Seed inocula for biohydrogen production from biodiesel solid residues

This study aimed to optimize the hydrogen production from various seed sludges (two kinds of sewage sludges (S1, S2), cow dung (S3), granular sludge (S4) and effluent from condensed soluble molasses H₂ fermenter (S5)) and enhancement of hydrogen production via heat treatment for substrate and seed sludge by using the solid residues of biodiesel production (BDSR). Two batch assay tests were operated at a biodiesel solid residue concentration of 10 g/L, temperature of 55 °C and an initial cultivation pH of 8. The results showed that the peak hydrogen yield (HY) of 94.6 mL H₂/g volatile solid (VS) (4.1 mmolH₂/g VS) was obtained from S1 when substrate and seed sludge were both heat treated at 100 °C for 1 h. However, the peak hydrogen production rate (HPR) and specific hydrogen production rate (SHPR) of 1.48 L H₂/L-d and 0.30 L H₂/g VSS-d were obtained from S2 without any treatment. The heat treatment was found to increase the HY in both the cases of sewage sludges S1 and S2.The HY of 89.5 mL H₂/g VS (without treatment) was increased to 94.6 mL H₂/g VS and 82.6 mL H₂/g VS (without treatment) was increased to 85.7 mL H₂/g VS for S1 and S2. The soluble metabolic product (SMP) analysis showed that the fermentation followed mainly acetate–butyrate pathway with considerable production of ethanol. The total bioenergy production was calculated as 2.8 and 2.9 kJ/g VS for favorable hydrogen and ethanol production, respectively. The BDSR could be used as feedstock for dark fermentative hydrogen production.     

Bio-hydrogen production by photo-fermentation of dark fermentation effluent with intermittent feeding and effluent removal

Pure culture of Rhodobacter sphaeroides (NRRL- B1727) was used for continuous photo-fermentation of volatile fatty acids (VFA) present in the dark fermentation effluent of ground wheat starch. The feed contained 1950 ± 50 mg L⁻¹ total VFA with some nutrient supplementation. Hydraulic residence time (HRT) was varied between 24 and 120 hours. The highest steady-state daily hydrogen production (55 ml d⁻¹) and hydrogen yield (185 ml H₂ g⁻¹ VFA) were obtained at HRT = 72 hours (3 days). Biomass concentration increased with increasing HRT. Volumetric and specific hydrogen formation rates were also maximum at HRT = 72 h. High extent of TVFA fermentation at HRT = 72 h resulted in high hydrogen gas production.     

Anaerobic fluidized bed reactor with expanded clay as support for hydrogen production through dark fermentation of glucose

This study evaluated hydrogen production in an anaerobic fluidized bed reactor (AFBR) fed with glucose-based synthetic wastewater. Particles of expanded clay (2.8–3.35 mm) were used as a support material for biomass immobilization. The reactor was operated with hydraulic retention times (HRT) ranging from 8 to 1 h. The hydrogen yield production increased from 1.41 to 2.49 mol H₂ mol⁻¹ glucose as HRT decreased from 8 to 2 h. However, when HRT was 1 h, there was a slight decrease to 2.41 mol H₂ mol⁻¹ glucose. The biogas produced was composed of H₂ and CO₂, and the H₂ content increased from 8% to 35% as HRT decreased. The major soluble metabolites during H₂ fermentation were acetic acid (HAc) and butyric acid (HBu), accounting for 36.1–53.3% and 37.7–44.9% of total soluble metabolites, respectively. Overall, the results demonstrate the potential of using expanded clay as support material for hydrogen production in AFBRs.     

Hydrogen gas production from acid hydrolyzed wheat starch by combined dark and photo-fermentation with periodic feeding

Hydrogen gas production from acid hydrolyzed waste wheat starch by combined dark and photo-fermentation was investigated in continuous mode with periodic feeding and effluent removal. A mixture of heat treated anaerobic sludge and Rhodobacter sphaeroides (NRRL-B 1727) were used as the seed culture for dark and light fermentations, respectively with biomass ratio of Rhodobacter/sludge = 3. Hydraulic residence time (HRT) was changed between 1 and 8 days by adjusting the feeding periods. Ground waste wheat was acid hydrolyzed at pH = 3 and 121 °C for 30 min using an autoclave and the resulting sugar solution was used as the substrate for combined fermentation after pH adjustment and nutrient addition. The highest daily hydrogen gas production (41 ml d⁻¹), hydrogen yield (470 ml g⁻¹ total sugar = 3.4 mol H₂ mol⁻¹glucose), volumetric and specific hydrogen production rates were obtained at the HRT of 8 days. The highest biomass and the lowest total volatile fatty acids (TVFA) concentrations were also realized at HRT = 8 days indicating VFA fermentation by Rhodobacter sp. at high HRTs. The lowest total sugar loading rate of 0.625 g L⁻¹ d⁻¹ resulted in the highest hydrogen yield and formation rate. Hydrogen gas production by combined fermentation with periodic feeding was proven to be an effective method resulting in high hydrogen yields at long HRTs.     

