Hydrogenases and Hydrogen Metabolism of Cyanobacteria

Cyanobacteria may possess several enzymes that are directly involved in dihydrogen metabolism: nitrogenase(s) catalyzing the production of hydrogen concomitantly with the reduction of dinitrogen to ammonia, an uptake hydrogenase (encoded by hupSL) catalyzing the consumption of hydrogen produced by the nitrogenase, and a bidirectional hydrogenase (encoded by hoxFUYH) which has the capacity to both take up and produce hydrogen. This review summarizes our knowledge about cyanobacterial hydrogenases, focusing on recent progress since the first molecular information was published in 1995. It presents the molecular knowledge about cyanobacterial hupSL and hoxFUYH, their corresponding gene products, and their accessory genes before finishing with an applied aspect—the use of cyanobacteria in a biological, renewable production of the future energy carrier molecular hydrogen. In addition to scientific publications, information from three cyanobacterial genomes, the unicellular Synechocystis strain PCC 6803 and the filamentous heterocystous Anabaena strain PCC 7120 and Nostoc punctiforme (PCC 73102/ATCC 29133) is included.     

Cyanobacterial H<Subscript>2</Subscript> production — a comparative analysis

Several unicellular and filamentous, nitrogen-fixing and non-nitrogen-fixing cyanobacterial strains have been investigated on the molecular and the physiological level in order to find the most efficient organisms for photobiological hydrogen production. These strains were screened for the presence or absence of hup and hox genes, and it was shown that they have different sets of genes involved in H₂ evolution. The uptake hydrogenase was identified in all N₂-fixing cyanobacteria, and some of these strains also contained the bidirectional hydrogenase, whereas the non-nitrogen fixing strains only possessed the bidirectional enzyme. In N₂-fixing strains, hydrogen was mainly produced by the nitrogenase as a by-product during the reduction of atmospheric nitrogen to ammonia. Therefore, hydrogen production was investigated both under non-nitrogen-fixing conditions and under nitrogen limitation. It was shown that the hydrogen uptake activity is linked to the nitrogenase activity, whereas the hydrogen evolution activity of the bidirectional hydrogenase is not dependent or even related to diazotrophic growth conditions. With regard to large-scale hydrogen evolution by N₂-fixing cyanobacteria, hydrogen uptake-deficient mutants have to be used because of their inability to re-oxidize the hydrogen produced by the nitrogenase. On the other hand, fermentative H₂ production by the bidirectional hydrogenase should also be taken into account in further investigations of biological hydrogen production.Abbreviations Chl chlorophyll - MV methyl viologen     

藻青菌产氢研究

目前全球都在寻找一种可替代化石燃料的能源物质。H_2作为能源是未来的希望。藻青菌是生物光合产氢具有很大前景的微生物。综述了藻青菌生物产氢的进展。     

Carbon-negative synthetic biology: challenges and emerging trends of cyanobacterial technology

Global warming and climate instability have spurred interest in using renewable carbon resources for the sustainable production of chemicals. Cyanobacteria are ideal cellular factories for carbon-negative production of chemicals owing to their great potentials for directly utilizing light and CO₂ as sole energy and carbon sources, respectively. However, several challenges in adapting cyanobacterial technology to industry, such as low productivity, poor tolerance, and product harvesting difficulty, remain. Synthetic biology may finally address these challenges. Here, we summarize recent advances in the production of value-added chemicals using cyanobacterial cell factories, particularly in carbon-negative synthetic biology and emerging trends in cyanobacterial applications. We also propose several perspectives on the future development of cyanobacterial technology for commercialization.     

Integrating photobiological hydrogen production with dye-metal bioremoval from simulated textile wastewater

The study reports production of hydrogen in photobioreactors with free (PBR_Fr) and immobilized (PBR_Imm) Nostoc biomass at enhanced and sustained rates. Before running the photobioreactors, effects of different immobilization matrices and cyanobacterial dose on hydrogen production were studied in batch mode. As hydrogen production in the PBRs declined spent biomass from the photobioreactors were collected and utilized further for column biosorption of highly toxic dyes (Reactive Red 198 + Crystal Violet) and metals (hexavalent chromium and bivalent cobalt) from simulated textile wastewater. Breakthrough time, adsorption capacity and exhaustion time of the biosorption column were studied. The photobioreactors with free and immobilized cyanobacterium produced hydrogen at average rates of 101 and 151 μmol/h/mg Chl a, respectively over 15 days, while the adsorption capacity of the spent biomass was up to 1.4 and 0.23 mg/g for metals and 15 and 1.75 mg/g for the dyes, respectively in continuous column mode.     

