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.     

Enhancing biohydrogen production from chemical wastewater treatment in anaerobic sequencing batch biofilm reactor (AnSBBR) by bioaugmenting with selectively enriched kanamycin resistant anaerobic mixed consortia

The basic aim of this study was to investigate the feasibility of bioaugmentation strategy in the process of enhancing biohydrogen (H₂) production from chemical wastewater treatment (organic loading rate (OLR)—6.3 kg COD/m³-day) in anaerobic sequencing batch biofilm reactor (AnSBBR) operated at room temperature (28±2$28\pm 2$ °C) under acidophilic microenvironment (pH 6) with a total cycle period of 24 h. Parent augmented inoculum (kanamycin resistant) was acquired from an operating upflow anaerobic sludge blanket (UASB) reactor treating chemical wastewater and subjected to selective enrichment by applying repetitive/cyclic pre-treatment methods [altering between heat-shock treatment (100 °C; 2 h) and acid treatment (pH 3; 24 h)] to eliminate non-spore forming bacteria and to inhibit the growth of methanogenic bacteria (MB). Experimental data revealed the positive influence of bioaugmentation strategy on the overall H₂ production. Specific H₂ production almost doubled after augmentation from 0.297 to 0.483 mol H₂/kg COD_R-day. Chemical wastewater acted as primary carbon source in the metabolic reactions involving molecular H₂ generation leading to substrate degradation. The augmented culture persisted in the system till the termination of the experiments. The survival and retention of the augmented inoculum and its positive effect on process enhancement may be attributed to the adopted reactor configuration and operating conditions. Scanning electron microscope (SEM) images documented the selective enrichment of morphologically similar group of bacteria capable of producing H₂ under acidophilic conditions in anaerobic microenvironment. This depicted work corroborated successful application of bioaugmentation strategy to improve H₂ production rate from anaerobic chemical wastewater treatment.     

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.     

Biocatalytic membrane reactor modeling for fermentative hydrogen production from wastewater: A review

Biologically produced hydrogen is a valuable energy carrier generated from renewable sources such as wastewater. This review focuses on modeling a submerged membrane bioreactor, namely the whole-cell biocatalytic membrane reactor (BMR), for simultaneous cell immobilization and gas recovery in fermentative hydrogen production. This technology is presented as an alternative for improved gas separation, and its fundamentals are comprehensively overviewed. We discuss models and modeling strategies that can partially represent the phenomenology involved in the BMR of interest. Empirical or semiempirical models are a well-established tool for fermentative bioreactors. Although these might not be suitable for BMRs due to spatial heterogeneity and process complexity, we compare different kinetic expressions for hydrogen formation, focusing on reaction and mass transfer limiting steps. Moreover, models of BMRs or similar for wastewater treatment are analyzed by classifying the phenomena, variables, and gaps that they have not covered to describe the internal design appropriately.     

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

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

Dark fermentative biohydrogen production from confectionery wastewater in continuous-flow reactors

The production of biohydrogen from industrial wastewater through the dark fermentation (DF) process has attracted increased interest in recent years. To implement a DF process on a large scale, a thorough knowledge of laboratory scale process control is required. The operating parameters and design features of the reactors have a great influence on the efficiency of the process. In this work, the possibility of continuous production of biohydrogen from confectionery wastewater was evaluated. The DF process was carried out at 37 ± 1 °C in two different reactors: an upflow anaerobic filter (AF) and a fluidized bed reactor (AFB). Polyurethane foam (PU) was used to immobilize the biomass. The DF process was studied at four hydraulic retention times (HRT) (1.5, 2.5, 7.5 and 15 days) and the corresponding organic loading rates (OLR) (9.21, 6.12, 2.04 and 1.02 g COD_init/(L day)). The highest hydrogen yield (HY) (44.73 ml/g COD_init) and hydrogen production rate (HPR) (92.5 ml/(L day)) was observed in AFB at HRT of 7.5 days and 2.5 days, respectively. The highest concentration of hydrogen in biogas was 34% in AF and 36% in AFB at HRT of 7.5 days. In contrast to AF, the COD removal efficiency in AFB increased with increasing HRT. The pH of the effluent was low (3.95–4.38). However, due to the use of PU for biomass immobilization, it is possible that there were local zones in the reactor that were optimal for the functioning of not only acidogens, but also methanogens. This was evidenced by a rather high content of methane in biogas (2.5% in AF and 9.6% in AFB at HRT of 15 days). These results provide valuable data for optimizing the continuous DF of wastewater from confectionery and other food industries to produce biohydrogen or biohythane.     

