Harnessing of biohydrogen from wastewater treatment using mixed fermentative consortia: Process evaluation towards optimization

Utilizing wastewater as a potential source for renewable energy generation through biological routes has instigated considerable interest recently due to its sustainable nature. An attempt was made in this communication to review and summarize the work carried out in our laboratory on dark fermentation process of biohydrogen (H₂) production utilizing wastewater as primary substrate under acidogenic mixed microenvironment towards optimization of dynamic process. Process was evaluated based on the nature and composition of wastewater, substrate loading rates, reactor configuration, operation mode, pH microenvironment and pretreatment procedures adopted for mixed anaerobic culture to selectively enrich acidogenic H₂ producing consortia. The fermentative conversion of the substrate to H₂ is possible by a series of complex biochemical reactions manifested by selective bacterial groups. In spite of striking advantages, the main challenge of fermentative H₂ production is that, relatively low energy from the organic source was obtained in the form of H₂. Further utilization of unutilized carbon sources present in wastewater for additional H₂ production will sustain the practical applicability of the process. In this direction, enhancing H₂ production by adapting various strategies, viz., self-immobilization of mixed consortia (onto mesoporous material and activated carbon), integration with terminal methanogenic and photo-biological processes and bioaugmentation with selectively enriched acidogenic consortia were discussed. Application of acidogenic microenvironment for in situ production of bioelectricity through wastewater treatment employing microbial fuel cell (MFC) was also presented.     

Integration of acidogenic and methanogenic processes for simultaneous production of biohydrogen and methane from wastewater treatment

Feasibility of integrating acidogenic and methanogenic processes for simultaneous production of biohydrogen (H₂) and methane (CH₄) was studied in two separate biofilm reactors from wastewater treatment. Acidogenic bioreactor (acidogenic sequencing batch biofilm reactor, AcSBBR) was operated with designed synthetic wastewater [organic loading rate (OLR) 4.75 kg COD/m³-day] under acidophilic conditions (pH 6.0) using selectively enriched acidogenic mixed consortia. The resultant outlet from AcSBBR composed of fermentative soluble intermediates (with residual carbon source), was used as feed for subsequent methanogenic bioreactor (methanogenic/anaerobic sequencing batch biofilm reactor, AnSBBR, pH 7.0) to generate additional biogas (CH₄) utilizing residual organic composition employing anaerobic mixed consortia. During the stabilized phase of operation (after 60 days) AcSBBR showed H₂ production of 16.91 mmol/day in association with COD removal efficiency of 36.56% (SDR_A—1.736 kg COD/m³-day). AnSBBR showed additional COD removal efficiency of 54.44% (SDR_M—1.071 kg COD/m³-day) along with CH₄ generation. Integration of the acidogenic and methanogenic processes enhanced substrate degradation efficiency (SDR_T—4.01 kg COD/m³-day) along with generation of both H₂ and CH₄ indicating sustainability of the process.     

Metabolic shift and electron discharge pattern of anaerobic consortia as a function of pretreatment method applied during fermentative hydrogen production

We have made an attempt to evaluate the variation in the electron discharge (ED) pattern of anaerobic consortia as a function of pretreatment viz., chemical, heat-shock, acid and oxygen-shock in comparison with untreated mixed consortia during fermentative hydrogen (H₂) production. Experiments were performed with dairy wastewater as substrate using anaerobic mixed consortia as biocatalyst (pretreated individually and in combination). Cyclic voltammetry (CV) elucidated significant variation in the ED pattern of mixed consortia along with H₂ production and substrate degradation (SD) as a function of pretreatment method applied. Higher ED was observed with all pretreated consortia which can be attributed to the stable proton (H⁺) shuttling due to the suppression of methanogenic activity. Oxygen-shock method and untreated consortia showed lower H₂ production and higher SD among the variations studied, while, combined pretreated consortia resulted higher H₂ production and lower SD. Lower ED observed with untreated consortia suggests the H⁺ reduction during methanogenesis rather than the inter-conversion of metabolites, which is presumed to be necessary for H₂ production. ED observed with combined pretreated consortia corroborated well with the observed H₂ production. Redox pairs were visualized on the voltammograms with almost all the experimental variations studied except untreated consortia. The potentials (E₀) of redox pairs observed were corresponding to intracellular electron carriers viz., NAD⁺/NADH (E₀ −0.32 V) and FAD⁺/FADH₂ (E₀ −0.24 V).     

