Enhanced fermentative hydrogen production from cassava stillage by co-digestion: The effects of different co-substrates

In this study, an efficient strategy to increase the hydrogen yield from cassava stillage (CS), by its co-digestion with organic wastes, was developed. The effects of different co-substrates on hydrogen yield were evaluated and compared, with increases obtained in all co-digestion reactors. The maximal hydrogen yield was achieved by CS co-digestion with cassava excess sludge (CES), which resulted in a 46% increase over the yield from CS alone. Improved hydrolysis and acidification performances, and thus increased hydrogen production, were additional benefits of co-digestion, especially with CES. Also, the presence of a co-substrate promoted butyrate over lactate generation. The most influential advantage of co-substrate addition was the enhanced pH buffering capacity, which correlated linearly with hydrogen yield (R² = 0.98). Moreover, the co-substrates addition resulting in more favorable carbohydrate-COD/protein-COD ratio, C/N and C/P ratios, all of which also correlated linearly with hydrogen yield (R² = 0.86, 0.71, and 0.75 respectively). Polymerase chain reaction–denaturing gradient gel electrophoresis analysis of the role of microorganisms indicated that the addition of even small amounts of co-substrate altered the bacterial communities within the reactors, enriching the proportion of hydrogen-producing bacteria. A species detected only in the co-digestion reactors was shown to be related to Clostridium cellulosi, which is an efficient hydrogen-producer, while some bacteria not involved in hydrogen production and present in the CS alone reactor were not detectable in any of the co-substrate-containing reactors.     

Fermentative hydrogen production from cassava stillage by mixed anaerobic microflora: Effects of temperature and pH

Fermentative hydrogen production from cassava stillage was conducted to investigate the influences of temperature (37 °C, 60 °C, 70 °C) and initial pH (4–10) in batch experiments. Although the seed sludge was mesophilic anaerobic sludge, maximum hydrogen yield (53.8 ml H₂/gVS) was obtained under thermophilic condition (60 °C), 53.5% and 198% higher than the values under mesophilic (37 °C) and extreme-thermophilic (70 °C) conditions respectively. The difference was mainly due to the different VFA and ethanol distributions. Higher hydrogen production corresponded with higher ratios of butyrate/acetate and butyrate/propionate. Similar hydrogen yields of 66.3 and 67.8 ml H₂/gVS were obtained at initial pH 5 and 6 respectively under thermophilic condition. The total amount of VFA and ethanol increased from 3536 to 7899 mg/l with the increase of initial pH from 4 to 10. Initial pH 6 was considered as the optimal pH due to its 19% higher total VFA and ethanol concentration than that of pH 5. Homoacetogenesis and methonogenesis were very dependent on the initial pH and temperature even when the inoculum was heat-pretreated. Moreover, a difference between measured and theoretical hydrogen was observed in this study, which could be attributed to homoacetogenesis, methanogenesis and the degradation of protein.     

An online-monitored thermophilic hydrogen production UASB reactor for long-term stable operation

In this study, the H₂ production and chemical oxygen demand (COD) removal performances of a thermophilic upflow anaerobic sludge blanket (UASB) reactor with online monitoring system were investigated. The online monitoring system enabled a rapid monitoring and timely control of the process. As a consequence, high operating stability was achieved despite of the varied hydraulic retention time (HRT) during 310-day operation. The COD efficiency remained at above 98%, and the hydrogen yield fluctuated slightly within the range of 2.42–3.06 mol H₂ mol⁻¹ sucrose. Thermophilic H₂-producing granules were successfully cultivated in this reactor, which showed better physical and microbial properties than floc sludge and higher H₂ production rate than mesophilic granules. An analysis of the microbial growth kinetics further demonstrated a possibly higher synthesis and metabolism activity of microbes in the thermophilic granule state.     

