Maximizing the hydrogen photoproduction yields in Chlamydomonas reinhardtii cultures: The effect of the H2 partial pressure

Photoproduction of H₂ gas has been examined in sulfur/phosphorus-deprived Chalmydomonas reinhardtii cultures, placed in photobioreactors (PhBRs) with different gas phase to liquid phase ratios (V_g.p./V_l.p.). The results demonstrate that an increase in the ratio stimulates H₂ photoproduction activity in both algal suspension cultures and in algae entrapped in thin alginate films. In suspension cultures, a 4× increase (from ∼0.5 to ∼2) in V_g.p./V_l.p results in a 2× increase (from 10.8 to 23.1 mmol l⁻¹ or 264–565 ml l⁻¹) in the total yield of H₂ gas. Remarkably, 565 ml of H₂ gas per liter of the suspension culture is the highest yield ever reported for a wild-type strain in a time period of less than 190 h. In immobilized algae, where diffusion of H₂ from the medium to the PhBR gas phase is not affected by mixing, the maximum rate and yield of H₂ photoproduction occur in PhBRs with V_g.p./V_l.p above 7 or in a PhBR with smaller headspace, if the H₂ is effectively removed from the medium by continuous flushing of the headspace with argon. These experiments in combination with studies of the direct inhibitory effect of high H₂ concentrations in the PhBR headspace on H₂ photoproduction activity in algal cultures clearly show that H₂ photoproduction in algae depends significantly on the partial pressure of H₂ (not O₂ as previously thought) in the PhBR gas phase.     

Strong pH dependence of hydrogen production from glucose by Rhodobacter sphaeroides

Purple non-sulfur bacteria (PNSB) are well known for converting short-chain organic acids to H₂, however, a decrease in pH caused by metabolic acids production limited H₂ production during the photo-fermentation from glucose. Here we address why volatile fatty acids (VFA) excreted as fermentation products cannot be further degraded by R. sphaeroides that readily use them. We found that the photo-fermentation with pH controlled at 6.9 ± 0.1 resulted in a 90% increase of H₂ yield and a 107.6% increase in volume H₂ production relative to the pH-uncontrolled culture. Comparative fermentations on glucose at pH 5.8 and pH 7.1 using culture medium supplemented with 50% spent fermentation broth demonstrated that low pH alone is not the limiting factor and compounds present in the supernatants along with pH decrease were the most inhibitory to H₂ production. The impact of byproducts VFA on phototrophic H₂ production was dependent on both the pH and VFA concentrations; even 7 mM VFA addition totally inhibited H₂ production from glucose at pH 5.4. H₂ production with pH control for the Δhup strain was not discernibly different from the parent strain, which are all significantly higher than high-performance strains by metabolic engineering. These results demonstrate that pH dependent VFA inhibition can be turned into a driving force for enhanced H₂ production from glucose by pH regulation.     

Effect of iron and molybdenum addition on photofermentative hydrogen production from olive mill wastewater

Photofermentative hydrogen production from olive mill wastewater (OMW) by Rhodobacter sphaeroides O.U.001 was assessed under iron and molybdenum supplementation. Control cultures were only grown with 2% OMW containing media. The analysis included measurements of biomass accumulation, hydrogen production, pH variations of the medium, and changes in the chemical oxygen demand (COD) of the wastewater. Growth under control and Mo-supplemented experiments yielded about the same amount of biomass (∼0.4 g dry cell weight per L culture). On the other hand, Mo addition slightly enhanced the total volume of H₂ gas production (62 mL H₂), in comparison with the control reactor (40 mL H₂). Fe-supplemented cultures showed a significant increase on H₂ production (125 mL H₂), tough having a longer lag time for the observation of the first H₂ bubbles (24 h), compared to the control (15 h) and Mo-supplemented ones (15 h). Fe-added cultures also yielded better wastewater treatment by achieving 48.1% degradation of the initial chemical oxygen demand (COD) value compared to the control reactor having 30.2% COD removal efficiency. Advances described in this work have the potential to find applications in hydrogen industry while attempting an effective management of cheap feedstock utilization.     