Acidogenic hydrogen production from wastewater: Process analysis with the function of influencing parameters

Current communication reports the application of kinetic models viz., modified Gompertz, modified Logistic, Ratkowsky and Andrew model to study the acidogenic hydrogen (H₂) production along with volatile fatty acids (VFA) production and substrate degradation from various wastewater (dairy, distillery, chemical and designed synthetic wastewater) using mixed consortia. Influence of fermentation time was specifically evaluated by modified Gompertz and modified Logistic models on H₂ and VFA production. Influence of system redox condition on process was evaluated by Ratkowsky and Andrew models. The modified Gompertz model showed best fit for H₂ production as well as substrate degradation while modified Logistic model showed good acceptability with VFA production. The Andrew model describes both H₂ and VFA production with respect to system redox condition relatively well. This information provides an understanding of the process behavior, which can help in the design and upscaling of the process for efficient H₂ production. Copyright © 2015 John Wiley & Sons, Ltd.     

Identification and cultivation of hydrogenotrophic methanogens from palm oil mill effluent for high methane production

By means of biorefinery, biogas production through anaerobic digestion is one of the most common treatments of wastewater in the palm oil industry. After biogas production, the treated palm oil mill effluent (POME) is generally discharged into the environment. However, certain level of hazardous compounds still exists in the treated wastewater, which can lead to the pollution of water bodies. In this study, we have investigated the dynamics of volatile organic acids dwelling in consecutive POME treatment lagoons as well as identified, and categorized, microbial species responsible for the treatment process. Bacteria and methanogens, both hydrogenotrophic and acetoclastic, related to methane production were identified using mcrA and 16S rRNA genes specific primers. Two hydrogenotrophic methanogens, Methanoculleus marisnigri and Methanoculleus chikugoensis, were found abundant in accordance with high formate concentration throughout the process of anaerobic digestion. This study has also isolated eight consortia of microbes that yielded different methane productions by utilizing formate as the substrate in the synthetic medium. The consortia of a group, containing M. marisnigri, M. chikugoensis, uncultured bacteria, Aminobacterium sp., and Ruminobacillus xylanolyticum, produced the highest methane yield of 259 mL/g COD after 25 days of incubation in the laboratory. The findings from this study are contributing to optimize and increase biogas production in POME, which will allow higher efficiency in palm oil mill wastewater treatment.     

A rapid and simple protocol for evaluating biohydrogen production potential (BHP) of wastewater with simultaneous process optimization

Biohydrogen production utilizing negative valued waste through dark-fermentation process is one of the emerging areas. Reported conditions for H₂ production are significantly variable and comparative analysis of data is major problem for unified understanding. A simple, rapid and generalized two phase methodology/protocol was developed to evaluate the biohydrogen production potential (BHP) of negative valued wastewater as substrate/feed-stock for renewable biohydrogen production using mixed consortia. Critical factors that can influence the overall process viz., redox condition, organic load and biocatalyst were considered in the designing the methodology. Feasibility of protocol was initially evaluated with synthetic wastewater and further validated with real field composite food and slaughter house wastewaters. The selected operational factors showed marked influence on both H₂ production and wastewater treatment. The reported methodology/protocol not only provides the ability of selected wastewater to generate H₂ but also facilitates process understanding based on selected factors and finally acquiesce optimum conditions.     