Adaptation of biohydrogen producing reactor to higher substrate load: Redox controlled process integration strategy to overcome limitations

Adaptation of acidogenic sequencing batch biofilm reactor (AcSBBR) to higher loading conditions of vegetable waste extract was studied during biohydrogen production at pH 6.0 under ambient conditions. H₂ production rate (HPR) and cumulative H₂ production (CHP) were found to improve with increase in organic load from 4.50 to 26.44 kg COD/m³ and later at 35.25 kg COD/m³ stabilization was observed. Acid metabolic intermediates production tends to lower the system pH which limits the substrate degradation and H₂ production at higher loading conditions. To overcome these limitations, redox controlled strategy (pH 7.0) was applied by integrating another AcSBBR. Upon redox controlled integration, CHP and substrate degradation were found to improve by 42.81% and 36.82% respectively. This approach helped to maintain the favorable redox microenvironment for fermentation at higher VFA concentrations. This process integration methodology will help to overcome some persistent limitation observed during biohydrogen production and make the process sustainable especially with high strength waste/wastewaters.     

Feasible pretreatment of textile wastewater for dark fermentative hydrogen production

In this study, the yield of hydrogen production was investigated under different feedstock pretreatment conditions. The feedstock for dark fermentative hydrogen production was textile wastewater which was obtained from the de-sizing process in a textile factory, located in northern Taiwan. The wastewater was pretreated with activated carbon, cation exchange resin or was not pretreated before being fed into the batch bottles. Biohydrogen production was carried out in a batch reactor with the sludge of mixed-culture using the feedstock from the pretreated wastewater. The sludge was obtained from the Taichung municipal wastewater treatment plant. The yield of hydrogen production using the two pretreatment methods or non – treatment were compared.     

The influence of the degree of back-mixing on hydrogen production in an anaerobic fixed-bed reactor

This paper reports on results obtained from experiments carried out in an acidogenic anaerobic reactor aiming at the optimization of hydrogen production by altering the degree of back-mixing. It was hypothesized that there is an optimum operating point that maximizes the hydrogen yield. Experiments were performed in a packed-bed bioreactor by covering a broad range of recycle ratios (R) and the optimum point was obtained for an R value of 0.6. In this operating condition the reactor behaved as 8 continuous stirred-tank reactors in series and the maximum yield was 4.22 mol H₂ mol sucrose⁻¹. Such optimum point was estimated by deriving a polynomial function fitted to experimental data and it was obtained as the conjugation of three factors related to the various degrees of back-mixing applied to the reactor: mass transfer from the bulk liquid to the biocatalyst, liquid-to-gas mass transfer and the kinetic behavior of irreversible reactions in series.     

Influence of pH on fermentative hydrogen production from sweet sorghum extract

The present study focused on the influence of pH on the fermentative hydrogen production from the sugars of sweet sorghum extract, in a continuous stirred tank bioreactor. The reactor was operated at a Hydraulic Retention Time of 12 h and a pH range of 3.5–6.5. The maximum hydrogen production rate and yield were obtained at pH 5.3 and were 1752 ± 54 mL H₂/d or 3.50 ± 0.07 L H₂/L reactor/d and 0.93 ± 0.03 mol H₂/mol glucose consumed or 10.51 L H₂/kg sweet sorghum, respectively. The main metabolic product at this pH value was butyric acid. The hydrogen productivity and yield were still at high levels for the pH range of 5.3–4.7, suggesting a pH value of 4.7 as optimum for hydrogen production from an economical point of view, since the energy demand for chemicals is lower at this pH. At this pH range, the dominant fermentation product was butyric acid but when the pH culture sharply decreased to 3.5, hydrogen evolution ceased and the dominant metabolic products were lactic acid and ethanol.     

Optimization and microbial community analysis for production of biohydrogen from palm oil mill effluent by thermophilic fermentative process

The optimum values of hydraulic retention time (HRT) and organic loading rate (OLR) of an anaerobic sequencing batch reactor (ASBR) for biohydrogen production from palm oil mill effluent (POME) under thermophilic conditions (60 °C) were investigated in order to achieve the maximum process stability. Microbial community structure dynamics in the ASBR was studied by denaturing gradient gel electrophoresis (DGGE) aiming at improved insight into the hydrogen fermentation microorganisms. The optimum values of 2-d HRT with an OLR of 60 gCOD l⁻¹ d⁻¹ gave a maximum hydrogen yield of 0.27 l H₂ g COD⁻¹ with a volumetric hydrogen production rate of 9.1 l H₂ l⁻¹ d⁻¹ (16.9 mmol l⁻¹ h⁻¹). The hydrogen content, total carbohydrate consumption, COD (chemical oxygen demand) removal and suspended solids removal were 55 ± 3.5%, 92 ± 3%, 57 ± 2.5% and 78 ± 2%, respectively. Acetic acid and butyric acid were the major soluble end-products. The microbial community structure was strongly dependent on the HRT and OLR. DGGE profiling illustrated that Thermoanaerobacterium spp., such as Thermoanaerobacterium thermosaccharolyticum and Thermoanaerobacterium bryantii, were dominant and probably played an important role in hydrogen production under the optimum conditions. The shift in the microbial community from a dominance of T. thermosaccharolyticum to a community where also Caloramator proteoclasticus constituted a major component occurred at suboptimal HRT (1 d) and OLR (80 gCOD l⁻¹ d⁻¹) conditions. The results showed that the hydrogen production performance was closely correlated with the bacterial community structure. This is the first report of a successful ASBR operation achieving a high hydrogen production rate from real wastewater (POME).     

