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Antibiotic substances produced by a marine green alga, Dunaliella primolecta
Bioresource Technology 44 (1993) 149-153
ANTIBIOTIC SUBSTANCES PRODUCED BY A MARINE GREEN ALGA, D UNALIELLA PRIMOLE CTA Thomas Chang, a Souichi Ohta, b, Naoto Ikegami, b Hideaki Miyata, b Takashi Kashimoto b & Masaomi Kondo b aFaculty of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565, Japan bFaculty of Pharmaceutical Sciences, Setsunan University, 45-1 Nagaotoge-cho, Hirakata, Osaka 573-01, Japan (Received 2 July 1992; revised version received 28 September 1992; accepted 5 October 1992) Abstract From among 84 marine algae, the green alga, Dunaliella primolecta C-525, exhibiting the highest antibiotic activity, was selected. A crude extract from this alga strongly inhibited the growth of Staphylococcus aureus, Bacillus cereus, Bacillus subtilis and Enterobacter aerogenes. From experiments on pH, solvent, and an inhibitor of the crude extract of this alga, the antibiotic substance in the algal cells was observed as a basestable, nonprotein material. To purify this antibiotic substance, a methanol extract of this alga was eluted successively on a DEAE-Sepharose CI-6B, silica and Sephadex LH-20 gel column. Three peaks exhibiting antibiotic activity from the final column were observed, indicating peak No. I to have a molecular weight of over 1300 and peaks Nos 2 and 3 to have molecular weights of about 300-400. The main fractions X, Y and Z of the three peaks were investigated for temperature stability. The activities of fractions X and Z were Stable up to 80°C, whereas the activity of fraction Y was unstable above 40°C. The above results indicate that algal cells of D. primolecta contained a number of different antibiotic substances.
aceutical products in microalgae (Matsueda et al., 1988; Shklar & Schwartz, 1988). In order to compete with other organisms in nature, algae are known to secrete toxic and/or growth-inhibitory substances under certain circumstances. This was first observed by Harder in 1917. Later, Pratt (1942) found antibacterial activity in a freshwater green alga, Chlorella sp. Since then, many bioactive compounds have been isolated from various algae (Berland, 1972; Mason et al., 1982) including fatty acids (Rosell & Strivastava, 1987), terpenes (Ishitsuka et al., 1988), bromophenols (Fujimoto et al., 1985) and halogenated compounds (Higgs, 1981). However, most of these compounds have been purified mainly from red or brown algae, and few have been isolated from green algae. Lustigman (1988) compared four ecotypes of Dunaliella sp. and observed antibiotic production by D. salina alone isolated from the seawaters of the high pollution area of San Francisco Bay, CA and Long Island Sound, New York in the USA. Here, we report the properties of antibiotic substances extracted from a marine green alga, Dunaliella primolecta.
Key words: Antibiotic substances, marine green alga, Dunaliella primolecta.
METHODS Algal strain and growth conditions Sixty-eight strains of marine algae were isolated from an enriched culture of marine samples (seawater, seaweeds and sediment) collected from the coastal areas of western Japan, and used throughout this study. These algae were grown in enriched seawater medium, and purified by agar-plate methods. Sixteen other strains of marine algae were obtained from three stock culture centres, the Institute of Applied Microbiology, University of Tokyo (IAM), Japan, Global Environmental Forum (GEF), Japan and American Type Culture Collection (ATCC), USA. A marine green alga, Dunaliella primolecta C-525 which was used in this study, was obtained from the IAM. For reproducible laboratory studies, the medium (ATCC medium No. 616) for D. primolecta C-525 was a modification of BG-11 medium (pH 8"0), made by the addition of NaCI, 20 g, vitamin Bt, 100/zg and vitamin B12, 1/zg in
INTRODUCTION Owing to their high growth rate, microalgae have long been recognized as a promising biomass source for the production of drugs, food and energy from light, CO 2 and minerals. The unicellular green algae, Chlorella and Dunaliella, as novel health food items, are cultivated commercially in large outdoor ponds (Borowitzka et al., 1984; Richmond, 1986). However, there are few studies which focus on the utilization of pharm*To whom correspondence should be addressed. Bioresource Technology 0960-8524/93/S06.00 © 1993 Elsevier Science Publishers Ltd, England. Printed in Great Britain 149
150
Thomas Chang, Souichi Ohm, Naoto Ikegami, Hideaki Miyata, Takashi Kashimoto, Masaomi Kondo
1-0 litre of the BG- 11 medium. The alga was grown at 25°C in 1.5-litre Roux flasks containing 1"0 litre of the medium, and harvested at late-log phase. The algal cultures were continuously illuminated by a bank of fluorescent lamps at a light intensity of 25 W/m 2. The cultures were continuously sparged with air containing 1% CO2 at a flow rate of about 300 ml/min. Preparation of crude extracts Crude extracts were prepared for activity testing from 84 algal cultures. Each algal culture was centrifuged (4000 g, 10 min) at 0°C. By the addition of distilled water to the pellets, the algal concentration was adjusted to 0"1 g fresh weight/mE Then, the algal suspension was sonicated (100 #A, 3 min) on ice by using an ultrasonic generator, and centrifuged. The supernatant was filtered through a membrane filter (0-22/~m, Millipore). The filtrate was designated as the crude extract. Antibacterial test The crude extracts were examined for antibacterial activity towards eight bacteria. This was determined by observing inhibition of their growth at 25°C on nutrient broth (Difco) agar plate for 24-48 h; 40 /~1 of algal crude extract (0-1 g fresh weight/ml) was transferred to an 8 mm paper disc, which was then applied to a plate seeded with a test strain. After incubation at 25°C for 24 h, the resulting zone of inhibition was measured. Distilled water was used as a control, as well as samples treated under various experimental conditions. No inhibition zone was observed with any of the control discs. Effects of pH, temperature and inhibitors To investigate the pH stability of the antimicrobial activity, sonicated algal cells were exposed to pH 2, 4, 6, 7, 9 and 12 at 25°C for 1 h, respectively. The samples were brought to pH 7, adjusted to the initial concentration (0" 1 g fresh wt/ml). After centrifugation and filtration, the antibiotic activity of the filtrate was measured by means of the disc test described above. The temperature stability was also investigated. Each sample of sonicated algal cells was incubated at 15, 25, 40, 60, 70, 80 and 100°C for 1 h, followed by centrifugation, filtration and testing as described above. To determine the effects of various inhibitors, an equal volume of the samples of sonicated algal cells and four inhibitors, ethylenediaminetetraacetate (EDTA), diethylpyrocarbonate (DEP), p-chloromercuribenzonic acid (PCMB) or HgC12 was mixed, adjusting to the concentration of algal cells adjusted to 0"1 g fresh wt/ml and the mixtures incubated at 25°C for 1 h. At this time, DEP was dissolved in 0" 1 U Tris-HC1 buffer (pH 7"0) adjusted to contain 50% dimethylsulphoxide, and other inhibitors were dissolved in 0"1 M Tris-HC1 buffer (pH 7-0). Each solvent buffer was also used as a control. The procedure after that was the same as for the pH-stability experiment.