Influence of reactor configuration on fermentative hydrogen production during wastewater treatment

Influence of reactor configuration [biofilm/suspended growth] on fermentative hydrogen (H₂) production and substrate degradation was evaluated employing anaerobic mixed consortia. Reactors were operated at acidophilic (pH 6.0) condition employing designed synthetic wastewater as substrate at an organic loading rate of 3.4 Kg COD/m³-day with a retention time of 24 h at 28 ± 2 °C. Experimental data enumerated the influence of reactor configuration on both H₂ production and wastewater treatment. Biofilm reactor (28.98 mmol H₂/day; 1.25 Kg COD/m³-day) showed relatively efficient performance over the corresponding suspended growth configuration (20.93 mmol H₂/day; 1.08 Kg COD/m³-day). Specific H₂ yields of 6.96 mmol H₂/g-COD_L-day (19.32 mmol H₂/g-COD_R-day) and 5.03 mmol H₂/g-COD_L-day (16.10 mmol H₂/g-COD_R-day) were observed during stabilized phase of operation of biofilm and suspended growth reactors respectively. Higher concentration of VFA generation was observed in the biofilm reactor. Both the configurations recorded higher acetate concentration over other soluble metabolites indicating the dominance of acid-forming metabolic pathway during the H₂ production process.     

Bio-electrochemical evaluation of fermentative hydrogen production process with the function of feeding pH

Fermentative hydrogen (H₂) production process in concurrence with feeding pH [aciodophilic (pH 6.0) and neutral (pH 7.0)] and reactor operation mode (continuous and fed-batch) was evaluated in a biofilm configured reactor [upflow mode; retention time, 24 h; operating temperature, 28 ± 2 °C; organic loading rate, 3.4 kg COD/m³ day] using anaerobic mixed consortia. Acidophilic pH showed relatively effective performance with respect to H₂ production compared to neutral operation. Neutral pH illustrated effective substrate removal efficiency over the corresponding acidophilic operation. Fed-batch mode of operation with acidophilic pH showed highest H₂ production among the studied experimental variations. The pattern of soluble metabolites distribution showed the persistence of acid-forming metabolic flow associated with acidogenesis which may be considered as optimum microenvironment for effective H₂ production. Bio-electrochemical behavior of mixed anaerobic consortia (whole cell) during H₂ production process was evaluated employing cyclic voltammetry (CV) in electrochemical cell [platinum as working electrode; Ag/AgCl as reference electrode; graphite rod as counter electrode; wastewater as electrolyte] to gain insight into the possible mechanism based on intracellular electron transfer involved in the fermentative metabolic process. Voltammogram profiles visualized well defined redox pairs in forward and reverse scans at both pH conditions and the signals corresponded to intracellular electron carrier, NADH/NAD⁺ (E^0′, −0.32 V). Relatively higher energy output was observed in acidophilic operation which might be attributed to the possibility of efficient proton (H⁺) transfer between metabolic intermediates.     

Fermentative hydrogen production - An alternative clean energy source

Hydrogen generation from wastewater is one of the promising approaches through biological route. So, exploitation of wastewater as substrate for hydrogen production with concurrent wastewater treatment is an attractive and effective way of tapping clean energy from renewable resources in a sustainable approach. In this direction, considerable interest is observed on various biological routes of hydrogen production using bio-photolysis, photo fermentation and heterotrophic dark fermentation process or by a combination of these processes. Therefore, in this communication, utilizing industrial wastewater as primary substrate for dark fermentation process is reviewed and different parametric aspects associated with this sustainable approach for better energy production is discussed. The industrial wastewaters that could be the source for bio hydrogen generation, such as rice slurry wastewater, food and domestic wastewaters, citric acid wastewater and paper mill wastewater, are also discussed in this article.     

Biological hydrogen production from probiotic wastewater as substrate by selectively enriched anaerobic mixed microflora

Biohydrogen production from probiotic wastewater using mixed anaerobic consortia is reported in this paper. Batch tests are carried out in a 5.0 L batch reactor under constant mesophillic temperature (37 °C). The maximum hydrogen yield 1.8 mol-hydrogen/mol-carbohydrate is obtained at an optimum pH of 5.5 and substrate concentration 5 g/L. The maximum hydrogen production rate is 168 ml/h. The hydrogen content in the biogas is more than 65% and no significant methane is observed throughout the study. In addition to hydrogen, acetate, propionate, butyrate and ethanol are found to be the main by-products in the metabolism of hydrogen fermentation.     