The enhancement of hydrogen photoproduction in Chlorella protothecoides exposed to nitrogen limitation and sulfur deprivation

H₂ photoproduction, hydrogenase activities and PSII photochemical activities in Chlorella protothecoides under sulfur (S–) or nitrogen (N–) deprivation or simultaneous N-limitation and S-deprivation were studied. C. protothecoides pre-cultured in full nutrient TAP medium containing 7 mM NH₄Cl was found to produce a detectable but low level of H₂, once the cells were inoculated either in S-free or N-free medium. However, cells pre-grown in a low concentration of NH₄Cl (0.35 and 0.7 mM) generated a large amount of H₂ after transfer to N-limited and S-free medium. The maximal H₂ outputs of ∼233.7 and ∼129.1 ml/l were obtained within 100 h in the cultures exposed to S-deprived medium containing 0.35 mM and 0.7 mM NH₄Cl, with the average H₂ production rates being ∼2.19 and ∼1.37 ml/l/h, respectively. Our studies further indicated that N-limitation resulted in considerable starch accumulation, chlorophyll synthesis reduction, photosynthetic electron transfer block and oxygen evolving complex (OEC) injury, as well as attenuation in PSII oxygenic activity. Significant starch degradation was not observed during the H₂ photoevolution process. Attenuation of PSII O₂ evolution favored a rapid establishment of anaerobiosis for hydrogenase induction. Meanwhile, a constant high level of hydrogenase activities in C. protothecoides exposed to simultaneous N-limitation and S-deprivation were measured. Based on the above results, a possible mechanism of high H₂ photoproduction in C. protothecoides exposed to N-limitation and S-deprivation was discussed. Low net photosynthetic oxygenic rates, together with high hydrogenase activities were thought to contribute to the enhancement of H₂ photoproduction by C. protothecoides.     

A truncated antenna mutant of Chlamydomonas reinhardtii can produce more hydrogen than the parental strain

Photoproduction of H₂ gas was examined in the Chlamydomonas reinhardtii tla1 strain, CC-4169, containing a truncated light-harvesting antenna, along with its parental CC-425 strain. Although enhanced photosynthetic performance of truncated antenna algae has been demonstrated previously (Polle et al. Planta 2003; 217:49-59), improved H₂ photoproduction has yet to be reported. Preliminary experiments showed that sulfur-deprived, suspension cultures of the tla1 mutant could not establish anaerobiosis in a photobioreactor, and thus, could not photoproduce H₂ gas under conditions typical for the sulfur-deprived wild-type cells (Kosourov et al. Biotech Bioeng 2002; 78:731-40). However, they did produce H₂ gas when deprived of sulfur and phosphorus after immobilization within thin (∼300 μm) alginate films. These films were monitored for long-term H₂ photoproduction activity under light intensities ranging from 19 to 350 μE m⁻² s⁻¹ PAR. Both the tla1 mutant and the CC-425 parental strain produced H₂ gas for over 250 h under all light conditions tested. Relative to the parental strain, the CC-4169 mutant had lower maximum specific rates of H₂ production at low and medium light intensities (19 and 184 μE m⁻² s⁻¹), but it exhibited a 4-times higher maximum specific rate at 285 μE m⁻² s⁻¹ and an 8.5-times higher rate at 350 μE m⁻² s⁻¹ when immobilized at approximately the same cell density as the parental strain. As a result, the CC-4169 strain accumulated almost 4-times more H₂ than CC-425 at 285 μE m⁻² s⁻¹ and over 6-times more at 350 μE m⁻² s⁻¹ during 250-h experiments. These results are the first demonstration that truncating light-harvesting antennae in algal cells can increase the efficiency of H₂ photoproduction in mass culture at high light intensity.     

Prevention of substrate and product inhibitions by using a dilution strategy during dark fermentative hydrogen production from molasses

Substrate and product inhibitions have a significant effect on dark fermentative hydrogen gas production. Particularly, rapid formation of volatile fatty acids leads to fast pH decreases shifting the metabolic pathway. Therefore, controlling volatile fatty acid accumulation has great importance in maintaining effective hydrogen production. In this context, a dilution strategy was applied to regulate volatile fatty acids levels within the desired concentration range. A three-factor Box-Behnken statistical experiment design was established to assess the effects of dilution time, dilution percentage and initial COD concentration on hydrogen formation yield and rate. Highest hydrogen yield (7.7 mL H₂/mL_reactor) and rate (21. 47 mL H₂/h or 9.38 mmol/L_reactor.h) were achieved when 85 gCOD/L containing fermentation media was diluted with a percentage of 130 of the initial working volume at the 3rd hour of the fermentation period. Moreover, this strategy enabled to start fermentation with 55 g glucose/L.     