Selection of solvents Algal cells (0-1 g fresh weight/ml) of D. primolecta were sonicated and freeze-dried. The antibiotic substance in the freeze-dried samples was then tested for solubility in different solvents. After centrifugation, the supernatants were freeze-dried again, and dissolved in distilled water, followed by filtration and testing as described in the case of the pH-stability experiment. Partial purification To purify the antibiotic substance in D. primolecta, algal cells were sonicated in a methanol solution. After centrifugation, the supernatants were partitioned with n-hexane to remove lipophilic compounds. The antibiotic substance in the methanol phase was concentrated and separated successively on three different columns filled with different gels, DEAE-Sepharose C1-6B (Pharmacia), silica (Merck) and Sephadex LH20 (Pharmacia). Each fraction eluted from the three columns was freeze-dried and dissolved in distilled water, followed by filtration and testing as described above. To investigate the temperature stability of the three fractions X, Y and Z containing antibiotic substances eluted from a Sephadex LH-20 column, they were incubated at 15, 25, 40, 60, 70 and 80°C for 1 h. The procedure after that was the same as that used for the temperature stability determination of the crude extract.
RESULTS AND DISCUSSION Antibiotic activity for bacteria In order to search for new antibiotic substances, 84 marine microalgae comprising 68 of isolated strains and 16 strains of stock culture were used; the algae could be grouped into seven classes, 35 strains of Cyanophyceae, 28 strains of Chlorophyceae, 11 strains of Bacilariophyceae, four strains of Dinophyceae, three strains of Rhaphidophyceae, two strains of Rhodophyceae, and one strain of Chryptophyceae. The antibiotic activity in the crude extract of all strains was tested against a Gram-negative bacterium, Escherichia Coli and a Gram-positive bacterium, Staphylococcus aureus. Although all crude extracts of the 84 strains exhibited no activity toward E. coil, the crude extracts of seven strains exhibited antibiotic activity toward S. aureus (Table 1). In particular, high activities were observed in crude extracts of Dunaliella bioculata C-523, Dunaliella primolecta C-525 (a stock culture strain) and our isolate Chlorococcum HS-101. Of these three strains, we finally chose the green alga D. primolecta C-525 because of its high activity and high growth rate. As shown in Table 2, crude extract from D. primolecta was tested against eight bacteria. Marked inhibition of the growth was observed for S. aureus, Bacillus subtilis, Bacillus cereus and Enterobacter aerogenes. Growth of four fungi, Cladosporium cladosporides, Penicillium funculosum, Paecilomyces variotii and Aspergillus niger was not inhibited (data not
Antibiotic substances ofDunaliella primolecta
shown). As strong growth inhibition of S. aureus was observed, antibiotic activity was compared with that of a commercial antibiotic substance, ampicillin. By using this relationship, the activity of crude extracts for S. aureus was represented in terms of an equivalent amount of ampicillin. Effects of pH and temperature on antibiotic activity T h e optimum conditions for activity of the antibiotic substance in D. primolecta C-525 were investigated by using S. aureus. Figure 1 shows the effect of p H on the stability of antibiotic activity in the crude extract of this alga. T h e activity was stable in the range of p H 6 - 1 2 , whereas that in the acidic range of p H 2 - 4 was depressed. T h e effect of temperature on the stability of the antibiotic activity in the crude extract of this strain was also investigated (Fig. 2). Activity of this substance was observed up to 80°C with an optimum between 25 and 40°C. No antibacterial activity was observed at 100°C. Effect of inhibitors on antibiotic activity Table 3 shows the effect of inhibitors on the antibiotic activity of crude extract of this strain. A chelator of metal ions ( E D T A ) and SH-blocking agents ( D E E PCMB, H g C l J were used as inhibitors. T h e activity was inhibited to 46% by 2 mM E D T A , but the effect of the other agents was not significant. This indicates that the expression of antibiotic activity involves a metal ion, and is probably not due to an enzyme reaction.
