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.     

Biohydrogen production from oil palm frond juice and sewage sludge by a metabolically engineered Escherichia coli strain

Biohydrogen is considered a promising and environmentally friendly energy source. Escherichia coli BW25113 hyaB hybC hycA fdoG frdc ldhA aceE has been previously engineered for elevated biohydrogen production from glucose. In this study, we show that this strain can also use biomass from oil palm frond (OPF) juice and sewage sludge as substrates. Substrate improvement was accomplished when hydrogen productivity increased 8-fold after enzymatic treatment of the sludge with a mixture of amylase and cellulase. The OPF juice with sewage sludge provided an optimum carbon/nitrogen ratio since the yield of biohydrogen increased to 1.5 from 1.3 mol H₂/mol glucose compared to our previous study. In this study, we also reveal that our engineered strain improved 200-fold biohydrogen productivity from biomass sources compared to the unmodified host. In conclusion, we determined that our engineered strain can use biomass as an alternative substrate for enhanced biohydrogen production.     

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.     

Mesophilic biohydrogen production from calcium hydroxide treated wheat straw

The use of Ca(OH)₂ pre-treatment to improve fermentative biohydrogen yields, from wheat straw was investigated. Wheat straw was pre-treated with 7.4% (w/w) Ca(OH)₂ at ambient temperature (20 °C) for 2, 5, 8, and 12 days, prior to 35 °C fermentation with sewage sludge inoculum. Biohydrogen yields were evaluated during dark fermentation and simultaneous saccharification fermentation (SSF) of total pre-treated straw material and compared to those from separated solid and hydrolysate fractions. Ca(OH)₂ pre-treatment followed by SSF, exhibited a synergetic relationship. On average, 58.78 mL-H₂ g-VS⁻¹ was produced from SSF of pre-treated and filtered solids. This was accompanied by approximately a 10-fold increase in volatile fatty acid production, compared to the untreated control. By omitting pre-treatment hydrolysate liquors from SSF, H₂ production increased on average by 35.8%, per VS of harvested straw. Additional inhibition studies indicated that CaCO₃, formed as a result of pre-treatment pH control, could promote homoacetogenesis and reduce biohydrogen yields.     

Effects of process,operational and environmental variables on biohydrogen production using palm oil mill effluent (POME)

A batch study for biohydrogen production was conducted using raw palm oil mill effluent (POME) and POME sludge as a feed and inoculum respectively. Response Surface Methodology (RSM) was used to design the experiments. Experiments were conducted at different reaction temperatures (30–50 °C), inoculum size to substrate ratios (I:S) and reaction times (8–24 h). An optimum condition of biohydrogen production was achieved with COD removal efficiency of 21.95% with hydrogen yield of 28.47 ml H₂ g^?1 COD removed. The I:S ratio was 40:60, with reaction temperature of 50 °C at 8 h of reaction time. The study showed that a lower substrate concentration (less than 20 g L^?1) for biohydrogen production using pre-settled POME was achievable, with optimum HRT of 8 h under thermophilic condition (50 °C). This study also found that pre-settled POME is feasible to be used as a substrate for biohydrogen production under thermophilic condition.     

Biogas production from municipal sewage sludge (MSS)

Biogas is produced by anaerobic (oxygen free) digestion of organic materials such as sewage sludge, animal waste, and municipal solid wastes (MSW). As sustainable clean energy carrier biogas is an important source of energy in heat and electricity generation, it is one of the most promising renewable energy sources in the world. Biogas is produced from the anaerobic digestion (AD) of organic matter, such as manure, MSW, sewage sludge, biodegradable wastes, and agricultural slurry, under anaerobic conditions with the help of microorganism. Biogas is composed of methane (55–75%), carbon dioxide (25–45%), nitrogen (0–5%), hydrogen (0–1%), hydrogen sulfide (0–1%), and oxygen (0–2%). The sewage sludge contains mainly proteins, sugars, detergents, phenols, and lipids. Sewage sludge also includes toxic and hazardous organic and inorganic pollutants sources. The digestion of municipal sewage sludge (MSS) occurs in three basic steps: acidogen, methanogens, and methanogens. During a 30-day digestion period, 80–85% of the biogas is produced in the first 15–18 days. Higher yields were observed within the temperature range of 30–60°C and pH range of 5.5–8.5. The MSS contains low nitrogen and has carbon-to-nitrogen (C/N) ratios of around 40–70. The optimal C/N ratio for the AD should be between 25 and 35. C/N ratio of sludge in small-scale sewage plants is often low, so nitrogen can be added in an inorganic form (ammonia or in organic form) such as livestock manure, urea, or food wastes. Potential production capacity of a biogas plant with a digestion chamber size of 500 m³ was estimated as 20–36 × 10³ Nm³ biogas production per year.     