Optimization and evaluation of fermentative hydrogen production and wastewater treatment processes using data enveloping analysis (DEA) and Taguchi design of experimental (DOE) methodology

The combined process efficiency with respect to fermentative hydrogen (H₂) production and wastewater treatment was evaluated in a series of batch experiments to enumerate the role of selected factors viz., origin of inoculum, pre-treatment, inlet pH and feed composition under anaerobic microenvironment using mixed culture. H₂ production and substrate degradation were found to depend significantly on the selected factors with individual conditions to achieve effective process performance. Significantly diverse operational conditions were observed for both H₂ production and substrate degradation with respect to process efficiency. However, while dealing with H₂ production in association with wastewater treatment, both the parameters are important and balancing the conditions for combined effective performance is critical. Data enveloping analysis (DEA) was applied to evaluate the combined system performance with respect to the two output parameters (H₂ production and substrate degradation) based on relative efficiency. Among various experimental combinations studied, those with untreated anaerobic mixed inoculum under acidophilic conditions (inlet pH 5.5) using simple wastewater as fermentative substrate illustrated combined process efficiency with respect to H₂ production (1.919 m mol H₂/day; 0.52 mol H₂/kg COD_R-day) and substrate degradation (substrate degradation rate, 4.56 kg COD/m³-day). DEA methodology provide the relative efficiency of the system by integrating two output parameters. Further, design of experimental methodology (DOE) by Taguchi approach was applied to enumerate the role of selected factors on the H₂ production and substrate degradation with the final aim of optimizing the process. By adapting the derived optimum conditions, the performance with respect to H₂ production and substrate degradation could be enhanced by three fold.     

Influence of sulfate-reducing bacteria,sulfide and molybdate on hydrogen photoproduction by purple nonsulfur bacteria

The interaction between bacterial species is of great importance for H₂ production using microbial consortia or non-sterile conditions. Sulfate reducing bacteria were found in anaerobic starch-hydrolyzing consortium and their inhibitory effect on the following H₂ photoproduction by purple nonsulfur bacteria was shown. This inhibition was clearly demonstrated in the mixed culture of Rhodobacter sphaeroides and Desulfomicrobium baculatum using the synthetic medium. This effect was conditioned by sulfide production rather than H₂ consumption or competition for organic substrate. Actually, the addition of equivalent sulfide concentration brought about the similar effects: inhibition of H₂ production without growth inhibition, cells aggregation, and the increase of carbohydrate content as an alternative way of expenditure of organic acids. In the long-term experiments the average sulfide concentration of about 0.3 mM was detrimental while in short-terms the H₂ production was not inhibited even at 3.2 mM. The protective effect of molybdates against sulfate reducers and sulfide was discussed.     

Fermentative biohydrogen production by mixed anaerobic consortia: Impact of glucose to xylose ratio

Glucose and xylose are the dominant monomeric carbohydrates present in agricultural materials which can be used as potential building blocks for various biotechnological products including biofuels production. Hence, the imperative role of glucose to xylose ratio on fermentative biohydrogen production by mixed anaerobic consortia was investigated. Microbial catabolic H₂ and VFA production studies revealed that xylose is a preferred carbon source compared to glucose when used individually. A maximum of 1550 and 1650 ml of cumulative H₂ production was observed with supplementation of glucose and xylose at a concentration of 5.5 and 5.0 g L⁻¹, respectively. A triphasic pattern of H₂ production was observed only with studied xylose concentration range. pH impact data revealed effective H₂ production at pH 6.0 and 6.5 with xylose and glucose as carbon sources, respectively. Co-substrate related biohydrogen fermentation studies indicated that glucose to xylose ratio influence H₂ and as well as VFA production. An optimum cumulative H₂ production of 1900 ml for 5 g L⁻¹ substrate was noticed with fermentation medium supplemented with glucose to xylose ratio of 2:3 at pH 6. Overall, biohydrogen producing microbial consortia developed from buffalo dung could be more effective for H₂ production from lignocellulosic hydrolysates however; maintenance of glucose to xylose ratio, inoculum concentration and medium pH would be essential requirements.     

