Effects of linoleic acid and its degradation by-products on mesophilic hydrogen production using flocculated and granular mixed anaerobic cultures

The effects of linoleic acid (LA (C18:2)) and its degradation by-products on hydrogen (H₂) production were examined at 37 °C and an initial pH value of 5.0 using granular and flocculated mixed anaerobic cultures from the same source. In the flocculated cultures, the H₂ consumers were inhibited to a greater extent when compared to the granular cultures. The maximum H₂ yields were 2.52 ± 0.2 and 1.9 ± 0.2 mol mol⁻¹ glucose in the flocculated and granular cultures, respectively. The major long chain fatty acids (LCFAs) detected at which H₂ attained a maximum value were LA (750 mg L⁻¹) and myristic acid (MA) (500 mg L⁻¹).     

Flux balance analysis of mixed anaerobic microbial communities: Effects of linoleic acid (LA) and pH on biohydrogen production

The internal fluxes of mixed anaerobic cultures fed 2000 mg l⁻¹ linoleic acid (LA) plus glucose at 6 initial pH conditions and maintained at 37 °C were estimated using a flux balanced analysis (FBA). In cultures fed LA at pH 7, less than 8% of the flux was diverted to CH₄. At an initial pH ≥ 5.5, the quantity of glucose removed was greater than 95%; however, at pH 4.5 and 5.0 the quantity consumed were 38% and 75%, respectively. The FBA output showed that the acetogenic H₂-consumers were responsible for more than 20% of the H₂ consumed. Adding LA and decreasing the pH was ineffective in reducing the activity of acetogenic H₂-consumers. As the initial pH decreased, the acetogenic H₂-consuming flux decreased in the presence of 2000 mg l⁻¹ LA. A maximum H₂ yield of 1.55 mol mol⁻¹ glucose consumed (peak hydrogenase flux (R12)) was attained when the acetogenic H₂-consuming flux reached 0.42 mol at a pH of 5.5.     

Enhanced biohydrogen production from corn stover hydrolyzate by pretreatment of two typical seed sludges

Five individual pretreatment methods (heat, ultrasonic, ultraviolet, acid, and base) were performed on two typical seed sludges (river sediments and anaerobic granular sludge) to evaluate their effectiveness on enriching efficient hydrogen (H₂)-producing bacteria and enhancing H₂ production using corn stover hydrolyzate. Results indicated that pretreatment processes caused more remarkable improvements for river sediments than anaerobic granular sludge. Among the five protocols, heat pretreatment reached high H₂ yield for both river sediments (4.17 mmol H₂/g utilized sugar) and anaerobic granular sludge (2.84 mmol H₂/g utilized sugar). Ultraviolet and ultrasonic pretreatments were conditionally effective for river sediments and anaerobic granular sludge, respectively. In most cases, pretreatment processes altered soluble metabolites distribution towards more acetate and less ethanol production. Microbial community analysis indicated that heat and ultrasonic pretreatments can respectively lead to significant and indistinctive change on original microbial community. Besides frequently detected Escherichia spp., Serratia spp., and Klebsiella spp., some species of Clostridium spp. and Bacillus spp. might be efficient H₂ producer responsible for better H₂-producing performances.     

Regulating biohydrogen production from wastewater by applying organic load-shock: Change in the microbial community structure and bio-electrochemical behavior over long-term operation

Detailed experiments were designed to evaluate the function of load-shock treatment strategy (50 g COD/l; 3 days) for selective enrichment of acidogenic hydrogen (H₂) producing consortia in comparison with untreated anaerobic consortia. Experiments performed in suspended-batch mode bioreactors for 520 days illustrated the relative efficiency of load-shock treated consortia in enhancing H₂ production (16.64 mol/kg COD_R) compared to untreated-parent consortia (3.31 mol/kg COD_R). On the contrary, substrate degradation was higher with control operation (ξ_COD, 62.86%; substrate degradation rate (SDR), 1.10 kg COD_R/m³-day) compared to load-shock culture (52.33%; 0.78 kg COD_R/m³-day). Fatty acid composition documented a shift in the metabolic pathway towards acetate formation after applying load-shock, which manifests higher H₂ production. Microbial profiling documented a significant alteration in species composition of microbial communities after repeated load-shock applications specific to enrichment of Firmicutes which are favourable for H₂ production. Dehydrogenase activity was stabilized with each re-treatment, signifying the adaptation inclination of the biocatalyst towards increased proton shuttling between metabolic intermediates, leading to higher H₂ production. Voltammograms of load-shock treated cultures showed a marked shift in oxidation and reduction catalytic currents towards more positive and negative values respectively with increasing scan rate evidencing simultaneous redox-conversion reactions, facilitating proton gradient in the cell towards increased H₂ production. Load-shock treatment facilitates direct cultivation of inoculums at higher substrate load without any chemical pretreatment. This study documented the feasibility of controlling microbial metabolic function by application of load-shock treatment either for preparing inoculum for startup of the reactor or to the reactor resident microflora (in situ) during operation whenever required to regain the process performance.     