Solubility and stability of antibiotic substance for various solvents It is essential to choose a suitable solvent for extracting and further purifying the antibiotic substance in this strain. As shown in Fig. 3, the solubility and stability of
"2
150
T-
U 100
50 Z 0
Stock strains Dunafiella bioculata C-523 Dunaliella primolecta C- 525 Our isolates Chlorococcum sp. HS- 101 Chlorella sp. HS-109 Chlorella sp. HS-110 Synechococcus sp. HS-364 Phorphyridium sp. HS-366
Inhibition zone (mm)
17'8±2'6 11"8±1"6 14"8±3"6 11"9±2'1 11-5±1'7
Seven marine algae were grown at 25°C in a modified BG-11 medium. Dunaliella sp. C-523 and C-525 were obtained from the culture collection of Applied Microbiology, University of Tokyo. Values are the mean + s.d. of three experiments.
41
I
I
6
7
I9
| 12
pH
Fig. 1. Effect of pH on antibiotic activity of crude extract from the marine green alga, Dunaliella primolecta. Points are the mean + s.d. of three experiments.
"2
75
7 a~
17'4±3"3 18.7±2"1
I 2
Table 1. Comparison of antibiotic acitivity in the crude extracts towards S. aureus produced by seven marine algae
Strain
151
50
v
.; ;5 25 g Z L 15
25
40
60
70
80
100
Temperoture (*C)
Fig. 2. Effect of temperature on antibiotic activity of crude extract from the marine green alga, Dunaliella primolecta. Points are the mean + s.d. of three experiments.
Table 2. Antibacterial activities of crude extract from the marine green alga, Dunaliella primolecta
Gram-negative bacterium Escherichia col± Salmonella typhimurium Enterobacter aerogenes Vibrio parahaemolyticus
Inhibition zone (mm) NA NA 13'5 -+ 2.4 NA
Gram-positive bacterium Staphylococcus aureus Bacillus cereus Bacillus subtilis Micrococcus luteus
Inhibition zone (mm) 18"5 _+2"3 15"5 _+3"2 16"0 _+2"8 NA
40/~1 of algal crude extracts (0"1 g fresh weight/ml) were examined. Values are the mean + s.d. of three or more experiments. NA, no activity.
152
Thomas Chang, Souichi Ohta, Naoto Ikegami, Hideaki Miyata, Takashi Kashimoto, Masaomi Kondo
this substance were tested in various solvents. Independent of the solvent polarity, the activities were observed in all solvents except n-hexane. High activities were observed in alcoholic solvents such as methanol, iso-propanol, iso-butanol and n-butanol. Lustigman (1988) tested the solvent activity of the antibiotic substance of D. salina, and the antibiotic activities were observed in propanol and butanol, not methanol and ethanol. Further, with respect to the antibacterial spectrum or the stability of pH and temperature, our results also were not in accord with his results. Therefore, it was suggested that Dunaliella sp. can produce diverse antibiotic substances according to the algal growth environment. Finally, methanol was chosen for further extraction and purification of the antibiotic substance, because its boiling point is the lowest of the alcoholic solvents exhibiting high activity.
Table 3. Effect of inhibitors on antibiotic activity of crude extract from the marine green alga, Dunaliella primolecta Inhibitor None EDTA" DEP" PCMB" HgCI2
Conc. (mM)
Residual activity (%) h
1.0 2.0 1.0 2.0 1'0 2.0 0.04 0.1
100 + 11"3 83 + 7"9 46 + 12.8 92 + 11'4 108+9.5 92 + 9.7 100 + 10.3 100 + 12-5 100 _+13.1
"EDTA, ethylenediaminetetraacetate; DEE diethyl pyrocarbonate; PCMB, p-chloromercuribenzoic acid. b100% activity is equivalent to 124.7 Mg ampicillin/g fresh wt. Values are the mean + s.d. of three experiments.
Partial purification of antibiotic substance The antibiotic substance in the methanol extract of this alga was separated by D E A E Sepharose CI-6B, and then by silica gel chromatography. In both cases, the antibiotic activity was observed in the eluted fraction of acetone: methanol ( 10: 3). Further purification of the elute fractions from the silica gel column was carried out on a column of Sephadex LH-20 gel permeation (Fig. 4). Tests of Chloramphenicol (MW:323) and vitamin B~2 (MW: 1355) as molecular weight markers were also performed under the same elution conditions. Figure 4 shows antibiotic activity of each elute (2 ml) from the column. Three peaks exhibiting antibiotic activity were observed, indicating peak No. 1 to have a molecular weight over 1300. Peaks No. 2 and 3 have molecular weights of about 300-400. Temperature stability of partial purified three fractions exhibiting antibiotic activity The main fractions X, Y and Z of the three peaks shown in Fig. 4 were investigated for their temperature stability. Figure 5 shows the effect of temperature on antibiotic activity in the fractions X, Y and Z after Sephadex LH-20 gel permeation column chromatography. The activity of the fractions X and Z was changed in a similar manner, indicating they are heatstable below 80°C with an optimum temperature at 60°C. However, the fraction Y had a different heat stability from the above two fractions. The antibiotic activity was constant between 15 and 40°C, but rapidly decreased above 40°C and completely disappeared above 70°C. From the above results, we concluded that D. primolecta has several different antibiotic substances toward S. aureus. Interestingly, each fraction of X, Y and Z also had an antibiotic activity toward B. subtilis, B.
1
Water
I
Methanol
I
Acetonitrile Acetone
I I
Ethanol Dioxane Chloroform
Ethyl acetate iso-Propanol
4'
iso-Butanol
I
1
N-Butanol
I
Methylene chloride
I
Benzene Ethyl ether N-Hexane 0
,
,
,
200
400
600
#
% 10 0
Antibiotic activity (pg ampicillin/g fresh wt.)
