Green algae, particularly *Chlamydomonas reinhardtii*, have emerged as a promising biological system for photobiological hydrogen production. Unlike conventional methods that rely on fossil fuels, this approach leverages photosynthesis to convert solar energy into hydrogen gas, offering a sustainable and carbon-neutral alternative. The process hinges on the unique metabolic flexibility of green algae, which can switch between oxygenic photosynthesis and anaerobic hydrogen production under specific conditions. The sulfur-deprivation method is a key technique used to induce hydrogen production, exploiting the algae's response to nutrient stress to create anaerobic conditions necessary for hydrogenase enzyme activity.

The mechanism of hydrogen production in *Chlamydomonas reinhardtii* involves a two-stage process. In the first stage, the algae perform normal oxygenic photosynthesis, using light energy to split water into oxygen, protons, and electrons. This stage sustains cell growth and builds up stored carbohydrates, primarily in the form of starch. The second stage is triggered by sulfur deprivation, which disrupts the repair cycle of Photosystem II (PSII), a critical component of the photosynthetic apparatus. As PSII activity declines, oxygen production ceases, and the algae transition to anaerobic metabolism. In the absence of oxygen, the hydrogenase enzymes, which are highly sensitive to oxygen, become active. These enzymes catalyze the recombination of protons and electrons to form molecular hydrogen, using reduced ferredoxin as an electron donor. The electrons are derived from the breakdown of stored carbohydrates through pathways such as glycolysis and the plastidial oxidative carbon metabolism.

Sulfur deprivation is a well-studied method to induce hydrogen production because sulfur is essential for the synthesis of PSII proteins, particularly the D1 subunit, which undergoes constant photodamage and repair. By removing sulfur from the growth medium, the algae cannot repair PSII, leading to its gradual inactivation. This creates a self-limiting system where oxygenic photosynthesis declines, and the culture becomes anaerobic. The transition typically occurs over 24 to 48 hours, depending on light intensity and cell density. Once anaerobic conditions are established, hydrogen production can continue for several days, though at declining rates as the stored carbohydrates are depleted.

The efficiency of hydrogen production in this system is influenced by multiple factors. Light intensity plays a critical role, as excessive light can cause photodamage, while insufficient light limits electron supply. Temperature and pH must be optimized to maintain enzyme activity and metabolic flux. The initial starch content of the cells is another determinant, as it provides the substrate for sustained hydrogen production. Under optimal conditions, *Chlamydomonas reinhardtii* can achieve hydrogen evolution rates of up to 2-4 mmol per liter of culture per hour, though these rates are still far below theoretical maxima.

Scalability remains a significant challenge for algal hydrogen production. One limitation is the inherent inefficiency of the process, as only a fraction of the absorbed light energy is converted into hydrogen. The requirement for anaerobic conditions also complicates large-scale cultivation, as maintaining uniform sulfur deprivation in open ponds or photobioreactors is difficult. Additionally, hydrogenase enzymes are highly sensitive to oxygen, and even trace amounts can irreversibly inhibit their activity. This sensitivity necessitates careful control of the culture environment, increasing operational complexity and cost.

Metabolic engineering offers potential solutions to improve the efficiency and scalability of algal hydrogen production. One strategy involves enhancing the electron supply to hydrogenases by redirecting metabolic fluxes. For example, reducing competing pathways such as cyclic electron flow around Photosystem I or alternative oxidase activity can increase the availability of electrons for hydrogen production. Another approach is to engineer more oxygen-tolerant hydrogenases, either by modifying native enzymes or introducing heterologous variants from other organisms. Advances in synthetic biology have enabled the design of algal strains with optimized starch accumulation, ensuring a sustained substrate supply during the hydrogen production phase.

Another promising direction is the decoupling of hydrogen production from growth phases. By separating the biomass accumulation and hydrogen production stages, it may be possible to achieve higher overall yields. For instance, algae could be grown under optimal conditions to maximize starch accumulation, then transferred to sulfur-deprived conditions for hydrogen production. This two-phase system could be further refined by using immobilized cells or biofilm-based cultures, which simplify the transition between stages and reduce energy inputs.

Despite these advancements, the economic viability of algal hydrogen production remains uncertain. The energy input required for cultivation, harvesting, and processing must be carefully balanced against the hydrogen output. Life cycle analyses suggest that significant improvements in efficiency and reductions in cost are needed to compete with other renewable hydrogen production methods, such as electrolysis powered by wind or solar energy. However, the potential for continuous hydrogen production using sunlight and water as the primary inputs makes this an area of ongoing research interest.

In summary, hydrogen production in *Chlamydomonas reinhardtii* relies on a carefully orchestrated interplay of photosynthesis, nutrient stress, and anaerobic metabolism. The sulfur-deprivation method provides a practical means to induce hydrogenase activity, though scalability and efficiency barriers persist. Metabolic engineering and process optimization hold promise for overcoming these challenges, but further research is needed to translate laboratory successes into commercially viable systems. The integration of algal hydrogen production into broader renewable energy frameworks will depend on advances in both biological and engineering disciplines, ensuring that this natural process can contribute meaningfully to a sustainable hydrogen economy.