Hydrogen and methane production from untreated rice straw and raw sewage sludge under thermophilic anaerobic conditions

Bioenergy produced from co-digestion of sewage sludge (SS) and rice straw (RS) as raw materials, without pretreatment and additional nutrients, was compared for the one-stage system for producing methane (CH₄) and the two-stage system for combined production of hydrogen (H₂) and CH₄ in batch experiments under thermophilic conditions. In the first stage H₂ fermentation process using untreated RS with raw SS, we obtained a high H₂ yield (21 ml/g-VS) and stable H₂ content (60.9%). Direct utilization of post-H₂ fermentation residues readily produced biogas, and significantly enhanced the CH₄ yield (266 ml/g-VS) with stable CH₄ content (75–80%) during the second stage CH₄ fermentation process. Overall, volatile solids removal (60.4%) and total bioenergy yield (8804 J/g-VS) for the two-stage system were 37.9% and 59.6% higher, respectively, than the one-stage system. The efficient production of bioenergy is believed to be due to a synergistically improved second stage process exploiting the well-digested post-H₂ generation residues over the one-stage system.     

Feasibility of biohydrogen production by anaerobic co-digestion of food waste and sewage sludge

Anaerobic co-digestion of food waste and sewage sludge for hydrogen production was performed in serum bottles under various volatile solids (VS) concentrations (0.5–5.0%) and mixing ratios of two substrates (0:100–100:0, VS basis). Through response surface methodology, empirical equations for hydrogen evolution were obtained. The specific hydrogen production potential of food waste was higher than that of sewage sludge. However, hydrogen production potential increased as sewage sludge composition increased up to 13–19% at all the VS concentrations. The maximum specific hydrogen production potential of 122.9 ml/g carbohydrate-COD was found at the waste composition of 87:13 (food waste:sewage sludge) and the VS concentration of 3.0%. The relationship between carbohydrate concentration, protein concentration, and hydrogen production potential indicated that enriched protein by adding sewage sludge might enhance hydrogen production potential. The maximum specific hydrogen production rate was 111.2 ml H₂/g VSS/h. Food waste and sewage sludge were, therefore, considered as a suitable main substrate and a useful auxiliary substrate, respectively, for hydrogen production. The metabolic results indicated that the fermentation of organic matters was successfully achieved and the characteristics of the heat-treated seed sludge were similar to those of anaerobic spore-forming bacteria, Clostridium sp.     

Effect of sludge recirculation on characteristics of hydrogen production in a two-stage hydrogen–methane fermentation process treating food wastes

The two-stage hydrogen–methane fermentation process with different patterns of recirculation was investigated. Operations with the circulation of heat-treated sludge performed considerably better than those with the recirculation of raw sludge with respect to both the hydrogen production rate and yield. In addition, the results of the batch tests demonstrated that circulated sludge was capable of consuming hydrogen under acidogenic pH while the heat-treated sludge was not. These results suggest that the recirculation of active methanogenic sludge had an inhibitive effect on the hydrogen production, which can likely be attributed to the high hydrogen-consuming activity of microorganisms present in the circulated sludge. On the other hand, operations without any sludge recirculation did not perform well in terms of hydrogen production or carbohydrates degradation compared to those with recirculation, perhaps due to a shortage of available nitrogen. This suggests that sludge recirculation in effect supplemented the NH₄⁺ in the hydrogen reactor.     