Fig. 3. Solubility and stability of antibiotic substance from the marine alga, Dunaliella primolecta for various solvents. Algal cells (0.1 g fresh wt/ml) of this alga were sonicated and freeze-dried. The antibiotic substance in the freeze-dried samples was then tested for solubility in different solvents. Values are the mean of two experiments.
Antibiotic substances ofDunaliella primolecta Vitamin B12 Chloramphenicol (MW: 1355 ) (MW:323 )
I ~
~0.4 ~'~
-
i~
/
~
~-
A _ _ _
Peak #i
7 O~
~
,~ Peak #2 ! /
,IN
T J: , :, k
I
0
_
Peak #3
IW :~
7_35
A
iTi
Fraction
#
i\
15.16 z:
1 .18
Fig. 4. Sephadex LH-20 column chromatography of antibiotic substances in the marine green alga, Dunaliella primolecta. The concentrated elute fraction of acetone-methanol (10:3) from silica gel column chromatography was put on a Sephadex LH-20 column (1.5 × 30 cm) equilibrated with methanol, and the antibiotic substances were eluted by methanol at a flow rate of 12 ml/h. Antibiotic activity for S. aureus of all elute fractions was tested. The fractions of chloramphenicol (MW:323) and vitamin B~2 (MW: 1355) were monitored by absorbance at 280 nm.
75
5O
~ 2s z 15
25
40
60
benefits of the biomass system by Dunaliella sp., one needs to recognize additional values such as the production of new pharmaceuticals. Therefore, our observation that it has been found to exhibit antibiotic activities is of added importance since it is already in commercial production. Hereafter, we plan to purify and identify these substances from D. primolecta, and to construct a new biomass system for antibiotic production by this alga.
![
10 20 Fraction number (2 ml/tube)
o
153
70
80
Temperature ('C)
Fig. 5. Effect of temperature on antibiotic activity of the partially purified fractions X, Y and Z after Sephadex LH-20 column chromatography of the marine green alga, Dunaliella primolecta. The main fractions X(o), Y(A ) and Z(o) are the three peaks shown in Fig. 4 which were investigated for their temperature stability. These fractions were incubated for 1 h at each temperature. Points are the mean of two experiments.
cereus and E. aerogenes. A crude extract of this alga strongly inhibited the growth of Methicillin-resistant S. aureus (MRSA) occurring in hospital infection (data not shown). At present, the most advanced large-scale biomass projects using microalgae are the production of /3caroten and glycerol by Dunaliella salina in outdoor ponds (Borowitzka et al., 1984). To further realize the
ACKNOWLEDGEMENTS The authors are particularly indebted to Associate Professor Y. Hara and graduate student Mr M. Kawachi of Tsukuba University, Japan for advice concerning the algal identification. We thank Dr H. Nakanishi of the Public Health Research Institute of Kobe, Japan for transferring the Methicillin-resistant S. aureus. REFERENCES Berland, B. R. (1972). The antibacterial substances of marine alga Sticocrysis immobilis (Crysophyta). J. Phycol., 8, 383-92. Borowitzka, L. J., Borowitzka, M. A. & Moulton, T. P. (1984). The mass culture of Dunaliella salina for fine chemicals: From laboratory to pilot plant. Hydrobiologia, 116/117, 115-21. Fujimoto, K., Ohmura, H. & Kaneda, T. (1985). Screening for antioxygenic compounds in marine algae and bromophenols as effective principles in a red alga, Polysiphonia ulceolate. Bull. Jap. Soc. Sci. Fish., 51, 1139-43. Harder, R. ( 1917). Ernarungsphysiologische untersuchungen an cyanophyceen hauptsachlich dem endophyticschen Nostoc punctiformis. Zeit. Bot., 9, 145-242. Higgs, M. D. (1981). Antimicrobial components of the red alga, Laurencia hybrida. Tetrahedron, 37, 4255-8. Ishitsuka, M., Kusami, T. & Kakisawa, H. (1988). Antitumor xenicane and norxenicane lactones from the brown alga, Dictyota dichotoma. J. Org. Chem., 53, 5010-13. Lustigman, B. (1988). Comparison of antibiotic production from four ecotypes of the marine alga, Dunaliella. Bull. Environ. Contain. Toxicol., 40, 18-22. Mason, C. P., Edwards, K. R., Carlson, R. E., Pignatello, J., Gleason, F. K. & Wood, J. M. (1982). Isolation of chlorinecontaining antibiotic from the fresh water cyanobacterium Scytonema hofmanni. Science, 215, 400-2. Matsueda, S., Shinpo, K., Abe, K., Karasawa, H. & Katsukura, Y. (1988). Studies of antitumor active glycoprotein from Chlorella vulgaris II -- Glycoprotein hydrolyzed with hydrolase. Yakugaku Zasshi, 107, 694-7. Pratt, R. H. (1942). Studies on Chlorella vulgaris V: Some properties of the growth inhibitor formed by Chlorella cells. Am. J. Bot., 29, 142-8. Richmond, A. (1986). Outdoor mass culture of microalgae. In CRC Handbook of Microalgal Mass Culture, ed. A. Richmond. CRC Press, Boca Raton, FL, pp. 285-329. Rosell, K. G. & Strivastava, L. M. (1987). Fatty acids as antimicrobial substances in brown algae. Hydrobiologia, 151 / 152,471-5. Shklar, G. & Schwartz, J. (1988). Tumor necrosis factor in experimental cancer regression with a-tocopherol, rcarotene, canthaxanthin and algal extract. Eur. J. Clin. Oncol., 24, 839-50.
