Co-digestion of untreated macro and microalgal biomass for biohydrogen production: Impact of inoculum augmentation and microbial insights

This study assessed the co-digestion of macro and microalgal biomass towards the improvement of hydrogen production. The red macroalgal biomass (Gelidium amansii) and green mixed microalgal biomass was mixed in a ratio of 8:2, with an initial substrate concentration of 10 g/L, and various amount of inoculum addition range from 3 to 15% (v/v) was evaluated to assess the feasible substrate to inoculum ratio for the effective co-digestion of the algal biomass. The results showed that the co-digestion with 6% inoculum addition provided the peak hydrogen yield of 45 mL/g dry biomass added with a high hydrogen content of 24% in the gas phase. The other tested conditions showed moderate hydrogen content in the range of 17–22%, respectively. These results suggest that anaerobic co-digestion of macro and microalgal biomass, with appropriate initial biomass loading (6%) is essential for enhanced hydrogen production.     

Pre-treatment study on two-stage biohydrogen and biomethane productions in a continuous co-digestion process from a mixture of swine manure and pineapple waste

This study involves continuous co-digestion of swine manure and pineapple waste mixture using two-stage anaerobic reactors and examines hydraulic retention time (HRT) and substrate heat pre-treatment. The maximum hydrogen and methane production rates of 1488.62 and 991.57 mL/L/d, respectively, reached optimal HRTs of 4.5 h in the hydrogen production fermenter (HPF) and 9 d in the methane production fermenter (MPF) using heat pre-treatment. Acetic acid is a dominant volatile fatty acid of the soluble metabolites with values 70%–73% under all the tested conditions and increased values under heat pre-treatment and high HRT. Firmicutes and Euryarchaeota are the main bacteria species detected in HPF and MPF, respectively. The optimal total energy of 196.47 kJ/L/d and chemical oxygen demand (COD) removal efficiency of 90% were obtained by a complete anaerobic co-digestion process at a high substrate concentration of 105 g COD/L and low HRT of 4.5 h. This shows that the two-stage co-digestion process could increase the COD removal efficiency, hydrogen production rate, and net energy gains and produce high quality biogas and significantly reduce fermentation time.     

Identification of new critical points for logistics model in cumulative methane yield curves after co-digestion of apple pulp and chicken manure with sulphuric acid pretreatment and a new modelling study

In this study, anaerobic co-digestion of apple pulp (AP) with high carbon content and chicken manure (CM) with high nitrogen content was evaluated with regard to sulphuric acid pretreatment conditions in the concentration range of 1%-6% v/v. The best mixing ratio of CM:AP was determined as 3:1 w/w under untreated conditions for which the methane yield was 255.88 ± 11.55 mL/g VS (volatile solid). Sulphuric acid pretreatments were applied to this mixture ratio. The highest methane yield after pretreatment was 466.01 ± 10.85 mL/g VS in the reactor, where the sulphuric acid pretreatment concentration was 3.0% v/v. Control of acid pretreatment results were achieved by analysis of lignocellulosic degradation and increase in soluble chemical oxygen demand. The highest cellulose, hemicellulose and lignin degradation after pretreatment were 27.5% ± 1.4%, 35.9% ± 2.9% and 14% ± 1.5% w/w, respectively. The cumulative methane yields (CMY) were analysed by the modified logistic model, modified Bertalanffy model (MBM), modified Gompertz model (MGM) and modified Holling model (MHM). The MBM and MGM were found to fit the experimental data better than other models. The MHM has not previously been applied to CMY. In addition to the kinetic studies, new critical points for CMYs were identified in the logistics model (lm). The importance and critical properties of these points with regard to CMY are introduced. The differences and superior properties of these critical points compared to other modelling methods are discussed.     

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.     

