# Biohydrogen from Microalgae: Production and Applications
## 3 The Role of Growth Parameters in Hydrogen Production  
Acting on cultivation parameters is the simplest way to produce immediate effects on the growth or the specific metabolite production. Regarding media components, the aforementioned S deprivation gives the expected effects only if preceded by a photosynthetic growth phase, providing all of the necessary elements for growth. More recently, alongside this strategy, which envisages separate growth and a deprivation step with several operational issues, a different modality that involves minimal quantities of S to simulate a condition of starvation has also been tested [25; 26]. Moreover, particular attention should be paid to the carbon source used: it has been observed that an increase in starch reserves during the phase preceding S deprivation can be crucial in supporting production. Mixotrophic conditions involving both inorganic and organic carbon sources appear to be preferred. [27; 28]. The less studied condition of nitrogen (N) deprivation induces hydrogen production in _Chlamydomonas reinhardtii_, albeit with a delayed effect and lower yields, suggesting a different mechanism in establishing process [29]. In _Scenedesmus obliquus_, N deprivation modulates metabolism towards lipids accumulation to be used as an energy reserve, suggesting that this strategy is not advantageous for the purposes of hydrogen production [30]. In ma rine _Chlorella_ spp., where S deprivation is inapplicable due to the sulfates-rich presence in seawater, the effect of phosphorus (P) deficiency was evaluated. Similarly, P deprivation was able to establish anaerobic metabolism with a sustained hydrogen photoproduction, even if low-density culture were required to reduce the effect linked to cellular P reservoirs [31]. Less common element depriations, such as magnesium (Mg), were also tested. Mg-deprived _Chlamydomonas reinhardtii_ cells exhibited a longer hydrogen production over time than the same cells in the case of S deprivation. This response may depend on the lesser Mg importance for cellular activities compared to S [32]. Various chemical compounds added in the cell media can affect protons and electrons mobilization inside the cell and, consequently, hydrogen metabolism. The protonophores carbonyl cyanide m-chlorophenylhydrazone (CCCP) and 2,4-dinitrophenol (DNF) increase hydrogen production, owing to proton mobilization [33]. The action of other similar decoupling agents, such as 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DMCU) and carbonyl cyanide p-trifluoromethyloremethoxy phenylhydrazone (FCCP) allow to stimulate hydrogen production through the PS II independent pathway, although the underlying mechanisms are not deeply understood [34]. Parameter changes to produce hydrogen require several operational steps, especially during cell transfer upon different media. To handle culture more easily, some attempts have demonstrated the feasibility of cell cultivation through different immobilization systems [35]. Most of the attempts have been applied in _Chlamydomonas reinhardtii_ through the use of fiberglass matrix, thin alginate films, or, more recently, sodium alginate beads. In particular, the latter study selected beads of few millimiters diameter to further investigate the process in the classical photobioreactors, bringing out the need to adapt bioreactors to new immobilization systems [36; 37; 38]. Indeed, photobioreactor design must take into account downstream applications and process needs. Closed systems are used in lab-scale hydrogen production experiments and represent a better alternative to open systems. Growth parameters, especially temperature and pH, are strictly monitored and, at the same time, the collection of the gas produced is less subject to dispersions. Particular importance is attached to the agitation mode and the intensity and quality of the light radiation. Vertical column, tubular, and flat panel, are the most investigated photobioreactor modalities for hydrogen production.