Comparison of fermentative hydrogen production from glycerol using immobilized and suspended mixed cultures

This study explored the fermentative hydrogen production by immobilized microorganisms from glycerol, which is the byproduct of biodiesel production, and compared it with suspended fermentation. The effect of immobilization on hydrogen production process was examined. Results showed that both cumulative hydrogen production (CHP) and hydrogen yield (HY) were enhanced by microbial immobilization. The highest CHP and HY of 64 mL/100 mL and 0.52 mol H₂/mol glycerol were obtained by immobilized microorganisms, compared to 9 mL/100 mL and 0.29 mol H₂/mol glycerol in suspended microorganisms. Immobilization enhanced CHP and HY by 611.1% and 79.3%. In addition, immobilized microorganisms showed stronger tolerance to high substrate concentration and higher capability in glycerol utilization, which is of great significance for hydrogen production from glycerol. The enhanced hydrogen production may be due to the favorable micro-environment for different microorganisms in immobilized beads.     

Enhancing photo fermentative hydrogen production using ethanol rich dark fermentation effluents

The present study demonstrates the feasibility of a two-phase biorefinery process applied to waste substrates producing ethanol rich effluents. The process includes a dark fermentation step followed by photo fermentation and it is able to optimize hydrogen production from waste biomass. The study was conducted using winery wastewater as feedstock. The results indicate that no additional treatments are required when an appropriate dilution of the initial waste is applied. Microbial consortia contained in the winery wastewater promoted a fermentative ethanol pathway. The ethanol rich effluent was converted into hydrogen by phototrophic microorganisms. Despite the presence of inhibiting compounds, the adoption of a mixed phototrophic culture allowed to obtain good results in terms of hydrogen production. Specifically, up to 310 mLH₂ gCOD_consumed^?1 were obtained in the photo fermentative stage. The effectiveness of ethanol rich dark fermentation effluents for hydrogen production enhancement was demonstrated. Noteworthy, polyhydroxybutyrate was also produced during the experiments. The work faces two of the major challenges in the sequential dark fermentation and photo fermentation technology applied to real waste substrates: the minimization of pre-treatments and the enhancement of the hydrogen production yields using ethanol rich DFEs.     

Thermophilic fermentative hydrogen production from various carbon sources by anaerobic mixed cultures

In the present work, various carbon sources, xylose, glucose, galactose, sucrose, cellobiose, and starch were tested for thermophilic (60 °C) fermentative hydrogen production (FHP) by using the anaerobic mixed culture. An inoculum was obtained from a continuously-stirred tank reactor (CSTR) operated at pH 5.5 and HRT 12 h, and fed with tofu processing waste. The dominant species in the CSTR were found to be Thermoanaerobacterium thermosaccharolyticum and Clostridium thermosaccharolyticum, which are well known thermophilic H₂-producers in anaerobic-state, and have the ability to utilize a wide range of carbohydrates. When initial pH was adjusted to 6.8 ± 0.1 but not controlled during fermentation, vigorous pH drop began within 5 h, and finally reached 4.0–4.5 in all carbon sources. Although over 90% of substrate removal was achieved for all carbon sources except cellobiose (71.7%), the fermentation performances were profoundly different with each other. Glucose, galactose, and sucrose exhibited relatively higher H₂ yields whereas lower H₂ yields were observed for xylose, cellobiose, and starch. On the other hand, when pH was controlled (pH ≥ 5.5), the fermentation performance was enhanced in all carbon sources but to a different extent. A substantial increase in H₂ production was observed for cellobiose, a 1.9-fold increase of H₂ yield along with a substrate removal increase to 93.8%, but a negligible increase for xylose. H₂ production capabilities of all carbon sources tested were as follows: sucrose > galactose > glucose > cellobiose > starch > xylose. The maximum H₂ yield of 3.17 mol H₂/mol hexose_added achieved from sucrose is equivalent to a 26.5% conversion of energy content in sucrose to H₂. Acetic and butyric acids were the main liquid-state metabolites of all carbon sources while lactic acid was detected only in cellobiose, starch and xylose exhibiting relatively lower H₂ yields.     