Enhancement of batch biohydrogen production from prehydrolysate of acid treated oil palm empty fruit bunch

Carbohydrates from hydrolyzed biomass has been a potential feedstock for fermentative hydrogen production. In this study, oil palm empty fruit bunch (OPEFB) was treated by sulfuric acid in different concentrations at 120 °C for 15 min in the autoclave. The optimal condition for pretreatment was obtained when OPEFB was hydrolyzing at 6% (w/v) sulfuric acid concentration, which gave the highest total sugar of 26.89 g/L and 78.51% of sugar production yield. However, the best conversion efficiency of OPEFB pretreatment was 39.47 at sulfuric acid concentration of 4%. A series of batch fermentation were performed to determine the effect of pH in fermentation media and the potential of this prehydrolysate was used as a substrate for fermentative hydrogen production under optimum pretreatment conditions. The prehydrolysate of OPEFB was efficiently converted to hydrogen via fermentation by acclimatized mixed consortia. The maximum hydrogen production was 690 mL H₂ L⁻¹ medium, which corresponded to the yield of 1.98 molH₂/mol_xylose achieved at pH 5.5 with initial total sugar concentration of 5 g/L. Therefore, the results implied that OPEFB prehydrolysate is prospective substrate for efficient fermentative hydrogen conducted at low controlled pH. No methane gas was detected throughout the fermentation.     

Effect of ammonia concentration on hythane (H2 and CH4) production in two-phase anaerobic digestion

Co-production of hydrogen and methane by two-phase anaerobic digestion (AD) may offer a sustainable solution for the centralized treatment of food waste (FW), while ammonia accumulation is potentially encountered. A mesophilic two-phase AD was investigated for hydrogen and methane production from FW at varying ammonia concentrations. The process achieved a hydrogen yield of 47.7 mL/g VS and a methane yield of 335 mL/g VS by optimizing the organic loading rate (OLR) and recirculation ratio. Total ammonia nitrogen (TAN) concentration of 4044 mg/L corresponded to a threshold in the hydrogen reactor, above which ammonia would initiate inhibition of hydrogenogenesis and acidogenesis. Methane yield was recovered in the methane reactor after acute inhibiting effects of TAN below 5800 mg/L, while TAN above 6200 mg/L caused chronic inhibition of methanogens. Adjusting hydraulic retention time (HRT) and recirculation ratio in hydrogen and methane reactors reduced TAN to 960 and 2105 mg/L respectively, resulting in successful recovery was achieved in the hydrogen reactor but not in the methane reactor. The two-phase AD for methane and hydrogen production can be a promising solution for ammonia accumulation in AD from FW.     

Nymphoides peltatum as a novel feedstock for biomethane production via anaerobic co-digestion with waste sludge

Nymphoides peltatum (NP) is exploited as a novel feedstock for biomethane production via anaerobic co-digestion with waste sludge (WS). Batch experiments are conducted under mesophilic condition at NP/WS of 1/3, 2/2, 3/1, 0/4 and 4/0 based on volatile solids (VS). Prior to anaerobic digestion (AD), NP undergoes only natural drying and grinding. The maximum net cumulative methane yield (265.16 mL CH₄·g VS_added^?1) and the highest gross VS removal rate (56.12%) are obtained at NP/WS of 1/3. The kinetic analysis by the modified Gompertz model fit hinted that 28 days is adequate for methane recovery and co-digestion significantly accelerates the digestion rate. Synergetic effect is corroborated to exist in co-digestion due to amiable conditions in term of total ammonia nitrogen, free ammonia, pH, volatile fatty acids and total alkalinity. High-throughput 16S rRNA pyrosequencing reveals that Bacteroidetes, Firmicutes, Methanosarcina and Methanosaeta are conducive to AD of NP.     

Improvement of biohythane production from Chlorella sp. TISTR 8411 biomass by co-digestion with organic wastes in a two-stage fermentation

The suitability of molasses, Napier grass (Pennisetum purpureum), empty fruit bunches (EFB), palm oil mill effluent (POME), and glycerol waste as a co-substrate with Chlorella sp. TISTR 8411 biomass for biohythane production was investigated. Mono-digestion of Chlorella biomass had hydrogen and methane yield of 23–35 and 164–177 mL gVS⁻¹, respectively. Co-digestion of Chlorella biomass with 2–6% TS of organic wastes was optimized for biohythane production with hydrogen and methane yield of 17–75 and 214–577 mL gVS⁻¹, respectively. The hydrogen and methane yield from co-digestion of Chlorella biomass with molasses, POME, and glycerol waste was increased by 8–100% and 80–264%, respectively. The biohythane production of co-digestion of Chlorella was 6–11 L L-mixed waste⁻¹ with an optimal C/N ratio range of 19–41 and H₂/CH₄ ratio range of 0.06–0.3. Co-digestion of Chlorella biomass was significantly improved biohythane production in term of yield, production rate, and kinetics.     

