Hydrogen and methane production through two-stage mesophilic anaerobic digestion of olive pulp

The present study focused on the anaerobic biohydrogen production from olive pulp (two phase olive mill wastes, TPOMW) and the subsequent anaerobic treatment of the effluent for methane production under mesophilic conditions in a two-stage process. Biohydrogen production from water-diluted (1:4) olive pulp was investigated at hydraulic retention times (HRT) of 30 h, 14.5 h and 7.5 h while methane production from the effluent of hydrogenogenic reactor was studied at 20 d, 15 d, 10 d and 5 d HRT. In comparison with previous studies, it has been shown that the thermophilic hydrogen production process was more efficient than the mesophilic one in both hydrogen production rate and yield. The methanogenic reactor was successfully operated at 20, 15 and 10 days HRT while it failed when an HRT of 5 days was applied. Methane productivity reached the maximum value of 1.13 ± 0.08 L/L/d at 10 days HRT whereas the methane yield increased with the HRT. The Anaerobic Digestion Model no. 1 (ADM1) was applied to the obtained experimental data from the methanogenic reactor to simulate the digester response at all HRT tested. The ability of the model to predict the experimental results was evident even in the case of the process failure, thus implying that the ADM1 could be a valuable tool for process design even in the case of a complex feedstock. In general, the two-stage anaerobic digestion proved to be a stable, reliable and effective process for energy recovery and stabilization treatment of olive pulp.     

Using cheese whey for hydrogen and methane generation in a two-stage continuous process with alternative pH controlling approaches

This study focuses on the exploitation of cheese whey as a source for hydrogen and methane, in a two-stage continuous process. Mesophilic fermentative hydrogen production from undiluted cheese whey was investigated at a hydraulic retention time (HRT) of 24 h. Alkalinity addition (NaHCO₃) or an automatic pH controller were used, to maintain the pH culture at a constant value of 5.2. The hydrogen production rate was 2.9 ± 0.2 L/Lreactor/d, while the yield of hydrogen produced was approximately 0.78 ± 0.05 mol H₂/mol glucose consumed, with alkalinity addition, while the respective values when using pH control were 1.9 ± 0.1 L/Lreactor/d and 0.61 ± 0.04 mol H₂/mol glucose consumed. The corresponding yields of hydrogen produced were 2.9 L of H₂/L cheese whey and 1.9 L of H₂/L cheese whey, respectively. The effluent from the hydrogenogenic reactor was further digested to biogas in a continuous mesophilic anaerobic bioreactor. The anaerobic digester was operated at an HRT of 20d and produced approximately 1 L CH₄/d, corresponding to a yield of 6.7 L CH₄/L of influent. The chemical oxygen demand (COD) elimination reached 95.3% demonstrating that cheese whey could be efficiently used for hydrogen and methane production, in a two-stage process.     

Effects of initial lactic acid concentration, HRTs, and OLRs on bio-hydrogen production from lactate-type fermentation

A batch test and continuous operation were performed to identify the effect of lactate on hydrogen production at pH 4.5. When the initial lactic acid concentration was increased from 0 to 8 g/L in the batch test, the hydrogen yield also increased from 1.41 to 1.72 mol-H₂/mol-glucose. The system exhibited a long lag time and an insignificant hydrogen yield with 16 g-lactic acid/L. A continuous stirred tank reactor (CSTR) was operated at different organic loading rates (OLRs: 10, 15, 20 and 40 g/L/day) and hydraulic retention times (HRTs: 6, 12 and 24 h). At an OLR of 20 g-glucose/L/day and 12 h of HRT, the hydrogen yield was 1.2 mol-H₂/mol-glucose. The yield decreased with a 24 h HRT. Even though lactate was one of the major constituents of volatile fatty acids (VFAs), hydrogen production was feasible throughout the operation. Clostridium sp. was the dominant hydrogen-producing bacteria in the system.     

