Dark Fermentative H2 Production Research Articles

Cleaner production of biohydrogen using poplar leaves: Experimental and optimization studies

Biohydrogen (bioH2) is recognized as a potential carbon-neutral energy vector, and developing novel methods has received increasing attention with a prime goal of producing H2 more efficient and cost effective manner. This study aimed to develop a unique reactor to investigate dark fermentative H2 production from poplar biomass using commercially available and inexpensive microorganism cultures. Therefore, six factors of the Box-Behnken design (BBD) were performed to evaluate the individual and combined effects of operational parameters: acid concentration (2–10%), biomass concentration (2–10 g), initial pH (5–8), temperature (30–40 °C), mixing ratio (150–350 rpm), and microorganism concentration (2–6 g) on bioH2 production. Among the operational parameters, the acid concentration was the most effective parameter on bioH2 production. The bioH2 production increased from 11.33 to 18.15 mg/g biomass with increasing acid concentration from 6 to 10%. Moreover, the optimum levels of operational variables were as follows: acid concentration of 9.9%, biomass amount of 2 g, pH of 6.56, temperature of 35 °C, mixing ratio of 345 rpm, and microorganism amount of 4.5 g for the highest bioH2 production of 20 mg/g-biomass according to the experimental design. Consequently, the bioH2 production performance of the dark fermentation process showed that bioH2 production from poplar biomass using commercially available microorganisms had a competitive advantage.

Hydrogen production from Brewer's spent grain hydrolysate by dark fermentation

The aim of this study was to evaluate the production of H2 by dark fermentation of brewer's spent grain (BSG) hydrolysate. The process was evaluated under different conditions of temperature, initial pH, and inoculum ratio. The maximum cumulative H2 production (PH2), H2 production rate (RH2), and H2 yield (YH2) was 4160 mL/L of working volume, 760 mL/L.h, and 5154.26 mL/gglucose cons, respectively. The assay with initial pH 7.5, inoculum 30%, and 35 °C, resulted in the highest PH2, and assay with initial pH 7.5, inoculum 30%, and 45 °C resulted in the highest RH2 and YH2. It is observed that PH2 and RH2 were statistically influenced by the initial pH, showing the negative effect of low pH. The initial concentration of furfural (<0.0047 g/L) and 5-(hydroxymethyl)furfural (<0.0220 g/L) were low. Therefore, BSG hydrolysate would be a suitable substrate for dark fermentative H2 production.

Dark fermentative hydrogen production from cassava starch: A comprehensive evaluation of the effects of starch extrusion and enzymatic hydrolysis

Hydrolysis could be rate-limiting for the dark fermentative H2 production from cassava starch (CS) due to its semicrystalline nature. Therefore, the effects of CS extrusion and enzymatic hydrolysis on improving dark fermentation (DF) were studied. Preliminary DF experiments were conducted to investigate the effects of inoculum (sludge) heat-pretreatment, which increased H2 production from untreated-CS by over 154%. Likewise, 15 g/L starch was considered best for H2 production. Extruded-CS presented a maximum H2 yield of 1.58 mol H2/mol glucose with a 76% reduction in the fermentation adaptive phase and 43% increase in maximum H2 productivity compared to untreated-CS. Meanwhile, extruded-CS hydrolysate presented a maximum H2 yield of 1.8 mol H2/mol glucose with a 63% reduction in the fermentation adaptive phase and 18% increase in H2 productivity. A comprehensive assessment of the H2 yield, process time, production rate, and process simplification revealed extruded-CS as the most promising feedstock under the evaluated conditions.

Dark fermentative hydrogen production and transcriptional analysis of genes involved in the unicellular halotolerant cyanobacterium Aphanothece halophytica under nitrogen and potassium deprivation.

