Fermentative H2 Research Articles

Inhibitory effects of furan derivatives and phenolic compounds on dark hydrogen fermentation

The inhibitory effects of furan derivatives [i.e. furfural and 5-hydroxymethylfurfural (5-HMF)] and phenolic compounds (i.e. vanillin and syringaldehyde) on dark hydrogen fermentation from glucose were comparatively evaluated. Phenolic compounds exhibited stronger inhibition on hydrogen production and glucose consumption than furan derivatives under the same 15mM concentration. Furan derivatives were completely degraded after 72h fermentation, while over 55% of phenolic compounds remained unconverted after 108h fermentation. The inhibition coefficients of vanillin (14.05) and syringaldehyde (11.21) were higher than those of 5-HMF (4.35) and furfural (0.64). Vanillin exhibited the maximum decrease of hydrogen yield (17%). The consumed reducing power by inhibitors reduction from R-CHO to RCH2OH was a possible reason contributed to the decreased hydrogen yield. Vanillin exhibited the maximum delay of peak times of hydrogen production rate and glucose consumption. Soluble metabolites and carbon conversion efficiency decreased with inhibitors addition, which were consistent with hydrogen production.

Effects of three methane mitigation agents on parameters of kinetics of total and hydrogen gas production, ruminal fermentation and hydrogen balance using in vitro technique.

Methane (CH4 ) can be mitigated through directly inhibiting methanogen activity and starving methanogens by hydrogen (H2 ) sink. Three types of mechanism (i.e. bromoethanesulphonate (BES), nitrate and emodin) and doses of CH4 mitigation agents were employed to investigate their pathways of CH4 inhibition. Results indicated that both BES and emodin inhibited CH4 production and altered H2 balance, which could be accompanied by decreased dry matter disappearance (DMD), fractional rate of gH2 formation, volatile fatty acid (VFA) production, ability to produce and use reducing equivalences and molecular H2 , and increased final asymptotic gH2 production, time to the peak of gH2 , discrete lag time of gH2 production and fermentation efficiency. However, emodin decreased gas volume produced by rapidly fermentable components of substrate and the rate of fermentation at early stage of incubation, while BES supplementation inhibited gas volume produced by both rapidly and slowly fermentable components of substrate and the rate of fermentation at middle or late stage of incubation. The nitrate supplementation inhibited CH4 production without affecting VFA profile, because of its dual role as H2 sink and being toxic to methanogens. Nitrate supplementation had more complicated pattern of fermentation, VFA production and profile and H2 balance in comparison to BES and emodin supplementation.

Effective conversion of maize straw wastes into bio-hydrogen by two-stage process integrating H2 fermentation and MECs.

The enhanced H2 production from maize straw had been achieved through the two-stage process of integrating H2 fermentation and microbial electrolysis cells (MECs) in the present work. Several key parameters affecting hydrolysis of maize straw through subcritical H2O were optimized by orthogonal design for saccharification of maize straw followed by H2 production through H2 fermentation. The maximum reducing sugar (RS) content of maize straw reached 469.7 mg/g-TS under the optimal hydrolysis condition with subcritical H2O combining with dilute HCl of 0.3% at 230 °C. The maximum H2 yield, H2 production rate, and H2 content was 115.1 mL/g-TVS, 2.6 mL/g-TVS/h, and 48.9% by H2 fermentation, respectively. In addition, the effluent from H2 fermentation was used as feedstock of MECs for additional H2 production. The maximum H2 yield of 1060 mL/g-COD appeared at an applied voltage of 0.8 V, and total COD removal reached about 35%. The overall H2 yield from maize straw reached 318.5 mL/g-TVS through two-stage processes. The structural characterization of maize straw was also carefully investigated by scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), and X-ray diffraction (XRD) spectra.