Hydrogenases and hydrogen metabolism of cyanobacteria.

Cyanobacteria may possess several enzymes that are directly involved in dihydrogen metabolism: nitrogenase(s) catalyzing the production of hydrogen concomitantly with the reduction of dinitrogen to ammonia, an uptake hydrogenase (encoded by hupSL) catalyzing the consumption of hydrogen produced by the nitrogenase, and a bidirectional hydrogenase (encoded by hoxFUYH) which has the capacity to both take up and produce hydrogen. This review summarizes our knowledge about cyanobacterial hydrogenases, focusing on recent progress since the first molecular information was published in 1995. It presents the molecular knowledge about cyanobacterial hupSL and hoxFUYH, their corresponding gene products, and their accessory genes before finishing with an applied aspect--the use of cyanobacteria in a biological, renewable production of the future energy carrier molecular hydrogen. In addition to scientific publications, information from three cyanobacterial genomes, the unicellular Synechocystis strain PCC 6803 and the filamentous heterocystous Anabaena strain PCC 7120 and Nostoc punctiforme (PCC 73102/ATCC 29133) is included.     

蓝细菌细胞工厂合成聚合物单体的研究进展

蓝细菌是当前合成生物学研究的热门底盘生物之一,是光合自养底盘微生物的典型代表。随着化石资源的逐渐枯竭和碳排放所导致的全球变暖问题的加剧,以CO2为碳源的蓝细菌细胞工厂的研究又迎来了一次新的浪潮。长期以来,人们对于蓝细菌细胞工厂的关注点主要是在生物能源的生产,比如液体燃料及氢气等。蓝细菌细胞工厂研究的主要瓶颈之一是其低效率导致的经济性问题。这一问题对于成本异常敏感的能源产品而言尤其突出。聚合物作为人类生产生活的重要基础,属于附加值较大的大宗化学品,对克服蓝细菌细胞工厂商业化所面临的经济性问题具有优势,近来得到了越来越多的关注。本文对蓝细菌的聚合物单体生产的相关研究进行了系统综述,阐述了各类单体的增产策略,并回顾了蓝细菌细胞工厂应用的相关技术,提出了蓝细菌合成生物学的应用领域所存在的问题并对未来的研究进行了展望。     

Selective effects of H<Subscript>2</Subscript>O<Subscript>2</Subscript> on cyanobacterial photosynthesis

The sensitivity of phytoplankton species for hydrogen peroxide (H₂O₂) was analyzed by pulse amplitude modulated (PAM) fluorometry. The inhibition of photosynthesis was more severe in five tested cyanobacterial species than in three green algal species and one diatom species. Hence the inhibitory effect of H₂O₂ is especially pronounced for cyanobacteria. A specific damage of the photosynthetic apparatus was demonstrated by changes in 77 K fluorescence emission spectra. Different handling of oxidative stress and different cell structure are responsible for the different susceptibility to H₂O₂ between cyanobacteria and other phytoplankton species. This principle may be potentially employed in the development of new agents to combat cyanobacterial bloom formation in water reservoirs.     

Light Driven CO2 Fixation by Using Cyanobacterial Photosystem I and NADPH-Dependent Formate Dehydrogenase

The ultimate goal of this research is to construct a new direct CO₂ fixation system using photosystems in living algae. Here, we report light-driven formate production from CO₂ by using cyanobacterial photosystem I (PS I). Formate, a chemical hydrogen carrier and important industrial material, can be produced from CO₂ by using the reducing power and the catalytic function of formate dehydrogenase (FDH). We created a bacterial FDH mutant that experimentally switched the cofactor specificity from NADH to NADPH, and combined it with an in vitro-reconstituted cyanobacterial light-driven NADPH production system consisting of PS I, ferredoxin (Fd), and ferredoxin-NADP⁺-reductase (FNR). Consequently, light-dependent formate production under a CO₂ atmosphere was successfully achieved. In addition, we introduced the NADPH-dependent FDH mutant into heterocysts of the cyanobacterium Anabaena sp. PCC 7120 and demonstrated an increased formate concentration in the cells. These results provide a new possibility for photo-biological CO₂ fixation.     