Influence of sulfate-reducing bacteria,sulfide and molybdate on hydrogen photoproduction by purple nonsulfur bacteria

The interaction between bacterial species is of great importance for H₂ production using microbial consortia or non-sterile conditions. Sulfate reducing bacteria were found in anaerobic starch-hydrolyzing consortium and their inhibitory effect on the following H₂ photoproduction by purple nonsulfur bacteria was shown. This inhibition was clearly demonstrated in the mixed culture of Rhodobacter sphaeroides and Desulfomicrobium baculatum using the synthetic medium. This effect was conditioned by sulfide production rather than H₂ consumption or competition for organic substrate. Actually, the addition of equivalent sulfide concentration brought about the similar effects: inhibition of H₂ production without growth inhibition, cells aggregation, and the increase of carbohydrate content as an alternative way of expenditure of organic acids. In the long-term experiments the average sulfide concentration of about 0.3 mM was detrimental while in short-terms the H₂ production was not inhibited even at 3.2 mM. The protective effect of molybdates against sulfate reducers and sulfide was discussed.     

Sustained hydrogen photoproduction by phosphorus-deprived Chlamydomonas reinhardtii cultures

This study demonstrates that, besides sulfur deprivation, sustained H₂ photoproduction in Chlamydomonas reinhardtii cultures can be generated by incubating algae under phosphorus-deprived (−P) conditions. However, phosphorus deficiency in algal cells could not be obtained by resuspension of algae in −P medium, evidently due to a significant reserve of phosphorus in cells. In this study, phosphorus deficiency was accomplished by inoculating the washed algae into the −P medium at low initial cell densities (below 2 mg Chl l⁻¹). After the initial growth period, where cells utilize intracellular phosphorus, algae established anaerobic environment followed by the period of H₂ photoproduction. The maximum H₂ output (∼70 ml l⁻¹) was obtained in cultures with the initial Chl content ∼1 mg l⁻¹. Cultures with Chl above 2 mg l⁻¹ did not produce H₂ gas. The physiological response of algal cultures to phosphorus deprivation demonstrated significant similarities with the response of algae to sulfur depletion.     

Enhancement in hydrogen production by co-cultures of Bacillus and Enterobacter

Defined co-cultures of hydrogen (H₂) producers belonging to Citrobacter, Enterobacter, Klebsiella and Bacillus were used for enhancing the efficiency of biological H₂ production. Out of 11 co-cultures consisting of 2–4 strains, two co-cultures composed of Bacillus cereus EGU43, Enterobacter cloacae HPC123, and Klebsiella sp. HPC793 resulted in H₂ yield up to 3.0 mol mol⁻¹ of glucose. Up-scaling of the reactor by 16-fold resulted in a corresponding increase in H₂ production with an actual evolution of 7.44 L of H₂. It constituted 58.2% of the total biogas. Continuous culture evolution of H₂ by co-cultures (B. cereus EGU43 and E. cloacae HPC123) immobilized on ligno-cellulosic materials resulted in 6.4-fold improvement in H₂ yield compared to free floating bacteria. This synergistic influence of B. cereus and E. cloacae can offer a better strategy for H₂ production than undefined or mixed cultures.     

Fermentative hydrogen production by Clostridium butyricum and Escherichia coli in pure and cocultures

The effect of coculture of Clostridium butyricum and Escherichia coli on hydrogen production was investigated. C. butyricum and E. coli were grown separately and together as batch cultures. Gas production, growth, volatile fatty acid production and glucose degradation were monitored. Whilst C. butyricum alone produced 2.09 mol-H₂/mol-glucose the coculture produced 1.65 mol-H₂/mol-glucose. However, the coculture utilized glucose more efficiently in the batch culture, i.e., it was able to produce more H₂ (5.85 mmol H₂) in the same cultivation setting than C. butyricum (4.62 mmol H₂), before the growth limiting pH was reached.     

Relationship between molecular hydrogen production,proton transport and the F0F1-ATPase activity in Rhodobacter sphaeroides strains from mineral springs

This article aims to study hydrogen production and proton transport in two strains of purple non-sulfur bacterium Rhodobacter sphaeroides isolated from mineral springs of Armenia. This bacterium is able to grow and produce molecular hydrogen (H₂) in anaerobic conditions upon illumination. Along with H₂ production, a marked decrease in redox potential and the alkalization of the medium have been observed; the latter might be the evidence of proton influx. H₂ production and alkalization of the medium by whole cells both are suppressed by the F₀F₁-ATPase inhibitors – N,N′-dicyclohexylcarbodiimide (DCCD), sodium azide (NaN₃) and protonophore – carbonyl cyanide m-chlorophenylhydrazone (CCCP). Membrane vesicles of two strains of R. sphaeroides demonstrate ATPase activity, inhibited by DCCD and NaN₃, but not by CCCP. These results indicate a relationship between H₂ production, proton transport and the F₀F₁-ATPase activity that might be a pathway to regulate bacterial activity under anaerobic conditions.     