Continuous hydrogen and butyric acid fermentation by immobilized Clostridium tyrobutyricum ATCC 25755: Effects of the glucose concentration and hydraulic retention time

The effects of the hydraulic retention time (HRT = 8, 10, 12 or 16.7 h) and glucose concentration (30, 40 or 50 g/L) on the production of hydrogen and butyrate by an immobilized Clostridium tyrobutyricum culture, grown under continuous culturing conditions, were evaluated. With 30 g/L glucose, the higher HRTs tested led to greater butyrate concentrations in the culture, i.e., 9.3 g/L versus 12.9 g/L with HRTs of 8 h and 16.7 h, respectively. In contrast, higher biogas and hydrogen production rates were generally seen when the HRT was lower. Experiments with different glucose concentrations saw a significant amount of glucose washed out when 50 g/L was used, the highest being 22.7 g/L when the HRT was 16.7 h. This study found the best conditions for the continuous production of hydrogen and butyric acid by C. tyrobutyricum to be with an HRT of 12 h and a glucose concentration of 50 g/L, respectively.     

Enhancement of bioenergy production from organic wastes by two-stage anaerobic hydrogen and methane production process

The present study investigated a two-stage anaerobic hydrogen and methane process for increasing bioenergy production from organic wastes. A two-stage process with hydraulic retention time (HRT) 3 d for hydrogen reactor and 12 d for methane reactor, obtained 11% higher energy compared to a single-stage methanogenic process (HRT 15 d) under organic loading rate (OLR) 3 gVS/(L d). The two-stage process was still stable when the OLR was increased to 4.5 gVS/(L d), while the single-stage process failed. The study further revealed that by changing the HRT_hydrogen:HRT_methane ratio of the two-stage process from 3:12 to 1:14, 6.7%, more energy could be obtained. Microbial community analysis indicated that the dominant bacterial species were different in the hydrogen reactors (Thermoanaerobacterium thermosaccharolyticum-like species) and methane reactors (Clostridiumthermocellum-like species). The changes of substrates and HRT did not change the dominant species. The archaeal community structures in methane reactors were similar both in single- and two- stage reactors, with acetoclastic methanogens Methanosarcina acetivorans-like organisms as the dominant species.     

Effects of key operational parameters on biohydrogen production via anaerobic fermentation in a sequencing batch reactor

In this study, a series of tests were conducted in a 6 L anaerobic sequencing batch reactor (ASBR) to investigate the effect of pH, hydraulic retention time (HRT) and organic loading rate on biohydrogen production at 28 °C. Sucrose was used as the main substrate to mimic carbohydrate-rich wastewater and inoculum was prepared from anaerobic digested sludge without pretreatment. The reactor was operated initially with nitrogen sparging to form anaerobic condition. Results showed that methanogens were effectively suppressed. The optimum pH value would vary depending on the HRT. Maximum hydrogen production rate and yield of 3.04 L H₂/L reactor d and 2.16 mol H₂/mol hexose respectively were achieved at pH 4.5, HRT 30 h, and OLR 11.0 kg/m³ d. Two relationships involving the propionic acid/acetic acid ratio and ethanol/acetic acid ratio were derived from the analysis of the metabolites of fermentation. Ethanol/acetic acid ratio of 1.25 was found to be a threshold value for higher hydrogen production.     