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

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

Optimization of phototrophic hydrogen production by Rhodopseudomonas palustris PBUM001 via statistical experimental design

Phototrophic hydrogen production by indigenous purple non-sulfur bacteria, Rhodopseudomonas palustris PBUM001 from palm oil mill effluent (POME) was optimized using response surface methodology (RSM). The process parameters studied include inoculum sizes (% v/v), POME concentration (% v/v), light intensity (klux), agitation (rpm) and pH. The experimental data on cumulative hydrogen production and COD reduction were fitted into a quadratic polynomial model using response surface regression analysis. The path to optimal process conditions was determined by analyzing response surface three-dimensional surface plot and contour plot. Statistical analysis on experimental data collected following Box-Behnken design showed that 100% (v/v) POME concentration, 10% (v/v) inoculum size, light intensity at 4.0 klux, agitation rate at 250 rpm and pH of 6 were the best conditions. The maximum predicted cumulative hydrogen production and COD reduction obtained under these conditions was 1.05 ml H₂/ml POME and 31.71% respectively. Subsequent verification experiments at optimal process values gave the maximum yield of cumulative hydrogen at 0.66 ± 0.07 ml H₂/ml POME and COD reduction at 30.54 ± 9.85%.     

Co-digestion of waste glycerol and glucose to enhance biogas production

Co-digestion in anaerobic fermentation has been widely used to improve biogas production. The biogas production from co-digestion of glucose and glycerol was studied in laboratory-scale batch reactors under mesophilic temperatures, pH 7. The batch experiments involved a variation of glycerol/glucose ratios with initial chemical oxygen demand (COD) for all conditions was fixed at 5,200 mg L⁻¹. The highest yield of biogas production was obtained from glycerol/glucose with 5:5 ratio. The cumulative biogas production was 298.2 mL, and the maximum production rate was 8 mL hr⁻¹. The findings suggested that co-digestion is a potential method to achieve glycerol waste treatment and energy recovery at the same time.     

Effects of harvest month on biochemical composition of alligator weed for biohydrogen and biomethane cogeneration：Identifying critical variations in microbial communities

To determine the optimal harvest period for alligator weed used in fermentation-based gaseous biofuel production, the effects of harvest months on the biochemical composition and fermentative hydrogen and methane co-production from alligator weed were evaluated. Compositional analysis indicates that alligator weed harvested in September was most suitable for fermentative hydrogen production because it exhibited an appropriate carbon to nitrogen molar ratio (26.75), the highest cellulose content (20.34%) and the lowest weight ratio of ash to volatiles (0.09). Biohydrogen production was significantly affected by the harvest month, peaking at 48.4 mL/g VS in September. Microbial community analysis suggests that the abundance of Clostridium sensu stricto, the dominating hydrogen producer in dark fermentation reactors, was greatly enriched with feedstock harvested in September. Moreover, the expression levels of functional genes involved in carbohydrate metabolism and microbial proliferation were highest in the reactor fed with feedstock harvested in September. During the second-stage anaerobic digestion (AD), biomethane yield mainly depended on the soluble COD content in the hydrogenogenic effluent and was highest in August (214.6 mL/g VS). A maximum total energy conversion efficiency of 44.8% was obtained in September through two-stage process combining dark fermentation and AD.     

