Hydrogen production from biological systems under different illumination conditions

Hydrogen is a natural by-product of several microbial driven biochemical reactions, mainly in anaerobic fermentation processes. In addition, certain microorganisms produce enzymes by which H₂ from water may be obtained if an outside energy source, like sunlight, is provided. Biophotolysis is a biological process which involves solar energy and algae clusters to convert water into hydrogen. Algae pigments absorb solar energy and enzymes in the cell act as catalysts to split water into hydrogen and oxygen. There are many research activities studying hydrogen production from biological systems cyanobacteria and green algae and some studies present a complete outline of the main available pathways to improve the photosynthetic H₂ production [1] and [2].Efficiency (energy produced from hydrogen divided by solar energy) of such processes can be estimated up to 10%. This value has to be increased for a large-scale hydrogen production. The effect of different artificial illumination conditions on H₂ production was studied for green algae cultures (Chlamydomonas reinhardtii). Results will be used to design a high-efficiency photobioreactor for a large-scale hydrogen production.     

Introducing pyruvate oxidase into the chloroplast of Chlamydomonas reinhardtii increases oxygen consumption and promotes hydrogen production

In an anaerobic environment, the unicellular green algae Chlamydomonas reinhardtii can produce hydrogen (H₂) using hydrogenase. The activity of hydrogenase is inhibited at the presence of molecular oxygen, forming a major barrier for large scale production of hydrogen in autotrophic organisms. In this study, we engineered a novel pathway to consume oxygen and correspondingly promote hydrogen production in Chlamydomonas reinhardtii. The pyruvate oxidase from Escherichia coli and catalase from Synechococcus elongatus PCC 7942 were cloned and integrated into the chloroplast of Chlamydomonas reinhardtii. These two foreign genes are driven by a HSP70A/RBCS2 promoter, a heat shock inducing promoter. After continuous heat shock treatments, the foreign genes showed high expression levels, while the growth rate of transgenic algal cells was slightly inhibited compared to the wild type. Under low light, transgenic algal cells consumed more oxygen than wild type. This resulted in lower oxygen content in sealed culture conditions, especially under low light condition, and dramatically increased hydrogen production. These results demonstrate that pyruvate oxidase expressed in Chlamydomonas reinhardtii increases oxygen consumption and has potential for improving photosynthetic hydrogen production in Chlamydomonas reinhardtii.     

Improvement of hydrogen yield of lba-transgenic Chlamydomonas reinhardtii caused by increasing respiration and impairing photosynthesis

The transgenic alga lba of Chlamydomonas reinhardtii yielded H₂ with 50%–180% higher than the control strain. Further experiments showed that photosynthetic rates and photosynthetic reaction center II's photochemical capacities of the transgenic algae obviously decreased 33.4%–85.9% and 30.0%–51.7%, respectively, compared with those of the control. On the contrary, respiration rates of the transgenic algae significantly increased, with 40.0%–200.0% higher than those of the control. Furthermore, starch contents of the transgenic algae were also improved significantly by 79.1%–592.8% compared with the control. Therefore, the reason of H₂ yield improvement of the transgenic alga lba is not only due to its decrease of photosynthetic capacity and increase of the respiration rate, but also due to the metabolic changes related to starch metabolism, photosynthesis and respiration which is possibly caused by hetero-expression of lba gene in chloroplasts of C. reinhardtii, indicating the potential of utilization of lba gene to improve hydrogen yield of micro-green algae.     