Renewable biomass production by mixotrophic algae in the presence of various carbon sources and wastewaters

This study evaluated mixotrophic growth potential of native microalgae in media supplemented with different organic carbon substrates and wastewaters. Three robust mixotrophic microalgae viz. Chlamydomonas globosa, Chlorella minutissima and Scenedesmus bijuga were isolated after long-term enrichments from industrial wastewater. The mixotrophic growth of these microalgae resulted in 3–10 times more biomass production relative to phototrophy. Glucose, sucrose and acetate supported significant mixotrophic growth. Poultry litter extract (PLE) as growth medium recorded up to 180% more biomass growth compared to standard growth medium BG11, while treated and untreated carpet industry wastewaters also supported higher biomass, compared to BG11 growth with no significant effect of additional nitrogen supplementation. Supplementing treated wastewater and PLE with glucose and nitrogen resulted in 2–7 times increase in biomass relative to the unamended wastewaters or PLE. The consortia of Chlamydomonas–Chlorella and Scenedesmus–Chlorella were the best for PLE and untreated wastewater respectively, while a combination all three strains was suitable for both PLE and wastewater. These algae can be good candidates for biofuel feedstock generation as they would not require freshwater or fertilizers. Such mixotrophic algal consortia offer great promise for production of renewable biomass for bioenergy applications using wastewaters.     

Pilot-scale high solids thermophilic anaerobic digestion of municipal solid waste with an emphasis on nutrient requirements

A pilot study was conducted to assess the biodegradable organic fraction of municipal solid waste (BOF/MSW) as a substrate in a high-solids anaerobic digestion process. Results obtained indicate that a typical BOF/MSW in the United States is deficient in most macro and micro-nutrients required for robust and stable digestion. The BOF/MSW was supplemented with nutrient-rich organic wastes such as wastewater treatment plant sludges, dairy manure, and synthetic chemical solutions to correct nutritional deficiencies. The combined addition of wastewater treatment plant sludge and dairy manure to a typical BOF/MSW significantly elevated the gas production rate and enhanced the process stability. Microbial nutrient requirements are identified and nutrient concentrations for stable operation are quantified.     

Biohydrogen production,sludge granulation,and microbial community in an anaerobic inner cycle biohydrogen production (AICHP) reactor at different hydraulic retention times

This study investigated the continuous biohydrogen production in an anaerobic inner cycle biohydrogen production (AICHP) reactor fed with synthetic molasses wastewater as the model substrate under mesophilic conditions (37 ± 1 °C). The hydraulic retention times (HRTs) were set as 6.12, 4.90, 4.08, 3.50, and 3.06 h. Both maximum hydrogen production rate (HPR) (8.08 ± 0.48 L/L/d) and maximum granule formation were achieved at the HRT of 3.50 h. Acetic acid and butyric acid were the dominant metabolites in all tested HRTs throughout the experiment. Microbial community analysis showed that shortening the HRT promoted hydrogen production. This was mainly achieved by enhancing the growth of acetogenic bacteria in the AICHP reactor, rather than the growth of hydrogen-producing bacteria.     

Effect of arabinose concentration on dark fermentation hydrogen production using different mixed cultures

Dark fermentation hydrogen production from arabinose at concentrations ranging between 0 and 100 g/L was examined in batch assays for three different mixed anaerobic cultures, two suspended sludges (S1, S2) obtained from two different sludge digesters and one granular sludge (G) obtained from a brewery wastewater treatment plant. After elimination of the methanogenic activity by heat treatment, all mixed cultures produced hydrogen, and optimal hydrogen rates and yields were generally observed for concentrations between 10 and 40 g/L of substrate. Higher concentrations of arabinose up to 100 g/L inhibited hydrogen production, although the effect was different from inoculum to inoculum. It was evident that the granular biomass was less affected by increased initial arabinose concentrations when calculating the rate of decrease in hydrogen yields versus arabinose concentrations, compared against the two suspended sludges.     