Fermentative hydrogen production by new marine Clostridium amygdalinum strain C9 isolated from offshore crude oil pipeline

The present study investigated hydrogen production potential of novel marine Clostridium amygdalinum strain C9 isolated from oil water mixtures. Batch fermentations were carried out to determine the optimal conditions for the maximum hydrogen production on xylan, xylose, arabinose and starch. Maximum hydrogen production was pH and substrate dependant. The strain C9 favored optimum pH 7.5 (40 mmol H₂/g xylan) from xylan, pH 7.5–8.5 from xylose (2.2–2.5 mol H₂/mol xylose), pH 8.5 from arabinose (1.78 mol H₂/mol arabinose) and pH 7.5 from starch (390 ml H₂/g starch). But the strain C9 exhibited mixed type fermentation was exhibited during xylose fermentation. NaCl is required for the growth and hydrogen production. Distribution of volatile fatty acids was initial pH dependant and substrate dependant. Optimum NaCl requirement for maximum hydrogen production is substrate dependant (10 g NaCl/L for xylose and arabinose, and 7.5 g NaCl/L for xylan and starch).     

New tolerant strains of purple nonsulfur bacteria for hydrogen production in a two-stage integrated system

In this study we described the isolation of eight new strains of purple non-sulfur bacteria resistant to salinity ≥30 g L⁻¹ and high concentration of VFAs (200 mM). These strains were characterized by their general physiological properties and the occurrence of hupSL genes. Some correlation was observed between the rate of H₂ photoproduction, the absence of hupSL genes and hydrogenase activity. Two fast-growing strains without hupSL genes showed high nitrogenase activity and hydrogen accumulation during growth on Ormerod medium. These strains were capable of H₂ photoproduction using non-treated dark culture (75% in water) after dark fermentation of starch at 30 g L⁻¹, unlike control strains, Rhodobacter capsulatus B10 and Rb. sphaeroides GL. New N7 and 13 strains identified as Rb. sphaeroides can be recommended for application in a two-stage H₂ production system.     

Influence of linoleic acid,pH and HRT on anaerobic microbial populations and metabolic shifts in ASBRs during dark hydrogen fermentation of lignocellulosic sugars

In this study, controlling an anaerobic microbial community to increase the hydrogen (H₂) yield during the degradation of lignocelluosic sugars was accomplished by adding linoleic acid (LA) at low pH and reducing the hydraulic retention time (HRT) of an anaerobic sequencing batch reactor (ASBR). At pH 5.5 and a 1.7 d HRT, the maximum H₂ yield for LA treated cultures fed glucose or xylose reached 2.89 ± 0.18 mol mol⁻¹ and 1.94 ± 0.17 mol mol⁻¹, respectively. The major soluble metabolites at pH 5.5 with a 1.7 day HRT differed between the control and LA treated cultures. A metabolic shift toward H₂ production resulted in increased hydrogenase activity in both the xylose (13%) and glucose (34%) fed LA treated cultures relative to the controls. In addition, the Clostridia population and the H₂ yield were elevated in cultures treated with LA. A flux balance analysis for the LA treated cultures showed a reduction in homoacetogenic activity which was associated with reducing the Bacteriodes levels from 12% to 5% in the glucose fed cultures and 16% to 10% in the xylose fed cultures. Strategies for controlling the homoacetogens and optimal hydrogen production from glucose and xylose are proposed.     

Effect of biochar in enhancing hydrogen production by mesophilic anaerobic digestion of food wastes: The role of minerals

The role of minerals in biochar in promoting hydrogen (H₂) production by anaerobic digestion of food waste was investigated. The cultures with the addition of biochar, leached biochar, metal sulphate solution and leached biochar combined with metal sulphate solution, respectively, were placed in bench-scale reactors and incubated at incubator at 32 °C. Daily H₂ production and volatile fatty acids (VFAs) were measured and the cumulative H₂ yield (Y_H) and maximum H₂ production rate (R_H) were calculated. The microbial analysis was performed using Illumina MiSeq sequencing. Biochar addition significantly increased the maximum Y_H by 107% and R_H by 54%. However, the addition of leached biochar only increased the maximum Y_H by 39% and R_H by 45% than control. The primary elements in biochars that contribute to H₂ production (Fe, K and Ca) were shown to increase the acetic acid, butyric acid and prevalence of the H₂ producing bacteria Clostridium butyricum.     