Artificial chloroplast: Au/chloroplast-morph-TiO2 with fast electron transfer and enhanced photocatalytic activity

The unique structures and functional features of chloroplasts in green plants provide a promising blueprint for greatly improving solar energy utilization efficiency. In this paper, a prototype of artificial chloroplast, Au/chloroplast-morph-TiO₂ with natural chloroplasts' nanostructure and analogous functional features, is provided. The nano-layered structures of chloroplast template inherited in the chloroplast-morph-TiO₂ lead to a large reaction area and fast photo-induced electron transfer; cocatalyst Au nanoparticles which work as reaction centers promote photo-induced charge separation and improve the overall photocatalytic activity in hydrogen production; Nitrogen and phosphorus self-doped from the bio-template increase visible light absorption, similar to the antenna pigment. With this new inorganic artificial photosynthetic system, we achieve effective light utilization, fast photo-induced charge separation, high electron transfer, enhanced photocatalytic activity for dye degradation rate and improved H₂ evolution efficiency. This concept provides the inspiration for constructing efficient photocatalysts by imitating the photosynthesis process from both structures and functions.     

Hydrogen gas production by combined systems of Rhodobacter sphaeroides O.U.001 and Halobacterium salinarum in a photobioreactor

Rhodobacter sphaeroides O.U.001 is a photosynthetic non-sulfur bacterium which produces hydrogen from organic compounds under anaerobic conditions. Halobacterium salinarum is an archaeon and lives under extremely halophilic conditions (4 M NaCl). H. salinarum contains a retinal protein bacteriorhodopsin in its purple membrane which acts as a light-driven proton pump. In this study the Rhodobacter sphaeroides O.U.001 culture was combined with different amounts of packed cells of H. salinarum S9 or isolated purple membrane fragments in order to increase the photofermentative hydrogen gas production. The packed cells of H. salinarum have the ability to pump protons upon illumination due to the presence of bacteriorhodopsin. The proton gradient produced may be used for the formation of ATP or protons may be used for H₂ production by R. sphaeroides. Similar to intact cells purple membrane fragments may also form vesicles around certain ions and may act like closed systems.     

Efficient photochemical hydrogen production under visible-light over artificial photosynthetic systems

Photosynthesis of green plants provides an effective blueprint for transform solar energy into useful hydrogen energy. Thereinto, their hierarchical structures are favorable to the light-harvesting. Meanwhile, the functional components (light-harvesting pigments) can absorb visible wavelengths of sunlight, and offer reaction center for the energy transform. Inspired by these, we contrive an artificial photosynthetic system for the high efficiency of H₂-production rate by introducing a similar functional structure (reticular hierarchical structure) and component (CdS/Pt–TiO₂). The CdS/Pt–TiO₂ with hierarchically reticular structure is prepared by transforming wings into TiO₂ via a sol–gel process, and depositing Pt and CdS nanoparticles onto the TiO₂ substrate by photoreduction and chemical bath deposition method, respectively. Contributing to the couple effect of reticular hierarchical structure and ternary hybrid composition, CdS/Pt–TiO₂ nanocomposites exhibit high visible-light photocatalytic H₂-production rate (12.7% apparent quantum efficiency obtained at 420 nm). This concept provides a new horizon to exploit solar energy for sustainable energy by imitating the photosynthesis process from structure and ingredients.     

Improvement of hydrogen production of Chlamydomonas reinhardtii by co-cultivation with isolated bacteria

Three bacteria, named L2, L3 and L4, were isolated from contaminated cultures of Chlamydomonas reinhardtii strain cc849 in laboratory. The phylogenetic analysis based on 16S rDNA sequences showed that L2, L3 and L4 belonged to genus Stenotrophomonas, Microbacterium and Pseudomonas, respectively. The co-cultivation of isolated L2, L3 and L4 with purified algae, respectively, demonstrated that moderate bacterial concentration did not affect algal growth significantly but improved algal H₂ production obviously. The maximal H₂ yields were gained by the co-culture of algae with L2 or L4, about 4.0 times higher than that of the single algal culture. Increased respiration rate or O₂ consumption was the main reason for the enhancement of H₂ yield of the co-cultures.     