ANTIBIOTIC SUBSTANCES PRODUCED BY A MARINE GREEN ALGA, D UNALIELLA PRIMOLE CTA Thomas Chang, a Souichi Ohta, b, Naoto Ikegami, b Hideaki Miyata, b Takashi Kashimoto b & Masaomi Kondo b aFaculty of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565, Japan bFaculty of Pharmaceutical Sciences, Setsunan University, 45-1 Nagaotoge-cho, Hirakata, Osaka 573-01, Japan (Received 2 July 1992; revised version received 28 September 1992; accepted 5 October 1992) Abstract From among 84 marine algae, the green alga, Dunaliella primolecta C-525, exhibiting the highest antibiotic activity, was selected. A crude extract from this alga strongly inhibited the growth of Staphylococcus aureus, Bacillus cereus, Bacillus subtilis and Enterobacter aerogenes. From experiments on pH, solvent, and an inhibitor of the crude extract of this alga, the antibiotic substance in the algal cells was observed as a basestable, nonprotein material. To purify this antibiotic substance, a methanol extract of this alga was eluted successively on a DEAE-Sepharose CI-6B, silica and Sephadex LH-20 gel column. Three peaks exhibiting antibiotic activity from the final column were observed, indicating peak No. I to have a molecular weight of over 1300 and peaks Nos 2 and 3 to have molecular weights of about 300-400. The main fractions X, Y and Z of the three peaks were investigated for temperature stability. The activities of fractions X and Z were Stable up to 80°C, whereas the activity of fraction Y was unstable above 40°C. The above results indicate that algal cells of D. primolecta contained a number of different antibiotic substances.
aceutical products in microalgae (Matsueda et al., 1988; Shklar & Schwartz, 1988). In order to compete with other organisms in nature, algae are known to secrete toxic and/or growth-inhibitory substances under certain circumstances. This was first observed by Harder in 1917. Later, Pratt (1942) found antibacterial activity in a freshwater green alga, Chlorella sp. Since then, many bioactive compounds have been isolated from various algae (Berland, 1972; Mason et al., 1982) including fatty acids (Rosell & Strivastava, 1987), terpenes (Ishitsuka et al., 1988), bromophenols (Fujimoto et al., 1985) and halogenated compounds (Higgs, 1981). However, most of these compounds have been purified mainly from red or brown algae, and few have been isolated from green algae. Lustigman (1988) compared four ecotypes of Dunaliella sp. and observed antibiotic production by D. salina alone isolated from the seawaters of the high pollution area of San Francisco Bay, CA and Long Island Sound, New York in the USA. Here, we report the properties of antibiotic substances extracted from a marine green alga, Dunaliella primolecta.
Key words: Antibiotic substances, marine green alga, Dunaliella primolecta.
METHODS Algal strain and growth conditions Sixty-eight strains of marine algae were isolated from an enriched culture of marine samples (seawater, seaweeds and sediment) collected from the coastal areas of western Japan, and used throughout this study. These algae were grown in enriched seawater medium, and purified by agar-plate methods. Sixteen other strains of marine algae were obtained from three stock culture centres, the Institute of Applied Microbiology, University of Tokyo (IAM), Japan, Global Environmental Forum (GEF), Japan and American Type Culture Collection (ATCC), USA. A marine green alga, Dunaliella primolecta C-525 which was used in this study, was obtained from the IAM. For reproducible laboratory studies, the medium (ATCC medium No. 616) for D. primolecta C-525 was a modification of BG-11 medium (pH 8"0), made by the addition of NaCI, 20 g, vitamin Bt, 100/zg and vitamin B12, 1/zg in
INTRODUCTION Owing to their high growth rate, microalgae have long been recognized as a promising biomass source for the production of drugs, food and energy from light, CO 2 and minerals. The unicellular green algae, Chlorella and Dunaliella, as novel health food items, are cultivated commercially in large outdoor ponds (Borowitzka et al., 1984; Richmond, 1986). However, there are few studies which focus on the utilization of pharm*To whom correspondence should be addressed. Bioresource Technology 0960-8524/93/S06.00 © 1993 Elsevier Science Publishers Ltd, England. Printed in Great Britain 149
150
Thomas Chang, Souichi Ohm, Naoto Ikegami, Hideaki Miyata, Takashi Kashimoto, Masaomi Kondo
1-0 litre of the BG- 11 medium. The alga was grown at 25°C in 1.5-litre Roux flasks containing 1"0 litre of the medium, and harvested at late-log phase. The algal cultures were continuously illuminated by a bank of fluorescent lamps at a light intensity of 25 W/m 2. The cultures were continuously sparged with air containing 1% CO2 at a flow rate of about 300 ml/min. Preparation of crude extracts Crude extracts were prepared for activity testing from 84 algal cultures. Each algal culture was centrifuged (4000 g, 10 min) at 0°C. By the addition of distilled water to the pellets, the algal concentration was adjusted to 0"1 g fresh weight/mE Then, the algal suspension was sonicated (100 #A, 3 min) on ice by using an ultrasonic generator, and centrifuged. The supernatant was filtered through a membrane filter (0-22/~m, Millipore). The filtrate was designated as the crude extract. Antibacterial test The crude extracts were examined for antibacterial activity towards eight bacteria. This was determined by observing inhibition of their growth at 25°C on nutrient broth (Difco) agar plate for 24-48 h; 40 /~1 of algal crude extract (0-1 g fresh weight/ml) was transferred to an 8 mm paper disc, which was then applied to a plate seeded with a test strain. After incubation at 25°C for 24 h, the resulting zone of inhibition was measured. Distilled water was used as a control, as well as samples treated under various experimental conditions. No inhibition zone was observed with any of the control discs. Effects of pH, temperature and inhibitors To investigate the pH stability of the antimicrobial activity, sonicated algal cells were exposed to pH 2, 4, 6, 7, 9 and 12 at 25°C for 1 h, respectively. The samples were brought to pH 7, adjusted to the initial concentration (0" 1 g fresh wt/ml). After centrifugation and filtration, the antibiotic activity of the filtrate was measured by means of the disc test described above. The temperature stability was also investigated. Each sample of sonicated algal cells was incubated at 15, 25, 40, 60, 70, 80 and 100°C for 1 h, followed by centrifugation, filtration and testing as described above. To determine the effects of various inhibitors, an equal volume of the samples of sonicated algal cells and four inhibitors, ethylenediaminetetraacetate (EDTA), diethylpyrocarbonate (DEP), p-chloromercuribenzonic acid (PCMB) or HgC12 was mixed, adjusting to the concentration of algal cells adjusted to 0"1 g fresh wt/ml and the mixtures incubated at 25°C for 1 h. At this time, DEP was dissolved in 0" 1 U Tris-HC1 buffer (pH 7"0) adjusted to contain 50% dimethylsulphoxide, and other inhibitors were dissolved in 0"1 M Tris-HC1 buffer (pH 7-0). Each solvent buffer was also used as a control. The procedure after that was the same as for the pH-stability experiment.