Influence of butyrate on fermentative hydrogen production and microbial community analysis

The effect of butyrate on hydrogen production and the potential mechanism were investigated by adding butyric acid into dark fermentative hydrogen production system at different concentrations at pH range of 5.5–7.0. The results showed that under all the tested pH from 5.5 to 7.0, the addition of butyric acid can inhibit the hydrogen production, and the inhibitory degree (from 10.5% to 100%) increased with the increase of butyric acid concentration and with the decrease of pH values, which suggested that the inhibition effect is highly associated with the concentration of undissociated acids. Substrate utilization rate and VFAs accumulation also decreased with the addition of butyric acid. The microbial community analysis revealed that butyrate addition can decrease the dominant position of hydrogen-producing microorganisms, such as Clostridium, and increase the proportion of other non-hydrogen-producing bacteria, including Pseudomonas, Klebsiella, Acinetobacter, and Bacillus.     

Mesophilic fermentative hydrogen production from sago starch-processing wastewater using enriched mixed cultures

Biohydrogen (bioH₂) production from starch-containing wastewater is an energy intensive process as it involves thermophilic temperatures for hydrolysis prior to dark fermentation. Here we report a low energy consumption bioH₂ production process with sago starch powder and wastewater at 30 °C using enriched anaerobic mixed cultures. The effect of various inoculum pretreatment methods like heat (80 °C, 2 h), acid (pH 4, 2.5 N HCl, 24 h) and chemical (0.2 g L⁻¹ bromoethanesulphonic acid, 24 h) on bioH₂ production from starch powder (1% w/v) showed highest yield (323.4 mL g⁻¹ starch) in heat-treatment and peak production rate (144.5 mL L⁻¹ h⁻¹) in acid-treatment. Acetate (1.07 g L⁻¹) and butyrate (1.21 g L⁻¹) were major soluble metabolites of heat-treatment. Heat-treated inoculum was used to develop mixed cultures on sago starch (1% w/v) in minimal medium with 0.1% peptone-yeast extract (PY) at initial pH 7 and 30 °C. The effect of sago starch concentration, pH, inoculum size and nutrients (PY and Fe ions) on batch bioH₂ production showed 0.5% substrate, pH 7, 10% inoculum size and 0.1% PY as the best H₂ yielding conditions. Peak H₂ yield and production rate were 412.6 mL g⁻¹ starch and 78.6 mL L⁻¹ h⁻¹, respectively at the optimal conditions. Batch experiment results using sago-processing wastewater under similar conditions showed bioH₂ yield of 126.5 mL g⁻¹ COD and 456 mL g⁻¹ starch. The net energy was calculated to be +2.97 kJ g⁻¹ COD and +0.57 kJ g⁻¹ COD for sago starch powder and wastewater, respectively. Finally, the estimated net energy value of +2.85 × 10¹³ kJ from worldwide sago-processing wastewater production indicates that this wastewater can serve as a promising feedstock for bioH₂ production with low energy input.     

Upscaling of biohydrogen production process in semi-pilot scale biofilm reactor: Evaluation with food waste at variable organic loads

The present account focuses on upscaling of biohydrogen (H₂) production at semi-pilot scale bioreactor using composite food waste. Experiments were conducted at different organic load (6, 12, 18, 30, 40, 50 and 66 g COD/l) conditions. H₂ production increased with an increasing organic load up to 50 g COD/l (9.67 l/h) followed by 40 g COD/l (6.48 l/h), 30 g COD/l (1.97 l/h), 18 g COD/l (0.90 l/h), 12 g COD/l (0.78 l/h) and 6 g COD/l (0.32 l/h). H₂ production was affected by acidification (pH drop to 3.96) at 66 g COD/l operation due to the excess accumulation of soluble metabolites (5696 mg VFA/l). Variation in organic load of food waste influenced the overall hydrogen production efficiency.     

