폐쇠 생태계 내에서 소포로리피드에 의한 Cochlodinium polykrikoides 저해 효과

백승학*, Xiao-Xia Sun**, 최중기***, 한경남***, 김은기*

*인하대학교 생물공학과

**Institute of Oceanology, Chinese Academy of Sciences

***인하대학교 해양학과

Mitigation of Cochlodinium polykrikoides bloom

by sophorolipid on mesocosm level

Seung-Hak Baek*, Xiao-Xia Sun**, Joong-Ki Choi***, Kyoung-Nam Han***

and Eun-Ki Kim*

*Department of Biological Engineering, Inha University, Incheon, Korea

**Institute of Oceanology, Chinese Academy of Sciences, Qingdao, China

***Department of Oceanography, Inha University, Incheon, Korea

1. Introduction

As a dominant harmful algal species, Cochlodinium polykrikoides has bloomed almost every year since 1995 in Korea, which brought great economic loss on the fishery industry. Previous studies on harmful algal blooms mitigation indicated that most of the traditional HAB mitigation methods were not applicable due to high cost, second pollution or impracticability (Anderson, 1997). Therefore, it is necessary to find an efficient and environment friendly way to mitigate the harmful effect of C. polykrikoides blooms. In a previous paper, a new method of using biosurfactant sophorolipid to mitigate harmful algal blooms was proposed and good effect was achieved in laboratory studies, which was considered as an promising strategy in future HABs mitigation due to the advantage of lower toxicity, biodegradability and ecological acceptability possessed by sophorolipid (Garcia-Ochoa et al. 1999). In order to investigate the mitigation effect of sophorolipid on mesocosm scale and explore the problems which may be occurred in field application, enclosed experiments were conducted during the bloom of Cochlodinium polykrikoides on Namhae sea in Korea in August, 2000. The results of mesocosm experiments further demonstrated the potential of sophorolipid in HABs mitigation.

Theories and Applications of Chem. Eng., 2002, Vol. 8, No. 1

2. Materials and Methods

화학공학의 이론과 응용 제8권 제1호 2002년

2.1 Production of sophorolipid

The yeast Candida bombicola ATCC22214 was maintained on agar slant and was transferred regularly. The standard medium for batch cultivation contained (per liter deionized water): glucose 100g, yeast extract 0.5g, KH2PO4 0.1g, MgSO4.7H2O 0.05g, CaCl2.2H2O 0.01g, NaCl 0.01g, peptone 0.07g, corn oil 100g. Cells were cultivated for 8 days in 2.5 liter jar fermentor at 25∩, 1vvm aeration rate and  pH 3.5. Sophorolipid was obtained by extracting the culture supernatant with equal volume of ethyl acetate.

2.2 Mesocosm design

As shown in Fig. 1, the enclosed mesocosm system was a cylinder with 1m in diameter and 2m in height. 5um mesh was covered around the cylinder so that the flow of seawater would be permitted and the dispersion of Cochlodinium polykrikoides would be prevented. Four plumbs weighed 2 kg respectively were attached symmetrically on the bottom of mesocosm system in order to make it balance in the seawater and the top of mesocosm equipment was hanged on the shipboard.

2.3 Experimental procedures

Surface tension: Surface tension was determined by surface tension meter at regular intervals on various layers after sophorolipid was spread on the surface layer in enclosed mesocosm system.

Motility inhibition: Immotile cells were counted with 1ml counting chamber under light microscope. Total cell numbers were determined by fixing the algal cells with Lugol solution. The motility inhibition rate was calculated as immotile/total cells.

Algicidal effect: Two mesocosm systems were used for determination of algicidal effect. After sophorolipid was spread, the numbers of normal, swelling and total cells were determined respectively.

Primary production: Primary production was determined through 14C assimilation method. Within in 10 minutes from the time the samples arrived on deck, they were subdivided into 60 ml clear and opaque bottles of polycarbonate and mixed with NaH14CO3 solution with an activity of 1uCi/ml. The bottles were then placed in surface water for 1h cultivation. Then the light and dark samples were filtered using GF/F filter paper. Filters were transferred to scintillation vials with 15 ml scintillation cocktail to determine dpm using Scintillation Counter. Total CO2 was determined according to Parsons et al. (1984).