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

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

A new approach for improving microalgal biohydrogen photoproduction based on safe & fast oxygen consumption

Microalgal H₂ photoproduction has the potential of being an affordable method for producing this alternative fuel; however extreme sensitivity of hydrogenase enzyme to photosynthetic O₂ naturally prevents large-scale H₂ production upon illumination. Although a two-phase sulfur deficiency method has been established to deal with this incompatibility, its time and cost demanding, so that this model is not commercially scalable. Despite much research has been conducted, no proper economic alternative for the sulfur deprivation model, with higher or even same productivity and sustainability, has been presented till now. Herein we propose a simple and viable alternative, through introducing a chemical O₂ scavenger system, called oxysorb, to algal cultures.Oxysorb, in non-cytotoxic concentrations (including 50 or 100 mM sodium ascorbate and 5 ppm cupric sulfate) for CC124 as well as pgr5 cultures (containing 30 μg/ml chlorophyll) showed a fast, safe and persisted O₂ removal capacity, initiating H₂ production in the sulfur-containing cultures, either in photoheterotrophic or in autotrophic conditions. Total H₂ production obtained with CC124 and pgr5 cultures, containing 100 mM oxysorb, was 2–5.5 times higher than sulfur-deprived ones (measured in a small closed system). This higher H₂ productivity in the oxysorb approach was achieved due to anoxia establishment with no ROS production and without impacting PSII activity.     

A novel method for increasing biohydrogen production from food waste using electrodialysis

The impact of continuous removal of volatile fatty acids on fermentative hydrogen production from food waste (FW) in a Continuously Stirred Tank Reactor (CSTR) was evaluated. Two experimental phases were conducted, a control phase and one in which volatile fatty acids were removed continuously from the reactor for the first time by electrodialysis (ED). Hydrogen yields were 64.7 cm³ H₂/g VS and 227.3 cm³ H₂/g VS for control phase, and ED phase respectively. Continuous removal of volatile fatty acids during fermentation not only increased H₂ yields but increased the production of volatile fatty acids (a valuable chemical feedstock) from 0.14 g/g VS to 0.34 g/g VS_.     

Bioaugmentation on hydrogen production from food waste

The aim of this work was to evaluate the effect of two hydrolytic (Paenibacillus polymyxa and Bacillus subtilis) and two fermentative (Clostridium saccharobutylicum and Clostridium beijerinckii) strains on hydrogen (H₂) production in dark fermentation by batch testing. Food waste was used as a substrate, pretreated anaerobic sludge was used as the inoculum, and different concentrations of the evaluated microorganisms were used. Bioaugmentation with 3.5 × 10⁹ CFU/mL/L_reactor B. subtilis showed the best performance, obtaining a production of 84.5 mL H₂/g SV and a reduction in the lag phase (from 7.9 h in control to 3.5 h). Bioaugmentation with B. subtilis in an anaerobic sequencing batch reactor exhibited a significant effect on volumetric productivity, reaching a maximal increase of 344% of H₂ production in comparison with that obtained without the addition of the strain. The increase in H₂ was observed in a short period of time (4 cycles), after which H₂ production returned to the original H₂ production baseline. During all reactor operations, the main volatile fatty acids produced were acetic acid and butyric acid. Microbial community analysis when bioaugmentation was applied showed an importance of lactic acid bacteria abundance, such as that of Bifidobacterium and Lactobacillus, whose metabolic activity was crucial in reactor performance. The added concentration of microorganisms is a critical parameter for the bioaugmentation process.     

Changes in hydrogenase genetic diversity and proteomic patterns in mixed-culture dark fermentation of mono-, di- and tri-saccharides

Dark fermentation using mixed cultures is a promising biotechnology for producing hydrogen (H₂) from renewable organic waste at a low cost. The impact of the characteristics of carbohydrates was evaluated on H₂ production and the associated changes in clostridial populations. A series of H₂-producing batch experiments was performed from mono-, di- to tri-saccharides (i.e. fructose, glucose, sucrose, maltose, cellobiose, maltotriose). Both chain length and alpha- or beta-linkage of carbohydrates impacted H₂ production performance as well as the patterns of hydrogenases. The H₂ yield, ranging from 1.38 to 1.84 mol-H₂/mol-hexose, decreased with the increasing chain length of the carbohydrates, showing a negative effect of the hydrolysis step on H₂ production efficiency. Changes in H₂ yield were associated with a specialization of clostridial species, which used different metabolic routes. The rise in H₂ production was associated with butyrate and acetate increases while H₂ consumption was related to caproate formation. Both clostridial [FeFe]- and [NiFe]-hydrogenases were identified in cellobiose cultures by a proteomic approach. This is the first study that combines genetic and proteomic analyses focused on H₂-producing bacteria under various conditions and it opens very interesting perspectives to better understand and optimize H₂ production using mixed cultures.