Fermentative H2 Research Articles

Evaluation of a novel pretreatment of NaOH/Urea at outdoor cold-winter conditions for enhanced enzymatic conversion and hythane production from rice straw

A novel pretreatment using NaOH/Urea (NU) solution at outdoor cold-winter conditions was developed to enhance the enzymatic saccharification and hythane production from rice straw (RS). Results revealed that the reducing sugar conversion of RS reached 90.02% after NU pretreatment at outdoor freezing temperature. Chemical composition analysis showed that the lignin removal was up to 62.74% with cellulose and hemicellulose loss of 0.56% and 18.87% after 3%–6% NU pretreatment at 100% solid loading for 3 months. Scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR) and X-ray photoelectron spectroscopy (XPS) analysis confirmed that the surface of pretreated RS exposed more cellulose and hemicellulose due to the disruption of resistant structure of lignocellulose. Subsequently, the enzymatic hydrolysate of pretreated RS was used as substrate to produce hythane by two-stage fermentation with the yield of 225.1 mL H2/g sugar and 112.8 mL CH4/g sugar. The energy conversion efficiency of hythane fermentation attained 10.4%, which was 22.8% and 190.5% higher than that for single H2 and CH4 fermentation. These results demonstrated that NU pretreatment at outdoor cold-winter conditions was practically and feasible way for improved hythane recovery from lignocellulosic biomass.

Valorization of apple pomace using bio-based technology for the production of xylitol and 2G ethanol.

Apple pomace was studied as a raw material for the production of xylitol and 2G ethanol, since this agroindustrial residue has a high concentration of carbohydrate macromolecules, but is still poorly studied for the production of fermentation bioproducts, such as polyols. The dry biomass was subjected to dilute-acid hydrolysis with H2SO4 to obtain the hemicellulosic hydrolysate, which was concentrated, detoxified and fermented. The hydrolyzate after characterization was submitted to submerged fermentations, which were carried out in Erlenmeyer flasks using, separately, the yeasts Candida guilliermondii and Kluyveromyces marxianus. High cellulose (32.62%) and hemicellulose (23.60%) contents were found in this biomass, and the chemical hydrolysis yielded appreciable quantities of fermentable sugars, especially xylose. Both yeasts were able to metabolize xylose, but Candida guilliermondii produced only xylitol (9.35gL-1 in 96h), while K. marxianus produced ethanol as the main product (10.47gL-1 in 24h) and xylitol as byproduct (9.10gL-1 xylitol in 96h). Maximum activities of xylose reductase and xylitol dehydrogenase were verified after 24h of fermentation with C. guilliermondii (0.23 and 0.53U/mgprot, respectively) and with K. marxianus (0.08 e 0.08U/mgprot, respectively). Apple pomace has shown potential as a raw material for the fermentation process, and the development of a biotechnological platform for the integrated use of both the hemicellulosic and cellulosic fraction could add value to this residue and the apple production chain.

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.

Dynamics of changes in organic acids, sugars and phenolic compounds and antioxidant activity of sea buckthorn and sea buckthorn-apple juices during malolactic fermentation.

Sea buckthorn (Hippophaë rhamnoides L.) berries have high biological value as a rich source of phenolic compounds, fatty acids and vitamins A, C, E. Due to the high organic acid content and sour taste, the fruits are rarely used in juice production. Therefore, the study aimed to determine the metabolic activity of Lactobacillus plantarum, Lactobacillus plantarum subsp. argentoratensis and Oenococcus oeni strains along with the dynamics of changes in organic acids, sugars, phenolic compounds, and antioxidant activity during 72-h fermentation of 100% sea buckthorn and mixed with apple (1:1) juices. The strongest malolactic conversion was in mixed juices (to 75.0%). The most efficient strains were L. plantarum DSM 10492, 20174 and 6872. L. plantarum strains caused an increase in flavonols and antioxidant activity of sea buckthorn-apple juices. The results can be used to select conditions and strains in industrial-scale fermentation, to produce novel sea buckthorn products and increase their consumption.

