Several unicellular green algae are capable of generating hydrogen through their [FeFe]-hydrogenase enzyme by reducing water protons to molecular hydrogen. However, given the sensitivity of [FeFe]-hydrogenase to oxygen, which is gener­ated by photosystem II (PSII), new approaches have been developed for increasing the practical application of microalgae for biohydrogen production (Laurinavichene et al. 2008). For instance, higher hydrogen production efficiencies were achieved by growing the microalgal cells under sulfur-deprived conditions (Melis et al. 2000). Sulfur deprivation causes partial inactivation of PSII, which is responsible for O2 generation, resulting an enhanced synthesis of [FeFe]-hydrogenase enzyme (Laurinavichene et al. 2008).

Immobilization processes have been proposed by several researchers for enhancing hydrogen production by sulfur-deprived microalgae and also allowing an easy exchange step between “sulfur-replete” and “sulfur-depleted” stages of the experiment (Laurinavichene et al. 2006, 2008). Several challenges require addressing for scaling up the current hydrogen production systems, while immo­bilization processes offer an alternative approach to the current technology. Immobilized cells were also reported to have higher light utilization efficiencies per area and higher cell densities (Kosourov and Seibert 2009).

In a study by Kosourov and Seibert (2009), C. reinhardtii cells were immobi­lized inside alginate films for the photoproduction of hydrogen. The cells were previously deprived of sulfur and phosphorus nutrients before being entrapped inside the alginate films. They observed higher cell densities and specific hydrogen production rates after the immobilization process. An immobilization strategy also provided easy protection of the hydrogenase enzyme from oxygen inhibition, yielding higher hydrogen production rates compared to the free cells.

Laurinavichene et al. (2006, 2008) used immobilized C. reinhardtii cells on a fiber glass matrix under sulfur-deprived conditions and observed a prolonged hydrogen production phase for the immobilized cells, while the specific hydrogen production rate was similar to the free-cell counterparts. In another study, algal cells were immobilized on fumed silica particles, which had similar hydrogen production rates with the suspended cultures (Hahn et al. 2007). Song et al. (2011) recently used agar-immobilized Chlorella sp. cells for a two-stage cyclic hydrogen pro­duction involving the oxygenic photosynthesis followed by anaerobic incubation under sulfur-deprived conditions.