Conversion of food waste into hydrogen by thermophilic acidogenesis

Conversion of food waste into hydrogen by thermophilic acidogenesis was investigated as a function of organic loading rate (OLR), hydraulic retention time (HRT) and pH in a continuous stirred tank reactor. In order to identify hydrogen-producing microorganisms, denaturing gradient gel electrophoresis (DGGE) of the polymerase chain reaction (PCR) – amplified V3 region of 16S rDNA analysis was conducted at each tested pH. The conversion of food waste into hydrogen was strongly influenced by the operational conditions. The hydrogen production was increased as OLR increased up to 8gVSl^-1d^-1, but drastically decreased at 10gVSl^-1d^-1. The yield of hydrogen was decreased from 2.2 to 1.0mol-H₂/mol-hexose consumed as HRT decreased from 5 to 2days. More carbohydrates in the food waste were decomposed at longer HRT, 76–90%, at HRT of 2–5days. The hydrogen production peaked at pH 5.5±0.1 and significantly decreased at pH 5.0±0.1. The biogas produced was composed of hydrogen and carbon dioxide, but no methane was detected at all tested conditions. The hydrogen contents in the gas produced were more than 55% (v/v) and not sensitive to all tested conditions. The optimum operational condition for continuous hydrogen production from the food waste was obtained at 8gVSl^-1d^-1, 5 days HRT and pH 5.5±0.1 where the hydrogen production rate, content, yield and the efficiency of carbohydrate decomposition were 1.0l H₂/l-d, 60.5% (v/v), 2.2mol-H₂/mol-hexose consumed and 90%, respectively. The hydrogen production was related with the concentration of total organic acids (TOA) which was strongly dependent on that of butyrate indicating that the reaction was mainly butyrate fermentation. The hydrogen-producing microorganism of Thermoanaerobacterium thermosaccharolyticum that involved in acetate/butyrate fermentation, was detected with strong intensity at all tested pHs by denaturing gradient gel electrophoresis (DGGE) of the polymerase chain reaction (PCR) – amplified V3 region of 16S rDNA analysis and sensitive to the tested pHs. The experimental results indicated that effective hydrogen production from the food waste could be obtained continuously by thermophilic acidogenesis at proper operational condition.     

Characteristics of hydrogen and methane production from cornstalks by an augmented two- or three-stage anaerobic fermentation process

This paper presents the co-production of hydrogen and methane from cornstalks by a two- or three-stage anaerobic fermentation process augmented with effective artificial microbial community. Two-stage fermentation by using the anaerobic sludge and DGGE analysis showed that effective and stable strains should be introduced into the system. We introduced Enterobacter aerogens or Clostridium paraputrificum into the hydrogen stage, and C. paraputrificum was proven to be more effective. In the three-stage process consisting of the improved hydrolysis, hydrogen and methane production stages, the highest soluble sugars (0.482 kg/kg cornstalks) were obtained after the introduction of Clostridium thermocellum in the hydrolysis stage, under the thermophilic (55 °C) and acidic (pH 5.0) conditions. Hydrolysates from 1 kg of cornstalks could produce 2.61 mol (63.7 l) hydrogen by augmentation with C. paraputrificum and 4.69 mol (114.6 l) methane by anaerobic granular sludge, corresponding to 54.1% energy recovery.     

Effect of operating conditions on the extraction of ovomucin

Ovomucin, mainly responsible for the gelatinous property of egg white, has potential applications as a functional food and nutraceutical ingredient. A 2-step method for ovomucin preparation was recently developed. The purpose of this study was to determine the effects of various operating conditions, such as pH, NaCl concentrations and extraction volume at the second extraction, temperature, and centrifugation force, on the purity and yield of ovomucin. Our results showed that pH has a significant effect on the purity and yield of ovomucin extracts. Increasing the extraction pH from 4.0 to 5.0 could significantly (p < 0.05) increase the purity and yield of ovomucin; at pHs higher than 5.0, the purity was not affected but the yield was significantly decreased. The highest yield of ovomucin extract (308 mg/100 g of egg white) was achieved at pH 5.0 while the highest purity was achieved at pH 7.0. There is a trend that the purity of ovomucin increased (p < 0.05) but the yield of ovomucin decreased (p > 0.05) at increasing salt concentrations. Reducing extraction volume did not affect the yield of ovomucin whereas its purity was significantly decreased. The yield of ovomucin however was significantly increased at increasing settling time or centrifugation force, but the purity was less affected. Extraction of ovomucin at room temperature could significantly reduce the extraction yield compared to that at lower temperature (4 °C) but the purity was not affected.     