혐기성 수소발효를 결합한 생물학적 2단공정의 유기성폐자원 처리 및 바이오에너지 생산

This study was performed to investigate the application of dark H2 fermentation to two-stage bioprocesses for organic waste treatment and energy production. We reviewed information about the two-stage bioprocesses combining dark H2 fermentation with CH4 fermentation, photo H2 fermentation, microbial fuel cells (MFCs), or microbial electrolysis cells (MECs) by using academic information databases and university libraries. Dark fermentative bacteria use organic waste as the sole source of electrons and energy, converting it into H2. The reactions related to dark H2 fermentation are rapid and do not require sunlight, making them useful for treating organic waste. However, the degradation is not complete and organic acids remain. Thus, dark H2 fermentation should be combined with a post-treatment process, such as CH4 fermentation, photo H2 fermentation, MFCs, or MECs. So far, dark H2 fermentation followed by CH4 fermentation is a promising two-stage bioprocess among them. However, if the problems of manufacturing expenses, operational cost, scale-up, and practical applications will be solved, the two-stage bioprocesses combining dark H2 fermentation with photo H2 fermentation, MFCs, or MECs have also infinite potential in organic waste treatment and energy production. This paper demonstrated the feasibility of two-stage bioprocesses combining dark H2 fermentation as a novel system for organic waste treatment and energy

Co-production of S-adenosyl-L-methionine and L-isoleucine in Corynebacterium glutamicum

In this study, production of S-adenosyl-L-methionine in Corynebacterium glutamicum was investigated by overexpressing genes metK and vgb. Compared with vector control, overexpression of metK alone in C. glutamicum ATCC13032 and IWJ001 increased SAM production 5.11 and 11.65 times, respectively; while overexpression of metK and vgb in C. glutamicum ATCC13032 and IWJ001 increased SAM production 5.83 and 14.95 times, respectively. Further studies on IWJ001/pDXW-8-metk-vgb showed that the limiting factor for SAM production is intracellular ATP supply. Since IWJ001 is an L-isoleucine production strain, IWJ001/pDXW-8-metk-vgb could produce both SAM and L-isoleucine. After 72h fermentation, SAM and L-isoleucine in IWJ001/pDXW-8-metk-vgb reached 0.67g/L and 13.8g/L, respectively. The results demonstrate the potential application of C. glutamicum for co-production of SAM and amino acids.

Fermentative hydrogen production from agroindustrial lignocellulosic substrates.

To achieve economically competitive biological hydrogen production, it is crucial to consider inexpensive materials such as lignocellulosic substrate residues derived from agroindustrial activities. It is possible to use (1) lignocellulosic materials without any type of pretreatment, (2) lignocellulosic materials after a pretreatment step, and (3) lignocellulosic materials hydrolysates originating from a pretreatment step followed by enzymatic hydrolysis. According to the current literature data on fermentative H2 production presented in this review, thermophilic conditions produce H2 in yields approximately 75% higher than those obtained in mesophilic conditions using untreated lignocellulosic substrates. The average H2 production from pretreated material is 3.17 ± 1.79 mmol of H2/g of substrate, which is approximately 50% higher compared with the average yield achieved using untreated materials (2.17 ± 1.84 mmol of H2/g of substrate). Biological pretreatment affords the highest average yield 4.54 ± 1.78 mmol of H2/g of substrate compared with the acid and basic pretreatment - average yields of 2.94 ± 1.85 and 2.41 ± 1.52 mmol of H2/g of substrate, respectively. The average H2 yield from hydrolysates, obtained from a pretreatment step and enzymatic hydrolysis (3.78 ± 1.92 mmol of H2/g), was lower compared with the yield of substrates pretreated by biological methods only, demonstrating that it is important to avoid the formation of inhibitors generated by chemical pretreatments. Based on this review, exploring other microorganisms and optimizing the pretreatment and hydrolysis conditions can make the use of lignocellulosic substrates a sustainable way to produce H2.

