Enhanced H2 photoproduction by down-regulation of ferredoxin-NADP reductase (FNR) in the green alga Chlamydomonas reinhardtii

Renewable H₂ photoproduction by green algae such as Chlamydomonas reinhardtii is a promising system for solar fuels. However, large-scale application of the system has lagged virtually due to lack of high H₂-producing strains. We previously identified ferredoxin-NADP⁺ reductase (FNR) among the 105 proteins differentially expressed in Chlamydomonas during sulfur-deprived H₂ photoproduction. In this work, we used an RNA interference (RNAi) approach to generate Chlamydomonas mutant strains with reduced levels of FNR. We found that fnr-RNAi strains exhibited higher rates of H₂ photoproduction (2.5-fold) than wild type under sulfur-deprived condition. To elucidate the basis for this increase, we analyzed the physiological characteristics of the fnr-RNAi strains under such condition. Major changes, due to the down-regulation of FNR, included the lower rates of photosynthetic O₂ evolution (44%), greater reduction of Rubisco amounts (60%) and higher rates of starch degradation (140%). These may result in an earlier onset of anaerobiosis and increased electron supply to the hydrogenases in the mutant strains. The results provide new information of FNR in regulating H₂ metabolism as well as potential strains for further improvement of the organism toward application in solar-powered systems.     

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.     

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.     

Hydrogen photoproduction under continuous illumination by sulfur-deprived, synchronous Chlamydomonas reinhardtii cultures

Unsynchronized Chlamydomonas reinhardtii cells subsequently deprived of sulfur produce H₂ under continuous illumination in the laboratory for 3–4 days. However, cultures grown outdoors will be exposed to day-and-night cycles that may synchronize their growth and cell division. While it is clear that only insignificant amounts of H₂ can be produced by sulfur-deprived cells during the night period, little work has been done to examine the effects of the light/dark cycles preceding sulfur deprivation on subsequent H₂ photoproduction. We show that (a) C. reinhardtii cells exhibit synchronized growth and cell division in the presence of acetate, (b) cells with the highest specific rates of H₂ photoproduction also have the highest rates of biomass accumulation, and (c) the highest rates of starch and protein degradation coincide with the highest rates of formate and acetate accumulation, but not with H₂ photoproduction. This work shows that it is possible to maximize the production of H₂ by sulfur-depriving synchronized cultures at about 4 h after the beginning of the light period.     

Relationships between PSII-independent hydrogen bioproduction and starch metabolism as evidenced from isolation of starch catabolism mutants in the green alga Chlamydomonas reinhardtii

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.     

Relationships between H2 photoproduction and different electron transport pathways in sulfur-deprived Chlamydomonas reinhardtii

In this study the relationships between photosystem (PS) II dependent and independent pathways of H₂ photoproduction, cyclic electron transport around PS I, chloro- and mitorespiration, and transmembrane ΔpH were examined by inhibitor analysis in S deprived Chlamydomonas reinhardtii. The rate of non-photochemical reduction of plastoquinones in photosynthetic membranes was significantly diminished under starvation which may explain the minor contribution of the PS II independent pathway of H₂ photoproduction in starved cells. The suppressive effect of the herbicide 3-(3,4-dichlorophenyl)-1,1-dimethylurea on the long-term H₂ photoproduction was shown to be entirely attributed to the inhibition of electron transport in PS II, whereas non-specific interactions did not take place. Ferredoxin-quinone reductase – dependent cyclic electron transport around PS I slowed down H₂ photoproduction more than two fold. This result was related to the competition between ferredoxin-quinone reductase and hydrogenase for the reduced ferredoxin and to the decrease in transmembrane ΔpH induced by the cyclic electron flow. The ΔpH gradient was shown to down regulate the PS II independent pathway of H₂ photoproduction in starved cells.     

Photochemical characteristics of Chlamydomonas mutant hpm91 lacking proton gradient regulation 5 (PGR5) during sustained H2 photoproduction under sulfur deprivation

Renewable H₂ photoproduction by Chlamydomonas reinhardtii offers a desirable bio-system for solar fuels. However, its large-scale application is hindered mainly due to lack of ideal strains. We previously isolated a mutant hpm91 which lacks PGR5 and sustains H₂ photoproduction for 25 days. To understand the photosynthetic basis for this remarkable phenotype, we hereby investigated its photochemical characteristics during sulfur-deprived H₂ photoproduction using in vivo chlorophyll fluorescence spectroscopy. Compared to wild type, effective quantum yield of PSII and PSI of hpm91 increased upto 78.9% and 147.6%, respectively. Electron transport rate of each photosystem is closely correlated with the increase of quantum yield, suggesting overall enhanced photochemistry of hpm91 under such condition. Moreover, ATP synthase activity decays slower and remains higher in this mutant. These are in vivo evidence demonstrating increased photosynthetic efficiency of hpm91 promotes its H₂ photoproduction. Together with its competent photoheterotrophic growth in a larger photobioreactor, we propose that hpm91 is a valuable strain for re-engineering Chlamydomonas towards improving light energy efficiency in a large-scale system.     