Selection of solvents Algal cells (0-1 g fresh weight/ml) of D. primolecta were sonicated and freeze-dried. The antibiotic substance in the freeze-dried samples was then tested for solubility in different solvents. After centrifugation, the supernatants were freeze-dried again, and dissolved in distilled water, followed by filtration and testing as described in the case of the pH-stability experiment. Partial purification To purify the antibiotic substance in D. primolecta, algal cells were sonicated in a methanol solution. After centrifugation, the supernatants were partitioned with n-hexane to remove lipophilic compounds. The antibiotic substance in the methanol phase was concentrated and separated successively on three different columns filled with different gels, DEAE-Sepharose C1-6B (Pharmacia), silica (Merck) and Sephadex LH20 (Pharmacia). Each fraction eluted from the three columns was freeze-dried and dissolved in distilled water, followed by filtration and testing as described above. To investigate the temperature stability of the three fractions X, Y and Z containing antibiotic substances eluted from a Sephadex LH-20 column, they were incubated at 15, 25, 40, 60, 70 and 80°C for 1 h. The procedure after that was the same as that used for the temperature stability determination of the crude extract.
RESULTS AND DISCUSSION Antibiotic activity for bacteria In order to search for new antibiotic substances, 84 marine microalgae comprising 68 of isolated strains and 16 strains of stock culture were used; the algae could be grouped into seven classes, 35 strains of Cyanophyceae, 28 strains of Chlorophyceae, 11 strains of Bacilariophyceae, four strains of Dinophyceae, three strains of Rhaphidophyceae, two strains of Rhodophyceae, and one strain of Chryptophyceae. The antibiotic activity in the crude extract of all strains was tested against a Gram-negative bacterium, Escherichia Coli and a Gram-positive bacterium, Staphylococcus aureus. Although all crude extracts of the 84 strains exhibited no activity toward E. coil, the crude extracts of seven strains exhibited antibiotic activity toward S. aureus (Table 1). In particular, high activities were observed in crude extracts of Dunaliella bioculata C-523, Dunaliella primolecta C-525 (a stock culture strain) and our isolate Chlorococcum HS-101. Of these three strains, we finally chose the green alga D. primolecta C-525 because of its high activity and high growth rate. As shown in Table 2, crude extract from D. primolecta was tested against eight bacteria. Marked inhibition of the growth was observed for S. aureus, Bacillus subtilis, Bacillus cereus and Enterobacter aerogenes. Growth of four fungi, Cladosporium cladosporides, Penicillium funculosum, Paecilomyces variotii and Aspergillus niger was not inhibited (data not
Antibiotic substances ofDunaliella primolecta
shown). As strong growth inhibition of S. aureus was observed, antibiotic activity was compared with that of a commercial antibiotic substance, ampicillin. By using this relationship, the activity of crude extracts for S. aureus was represented in terms of an equivalent amount of ampicillin. Effects of pH and temperature on antibiotic activity T h e optimum conditions for activity of the antibiotic substance in D. primolecta C-525 were investigated by using S. aureus. Figure 1 shows the effect of p H on the stability of antibiotic activity in the crude extract of this alga. T h e activity was stable in the range of p H 6 - 1 2 , whereas that in the acidic range of p H 2 - 4 was depressed. T h e effect of temperature on the stability of the antibiotic activity in the crude extract of this strain was also investigated (Fig. 2). Activity of this substance was observed up to 80°C with an optimum between 25 and 40°C. No antibacterial activity was observed at 100°C. Effect of inhibitors on antibiotic activity Table 3 shows the effect of inhibitors on the antibiotic activity of crude extract of this strain. A chelator of metal ions ( E D T A ) and SH-blocking agents ( D E E PCMB, H g C l J were used as inhibitors. T h e activity was inhibited to 46% by 2 mM E D T A , but the effect of the other agents was not significant. This indicates that the expression of antibiotic activity involves a metal ion, and is probably not due to an enzyme reaction.