Modeling fermentative hydrogen production from sucrose supplemented with dairy manure

Our previous studies had shown that fermentative hydrogen production from sucrose could be improved with dairy manure as a supplement. In addition to contributing to nearly 10% more hydrogen yield at ambient temperature, dairy manure was shown to be capable of providing the required nutritional needs, buffering capacity, and hydrogen-producing organisms, improving the practical viability of fermentative hydrogen production. In this report, we present a kinetic model for fermentative hydrogen production from sucrose supplemented with dairy manure. This model includes hydrogen production from sucrose as well as from the soluble products hydrolyzed from particulate manure. The integrated model was calibrated using experimental data from one batch reactor and validated with dissolved COD, hydrogen, and volatile fatty acid data from four other reactors. Predictions by this model agreed well with the temporal trends in the experimental data, with r² averaging 0.85 for dissolved COD; 0.94 for total COD; 0.84 for hydrogen; 0.84 for acetic acid; and 0.89 for butyric acid; quality of fit in the case of propionic acid was lower with r² averaging 0.57.     

Fermentative bio-hydrogen production from cellulose by cow dung compost enriched cultures

The performance of hydrogen production from cellulose by the cow dung compost enriched continuously in defined medium containing cellulose was investigated. In the initial experiments, batch-fermentation was carried out to observe the effects of different substrate concentration conditions on the rate of cellulose-degrading, growth of bacteria and the capability of hydrogen-producing from cellulose. The result showed that the cellulose degradation decreased from 55% at 5 g/l to 22% at 30 g/l. The maximum cumulative hydrogen production and the rate of hydrogen production first increased from 828 ml/l at 5 g/l to 1251 ml/l at 10 g/l then remained constant beyond 10 g/l. The maximum hydrogen production potential, the rate of hydrogen production and the yield of hydrogen was 1525 ml/l, 33 ml/l.h, and 272 ml/g-cellulose (2.09 mol/mol-hexose) was obtained at substrate concentration 10 g/l, the hydrogen concentration in biogas was 47–50%(v/v) and there was no methane observed. During the conversion of cellulose into hydrogen, acetate and butyrate were main liquid end-products in the metabolism of hydrogen fermentation. These results proposed that cow dung compost enriched cultures were ideal microflora for hydrogen production from cellulose.     

Hydrogen production from xylose by moderate thermophilic mixed cultures using granules and biofilm up-flow anaerobic reactors

Continuous H₂ production from xylose by granules and biofilm up-flow anaerobic reactor using moderate thermophilic mixed cultures was investigated. The maximum H₂ yield of 251 mL H₂/g-xylose with H₂production rate of 15.1 L H₂/L⋅d was obtained from granules reactor operating at the organic loading rate (OLR) of 60 g-xylose/L⋅d and hydraulic retention time (HRT) of 4 h. Meanwhile the highest H₂ production rate of 13.3 L H₂/L⋅d with an H₂ yield of 221 mL H₂/g–xylose was achieved from the biofilm reactor. Both reactors were dominated by Thermoanaerobacterium species with acetate and butyrate as main fermentation products. The microbial community of the biofilm reactor was composed of Thermoanaerobacterium species, while granules reactor was composed of Clostridium sp., Thermoanaerobacterium sp. and Caloramator sp. The granular reactor was more microbial diversity and more balance between economic efficiency in term of the hydrogen production rate and technical efficiency in term of hydrogen yield.     