The unicellular halotolerant cyanobacterium Aphanothece halophytica is known as a potential hydrogen (H2) producer. This study aimed to investigate the enhancement of H2 production under nutrient deprivation. The results showed that nitrogen and potassium deprivation induced dark fermentative H2 production by A. halophytica, while no differences in H2 production were found under sulfur and phosphorus deprivation. In addition, deprivation of nitrogen and potassium resulted in the highest H2 production in A. halophytica due to the stimulation of hydrogenase activity. The effect of adaptation time under nitrogen and potassium deprivation on H2 production was investigated. The results showed that the highest H2 accumulation of 1,261.96 ± 96.99µmol H2 g dry wt-1 and maximum hydrogenase activity of 179.39 ± 8.18µmol H2 g dry wt-1 min-1 were obtained from A. halophytica cells adapted in the nitrogen- and potassium-deprived BG11 medium supplemented with Turk Island salt solution (BG110-K) for 48h. An increase in hydrogenase activity was attributed to the decreased O2 concentration in the system, due to a reduction of photosynthetic O2 evolution rate and a promotion of dark respiration rate. Moreover, nitrogen and potassium deprivation stimulated glycogen accumulation and decreased specific activity of pyruvate kinase. Transcriptional analysis of genes involved in H2 metabolism using RNA-seq confirmed the above results. Several genes involved in glycogen biosynthesis (glgA, glgB, and glgP) were upregulated under both nitrogen and potassium deprivation, but genes regulating enzymes in the glycolytic pathway were downregulated, especially pyk encoding pyruvate kinase. Interestingly, genes involved in the oxidative pentose phosphate pathway (OPP) were upregulated. Thus, OPP became the favored pathway for glycogen catabolism and the generation of reduced nicotinamide adenine dinucleotide phosphate (NADPH), which resulted in an increase in H2 production under dark anaerobic condition in both nitrogen- and potassium-deprived cells.

Pretreatment in Vortex Layer Apparatus Boosts Dark Fermentative Hydrogen Production from Cheese Whey

Dark fermentation (DF) is a promising process for mitigating environmental pollution and producing “green” H2. However, wider implementation and scaling of this technology is hampered by insufficient process efficiency. In this work, for the first time, the effect of innovative pretreatment of cheese whey (CW) in a vortex layer apparatus (VLA) on characteristics and DF of CW was studied. Pretreatment in VLA resulted in a heating of the CW, slight increase in pH, volatile fatty acids, iron, and reduction in fat, sugar, and chemical oxygen demand (COD). The biochemical hydrogen potential test and analysis of H2 production kinetics confirmed the significant potential of using VLA in enhancement of dark fermentative H2 production. The maximum potential H2 yield (202.4 mL H2/g COD or 3.4 mol H2/mol hexose) was obtained after pretreatment in VLA for 45 s and was 45.8% higher than the control. The maximum H2 production rate after 5 and 45 s of pretreatment was 256.5 and 237.2 mL H2/g COD/d, respectively, which is 8.06 and 7.46 times higher than in the control. The lag phase was more than halved as a function of the pretreatment time. The pretreatment time positively correlated with the total final concentration of Fe2+ and Fe3+ and negatively with the lag phase, indicating a positive effect of pretreatment in VLA on the start of H2 production.

Factor-based assessment of continuous bio-H2 production from cheese whey

Despite having been widely investigated, dark fermentative H2 production from organic residues is still limited by process-related issues which may hamper the perspectives of full-scale process implementation. Such constraints are mainly due to the process complexity, which is largely affected by multiple and often mutually interacting factors. In the present work, the results of continuous fermentative H2 production experiments using synthetic cheese whey as the input substrate were used to gain detailed knowledge of the process features and identify suitable and critical operating conditions. Specifically, innovative process interpretation involved a combination of analytical characterization of the fermentation broth, mass balance calculations and statistical methods (correlation and principal component analyses) to derive systematic considerations for process characterization and scale-up. The metabolic products mainly included acetate and butyrate, which however were likely to derive (in different proportions depending on the operating conditions) from both hydrogenogenic and competing pathways. For some tests, lactate and succinate were also found to have been formed. It was observed that the main features of the process (H2 yield and rate, stability condition) were correlated with the operational and analytical parameters. The first three principal components identified by the statistical analysis were able to account for: 1) the effect of retention time and total metabolites produced; 2) biogas (H2 and CO2) generation, butyrate production and stability condition; and 3) organic loading rate and propionate production. The results suggested that the main features of hydrogenogenic fermentation can be described by a reduced set of factors that may be usefully adopted for both process monitoring and prediction purposes.

Simazine Enhances Dark Fermentative H2 Production by Unicellular Halotolerant Cyanobacterium Aphanothece halophytica.