Evaluation of bioenergy recovery processes treating organic residues from ethanol fermentation process

This study evaluates a two-stage bioprocess for recovering bioenergy in the forms of hydrogen and methane while treating organic residues of ethanol fermentation from tapioca starch. A maximum hydrogen production rate of 0.77 mmol H₂/g VSS/h can be achieved at volumetric loading rate (VLR) of 56 kg COD/m³/day. Batch results indicate that controlling conditions at S₀/X₀ = 12 with X₀ = 4000 mg VSS/L and pH 5.5-6 are important for efficient hydrogen production from fermentation residues. Hydrogen-producing bacteria enriched in the hydrogen bioreactor are likely utilizing lactate and acetate for biohydrogen production from ethanol-fermentation residues. Organic residues remained in the effluent of hydrogen bioreactor can be effectively converted to methane with a rate of 0.37 mmol CH₄/g VSS/h at VLR of 8 kg COD/m³/day. Approximately 90% of COD in ethanol-fermentation residues can be removed and among that 2% and 85.1% of COD can be recovered in the forms of hydrogen and methane, respectively.     

Acclimatization of Methanogenic Consortia for Low pH Biomethanation Process

Methanogenic cultures were acclimatized to operate at low pHs and the efficiency of methane production at pH 5.0, 4.5 and 4.0 was 67, 37 and 34% respectively, compared to biomethanation at pH 7.0 (100% yield) with 55–65% methane in the biogas composition. Above pH 5.0, the efficiency of methane production was more than 75%. The data for most of the experiments are based on the observations carried out for more than 60 and 100 days. The control experiments of direct pH shock of 4.5 and 4.0 did not show any gas production and even at pH 5.5 production of methane was marginal.     

Effect of hydraulic retention time on anaerobic hydrogenesis in CSTR

The objective of this work was to evaluate the production of hydrogen in a continuous system as a function of hydraulic retention time (HRT). The intermediates accumulated and other parameters of pH, oxidation-reduction potential were quantified. The heat treatment (103 degrees C for 24 h) of the compost from a cattle dung composting facility was able to select H2-producing spores; this product was used as a seed for continuous systems. The brewery waste was used as substrate. For the eight runs with combinations of five HRTs and four pHs, the results indicate that at pH=5.5, a maximum H2 production of 47% H2 concentration, 43 ml H2/g COD(added), and 3.1 l H2/l reactor d was achieved at HRT=18 h. Nevertheless, at HRT=18 h, pH 5.5 was also the optimum pH for the maximum H2 production among four pHs evaluated from 5 to 6.5. There was a significant accumulation of volatile acid and alcohols during the entire study.     

Hydrogen production by Rhodopseudomonas palustris WP 3-5 in a serial photobioreactor fed with hydrogen fermentation effluent

In this study, a lab-scale serial photobioreactor composed of three column reactors was constructed and continuously operated to investigate several parameters influencing photohydrogen production when using the synthetic wastewater and the anaerobic hydrogen fermentation effluents as the influents. The results indicated that better hydrogen production rate was obtained when the serial photobioreactor was operated under cellular recycling at a short HRT of 8 h. The serial photobioreactor maintained high hydrogen content ca. 80% in the produced gas and 0.4× dilution ratio was the suitable ratio for hydrogen production. When the photobioreactor fed with the real wastewater (Effluent 1) containing 100 mg/L NH₄Cl, Column 1 reactor successfully reduced ammonia concentration to about 60 mg/L for cell synthesis, resulting in a steady hydrogen production in the following two column reactors. The average hydrogen production rate was 205 mL-H₂/L/d.     