Comparative analysis of thermophilic immobilized biohydrogen production using packed materials of ceramic ring and pumice stone

Ceramic ring and pumice stone were used as a support matrix for the enhancement of biohydrogen production in immobilized cell culture systems. The reactors were continuously operated for the hydrogen fermentation using sucrose as the major carbon source at varying hydraulic retention times (HRT) as an important operational factor. In terms of volumetric hydrogen production, the best value was obtained with ceramic ring at 1.5 h HRT (2.98 l H₂/l/d), on the other hand, the pumice stone packed reactor resulted in 30% less volumetric hydrogen production (2.28 l H₂/l/d) at two fold longer retention time (HRT 3 h). It was demonstrated that volumetric hydrogen production with the immobilized bioreactor configurations was 6 fold better than the suspended culture bioreactor configuration (CSTR). Furthermore, up to 4 mol and 5 mol hydrogen yields per mole of sucrose used (which are 62.5% and 50% of the theoretical values) were achieved by pumice stone and ceramic ring packed reactors, respectively, whereas suspended culture system yielded only 0.5 mol H₂/mol sucrose.     

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.     

Evaluation and simultaneous optimization of bio-hydrogen production using 3 factorial design and the desirability function

Various mixtures incorporating a simulated organic fraction of municipal solid wastes and blood from a poultry slaughterhouse were used as substrate in a dark fermentation process for the production of hydrogen. The individual and interactive effects of hydraulic retention time (HRT), solid content in the feed (%TS) and proportion of residues (%Blood) on bio-hydrogen production were studied in this work. A central composite design and response surface methodology were employed to determine the optimum conditions for the hydrogen production process. Experimental results were approximated to a second-order model with the principal effects of the three factors considered being statistically significant (P < 0.05). The production of hydrogen obtained from the experimental point at conditions close to best operability was 0.97 L Lr⁻¹ day⁻¹. Moreover, a desirability function was employed in order to optimize the process when a second, methanogenic, phase is coupled with it. In this last case, the optimum conditions lead to a reduction in the production of hydrogen when the optimization process involves the maximization of intermediary products.     

A bench scale study of fermentative hydrogen and methane production from food waste in integrated two-stage process

A two-stage fermentation process combining hydrogen and methane production for the treatment of food waste was investigated in this paper. In hydrogen fermentation reactor, the indigenous mixed microbial cultures contained in food waste were used for hydrogen production. No foreign inoculum was used in the hydrogen fermentation stage, the traditional heat treatment of inoculum was not applied either in this bench scale experiment. The effects of the stepwise increased organic loading rate (OLR) and solid retention time (SRT) on integrated two-stage process were investigated. At steady state, the optimal OLR and SRT for the integrated two-stage process were found to be 22.65 kg VS/m³ d (160 h) for hydrogen fermentation reactor and 4.61 (26.67 d) for methane fermentation reactor, respectively. Under the optimum conditions, the maximum yields of hydrogen (0.065 m³ H₂/kg VS) and methane (0.546 m³ CH₄/kg VS) were achieved with the hydrogen and methane contents ranging from 29.42 to 30.86%, 64.33 to 71.48%, respectively. Biodegradability analysis showed that 5.78% of the influent COD was converted to the hydrogen in H₂-SCRD and 82.18% of the influent COD was converted to the methane in CH₄-SCSTR under the optimum conditions.     