The halotolerant cyanobacterium Aphanothece halophytica is a potential H2 producer that induces H2 evolution under nitrogen deprivation. H2 is mainly produced via the catabolism of stored glycogen under dark anaerobic condition. H2 evolution is catalyzed by O2-sensitive bidirectional hydrogenase. The aim of this study was to improve H2 production by A. halophytica using various kinds of inhibitors. Among all types of inhibitors, simazine efficiently promoted the highest H2 production under dark conditions. High simazine concentration and long-term incubation resulted in a decrease in cell and chlorophyll concentrations. The optimal simazine concentration for H2 production by A. halophytica was 25 µM. Simazine inhibited photosynthetic O2 evolution but promoted dark respiration, resulting in a decrease in O2 level. Hence, the bidirectional hydrogenase activity and H2 production was increased. A. halophytica showed the highest H2 production rate at 58.88 ± 0.22 µmol H2 g−1 dry weight h−1 and H2 accumulation at 356.21 ± 6.04 μmol H2 g−1 dry weight after treatment with 25 µM simazine under dark anaerobic condition for 2 and 24 h, respectively. This study demonstrates the potential of simazine for the enhancement of dark fermentative H2 production by A. halophytica.

Scale-Up of Dark Fermentative Biohydrogen Production by Artificial Microbial Co-Cultures

As a renewable energy carrier, dark fermentative biohydrogen (H2) represents a promising future alternative to fossil fuels. Recently, the limited H2 yield of 4 moles of H2 per mole glucose, the so-called “Thauer limit”, was surpassed by a defined artificial consortium. In this article, we demonstrate the upscaling of this drawing board design, from serum bottles to laboratory scale bioreactors. Our results illustrate that this designed microbial co-culture can be successfully implemented in batch mode, with maximum H2 yields of 6.18 and 4.45 mol mol−1 substrate. Furthermore, we report volumetric H2 productivities of 105.6 and 80.8 mmol H2 L−1 h−1. These rates are higher than for any other dark fermentative H2 production system using a synthetic microbial co-culture applied in batch mode on a defined medium. Our study is an important step forward for the application of artificial microbial consortia in future biotechnology and energy production systems.

Modeling dark fermentation of cheese whey for H2 and n-butyrate production considering the chain elongation perspective

This study aimed to develop and compare different Anaerobic Digestion Model no. 1 extensions to represent hydrogen and n-butyrate synthesis (ADM-CE). For developed models' calibration, experimental data were gathered from a previous study investigating dark fermentative H2 production from cheese whey in mesophilic batch mode. The Markov chain Monte Carlo method was applied to determine free parameters values under limited iterations until reaching convergence assisted by Geweke's diagnostic. Each developed model considered different aspects of investigation such as: pH inhibition, phase transfer limitation mechanisms, MCMC sample sizes and approaches, competitive inhibition, hydrolysis step and chain elongation biomass discretization. The estimation of all parameters showed upright MCMC. Among the proposed models, the ADM-CE could predict about 73% accumulated H2 and 87% of total n-butyrate obtained experimentally. In effect, the consideration of self-regulation mechanism between fermentative organisms represents a possibility not only for H2 recovery, but also a high prospect for n-butyrate improvement.

High-rate biohydrogen production from xylose using a dynamic membrane bioreactor

This study aimed a high-rate dark fermentative H2 production from xylose using a dynamic membrane module bioreactor (DMBR) with a 444-μm pore polyester mesh. 20 g xylose/L was fed continuously to the DMBR at different hydraulic retention times (HRTs) from 12 to 3 h at 37 °C. The maximum average H2 yield (HY) and H2 production rate (HPR) at 3 h HRT were found to be 1.40 ± 0.07 mol H2/mol xyloseconsumed and 30.26 ± 1.19 L H2/L-d, respectively. The short HRT resulted in the maximum suspended biomass concentration (8.92 ± 0.40 g VSS/L) along with significant attached biomass retention (7.88 ± 0.22 g VSS/L). H2 was produced by both butyric and acetic acid pathways. Low HY was concurrent with lactic acid production. The bacterial population shifted from non-H2 producers, such as Lactobacillus and Sporolactobacillus spp., to Clostridium sp., when HY increased. Thus, xylose from lignocellulose is a feasible substrate for dark fermentative H2 production using DMBR.

