Environmental conditions such as temperature, pH, and salinity play a critical role in the efficiency and stability of photobiological hydrogen production by algae and cyanobacteria. These microorganisms rely on photosynthesis to drive hydrogen generation, and their metabolic pathways are highly sensitive to external factors. Understanding how these variables influence hydrogen yield is essential for optimizing production systems and scaling up sustainable biohydrogen technologies.

Temperature is a key factor affecting the growth and hydrogen production of photosynthetic microorganisms. Each species has an optimal temperature range for metabolic activity, and deviations can significantly impact performance. For example, green algae such as Chlamydomonas reinhardtii exhibit peak hydrogen production between 25°C and 30°C. Temperatures above 35°C can inhibit photosynthesis by damaging the photosystem II (PSII) complex, reducing electron flow to hydrogenase enzymes. Conversely, low temperatures slow enzymatic activity, decreasing the overall metabolic rate. Cyanobacteria like Synechocystis sp. PCC 6803 show similar trends, with optimal hydrogen production occurring around 30°C. Some species have evolved heat-shock proteins (HSPs) to mitigate thermal stress, stabilizing enzymes and cellular structures under suboptimal conditions.

pH levels influence the proton gradient across thylakoid membranes, directly affecting photosynthetic efficiency and hydrogenase activity. Most algae and cyanobacteria prefer neutral to slightly alkaline conditions (pH 7–9). Acidic environments (pH < 6) disrupt membrane integrity and inhibit the function of hydrogen-producing enzymes. In Chlamydomonas, low pH reduces the activity of [FeFe]-hydrogenases, which are responsible for catalyzing proton reduction. Alkaline conditions (pH > 9) can also impair growth by altering nutrient availability and inducing oxidative stress. Some cyanobacteria, such as Nostoc sp., employ pH homeostasis mechanisms, including proton pumps and buffering molecules, to maintain internal stability under fluctuating external pH.

Salinity stress affects osmotic balance and ion homeostasis, which are critical for maintaining photosynthetic activity. High salt concentrations can lead to water loss, plasmolysis, and oxidative damage. For instance, Dunaliella salina, a halotolerant green alga, produces glycerol as an osmoprotectant to counteract high salinity. However, excessive salt levels (> 1.5 M NaCl) still suppress hydrogen production by disrupting electron transport chains. Freshwater species like Chlamydomonas moewusii show severe growth inhibition at salt concentrations above 0.2 M NaCl. Cyanobacteria such as Arthrospira platensis possess salt-responsive genes that regulate compatible solute synthesis (e.g., glycine betaine) to mitigate osmotic stress.

Light intensity interacts with these environmental factors, further modulating hydrogen output. Under high light, excess energy can lead to photoinhibition, damaging PSII and reducing electron supply for hydrogenase. Many algae activate non-photochemical quenching (NPQ) to dissipate surplus energy as heat, protecting the photosynthetic apparatus. In contrast, low light limits the reducing equivalents available for hydrogen production. Some species adjust pigment composition or increase light-harvesting antennae to optimize light capture under suboptimal conditions.

Nutrient availability, particularly sulfur and nitrogen, also plays a role in stress responses. Sulfur deprivation is a common method to induce hydrogen production in algae by downregulating PSII repair and redirecting electrons to hydrogenases. However, prolonged sulfur deficiency leads to growth arrest and eventual culture collapse. Nitrogen limitation triggers similar metabolic shifts but may also induce lipid accumulation as a survival strategy, competing with hydrogen production for reducing power.

Oxidative stress is a common consequence of environmental fluctuations, generating reactive oxygen species (ROS) that damage cellular components. Algae and cyanobacteria employ antioxidant systems, including superoxide dismutase (SOD), catalase, and ascorbate peroxidase, to scavenge ROS. The balance between ROS production and detoxification influences hydrogen yield, as excessive oxidative stress can irreversibly damage hydrogenase enzymes.

In large-scale cultivation, diurnal and seasonal variations introduce additional challenges. Temperature and light fluctuations require robust strains capable of acclimating to dynamic conditions. Some researchers have explored mixed cultures or genetically modified strains with enhanced stress tolerance to improve resilience. For example, introducing heterologous genes for heat-shock proteins or osmoprotectant synthesis has shown promise in stabilizing hydrogen production under adverse conditions.

Despite these challenges, photobiological hydrogen production remains a promising avenue for sustainable energy. Advances in bioreactor design, such as temperature and pH control systems, can mitigate environmental stressors. Additionally, selecting or engineering strains with broad tolerance ranges could enhance performance in outdoor systems exposed to variable climates.

In summary, temperature, pH, and salinity profoundly influence photobiological hydrogen production by affecting photosynthetic efficiency, enzyme activity, and cellular homeostasis. Stress-response mechanisms in algae and cyanobacteria provide critical adaptations, but extreme conditions still pose significant barriers. Optimizing these environmental parameters, alongside genetic and engineering approaches, will be essential for advancing biohydrogen as a viable renewable energy source.