Biohydrogen and biogas production from mashed and powdered vegetable residues by an enriched microflora in dark fermentation

This study conducted the utilization of vegetable residues by an enriched microflora inoculum to produce biohydrogen via anaerobic batch reactor. Dark fermentation processes were carried out with 3 kinds of vegetable residue substrates including broccoli (Brassica oleracea var. italica.), onion (Alium cepa Linn.), and sweet potato (Ipomoea batatas (L.) Lam). Vegetable wastes were pretreated into 2 forms, i.e. mashed and powdered vegetable, prior to the fermentation. The substrate used for the biohydrogen production were vegetable residues and inoculum at the vegetable residues/inoculum ratio of 1:1 (based on TS). The digestion processes were performed under 120 rpm speed of shaking bottle in the incubator with control temperature of 35^?C. In this work, the maximum hydrogen production was achieved by anaerobic digestion at mashed onion with bioreactor inoculum that produced total hydrogen of 424.1 mL H₂ with hydrogen yield and hydrogen concentration of 151.67 mL H₂/g VS_added and 43.54%, respectively. In addition, the hydrogen production continues took only 7 days for the vegetables blended with the bioreactor inoculum. Finally, it was found that the high potential of degradation of vegetable wastes an enriched microflora in dark fermentation also showed alternative solution to eliminate agricultural wastes to produce green energy.     

Hydrogen production by sequential dark and photofermentation using wet biomass hydrolysate of Spirulina platensis: Response surface methodological approach

Microalgae and cyanobacteria can be used as a potential biomass to produce hydrogen from stored glycogen and starch through fermentation and photofermentation. In this study, the potential of algal biomass i.e. Spirulina platensis hydrolysate as a substrate for sequential fermentative (I-stage) and photo-fermentative (II-stage) biohydrogen production was evaluated. Response Surface Methodology (RSM) was employed to find the optimum photofermentation conditions. From the preliminary optimization experiments, it was found that the significantly affecting factors for H₂ production were pH, dilution fold (D.F.) of fermentate and Fe(II) sulfate concentration during photofermentation (second stage). In the present study, 1% (w/v) Spirulina platensis hydrolyzate produced 23.06 ± 3.63 mmol of H₂ with yield of 1.92 ± 0.20 mmol H₂/g COD reduced. In the second stage experiment 1510 ± 35 mL/l hydrogen was produced using inoculum volume-20.0% (v/v) and inoculum age-48 h of co-culture of Rhodobacter sphaeroides NMBL-01 and Bacillus firmus NMBL-03 under conditions pH-5.95, D.F. of dark fermentate-20.30 folds, Fe(II) sulfate concentration-0.412 μM, temperature-32±2 °C and light intensity-2.5 klux.     

Biohydrogen production from paper industry wastes by SSF: A study of the influence of temperature/enzyme loading

The present work focused in assessing the hydrogen production from pretreated wastes of the paper industry (PIW) by simultaneous saccharification and fermentation (SSF) using anaerobic biofilms developed in natural fibers (ixtle) at different conditions. Anaerobic sludge from brewery wastewater treatment was used for biofilms and they were developed in plastic spheres covered with fiber cord from ixtle. The solid wastes of paper industry were previously pretreated with H₂SO₄ at 2.5% (v/v) at 120 °C by 30 min. The solids pretreated were hydrolyzed and fermented in batch reactors. All reactors were kept at an initial pH of 5.0 and three levels of enzyme loadings (10, 40 and 70 FPU/mL) and temperatures (35, 45, 55 °C) were assessed. The maximum hydrogen obtained (60.75 mmol/h*g volatile solids) was at 45 °C and 70 FPU/mL, moreover, no methane was detected in all cases.     

Decreasing methane production in hydrogenogenic UASB reactors fed with cheese whey

One of the problems in fermentative hydrogen producing reactors, inoculated with pre-treated anaerobic granular sludge, is the eventual methane production by hydrogen-consuming methanogens. In this study, strategies such as reduction of pH and HRT, organic shock loads and repeated biomass heat treatment were applied to hydrogenogenic UASB reactors fed with cheese whey, that showed methane production after certain time of continuous operation (between 10 and 60 days). The reduction of pH to 4.5 not only decreased methane production but also hydrogen production. Organic shock load (from 20 to 30 g COD/L-d) was the more effective strategy to decrease the methane production rate (75%) and to increase the hydrogen production rate (172%), without stopping reactor operation. Repeated heat treatment of the granular sludge was the only strategy that inhibited completely methane production, leading to high volumetric hydrogen production rates (1.67 L H₂/L-d), however this strategy required stopping reactor operation; in addition homoacetogenesis, another hydrogen-consuming pathway, was not completely inhibited. This work demonstrated that it was possible to control the methane activity in hydrogen producing reactors using operational strategies.     