Co-fermentation of residual algal biomass and glucose under the influence of Fe3O4 nanoparticles to enhance biohydrogen production under dark mode

The present study reports Fe3O4 nanoparticles (Fe3O4 NPs) induced enhanced hydrogen production via co-fermentation of glucose and residual algal biomass (cyanobacteria Lyngbya limnetica). A significant enhancement of dark fermentative H2 production has been noticed under the influence of co-fermentation of glucose and residual algal biomass using Fe3O4 NPs as catalyst. Further, using the optimized ratio of glucose to residual algal biomass (10:4), ∼ 37.14 % higher cumulative H2 has been recorded in presence of 7.5 mg/L Fe3O4 NPs as compared to control at 37 °C. In addition, under the optimum conditions [glucose to residual algal biomass ratio (10:4)] presence of 7.5 mg/L Fe3O4 NPs produces ∼ 937 mL/L cumulative H2 in 168 h at pH 7.5 and at temperature 40 °C. Clostridum butyrium, employed for the dark fermentation yielded ∼ 7.7 g/L dry biomass in 168 h whereas acetate (9.0 g/L) and butyrate (6.2 g/L) have been recorded as the dominating metabolites.

Effect of genus Clostridium abundance on mixed-culture fermentation converting food waste into biohydrogen

This study examined the effect of various inocula on mixed-culture dark fermentative H2 production from food waste. Heat-treated and frozen H2-producing granular sludge (HPG) grown with monomeric sugars showed a higher H2 yield, production rate, and acidogenic efficiency along with a shorter lag phase than heat-treated methanogenic sludge. Among three different methods of methanogenic sludge inoculation, inoculation after centrifugation showed better H2 production performance. Propionic acid production and homoacetogenesis were regarded as major H2-consuming pathways when methanogenic sludge was used, whereas only homoacetogenesis was found in HPG-inoculated fermentation. During fermentation, the abundance of Clostridium increased greater than 48-fold for methanogenic sludge and greater than 108-fold for HPG, respectively. The initial abundance of Clostridium showed a linear relationship with the H2 production rate and lag-phase time. The use of inoculum with a high abundance of Clostridium is essential for H2 production from food waste.

Biochar boosts dark fermentative H2 production from sugarcane bagasse by selective enrichment/colonization of functional bacteria and enhancing extracellular electron transfer

The influence of biochar (BC) on anerobic digestion (AD) of organic wastes have been widely studied. However, the effect of BC on rate-limiting step during AD of lignocellulosic waste, i.e. the hydrolysis and acidogenesis step, is rarely studied and the underlying mechanisms have not been investigated. In this study, the benefits of BC with respect to dark fermentative hydrogen production were explored in a fermentation system by a heat-shocked consortium from sewage sludge (SS) with pretreated sugarcane bagasse (PSCB) as carbon source. The results showed that biochar boosted biohydrogen production by 317.1% through stimulating bacterial growth, improving critical enzymatic activities, manipulating the ratio of NADH/NAD+ and enhancing electron transfer efficiency of fermentation system. Furthermore, cellulolytic Lachnospiraceae was efficiently enriched and electroactive bacteria were selectively colonized and the ecological niche was formed on the surface of biochar. Synergistic effect between functional bacteria and extracellular electron transfer (EET) in electroactive bacteria were assumed to be established and maintained by biochar amendment. This study shed light on the underlying mechanisms of improved performance of biohydrogen production from lignocellulosic waste during mesophilic dark fermentation by BC supplementation.

Parametric optimization of the dark fermentation process for enhanced biohydrogen production from the organic fraction of municipal solid waste using Taguchi method

In this work, the Taguchi method was used to optimize the dark fermentative H2 production from the organic fraction of municipal solid waste (OFMSW). The experiments were planned using the L16 orthogonal array design with each trial conducted at different levels of substrate concentration, inoculum-to-substrate ratio (ISR), and temperature. Based on the results, the optimal setting of the process parameters was the substrate concentration of 6 g-VS/L, ISR 0.5, and temperature of 55 °C. Furthermore, substrate concentration was the most important parameter affecting bio-H2 production among the three process parameters considered. Finally, a confirmation experiment under optimal conditions yielded 62.5 mL H2/g-VSadded, which was higher than all the bio-H2 yield values obtained in the other conditions tested in this study. The measured and predicted bio-H2 yields in the verification test were also very close to each other, confirming the reliability of the Taguchi method in optimizing the bio-H2 production process.