Continuous fermentative hydrogen production from cheese whey wastewater under thermophilic anaerobic conditions

Hydrogen (H₂) production from cheese processing wastewater via dark anaerobic fermentation was conducted using mixed microbial communities under thermophilic conditions. The effects of varying hydraulic retention time (HRT: 1, 2 and 3.5 days) and especially high organic load rates (OLR: 21, 35 and 47 g chemical oxygen demand (COD)/l/day) on biohydrogen production in a continuous stirred tank reactor were investigated. The biogas contained 5–82% (45% on average) hydrogen and the hydrogen production rate ranged from 0.3 to 7.9 l H₂/l/day (2.5 l/l/day on average). H₂ yields of 22, 15 and 5 mmol/g COD (at a constant influent COD of 40 g/l) were achieved at HRT values of 3.5, 2, and 1 days, respectively. On the other hand, H₂ yields were monitored to be 3, 9 and 6 mmol/g COD, for OLR values of 47, 35 and 21 g COD/l/day, when HRT was kept constant at 1 day. The total measurable volatile fatty acid concentration in the effluent (as a function of influent COD) ranged between 118 and 27,012 mg/l, which was mainly composed of acetic acid, iso-butyric acid, butyric acid, propionic acid, formate and lactate. Ethanol and acetone production was also monitored from time to time.To characterize the microbial community in the bioreactor at different HRTs, DNA in mixed liquor samples was extracted immediately for PCR amplification of 16S RNA gene using eubacterial primers corresponding to 8F and 518R. The PCR product was cloned and subjected to DNA sequencing. The sequencing results were analyzed by using MegaBlast available on NCBI website which showed 99% identity to uncultured Thermoanaerobacteriaceae bacterium.     

Anaerobic treatment of cassava stillage for hydrogen and methane production in continuously stirred tank reactor (CSTR) under high organic loading rate (OLR)

Anaerobic hydrogen and methane production from cassava stillage in continuously stirred tank reactor (CSTR) were investigated in this study. Results showed that the heat-pretreatment of inoculum did not enhance hydrogen yield compared to raw inoculum under mesophilic condition after continuous operation. However, the hydrogen yield increased from about 14 ml H₂/gVS under mesophilic condition to 69.6 ml H₂/gVS under thermophilic condition due to the decrease of propionate concentration and inhibition of homoacetogens. Therefore, temperature was demonstrated to be more important than pretreatment of inoculum to enhance the hydrogen production. Under high organic loading rate (OLR) (>10 gVS/(L·d)), the two-phase thermophilic CSTR for hydrogen and methane production was stable with hydrogen and methane yields of 56.6 mlH₂/gVS and 249 mlCH₄/gVS. The one-phase thermophilic CSTR for methane production failed due to the accumulation of both acetate and propionate, leading to the pH lower than 6. Instead of propionate alone, the accumulations of both acetate and propionate were found to be related to the breakdown of methane reactor.     

Continuous hydrogen production from cofermentation of sugarcane vinasse and cheese whey in a thermophilic anaerobic fluidized bed reactor

The co-fermentation of vinasse and cheese whey (CW) was evaluated in this study by using two thermophilic (55° C) anaerobic fluidized bed reactors (AFBRs). In AFBR using vinasse and CW (AFBR-V-CW), the CW was added in increasing proportions (2, 4, 6, 8, and 10 g COD.L^?1) to vinasse (10 g COD.L^?1) to assess the advantage of adding CW to vinasse. By decreasing the hydraulic retention time (HRT) from 8 h to 1 h in AFBR-V, maximum hydrogen yield (HY), production rate (HPR), and H₂ content (H₂%) of 1.01 ± 0.06 mmol H₂.g COD^?1, 2.54 ± 0.39 L H₂.d^?1.L^?1, and 47.3 ± 2.9%, respectively, were observed at an HRT of 6 h. The increase in CW concentration to values over 2 g COD.L^?1 in AFBR-V-CW decreased the HY, PVH, and H₂%, with observed maximum values of 0.82 ± 0.07 mmol H₂.g COD^?1, 1.41 ± 0.24 L H₂.d^?1.L^?1, and 55.5 ± 3.7%, respectively, at an HRT of 8 h. The comparison of AFBR-V-CW and AFBR-V showed that the co-fermentation of vinasse with 2 g COD.L^?1 of CW increased the HPR, H₂%, and HY by 117%, 68%, and 82%, respectively.     

The effect of hydraulic retention time on thermophilic dark fermentative biohydrogen production in the continuously operated packed bed bioreactor