Solubility and stability of antibiotic substance for various solvents It is essential to choose a suitable solvent for extracting and further purifying the antibiotic substance in this strain. As shown in Fig. 3, the solubility and stability of
"2
150
T-
U 100
50 Z 0
Stock strains Dunafiella bioculata C-523 Dunaliella primolecta C- 525 Our isolates Chlorococcum sp. HS- 101 Chlorella sp. HS-109 Chlorella sp. HS-110 Synechococcus sp. HS-364 Phorphyridium sp. HS-366
Inhibition zone (mm)
17'8±2'6 11"8±1"6 14"8±3"6 11"9±2'1 11-5±1'7
Seven marine algae were grown at 25°C in a modified BG-11 medium. Dunaliella sp. C-523 and C-525 were obtained from the culture collection of Applied Microbiology, University of Tokyo. Values are the mean + s.d. of three experiments.
41
I
I
6
7
I9
| 12
pH
Fig. 1. Effect of pH on antibiotic activity of crude extract from the marine green alga, Dunaliella primolecta. Points are the mean + s.d. of three experiments.
"2
75
7 a~
17'4±3"3 18.7±2"1
I 2
Table 1. Comparison of antibiotic acitivity in the crude extracts towards S. aureus produced by seven marine algae
Strain
151
50
v
.; ;5 25 g Z L 15
25
40
60
70
80
100
Temperoture (*C)
Fig. 2. Effect of temperature on antibiotic activity of crude extract from the marine green alga, Dunaliella primolecta. Points are the mean + s.d. of three experiments.
Table 2. Antibacterial activities of crude extract from the marine green alga, Dunaliella primolecta
Gram-negative bacterium Escherichia col± Salmonella typhimurium Enterobacter aerogenes Vibrio parahaemolyticus
Inhibition zone (mm) NA NA 13'5 -+ 2.4 NA
Gram-positive bacterium Staphylococcus aureus Bacillus cereus Bacillus subtilis Micrococcus luteus
Inhibition zone (mm) 18"5 _+2"3 15"5 _+3"2 16"0 _+2"8 NA
40/~1 of algal crude extracts (0"1 g fresh weight/ml) were examined. Values are the mean + s.d. of three or more experiments. NA, no activity.
152
Thomas Chang, Souichi Ohta, Naoto Ikegami, Hideaki Miyata, Takashi Kashimoto, Masaomi Kondo
this substance were tested in various solvents. Independent of the solvent polarity, the activities were observed in all solvents except n-hexane. High activities were observed in alcoholic solvents such as methanol, iso-propanol, iso-butanol and n-butanol. Lustigman (1988) tested the solvent activity of the antibiotic substance of D. salina, and the antibiotic activities were observed in propanol and butanol, not methanol and ethanol. Further, with respect to the antibacterial spectrum or the stability of pH and temperature, our results also were not in accord with his results. Therefore, it was suggested that Dunaliella sp. can produce diverse antibiotic substances according to the algal growth environment. Finally, methanol was chosen for further extraction and purification of the antibiotic substance, because its boiling point is the lowest of the alcoholic solvents exhibiting high activity.
Table 3. Effect of inhibitors on antibiotic activity of crude extract from the marine green alga, Dunaliella primolecta Inhibitor None EDTA" DEP" PCMB" HgCI2
Conc. (mM)
Residual activity (%) h
1.0 2.0 1.0 2.0 1'0 2.0 0.04 0.1
100 + 11"3 83 + 7"9 46 + 12.8 92 + 11'4 108+9.5 92 + 9.7 100 + 10.3 100 + 12-5 100 _+13.1
"EDTA, ethylenediaminetetraacetate; DEE diethyl pyrocarbonate; PCMB, p-chloromercuribenzoic acid. b100% activity is equivalent to 124.7 Mg ampicillin/g fresh wt. Values are the mean + s.d. of three experiments.
Partial purification of antibiotic substance The antibiotic substance in the methanol extract of this alga was separated by D E A E Sepharose CI-6B, and then by silica gel chromatography. In both cases, the antibiotic activity was observed in the eluted fraction of acetone: methanol ( 10: 3). Further purification of the elute fractions from the silica gel column was carried out on a column of Sephadex LH-20 gel permeation (Fig. 4). Tests of Chloramphenicol (MW:323) and vitamin B~2 (MW: 1355) as molecular weight markers were also performed under the same elution conditions. Figure 4 shows antibiotic activity of each elute (2 ml) from the column. Three peaks exhibiting antibiotic activity were observed, indicating peak No. 1 to have a molecular weight over 1300. Peaks No. 2 and 3 have molecular weights of about 300-400. Temperature stability of partial purified three fractions exhibiting antibiotic activity The main fractions X, Y and Z of the three peaks shown in Fig. 4 were investigated for their temperature stability. Figure 5 shows the effect of temperature on antibiotic activity in the fractions X, Y and Z after Sephadex LH-20 gel permeation column chromatography. The activity of the fractions X and Z was changed in a similar manner, indicating they are heatstable below 80°C with an optimum temperature at 60°C. However, the fraction Y had a different heat stability from the above two fractions. The antibiotic activity was constant between 15 and 40°C, but rapidly decreased above 40°C and completely disappeared above 70°C. From the above results, we concluded that D. primolecta has several different antibiotic substances toward S. aureus. Interestingly, each fraction of X, Y and Z also had an antibiotic activity toward B. subtilis, B.
1
Water
I
Methanol
I
Acetonitrile Acetone
I I
Ethanol Dioxane Chloroform
Ethyl acetate iso-Propanol
4'
iso-Butanol
I
1
N-Butanol
I
Methylene chloride
I
Benzene Ethyl ether N-Hexane 0
,
,
,
200
400
600
#
% 10 0
Antibiotic activity (pg ampicillin/g fresh wt.)