Hydrogenase activity monitoring in the fermentative hydrogen production using heat pretreated sludge: A useful approach to evaluate bacterial communities performance

The conversion of organic compounds into H₂ has received increasing attention. Enrichment of inocula by heat pretreatment eliminates non-spore forming hydrogen consuming microorganisms and promotes spore germination in genus Clostridium, which is known as one of the key hydrogen producers. Useful information about metabolic pathway is provided by some intermediate metabolites, such as: acetic, propionic, butyric and formic acids. The increase of acetic/butyric acids ratio indicates H₂ production in heat pretreated inoculum when compared to untreated inoculum in the same cultivation conditions. The effect of heat pretreatment on inocula and consequently on the performance of bacterial communities responsible for H₂ production was monitored through the measurement of the level of hydrogenase gene expression, as well as through the content and distribution of volatile fatty acids. The acetic acid type fermentation was followed by the microorganisms presented in untreated and heat pretreated sludge. The medium containing untreated sludge presented a ratio of acetic/butyric acid of approximately 4, the same parameter was 7 when heat pretreated sludge was employed. The level of hydrogenase gene expression tripled when heat pretreated inoculum was used, indicating a higher production of H₂.     

Cladophora sp. as a sustainable feedstock for dark fermentative biohydrogen production

Macroalgae are rich in carbohydrates which can be used as a promising substrate for fermentative biohydrogen production. In this study, Cladophora sp. biomass was fermented for biohydrogen production at various inoculum/substrate (I/S) ratios against a control of inoculum without substrate in laboratory-scale batch reactors. The biohydrogen production yield ranged from 40.8 to 54.7 ml H₂/g-VS, with the I/S ratio ranging from 0.0625 to 4. The results indicated that low I/S ratios caused the overloaded accumulation of metabolic products and a significant pH decrease, which negatively affected hydrogen production bacteria's metabolic activity, thus leading to the decrease of hydrogen fermentation efficiency. The overall results demonstrated that Cladophora sp. biomass is an efficient fermentation feedstock for biohydrogen production.     

Photofermentative molecular biohydrogen production by purple-non-sulfur (PNS) bacteria in various modes: The present progress and future perspective

Hydrogen is green fuel for the future, mainly due to its recyclability. Biohydrogen production processes are less energy intensive and environmental friendly in compared to chemical processes. Fermentative biohydrogen production can be broadly classified as: dark and photo fermentation. Two enzymes, nitrogenase and hydrogenase play important role in biohydrogen production. Purple Non-Sulfur bacteria (PNS) are mainly used in photofermentative hydrogen production through which the overall yield can be improved manifolds. The scope and objective of this review paper is to investigate the performance of various light driven photofermentative hydrogen production by PNS bacteria along with several developmental works related to batch, repeated batch, feed batch and continuous operation. However the study of Photobiological process by microalgae or cyanobacteria is outside the scope of this review. Optimization of suitable process parameters such as carbon and nitrogen ratio, illumination intensity, bioreactor configuration, immobilization of active cells in specific continuous mode and inoculum age may lead to higher yield of hydrogen generation.     

Fermentative biohydrogen production from starch-containing textile wastewater

The present study deals with the biohydrogen production from starch-containing wastewater collected from the textile industry in Taiwan. The effects of inoculums collected from different sources (sewage sludge, soil and cow dung), substrate concentrations (5–25 g COD/L) and pH (4.0–8.0) on hydrogen production from wastewater were investigated.     

Hydrogen production by Clostridium acetobutylicum ATCC 824 and megaplasmid-deficient mutant M5 evaluated using a large headspace volume technique