木糖厌氧发酵产氢特性研究

以木糖作为厌氧发酵产氢底物,热预处理(100℃,处理20 min)的厌氧颗粒污泥作为接种物,研究了中温条件(37℃)下厌氧发酵产氢特性.结果表明,当反应进行至50 h时,累积产氢量最大,为81.11 mL;乙酸、丁酸和乙醇是液相末端产物中的主要物质,其中乙酸和丁酸的浓度分别为1290 mg/L和1225 mg/L,发酵类型是典型的丁酸型发酵;反应体系的pH值开始降低,最后稳定在4.40左右,形成一个稳定的缓冲体系.     

Thermophilic hydrogen production from cellulose with rumen fluid enrichment cultures: Effects of different heat treatments

Elevated temperatures (52, 60 and 65 °C) were used to enrich hydrogen producers on cellulose from cow rumen fluid. Methanogens were inhibited with two different heat treatments. Hydrogen production was considerable at 60 °C with the highest H₂ yield of 0.44 mol-H₂ mol-hexose⁻¹ (1.93 mol-H₂ mol-hexose-degraded⁻¹) as obtained without heat treatment and with acetate and ethanol as the main fermentation products. H₂ production rates and yields were controlled by cellulose degradation that was at the highest 21%. The optimum temperature and pH for H₂ production of the rumen fluid enrichment culture were 62 °C and 7.3, respectively. The enrichments at 52 and 60 °C contained mainly bacteria from Clostridia family. At 52 °C, the bacterial diversity was larger and was not affected by heat treatments. Bacterial diversity at 60 °C remained similar between heat treatments, but decreased during enrichment. At 60 °C, the dominant microorganism was Clostridium stercorarium subsp. leptospartum.     

Fermentative hydrogen production from macro-algae Laminaria japonica using anaerobic mixed bacteria

In this study, one macro-alga (Laminaria japonica) was used for fermentative hydrogen production by anaerobic mixed bacteria. The saccharification efficiency and hydrogen production by L. japonica with four different pretreatment methods, including heat, acid, alkaline and ultrasonic treatment, were investigated. The results showed that the saccharification efficiency from L. japonica that was pretreated with acid was the highest among the four methods. The saccharification efficiency for the total reducing sugars in the acid-pretreated L. japonica was 350.54 ± 19.89 mg/g (mean ± S.E.). The cumulative hydrogen production was 66.68 ± 5.68 mL/g from the heat-pretreated L. japonica, whereas that of L. japonica that was subjected to acid, alkaline, and ultrasonic pretreatment and the control was 43.65 ± 6.87 mL/g, 15.00 ± 3.89 mL/g, 23.56 ± 4.56 mL/g and 10.00 ± 1.21 mL/g, respectively. In addition, the effects of substrate concentration and initial pH on hydrogen production from heat-pretreated L. japonica were also analyzed. The results showed that the maximum hydrogen production was 83.45 ± 6.96 mL/g with a hydrogen concentration of approximately 28.4% from heat-pretreated L. japonica when the initial pH and substrate concentration were determined to be 6.0 and 2%, respectively. Heat pretreatment was the most effective method for increasing fermentative hydrogen production when L. japonica was used as the only substrate.     

Biohydrogen production from mixed xylose/arabinose at thermophilic temperature by anaerobic mixed cultures in elephant dung

Biohydrogen production by batch fermentation of mixed xylose/arabinose at thermophilic temperature using anaerobic mixed cultures in elephant dung as the seed inoculums was investigated. Elephant dung was heat-treated in boiling water for 2 h before used as the seed inoculum in order to inhibit methanogenic activity. Biohydrogen was successfully produced from mixed xylose/arabinose. The optimum conditions for hydrogen production were the initial concentration of mixed xylose/arabinose 5 g/L each, initial cultivation pH 5.5 and temperature 55 °C. Under the optimum conditions, a maximum hydrogen yield of 2.49 mol-H₂/mol-sugar consumed was obtained. The optimum conditions were then used to produce hydrogen from sugar derived from acid-hydrolysed sugarcane bagasse (SCB) at a reducing sugar concentration of 10 g/L in which a lower hydrogen yield of 1.48 mol-H₂/mol-sugar consumed was achieved. Main soluble product was acetate suggesting the hydrogen fermentation from mixed xylose/arabinose is the acetate type. The dominant hydrogen producers found in both fermentation broth were Thermoanaerobacterium thermosaccharolyticum and Clostridium sp. Lower hydrogen yield in the SCB hydrolysate fermentation broth may be due to the present of Clostridium ragsdalei and microorganisms in the class Bacilli viz. Lactococus lactis subsp., Lactobacillus delbrueckii, and Sporolactobacillus sp. as well as the inhibitors (acetic acid and furfural) contained in the SCB hydrolysate.     