Impacts of rumen fluid, refrigerated or reconstituted from a refrigerated pellet, on gas production measured at 24h of fermentation

Rumen fluid is used as fresh inoculum for gas production fermentations to predict the nutritional value of feeds and rations for ruminants. However, collection of rumen fluid from animal donors is invasive, expensive, time consuming and results in fluids of variable quality. The general aim was to identify a procedure to manipulate rumen inoculum in order to facilitate its storage and transfer between laboratories. This strategy would also limit fluid collections from animals. Two experiments were completed based on gas production from graduated 100 mL glass syringe with five feeds as substrates.In experiment 1, the gas production and some fermentation parameters of fresh rumen fluids were compared with those preserved at 4 °C for 24, 48, 72 and 96 h. Refrigeration did not modify concentration of volatile fatty acids and pH, but ammonia in liquids refrigerated for 48–96 h was higher (P < 0.05) compared to fresh. In contrast, rumen fluid refrigeration for 24, 48 or 72 h did not depress gas production at 24 h, but it was lower at 96 h. In experiment 2, the rumen fluid was centrifugated at 13,000 x g and sedimented material (i.e., pellet) was refrigerated for 48 h at 4 °C. The asymptote of gas production kinetics from rumen fluid regenerated from the pellet was 8 % lower (P < 0.05) than that from fresh. However for 24 h gas production, the correlation between fresh liquid and pellet inoculum, calculated for five ingredients, was high (R2 = 0.94). Results support the use of rumen fluid preserved by refrigeration for up to 72 h, and rumen fluid reconstituted from refrigerated pellet, as an alternative to fresh. This would reduce the need for laboratories to maintain animal donors and/or frequently collect rumen fluid.

Metabolic engineering of Corynebacterium glutamicum WM001 to improve l-isoleucine production.

In this study, l-isoleucine production in Corynebacterium glutamicum WM001 was improved by deleting three genes in the genome, replacing the native promoter of ilvA in the genome, and overexpression of five genes in an alr-based auxotrophic complementation expression system. The three genes deleted in the genome are alaT, brnQ, and alr. Deletion of alaT improved l-isoleucine production by increasing the supply of pyruvate, whereas deletion of brnQ improved l-isoleucine production by blocking the uptake of extracellular l-isoleucine. Exchange of the native promoter of ilvA with promoter tac or tacM could contribute to l-isoleucine production by increasing 2-ketobutyric acid; tac is better than tacM for improving l-isoleucine yield. Different combinations of the genes ilvBN, ppnK, lrp, and brnFE were overexpressed in an alr-based auxotrophic complementation expression system to further improve l-isoleucine production, and the best yield after 72-H flask fermentation was obtained from the strain WM005/pYCW-1-ilvBN2-ppnK1. Without addition of any antibiotics, WM005/pYCW-1-ilvBN2-ppnK1 could produce 32.1g/L l-isoleucine after 72-H fed-batch fermentation, which is 34.3% increase compared with the original strain WM001.

Utilisation of raw palm oil mill effluent (POME) as a renewable substrate for fermentative H2 production: Optimisation using statistical approach

Fermentative H 2 production was studied using raw POME as the substrate with heat-shock pre-treated POME anaerobic sludge acted as the inoculum. The effect of crucial operating variables (initial pH of medium, incubation temperature, and inoculum size) on H 2 production was studied using Box−Behnken Design. A second-order polynomial regression model was generated to evaluate H 2 production trend under conditions tested. The model analysis revealed the high significance of linear effects of initial pH, incubation temperature, and inoculum size ( P < 0.01) towards H 2 production. Similar results indicated that the interaction effect between initial pH and inoculum size, and interaction effect between incubation temperature and inoculum size were highly significant ( P < 0.01). The regression model suggested that the optimum conditions were set to a pH value, incubation temperature and inoculum size of 6.4, 58.0°C and 8.0% v/v, respectively. In order to validate the optimum conditions determined by the model, heat-shock pre-treated POME anaerobic sludge was incubated with raw POME under optimum conditions. Validation experiment showed that a cumulative H 2 volume of 239.0 mL was produced. Microbial community analysis of inoculum showed that mixed consortia between Clostridium sp. and other obligate anaerobic non-spore forming bacteria, mostly belonging to the Firmicutes and Bacteroidetes phyla were identified as the major H 2 -producers and were hugely responsible towards fermentative H 2 production.