Biohydrogen production is measured using a variety of techniques, ranging from low cost intermittent gas release methods where yields are usually reduced due to high partial pressures of hydrogen, to expensive respirometers that can eliminate pressure buildup. A new large headspace volume technique was developed that reduces the potential for hydrogen gas inhibition without the need for a respirometer. We tested this method with two strains of clostridia, Clostridium acetobutylicum ATCC 824 and its mutant M5 that lacks a megaplasmid responsible for butanol and acetone production, and a mixed culture (heat-treated sludge). The hydrogen yield using M5 (2.64 mol-H₂/mol-glucose) was 47% higher than that of the parent strain (1.79 mol-H₂/mol-glucose), and 118% larger than that obtained in tests with the sludge inoculum (1.21 mol-H₂/mol-glucose). The increased yield for M5 was primarily due to a decrease in biomass synthesis (38%) compared to the parent strain. Hydrogen yields measured using this new method were on average 14% higher than those obtained using a conventional respirometric method. These findings indicate enhanced biohydrogen production from the megaplasmid-deficient mutant of C. acetobutylicum ATCC 824, and that an intermittent gas-sampling technique can effectively measure high hydrogen gas by using a large headspace volume.     

厨余和污泥不同混合比例碱处理产氢特性研究

以厨余垃圾和污泥为反应底物,加热预处理的污泥为发酵接种物,考察了碱处理下厨余与污泥不同混合比例的发酵产氢特性。结果表明:不同pH碱液对厨余垃圾进行预处理后,其效果以pH=13时最佳,预处理3h后SCOD和还原糖含量分别为31316.8mg/L和5.54mg/mL;碱预处理后的污泥与厨余联合发酵能够改善物料的营养平衡,缩短反应延迟时间到1h内;当厨余与污泥混和比例为5:1时为本试验最佳的试验条件,其氢气含量、比产氢速率峰值和氢产率分别为52.69%,1.73mL H_2/(h·gVS)和50.27mL H_2/gVS。     

Development of a method for biohydrogen production from wheat straw by dark fermentation

Lignocellulosic biomass contains approximately 70-80% carbohydrates. If properly hydrolyzed, these carbohydrates can serve as an ideal feedstock for fermentative hydrogen production. In this research, batch tests of biohydrogen production from acid-pretreated wheat straw were conducted to analyze the effects of various associated bioprocesses. The objective of the pretreatment phase was to investigate the effects of various sulfuric acid pretreatments on the conversion of wheat straw to biohydrogen. When sulfuric acid-pretreated solids at a concentration of 2% (w/v) were placed in an oven for 90 min at 120 °C, they degraded substantially to fermentative gas. Therefore, wheat straw that is pre-treated under the evaluated conditions is suitable for hydrolysis and fermentation in a batch test apparatus. Five different conditions were evaluated in the tests, which were conducted in accordance with standard batch test procedures (DIN 38414 S8): fresh straw, pre-treated straw, supernatants derived from acid hydrolyzation, Separate Hydrolysis and Fermentation (SHF) and Simultaneous Saccharification and Fermentation (SSF). The SSF method proved to be the most effective and economical way to convert wheat straw to biohydrogen. The hydrogen yield by this method was 1 mol H₂/mol glucose, which resulted from 5% carbon degradation (η_{C, gas}) or the equivalent of 64% of the hydrogen volume that was produced in the reference test (glucose equivalent test). This method also proved to have the shortest lag phase for gas production. The supernatants derived from acid hydrolysis were very promising substances for continuous tests and presented excellent characteristics for the mass production of biohydrogen. For example, a 1.19 mol H₂/mol glucose (76% glucose equivalent) yield was achieved along with a 52% carbon degradation.     

Enzymatic characterization of acid tolerance response (ATR) during the enhanced biohydrogen production process from Taihu cyanobacteria via anaerobic digestion

Enhancement of biohydrogen production via anaerobic digestion from Taihu cyanobacteria (blue algae) after acid stress on anaerobic sludge, and the enzymatic characterization of the acid tolerance response (ATR) during the enhanced biohydrogen production process were investigated in this study. Comparing to those of the control, biohydrogen accumulation and hydrogen content increased by 1.9 and 1.7 times, when 12.5 and 7.5 g/L of acid stress on anaerobic sludge were performed respectively. Other than that, activities of hydrolytic enzymes, such as β-glucosidase, BAA-proteolytic enzyme and phosphatase were all improved during the enhanced biohydrogen process after appropriate acid stress. Significantly, activity of glutamate decarboxylase (GAD), the main microbial ATR stimulated by excessive acids, was increased consistently with the biohydrogen accumulation. Therefore, acid stress might be a practical approach to improving the biochemical traits of the anaerobic sludge. In turn, improved hydrolysis of organic substances would help the anaerobic sludge better survive excessive organic acids, and then enhance biohydrogen production from Taihu cyanobacteria.     