The main objective of the study is to investigate the effect of hydraulic retention times on continuous dark fermentative biohydrogen production in an up-flow packed bed reactor (UPBR) containing a novel microorganism immobilization material namely polyester fiber beads. The hydrogen producing dark fermentative microorganisms were obtained by heat-pretreatment of anaerobic sludge from the acidogenic phase of an anaerobic wastewater treatment plant. Glucose was the sole carbon source and the initial concentration was 15 ± 1 g/L throughout the continuous feeding. UPBR was operated under the thermophilic condition at T = 48 ± 2 °C and at varying HRTs between 2 h and 6 h. The hydrogen productivity of continuously operated UPBR increased with increasing HRT. Hydrogen production volume varied between 4331 and 6624 ml/d, volumetric hydrogen production rates (VHPR) were obtained as 3.09–4.73 L H₂/L day, and hydrogen production yields (HY) were 0.49 mol/mol glucose-0.89 mol/mol glucose depending on HRT. Maximum daily hydrogen volume (6624 ml/d), the yield (0.89 mol/mol glucose) and VHPR (4.73 L H₂/L day) were obtained at HRT = 6 h. The production rate and the yield decreased with increasing organic loading rate due to substrate inhibition.     

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.     

Continuous hydrogen production from tofu processing waste using anaerobic mixed microflora under thermophilic conditions

We carried out continuous fermentative H₂ production from tofu (soybean curd)-processing waste (TPW) using anaerobic mixed microflora under thermophilic (60 °C) conditions and compared the rates and yields of H₂ production in a continuous stirred-tank reactor (CSTR) and a membrane bioreactor (MBR), wherein the membrane filtration unit was coupled to the CSTR. The TPW was diluted with tap water and then hydrolyzed by blending for 5 min in the presence of 0.5% HCl, and it was found that this protocol significantly increased the amount of soluble material in the mixture. The soluble chemical oxygen demand (SCOD)-to-total COD (TCOD) ratio jumped from 14% to 60%, and the soluble carbohydrate concentration was increased threefold, from 2.4 g/L to 7.2 g/L. Accordingly, H₂ production potential was increased 2.8-fold. In a CSTR operation using pretreated TPW as the substrate, a stable volumetric H₂ production rate (VHPR) of 8.17 ± 0.32 L H₂/L/d and a H₂ yield of 1.20 ± 0.05 mol H₂/mol hexose_added at 8-h HRT were achieved. Substantial increases in the VHPR and H₂ yield over those obtained with the CSTR were observed in the MBR operation. The role of the MBR was to increase the retention time of the solid substrate and the concentration of microorganisms, thereby enhancing the substrate utilization rate for H₂ production. Acetic and butyric acids were the main liquid-state metabolites produced during the fermentation process, thus indicating that the thermophilic operation provided favorable conditions for H₂ production from TPW. A maximum H₂ yield of 1.87 mol H₂/mol hexose_added was achieved at 8-h HRT and then gradually decreased to 1.00 mol H₂/mol hexose-equivalent at 2-h HRT. Meanwhile, the VHPR continuously increased to a maximum of 19.86 L H₂/L/d at 4-h HRT and then decreased with a high dilution rate as the HRT was lowered to 2 h (minimum). At 2-h HRT, the degradation of soluble carbohydrate was limited.     

Biohydrogen production from cassava starch processing wastewater by thermophilic mixed cultures

Natural microbial consortia from hot spring samples were used to developed thermophilic mixed cultures for biohydrogen production from cassava starch processing wastewater (CSPW). Significant hydrogen production potentials were obtained from three thermophilic mixed cultures namely PK, SW and PR with maximum hydrogen production yields of 249.3, 180 and 124.9 mL H₂/g starch, respectively from raw cassava starch and 252.4, 224.4 and 165.4 mL H₂/g starch, respectively from gelatinized cassava starch. Acetic acid-ethanol and acetic-lactic acid type fermentation were observed in cassava starch fermentation, based on three thermophilic mixed cultures performance. The thermophilic mixed cultures PK, SW and PR exhibited the maximum hydrogen yield of 287, 264 and 232 mL H₂/g starch in CSPW, respectively corresponding to 53%, 48.7% and 42.8% of the theoretical values. Phylogenetic analysis of thermophilic mixed cultures revealed that members involved cassava starch degrading bacteria and hydrogen producers in both raw cassava starch and CSPW were phylogenetically related to the Thermoanaerobacterium saccharolyticum, Thermoanaerobacterium thermosaccharolyticum, Anoxybacillus sp., Geobacillus sp. and Clostridium sp.     