Response surface methodology to optimize the conditions for Enterococcus faecium YA002 producing H2 from xylose

A bacterium was newly isolated from cow dung, and identified as Enterococcus faecium YA002 by 16S rRNA gene sequence analyzing. It showed high H2 production performance in dark fermentation with xylose as substrate, the fermentative conditions including xylose concentration, ratio of nitrogen source to carbon source in mass (N/C ratio), temperature, and initial pH were optimized by response surface methodology (RSM) with a Box-Behnken design (BBD). After regression analyzing, it could be found that H2 production was well fitted by a quadratic polynomial equation, and significantly affected by the four studied factors. The optimal conditions were xylose concentration of 22.69 g/L, N/C ratio of 0.127, temperature of 37.2 °C, and initial pH of 8.0, and the experimental H2 production under the conditions was 2918.91 ± 125.49 mL/L, which was 97.91% of the predicted H2 production. The results indicated that Enterococcus faecium YA002 was an ideal inoculum for dark fermentative H2 production from xylose, and the conditions for the H2 production were successfully optimized by response surface methodology.

Impact of microbial inoculum storage on dark fermentative H2 production

Complex organic substrates represent an important and relevant feedstock for producing hydrogen by Dark Fermentation (DF). Usually, an external microbial inoculum originated from various natural environments is added to seed the DF reactors. However, H2 yields are significantly impacted by the inoculum origin and the storage conditions as microbial community composition can fluctuate. This study aims to determine how the type and time of inoculum storage can impact the DF performances. Biochemical Hydrogen Potential tests were carried out using three substrates (glucose, the organic fraction of municipal solid waste, and food waste), inocula of three different origins, different storage conditions (freezing or freeze-drying) and duration. As a result, H2 production from glucose with the differently stored inocula was significantly impacted (positively or negatively) and was inoculum-origin-dependent. For complex substrates, hydrogen yields with the stored inocula were not statistically different from the fresh inocula, offering the possibility to store an inoculum.

Optimization of dark fermentative hydrogen production from organic wastes using acidogenic mixed consortia

The present study investigates the improvement of dark fermentative H2 production from organic wastes using acidogenic mixed consortia. A preliminary study was conducted to find out the suitability of the wastes as a feedstock for H2 production using acidogenic mixed consortia, which was developed by heat-shock pretreatment of anaerobically digested sludge. In the present study, starchy wastewater supplemented with groundnut de-oiled cake (GDOC) was found most suitable for H2 production. Single parameter optimization studies were performed, followed by Taguchi analysis. pH was found to be the most influential factor as compared to other physicochemical parameters. A central composite design was implemented to maximize H2 production. Additionally, the experimental results were analyzed by response surface methodology (RSM) and artificial intelligence (AI) techniques such as artificial neural network (ANN) and support vector machine (SVM). SVM was found to be better as compared to ANN and RSM in terms of prediction capability. R2 and root mean square error (RMSE) for the SVM model were estimated to be 0.988 and 0.0103, respectively. Subsequently, these AI-based models were further coupled with genetic algorithm (GA) and particle swarm optimization (PSO) for the determination of optimal process parameters. GA and PSO both offered a similar optimal value of the parameters. However, PSO was found to be faster than GA. The maximum H2 yield of 8.28 mol H2 kg−1 CODremoved was achieved under the optimized condition of pH 6.75, GDOC concentration 16.16 g L−1 and temperature 37.55 °C determined by SVM-GA and SVM-PSO models. H2 yield was improved by 2.1 folds using SVM based model as compared to un-optimized condition. This is the first attempt to implement the SVM model in biological process optimization and to compare it with ANN and RSM along with validation study.

Effects of the Photosystem II Inhibitors CCCP and DCMU on Hydrogen Production by the Unicellular Halotolerant Cyanobacterium Aphanothece halophytica.