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.     

Effect of temperature on anaerobic fermentative hydrogen gas production from feedlot cattle manure using mixed microflora

Hydrogen production from the anaerobic fermentation of feedlot cattle manure was examined in batch cultures over a temperature range from 36 to 60 °C at a pH of 5.2. The amount of hydrogen produced increased with temperature to a maximum of 65 L H₂ kg TS⁻¹ at 52 °C. At temperatures > 52 °C, acetate was the main volatile fatty acid (VFA) accumulated, while at <52 °C butyrate accumulated the most. Formate was detected in the 56 and 60 °C treatments but was absent in all others. Thermophilic conditions resulted in the highest hydrogen production rates, with maximum hydrogen production occurring 52 °C. Changing incubation temperature by small (4 °C) increments up or down from 52 °C resulted in changes in the metabolic flux (conversion of substrate to VFA and gaseous products) of the anaerobic digestion system. These findings indicate that the hydrogen production potential of anaerobic systems utilizing heat treated cattle manure as inoculum is affected greatly by incubation temperature.     

Effects of N/C,P/C and Fe/C ratios on dark fermentative hydrogen gas production from waste paper towel hydrolysate

The effects of N/C, P/C and Fe/C ratios on dark fermentative hydrogen gas production from activated carbon treated WPT hydrolysate were investigated using Box–Behnken statistical experiment design. N/C, P/C and Fe/C ratios were chosen as independent variables while the H₂ yield and SHPR were set as the objective functions. H₂ yield and SHPR functions were described by two quadratic model functions. The addition of a proper amount of N, P and Fe to the fermentation media was found to be essential to enhance the H₂ production performance. Linear and interaction terms of N/C and Fe/C did have a significant effect on the H₂ yield in the model function. However, the SHPR was significantly affected by the linear and interaction terms of N/C and P/C. The most convenient N/C, P/C and Fe/C ratios resulting maximum H₂ yield (0.656 mol H₂/mol glucose) and SHPR (241.64 mL H₂/g biomass.h) were determined as 0.05, 0.09 and 0.003 (w/w), respectively.     

Fermentative hydrogen production from starch using natural mixed cultures

Batch and continuous tests were conducted to evaluate fermentative hydrogen production from starch (at a concentration of chemical oxygen demand (COD) 20 g/L) at 35 °C by a natural mixed culture of paper mill wastewater treatment sludge. The optimal initial cultivation pH (tested range 5–7) and substrate concentration (tested range 5–60-gCOD/L) were evaluated by batch reactors while the effects of hydraulic retention time (HRT) on hydrogen production, as expressed by hydrogen yield (HY) and hydrogen production rate (HPR), were evaluated by continuous tests. The experimental results indicate that the initial cultivation pH markedly affected HY, maximum HPR, liquid fermentation product concentration and distribution, butyrate/acetate concentration ratio and metabolic pathway. The optimal initial cultivation pH was 5.5 with peak values of HY 1.1 mol-H₂/mol-hexose maximum HPR 10.4 mmol-H₂/L/h and butyrate concentration 7700 mg-COD/L. In continuous hydrogen fermentation, the optimal HRT was 4 h with peak HY of 1.5 mol-H₂/mol-hexose, peak HPR of 450 mmol-H₂/L/d and lowest butyrate concentration of 3000 mg-COD/L. The HPR obtained was 280% higher than reported values. A shift in dominant hydrogen-producing microbial population along with HRT variation was observed with Clostridium butyricum, C. pasteurianum, Klebshilla pneumoniae, Streptococcus sp., and Pseudomonas sp. being present at efficient hydrogen production at the HRTs of 4–6 h. Strategies based on the experimental results for optimal hydrogen production from starch are proposed.     