皇竹草厌氧发酵特性及稀土元素溶出研究

为探讨稀土尾矿区修复用皇竹草的厌氧发酵特性和发酵过程稀土元素溶出情况,开展不同发酵浓度（总固体浓度分别为2%、4%、6%、8%、10%）的中温（37±1℃）批式厌氧发酵实验。结果表明,总固体（TS）浓度为4%条件下皇竹草产甲烷性能最佳,日产甲烷率和累积产甲烷率分别为25.56 mL/g和197.33 mL/g。采用修正Gompertz方程能较好地模拟不同TS下皇竹草发酵累积产甲烷率的变化。分析发酵液中稀土元素浓度变化,结果表明,皇竹草内的稀土元素在厌氧发酵过程中只发生小部分溶出,主要包括La、Ce、Nd、Sc、Y等,各处理发酵液中稀土元素浓度并未超出文献报道的抑制浓度。相关性分析结果表明,稀土元素溶出率与发酵体系的TS浓度、化学需氧量（COD）呈负相关,与pH呈正相关。研究可为稀土矿区皇竹草沼气工程应用和发酵剩余物的肥料化利用提供指导。     

Integrated process to produce biohydrogen from wheat straw by enzymatic saccharification and dark fermentation

In this study, different pretreatment methods, including lyophilization, hydrothermal pretreatment, and ultrasound combined with dilute alkali post-cooking, were investigated to enhance the efficiency of enzymatic saccharification and biohydrogen production of the wheat straw. All pretreatment methods could effectively remove lignin and hemicellulose while retaining cellulose, further enhancing the biomass accessibility for subsequently enzymatic saccharification and biohydrogen production. A reducing sugar concentration of 13.18 g/L was acquired when wheat straw was treated with ultrasound and dilute alkali cooking (R_U). The sequential fermentative hydrogen yield of the substrate R_U was 133.6 mL/g total solids (TS), which was 5.6-fold larger than that of the raw material (23.9 mL/g TS). The study confirmed that ultrasound combined with dilute alkali cooking was an effective method, which not only provided significant guideline for improving biohydrogen production but also presented helpful direction for the efficient pretreatment of other lignocellulosic biomass.     

A robust and efficient bioprocess of hydrogen production from crude glycerol by Clostridium beijerinckii G117

Clostridia can produce hydrogen (a renewable-biofuel) from crude glycerol (CG). Reportedly, the indigenous CG-impurities (eg.methanol, soap and salt) interfere with clostridial-process, and reduces hydrogen production. Thus, it is important to develop a robust bioprocess to directly utilize native-CG for hydrogen production. In this study, among four Clostridia tested, Clostridium beijerinckii G117 is selected based on its high hydrogen (>290 mL/g) production from CG. Furthermore, optimization of process-parameters enhances hydrogen production from strain G117 by ～5 fold from 1195 ± 45 to 5893 ± 25 mL/L with 2.16 mol/mol yield. The order of impact of process-parameters (optimum value in parenthesis) on hydrogen production is: CG concentration (12.5 g/L)>inoculum size (10%v/v)>inoculum age (12 h)>temperature (39 °C)>initial medium-pH (6.4)>L-cysteine (1 g/L)>operational volume (30 mL)>agitation rate (150 rpm). Notably, this optimized bioprocess records >90% efficiency by determining total mass and electron balances. Interestingly, this optimized bioprocess retains adequate robustness to yield 1.94–2.02 mol/mol hydrogen with elevated CG-impurities including alcohols/salts/surfactants.     

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.