Enhancing thermophilic dark fermentative hydrogen production at high glucose concentrations via bioaugmentation with Thermotoga neapolitana

The aim of the present study was to investigate the effect of gradually increasing glucose concentrations (from 5.6 to 111 mmol L−1) on the fermentative H2 production with and without bioaugmentation. A stirred tank reactor (STR) was operated at 70 °C and inoculated with a hyperthermophilic mixed culture or a hyperthermophilic mixed culture bioaugmented with Thermotoga neapolitana. With both the unaugmented (control) and augmented cultures, the H2 production rate was improved when the initial glucose concentration was increased. In contrast, the highest H2 yield (1.68 mol H2 mol−1 glucose consumed) was obtained with the augmented culture at the lowest glucose concentration of 5.6 mmol L−1 and was 37.5% higher than that obtained with the unaugmented culture at the same feed glucose concentration. Overall, H2 production rates and yields were higher in the bioaugmented cultures than in the unaugmented cultures whatever the glucose concentration. Quantitative polymerase chain reaction targeting T. neapolitana hydA gene and MiSeq sequencing proved that Thermotoga was not only present in the augmented cultures but also the most abundant at the highest glucose concentrations.

Effects of high-level expression of A1-ATPase on H2 production in Thermococcus kodakarensis

The hyperthermophilic archaeon Thermococcus kodakarensis can grow on pyruvate or maltooligosaccharides through H2 fermentation. H2 production levels of members of the Thermococcales are high, and studies to improve their production potential have been reported. Although H2 production is primary metabolism, here we aimed to partially uncouple cell growth and H2 production of T.kodakarensis. Additional A1-type ATPase genes were introduced into T.kodakarensis KU216 under the control of two promoters; the strong constitutive cell surface glycoprotein promoter, Pcsg, and the sugar-inducible fructose-1,6-bisphosphate aldolase promoter, Pfba. Whereas cells with the A1-type ATPase genes under the control of Pcsg displayed only trace levels of growth, cells with Pfba (strain KUA-PF) displayed growth sufficient for further analysis. Increased levels of A1-type ATPase protein were detected in KUA-PF cells grown on pyruvate or maltodextrin, when compared to the levels in the host strain KU216. The growth and H2 production levels of strain KUA-PF with pyruvate or maltodextrin as a carbon and electron source were analyzed and compared to those of the host strain KU216. Compared to a small decrease in total H2 production, significantly larger decreases in cell growth were observed, resulting in an increase in cell-specific H2 production. Quantification of the substrate also revealed that ATPase overexpression led to increased cell-specific pyruvate and maltodextrin consumptions. The results clearly indicate that ATPase production results in partial uncoupling of cell growth and H2 production in T.kodakarensis.

Enhancing CO2-Valorization Using Clostridium autoethanogenum for Sustainable Fuel and Chemicals Production.