Commercial application scenario using patent analysis: Fermentative hydrogen production from biomass

The main purpose of this study is to use patent analysis to investigate scenarios for future commercial applications of dark fermentation or anaerobic fermentation using biomass or organic matter as feedstock materials. The first step in this study includes a patent search procedure and patent content interpretation, in which 29 technology patents were identified from the US patent database and divided into five groups in accordance with the scope of their technical applications. The following five scenarios of commercial applications of biomass fermentation for hydrogen production were established through a combination of group applications: screening and cultivation of hydrogen-producing bacteria, biomass waste sources, biomass energization application, value enhancement of waste or wastewater treatment systems, and the application of a multi-functional hydrogen production system integrated with other technologies.     

Homoacetogenesis during hydrogen production by mixed cultures dark fermentation: Unresolved challenge

This paper is a comprehensive review of H₂ consumption during anaerobic mixed culture H₂ dark fermentation with a focus on homoacetogenesis. Homoacetogenesis consumed from 11% to 43% of the H₂ yield in single and repeated batch fermentations, respectively. However, its quantification and extent during continuous fermentation are still not well understood. Models incorporating thermodynamic and kinetic controls are required to provide insight into the dynamic of homoacetogenesis during H₂ dark fermentation. Currently, no adequate method exists to eliminate homoacetogenesis because it does not depend on the culture's source, pre-treatment, substrate, type of reactor, or operation conditions. Controlling CO₂ concentrations during dark fermentation needs further investigation as a potential strategy towards controlling homoacetogenesis. Incorporating radioactive labeling technique in H₂ fermentation research could provide information on simultaneous production and consumption of H₂ during dark fermentation. Genetic studies investigating blocking H₂ consuming pathways and enhancing H₂ evolving hydrogenases are suggested towards controlling homoacetogenesis during dark fermentation.     

Towards the integration of dark- and photo-fermentative waste treatment. 2. Optimization of starch-dependent fermentative hydrogen production

Eight natural microbial consortia collected from different sites were tested for dark, hydrogen production during starch degradation. The most active consortium was from silo pit liquid under mesophilic (37 °C) conditions. The fermentation medium for this consortium was optimized (Fe, NH₄⁺, phosphates, peptone, and starch content) for both dark fermentation and for subsequent purple photosynthetic bacterial H₂ photoproduction [Laurinavichene TV, Tekucheva DN, Laurinavichius KS, Ghirardi ML, Seibert M, Tsygankov AA. Towards the integration of dark and photo fermentative waste treatment. 1. Hydrogen photoproduction by purple bacterium Rhodobacter capsulatus using potential products of starch fermentation. Int J Hydrogen Energy 2008;33(23):7020–26], in the presence of the spent dark, fermentation effluent. The addition of Zn (10 mg L⁻¹), as a methanogenesis inhibitor that does not inhibit purple bacteria at this concentration, also did not inhibit dark, fermentative H₂ production. The influence of various fermentation end products at different concentrations (up to 30 g L⁻¹) on dark, H₂ production was also examined. Added lactate stimulated, but added isobutyrate and butanol strongly inhibited gas production. Under optimal conditions the fermentation of starch (30 g L⁻¹) resulted in 5.7 L H₂ L⁻¹ of culture (1.6 mol H₂ per mole of hexose) with the co-production mainly of butyrate and acetate.