Towards the integration of dark- and photo-fermentative waste treatment. 2. Optimization of starch-dependent fermentative hydrogen production

Eight natural microbial consortia collected from different sites were tested for dark, hydrogen production during starch degradation. The most active consortium was from silo pit liquid under mesophilic (37 °C) conditions. The fermentation medium for this consortium was optimized (Fe, NH₄⁺, phosphates, peptone, and starch content) for both dark fermentation and for subsequent purple photosynthetic bacterial H₂ photoproduction [Laurinavichene TV, Tekucheva DN, Laurinavichius KS, Ghirardi ML, Seibert M, Tsygankov AA. Towards the integration of dark and photo fermentative waste treatment. 1. Hydrogen photoproduction by purple bacterium Rhodobacter capsulatus using potential products of starch fermentation. Int J Hydrogen Energy 2008;33(23):7020–26], in the presence of the spent dark, fermentation effluent. The addition of Zn (10 mg L⁻¹), as a methanogenesis inhibitor that does not inhibit purple bacteria at this concentration, also did not inhibit dark, fermentative H₂ production. The influence of various fermentation end products at different concentrations (up to 30 g L⁻¹) on dark, H₂ production was also examined. Added lactate stimulated, but added isobutyrate and butanol strongly inhibited gas production. Under optimal conditions the fermentation of starch (30 g L⁻¹) resulted in 5.7 L H₂ L⁻¹ of culture (1.6 mol H₂ per mole of hexose) with the co-production mainly of butyrate and acetate.     

Inoculum density and buffer capacity are crucial for H2 photoproduction from acetate by purple bacteria

The increase of inoculum density of Rhodobacter sphaeroides B-3059 above 2.0 mg bacteriochlorophyll/l in 15 mM K-phosphate buffer inhibited hydrogen photoproduction but favored PHB synthesis. This inhibition was substrate-specific and was observed on media containing acetate. The inhibition resulted from sharp increase in pH above 10 due evidently to fast acetate consumption. The lower the buffer concentration, the lower the inhibitory inoculum density was. The dependence of H₂ photoproduction from acetate on buffer concentration at low and high inoculum density was different for different strains of purple bacteria. However, conditions were found to provide H₂ production by each of 9 tested strains. Acetate-dependent hydrogen photoproduction in 10 mM K-phosphate buffer was unreliable even with low inoculum density. Ammonium traces and high acetate concentration also facilitated medium alkalization and, consequently, inhibited H₂ production. The increase of buffer concentration above 20 mM helped to prevent pH rise independent of the triggering factor.     

The enhancement mechanism of hydrogen photoproduction in Chlorella protothecoides under nitrogen limitation and sulfur deprivation

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.     

Enhancement of fermentative hydrogen production from green algal biomass of Thermotoga neapolitana by various pretreatment methods

Biomass of the green algae has been recently an attractive feedstock source for bio-fuel production because the algal carbohydrates can be derived from atmospheric CO₂ and their harvesting methods are simple. We utilized the accumulated starch in the green alga Chlamydomonas reinhardtii as the sole substrate for fermentative hydrogen (H₂) production by the hyperthermophilic eubacterium Thermotoga neapolitana. Because of possessing amylase activity, the bacterium could directly ferment H₂ from algal starch with H₂ yield of 1.8–2.2 mol H₂/mol glucose and the total accumulated H₂ level from 43 to 49% (v/v) of the gas headspace in the closed culture bottle depending on various algal cell-wall disruption methods concluding sonication or methanol exposure. Attempting to enhance the H₂ production, two pretreatment methods using the heat-HCl treatment and enzymatic hydrolysis were applied on algal biomass before using it as substrate for H₂ fermentation. Cultivation with starch pretreated by 1.5% HCl at 121 °C for 20 min showed the total accumulative H₂ yield of 58% (v/v). In other approach, enzymatic digestion of starch by thermostable α-amylase (Termamyl) applied in the SHF process significantly enhanced the H₂ productivity of the bacterium to 64% (v/v) of total accumulated H₂ level and a H₂ yield of 2.5 mol H₂/mol glucose. Our results demonstrated that direct H₂ fermentation from algal biomass is more desirably potential because one bacterial cultivation step was required that meets the cost-savings, environmental friendly and simplicity of H₂ production.     