生物强化菌系添加量对不同食微比餐厨垃圾厌氧发酵性能影响

为考察生物强化菌系添加剂量对餐厨垃圾厌氧发酵性能影响,确定不同食微比（F/M）下最佳菌剂添加量,进行了批式厌氧发酵实验。以实验室已获得的丙酸产甲烷菌系为生物强化菌系,对不同F/M（0.5、1.0、2.0）进行生物强化,菌剂添加量设置为5%、10%、15%、25%、35%比,通过产气性能及中间代谢产物的对比,结合产甲烷动力学评价生物强化效果。结果表明：各剂量的生物强化均可促进餐厨垃圾产气,提高累积产甲烷率1 ~ 3倍;各F/M下,累积产甲烷率均随生物强化剂量增加而增大,35%的添加量产气效果最佳。就生物强化效率而言,3组F/M发酵中,F/M为1.0且菌系添加量为15%时,单位质量菌剂获得最大甲烷提升效率（1 706 mL/gVS_菌剂）;中间代谢产物分析显示,生物强化可促进丙酸和乙酸的降解,避免酸抑制,从而提高产甲烷能力。修正Gompertz模型对产甲烷潜力动力学分析表明,生物强化可以缩短不同食微比下的发酵延滞期,加快反应进程。     

Harnessing of biohydrogen by acidogenic fermentation of Citrus limetta peelings: Effect of extraction procedure and pretreatment of biocatalyst

The extracts of Citrus limetta (sweet lime) peelings were evaluated as a fermentable substrate for hydrogen (H₂) production by dark-fermentation (acidogenic) using both anaerobic mixed consortia and selectively enriched acidogenic mixed consortia. Extraction was carried by pretreating sweet lime peelings at 121 °C (1 bar pressure) at variable pH (6 and 7) and digestion time (20 and 40 min). Maximum organic matter extraction was observed at pH 7.0 (40 min). Fermentation was performed at different organic loading conditions [OL1, 1.17 kg COD/m³; OL2, 2.35 kg COD/m³; OL3, 4.69 kg COD/m³] under acidophilic microenvironment. H₂ production was found to depend on the concentration of the substrate and composition. Increase in organic load showed consistent improvement in H₂ production. Operation at OL3 employing selectively enriched inoculum documented higher cumulative H₂ production (10.07 mmol) and H₂ production rate (0.345 mmol/h) (pH 7; 40 min). Substrate degradation was also found to increase with increase in organic loading. Maximum substrate degradation (SD) was registered at pH 6 (40 min) with anaerobic culture (2.80 kg COD_R/m³; ξ_COD 31.82%) and at pH 7 (40 min) with selectively enriched acidogenic culture (3.20 kg COD_R/m³; ξ_COD 36.36%). Concentration of volatile fatty acids (VFAs) also improved with increase in organic load. Maximum VFA concentration (1098 mg/l) was observed with OL3 (pH 7; 40 min) by using selectively enriched culture.     

Hydrogen production from rice winery wastewater in an upflow anaerobic reactor by using mixed anaerobic cultures

Continuous production of hydrogen from the anaerobic acidogenesis of a high-strength rice winery wastewater by a mixed bacterial flora was demonstrated. The experiment was conducted in a 3.0-l upflow reactor to investigate individual effects of hydraulic retention time (HRT) (2–24 h), chemical oxygen demand (COD) concentration in wastewater (14–36 g COD/l), pH (4.5–6.0) and temperature (20–55°C) on bio-hydrogen production from the wastewater. The biogas produced under all test conditions was composed of mostly hydrogen (53–61%) and carbon dioxide (37–45%), but contained no detectable methane. Specific hydrogen production rate increased with wastewater concentration and temperature, but with a decrease in HRT. An optimum hydrogen production rate of 9.33 lH₂/gVSSd was achieved at an HRT of 2 h, COD of 34 g/l, pH 5.5 and 55°C. The hydrogen yield was in the range of 1.37–2.14 mol/mol-hexose. In addition to acetate, propionate and butyrate, ethanol was also present in the effluent as an aqueous product. The distribution of these compounds in the effluent was more sensitive to wastewater concentration, pH and temperature, but was less sensitive to HRT. This upflow reactor was shown to be a promising biosystem for hydrogen production from high-strength wastewaters by mixed anaerobic cultures.