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Molasses are a source of fermentable sugars, the substrate utilized for this study, as it is a subproduct from sugarcane processing for sucrose recovery. These sugars can be metabolized for bioH 2 production by bacterial strains, particularly strict-anaerobe Clostridium acetobutylicum (Clac) and its media preparation requires chemical of O 2 removal and N 2 flushing to promote anaerobiosis. Bacillus subtilis (Bsub) can deplete O 2 from culture media and promotes anaerobiosis, which is an aspect that would benefit from further research. Clac was grown in an anaerobic medium and produced 269 mL of hydrogen at 72 h. By co-culturing Clac and Bsub , 502 mL of bioH 2 were produced in a cysteine/N 2 flushed medium, while 203 mL of bioH 2 at 72 h were produced by aerobic co-culture. Additionally, in an untreated/aerobic conventional growth culture for Clac , no production of H 2 was detected, reinforcing the utility of Bsub for anaerobiosis generation. • Clostridium acetobutylicum consumes substrate approximately 6x faster than Bacillus subtilis. • Clostridium acetobutylicum produced 202 mL of bioH 2 in a culture with anaerobiosis assisted by Bacillus subtilis. • Bacillus subtilis CBB5555 can act as a biological O 2 depleter for anaerobic bioprocesses. [ABSTRACT FROM AUTHOR]

Abstract Low yield and inhibition of hydrogenase by oxygen are the main limitations for hydrogen production by microalgae. Considering the role of electron flow in the metabolism for hydrogen production, a genome-scale metabolic model (named i MM627) was reconstructed for Auxenochlorella protothecoides. i MM627 was evaluated using experimental data for growth and flux distribution. Then, considering the well-known degeneracy of FBA solutions, a new method of finding effective reactions based on multiple optimal solutions was developed. At a constant growth rate, flux distributions for maximal and minimal hydrogen production under anaerobiosis and for maximal oxygen production were compared to identify target reactions for improvement of hydrogen production and for providing anaerobiosis. Existing researches on some reactions truly confirm the predicted changes by i MM627. The main proposed strategies for improvement of hydrogen production include changes in metabolism to provide NADH. Consumption of oxygen by mitochondrial respiration and energy dissipation was proposed to provide anaerobiosis. Graphical abstract Image 1 Highlights • The first genome-scale metabolic model was reconstructed for A. protothecoides. • The model was employed as a framework to study hydrogen and oxygen production. • A new method of finding effective reactions was developed by optimal solutions. • The method was applied to find strategies for improvement of hydrogen production. [ABSTRACT FROM AUTHOR]

Previously, we found that marine Chlorella pyrenoidosa strain IOAC707S could generate hydrogen (H 2 ) under nitrogen deprivation in seawater medium, without dark incubation, N 2 flushing or addition of protonophore. In this work, the H 2 yield of C. pyrenoidosa was enhanced, and the mechanism of H 2 photoproduction was investigated. It was found that C. pyrenoidosa could produce H 2 under hypoxic conditions, which indicated the hydrogenase of C.pyrenoidosa could tolerate a low concentration of O 2 . Nitrogen deprivation could lower the efficiency of photosyntem (PS) II photochemical activity and PSII oxygenic activities, which were favorable for rapid establishment of anoxia and efficient H 2 photoproduction, and thus significantly enhance H 2 photoproduction of C. pyrenoidosa . Pre-culture in nitrogen-deprived medium could further increase the H 2 yield of C. pyrenoidosa , for its more severe inhibition of the oxygen-evolving complex, electron transport and photochemical efficiency of PSII, and PSII oxygenic activities, which facilitated the establishment of anaerobiosis and activation of hydrogenase. [ABSTRACT FROM AUTHOR]

Abstract: The aim of this study was to understand the enhancement mechanism of H2 photoproduction in Chlorella protothecoides under simultaneous nitrogen limitation and sulfur deprivation (LNS). Nitrogen limitation (LN) rather than sulfur deprivation significantly inhibited relative variable fluorescence at K-step (W K) and J-step (V J), photochemical efficiency of PSII (photosystem II), F v/F m, during the process of incubation in the light. Under such conditions, photosynthetic O2 evolution decreased and the anaerobiosis was established after 12 h of incubation. The algae generated large amounts of H2 under nitrogen limitation but generated only trace amounts under sulfur deprivation. Obviously, nitrogen limitation rather than sulfur deprivation was the decisive factor that induced H2 photoproduction in C. protothecoides under LNS. The LNS culture generated much more H2 than the LN culture in the presence of DCMU during incubation, suggesting that a PSII-independent electron source contributed many more electrons for transfer to hydrogenase in the LNS culture. PSII electron transport includes linear electron flow (LEF) and cyclic electron flow (CEF) of PSII in C. protothecoides. In the PSII-dependent electron source for H2 photoproduction, PSII supplies electrons to hydrogenase through the LEF. The LNS culture showed much higher LEF and lower CEF than the LN culture during the H2 photoproduction phase, as indicated by the large lower quantum yield of PSII electron transport (Φ PSII) in the LNS culture in the presence of DCMU. Therefore, compared with nitrogen limitation, simultaneous nitrogen limitation and sulfur deprivation enhanced H2 photoproduction in C. protothecoides mainly due to enhanced PSII-dependent and -independent electron sources. [Copyright &y& Elsevier]