A high yield mutant of Chlamydomonas reinhardtii for photoproduction of hydrogen

To identify genes important for the photoproduction of H₂ of Chlamydomonas reinhardtii, random insertional mutants, by the ble gene encoding the enzyme with zeomycin resistant properties, were screened for clones with changed H₂ production. One of the mutants, denoted T1, with 7.8-fold of H₂ yield and about 23% of respiration rate increased compared with the parental strain cc849 was obtained among the zeomycin-resistant transformants. The photosynthetic rate and Fv/Fm, the efficiency of PSII primary photochemistry of T1 declined. Therefore, the photosynthesis/respiration capacity ratio (P/R ratio) of T1 was reduced compared with strain cc849. However, the growth of the mutant T1 was not inhibited, indicating that T1 was a good mutant for further studies of the genes improving H₂ yield by lowering the photosynthetic capacity and/or enhancing the respiration capacity in C. reinhardtii, and had potential to be used in further improvement of H₂ generation by further biotechnological approaches.     

Towards the integration of dark- and photo-fermentative waste treatment. 3. Potato as substrate for sequential dark fermentation and light-driven H2 production

The goal of the study was to characterize H₂ production in an integrated process utilizing potato homogenate (PH) for dark, fermentative H₂ production followed by H₂ photoproduction using purple non-sulfur bacteria. Emphasis was placed on (a) examining potato fermentation effluent (FE) as substrate for H₂ photoproduction, (b) estimating the yield and efficiency of both processes, and (c) elucidating the physiological factors influencing the integrated system as a whole. In the dark stage maximal production of gas (11.5 L L⁻¹ of the culture) and VFA (350 mM) were observed with a PH concentration of 400 g L⁻¹ of medium, but higher yields (0.05 L g⁻¹ PH; 1.9 mmol g⁻¹ PH) were obtained at PH concentrations of 50–100 g L⁻¹. H₂ photoproduction by purple bacteria was inhibited at high FE content. Upon suitable dilution, adequate illumination, and supplementation with Fe/Mg/phosphate nutrients, H₂ photoproduction reached 40 L L⁻¹ of non-diluted FE, with a total H₂ yield of 5.6 mol mol⁻¹ glucose equivalent for the two-stage integrated process.     

Integration of purple non-sulfur bacteria into the starch-hydrolyzing consortium

Purple non-sulfur bacteria Rhodobacter sphaeroides N7 was integrated into the heterotrophic starch-hydrolyzing consortium containing Clostridium butyricum as the main component. The resulting light-dependent consortium with Bchl ∼9 μg/ml was maintained during 12 months of regular transfers. The protein content in this consortium was four-fold higher compared to the heterotrophic consortium, suggesting that purple bacteria became its important component. Under illumination, the starch hydrolysis by the light-dependent consortium (as opposed to heterotrophic consortium) resulted in the absence of VFAs, hydrogen production being quite similar and residual hexose higher. Fermentation of glucose (unlike to starch) by the light-dependent consortium or Rb. sphaeroides alone resulted in ∼3 fold increase of hydrogen production compared to the heterotrophic consortium. Apparently, hydrolysis of starch to hexose was the bottleneck in H₂ photoproduction by the light-dependent consortium. Availability of residual hexose after photofermentation of starch/glucose by the light-dependent consortium or Rb. sphaeroides is discussed.     

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.     

Accumulation of O2-tolerant phenotypes in H2-producing strains of Chlamydomonas reinhardtii by sequential applications of chemical mutagenesis and selection

The photoproduction of hydrogen by anaerobically induced algae is catalyzed by a bidirectional hydrogenase that is rapidly inactivated by oxygen. We isolated two generations of Chlamydomonas reinhardtii strains with H₂-evolving activities of up to 10 times the O₂-tolerance seen in the wild-type (WT). These isolates were generated by two sequential selections, consisting of random chemical mutagenesis, enrichment for H₂-metabolism clones following exposure to increasing amounts of O₂, and screening using a chemochromic sensor. The selected strains were characterized by two types of assays and classified as those that (a) can evolve H₂ following exposure to O₂ concentrations that inactive the WT strain and (b) in addition, are able to quickly reactivate H₂-production activity once O₂ is removed. These results suggest that O₂-tolerance can be increased by successive rounds of mutagenesis, selection, and screening, demonstrating that the WT phenotype can be improved by genetic means. Other results show that the hydrogenase is less sensitive to O₂ when it is actively catalyzing H₂ evolution.     