Effect of hydraulic retention time on hydrogen production and chemical oxygen demand removal from tapioca wastewater using anaerobic mixed cultures in anaerobic baffled reactor (ABR)

This study evaluated hydrogen production and chemical oxygen demand removal (COD removal) from tapioca wastewater using anaerobic mixed cultures in anaerobic baffled reactor (ABR). The ABR was conducted based on the optimum condition obtained from the batch experiment, i.e. 2.25 g/L of FeSO₄ and initial pH of 9.0. The effects of the varying hydraulic retention times (HRT: 24, 18, 12, 6 and 3 h) on hydrogen production and COD removal in a continuous ABR were operated at room temperature (32.3 ± 1.5 °C). Hydrogen production rate (HPR) increased with a reduction in HRT i.e. from 164.45 ± 4.14 mL H₂/L.d (24 h HRT) to 883.19 ± 7.89 mL H₂/L.d (6 h HRT) then decreased to 748.54 ± 13.84 mL H₂/L.d (3 h HRT). COD removal increased with reduction in HRT i.e. from 14.02 ± 0.58% (24 h HRT) to 29.30 ± 0.84% (6 h HRT) then decreased to 21.97 ± 0.94% (3 h HRT). HRT of 6 h was the optimum condition for ABR operation as indicated.     

Continuous production of hydrogen from oat straw hydrolysate in a biotrickling filter

Hydrogen was produced in a biotrickling filter (BF) packed with perlite and fed with oat straw acid hydrolysate at 30 °C. Inlet chemical oxygen demand (COD) from 1.2 to 35 g/L and hydraulic retention time (HRT) between 24 h and 6 h were assayed. With increasing inlet COD or decreasing HRT, H₂ production rate (HPR) increased but H₂ production yield (HY) decreased. Maximum HPR of 81.4 mL H₂/L_reactor h (3.3 mmol H₂/L_reactor h) and HY of 2.9 mol H₂/mol_{hexose consumed} were found at an inlet COD of 0.05 g_COD/L h (HRT 24 h) and 2.9 g_COD/L h (HRT 12 h), respectively. Maximum hydrogen composition in gas was 45 ± 4% (v/v) with CO₂ as balance. Methane was not detected. Maximum HPR and inlet COD used in this work were higher than others reported for reactors with suspended or fixed biomass. However, implementation of strategies for biomass control to avoid reactor clogging is needed.     

Bioproduction of hydrogen from food waste by pilot-scale combined hydrogen/methane fermentation

A pilot-scale two-phase hydrogen/methane fermentation system generated 3.9 L biogas per unit time and reactor volume from food waste, of which the fraction of H₂ was approximately 60% at a hydraulic retention time (HRT) of 21 h. As substrate, 90% of the carbohydrates in the organic compounds were consumed, based on COD removal efficiency, and the hydrogen yield was approximately 1.82 (H₂-mol/glucose-mol). The maximum decomposition rate coefficient of hydrogen fermentation was observed at an HRT of 21 h, indicating that reducing HRTs improves hydrogen production. Over 80% of the methane was produced in the methane fermentation tank and the predominant fraction of organic acids after methane fermentation comprised acetic acid. Based on our economic evaluation, two-phase hydrogen/methane fermentation has greater potential for recovering energy than methane-only fermentation.     

Effect of hydraulic retention time on hydrogen production from sewage sludge and wine vinasse in a thermophilic acidogenic CSTR: A promising approach for hydrogen production within the biorefinery concept

The aim of the present study was to investigate the effect of hydraulic retention time (HRT) on hydrogen production from sewage sludge:wine vinasse (50:50 v/v) in a laboratory-scale continuously stirred tank reactor under thermophilic conditions. For this purpose, nine HRT ranging from 5.0 to 0.25 days were tested. Maximum hydrogen production and specific hydrogen production of 0.90 LH₂/L_reactor/d and 35.19 mLH₂/g VS_added were respectively obtained at a HRT of 0.5 days. Eubacteria was the main group (65–79%) for all the tested HRT. Decreasing HRT was inversely correlated with hydrogen production and microbial population. HRT of 0.5 days is optimal for the growth of the acidogenic population and therefore this population is more active and maximum microbial activity (15.28·10^?10 LH₂/cells) was also achieved at this HRT.     

Value analysis for commercialization of fermentative hydrogen production from biomass

In addition to producing hydrogen gas, biohydrogen production is also used to process wastewater. Therefore, this study specifically conducted value analyses of two different scenarios of fermentative hydrogen production from a biomass system: to increase the value of a wastewater treatment system and to specifically carry out hydrogen production. The analytical results showed that fermentative hydrogen production from a biomass system would increase the value of a wastewater treatment system and make its commercialization more feasible. In contrast, fermentative hydrogen production from a biomass system designed specifically for producing hydrogen gas would have a lower system value, which indicated that it is not yet ready for commercialization. The main obstacle to be overcome in promoting biohydrogen production technology and system application is the lack of sales channels for the system's products such as hydrogen gas and electricity. Thus, in order to realize its commercialization, this paper suggests that governments provide investment subsidies for the use of biohydrogen production technology and establish a buy-back tariff system for fuel cells.     