Dynamic modelling of high biomass density cultivation and biohydrogen production in different scales of flat plate photobioreactors

This paper investigates the scaling‐up of cyanobacterial biomass cultivation and biohydrogen production from laboratory to industrial scale. Two main aspects are investigated and presented, which to the best of our knowledge have never been addressed, namely the construction of an accurate dynamic model to simulate cyanobacterial photo‐heterotrophic growth and biohydrogen production and the prediction of the maximum biomass and hydrogen production in different scales of photobioreactors. To achieve the current goals, experimental data obtained from a laboratory experimental setup are fitted by a dynamic model. Based on the current model, two key original findings are made in this work. First, it is found that selecting low‐chlorophyll mutants is an efficient way to increase both biomass concentration and hydrogen production particularly in a large scale photobioreactor. Second, the current work proposes that the width of industrial scale photobioreactors should not exceed 0.20 m for biomass cultivation and 0.05 m for biohydrogen production, as severe light attenuation can be induced in the reactor beyond this threshold. Biotechnol. Bioeng. 2015;112: 2429–2438. © 2015 The Authors. Biotechnology and Bioengineering Published by Wiley Peiodicals, Inc.     

Promoting R &; D in Photobiological Hydrogen Production Utilizing Mariculture-Raised Cyanobacteria

This review article explores the potential of using mariculture-raised cyanobacteria as solar energy converters of hydrogen (H₂). The exploitation of the sea surface for large-scale renewable energy production and the reasons for selecting the economical, nitrogenase-based systems of cyanobacteria for H₂ production, are described in terms of societal benefits. Reports of cyanobacterial photobiological H₂ production are summarized with respect to specific activity, efficiency of solar energy conversion, and maximum H₂ concentration attainable. The need for further improvements in biological parameters such as low-light saturation properties, sustainability of H₂ production, and so forth, and the means to overcome these difficulties through the identification of promising wild-type strains followed by optimization of the selected strains using genetic engineering are also discussed. Finally, a possible mechanism for the development of economical large-scale mariculture operations in conjunction with international cooperation and social acceptance is outlined.     

Efficacy of New Inexpensive Cyanobacterial Biofertilizer Including its Shelf-life

Summary Four cyanobacterial inoculants all significantly increased grain and straw yield of rice either alone or in combination with chemical fertilizer. A saving of 25 kg N ha⁻¹ can be attained through cyanobacterial fertilization. Tobacco waste-based cyanobacterial biofertilizer was best in performance. Cyanobacterial acetylene reducing activity in vivo varied from 144 to 255 μmol C₂H₄ m⁻² h⁻¹ in different treatments, being highest for tobacco-based cyanobacterial biofertilizer integrated with 50% chemical N. The nutrient balance for total N, available N, total P and available P was found positive in biofertilizer- and chemical fertilizer-treated plots. The total and available K showed negative balance in all the treatments. The shelf-life of cyanobacterial biofertilizer can be augmented by selecting translucent packing material, dry mixing and paddy straw as a carrier. Dry mixing and a mixing ratio of 50:50 (carrier:cyanobacteria) gave better inoculum loading and shelf-life. Decrease in cyanobacterial population was least in dried cyanobacterial flacks, indicating a possibility of developing cyanobacterial biofertilizer without carrier mixing at the time of production.     

Acetylene reduction and hydrogen metabolism by a cyanobacterial/sulfate‐reducing bacterial mat ecosystem

We report a study of nitrogenase activity (acetylene reduction) and hydrogen gas metabolism in intact smooth cyanobacterial mats from Hamelin Pool, Shark Bay, Western Australia. The predominant cyanobacterial population in these mats is Microcoleus chthonoplastes. The mats had a significant capacity for nitrogen fixation, predominantly attributable to the photosyn‐thetic component. By physical and chemical perturbation we revealed an active hydrogen metabolism within the mats. Most of the H₂ formation was attributed to fermentative processes, whereas hydrogen was consumed in light‐dependent, together with oxygen‐ and sulfate‐dependent respiratory processes. It was concluded that H₂ formed by fermentative bacteria in the dark drives a significant proportion of sulfate reduction in the mats, but there was little H₂ transfer from the cyanobacteria to the sulfate‐reducing bacteria. Thus photosynthetically produced H₂ gas is unlikely to significantly alter the previously measured carbon: sulfur ratio relating photosynthesis to sulfate reduction.     