Maximizing the solar to H2 energy conversion efficiency of outdoor photobioreactors using mixed cultures

A numerical study is presented aiming to maximize the solar to hydrogen energy conversion efficiency of a mixed culture containing microorganisms with different radiation characteristics. The green algae Chlamydomonas reinhardtii CC125 and the purple non-sulfur bacteria Rhodobacter sphearoides ATCC 49419 are chosen for illustration purposes. The previously measured radiation characteristics of each microorganism are used as input parameters in the radiative transport equation for calculating the local spectral incident radiation within a flat panel photobioreactor. The specific hydrogen production rate for each microorganism as a function of the available incident radiation is recovered from data reported in the literature.     

Enhancing biological hydrogen production through complementary microbial metabolisms

Hydrogen (H₂) production through biological routes is an eco-friendly process. Dark-fermentative or photosynthetic pathways have been widely studied to produce H₂. In these cases yields up to 3.8 mol H₂/mol glucose have been reported to be produced under dark-fermentative conditions by Enterobacter, Caldicellulosiruptor and Thermotoga spp., or through photo-fermentative route by bacteria such as Rhodopseudomonas spp. A combination of the two systems allows a 2-fold increase in H₂ yield. The need is to increase the efficiency of the whole process to yield 12 mol H₂/mol glucose. In this review, we propose that metabolic activities of bacteria may be complemented to use biowaste as feed for increasing the efficiency of H₂ production process. Further utilization of intermediates of these two processes for polyhydroxyalkanoate and methane production is likely to increase the feasibility of meeting the ever increasing energy demand.     

Comparative study of biohydrogen production by four dark fermentative bacteria

Dark fermentation is a promising biological method for hydrogen production because of its high production rate in the absence of light source and variety of the substrates. In this study, hydrogen production potential of four dark fermentative bacteria (Clostridium butyricum, Clostridium pasteurianum, Clostridium beijerinckii, and Enterobacter aerogenes) using glucose as substrate was investigated under anaerobic conditions. Batch experiments were conducted to study the effects of initial glucose concentration on hydrogen yield, hydrogen production rate and concentration of volatile fatty acids (VFA) in the effluents. Among the four different fermentative bacteria, C. butyricum showed great performance at 10 g/L of glucose with hydrogen production rate of 18.29 mL-H₂/L-medium/hand specific hydrogen production rate of 3.90 mL-H₂/g-biomass/h. In addition, it was found that the distribution of volatile fatty acids was different among the fermentative bacteria. C. butyricum and C. pasteurianum had higher ratio of acetate to butyrate compared to the other two species, which favored hydrogen generation.     

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.     

The turnover times and pool sizes of photosynthetic hydrogen production by green algae

An investigation of the turnover times of photobiological production of hydrogen gas by green algae indicate that the photoreactions associated with molecular hydrogen production have promising properties for solar energy conversion and storage. Our results indicate that (a) the intrinsic kinetic rate capability of the hydrogen photoapparatus in green algae can keep pace with the incidence rate of light quanta, even in full sunlight; (b) the photogenerated electrons for hydrogen production probably lie in the mainstream of the electron transport chain of photosynthesis.These results have been obtained by performing the first measurements on the turnover times and pool sizes of photosynthetic hydrogen production. For the three species of green algae studied, the turnover times range from 0.1 to 3 ms. The turnover time for photosynthetic hydrogen production is, therefore, comparable to that for oxygen production.Rapid multiple flash experiments have been performed which indicate that the immediate source of reductant for photosynthetic hydrogen production is derived from a pool of 5–20 equivalents, depending on the alga. This pool is probably the plastoquinone pool linking the two photosystems of photosynthesis.     