Efficient production of succinic acid from macroalgae hydrolysate by metabolically engineered Escherichia coli

In this study, microbial production of succinic acid from macroalgae (i.e., Laminaria japonica) was investigated for the first time. The engineered Escherichia coli BS002 exhibited higher molar yield of succinic acid on mannitol (1.39±0.01mol/mol) than glucose (1.01±0.05mol/mol). After pretreatment and enzymatic hydrolysis, L. japonica hydrolysate was mainly glucose (10.31±0.32g/L) and mannitol (10.12±0.17g/L), which was used as the substrate for succinic acid fermentation with the recombinant BS002. A final 17.44±0.54g/L succinic acid was obtained from the hydrolysate after 72h dual-phase fermentation. The yield was as high as 1.24±0.08mol/mol total sugar, which reached 73% of the maximum theoretical yield. The results demonstrate that macroalgae biomass represents a novelty and economical alternative feedstock for biochemicals production.

Overexpression of ribosome elongation factor G and recycling factor increases L-isoleucine production in Corynebacterium glutamicum.

Ribosome elongation factor G encoded by fusA promotes the translocation step of protein synthesis in bacteria; ribosome recycling factor encoded by frr, together with the elongation factor G, dissociates ribosomes from messenger RNA after the termination of translation. Both factors play important roles during protein synthesis in bacteria. In this study, we found that overexpression of fusA and/or frr led to the increase of L-isoleucine production in Corynebacterium glutamicum IWJ001, an L-isoleucine production strain generated by random mutagenesis. Reverse transcription polymerase chain reaction analysis showed that transcriptional levels of genes lysC, hom, thrB, ilvA, ilvBN, and ilvE encoding the key enzymes in the biosynthetic pathway of L-isoleucine increased in C. glutamicum IWJ001 when fusA and/or frr were overexpressed. Co-overexpression of fusA and frr, together with genes ilvA, ilvB, ilvN, and ppnk in C. glutamicum IWJ001, led to 76.5% increase of L-isoleucine production in flask cultivation and produced 28.5g/L L-isoleucine in 72-h fed-batch fermentation. The results demonstrate that overexpressing ribosome elongation factor G and ribosome recycling factor is an efficient approach to enhance L-isoleucine production in C. glutamicum.

PH shift-mediated dehydrogenation and hydrogen production are responsible for microbial iron(III) reduction in submerged paddy soils

pH shift-induced responses of iron(III) [Fe(III)] reduction and associated dehydrogenation and hydrogen (H2) production in submerged paddy soils were investigated to improve our understanding of the contribution of fermentative processes to microbial Fe(III) reduction. A 40-day anaerobic incubation experiment was conducted using two paddy soils from geographically distinct regions in northern (Hapli-Stagnic Anthosol, HSA; pH 8.14) and southern China (Fe-accumili-Stagnic Anthosol, FSA; pH 5.00), respectively. The initial soil pH was adjusted with aluminum sulfate or sodium carbonate to strongly acidic, acidic, neutral, alkaline, and strongly alkaline levels. During anaerobic incubation, as the initial soil pH shifts to acidic or lower levels, dehydrogenase activity (DHA) was greatly inhibited. However, increased acid-soluble Fe(III) level was accompanied by decreased maximum Fe(III) reduction potential (a) and prolonged time to maximum Fe(III) reduction rate (T Vmax ). Decreased pH led to a significantly lower peak partial pressure of H2 (ppH 2max ) in HSA and an increase in FSA. The initial shifts in pH to neutral and higher levels had no significant effect on DHA or acid-soluble Fe(III); however, ppH 2max declined in the neutral treatment of HSA, while T Vmax decreased slightly in the presence of a low ppH2 in the neutral to strongly alkaline FSA. DHA was correlated with Fe(II) accumulation in both paddy soils during the rapid Fe(III) reduction stage (p < 0.05). DHA is related to microbially mediated Fe(III) reduction in anaerobic paddy soils. pH shift-mediated enzymatic dehydrogenation and fermentative H2 production are responsible for microbial Fe(III) reduction in flooded rice fields, with initial pH as the main driving force.

