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Poly-β-hydroxybutyrate (PHB) in cyanobacteria, which accumulates as energy and carbon sources through the action of photosynthesis, is expected to substitute for petroleum-based plastics. This study first demonstrated that PHB accumulation was induced, with the appearance of lipid droplets, in sulfur (S)-starved cells of a cyanobacterium, Synechocystis sp. PCC 6803, however, to a lower level than in nitrogen (N)- or phosphorus (P)-starved cells. Concomitantly found was repression of the accumulation of total cellular proteins in the S-starved cells to a similar level to that in N-starved cells, and a severer level than in P-starved cells. Intriguingly, PHB accumulation was induced in Synechocystis even under nutrient-replete conditions, upon repression of the accumulation of total cellular proteins through treatment of the wild type cells with a protein synthesis inhibitor, chloramphenicol, or through disruption of the argD gene for Arg synthesis. Meanwhile, the expression of the genes for PHB synthesis was hardly induced in S-starved cells, in contrast to their definite up-regulation in N- or P-starved cells. It therefore seemed that PHB accumulation in S-starved cells is achieved through severe repression of protein synthesis, but is smaller than in N- or P-starved cells, owing to little induction of the expression of PHB synthesis genes.

Triacylglycerols (TG), the major components of crop oils used for the production of food oils and biodiesel fuel, accumulate in algal species under single environmental stress conditions including nitrogen-starvation. In a freshwater green alga, Chlorella kessleri, its TG content reached 24.7% in dry cell weight in 72 h, on cultivation in 3-fold diluted seawater (1/3 SW), through the synergetic stimulating effects of hyperosmosis and nutrient-limitation. These observations would indicate the importance of combinatory stresses for strong induction of TG accumulation [Hirai et al. 2016]. We here searched for novel stresses in C. kessleri to elevate the seawater-based TG accumulation level through their additional actions. A higher light intensity (moderate light, ML) caused a small, but definite increase of TG, whereas a lowered growth temperature (LT) led to a large, but temporal increase of it until 48 h, followed by severe degradation. The decrease of TG in LT cells was abolished on simultaneous imposition of ML. C. kessleri cells were then cultured in 1/3 SW under combinatory conditions of ML/LT, the TG content consequently increasing to as high as 48.5% in dry cells. These results indicated the validity of combinatory stresses for strong induction of algal TG accumulation. As to the fatty acid composition of TG, C. kessleri was intermediate between palm and the other three major oil crops including rapeseed. The rapid and abundant accumulation of TG with fatty acid composition suitable for industrial use demonstrated the great potential of development of the industrial algal TG production system with the use of the combinatory stresses in C. kessleri. TG of C. kessleri was also characteristic to contain C18 acids as representing 81 mol% of its constituent fatty acids or 90% of fatty acids at its sn-2 position, which suggested a peculiar TG synthetic pathway in this oleaginous alga.

Triacylglycerols of oleaginous algae are promising for production of food oils and biodiesel fuel. Air-drying of cells induces triacylglycerol accumulation in a freshwater green alga, Chlorella kessleri, therefore, it seems that dehydration, i.e., intracellular hyperosmosis, and/or nutrient-limitation are key stressors. We explored this possibility in liquid-culturing C. kessleri cells. Strong hyperosmosis with 0.9 M sorbitol or 0.45 M NaCl for two days caused cells to increase the triacylglycerol content in total lipids from 1.5 to 48.5 and 75.3 mol%, respectively, on a fatty acid basis, whereas nutrient-limitation caused its accumulation to 41.4 mol%. Even weak hyperosmosis with 0.3 M sorbitol or 0.15 M NaCl, when nutrient-limitation was simultaneously imposed, induced triacylglycerol accumulation to 61.9 and 65.7 mol%, respectively. Furthermore, culturing in three-fold diluted seawater, the chemical composition of which resembled that of the medium for the combinatory stress, enabled the cells to accumulate triacylglycerol up to 24.7 weight% of dry cells in only three days. Consequently, it was found that hyperosmosis is a novel stressor for triacylglycerol accumulation, and that weak hyperosmosis, together with nutrient-limitation, exerts a strong stimulating effect on triacylglycerol accumulation. A similar combinatory stress would contribute to the triacylglycerol accumulation in air-dried C. kessleri cells.