Effect of furans and linoleic acid on hydrogen production

The effects of furans (furfural and 5-hydroxymethylfurfural (HMF)) on hydrogen (H₂) production using mixed anaerobic cultures were evaluated by conducting batch experiments. Two mixed anaerobic cultures (culture A and B) fed furans plus glucose and treated with and without linoleic acid (LA) at pH 5.5 were maintained at 37 °C. In the LA inhibited cultures A and B fed 0.75 g L⁻¹ furfural and 0.25 g L⁻¹ HMF, the maximum H₂ yields observed were 1.89 ± 0.27 mol mol⁻¹ glucose and 1.75 ± 0.22 mol mol⁻¹ glucose, respectively. In cultures with maximum H₂ yields, Clostridium sp. and Flavobacterium sp. were dominant. Acetate, butyrate and ethanol were the major soluble metabolites detected in cultures A and B whereas propionate was also dominant in culture B. A canonical correspondence analysis based on the byproducts and the relative abundance of the terminal-restriction fragments revealed less variation between cultures treated with LA and low correlation value between the factors and the species composition.     

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.     

Biohydrogen production from pig slurry in a CSTR reactor system with mixed cultures under hyper-thermophilic temperature (70 °C)

A continuous stirred tank reactor (CSTR) (750 cm³ working volume) was operated with pig slurry under hyper-thermophilic (70 °C) temperature for hydrogen production. The hydraulic retention time (HRT) was 24 h and the organic loading rate was 24.9 g d⁻¹ of volatile solid (VS). The inoculum used in the hyper-thermophilic reactor was sludge obtained from a mesophilic methanogenic reactor. The continuous feeding with active biomass (inoculum) from the mesophilic methanogenic reactor was necessary in order to achieve hydrogen production. The hyper-thermophilic reactor started to produce hydrogen after a short adapted period of 4 days. During the steady state period the mean hydrogen yield was 3.65 cm³ g⁻¹ of volatile solid added. The high operation temperature of the reactor enhanced the hydrolytic activity in pig slurry and increased the volatile fatty acids (VFA) production. The short HRT (24 h) and the hyper-thermophilic temperature applied in the reactor were enough to prevent methanogenesis. No pre-treatment methods or other control methods for preventing methanogenesis were necessary. Hyper-thermophilic hydrogen production was demonstrated for the first time in a CSTR system, fed with pig slurry, using mixed culture. The results indicate that this system is a promising one for biohydrogen production from pig slurry.     

Statistical optimization of factors affecting biohydrogen production from xylose fermentation using inhibited mixed anaerobic cultures

The role of different chemical and physical factors in enhancing biohydrogen production from xylose using a mixed anaerobic culture was examined under mesophilic conditions. A fractional factorial design (FFD) 3^(k–p) was used to optimize pH, the oleic acid (OA) concentration and the biomass concentration. The FFD analysis indicated that the hydrogen (H₂) yield was affected by 3 single factors as well as by 2 factor interactions. Under optimum conditions (1600 mg L⁻¹ of oleic acid (OA) and 1900 mg L⁻¹ VSS and pH 6.7), the H₂ yield reached 2.64 ± 0.12 mol mol⁻¹ of xylose (80% of the theoretical yield). Based on the ANOVA and Pareto chart analysis, the linear and quadratic OA and pH terms were significant and the linear and quadratic VSS terms were insignificant. Normally distribution of the residuals was confirmed from the Anderson-Darling (AD) plot. The studentized residuals versus the predicted values plot clearly demonstrated that the data points were randomly scattered.     