Inhibition of fermentative H2 production by hydrolysis byproducts of lignocellulosic substrates

Lignocellulosic materials are potential renewable substrates for fermentative H2 production; however, most of the methods available to hydrolyze these materials produce fermentation inhibitors. This study assessed the effect of three different groups of inhibitors on fermentative H2 production by a mixed culture: (1) acetic acid; (2) furan derivatives, such as furfural and 5-hydroxymethylfurfural (HMF); and (3) phenolic monomers, such as vanillin, syringaldehyde, and 4-hydroxybenzoic acid (HBA). Conduction of batch assays in the presence of glucose and different concentrations of inhibitors helped to assess how the inhibitors affected the kinetic parameters of the modified Gompertz model (Rm, Hmax, and λ). The concentrations of inhibitors that reduced 50% of the maximum H2 production rate (IC50) were estimated. In terms of IC50, HBA provided the largest inhibition, 0.38 g L−1, which is a novel result in the literature. HBA was followed by HMF and furfural, 0.48 and 0.62 g L−1, respectively. Vanillin, syringaldehyde, and acetic acid at 0.71; 1.05; and 5.14 g L−1 provided the same inhibition level, respectively. Knowledge about the degree of inhibition of these compounds shall contribute to sustainable H2 production from lignocellulosic substrates.

Phosphate enhancing fermentative hydrogen production from substrate with municipal solid waste composting leachate as a nutrient

To overcome phosphorus deficiency in municipal solid waste composting leachate, orthophosphate (OP) and pyrophosphate (PP) were separately added into leachate to evaluate the possibility of fermentative H2 production with leachate and phosphorus-rich streams as a full nutrient source. Results indicate H2 production is significantly promoted by OP addition but slightly facilitated by PP in some cases, depending on initial pH and P dosage. The highest hydrogen yield (1.95±0.07mol H2/mol glucose) was achieved at a COD/P ratio of 27.64 (mg/mg) with OP as phosphorus source at initial pH 5. For PP, a maximum yield of 1.58±0.09mol H2/mol glucose can be attained at the optimal COD/P ratio of 221.12 (mg/mg) and initial pH 5. OP promotes H2 production via dual approaches: supplying nutrient and relieving inhibition from excessive Ca2+ on granule sludge. However, both the roles in nutrient supply and Ca2+ removal by PP addition are limited.

Water-insoluble material from apple pomace makes changes in intracellular NAD+/NADH ratio and pyrophosphate content and stimulates fermentative production of hydrogen

Apple pomace is one of the major agricultural residues in Aomori prefecture, Japan, and it would be useful to develop effective applications for it. As apple pomace contains easily fermentable sugars such as glucose, fructose and sucrose, it can be used as a feedstock for the fermentation of fuels and chemicals. We previously isolated a new hydrogen-producing bacterium, Clostridium beijerinckii HU-1, which could produce H2 at a production rate of 14.5 mmol of H2/L/h in a fed-batch culture at 37 °C, pH 6.0. In this work we found that the HU-1 strain produces H2 at an approximately 20% greater rate when the fermentation medium contains the water-insoluble material from apple pomace. The water-insoluble material from apple pomace caused a metabolic shift that stimulated H2 production. HU-1 showed a decrease of lactate production, which consumes NADH, accompanied by an increase of the intracellular pyrophosphate content, which is an inhibitor of lactate dehydrogenase. The intracellular NAD(+)/NADH ratios of HU-1 during H2 fermentation were maintained in a more reductive state than those observed without the addition of the water insoluble material. To correct the abnormal intracellular redox balance, caused by the repression of lactate production, H2 production with NADH oxidation must be stimulated.

Effect of the accuracy of pH control on hydrogen fermentation

pH, known as the most important parameter in H2 fermentation, cannot be precisely controlled in a scaled-up fermenter as in a lab fermenter. In the preset work, to assess the effect of pH control accuracy on H2 fermentation, the pH was controlled at 6.0±0.1, 6.0±0.3, 6.0±0.5, 6.0±0.7, and 6.0±0.9 during batch fermentation of food waste. Up to deviation of ±0.3, a high H2 yield of 1.67–1.73molH2/mol hexoseadded was attained with producing butyrate as a major metabolite (>70% of total organic acids produced). A huge drop of H2 production, however, was observed at deviation >±0.5 with lowered substrate utilization and increased production of lactate. Next generation sequencing results showed that Clostridium was found to be the dominant genus (76.4% of total number of sequences) at deviation of ±0.1, whereas the dominant genus was changed to lactic acid bacteria such as Streptococcus and Lactobacillus with increase of deviation value.