Evaluation of performance and microbial community in a two-stage UASB reactor pretreating acrylic fiber manufacturing wastewater

A two-stage UASB reactor was employed to pretreat acrylic fiber manufacturing wastewater. Mesophilic operation (35 ± 0.5 °C) was performed with hydraulic retention time (HRT) varied between 28 and 40 h. Mixed liquor suspended solids (MLSS) in the reactor was maintained about 8000 mg/L. The results showed COD and sulfate removal could be kept at 51% and 75%, respectively, when the HRT was no less than 38 h. Sulfate reduction mainly occurred in the acidification-stage reactor while methane production mainly occurred in the methane-stage reactor. The size of granule formed in the acidification-stage reactor ranged between 1 and 5 mm while the largest size of granule in the methane-stage reactor ranged from 0.5 to 2 mm. Compared to microbial populations in the acidification-stage reactor, the microbial diversity in methane-stage reactor was more abundant. In the acidification-stage reactor, the Syntrophobacter sulfatireducens devoted to both sulfate reduction and acetate production.     

Kinetics of an extracellular exo-inulinase production from a 5-flourocytosine resistant mutant of Geotrichum candidum using two-factorial design

In the present study, eight different strains of Geotrichum candidum were isolated and screened for an extracellular exo-inulinase production using chemically enriched sucrose–mineral media. The isolate (Zool-3i) with a better enzyme activity (1.38 IU/ml) was subjected to induced mutagenesis using methyl methane sulphonate (MMS) and a mutant with an enzyme activity of 32.06 IU/ml was obtained. Further exposure to ethyl methane sulphonate (EMS) and ultraviolet (UV) radiations yielded a mutant exhibiting an improved activity of 39.34 IU/ml. The potential mutant was cultured overnight and plated on 5fc–YPR agar medium and thus made resistant against 5-flourocytocine. Over 50-fold enhancement in enzyme production (71.85 IU/ml) was achieved when the process parameters including incubation period (48 h), sucrose concentration (5.0 g/L), pH (6.0), inoculum size (2.0%, 16 h old) and urea (0.2%) were identified using Plackett–Burman design. On the basis of kinetic variables, notably Q_p (0.723 U/g/h), Y_p/s (2.036 U/g) and q_p (0.091 U/g cells/h), the mutant MEU-5fc-6 was found to be a hyper producer of exo-inulinase (HS, LSD 0.045, p ? 0.05).     

A thermotolerant and cold-active mannan endo-1,4-β-mannosidase from Aspergillus niger CBS 513.88: Constitutive overexpression and high-density fermentation in Pichia pastoris

The mannan endo-1,4-β-mannosidase gene man26A from Aspergillus niger CBS 513.88 was optimized according to the codon usage bias in Pichia pastoris and synthesized by splicing overlap extension PCR. It was successfully expressed in P. pastoris using constitutive expression vector pGAPzαA. The recombinant endo-beta-1,4-mannanase could work in an extremely board temperature range and over 30% relative activity were retained in the temperature range of 5-60 °C. The optimal pH value and temperature for activity were 5.0 and 45 °C, respectively. It was highly thermotolerant with a half-life time of 15 min at 90 °C. A novel fed-batch strategy was developed successfully for high cell-density fermentation and mannanase activity reached 5069 U/mL after cultivation for 56 h in 50 L fermenter. The broad working temperature range, high thermotolerance and efficient expression made this enzyme possible to be applied in food, animal feed and the production of biofuels.     

Production of leucine amino peptidase in lab scale bioreactors using Streptomyces gedanensis

Studies were conducted on the production of leucine amino peptidase (LAP) by Streptomyces gedanensis to ascertain the performance of the process in shake flask, parallel fermenter and 5-L fermenter utilizing soy bean meal as the carbon source. Experiments were conducted to analyze the effects of aeration and agitation rate on cell growth and LAP production. The results unveiled that an agitation rate of 300 rpm, 50% dissolved oxygen (DO) upholding and 0.15 vvm strategies were the optimal for the enzyme production, yielding 22.72 ± 0.11 IU/mL LAP in parallel fermenter which was comparable to flask level (24.65 ± 0.12 IU/mL LAP) fermentation. Further scale-up, in 5-L fermenter showed 50% DO and 1 vvm aeration rate was the best, producing optimum and the production was 20.09 ± 0.06 IU/mL LAP. The information obtained could be useful to design a strategy to improve a large-scale bioreactor cultivation of cells and production of LAP.     