Utilization of cyanobacterial biomass from water bloom for bioproduction of lactic acid

Cyanobacterial biomass obtained from water blooms was successfully utilized as a material for lactic acid production. The starch contained in the biomass could be converted to D- and L-lactic acid with 80–90% yield by Lactobacillus amylovorus, in a manner similar to that contained in laboratory-cultured cyanobacterial biomass. The starch was also available for L-lactic acid production with similar high yields by L. agilis and L. ruminis that specifically produce L-lactic acid. The lactic acid production from the cyanobacterial biomass did not require any supplements such as yeast extract which are essential for lactic acid production from reagent soluble starch, indicating that nutrients contained in the cyanobacterial biomass might be effectively used for the production instead of the supplements. The starch content of the fresh cyanobacterial biomass from water bloom was increased from 10 to 19 and 24% by cultivation in 1 and 5% CO₂ in air, respectively. Using such starch-rich biomass, the concentration of lactic acid produced was successfully increased without changes in the conversion yield. These results indicate that wastewater bloom cyanobacteria could be utilized for the production of a useful compound, lactic acid.     

Halophilic algal-bacterial and cyanobacterial communities and their role in carbonate precipitation

This work studies the diversity of cyanobacterial and algal-bacterial communities of saline water bodies in the Crimean Peninsula and Altai Region. Plant-bacterial communities are described for the first time. The dependence of the production and destruction on the season and salinity of the water body is shown. The development of planktonic cyanobacteria is related to the presence of zooplankton, the development of which is controlled by hydrogen sulfide. The high hydrogen sulfide tolerance of benthic cyanobacteria secures the integrity of cyanobacterial communities. Observations in nature and laboratory modeling show that the formation of mineral layers is restricted to conditions of supersaturation with mineral components. Carbonate precipitation can take place in cyanobacterial communities under conditions of mixing sea water enriched with Ca and Mg with continental water enriched with sodium carbonate. Cyanobacteria are able to form and transform various Ca-Mg-carbonates. Dolomite formation is a derived process that occurs in cyanobacterial mats in the presence of sulfate-reducing bacteria. Carbonatization of cyanobacterial cells is considered using the example of the unicellular halophilic-alkaliphilic cyanobacterium Euhalothece sp. The accomplished study is of certain interest for interpretation of geological and paleontological data in the context of the supposed analogy between cyanobacterial mats and ancient stromatolites.     

Engineering cyanobacteria to generate high-value products

Although many microorganisms have been used for the bioindustrial generation of valuable metabolites, the productive potential of cyanobacterial species has remained largely unexplored. Cyanobacteria possess several advantages as organisms for bioindustrial processes, including simple input requirements, tolerance of marginal agricultural environments, rapid genetics, and carbon-neutral applications that could be leveraged to address global climate change concerns. Here, we review recent research involving the engineering of cyanobacterial species for the production of valuable bioindustrial compounds, including natural cyanobacterial products (e.g. sugars and isoprene), biofuels (e.g. alcohols, alkanes and hydrogen), and other commodity chemicals. Biological and economic obstacles to scaled cyanobacterial production are highlighted, and methods for increasing cyanobacterial production efficiencies are discussed.     

Rational redesign of the ferredoxin-NADP+-oxido-reductase/ferredoxin-interaction for photosynthesis-dependent H2-production

Utilization of electrons from the photosynthetic water splitting reaction for the generation of biofuels, commodities as well as application in biotransformations requires a partial rerouting of the photosynthetic electron transport chain. Due to its rather negative redox potential and its bifurcational function, ferredoxin at the acceptor side of Photosystem 1 is one of the focal points for such an engineering. With hydrogen production as model system, we show here the impact and potential of redox partner design involving ferredoxin (Fd), ferredoxin-oxido-reductase (FNR) and [FeFe]?hydrogenase HydA1 on electron transport in a future cyanobacterial design cell of Synechocystis PCC 6803. X-ray-structure-based rational design and the allocation of specific interaction residues by NMR-analysis led to the construction of Fd- and FNR-mutants, which in appropriate combination enabled an about 18-fold enhanced electron flow from Fd to HydA1 (in competition with equimolar amounts of FNR) in in vitro assays. The negative impact of these mutations on the Fd-FNR electron transport which indirectly facilitates H₂ production (with a contribution of ≤42% by FNR variants and ≤23% by Fd-variants) and the direct positive impact on the Fd-HydA1 electron transport (≤23% by Fd-mutants) provide an excellent basis for the construction of a hydrogen-producing design cell and the study of photosynthetic efficiency-optimization with cyanobacteria.     