Engineered biosynthesis of medium-chain esters in Escherichia coli

Medium-chain esters such as isobutyl acetate (IBAc) and isoamyl acetate (IAAc) are high-volume solvents, flavors and fragrances. In this work, we engineered synthetic metabolic pathways in Escherichia coli for the total biosynthesis of IBAc and IAAc directly from glucose. Our pathways harnessed the power of natural amino acid biosynthesis. In particular, the native valine and leucine pathways in E. coli were utilized to supply the precursors. Then alcohol acyltransferases from various organisms were investigated on their capability to catalyze esterification reactions. It was discovered that ATF1 from Saccharomyces cerevisiae was the best enzyme for the formation of both IBAc and IAAc in E. coli. In vitro biochemical characterization of ATF1 confirmed the fermentation results and provided rational guidance for future enzyme engineering. We also performed strain improvement by removing byproduct pathways (Δldh, ΔpoxB, Δpta) and increased the production of both target chemicals. Then the best IBAc producing strain was used for scale-up fermentation in a 1.3-L benchtop bioreactor. 36g/L of IBAc was produced after 72h fermentation. This work demonstrates the feasibility of total biosynthesis of medium-chain esters as renewable chemicals.

Flux balance analysis of different carbon source fermentation with hydrogen producing Clostridium butyricum using Cell Net Analyzer

A metabolic network model for Clostridium butyricum was developed using six different carbon sources (sucrose, fructose, galactose, mannose, trehalose and ribose) to study the fermentative H2 production. The model was used for investigation of H2 production and the ability of growth on different substrates to predict the maximum abilities for fermentative H2 production that each substrate can support. NADH fluxes were calculated by the model as an important cofactor affecting on H2 production. Butyrate and acetate production were used as model assumptions and biomass formation was chosen as the objective function for flux analysis calculations. Among the substrates selected, sucrose and trehalose supported the maximum growth and H2 yields. The Cell Net Analyzer metabolic network model developed for H2 estimation revealed good correlation with experimental data and could be further used to study the effect of environmental conditions and substrates concentration on H2 yield.

Effects of 5-hydromethylfurfural, levulinic acid and formic acid, pretreatment byproducts of biomass, on fermentative H2 production from glucose and galactose

In this study, the effects of 5-hydromethylfurfural (5-HMF), levulinic acid, and formic acid on fermentative H2 production have been investigated. The major byproducts of physicochemical pretreatment of lignocellulosic or algal biomass inhibited H2 production from glucose and galactose. The half maximal inhibitory concentrations (IC50) of 5-HMF on glucose, levulinic acid on glucose, formic acid on glucose, levulinic acid on galactose and formic acid on galactose were 0.59, 1.55, 12.50, 1.33 and 2.99 g/L, respectively. The pretreatment byproducts levels should be mitigated by controlling the severity of a physicochemical treatment or an additional selective removal process prior to H2 fermentation.

Effect of acid-pretreatment on hydrogen fermentation of food waste: Microbial community analysis by next generation sequencing

This work presents the effect of acid-pretreatment on H2 fermentation of food waste with detailed microbial information by next generation sequencing. The pretreated food waste at pH 1.0–4.0 was cultivated under mesophilic conditions without external inoculum addition. From the food waste acid-pretreated at pH 1–3, H2 yields in the range of 1.37–1.74 mol H2/mol hexoseadded were achieved, attaining the highest value at pH 2. Clostridium sp. such as Clostridium acetobutylicum ATCC 824 and Clostridium perfringens occupied more than 70% of total number of sequences at pH 1–3. On the other hand, in the control (no pretreatment) and at pH 4, lactic acid bacteria such as Lactobacillus and Streptococcus were found to be the dominant genus (>90% of total number of sequences), resulting in a low H2 yield. In addition, the effect of substrate concentration on H2 fermentation was investigated, and the maximum H2 productivity was estimated to be 27.2 L H2/L/d by Andrew's model.