Determining optimum conditions for hydrogen production from glucose by an anaerobic culture using response surface methodology (RSM)

Hydrogen fermentation is a very complex process and is greatly influenced by many factors. Previous studies have demonstrated that temperature, pH and substrate are important factors controlling biological H₂ production. Response surface methodology with central composite design was used in this study to optimize H₂ production from glucose by an anaerobic culture. The individual and interactive effects of pH, temperature and glucose concentration on H₂ production were also evaluated. The optimum conditions for maximum H₂ yield of 1.75 mol-H₂ mol-glucose⁻¹ were found as temperature 38.8 °C, pH 5.7 and glucose concentration 9.7 g L⁻¹. The linear effects of temperature and pH as well as their quadratic effects on H₂ yield were significant, while the interactive effects of three parameters were minor.     

Bio-hydrogen production from cheese whey powder (CWP) solution: Comparison of thermophilic and mesophilic dark fermentations

Cheese whey powder (CWP) solution was used as the raw material for hydrogen gas production by mesophilic (35 °C) and thermophilic (55 °C) dark fermentations at constant initial total sugar and bacteria concentrations. Thermophilic fermentation yielded higher cumulative hydrogen formation (CHF = 171 mL), higher hydrogen yield (111 mL H₂ g⁻¹ total sugar), and higher hydrogen formation rate (3.46 mL H₂ L⁻¹ h⁻¹) as compared to mesophilic fermentation. CHF in both cases were correlated with the Gompertz equation and the constants were determined. Despite the longer lag phase, thermophilic fermentation yielded higher specific H₂ formation rate (2.10 mL H₂ g⁻¹cells h⁻¹). Favorable results obtained in thermophilic fermentation were probably due to elimination of H₂ consuming bacteria at high temperatures and selection of fast hydrogen gas producers.     

Optimization of sugar release from banana peel powder waste (BPPW) using box-behnken design (BBD): BPPW to biohydrogen conversion

Optimization of pre-treatment conditions has been achieved for total sugar release from banana peel powder waste (BPPW) feedstock modelled through a three-level Box-Behnken design (BBD) of the response surface methodology (RSM). A series of various runs were executed at varied acid (H₂SO₄) concentrations (0.05%–0.15% v/v), incubation periods (1 h–3 h) in water bath at 95 °C and alkali (NaOH) concentrations (0.05%–0.15% v/v) according to the Box-Behnken design (BBD). From RSM the significant values of incubation period, acid concentration and alkali concentration were obtained as 3 h, 0.095% v/v, and 0.05% v/v respectively. The maximum total sugar release was reported as 5243.62 μg/ml which was highly close to the predicted value (5010.07 μg/ml). The model P- value (0.001), R-sq (98.26%), (adj) R-sq (95.14%) and (pred) R-sq (79.56%) obtained through ANOVA justified the results. The mutual impact of alkali and incubation period had the highest effect on total sugar release from dried banana peel powder, followed by mutual impact of acid and incubation period based on ANOVA (Analysis of Variance) results.Under optimized conditions of pre-treatment six different substrate concentrations (1%, 3%, 5%, 7% and 9% w/v) of BPPW was hydrolyzed and used to obtain volumetric bio-hydrogen evolution. The highest cumulative volumetric bio hydrogen gas 43 ml H₂/30 ml media was achieved at 5% w/v of pretreated BPPW. The substrate concentration above 5% w/v resulted in lowered fermentation process owing to product and substrate inhibition.     

Acetate and butyrate as substrates for hydrogen production through photo-fermentation: Process optimization and combined performance evaluation

Organic acids viz., acetate and butyrate were evaluated as primary substrates for the production of biohydrogen (H₂) through photo-fermentation process using mixed culture at mesophilic temperature (34 °C). Experiments were performed by varying parameters like operating pH, presence/absence of initiator substrate (glucose) and vitamin solution, type of nitrogen source (mono sodium salt of glutamic acid and amino glutamic acid) and gas (nitrogen/argon) used to create anaerobic microenvironment. Experimental data showed the feasibility of H₂ production along with substrate degradation utilizing organic acids as metabolic substrate but was found to be dependent on the process parameters evaluated. Maximum specific H₂ production and substrate degradation were observed with acetic acid [3.51 mol/Kg COD_R-day; 1.22 Kg COD_R/m³-day (92.96%)] compared to butyric acid [3.33 mol/Kg COD_R-day; 1.19 Kg COD_R/m³-day (88%)]. Higher H₂ yield was observed under acidophilic microenvironment in the presence of glucose (co-substrate), mono sodium salt of glutamic acid (nitrogen source) and vitamins. Argon induced microenvironment was observed to be effective compared to nitrogen induced microenvironment. Combined process efficiency viz., H₂ production and substrate degradation was evaluated employing data enveloping analysis (DEA) methodology based on the relative efficiency. Integration of dark fermentation with photo-fermentation appears to be an economically viable route for sustainable biohydrogen production if wastewater is used as substrate.     

