Carbon metabolism plays a central role in photobiological hydrogen production by algae and cyanobacteria, as it directly influences the availability of electrons and ATP required for hydrogenase or nitrogenase activity. The interplay between carbon fixation, storage, and utilization determines the efficiency of hydrogen generation, with metabolic pathways such as the Calvin cycle and glycogen metabolism acting as key regulators. Understanding and manipulating these pathways through metabolic flux analysis and genetic engineering can enhance hydrogen yields by redirecting carbon flux away from storage and toward hydrogen-producing mechanisms.
In oxygenic photosynthetic microorganisms, light energy drives water splitting at Photosystem II, generating electrons, protons, and oxygen. These electrons are transferred through the photosynthetic electron transport chain to ferredoxin, which can donate them to hydrogenases or nitrogenases for hydrogen production. However, carbon metabolism competes for these reducing equivalents, as the Calvin cycle consumes ATP and NADPH to fix CO₂ into organic compounds. Under normal conditions, a significant portion of photosynthetic output is allocated to biomass accumulation or stored as glycogen or starch, limiting electron availability for hydrogen evolution.
Metabolic flux analysis reveals that carbon partitioning between the Calvin cycle and glycogen synthesis significantly impacts hydrogen production. In cyanobacteria, glycogen serves as a major carbon reserve, synthesized from glucose-1-phosphate via glycogen synthase. During the day, carbon is fixed and stored as glycogen, while at night, glycogen is catabolized to sustain respiration and other metabolic processes. This diurnal cycling creates a bottleneck for continuous hydrogen production, as glycogen breakdown does not inherently prioritize hydrogenase activity. Redirecting carbon flux away from glycogen synthesis and toward hydrogen generation requires suppressing glycogen formation or enhancing its catabolism under hydrogen-producing conditions.
Genetic engineering approaches have been employed to modulate carbon allocation. Deletion of glycogen synthase genes in cyanobacteria such as Synechocystis sp. PCC 6803 or Synechococcus elongatus PCC 7942 disrupts glycogen accumulation, forcing fixed carbon into alternative pathways. Studies show that glycogen-deficient mutants exhibit increased hydrogen production under certain conditions, as more reductants are available for hydrogenases. However, complete glycogen elimination can impair cell viability under stress, indicating a need for balanced metabolic engineering. Fine-tuning glycogen metabolism by partial suppression or conditional gene knockdowns may offer a more sustainable solution.
Another strategy involves manipulating the Calvin cycle to reduce carbon fixation competition. Overexpression of glycolytic enzymes or downregulation of Calvin cycle components can shift metabolic flux toward pathways that supply pyruvate or other substrates to hydrogen-producing enzymes. For example, reducing the activity of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) decreases carbon fixation, potentially freeing up NADPH for hydrogenases. However, this must be carefully balanced to avoid severe growth defects.
Photorespiration also influences carbon metabolism and hydrogen production. In algae, the oxygenase activity of RuBisCO produces 2-phosphoglycolate, which is metabolized through the photorespiratory pathway, consuming energy and releasing CO₂. Engineering strategies that minimize photorespiration, such as introducing alternative glycolate metabolic pathways, can improve overall photosynthetic efficiency and increase the availability of reductants for hydrogen evolution.
Metabolic flux analysis has identified key nodes where carbon diversion can enhance hydrogen yields. One critical node is the partitioning of pyruvate, which can either enter the tricarboxylic acid cycle for biomass production or serve as a substrate for pyruvate:ferredoxin oxidoreductase (PFOR), generating reduced ferredoxin for hydrogenases. Overexpressing PFOR or inhibiting competing pyruvate-consuming enzymes can direct more carbon toward hydrogen production. Similarly, enhancing the oxidative pentose phosphate pathway increases NADPH availability, which can indirectly support hydrogenase activity.
In green algae such as Chlamydomonas reinhardtii, hydrogen production occurs mainly under anaerobic conditions induced by sulfur deprivation, which arrests oxygen evolution and activates hydrogenase expression. Here, carbon metabolism shifts from aerobic respiration to fermentation, with starch degradation providing substrates for hydrogen generation. Metabolic engineering of starch metabolism—such as increasing starch breakdown rates or altering its composition—can improve hydrogen output. For instance, overexpression of amylases or debranching enzymes accelerates starch mobilization, supplying more carbon for hydrogen production.
Cyanobacteria employ nitrogenases for hydrogen production under nitrogen-limiting conditions, a process heavily dependent on carbon metabolism. Nitrogenase activity requires large amounts of ATP and reducing equivalents, which are supplied by glycogen catabolism and respiratory electron transport. Engineering cyanobacteria to optimize glycogen utilization for nitrogenase activity—such as by coupling glycogen breakdown directly to nitrogenase electron supply—can enhance hydrogen yields. Additionally, reducing the carbon sink into phycobilisomes or other non-essential polymers can increase metabolic flexibility for hydrogen production.
Dynamic metabolic modeling and synthetic biology tools are advancing the precision of carbon flux redirection. Systems biology approaches integrate transcriptomic, proteomic, and metabolomic data to identify regulatory nodes that control carbon partitioning. CRISPR-based gene editing enables targeted modifications of these nodes without introducing unintended metabolic imbalances. For example, tuning the expression of global regulators such as NtcA in cyanobacteria or PSR1 in algae can coordinately adjust carbon storage and utilization pathways to favor hydrogen production.
A major challenge in engineering carbon metabolism for hydrogen production is maintaining cellular energy balance. Overly aggressive redirection of carbon flux can deplete ATP or NADPH pools, impairing cell growth and viability. Strategies such as temporal separation of carbon fixation and hydrogen production, or the use of light-responsive genetic circuits, can help mitigate these issues. For instance, inducing hydrogenase expression only during high-light conditions, when excess reductants are available, can prevent metabolic overload.
Future directions include the development of hybrid pathways that merge synthetic and native metabolism to optimize carbon use. Introducing synthetic carbon fixation pathways with higher efficiency than the Calvin cycle could reduce energy expenditure and increase reductant availability. Similarly, engineering algae or cyanobacteria to secrete excess carbon as organic acids or other compounds could free up intracellular resources for hydrogen production without compromising cell health.
In summary, carbon metabolism is a critical determinant of photobiological hydrogen production, with glycogen storage and the Calvin cycle acting as major competing pathways. Metabolic flux analysis provides insights into key regulatory nodes, while genetic engineering enables targeted redirection of carbon toward hydrogen generation. Balancing these modifications to maintain cellular fitness remains a central challenge, but advances in systems and synthetic biology hold promise for unlocking higher hydrogen yields from photosynthetic microorganisms.