A review on fermentative hydrogen production from dairy industry wastewater

AbstractHydrogen (H2) is one of the most promising alternative fuel sources for satisfying future energy demand, and fermentative hydrogen production is advantageous over other processes. In recent decades, considerable research has been conducted on H2 production from carbohydrate‐rich materials since it is a cost‐effective approach and has the ability to generate intensive renewable energy from organic wastes. Dairy wastewater is a high volume industrial wastewater and with its immense carbohydrate content is an attractive candidate for sustainable fermentative H2 production. The present paper provides comprehensive evaluation of recent reports on fermentative H2 production from dairy wastewater. Important effective parameters and current bioreactor technologies for H2 production from dairy wastewater are discussed. © 2014 Society of Chemical Industry

Effect of Lineage-Specific Metabolic Traits of Lactobacillus reuteri on Sourdough Microbial Ecology

This study determined the effects of specific metabolic traits of Lactobacillus reuteri on its competitiveness in sourdoughs. The competitiveness of lactobacilli in sourdough generally depends on their growth rate; acid resistance additionally contributes to competitiveness in sourdoughs with long fermentation times. Glycerol metabolism via glycerol dehydratase (gupCDE) accelerates growth by the regeneration of reduced cofactors; glutamate metabolism via glutamate decarboxylase (gadB) increases acid resistance by generating a proton motive force. Glycerol and glutamate metabolisms are lineage-specific traits in L. reuteri; therefore, this study employed glycerol dehydratase-positive sourdough isolates of human-adapted L. reuteri lineage I, glutamate decarboxylase-positive strains of rodent-adapted L. reuteri lineage II, as well as mutants with deletions in gadB or gupCDE. The competitivenesses of the strains were quantified by inoculation of wheat and sorghum sourdoughs with defined strains, followed by propagation of doughs with a 10% inoculum and 12-h or 72-h fermentation cycles. Lineage I L. reuteri strains dominated sourdoughs propagated with 12-h fermentation cycles; lineage II L. reuteri strains dominated sourdoughs propagated with 72-h fermentation cycles. L. reuteri 100-23ΔgadB was outcompeted by its wild-type strain in sourdoughs fermented with 72-h fermentation cycles; L. reuteri FUA3400ΔgupCDE was outcompeted by its wild-type strain in sourdoughs fermented with both 12-h and 72-h fermentation cycles. Competition experiments with isogenic pairs of strains resulted in a constant rate of strain displacement of the less competitive mutant strain. In conclusion, lineage-specific traits of L. reuteri determine the competitiveness of this species in sourdough fermentations.

An anaerobic sequential batch reactor for enhanced continuous hydrogen production from fungal pretreated cornstalk hydrolysate

Anaerobic sequencing batch reactor (ASBR) process offers great potential for H2 production from wastewaters. In this study, an ASBR was used at first time for enhanced continuous H2 production from fungal pretreated cornstalk hydrolysate by Thermoanaerobacterium thermosaccharolyticum W16. The reactor was operated at different hydraulic retention times (HRTs) of 6, 12, 18, and 24 h by keeping the influent hydrolysate constant at 65 mmol sugars L−1. Results showed that increasing the HRT from 6 to 12 h led to the H2 production rate increased from 6.7 to the maximum of 9.6 mmol H2 L−1 h−1 and the substrate conversion reached 90.3%, although the H2 yield remained at the same level of 1.7 mol H2 mol−1 substrate. Taking into account both H2 production and substrate utilization efficiencies, the optimum HRT for continuous H2 production via an ASBR was determined at 12 h. Compared with other continuous H2 production processes, ASBR yield higher H2 production at relatively lower HRT. ASBR is shown to be another promising process for continuous fermentative H2 production from lignocellulosic biomass.