The unicellular halotolerant cyanobacterium Aphanothece halophytica is a potential dark fermentative producer of molecular hydrogen (H2) that produces very little H2 under illumination. One factor limiting the H2 photoproduction of this cyanobacterium is an inhibition of bidirectional hydrogenase activity by oxygen (O2) obtained from splitting water molecules via photosystem II activity. The present study aimed to investigate the effects of the photosystem II inhibitors carbonyl cyanide m-chlorophenyl hydrazone (CCCP) and 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) on H2 production of A. halophytica under light and dark conditions and on photosynthetic and respiratory activities. The results showed that A. halophytica treated with CCCP and DCMU produced H2 at three to five times the rate of untreated cells, when exposed to light. The highest H2 photoproduction rates, 2.26 ± 0.24 and 3.63 ± 0.26 μmol H2 g−1 dry weight h−1, were found in cells treated with 0.5 μM CCCP and 50 μM DCMU, respectively. Without inhibitor treatment, A. halophytica incubated in the dark showed a significant increase in H2 production compared with cells that were incubated in the light. Only CCCP treatment increased H2 production of A. halophytica during dark incubation, because CCCP functions as an uncoupling agent of oxidative phosphorylation. The highest dark fermentative H2 production rate of 39.50 ± 2.13 μmol H2 g−1 dry weight h−1 was found in cells treated with 0.5 μM CCCP after 2 h of dark incubation. Under illumination, CCCP and DCMU inhibited chlorophyll fluorescence, resulting in a low level of O2, which promoted bidirectional hydrogenase activity in A. halophytica cells. In addition, only CCCP enhanced the respiration rate, further reducing the O2 level. In contrast, DCMU reduced the respiration rate in A. halophytica.

Enhancement of mass transfer conditions to increase the productivity and efficiency of dark fermentation in continuous reactors

Hydrogen (H2) produced by dark fermentation is an alternative to fulfill the requirements of the transportation sector and to be a complementary source in the forthcoming electricity grid. However, the dark fermentative H2 production is limited by the accumulation of H2 in the fermentation broth. In continuous stirred-tank reactors (CSTR), such phenomenon is associated with poor mass transfer conditions. Nevertheless, this parameter has been scarcely considered to enhance H2 production. In this research, the effect of the H2 mass transfer conditions on the productivity and efficiency of continuous H2 production was evaluated using a series of CSTR operated at H2 mass transfer coefficients (kLa) ranging from 1.04 to 4.23 1/h. The results showed that volumetric H2 production rate (VHPR) and H2 yield increased 74 and 78%, respectively, due to enhanced mass transfer conditions. This behavior was driven by 53% decrease of the dissolved H2 concentration. More specifically, the maximum VHPR of 7.66 L/L-d with a H2 yield of 1.1 mol H2/mol hexose was obtained at a kLa = 4.23 1/h. Furthermore, 16S-DGGE analysis and sequencing revealed that Clostridium and Lactobacillus were the dominant bacterial genera in continuous operation. In particular, Clostridium increased its occurrence at kLa of 2.72–4.23 1/h as a response to lower dissolved H2 concentrations. The novelty of this work relies on the demonstration that mass transfer conditions control not only the H2 accumulation and reactor performance (VHPR and H2 yield), but they also influence the metabolic pathways and the composition of the microbial community.

Enhanced dark fermentative H2 production by agar-immobilized cyanobacterium Aphanothece halophytica

Cell immobilization is one of the techniques used to improve H2 productivity in cyanobacteria. In this study, H2 production by immobilized cells of unicellular halotolerant cyanobacterium Aphanothece halophytica was investigated and optimized. The results showed that immobilized cells of A. halophytica had higher H2 production than free cells under nitrogen-deprived condition. Among various support material types used, agar-immobilized cells showed the highest H2 production rate. Under nitrogen deprivation, the optimal conditions of cell immobilization for H2 production were 3% (w/v) agar concentration, 0.2 mg dry cell weight per mL of gel solution, and 0.125 cm3 of agar cube. The optimum pH of medium and incubation temperature for H2 production by agar-immobilized cells were pH 7.4 and 40 °C, respectively. Using a large glass vial and headspace volume resulted in enhancement of H2 production by agar-immobilized cells. Finally, H2 production by agar-immobilized cells was analyzed for three consecutive cycles. H2 production could be maintained at the highest level after two cycles when half of immobilized cells were replaced with fresh immobilized cells. These findings indicate that the enhanced H2 production of the unicellular halotolerant cyanobacterium A. halophytica can be achieved by immobilization method, thus providing the possibility to improve H2 production by cyanobacteria in the future.