Effects of process,operational and environmental variables on biohydrogen production using palm oil mill effluent (POME)

A batch study for biohydrogen production was conducted using raw palm oil mill effluent (POME) and POME sludge as a feed and inoculum respectively. Response Surface Methodology (RSM) was used to design the experiments. Experiments were conducted at different reaction temperatures (30–50 °C), inoculum size to substrate ratios (I:S) and reaction times (8–24 h). An optimum condition of biohydrogen production was achieved with COD removal efficiency of 21.95% with hydrogen yield of 28.47 ml H₂ g^?1 COD removed. The I:S ratio was 40:60, with reaction temperature of 50 °C at 8 h of reaction time. The study showed that a lower substrate concentration (less than 20 g L^?1) for biohydrogen production using pre-settled POME was achievable, with optimum HRT of 8 h under thermophilic condition (50 °C). This study also found that pre-settled POME is feasible to be used as a substrate for biohydrogen production under thermophilic condition.     

Efficiency and economic benefit of dark-fermentative biohydrogen production in Asian circular economies: Evaluation using soft-link methodology with data envelopment analysis (DEA) and computable general equilibrium model (CGE)

This study combines a data envelopment analysis, a dynamic computable general equilibrium model with estimated secondary material flows for circular economy, based on economist Joseph Schumpeter's macroeconomic theory, to develop a novel soft-link model to determine the efficiency of forty-three dark-fermentative technology of biohydrogen, and technology improvement impacts on biohydrogen output and supply price for six major emerging Asian countries. The integrated model is found to be feasible.This study finds that efficiency of continuous technology significantly exceeds that of batch technology. Biomass substrate concentration is the most important input in the generation of biohydrogen statistically; pH influences the efficiency of the batch technology, and the efficiency of continuous production technologies significantly exceeds that of batch technologies, but still have a gap to improve to full production efficiency for most of continuous technologies. India and China generate highest output growth of biohydrogen in baseline scenario. Japan and India can most benefit from improvements in batch and continuous biohydrogen production technology. The models and results of this study provides guidelines and references for decision-makers in industry and government who are responsible for reforming future energy policy.     

Assessing the impact of palmitic,myristic and lauric acids on hydrogen production from glucose fermentation by mixed anaerobic granular cultures

The effects of lauric (LUA), myristic (MA), palmitic (PA), and a mixture of myristic:palmitic (MA:PA) acids on hydrogen (H₂) production from glucose degradation using anaerobic mixed cultures were assessed at 37 °C with an initial pH set at 5.0 and 7.131 mM of each acid. The maximum H₂ yield (2.53 ± 0.18 mol mol⁻¹ glucose) was observed in cultures treated with PA. A principal component analysis (PCA) of the by-products and the microbial population data sets detected similarities between the controls and PA treated cultures; however, differences were observed between the controls and PA treated cultures in comparison to the MA and LUA treated cultures. The flux balance analysis (FBA) showed that PA decreased the quantity of H₂ consumed via homoacetogenesis compared to the other LCFAs. The control culture was dominated by Thermoanaerovibrio acidaminovorans (60%), Geobacillus sp. and Eubacterium sp. (28%), while Clostridium sp. was less than 1%. Treatment with PA, MA, MA:PA, or LUA increased the H₂ producers (Clostridium sp. and Bacillus sp.) population by approximately 48, 67, 86, and 86%, respectively.     

HRT control as a strategy to enhance continuous hydrogen production from sugarcane juice under mesophilic and thermophilic conditions in AFBRs

The objective of this study was to evaluate the effects of hydraulic retention time (HRT) (8–1 h) on H₂ production from sugarcane juice (5000 mg COD L⁻¹) in mesophilic (30 °C, AFBR-30) and thermophilic (55 °C, AFBR-55) anaerobic fluidized bed reactors (AFBRs). At HRTs of 8 and 1 h in AFBR-30, the H₂ production rates were 60 and 116 mL H₂ h⁻¹ L⁻¹, the hydrogen yields were 0.60 and 0.10 mol H₂ mol⁻¹ hexose, and the highest bacterial diversities were 2.47 and 2.34, respectively. In AFBR-55, the decrease in the HRT from 8 to 1 h increased the hydrogen production rate to 501 mL H₂ h⁻¹ L⁻¹ at the HRT of 1 h. The maximum hydrogen yield of 1.52 mol H₂ mol⁻¹ hexose was observed at the HRT of 2 h and was associated with the lowest bacterial diversity (0.92) and highest bacterial dominance (0.52).     