Applications of cyanobacteria in biotechnology

Cyanobacteria have gained a lot of attention in recent years because of their potential applications in biotechnology. We present an overview of the literature describing the uses of cyanobacteria in industry and services sectors and provide an outlook on the challenges and future prospects of the field of cyanobacterial biotechnology. Cyanobacteria have been identified as a rich source of biologically active compounds with antiviral, antibacterial, antifungal and anticancer activities. Several strains of cyanobacteria were found to accumulate polyhydroxyalkanoates, which can be used as a substitute for nonbiodegradable petrochemical-based plastics. Recent studies showed that oil-polluted sites are rich in cyanobacterial consortia capable of degrading oil components. Cyanobacteria within these consortia facilitated the degradation processes by providing the associated oil-degrading bacteria with the necessary oxygen, organics and fixed nitrogen. Cyanobacterial hydrogen has been considered as a very promising source of alternative energy, and has now been made commercially available. In addition to these applications, cyanobacteria are also used in aquaculture, wastewater treatment, food, fertilizers, production of secondary metabolites including exopolysaccharides, vitamins, toxins, enzymes and pharmaceuticals. Future research should focus on isolating new cyanobacterial strains producing high value products and genetically modifying existing strains to ensure maximum production of the desired products. Metagenomic libraries should be constructed to discover new functional genes that are involved in the biosynthesis of biotechnological relevant compounds. Large-scale industrial production of the cyanobacterial products requires optimization of incubation conditions and fermenter designs in order to increase productivity.     

Stratification,nitrogen fixation,and cyanobacterial bloom stage regulate the planktonic food web structure

Changes in the complexity of planktonic food webs may be expected in future aquatic systems due to increases in sea surface temperature and an enhanced stratification of the water column. Under these conditions, the growth of unpalatable, filamentous, N₂‐fixing cyanobacterial blooms, and their effect on planktonic food webs will become increasingly important. The planktonic food web structure in aquatic ecosystems at times of filamentous cyanobacterial blooms is currently unresolved, with discordant lines of evidence suggesting that herbivores dominate the mesozooplankton or that mesozooplankton organisms are mainly carnivorous. Here, we use a set of proxies derived from amino acid nitrogen stable isotopes from two mesozooplankton size fractions to identify changes in the nitrogen source and the planktonic food web structure across different microplankton communities. A transition from herbivory to carnivory in mesozooplankton between more eutrophic, near‐coastal sites and more oligotrophic, offshore sites was accompanied by an increasing diversity of microplankton communities with aging filamentous cyanobacterial blooms. Our analyses of 124 biotic and abiotic variables using multivariate statistics confirmed salinity as a major driver for the biomass distribution of non‐N₂‐fixing microplankton species such as dinoflagellates. However, we provide strong evidence that stratification, N₂ fixation, and the stage of the cyanobacterial blooms regulated much of the microplankton diversity and the mean trophic position and size of the metabolic nitrogen pool in mesozooplankton. Our empirical, macroscale data set consistently unifies contrasting results of the dominant feeding mode in mesozooplankton during blooms of unpalatable, filamentous, N₂‐fixing cyanobacteria by identifying the at times important role of heterotrophic microbial food webs. Thus, carnivory, rather than herbivory, dominates in mesozooplankton during aging and decaying cyanobacterial blooms with hitherto uncharacterized consequences for the biogeochemical functions of mesozooplankton.     

Engineered cyanobacteria: teaching an old bug new tricks

Cyanobacteria have played an important role in the development of the Earth and have long been studied as model organisms for photosynthesis and the circadian rhythm. Recent developments have led to increased interest in the use of engineered cyanobacteria for the production of protein and chemical products. This review highlights the genetic tools and strategies for manipulation of cyanobacteria as well as previous accomplishments in the development of engineered cyanobacteria for applied use. Particular attention is given to the engineering of cyanobacteria for biofuel production, including both hydrocarbon and hydrogen fuels. Genetic engineering efforts to enhance cyanobacterial fitness are reviewed with an emphasis on physiological improvements for large-scale production. Lastly, a future outlook on engineered cyanobacteria is presented, highlighting the future areas of focus and technical challenges in this field. With the uncertainty of future energy security, it is an exciting time in applied cyanobacterial research, but we must take the time to learn from these past accomplishments before we can capitalize on the potential of these photosynthetic microorganisms.