Bio-hydrogen production from ground wheat starch by continuous combined fermentation using annular-hybrid bioreactor

Continuous combined fermentation of ground wheat starch was realized in an annular-hybrid bioreactor (AHB) for hydrogen gas production. A mixture of pure cultures of Clostridium beijerinkii (DSMZ-791) and Rhodobacter sphaeroides-RV were used as seed cultures in combined fermentation. The feed contained 5 g L⁻¹ ground wheat with some nutrient supplementation. Effects of hydraulic residence time (HRT) on the rate and yield of hydrogen gas formation were investigated. Steady-state daily hydrogen production decreased but, hydrogen yield increased with increasing HRT. The highest hydrogen yield was 90 ml g⁻¹ starch at HRT of 6 days. Hydrolysis of starch and fermentation of glucose to volatile fatty acids (VFA) were readily realized at all HRTs. However, slow conversion of VFAs to H₂ and CO₂ by photo-fermentation caused accumulation of VFAs in the medium. Specific and volumetric rates of hydrogen formation also decreased with increasing HRT. High hydrogen yields obtained at high HRTs are due to partial fermentation of VFAs by Rhodobacter sp. The system should be operated at HRTs longer than 5 days for effective hydrogen gas formation by the dark and photo-fermentation bacteria.     

Biohydrogen production in anaerobic fluidized bed reactors: Effect of support material and hydraulic retention time

This study evaluated two different support materials (polystyrene and expanded clay) for biohydrogen production in an anaerobic fluidized bed reactor (AFBR) treating synthetic wastewater containing glucose (4000 mg L⁻¹). The AFBRs contained either polystyrene (R1) or expanded clay (R2) as support materials were inoculated with thermally pre-treated anaerobic sludge and operated at a temperature of 30 °C and a pH of approximately 5.5. The AFBRs were operated with a range of hydraulic retention times (HRTs) between 1 and 8 h. For R1 with an HRT of 2 h, the maximum hydrogen yield (HY) was 1.90 mol H₂ mol⁻¹ glucose, with 0.805 mg of biomass (as total volatile solids, or TVS) attached to each g of polystyrene. For R2 operated at an HRT of 2 h, the maximum HY was 2.59 mol H₂ mol⁻¹ glucose, with 1.100 mg of attached biomass (as TVS) g⁻¹ expanded clay. The highest hydrogen production rates (HPR) were 0.95 and 1.21 L h⁻¹ L⁻¹ for R1 and R2, respectively, using an HRT of 1 h. The H₂ content increased from 16–47% for R1 and from 22–51% for R2. No methane was detected in the biogas produced throughout the period of AFBR operation. These results show that the values of HY, HPR, H₂ content, and g of attached biomass g⁻¹ support material were all higher for AFBRs containing expanded clay than for reactors containing polystyrene.     

Effects of hydraulic retention time on anaerobic hydrogenation performance and microbial ecology of bioreactors fed with glucose–peptone and starch–peptone

This study evaluated anaerobic hydrogenation performance and microbial ecology in bioreactors operated at different hydraulic retention time (HRT) conditions and fed with glucose–peptone (GP) and starch–peptone (SP). The maximum hydrogen production rates for GP- and SP-fed bioreactors were found to be 1247 and 412 mmol-H₂/L/d at HRT of 2 and 3 h, respectively. At HRT > 8 h, hydrogen consumption due to peptone fermentation could occur and thus reduced hydrogen yield from carbohydrate fermentation. Results of cloning/sequencing and denaturant gradient gel electrophoresis (DGGE) indicated that Clostridium sporogenes and Clostridium celerecrescens were dominant hydrogen-producing bacteria in the GP-fed bioreactor, presumably due to their capability on protein hydrolysis. In the SP-fed bioreactor, Lactobacillus plantarum, Propionispira arboris, and Clostridium butyricum were found to be dominant populations, but the presence of P. arboris at HRT > 3 h might be responsible for a lower hydrogen yield from starch fermentation. As a result, optimizing HRT operation for bioreactors was considered an important asset in order to minimize hydrogen-consuming activities and thus maximize net hydrogen production. The limitation of simple parameters such as butyrate to acetate ratio (B/A ratio) in predicting hydrogen production was recognized in this study for bioreactors fed with multiple substrates. It is suggested that microbial ecology analysis, in addition to chemical analysis, should be performed when complex substrates and mixed cultures are used in hydrogen-producing bioreactors.     

