Two marine Chlorella strains, Chlorella pyrenoidosa 689S and C. pyrenoidosa 707S, were previously found to
generate H2 gas under sulfur (S) deprivation. In this work, their ability to generate H2 under sulfur-replete,
artificial and natural seawater conditions was further explored. C. pyrenoidosa 689S and 707S evolved 20.77
and 25.51 ml/l H2, respectively, in artificial seawater (ASW) without any nutrient deprivation, simply by adding
acetic acid. Sulfur or nitrogen (N) deprivation promoted H2 production by C. pyrenoidosa 689S and 707S incubated
in ASW, while phosphorous (P) deprivation did not improve H2 production effectively. Changes of culture
medium from ASW-S to TAP′-S induced an increase of ~14% and 200% in H2 yield in C. pyrenoidosa 689S and
707S respectively. However, the Chlorella cells did not grow well in TAP′ medium, which contains NH4Cl as nitrogen
source, at approximately the same molar concentration as the nitrogen in ASW medium, as indicated by the
low chlorophyll content in TAP′-S cultures. When natural seawater was used as the water source for culture
medium instead of the ASW prepared using distilled water, the H2 production by C. pyrenoidosa 689S and 707S
was significantly altered. Both Chlorella strains failed to transit to the anaerobic phase and evolved no H2 in L1
medium containing acetate prepared using natural seawater, implying that some unknown factor(s) in natural
seawater might hinder the establishment of anaerobiosis and activation of H2ase. Even N and P deprivation did
not induce H2 production in C. pyrenoidosa 689S. Although C. pyrenoidosa 707S was not able to evolve H2
under P-deprivation conditions in natural seawater medium, it was found, surprisingly, to generate comparable
amounts of H2 in N-free medium prepared using natural seawater as in N-free ASW medium, showing that the
strain might be useful for biological H2 photoproduction.