# Biohydrogen from Microalgae: Production and Applications
## 5 Fermentation Processes and Biomass-Applied Technologies  
Direct and indirect biophotolysis processes are intrinsically linked to the photosynthetic process and the connected electron transport chain. Together with these two photosynthetic pathways that contribute to the production of hydrogen, another one linked to fermentative metabolism has also been identified. In dark conditions, the enzymatic activity of pyruvate:ferredoxin oxidoreductase (PFR) in _Chlamydomonas reinhardtii_ is responsible for the reduction of Fd and the passage of electrons towards hydrogenase. Overall, this pyruvate-dependent hydrogen production acts in ways similar to those observed in bacteria, and though the yield is low, its contribution is not negligible [51]. The accumulation of complex carbohydrates, such as starch, or endogenous substrates, is positively associated with the production of hydrogen, while, it was also observed that exogenous carbon-rich media further stimulate its production in the early anaerobic stages. Several fermentative bacteria use anaerobic processes to transform carbon sources into various by-products, including hydrogen. Processes, such as photo- and dark- fermentation, are commonly exploited and widely investigated in bacteria species, such as _Escherichia coli_, _Clostridium_ spp., _Thermococales_ spp., _Rhodobacter_ spp., and _Rhodopesudomonas_ spp. [52; 53]. The consumption of organic substrates, also deriving from waste, by photodtermentation, include the transformation into organic acids, alcohols, CO\({}_{2}\), and H\({}_{2}\) in presence of light, but with a low overall yield of solar energy conversion. In a similar way, but without light, dark fermentation uses various substrates and waste too, leading to the release of different components and gaseous mixtures in which hydrogen is present [46; 54]. It has recently been observed that hydrogen production can be increased by up to 60% compared to _Chlamydomonas reinhardtii_ monoculture systems, by using co-culture systems with _Escherichia coli_. Growth media glucose-rich are exploited by bacteria that produce acetic acid, which can be used in algal metabolism [55]. Synergistically, different photobiological and fermentative microbial metabolisms may interact and cooperate increasing the hydrogen yields [56].  
Microalgae show an enormous biodiversity being present in different habitats, even extreme and hostile to most living organisms, suitably adapting their metabolism. It is therefore possible to modulate the growth conditions to obtain biomass of the desired composition, based on the requirements of the downstream process also using industrial and agricultural processing water and waste. In this perspective, various strategies have been applied by combining bioenergy production and bioremediation approaches [57; 58]. Unicellular green alga _Scenedesmus obliquus_ managed to biodegrade the phenolic content present in the olive oil mill wastewater. This strategy makes it possible to remedy a problem particularly encountered in the Mediterranean area and, since the biotransformation carried out consumes oxygen, favorable conditions to trigger a concomitant production of hydrogen are also generated [59]. A consortium of microalgae, mainly composed of _Scenedesmus_ and _Chorella_ species, grown in pig manure showed good growth, without the addition of external nutrients, and significant fermentative hydrogen production [60; 61].  
One of the main problems associated to these approaches is the elevated costs in terms of management and purification of the components obtained, which include the presence of several by-products, also toxic. Moreover, although the biomass of the microalgae contains a reduced lignin content compared to other lignocellulosic feedstock previously used for energy purposes, preliminary treatments are often necessary to facilitate the extraction and conversion of the microalgae content. Mechanical, thermal, chemical, or biological treatments are often applied to biomass separately or in combination as a preliminary step [57; 58].