Abstract: The green alga Chlamydomonas reinhardtii is capable of photoproducing molecular hydrogen following sulphur deprivation, which results in anaerobiosis and a suppression of oxygen evolution and thus an alleviation of the inhibitory effect of oxygen on the hydrogenase. At the same time it transiently maintains a limited supply of electrons arising from photosystem II (PSII) to the hydrogenase (Melis and Happe Plant Physiol 2001; 127:740–748). In this work, using fast chl a fluorescence and P700 measurements, we show that ascorbate (Asc), a naturally occurring PSII alternative electron donor, is capable of donating electrons to PSII in heat-treated and sulphur-deprived cells and this can be significantly accelerated by supplementing the culture with 10 mM Asc. It also enhances, about three-fold, the photoproduction of hydrogen in cells subjected to sulphur deprivation as shown by gas chromatography. Similar stimulation was obtained in the presence of diphenylcarbazide (DPC), an artificial PSII electron donor. Asc and DPC also facilitated the anaerobiosis of cells, probably via super reducing the oxygen evolving complex while feeding electrons to PSII reaction centres and the linear electron transport chain, and ultimately to the hydrogenase – as shown by the significant DCMU-sensitivity of the light-induced Asc- and DPC-dependent re-reduction of P700+ and hydrogen evolution. [Copyright &y& Elsevier]

Abstract: Microbes and enzymes deliver electrons from carbon sources under anaerobic condition. Electrons are generated as the microorganisms are actively catabolized organic substances. The liberated electrons travel to anode surface. Saccharomyces cerevisiae (PTCC 5269) was implemented as biocatalyst in the anaerobic anode compartment. Glucose was used as carbon source. Also mediator as electron promoter was incorporated in the anode. Among several mediators, methylene blue (MB) as electron promoter with concentration of 50, 100, 200, 300 and 400 μM was selected to shuttle the liberated electron to anode surface. Maximum power and current with MB concentration of 300 μM were obtained. Resistances were applied to control the electron flow from anode to cathode chambers. Data were recorded through an online data-logger. Polarization curves with and without mediator were analyzed in the fabricated cell. MB had good ability to enhance power generation. Maximum open circuit voltage of 250 mV was achieved; the voltage was stabled for the duration of 36 h. [Copyright &y& Elsevier]

Abstract: The key process parameters of solid state enzymolysis for the generation of soluble sugar (SS) and bio-hydrogen production from corn stalk were optimized by the response surface methodology (RSM) based on a three factor-five level central composite design (CCD), respectively. The result showed that the optimal solid state enzymolysis condition from corn stalk was 47.7 °C, SCED of 0.054 g/g and 10.3 days for the maximum SS yield of 526 mg/g-TVS. Correspondingly, the optimal enzymolysis conditions from corn stalk appeared at 46.3 °C, SCED of 0.049 g/g and 7.5 days for the maximum hydrogen yield of 205.5 mL/g-TVS from the hydrolyzed substrate by the next dark fermentation. In addition, the bio-hydrogen production mechanism from corn stalk was preliminary investigated by XRD and SEM analyses. The results suggested that the solid state enzymolysis of substrate played a vital role in the effective conversion of corn stalk into bio-hydrogen by dark fermentation. [Copyright &y& Elsevier]

Abstract: Nature and survivability of microalgal population during growth and photofermentative hydrogen (H2) production was evaluated using acid-rich effluents generated from dark-fermentation. Microalgae enriched in presence of glucose showed the dominance of H2 producing Chlorella sp. (Consortia-A), while consortia under starvation had the dominance of Scenedesmus sp. and Diatoms (Consortia-B) with negligible H2 production. Further experiments with Consortia-A using acid-rich effluents of designed synthetic wastewater (DSW) and food waste (FW) showed higher H2 production in the former than the latter in both dark (DSW, 1.21 mmol; FW, 0.16 mmol) and photo (DSW, 5.22 mmol; FW, 1.24 mmol) fermentation. High acetate concentrations in DSW effluents favored H2 production over FW effluents. Substrate degradation was also higher in case of DSW than the FW sets. Microalgal population that dominanted in Consortia-B appears to have opted the lipid storing metabolic pathway evidencing 2% lipid that can be converted to biodiesel. The data represent a novel approach to exploiting microalgae for biohydrogen production at the expense of cheaper raw material like dark-fermentation effluents. [Copyright &y& Elsevier]