Acetogenic bacteria can convert waste gases into fuels and chemicals. Design of bioprocesses for waste carbon valorization requires quantification of steady-state carbon flows. Here, steady-state quantification of autotrophic chemostats containing Clostridium autoethanogenum grown on CO2 and H2 revealed that captured carbon (460 ± 80 mmol/gDCW/day) had a significant distribution to ethanol (54 ± 3 C-mol% with a 2.4 ± 0.3 g/L titer). We were impressed with this initial result, but also observed limitations to biomass concentration and growth rate. Metabolic modeling predicted culture performance and indicated significant metabolic adjustments when compared to fermentation with CO as the carbon source. Moreover, modeling highlighted flux to pyruvate, and subsequently reduced ferredoxin, as a target for improving CO2 and H2 fermentation. Supplementation with a small amount of CO enabled co-utilization with CO2, and enhanced CO2 fermentation performance significantly, while maintaining an industrially relevant product profile. Additionally, the highest specific flux through the Wood-Ljungdahl pathway was observed during co-utilization of CO2 and CO. Furthermore, the addition of CO led to superior CO2-valorizing characteristics (9.7 ± 0.4 g/L ethanol with a 66 ± 2 C-mol% distribution, and 540 ± 20 mmol CO2/gDCW/day). Similar industrial processes are commercial or currently being scaled up, indicating CO-supplemented CO2 and H2 fermentation has high potential for sustainable fuel and chemical production. This work also provides a reference dataset to advance our understanding of CO2 gas fermentation, which can contribute to mitigating climate change.

Improve biotransformation of crude glycerol to ethanol of Enterobacter aerogenes by two-stage redox potential fed-batch process under microaerobic environment

Ethanol production from crude glycerol under microaerobic condition was optimized. The aeration rate in the reactor was controlled using redox potential (ORP) as to replace a conventional oxygen sensor that became insensitive at low oxygen concentration. The performance of Enterobacter aerogenes TISTR1468 in a batch reactor using a simple medium (crude glycerol and tuna condensate (GT) medium) was evaluated on various aeration strategies: continuous aeration at 0.5 vvm; controlled aeration at −350 mV and −400 mV ORP; and no aeration. The 72-h batch fermentation showed that a more reductive environment (lower aeration and ORP value) increased ethanol concentration and yield, but decreased the ethanol productivity. No-aeration fermentation with the lowest ORP value achieved the highest ethanol production (18.78 g L−1) and yield (0.94 g-ethanol.g-glycerol−1). A more oxidative environment (higher aeration and ORP value) fasten the specific growth rate (μ), but ethanol concentration and yield decreased. The optimum specific ethanol production rate (qp) and glycerol consumption rate occurred at ORP of −400 mV and −350 mV, respectively. When the ORP was very low, the μ decreased following substrate depletion, but this phenomenon was not observed at higher ORP value, an evident that adequate oxygen supply could maintain cell viability.Fed-batch with two-stage aeration strategy (−350 mV and −400 mV ORP) acquired higher ethanol production (30.31 g L−1) than fed-batch with single-stage aeration (25.95 g L−1) and batch process (12.33 g L−1).This study has revived the potential of crude glycerol biotransformation to ethanol and opened up opportunities for optimization of related microaerobic systems other than ethanol.

Development of probiotic vegetable juice using Lactobacillus Rhamnosus GR-1

PurposeThe purpose of this paper is to assess the growth and viability of Lactobacillus rhamnosus GR-1 (L. rhamnosus GR-1) in carrot juice (CJ), carrot apple juice (CAJ), carrot orange juice (COJ) and carrot beet juice (CBJ) over 72 h of fermentation and 30 days of refrigerated storage at 4°C. The secondary objective is to evaluate sensory properties.Design/methodology/approachFour vegetable juice samples were inoculated with the probiotic strain L. rhamnosus GR-1 and fermented for 72 h. To observe the samples’ storage ability, the samples were refrigerated for 30 days. Microbial enumeration was conducted throughout the fermentation and storage periods to determine the viability of L. rhamnosus GR-1. Sensory evaluation with 106 participants was also conducted to assess the consumer acceptability of the vegetable juices.FindingsAll tested samples achieved mean microbial counts of at least 109 CFU/ml. During the 72-h fermentation period significant differences in microbial counts in juices CJ (p = 0.001), CAJ (p = 0.031), COJ (p = 0.047) and CBJ (p = 0.001) were observed. Over the 30-day storage period, significant differences in microbial counts were only found in juices CJ (p = 0.001) and COJ (p = 0.019). A significant decline in pH (p = 0.001) was also observed during 72 h of fermentation and 30-days of cold storage. Sensory evaluation of all juices showed significant differences in sensory attributes such as appearance (p = 0.001), flavour (p = 0.001), texture (p = 0.001) and overall acceptability (p = 0.001). Sensory results showed that the probiotic CBJ and CJ had the highest hedonic scores for flavour, texture and overall acceptability (p = 0.001) among participants. This study demonstrated that non-dairy vegetable juices could be an alternative to dairy-based probiotic products.Originality/valueCommercially available probiotic dairy-based foods make up a large sector of the consumer market. However, the growing consumer interest in healthful eating has led to an increased demand for plant-based products. The probiotic L. rhamnosus GR-1 provides numerous therapeutic benefits, such as reducing the recurrence of bacterial vaginosis, yeast and urinary tract infections. The results of this study may have a significant influence on the health of individuals, especially in less economically developed countries.