Inhibition of photosystem 2 in starch-enriched Chlamydomonas reinhardtii cells prevents the efficient induction of H2 production in sulfur-depleted cultures

In sulfur-deprived Chlamydomonas reinhardtii cells the activity of photosystem 2 (PSII) has been shown to have a crucial role in the photosynthetic production of H₂, since it allows the synthesis of internal reserves such as starch. In the present investigation, the PSII inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) was added in starch-enriched and sulfur-depleted C. reinhardtii cultures 1) at the very end of the aerobic phase, and 2) soon after the culture started to evolve H₂. In the former case, production of H₂ on a volumetric basis was completely down-regulated, although starch mobilization was enhanced. In vitro tests showed that the hydrogenase enzyme was active, although its efficiency of utilization in vivo was lowered very soon in the experiment. When an inhibitor of Rubisco such as glycolaldehyde was added under the same conditions, no substantial improvement in H₂ production rates was noted. These findings indicate that, aside starch storage, PSII plays an active role in the induction of the H₂ production process.     

Pathways of hydrogen photoproduction by immobilized Chlamydomonas reinhardtii cells deprived of sulfur

The green algae Сhlamydomonas reinhardtii entrapped in a thin alginate film have been shown to sustain elevated rates of hydrogen photoproduction under anaerobic incubation in sulfur/phosphorus depleted tris-acetate medium. In the present work we studied mechanisms, underlying hydrogen photoproduction by the immobilized culture, particularly, the roles of PSII and starch accumulation/breakdown. DCMU, a specific inhibitor of electron transport in PSII, is known to suppress hydrogen evolution by circa 80% in suspension cultures of S-deprived C. reinhardtii. In immobilized cells DCMU caused successive stimulatory and inhibitory effects on hydrogen photoproduction, both depending on the deprivation status of the algal cell. The inhibitory effect of DCMU was 25% at 70 h of S deficiency when maximal rates of hydrogen photoproduction were observed. Measurements of the light-induced prompt and delayed chlorophyll fluorescence transients and reflectance at 820 nm (P₇₀₀ redox transitions) revealed very rapid decline of PSII activity in the entrapped S-deprived cells as compared with the suspension culture, whereas PSI suffered less. The immobilized culture showed a high capacity to accumulate starch during early stages of S deprivation and relatively high rates of anaerobic starch degradation during the following hydrogen evolution period. DCMU partly inhibited starch breakdown. Results of the present work brought us to the conclusion that PSII-independent pathway of hydrogen evolution is elevated in the immobilized S-deprived cells rather due to the rapid inactivation of PSII, efficient starch catabolism and non-photochemical PQ reduction.     

Batch and continuous biohydrogen production from starch hydrolysate by Clostridium species

In this study, hydrogen gas was produced from starch feedstock via combination of enzymatic hydrolysis of starch and dark hydrogen fermentation. Starch hydrolysis was conducted using batch culture of Caldimonas taiwanensis On1 able to hydrolyze starch completely under the optimal condition of 55 °C and pH 7.5, giving a yield of 0.46–0.53 g reducing sugar/g starch. Five H₂-producing pure strains and a mixed culture were used for hydrogen production from raw and hydrolyzed starch. All the cultures could produce H₂ from hydrolyzed starch, whereas only two pure strains (i.e., Clostridium butyricum CGS2 and CGS5) and the mixed culture were able to ferment raw starch. Nevertheless, all the cultures displayed higher hydrogen production efficiencies while using the starch hydrolysate, leading to a maximum specific H₂ production rate of 116 and 118 ml/g VSS/h, for Cl. butyricumCGS2 and Cl. pasteurianum CH5, respectively. Meanwhile, the H₂ yield obtained from strain CGS2 and strain CH5 was 1.23 and 1.28 mol H₂/mol glucose, respectively. The best starch-fermenting strain Cl. butyricum CGS2 was further used for continuous H₂ production using hydrolyzed starch as the carbon source under different hydraulic retention time (HRT). When the HRT was gradually shortened from 12 to 2 h, the specific H₂ production rate increased from 250 to 534 ml/g  VSS/h, whereas the H₂ yield decreased from 2.03 to 1.50  mol H₂/mol glucose. While operating at 2 h HRT, the volumetric H₂ production rate reached a high level of 1.5 l/h/l.