Effect of food to microorganism ratio on biohydrogen production from food waste via anaerobic fermentation

The effect of different food to microorganism ratios (F/M) (1–10) on the hydrogen production from the anaerobic batch fermentation of mixed food waste was studied at two temperatures, 35 ± 2 °C and 50 ± 2 °C. Anaerobic sludge taken from anaerobic reactors was used as inoculum. It was found that hydrogen was produced mainly during the first 44 h of fermentation. The F/M between 7 and 10 was found to be appropriate for hydrogen production via thermophilic fermentation with the highest yield of 57 ml-H₂/g VS at an F/M of 7. Under mesophilic conditions, hydrogen was produced at a lower level and in a narrower range of F/Ms, with the highest yield of 39 ml-H₂/g VS at the F/M of 6. A modified Gompertz equation adequately (R² > 0.946) described the cumulative hydrogen production yields. This study provides a novel strategy for controlling the conditions for production of hydrogen from food waste via anaerobic fermentation.     

Hydrogen production by combined dark and light fermentation of ground wheat solution

Ground waste wheat was subjected to combined dark and light batch fermentation for hydrogen production. The dark to light biomass ratio (D/L) was changed between 1/2 and 1/10 in order to determine the optimum D/L ratio yielding the highest hydrogen formation rate and the yield. Hydrogen production by only dark and light fermentation bacteria was also realized along with the combined fermentations. The highest cumulative hydrogen formation (CHF = 76 ml), hydrogen yield (176 ml H₂ g⁻¹ starch) and formation rate (12.2 ml H₂ g⁻¹ biomass h⁻¹) were obtained with the D/L ratio of 1/7 while the lowest CHF was obtained with the D/L ratio of 1/2. Dark–light combined fermentation with D/L ratio of 1/7 was faster as compared to the dark and light fermentations alone yielding high hydrogen productivity and reduced fermentation time. Dark and light fermentations alone also yielded considerable cumulative hydrogen, but slower than the combined fermentation.     

Towards the integration of dark- and photo-fermentative waste treatment. 3. Potato as substrate for sequential dark fermentation and light-driven H2 production

The goal of the study was to characterize H₂ production in an integrated process utilizing potato homogenate (PH) for dark, fermentative H₂ production followed by H₂ photoproduction using purple non-sulfur bacteria. Emphasis was placed on (a) examining potato fermentation effluent (FE) as substrate for H₂ photoproduction, (b) estimating the yield and efficiency of both processes, and (c) elucidating the physiological factors influencing the integrated system as a whole. In the dark stage maximal production of gas (11.5 L L⁻¹ of the culture) and VFA (350 mM) were observed with a PH concentration of 400 g L⁻¹ of medium, but higher yields (0.05 L g⁻¹ PH; 1.9 mmol g⁻¹ PH) were obtained at PH concentrations of 50–100 g L⁻¹. H₂ photoproduction by purple bacteria was inhibited at high FE content. Upon suitable dilution, adequate illumination, and supplementation with Fe/Mg/phosphate nutrients, H₂ photoproduction reached 40 L L⁻¹ of non-diluted FE, with a total H₂ yield of 5.6 mol mol⁻¹ glucose equivalent for the two-stage integrated process.     

Dark fermentation of ground wheat starch for bio-hydrogen production by fed-batch operation

Ground wheat solution was used for bio-hydrogen production by dark fermentation using heat-treated anaerobic sludge in a completely mixed fermenter operating in fed-batch mode. The feed wheat powder (WP) solution was fed to the anaerobic fermenter with a constant flow rate of 8.33 mL h⁻¹ (200 mL d⁻¹). Cumulative hydrogen production, starch utilization and hydrogen yields were determined at three different WP loading rates corresponding to the feed WP concentrations of 10, 20 and 30 g L⁻¹. The residual starch (substrate) concentration in the fermenter decreased with operation time while starch consumption was increasing. The highest cumulative hydrogen production (3600 mL), hydrogen yield (465 mL H₂ g⁻¹ starch or 3.1 mol H₂ mol⁻¹ glucose) and hydrogen production rate (864 mL H₂ d⁻¹) were obtained after 4 days of fed-batch operation with the 20 g L⁻¹ feed WP concentration corresponding to a WP loading rate of 4 g WP d⁻¹. Low feed WP concentrations (10 g L⁻¹) resulted in low hydrogen yields and rates due to substrate limitations. High feed WP concentrations (30 g L⁻¹) resulted in the formation of volatile fatty acids (VFAs) in high concentrations causing inhibition on the rate and yield of hydrogen 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.     

