve as the major bottleneck in the scaling up of the process as an industrial technology. Therefore, future research needs to be directed towards increasing optimization of the processes by reducing energy and resource demand, recycling metabolic wastes and process components, genetically engineered microalgae to adopt more efficient routes, and conducting pilot studies for commercialization.     

Biohydrogen production from immobilized cells and suspended sludge systems with condensed molasses fermentation solubles

The anaerobic fermentation using the condensed molasses fermentation solubles (CMS) as substrate in a continuously stirred anaerobic bioreactor (CSABR) was carried out for optimal hydrogen production performance of biohydrogen production rate and yield, where as two kinds of bioreactors used. One is a suspended sludge bioreactor (SSB) which used suspended seed sludge. The other bioreactor is an immobilized cell bioreactor (ICB) which used immobilized cells and mix the same seed sludge in the SSB as the source of the bacteria. It was found that the hydrogen production rate increased with a decrease in the hydraulic retention time (HRT), when substrate concentration was 40 g COD/L in an SSB as well as maximum hydrogen production rate of 14.04 ± 2.08 L/d/L obtained at HRT 0.5 h (ca. 5.78 times value of HRT 4 h) in the SSB system. The hydrogen production rate at low dilution rate (HRT > 4 h), in the ICB is better than SSB, meanwhile at a high dilution rate (HRT < 4 h), due to the presence of enriched granules in the SSB (12.30 g VSS/L), absent in the ICB (9.89 g VSS/L), the hydrogen production rate was 7.60 ± 1.05 L/d/L (ca. 1.23 times higher than HRT 4 h), which was lower than the rate in the SSB. Eventually, the hydrogen production rate increased by increasing the substrate concentrations from 40 to 60 g COD/L within the HRT range of 2–4 h in both the SSB as well as in ICB systems.     

Biohydrogen production from dual digestion pretreatment of poultry slaughterhouse sludge by anaerobic self-fermentation

Poultry slaughterhouse sludge from chicken processing wastewater treatment plant was tested for their suitability as a substrate and inoculum source for fermentation hydrogen production. Dual digestion of poultry slaughterhouse sludge was employed to produce hydrogen by batch anaerobic self-fermentation without any extra-seeds. The sludge (5% TS) was dual digested by aerobic thermophilic digestion at 55 °C with the varying retention time before using as substrate in anaerobic self-fermentation. The best digestion time for enriching hydrogen-producing seeds was 48 h as it completely repressed methanogenic activity and gave the maximum hydrogen yield of 136.9 mL H₂/g TS with a hydrogen production rate of 2.56 mL H₂/L/h. The hydrogen production of treated sludge at 48 h (136.9 mL H₂/g TS) was 15 times higher than that of the raw sludge (8.83 mL H₂/g TS). With this fermentation process, tCOD value in the activated sludge could be reduced up to 30%.     

The preparation and application of crude cellulase for cellulose-hydrogen production by anaerobic fermentation

Strategies were adopted to cost-efficiently produce cellulose-hydrogen by anaerobic fermentation in this paper. First, cellulase used for hydrolyzing cellulose was prepared by solid-state fermentation (SSF) on cheap biomass from Trichoderma viride. Several cultural conditions for cellulase production on cheap biomass such as moisture content, inoculum size and culture time were studied. And the components of solid-state medium were optimized using statistical methods to further improve cellulase capability. Second, the crude cellulase was applied to cellulose-hydrogen process directly. The maximal hydrogen yield of 122 ml/g-TVS was obtained at the substrate concentration of 20 g/L and cultured time of 53 h. The value was about 45-fold than that of raw corn stalk wastes. The hydrogen content in the biogas was 44–57%(v/v) and there was no significant methane gas observed.     

Characterization of cell growth and photobiological H2 production of Chlamydomonas reinhardtii in ASSF industry wastewater

Photobiological H₂ production in microalgae is a promising approach for the development of alternative clean and renewable energy. As a unicellular green alga, Chlamydomonas reinhardtii is regarded as an ideal candidate for sustainable photo-H₂ production. However, growth and photo-H₂ producing are still expensive and energy extensive. Wastewater has been suggested as an economical resource for microalgae growth and biofuels production. In this study, we characterized the cell growth and photo-H₂ production of C. reinhardtii CC503 cultured in waste water from pressing process of fermented sweet sorghum stalks during Advanced Solid State Fermentation (ASSF). The maximal cells concentration reached 8.9 × 10⁶ cells/mL in ASSF wastewater medium (AWM) with the fastest growth rate of 0.19 × 10⁶ cells/h, compared to 18.2 × 10⁶ cells/mL and 0.36 × 10⁶ cells/h in TAP medium and to 1.3 × 10⁶ cells/mL and 0.02 × 10⁶ cells/h in BGII medium respectively. The optimized concentration of wastewater for algae cells growth was determined to be 13.3% (7.5 folds dilution), under which, surprisingly the photosynthetic H₂ evolution was increased by more than 700% compared to the cells grown in TAP medium. This system appears to be a good strategy for the development of an economical microalgal photobiological H₂ production scheme. Finally, the possible mechanism for such an H₂ enhancement was identified as the reduction of PSII activity in AWM grown cells.     

