Optimization of DHA and astaxanthin production by Schizochytrium sp.

isolated from mangrove forests in Thailand

Rujiralai Poontawe1, Tsunehiro Aki

2 and Wichien Yongmanitchai

1

1Department of Microbiology, Faculty of Science, Kasetsart University 2Department 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 N-

methyl-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 ºC 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 ºC to 168 h at 12 ºC. 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 12ºC, 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).

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

Incubation Time (hrs)

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

Cell dry weight (g l-1)

0

2

4

6

8

10

12

14

16

18

Sugar remaining (%)

0

1

2

3

4

5

Asataxanthin production (m

g l-1)

0

1

2

3

4

5

6

DHA production (m

g l-1)

0

1000

2000

3000

4000

5000

6000

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. �)

Glucose concentration (%)

12°

C