Effects of pretreatment methods on cassava wastewater for biohydrogen production optimization

Batch production of biohydrogen from cassava wastewater pretreated with (i) sonication, (ii) OPTIMASH BG^® (enzyme), and (iii) α-amylase (enzyme) were investigated using anaerobic seed sludge subjected to heat pretreatment at 105 °C for 90 min. Hydrogen yield at pH 7.0 for cassava wastewater pretreated with sonication for 45 min using anaerobic seed sludge was 0.913 mol H₂/g COD. Results from pretreatment with OPTIMASH BG^® at 0.20% and pH 7 showed a hydrogen yield of 4.24 mol H₂/g COD. Superior results were obtained when the wastewater was pretreated with α-amylase at 0.20% at pH 7 with a hydrogen yield of 5.02 mol H₂/g COD. In all cases, no methane production was observed when using heat-treated sludge as seed inoculum. Percentage COD removal was found to be highest (60%) using α-amylase as pretreatment followed by OPTIMASH BG^® at 54% and sonication (40% reduction rate). Results further suggested that cassava wastewater is one of the potential sources of renewable biomass to produce hydrogen.     

A web-enabled software for real-time biogas fermentation monitoring – Assessment of dark fermentations for correlations between medium conductivity and biohydrogen evolution

A web-enabled software was developed for continuous online monitoring of biohydrogen, biomethane and carbon dioxide gas fractions, temperature, Dissolved Oxygen (DO), pH, conductivity and gas volume for biogas fermentations. The cumulative gas volumes are computed recursively at 1 min intervals. Process data streamed into a MySQL database are accessible on a Local Area Network in real-time. This software was evaluated via continuous monitoring of two batches of dark fermentations for biohydrogen production using anaerobic sludge as inoculum and glucose as substrate. The effect of sampling frequency on the accuracy of cumulative hydrogen volume determination and the possible correlations between medium conductivity and biohydrogen evolution were examined. Data showed an erroneous over estimation of biohydrogen production at 12.09% and 16.23% for 12 and 24 h sampling intervals, suggesting the adoption of a high sampling rate to derive reliable key process parameters from the Gompertz model. Partial correlations were observed between medium conductivity and hydrogen gas fractions with maximum conductivity changes of 5.0 and 21.1 S × cm⁻¹, corresponding to peaks of hydrogen concentrations of 33.53% and 44.86%, respectively. Accurate application of conductimetric techniques for real-time monitoring of biohydrogen production requires further understanding of all sources of conductivity changes. The implemented software could generate high throughput actionable information for biohydrogen process development and optimization.     

Thermophilic hydrogen fermentation from Korean rice straw by Thermotoga neapolitana

Rice straw, a low-cost lignocellulosic biomass was used as feedstock for thermophilic hydrogen fermentation by Thermotoga neapolitana. Hydrogen production, the growth and cellulose digestibility of the hyperthermophile in batch mode from untreated as well as chemically pretreated (ammonia and dilute sulfuric acid) Korean rice straws were investigated. Pretreatment method using combination of 10% ammonia and 1.0% dilute sulfuric acid was developed to increase the digestibility of rice straw for the hyperthermophilic H₂ fermentation and to decrease the time consumption. In a typical fermentation using raw rice straw, 29% of the substrate was digested and 2.3 mmol H₂/g straw of hydrogen yield was consistently obtained. Compared with the pretreatments using only ammonia or dilute sulfuric acid, the combined pretreatment method using both chemical agents significantly increases the digestibility of rice straw with 85.4% of substrate consumption. H₂ production on rice straw from this combined pretreatment showed the highest yield (2.7 mmol H₂/g straw) and the highest sugar conversions (72.9% of glucose and 95.7% of xylose).