| Summary: | The alga Dunaliella salina possesses the unique ability to accumulate up to 14 % of it's dry weight as β-carotene in response to stress conditions. This hyper-accumulation of β-carotene has led to the commercial exploitation of this alga for the biotechnological production of this important carotenoid. In order to maximise β-carotene production, a dual-stage process which separates the distinctive growth phase from the β-carotene accumulating stress phase has recently been patented. Preliminary laboratory studies showed that although stress factors such as high salinity and nutrient limitation enhance β-carotene accumulation in D. salina (± 10 pg.cell⁻¹), high light intensity was the single most important factor contributing to the induction of β-carotene accumulation in this alga (± 20 pg.cell⁻¹). Moreover, it was demonstrated that β-carotene accumulation can be further stimulated by exposing the alga to a combination of high light intensity, salt and nutrient stresses (± 30-60 pg.cell⁻¹). The response of D. salina to stress was shown to occur in two phases. The first phase occurred within 24 hours and was characterized most importantly by higher rates of β-carotene accumulation for all the stresses investigated. In cells exposed to multiple stress factors in mass culture, the β-carotene accumulation rate was as much as 9.5 pg.cell⁻¹.day⁻¹ in the first phase compared to only 3 pg.cell·day⁻¹ in the second phase. Since the rate of β-carotene accumulation was higher within the first 24 hours after exposure to stress, the first phase was considered crucial for stress-induced β-carotene accumulation. Characterization of this phase revealed that the stress response was multifaceted. Transition of cells from the growth stage to stress conditions was characterized by the following: 1) Change in cell volume. Hypersalinity caused cell shrinkage while cells exposed to nutrient limitation and/or high light intensity caused cells to swell. Restoration of cell volume was shown to occur within 8 hours for all stresses investigated. 2) Altered photosynthesis. Inhibition of both carbon fixation and oxygen evolution was demonstrated in cells immediately after exposure to multiple stress factors. 3) Production of abscisic acid. Intracellular ABA levels increased within 6-8 hours after exposure to all stresses investigated. The rise in intracellular ABA levels coincided with an increase or return to starting cell volume. High intracellular ABA levels were however transient and within 24 hours ABA began to partition into the culture medium. 4) Change in pigment composition. Changes in xanthophyll cycle pigment content was demonstrated soon after exposure to stress conditions. In hypersalinity shocked cells, initial epoxidation of zeaxanthin to violaxanthin and subsequent de-epoxidation to zeaxanthin occurred, whereas exposure to high stress resulted in an immediate and continued decrease in the epoxidation state indicating accumulation of zeaxanthin. A rapid rate of chlorophyll depletion was also demonstrated. In addition to the above responses a rapid decrease in growth rate during this phase was also noted. An interrelationship between cell volume change, ABA production, maintenance of xanthophyll cycle operation and β-carotene accumulation therefore appeared to exist. ABA production was shown to be stoichiometrically related to changes in xanthophyll content with r² = 0.84 and slope of the curve = 0.91 being achieved for high light stressed cells. This study therefore presents strong circumstantial evidence in support of a carotenoid origin for ABA in Dunaliella. In addition, enhanced β-carotene content was achieved by the application of exogenous ABA and related compounds suggesting a role for ABA as a regulator of the overall stress response. Furthermore, zeaxanthin accumulation was shown to be positively correlated ( r²≥ 0.81) to β-carotene accumulation for all the stresses investigated. The second phase was characterized by a return to homoeostasis of the physiological processes mentioned above, indicating acclimation of the cell to prevailing conditions. This stage was characterised by continued β-carotene accumulation and a decreased epoxidation state of the xanthophyll cycle which together appeared to sustain photosynthesis, allowing this organism to tolerate stress conditions.
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