Metabolic activity analyses demonstrate that Lokiarchaeon exhibits homoacetogenesis in sulfidic marine sediments.

The genomes of the Asgard superphylum of Archaea hold clues pertaining to the nature of the host cell that acquired the mitochondrion at the origin of eukaryotes1-4. Representatives of the Asgard candidate phylum Candidatus Lokiarchaeota (Lokiarchaeon) have the capacity for acetogenesis and fermentation5-7, but how their metabolic activity responds to environmental conditions is poorly understood. Here, we show that in anoxic Namibian shelf sediments, Lokiarchaeon gene expression levels are higher than those of bacterial phyla and increase with depth below the seafloor. Lokiarchaeon gene expression was significantly different across a hypoxic-sulfidic redox gradient, whereby genes involved in growth, fermentation and H2-dependent carbon fixation had the highest expression under the most reducing (sulfidic) conditions. Quantitative stable isotope probing revealed that anaerobic utilization of CO2 and diatomaceous extracellular polymeric substances by Lokiarchaeon was higher than the bacterial average, consistent with higher expression of Lokiarchaeon genes, including those involved in transport and fermentation of sugars and amino acids. The quantitative stable isotope probing and gene expression data demonstrate homoacetogenic activity of Candidatus Lokiarchaeota, whereby fermentative H2 production from organic substrates is coupled with the Wood-Ljungdahl carbon fixation pathway8. The high energetic efficiency provided by homoacetogenesis8 helps to explain the elevated metabolic activity of Lokiarchaeon in this anoxic, energy-limited setting.

Impact of the microbial inoculum source on pre-treatment efficiency for fermentative H2 production from glycerol

Hydrogen (H2) production by dark fermentation can be performed from a wide variety of microbial inoculum sources, which are generally pre-treated to eliminate the activity of H2-consuming species and/or enrich the microbial community with H2-producing bacteria. This paper aims to study the impact of the microbial inoculum source on pre-treatment behavior, with a special focus on microbial community changes. Two inocula (aerobic and anaerobic sludge) and two pre-treatments (aeration and heat shock) were investigated using glycerol as substrate during a continuous operation. Our results show that the inoculum source significantly affected the pre-treatment efficiency. In aerobic sludge no pre-treatment is necessary, while in anaerobic sludge the heat pre-treatment increased H2 production but aeration caused unstable H2 production. In addition, biokinetic control was key in Clostridium selection as dominant species in all microbial communities. Lower and unstable H2 production were associated with a higher relative abundance of Enterobacteriaceae family members. Our results allow a better understanding of H2 production in continuous systems and how the microbial community is affected. This provides key information for efficient selection of operating conditions for future applications.

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.