Hydrogen production from diluted and raw sugarcane vinasse under thermophilic anaerobic conditions

Two continuous anaerobic fluidized bed reactors (AFBRs) were operated under thermophilic (55 °C) temperature for 150 days to investigate the effect of dark H₂ fermentation of diluted and raw sugarcane vinasse on H₂ production using mixed seed sludge. Although effective H₂ production (52.8% of H₂; 0.80 L H₂ h⁻¹ L⁻¹; 0.79 mmol gCOD_added⁻¹) was observed using an elevated substrate concentration (30,000 mg COD L⁻¹), the optimal operational conditions were found for the AFBR₁ (10,000 mg COD L⁻¹) fed with diluted sugarcane vinasse (HRT 6 h; OLR 40 kg COD m⁻³ d⁻¹), achieving a H₂ yield of 2.86 mmol H₂ g COD_added⁻¹. H₂ production was inhibited by elevated volatile fatty acid (VFA) concentrations (butyric and acetic acids = 3.7 and 3.0 g L⁻¹, respectively) in the raw feedstock. Denaturing gradient gel electrophoresis (DGGE) analyses revealed changes in the bacterial population of the expanded clay biofilm as a function of the substrate concentration.     

发酵产氢面临的问题及对策

氢是一种高效、清洁、可再生的燃料，通过发酵的方式产生氢气，已经成为国内外研究的热点。目前，这一新兴技术的研究取得了可喜的成果。文章在综合国内外发酵产氢技术的基础上，通过比较沼气发酵和产氢发酵的技术成果，揭示了挥发性有机酸反馈抑制是制约生物法发酵产氢的关键因素，提出了如何提高氢的转化率和发酵稳定性的措施和对策。     

Effect of hydraulic retention time on the hydrogen production in a horizontal and vertical continuous stirred-tank reactor

Hydraulic retention time (HRT) is the main process parameter for biohydrogen production by anaerobic fermentation. This paper investigated the effect of the different HRT on the hydrogen production of the ethanol-type fermentation process in two kinds of CSTR reactors (horizontal continuous stirred-tank reactor and vertical continuous stirred-tank reactor) with molasses as a substrate. Two kinds of CSTR reactors operated with the organic loading rates (OLR) of 12kgCOD/m3•d under the initial HRT of the 8 h condition, and then OLR was adjusted as 6kgCOD/m3•d when the pH drops rapidly. The VCSTR and HCSTR have reached the stable ethanol-type fermentation process within 21 days and 24 days respectively. Among the five HRTs settled in the range of 2–8 h, the maximum hydrogen production rate of 3.7LH₂/Ld and 5.1LH₂/Ld were investigated respectively in the VCSTR and HCSTR. At that time the COD concentration and HRT were 8000 mg/L and 5 h for VCSTR, while 10000 mg/L and 4 h for HCSTR respectively.Through the analysis on the composition of the liquid fermentation product and biomass under the different HRT condition in the two kinds of CSTR, it can found that the ethanol-type fermentation process in the HCSTR is more stable than VCSTR due to enhancing biomass retention of HCSTR at the short HTR.     

Continuous fermentative hydrogen production from cheese whey – new insights into process stability

Continuous dark fermentation experiments for hydrogen production from synthetic cheese whey were conducted at different HRTs and OLRs. The study mainly aimed at developing a novel criterion to quantitatively assess stability and relating it to the evolution of microbial pathways and associated metabolic products.For HRTs = 6–8 h and OLRs = 65–97.5 g TOC/(L·d), the best hydrogen generation performance was attained, yielding 42–50 L H₂/kg TOC. Instead of using a stability index for the entire test length, accounting for the fluctuations of hydrogen production over 1-HRT periods (dynamic stability index) provided a more accurate assessment of process stability showing a clear correlation with the hydrogen yield. The analysis of the metabolic reactions provided evidence of a competition among acidogenic, hydrogen-consuming and hydrogen-neutral microbial species. This explained the lower process performance in comparison to the theoretical yield expected, pointing out at the need for further investigation on suitable strategies to effectively inhibit undesired metabolic pathways.