A limited LCA comparing large- and small-scale production of rape methyl ester (RME) under Swedish conditions

Production of rape methyl ester (RME) can be carried out with different systems solutions, in which the choice of system is usually related to the scale of the production. The purpose of this study was to analyse whether the use of a small-scale RME production system reduced the environmental load in comparison to a medium- and a large-scale system. To fulfil this purpose, a limited LCA, including air-emissions and energy requirements, was carried out for the three plant sizes. For small plants and physical allocation, the global warming potential was 40.3 g CO₂-eq/MJ_fuel, the acidification potential 236 mg SO₂-eq/MJ_fuel, the eutrophication potential 39.1 mg PO₄³⁻-eq/MJ_fuel, the photochemical oxidant creation potential 3.29 mg C₂H₄-eq/MJ_fuel and the energy requirement 295 kJ/MJ_fuel. It was shown that the differences in environmental impact and energy requirement between small-, medium- and large-scale systems were small or even negligible. The higher oil extraction efficiency and the more efficient use of machinery and buildings in the large-scale system were, to a certain degree, outweighed by the longer transport distances. The dominating production step was the cultivation, in which production of fertilisers, soil emissions and tractive power made major contributions to the environmental load. The results were, however, largely dependent on the method used for allocation of the environmental burden between the RME and the by-products meal and glycerine. This indicates that when different biofuels or production strategies are to be compared, it is important that the results are calculated with the same allocation strategies and system limitations.     

Simultaneous biohydrogen (H2) and bioplastic (poly-β-hydroxybutyrate-PHB) productions under dark,photo, and subsequent dark and photo fermentation utilizing various wastes

The present study is focused on bio hydrogen (H₂) and bioplastic (i.e., poly-β-hydroxybutyrate; PHB) productions utilizing various wastes under dark fermentation, photo fermentation and subsequent dark-photo fermentation. Potential bio H₂ and PHB producing microbes were enriched and isolated. The effects of substrate (rice husk hydrolysate, rice straw hydrolysate, dairy industry wastewater, and rice mill wastewater) concentration (10–100%) and pH (5.5–8.0) were examined in the batch mode under the dark and photo fermentation conditions. Using 100% rice straw hydrolysate at pH 7, the maximum bio H₂ (1.53 ± 0.04 mol H₂/mol glucose) and PHB (9.8 ± 0.14 g/L) were produced under dark fermentation condition by Bacillus cereus. In the subsequent dark-photo fermentation, the highest amounts of bio H₂ and PHB were recorded utilizing 100% rice straw hydrolysate (1.82 ± 0.01 mol H₂/mol glucose and 19.15 ± 0.25 g/L PHB) at a pH of 7.0 using Bacillus cereus (KR809374) and Rhodopseudomonas rutila. The subsequent dark-photo fermentative bio H₂ and PHB productions obtained using renewable biomass (i.e., rice husk hydrolysate and rice straw hydrolysate) can be considered with respect to the sustainable management of global energy sources and environmental issues.     

Effect of hydraulic retention time on hydrogen production and chemical oxygen demand removal from tapioca wastewater using anaerobic mixed cultures in anaerobic baffled reactor (ABR)

This study evaluated hydrogen production and chemical oxygen demand removal (COD removal) from tapioca wastewater using anaerobic mixed cultures in anaerobic baffled reactor (ABR). The ABR was conducted based on the optimum condition obtained from the batch experiment, i.e. 2.25 g/L of FeSO₄ and initial pH of 9.0. The effects of the varying hydraulic retention times (HRT: 24, 18, 12, 6 and 3 h) on hydrogen production and COD removal in a continuous ABR were operated at room temperature (32.3 ± 1.5 °C). Hydrogen production rate (HPR) increased with a reduction in HRT i.e. from 164.45 ± 4.14 mL H₂/L.d (24 h HRT) to 883.19 ± 7.89 mL H₂/L.d (6 h HRT) then decreased to 748.54 ± 13.84 mL H₂/L.d (3 h HRT). COD removal increased with reduction in HRT i.e. from 14.02 ± 0.58% (24 h HRT) to 29.30 ± 0.84% (6 h HRT) then decreased to 21.97 ± 0.94% (3 h HRT). HRT of 6 h was the optimum condition for ABR operation as indicated.