3. Results and Discussion

3.1 Motility inhibition

Fig. 2 showed the motility inhibition effect of sophorolipid on C. polykrikoides. Similar to the results in lab experiments, the inhibition rates increased with contact time and sophorolipid concentration. When the concentration of sophorolipid was 10mg/L, the motility inhibition rate achieved more than 80%, indicating the lost of the moving ability of most algal cells. The concentration of 10mg/L sophorolipid was considered as an effective concentration for inhibiting the motility of C. polykrikoides.

3.2 Algicidal effect

The algicidal effect of sophorolipid was studied in two concentration groups: 50mg/L and 10mg/L respectively. As shown in Fig. 3, the numbers of normal and total cells were both decreased evidently under the high concentration of 50mg/L. For example, the concentration of normal cells decreased from the original 6000cells/ml to lower than 1000 cells/ml in 40min, which indicated that most cells became lysis under such concentration. Under the low concentration of 10mg/L sophorolipid (Fig. 4), there was still a large part of cells becoming swelling although the cell lysis decreased, indicating the effectiveness of lower sophorolipid concentration on algal cells.

3.3 Primary production

In order to investigate the effect of sophorolipid on C. polykrikoides, the primary production was determined at the same time. According to Fig. 5, the assimilation number decreased from the original 6mgC mgChl-a-1h-1 to 3.47 and 0.84 mgC mgChl-a-1h-1 after 1h at the concentration of 10 and 50mg/l sophorolipid respectively, indicating the reduction on primary production of algal cells.

3.4 Surface tension

As shown in Fig. 6, sophorolipid diffused rapidly to the deeper layer in 1min after the spreading of sophorolipid, which indicated that sophorolipid could become effective and inhibit the growth of algal cells efficiently in short time. From 1 to 10min, the surface tension of seawater increased gradually, demonstrating the decrease and disappearance of sophorolipid with the process of biodegradation and the flow of seawater. Above results suggested the potential of sophorolipid as a kind of promising anti-HABs agent without second pollution.

4. Conclusions

According to the results on mesocosm study, sophorolipid showed great potential in HABs mitigation. The concentration of 10mg/L sophorolipid could inhibit the motility, cell concentration and primary production of C. polykrikoides effectively, and the advantage of biodegradability and non-pollution made sophorolipid more suitable in application.

References

Anderson, D. M., 1997, Turning back the harmful red tide, Nature, 38: 513-514.

Babin, M., A. Morel and R. Gagnon, 1994, An incubator designed for extensive and sensitivity measurements of phytoplankton photosynthetic parameters. Limnol. Oceanogr., 39(3): 694-702.

F. Garcia-Ochoa, J. A. Casas, 1999, Unstructured kinetic model for sophorolipid production by Candida bombicola, Enzyme and Microbial Technology, 25: 613-621.

Parsons, T. R., Y. Maita and C. M. Lalli, 1984, A manual of chemical and biological methods for seawater analysis, Pergamon Press, pp. 3-28.

Platt, T., C. Gallegos and W. G. Harrison, 1980, Photoinhibition of photosynthesis in natural assemblages of marine phytoplankton. J. Mar. Res., 38: 687-701.

Fig. 1. Schematic diagram of enclosed mesocosm system

Fig. 2. Motility inhibition of sophorolipid against C. polykrikoides

(-●-, 20mg/L; -○-, 10mg/L; -▼-, 5mg/L)

Fig. 4 Change of cell numbers after treatment by 10mg/L sophorolipid (■ : normal cell, □ : swelled cell)

Fig. 3 Change of cell numbers after treatment by 50mg/L sophorolipid (■ : normal cell, □ : swelled cell)

Fig. 5 Effect of sophorolipid on the assimilation number of C. polykrikoides (-●-, 50mg/L; -○-, 10mg/L)

Fig. 6. Change on surface tension of seawater after addition of sophorolipid (-●-, 0m; -○-, 0.5m; -▼-, 1m; -▽-, 1.5m; -■-, 2m)