A comparison of methods of ethanol production from sweet sorghum bagasse

Ethanol production from sweet sorghum bagasse (SSB) is reported. A two-step pretreatment with hydrogen peroxide followed by NaOH at 121 °C for 20 min, was used prior to enzymatic treatment. Enzymatic treatment was carried out using a commercial cellulase. Enzymatic hydrolysis for 72 h resulted in a hydrolysate containing xylose (5.1 g L−1), glucose (75.3 g L−1) and cellobiose (1.4 g L−1). This hydrolysate was used to produce ethanol by a 72-h fermentation with the yeast Saccharomyces cerevisiae TISTR 5606 at 30 °C. This separate hydrolysis and fermentation (SHF) method was compared with a simultaneous saccharification and fermentation (SSF) method. The SSF process used the pretreated SSB and was carried out in two different ways: (1) In the first method, the enzyme, the substrate and the yeast inoculum were mixed and SSF fermentation took place at 37 °C for 72-h, to produce a final ethanol concentration of 22.3 g L−1; (2) in the second method, termed the ‘delayed simultaneous-saccharification fermentation’ or DSSF, the pretreated SSB was enzymatically hydrolyzed at 50 °C for 6-h, cooled to 37 °C, and then inoculated with the yeast to commence a 66-h fermentation. This gave a final ethanol concentration of 28.3 g L−1. The ethanol productivity was 0.31 g L−1 h−1 for the SHF and SSF processes, but the DSSF had a ∼26% higher productivity.

Effects of different pre-treatment methods on anaerobic mixed microflora for hydrogen production and COD reduction from domestic effluent

Effects of different pretreatment methods on sludge inoculum were evaluated concerning hydrogen (H2) production enhancement and COD (chemical oxygen demand) reduction, using domestic effluent in a batch system. The sludge was taken from a recycled line of the activated sludge reactor. Two types of pretreatment were investigated, heat treatment and chloroform treatment. The experiment was conducted at pH 4-6 and inoculum sizes of microbes were 10%, 20%, and 30% respectively; and experiment without sludge pretreatment was also conducted as control. The result showed that 30% COD reduction was achieved for chloroform pretreatment at pH 3 and 10% inoculum size. For heat treatment, a maximum COD removal of 60% was achieved in the experiment at pH 6 and 10% inoculum size. In chloroform pretreatment, a maximum volume of gas evolved was 3.6 mL, at pH 3 and 20% inoculum size. For heat pretreatment, maximum biogas evolved was 2.1 mL, at pH 3 and 10% inoculum size. The experimental results showed that the pretreatment methods (heat treatment and chloroform treatment) at 35 °C and initial pH 5.5 had a positive influence on H2 production yield and COD removal efficiency during the fermentative H2 production as compared to the control experiments (without pretreatment). Heat treatment method was shown to be a simple and useful method for enhancing both H2 producing and COD removal processes from domestic effluent with highest H2 yield and COD removal efficiency at 0.314 mmol H2/g COD and 86%, respectively.

Effect of storage time and temperature on hydrogen fermentation of food waste

Practically, before being fed to the treatment plant, food waste (FW) is stored for up to a week in a storage tank under ambient temperature condition, which would have an impact on the bioenergy yield. In the present work, FW was stored at different temperatures (5 °C, 20 °C, and 35 °C) for 0 d, 1 d, and 2 d, and it was used as a feedstock for mesophilic H2 fermentation. H2 production curves were divided by three groups, finally attaining 1.7–1.8 mol H2/mol hexoseadded, 1.4–1.5 mol H2/mol hexoseadded, and 1.2 mol H2/mol hexoseadded, achieved from the (fresh, FW stored at 5 °C), (FW stored at 20 °C, and 35 °C for 1 d), and (FW stored at 35 °C for 2 d), respectively. The different performance was attributed to the growth of indigenous lactic acid bacteria such as Lactobacillus and Weissella during storage under high temperature condition. In addition, it was found that the activity of homoacetogenic reaction (R17, 4H2 + CO2 → Acetate) calculated by establishing metabolic flux balance was different depending on the H2 production performance. The flux of R17 ranged 0.03–0.06 under low H2 yield achieved conditions, while it increased to 0.10–0.17 those showing low H2 yields.

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.