Performance and microbial community analysis of two-stage process with extreme thermophilic hydrogen and thermophilic methane production from hydrolysate in UASB reactors

The two-stage process for extreme thermophilic hydrogen and thermophilic methane production from wheat straw hydrolysate was investigated in up-flow anaerobic sludge bed (UASB) reactors. Specific hydrogen and methane yields of 89 ml-H₂/g-VS (190 ml-H₂/g-sugars) and 307 ml-CH₄/g-VS, respectively were achieved simultaneously with the overall VS removal efficiency of 81% by operating with total hydraulic retention time (HRT) of 4 days . The energy conversion efficiency was dramatically increased from only 7.5% in the hydrogen stage to 87.5% of the potential energy from hydrolysate, corresponding to total energy of 13.4 kJ/g-VS. Dominant hydrogen-producing bacteria in the H₂-UASB reactor were Thermoanaerobacter wiegelii, Caldanaerobacter subteraneus, and Caloramator fervidus. Meanwhile, the CH₄-UASB reactor was dominated with methanogens of Methanosarcina mazei and Methanothermobacter defluvii. The results from this study suggest the two stage anaerobic process can be effectively used for energy recovery and for stabilization of hydrolysate at anaerobic conditions.     

Biohydrogen production in a three-phase fluidized bed bioreactor using sewage sludge immobilized by ethylene–vinyl acetate copolymer

Ethylene–vinyl acetate (EVA) copolymer was used to immobilize H₂-producing sewage sludge for H₂ production in a three-phase fluidized bed reactor (FBR). The FBR with an immobilized cell packing ratio of 10% (v/v) and a liquid recycle rate of 5 l/min (23% bed expansion) was optimal for dark H₂ fermentation. The performance of the FBR reactor fed with sucrose-based synthetic medium was examined under various sucrose concentration (C_so) and hydraulic retention time (HRT). The best volumetric H₂ production rate of 1.80 ± 0.02 H₂ l/h/l occurred at C_so = 40 g COD/l and 2 h HRT, while the optimal H₂ yield (4.26 ± 0.04 mol H₂/mol sucrose) was obtained at C_so = 20 g COD/l and 6 h HRT. The H₂ content in the biogas was stably maintained at 40% or above. The primary soluble metabolites were butyric acid and acetic acid, as both products together accounted for 74–83% of total soluble microbial products formed during dark H₂ fermentation.     

Performance evaluation and phylogenetic characterization of anaerobic fluidized bed reactors using ground tire and pet as support materials for biohydrogen production

This study evaluated two different support materials (ground tire and polyethylene terephthalate [PET]) for biohydrogen production in an anaerobic fluidized bed reactor (AFBR) treating synthetic wastewater containing glucose (4000 mg L⁻¹). The AFBR, which contained either ground tire (R1) or PET (R2) as support materials, were inoculated with thermally pretreated anaerobic sludge and operated at a temperature of 30 °C. The AFBR were operated with a range of hydraulic retention times (HRT) between 1 and 8 h. The reactor R1 operating with a HRT of 2 h showed better performance than reactor R2, reaching a maximum hydrogen yield of 2.25 mol H₂ mol⁻¹ glucose with 1.3 mg of biomass (as the total volatile solids) attached to each gram of ground tire. Subsequent 16S rRNA gene sequencing and phylogenetic analysis of particle samples revealed that reactor R1 favored the presence of hydrogen-producing bacteria such as Clostridium, Bacillus, and Enterobacter.