Fig. 3. Solubility and stability of antibiotic substance from the marine alga, Dunaliella primolecta for various solvents. Algal cells (0.1 g fresh wt/ml) of this alga were sonicated and freeze-dried. The antibiotic substance in the freeze-dried samples was then tested for solubility in different solvents. Values are the mean of two experiments.
Antibiotic substances ofDunaliella primolecta Vitamin B12 Chloramphenicol (MW: 1355 ) (MW:323 )
I ~
~0.4 ~'~
-
i~
/
~
~-
A _ _ _
Peak #i
7 O~
~
,~ Peak #2 ! /
,IN
T J: , :, k
I
0
_
Peak #3
IW :~
7_35
A
iTi
Fraction
#
i\
15.16 z:
1 .18
Fig. 4. Sephadex LH-20 column chromatography of antibiotic substances in the marine green alga, Dunaliella primolecta. The concentrated elute fraction of acetone-methanol (10:3) from silica gel column chromatography was put on a Sephadex LH-20 column (1.5 × 30 cm) equilibrated with methanol, and the antibiotic substances were eluted by methanol at a flow rate of 12 ml/h. Antibiotic activity for S. aureus of all elute fractions was tested. The fractions of chloramphenicol (MW:323) and vitamin B~2 (MW: 1355) were monitored by absorbance at 280 nm.
75
5O
~ 2s z 15
25
40
60
benefits of the biomass system by Dunaliella sp., one needs to recognize additional values such as the production of new pharmaceuticals. Therefore, our observation that it has been found to exhibit antibiotic activities is of added importance since it is already in commercial production. Hereafter, we plan to purify and identify these substances from D. primolecta, and to construct a new biomass system for antibiotic production by this alga.
![
10 20 Fraction number (2 ml/tube)
o
153
70
80
Temperature ('C)
Fig. 5. Effect of temperature on antibiotic activity of the partially purified fractions X, Y and Z after Sephadex LH-20 column chromatography of the marine green alga, Dunaliella primolecta. The main fractions X(o), Y(A ) and Z(o) are the three peaks shown in Fig. 4 which were investigated for their temperature stability. These fractions were incubated for 1 h at each temperature. Points are the mean of two experiments.
cereus and E. aerogenes. A crude extract of this alga strongly inhibited the growth of Methicillin-resistant S. aureus (MRSA) occurring in hospital infection (data not shown). At present, the most advanced large-scale biomass projects using microalgae are the production of /3caroten and glycerol by Dunaliella salina in outdoor ponds (Borowitzka et al., 1984). To further realize the
ACKNOWLEDGEMENTS The authors are particularly indebted to Associate Professor Y. Hara and graduate student Mr M. Kawachi of Tsukuba University, Japan for advice concerning the algal identification. We thank Dr H. Nakanishi of the Public Health Research Institute of Kobe, Japan for transferring the Methicillin-resistant S. aureus. REFERENCES Berland, B. R. (1972). The antibacterial substances of marine alga Sticocrysis immobilis (Crysophyta). J. Phycol., 8, 383-92. Borowitzka, L. J., Borowitzka, M. A. & Moulton, T. P. (1984). The mass culture of Dunaliella salina for fine chemicals: From laboratory to pilot plant. Hydrobiologia, 116/117, 115-21. Fujimoto, K., Ohmura, H. & Kaneda, T. (1985). Screening for antioxygenic compounds in marine algae and bromophenols as effective principles in a red alga, Polysiphonia ulceolate. Bull. Jap. Soc. Sci. Fish., 51, 1139-43. Harder, R. ( 1917). Ernarungsphysiologische untersuchungen an cyanophyceen hauptsachlich dem endophyticschen Nostoc punctiformis. Zeit. Bot., 9, 145-242. Higgs, M. D. (1981). Antimicrobial components of the red alga, Laurencia hybrida. Tetrahedron, 37, 4255-8. Ishitsuka, M., Kusami, T. & Kakisawa, H. (1988). Antitumor xenicane and norxenicane lactones from the brown alga, Dictyota dichotoma. J. Org. Chem., 53, 5010-13. Lustigman, B. (1988). Comparison of antibiotic production from four ecotypes of the marine alga, Dunaliella. Bull. Environ. Contain. Toxicol., 40, 18-22. Mason, C. P., Edwards, K. R., Carlson, R. E., Pignatello, J., Gleason, F. K. & Wood, J. M. (1982). Isolation of chlorinecontaining antibiotic from the fresh water cyanobacterium Scytonema hofmanni. Science, 215, 400-2. Matsueda, S., Shinpo, K., Abe, K., Karasawa, H. & Katsukura, Y. (1988). Studies of antitumor active glycoprotein from Chlorella vulgaris II -- Glycoprotein hydrolyzed with hydrolase. Yakugaku Zasshi, 107, 694-7. Pratt, R. H. (1942). Studies on Chlorella vulgaris V: Some properties of the growth inhibitor formed by Chlorella cells. Am. J. Bot., 29, 142-8. Richmond, A. (1986). Outdoor mass culture of microalgae. In CRC Handbook of Microalgal Mass Culture, ed. A. Richmond. CRC Press, Boca Raton, FL, pp. 285-329. Rosell, K. G. & Strivastava, L. M. (1987). Fatty acids as antimicrobial substances in brown algae. Hydrobiologia, 151 / 152,471-5. Shklar, G. & Schwartz, J. (1988). Tumor necrosis factor in experimental cancer regression with a-tocopherol, rcarotene, canthaxanthin and algal extract. Eur. J. Clin. Oncol., 24, 839-50.