Dark fermentation of acid hydrolyzed ground wheat starch for bio-hydrogen production by periodic feeding and effluent removal

Dark fermentation of acid hydrolyzed ground wheat starch for bio-hydrogen production by periodic feeding and effluent removal was investigated at different feeding intervals. Ground wheat was acid hydrolyzed at pH = 3 and T = 121 °C for 30 min using an autoclave. The resulting sugar solution was subjected to dark fermentation with periodic feeding and effluent removal. The feed solution contained 9 ± 0.5 g L⁻¹ total sugar supplemented with some nutrients. Depending on the feeding intervals hydraulic residence time (HRT) was varied between 6 and 60 h. Steady-state daily hydrogen production increased with decreasing HRT. The highest daily hydrogen production (305 ml d⁻¹) and volumetric hydrogen production rate (1220 ml H₂ L⁻¹ d⁻¹) were obtained at HRT of 6 h. Hydrogen yield (130 ml H₂ g⁻¹ total sugar) reached the highest level at HRT = 24 h. Effluent total sugar concentration decreased, biomass concentration and yield increased with increasing HRT indicating more effective sugar fermentation at high HRTs. Dark fermentation end product profile shifted from acetic to butyric acid with increasing HRT. High acetic/butyric acid ratio obtained at low HRTs resulted in high hydrogen yields.     

Effects of sludge pre-treatment method on bio-hydrogen production by dark fermentation of waste ground wheat

Three different pre-treatment methods were applied on two different anaerobic sludge cultures and their mixtures in order to investigate the effects of pre-treatment methods on bio-hydrogen production from dark fermentation of waste ground wheat solution. Repeated heat, chloroform and combinations of heat and chloroform pre-treatment methods were applied to anaerobic sludges from different sources. Repeated heat treatment (2 × 5 h) was found to be more effective in selecting hydrogen producing bacteria compared to the other treatment methods tested on the basis of cumulative hydrogen production. The highest hydrogen formation (652 ml) and specific hydrogen production rate (SHPR = 25.7 ml H₂ g⁻¹ cells h⁻¹) were obtained with the anaerobic sludge pre-treated by repeated boiling. Both the type of anaerobic sludge and the pre-treatment method had considerable effects on bio-hydrogen production from wheat powder solution (WPS) by dark fermentation.     

Continuous biohydrogen production from liquid swine manure supplemented with glucose using an anaerobic sequencing batch reactor

Liquid swine manure supplemented with glucose (10 g/L) was used as substrate for hydrogen production using an anaerobic sequencing batch reactor at 37 ± 1 °C and pH 5.0 under different hydraulic retention times (HRTs). Decreasing HRT from 24 to 8 h caused an increasing hydrogen production rate from 0.05 to 0.15 L/h/L. Production rates of both total biogas and hydrogen were linearly correlated to HRT with R² being 0.993 and 0.997, respectively. The hydrogen yield ranged between 1.18 and 1.63 mol-H₂/mol glucose and the 12 h HRT was preferred for high production rate and efficient yield. For all the five HRTs examined, the glucose utilization efficiency was over 98%. The biogas mainly consisted of carbon dioxide and hydrogen (up to 43%) with no methane detected throughout the experiment. Ethanol and organic acids were the major aqueous metabolites produced during fermentation, with acetic acid accounting for 56–58%. The hydrogen yield was found to be related to the acetate/butyrate ratio.     

Selection of yeast strains for alcoholic fermentation of sugar beet thick juice and green syrup

Presented work aimed at determination of effect of various strains of yeast Saccharomyces cerevisiae and concentration of fermentation worts on dynamics and efficiency of alcoholic fermentation. Fermentation worts contained either thick juice or green syrup.It was found that yeast strains designated as M₁, M₂ and D-2 most efficiently fermented thick juice worts inoculated with yeast cream at a rate of 2 kg m⁻³ of wort. Fermentation processes lasted for approximately 2 days and ethanol yield approached 92-94% of the theoretical yield. Fermentations of green syrup worts were most efficient (ethanol yield reached 90-92% of the theoretical yield) when these processes were carried out by yeast strains M₁, M₂, D-2 and As4 (inoculum - 2 kg m⁻³ of wort).S. cerevisiae strains M₁ and M₂ dynamically and efficiently fermented thick juice worts with extract of 200 g kg⁻¹ and 250 g kg⁻¹ (89-94% of the theoretical yield) while strain D-2 preferred less dense worts (extract of 200 g kg⁻¹) and produced ethanol with the yield of over 92% of the theoretical yield. The optimum green syrup worts extract was 200 g kg⁻¹.