Optimization of DHA and astaxanthin production by Schizochytrium sp.

isolated from mangrove forests in Thailand

Rujiralai Poontawe1, Tsunehiro Aki2 and Wichien Yongmanitchai1 1 Department of Microbiology, Faculty of Science, Kasetsart University 2 Department Molecular Biotechnology, ADSM, Hiroshima University

Abstract Three hundred and fifteen thraustochytrids isolated from mangrove forests of Thailand were screened for docosahexaenoic acid (DHA) and astaxanthin production. The isolate SP62 showed the ability to produce large amounts of both DHA and astaxanthin. Improvement of this strain for the carotenoid pigment by ultraviolet (UV) radiation and Nmethyl-N-nitro-N-nitroso-guanidine (NTG) were carried out. Results reveal that mutant D616 showed higher astaxanthin accumulation than wild type. Cultivation under optimal conditions, i.e., medium containing 4.0% glucose, 0.5% peptone, 0.5% yeast extract, 0.07% ammonium chloride (C:N ratio approximately 20:1), 2.25% NaCl (w/v), initial pH at 5.0, continuous of light and temperature at 12 C for 168 h, D616 yielded the highest astaxanthin concentration at 5.09 mg l-1 and 542.31 mg l-1 of DHA which was 65.9% of total fatty acid (TFA). Introduction Thraustochytrids are common marine microheterotrophs, taxonomically aligned with heterokont algae (Lewis et al., 1999). Thraustochytrids are being explored as potential producer of PUFAs, especially docosahexaenoic acid (DHA) for nutritional enrichment of food products and use as feed additive in aquaculture. However, there have been several reports on accumulation of carotenoid pigments, astaxanthin in particular, for example, Schizochytrium aggregatum (Valadon, 1976), Schizochytrium KH105 (Aki et al.,2003) and Thraustochytrium sp. CHN-1 (Carmona et al., 2003). Objective Optimization of culture conditions for DHA and astaxanthin production by Schizochytrium sp. isolated from mangrove forests in Thailand. Results, Discussion and Conclusions Three hundred and fifteen thraustochytrids were obtained from mangrove forests in central region, Andaman Sea and Gulf of Thailand. SP62 isolate produced astaxanthin pigment higher than other isolates. Strain improvement by UV and NTG results in 315 mutants. Among them the mutant D616 showed the ability to produced astaxanthin higher than wild type strain. Both wild type and mutant strains were light dependent to induce astaxanthin accumulation. They also required surprisingly low temperature to enhance pigment production (Fig. 1). However, negative effect on growth rate was clearly seen as cultivation time was increase from 48 h at 30 C to 168 h at 12 C. Optimal condition for highest astaxanthin production include culture medium containing 4.0% glucose, 0.5% peptone, 0.5% yeast extract, 0.07% ammonium chloride (C:N ratio approximately 20:1), 2.25% NaCl (w/v) with initial pH 5.0. After cultivation for 168 h in shaking flask with continuous illumination from fluorescents lamp and temperature at 12C, D616 could accumulate 5.09 mg l-1 of astaxanthin and 5.42 g l-1 of DHA which was 65.9% of TFA. Cultivation in 2L fermentor took longer time to 264 h to obtain similar results (Fig. 2).

12 C Fig. 1 Effect of incubation temperature (6.0-23.5C) on astaxanthin content of D616

18 16

6000

Glucose concentration (%)

14

As a ta x a nthin p ro du c tion (m g l -1 )

5000

12 10

4000

3 8 6 4 1 2 2

3000

2000

1000

0 0 24 48 72 96 120 144 168 192 216 240 264

Incubation Time (hrs)

Fig. 2 Fermentation time course of D616 in 2 liter fermentor at 12 C with continuous illumination in medium containing 4.0% glucose, 0.5% peptone, 0.5% yeast extract, 0.07% ammonium chloride (C:N ratio approximately 20:1), 2.25% NaCl (w/v) with initial pH 5.0. (Growth ; DHA content ; astaxanthin accumulation and glucose conc. )

D H A p rod uc tio n (m g l -1 )

C e ll d ry w e igh t (g l -1 )

S ug a r re m a ining (% )