Evaluation of hydrogen production by Rhodobacter sphaeroides O.U.001 and its hupSL deficient mutant using acetate and malate as carbon sources

Rhodobacter sphaeroides O.U.001 is one of the candidates for photobiological hydrogen production among purple non-sulfur bacteria. Hydrogen is produced by Mo-nitrogenase from organic acids such as malate or lactate. A hupSL in frame deletion mutant strain was constructed without using any antibiotic resistance gene. The hydrogen production potential of the R. sphaeroides O.U.001 and its newly constructed hupSL deleted mutant strain in acetate media was evaluated and compared with malate containing media. The hupSL⁻R. sphaeroides produced 2.42 l H₂/l culture and 0.25 l H₂/l culture in 15 mM malate and 30 mM acetate containing media, respectively, as compared to the wild type cells which evolved 1.97 l H₂/l culture and 0.21 l H₂/l culture in malate and acetate containing media, correspondingly. According to the results, hupSL⁻R. sphaeroides is a better hydrogen producer but acetate alone does not seem to be an efficient carbon source for photoheterotrophic H₂ production by R. sphaeroides.     

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.     

Hydrogen production and consumption of organic acids by a phototrophic microbial consortium

Alternative fuel sources have been extensively studied. Hydrogen gas has gained attention because its combustion releases only water, and it can be produced by microorganisms using organic acids as substrates. The aim of this study was to enrich a microbial consortium of photosynthetic purple non-sulfur bacteria from an Upflow Anaerobic Sludge Blanket reactor (UASB) using malate as carbon source. After the enrichment phase, other carbon sources were tested, such as acetate (30 mmol l⁻¹), butyrate (17 mmol l⁻¹), citrate (11 mmol l⁻¹), lactate (23 mmol l⁻¹) and malate (14.5 mmol l⁻¹). The reactors were incubated at 30 °C under constant illumination by 3 fluorescent lamps (81 μmol m⁻² s⁻¹). The cumulative hydrogen production was 7.8, 9.0, 7.9, 5.6 and 13.9 mmol H₂ l⁻¹ culture for acetate, butyrate, citrate, lactate and malate, respectively. The maximum hydrogen yield was 0.6, 1.4, 0.7, 0.5 and 0.9 mmol H₂ mmol⁻¹ substrate for acetate, butyrate, citrate, lactate and malate, respectively. The consumption of substrates was 43% for acetate, 37% for butyrate, 100% for citrate, 49% for lactate and 100% for malate. Approximately 26% of the clones obtained from the Phototrophic Hydrogen-Producing Bacterial Consortium (PHPBC) were similar to Rhodobacter, Rhodospirillum and Rhodopseudomonas, which have been widely cited in studies of photobiological hydrogen production. Clones similar to the genus Sulfurospirillum (29% of the total) were also found in the microbial consortium.     

Enhancement of phototrophic hydrogen production by Rhodobacter sphaeroides ZX-5 using a novel strategy — shaking and extra-light supplementation approach

Biohydrogen has gained attention due to its potential as a sustainable alternative to conventional methods for hydrogen production. In this study, the effect of light intensity as well as cultivation method (standing- and shaking-culture) on the cell growth and hydrogen production of Rhodobacter sphaeroides ZX-5 were investigated in 38-ml anaerobic photobioreactor with RCVBN medium. Thus, a novel shaking and extra-light supplementation (SELS) approach was developed to enhance the phototrophic H₂ production by R. sphaeroides ZX-5 using malate as the sole carbon source. The optimum illumination condition for shaking-culture by strain ZX-5 increased to 7000–8000 lux, markedly higher than that for standing-culture (4000–5000 lux). Under shaking and elevated illumination (7000–8000 lux), the culture was effective in promoting photo-H₂ production, resulting in a 59% and 56% increase of the maximum and average hydrogen production rate, respectively, in comparison with the culture under standing and 4000–5000 lux conditions. The highest hydrogen-producing rate of 165.9 ml H₂/l h was observed under the application of SELS approach. To our knowledge, this record is currently the highest hydrogen production rate of non-immobilized purple non-sulphur (PNS) bacteria. This optimal performance of photo-H₂ production using SELS approach is a favorable choice of sustainable and economically feasible strategy to improve phototrophic H₂ production efficiency.