Hydrogen is seen as one of the most promising fuels for the future owing to the fact that it is renewable and liberates large amounts of energy per unit weight without evolving CO2 when combusted. Biohydrogen production has several advantages over hydrogen pro­duction by photoelectrochemical or thermochemical processes. For example, whereas electrochemical hydrogen production requires the use of solar batteries with high energy requirements to split water and form the hydrogen product, biohydrogen production by photosynthetic microorganisms only requires simple PBRs with low energy requirements. A select group of green algae (including Chlamydomonas reinhardtii) and cyanobacteria offer an alternative route to renewable H2 production (Levin et al., 2004; Sakurai and Masukawa,

2007) . Cyanobacteria are able to diverge the electrons emerging from the two primary reactions of oxygenic photosynthesis directly into the production of H2, making them attractive for the production of renewable H2 from solar energy and water.

Cyanobacteria utilize two enzymatic pathways for H2 production, either nitrogenases or bidirectional hydroge — nases (Angermayr et al., 2009). Nitrogenases require ATP, whereas bidirectional hydrogenases do not require ATP for H2 production, hence making them more efficient and favorable for H2 production with a much higher turnover. The fundamental aspects of cyanobacterial hy — drogenases, and their more applied potential use as future producers of renewable H2 from sun and water, are receiving increased international attention. At the same time, significant progress is being made in the un­derstanding of the molecular regulation of the genes encoding both the enzymes and the accessory proteins needed for the correct assembly of an active hydrogenase. With the increasing interest of both scientific and public communities in clean and renewable energy sources, and consequent funding opportunities, rapid progress will likely be made in the fundamental understanding of the regulation of cyanobacterial hydrogenases at both genetic and proteomic levels. Bandyopadhyay et al.

(2010) have described Cyanothece sp. ATCC 51142, a uni­cellular, diazotrophic cyanobacterium with capacity to generate high levels of hydrogen under aerobic condi­tions. Wild-type Cyanothece sp. 51142 can produce hydrogen at rates as high as 465 mmol/mg of chloro — phyll/h in the presence of glycerol. Authors also report that hydrogen production in this strain is mediated by an efficient nitrogenase system, which can be manipu­lated to convert solar energy into hydrogen at rates that are several fold higher, compared to other previously described wild-type hydrogen-producing photosynthetic microbes. These strains have evolved the ability to use so­lar energy to produce H2 from water (Esquivel, 2011; Levin et al., 1961). The theoretical conversion efficiency from light to H2 is calculated to be as high as ~10% (Levin et al., 1961).

Photosystem II (PSII) drives the first stage of the pro­cess (Figure 10.10), by splitting H2O into protons (H2), electrons (e~), and O2.

H2 Production

H2O/2H++ 2e~ + У2 O2
2H+ + 2e~ /H2

H2 Combustion

H2 + У2 O2 / H2O + 285.8 kJ/mol

Normally, the photosynthetic light reactions and the Cal­vin cycle produce carbohydrates that fuel mitochondrial respiration and cell growth. Under anaerobic conditions, however, mitochondrial oxidative phosphorylation is largely inhibited, which leads some organisms (e. g. Chla- mydomonas reinhardtii) to reroute the energy stored in

carbohydrates to a chloroplast hydrogenase (HydA), likely using an NAD(P)H~PQ e~ transfer mechanism, to facilitate ATP production via photophosphorylation. Thus, hydrogenase reacts with H+ (from the medium) and e~ (from reduced ferredoxin) to produce H2 gas that is subsequently excreted from the cell. The combus­tion of the recovered H2 yields only heat and H2O and thus is a model green technology.

Several renewable energy laboratories have concluded that production efficiencies must be improved from 0.2% photon to H2 conversion efficiency at 20 W/m2 illumination to ~ 7—10% at 230 W/m2 illumination (day light) to make the process economically viable. Through extensive preliminary work, the efficiency of this process has been enhanced to ~1.0% from light to H2 and 2% to biomass. The H2 gas produced in such mutants has a purity of ~90—95% and typical yields are 500 ml H2 for a 11 culture (10 days; 110 W illumination). Without further purification, the H2 gas can used to power a small-scale fuel cell car.

In addition to work with Chlamydomonas, a large number of unicellular, filamentous, freshwater, and ma­rine cyanobacterial species have been reported to pro­duce large quantities of biohydrogen. Among other species, Anabaena azollae, Anabaena cylindrica, Anabaena variabilis, Arthrospira (Spirulina) platensis, Cyanothece, Gloeocapsa alpicola, and Nostoc muscorum have been re­ported to produce high levels of hydrogen gas (Jeffries et al., 1978; Aoyama et al., 1997; Antal and Lindblad, 2005). In particular, Anabaena sp. is reported to produce relatively large quantities of biohydrogen. Among these species, nitrogen-starved A. cylindrica cells produce the highest concentration of biohydrogen (30 ml H2/l/h) (Margheri et al., 1990).

These cyanobacterial strains use two sets of enzymes to generate hydrogen gas. The first enzyme is nitroge — nase, and it is found in the heterocysts of filamentous cyanobacteria when grown under nitrogen-limiting con­ditions. Hydrogen is produced as a by-product of fixa­tion of nitrogen into ammonia. The reaction consumes 16 ATP for fixation of 1 mol of N2, and results in forma­tion of 1 mol of H2. The other hydrogen-metabolizing or hydrogen-producing enzymes in cyanobacteria are hy- drogenases, which occur as two distinct types in different cyanobacterial species. The first type is uptake hydrogenase (encoded by hupSL), which has the ability to oxidize hydrogen via oxyhydrogenation or the Knall — gas reaction. The other type of hydrogenase is reversible or bidirectional hydrogenase (encoded by hoxFUYH), and it is capable of uptake and production of hydrogen (Schmitz et al., 1995; Tamagnini et al., 2002). Hydrogen is an important fuel source and is widely applied in fuel cells, coal liquefaction, upgrading of heavy oils, and several other operations. Hydrogen can be produced biologically by various means, including the steam reformation of bio-oils, dark — and photofermentation of organic materials, and photolysis of water catalyzed by special microalgal species (Kapdan and Kargi, 2006; Ran et al., 2006; Wang et al., 2008).

BIOCRUDE

In addition to direct combustion, there is growing attention to conversion of biomass into liquid energy carriers. Applying more traditional biofuel production processes (e. g. lipid extraction followed by transesterifi­cation, fast pyrolysis and gasification) to algal biomass requires that the algae be dried prior to use. Unless ac­cess to waste heat is available, the energy required to first concentrate the biomass to a paste followed by com­plete drying far exceeds to energy value of the produced biocrude. An alternative production pathway called hy­drothermal liquefaction (HTL) bypasses the drying step and converts the algal biomass into a hydrocarbon — based biocrude fuel in the aqueous phase. A simple comparison of the enthalpies of liquid water at 350 °C and water vapor at 50 °C (i. e. drying the biomass) indi­cates that processing in liquid water saves 921 kJ/kg.