Biohydrogen production from ricebran using Clostridium saccharoperbutylacetonicum N1-4

Treated ricebran hydrolysate was fermented anaerobically using Clostridium saccharoperbutylacetonicum N1-4 at an initial pH of 6 ± 0.2 and an operating temperature of 30 °C for production of hydrogen. The effects of different pretreatment methods on the liberation of sugar from 100 g of ricebran per litre of medium (distilled water) were investigated. In addition, the effects of the pretreatment method on ricebran hydrolysates of different initial ricebran concentrations on liberated sugar as well as the effects of the initial inoculum concentration, ricebran (substrate) concentration, and FeSO₄·7H₂O concentration on the yield as well as the productivity of hydrogen were investigated. The combination of enzymatic hydrolysis and a boiling pretreatment method produced the most fermentable sugar, 29.03 ± 0.0 g/L from 100 g of ricebran per litre of medium (distilled water), while the amount of sugar liberated by ricebran hydrolysates of different initial ricebran concentrations upon pretreatment monotonically increased with the initial ricebran concentration. The increment in substrate, inoculum, and FeSO₄·7H₂O concentrations had a significantly positive effect (p < 0.05) on both the yield and productivity of hydrogen. The maximum hydrogen gas yield (Y_P/S) and productivity of 3.37 mol-H₂ per mol-sugar consumed and 7.58 mmol/(L h), respectively, were obtained from ricebran hydrolysate with a 100 g/L ricebran concentration (equivalent to 28.59 ± 1.27 g sugar/L). In other experiments, 0.03 g/L FeSO₄·7H₂O and 1.5 g/L inoculum resulted in the best hydrogen gas yield and productivity from ricebran hydrolysates.     

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.     

Biohydrogen production by Ethanoligenens harbinense B49: Nutrient optimization

A new hydrogen-producing bacterial strain Ethanoligenens harbinense B49 was examined for its capability of H₂ production with glucose as sole carbon source. The H₂ production was significantly affected by the concentration of the yeast powder and phosphate in the synthetic medium. The optimized concentration of yeast powder was 0.3–0.5 g/L and the maximum hydrogen yield was obtained at the concentration of phosphate about 100–150 mmol/L. The dynamics of hydrogen production showed that rapid evolution of hydrogen appeared to start after the middle-phase of exponential growth (about 8 h). The maximum H₂ yield and specific hydrogen production rate were estimated to be 2.26 mol H₂/mol glucose and 27.74 mmol H₂/g cell, respectively, when 10 g/L of glucose was present in the medium. The possible pathway of hydrogen production by Ethanoligenens sp. B49 during glucose fermentation was oxidative decarboxylation of pyruvate and the NADH pathway.     

热处理对污泥厌氧发酵产氢的影响

通过对污泥进行热处理来提高污泥厌氧发酵产氢的能力.结果表明:热处理是一种有效的污泥融胞方法,热处理对糖和蛋白质的水解效果好,热处理后污泥中可溶蛋白质浓度为原污泥的6.4～8.9倍,可溶糖浓度为原污泥的1.6～7.9倍.75℃热处理10 min效果最好,最大累积产氢量可达20.3 ml,较原污泥提高了19倍;VS最大比产氢率为152.2 ml·(kg·h)-1.并用SGompertz方程对实验数据进行拟合,定量说明在不同的热处理温度和时间下,厌氧发酵的累积产氢量(y)和时间(x)的关系.污泥厌氧发酵产氢前后各指标都发生明显变化,NH4 -N和总挥发性脂肪酸(TVFA)的浓度都增加了,而可溶糖和可溶蛋白质的浓度都降低了.热处理后的污泥在厌氧发酵产氢过程中,主要降解的有机物为蛋白质,发酵后蛋白质可降解20%～41%.     

替硝唑片对污泥厌氧发酵产氢作用的研究

研究表明，在污泥中添加替硝唑片可使污泥中的有机物质含量明显增加，特别是可溶的有机物质；替硝唑片对提高污泥产氢效果显著，替硝唑片添加量为1．40g时，污泥的产氢效果最佳，其累积产氢量和产氢潜能分别为580．64mL和98．41mL／gVS；厌氧发酵属于典型的乙醇型发酵；发酵代谢过程主要降解的有机物质为糖类物质，总糖降解率高达70．32％，蛋白质降解率只有26.25％。     

Enhancing the performance of dark fermentative hydrogen production using a reduced pressure fermentation strategy

The partial pressure of hydrogen is an extremely important factor for hydrogen generation. This study investigated the effect of reduced pressure (via vacuum) on hydrogen production in a CSTR reactor. The results show that the reduced pressure condition is more effective in enhancing H₂ production at lower HRT (e.g., 8–4 h) than at higher HRT (e.g., 12 h). The optimal hydrogen yield and overall hydrogen production efficiency occurred at a HRT of 6 h with a value of 4.50 mol H₂/mol sucrose and 56.2%, respectively. Meanwhile, at HRT 6 h the hydrogen production rate was 0.937 mol/L/d. In addition, the HPR could be further improved to 1.196 mol/L/d when the HRT was shortened to 4 h, obtaining a 37–271% increase in HPR when compared with that described in the relevant reports. For all experiments, butyrate and acetate were the two primary soluble metabolites, accounting for 85–99% of total soluble microbial products. Predominant production of acetate and butyrate demonstrates the efficient H₂ fermentation with reduced pressure processes.