Responses of the methanogenic reactor to different effluent fractions of fermentative hydrogen production in a phase-separated anaerobic digestion system

Phase-separated anaerobic digestion (AD) system was suggested to recover energy in the form of CH₄ from different effluent fractions of fermentative hydrogen production. In the present study, a two-phase AD system consisting of an H₂-producing fixed-bed reactor (RH) and a CH₄-producing upflow anaerobic sludge bed reactor (RM) was employed to investigate the responses of RM to three types of pretreated RH effluents: membrane-filtered, centrifuged, and poorly settled. The results showed that RM easily converted soluble byproducts in the membrane-filtered effluent, but it had difficulties in degradation of the mixture of soluble and colloidal organic matters in the centrifuged effluent under high organic loadings. The colloidal matters originated from extracellular and intracellular macro-polymers were believed to have been adsorbed onto the surface of granular sludge and formed a film of increasing thickness which retarded the soluble substrate supply to the inner acetogens and methanogens. Due to the fact that the degradation of H₂ biomass residue in the slightly settled RH effluent was inefficient under a tested hydraulic retention time (HRT) of 20 h, they were either trapped in RM causing the expansion of sludge bed or washed out with the effluent. This study recommended the suspended solids in the RH effluent be removed from the feed to RM; be treated elsewhere or recycled. Extra care should be taken to fine tune RM to accommodate the degradation of colloidal solids.     

Changes in microbial community structure during dark fermentative hydrogen production

Microbial community structure plays a significant role in the efficiency of dark fermentative hydrogen production using mixed culture. However, the detailed evolutions in microbial community structure during dark fermentation process are still unclear. This study investigated the detailed evolution patterns of microbial community structure during dark fermentation process by high-throughput pyrosequencing. Results showed that microbial community structure changed significantly over time in dark fermentation. Microbial diversity showed a constant decreasing trend during the fermentation process. The analysis of microbial community composition showed that Clostridium sensu stricto 1, Paraclostridium, Romboutsia and Paeniclostridium, which were all rarely existed in the inoculum, dramatically became dominant genera in the system after 6 h fermentation, with total relative abundance of more than 99%. This interesting result revealed that how quickly hydrogen-producing genera overwhelmed the microbial community in dark fermentation. Spearman correlation analysis showed that Clostridium sensu stricto 1 contributed the most to hydrogen fermentation performances.     

Effect of pH on continuous biohydrogen production from liquid swine manure with glucose supplement using an anaerobic sequencing batch reactor

pH is considered as one of the most important factors governing the hydrogen fermentation process. In this project, five pH levels, ranging from 4.4 to 5.6 at 0.3 increments, were tested to evaluate the pH effect on hydrogen production from swine manure supplemented with glucose in an anaerobic sequencing batch reactor system with 16 h of hydraulic retention time (HRT). The optimal hydrogen yield (1.50 mol H₂/mol glucose) was achieved at pH 5.0 when the maximum production rate of 2.25 L/d/L was obtained. Continuous hydrogen production was achieved for over 3 weeks for pH 5.0, 4.7, and 4.4, with no significant methane produced. However, as pH increased to 5.3 and 5.6, methane production was observed in the biogas with concurrent reductions in hydrogen production, indicating that methanogens could become increasingly activated for pH 5.3 or higher. Acetate, propionate, butyrate, valerate, and ethanol were the main aqueous products whose distribution was significantly affected by pH as well.     

Critical assessment of biofilm and suspended growth reactor configurations for acidogenic biohydrogen production using wastewater as a function of redox microenvironment

Comprehensive experiments were designed to study the relative function of two bioreactor configurations viz., biofilm and suspended growth for dark-fermentative hydrogen (H₂) production along with wastewater treatment at two varying feed pH conditions (6 and 7). Both the reactors were operated in sequencing batch mode using anaerobic inoculum after pretreatment (combined treatment: acid-shock, heat-shock and chemical-shock). Biofilm system showed efficient H₂ production over the corresponding suspended growth operation at feeding pH 6. VFA profiles visualized high acetate fraction supporting feasible microenvironment for higher H₂ production. Voltammogram profiles visualized significant variation in the bio-electrochemical behavior with the function of operating pH and reactor configuration. It can be inferred from this study that biofilm systems are efficient for H₂ production particularly at acidiophilic microenvironment.     

Batch fermentative hydrogen production utilising sago (Metroxylon sp.) starch processing effluent by enriched sago sludge consortia

Sago starch processing effluent (SSPE) is an ideal bio-resource that can be utilised as a substrate for fermentative reactions due to its relatively high organic content. Annually in Malaysia, about 2.5 million tonnes of effluent are generated from the processing of sago starch. In this study, the potential use of SSPE as a substrate for fermentative hydrogen production was confirmed under all the experimental conditions studied. The maximum hydrogen production and volumetric hydrogen production rate were 575 mL H₂/L SSPE and 57.54 mL H₂/hr.L SSPE, respectively, from cultures with an initial pH of 7 and substrate concentration of 11 g soluble carbohydrate/L SSPE. The final soluble metabolites were comprised mainly of acetate (24–43%), butyrate (4–20%), propionate (1–7%) and ethanol (44–66%), suggesting an acetic acid-ethanol type fermentation pathway.