Abstract: In this paper, mitochondrial mutants of Chlamydomonas reinhardtii defective for respiratory complex I (NADH:ubiquinone oxidoreductase), complex III (ubiquinol cytochrome c oxidoreductase) and both complexes I and III were analyzed for H2 photoproduction. Several parameters were followed during the S-deficiency stage and the anaerobic stage leading to H2 photoproduction. At the early aerobic S-deficiency stage, starch and neutral lipids accumulated in all strains but their amount was significantly decreased in mutants compared to wild type. During the H2 photoproduction process, whereas starch content strongly decreased in all strains, neutral lipid amount remained nearly unchanged, suggesting that starch degraded by glycolysis is the preferential substrate for energy production during anaerobiosis. The mutants displayed a decrease in H2 photoproduction correlating to the number of active mitochondrial proton-pumping sites lost in the strains. Our results thus highlight the critical role of oxidative phosphorylation during the first (aerobic) stage of S-starvation when carbon resources are accumulated. [Copyright &y& Elsevier]

Abstract: Sulfur deprivation, which is considered as an efficient way to trigger long-term hydrogen photoproduction in unicellular green algae has two major effects: a decrease in PSII which allows anaerobiosis to be reached and carbohydrate (starch) storage. Starch metabolism has been proposed as one of the major factors of hydrogen production, particularly during the PSII-independent (or indirect) pathway. While starch biosynthesis has been characterized in the green alga Chlamydomonas reinhardtii, little remains known concerning starch degradation. In order to gain a better understanding of starch catabolism pathways and identify those steps likely to limit the starch-dependent hydrogen production, we have designed a genetic screening procedure aimed at isolating mutants of the green alga C. reinhardtii affected in starch mobilization. Using two different screening protocols, the first one based on aerobic starch degradation in the dark and the second one on anaerobic starch degradation in the light, eighteen mutants were isolated among a library of 15,000 insertion mutants, eight (std1-8) with the first screen and ten (sda1-10) with the second. Most of the mutant strains isolated in this study showed a reduction or a delay in the PSII-independent hydrogen production. Further characterization of these mutants should allow the identification of molecular determinants of starch-dependent hydrogen production and supply targets for future biotechnological improvements. [ABSTRACT FROM AUTHOR]

Abstract: The optimization of total solids in the feed (%TS) and alkalinity ratio (γ) for H2 production from organic solid wastes under thermophilic regime was carried out using response surface methodology based on a central composite design. The total solids levels were 20.9, 23.0, 28.0, 33.0 and 35.1% whereas the levels of alkalinity ratio (defined as g phosphate alkalinity/g dry substrate) were 0.15, 0.20, 0.30, 0.41 and 0.45. High levels of TS and γ affected in a negative way the H2 productivity and yield; both response variables significantly increased upon decreasing the TS content and alkalinity ratio. The highest H2 productivity and yield were 463.7NmL/kg-d and 54.8NmL/gVSrem, respectively, predicted at 20.9% TS and alkalinity ratio 0.25 (0.11gCaCO3/g dry substrate). The alkalinity requirements for hydrogenogenic processes were lower than those reported for methanogenic processes (0.11 vs. 0.30gCaCO3/gCOD). Adequate alkalinity ratio was necessary to maintain optimal biological activity for hydrogen production; however, excessive alkalinity negatively affected process performance probably due to an increase of osmotic pressure. Interestingly, reactor pH depended only on the alkalinity ratio, thus the buffer capacity was able to maintain a constant pH independently of TS levels. At γ =0.15–0.30 the pHs were in the range 5.56–5.95, which corresponded to the highest hydrogen productivities and yields. Finally, the highest metabolite accumulation corresponded with the highest removal efficiencies but not with high H2 productivities and yields. Therefore, it seems that organic matter removal was channeled toward solvent generation instead of hydrogen production at high TS and γ levels. This is the first study that shows the requirements of alkalinity in solid substrate fermentation conditions for H2 production processes and their interaction with the content of total solids in the feed. [Copyright &y& Elsevier]

Abstract: Microbial fermentation is a potentially important member of a diverse portfolio of methods available for renewable production of hydrogen using biomass as a substrate. A major factor currently limiting commercial viability of fermentative hydrogen production is poor stoichiometric yield per mole of sugar and end-product feedback inhibition. Although several approaches have been tried to enhance microbial hydrogen productivity, genetic engineering technologies for most promising microorganisms are either unavailable or severely limited. Further efforts to overcome existing issues will need to rely on the ability to analyze, predict and engineer microbial metabolism in native H2-producing strains as well as genetically engineerable strains with constructed H2-metabolism. A shift in the type of biomass substrate from starch-based food crops to ligno-cellulosic crops and wastes that are economically and environmentally less costly to produce, yet more difficult to chemically and biologically process, presents technical challenges that are shared by all emerging biofuel technologies. A brief overview of the current status of fermentative H2-production, including a discussion of directly and indirectly related emerging technologies, is presented. [Copyright &y& Elsevier]