World Journal of Microbiology & Biotechnology 10, 338-341
Bioactive compound production by thermophilic and thermotolerant cyanobacteria (blue-green algae) S.A. Fish and G.A. Codd* A thermotolerant species of Phormidium produced extracellular anti-microbial material during batch culture. Although this material was inactive when screened against a number of other cyanobacteria, it inhibited the growth of a wide range of Gram-positive and Gram-negative heterotrophic bacteria, Candida albicans and Cladosporium resinae. Key words: Anti-microbial products, bioactive products, blue-green algae, cyanobacteria, Phormidium.
Cyanobacteria have long been recognized to be capable of producing bioactive compounds (Lef6vre 1964). Recently, compounds from cyanobacteria have been isolated which display inhibitory effects on bacterial growth (Reichelt & Borowitzka 1984; Cannell et al. 1988), fungal growth (Kellam et al. 1988; De Mul6 et al. 1991), cancer cells (Kashiwagi et al. 1980; Carmeli et al, 1990), viruses (Starr et al. 1962; Gustafson et al. 1989) and enzymes (Cannell et al. 1987). Many mesophilic cyanobacteria have been screened for the above activities but before the present study, thermotolerant or thermophilic species had not been specifically targeted. The intention of the present investigation was to examine the production of bioactive compounds by cyanobacteria from thermal environments using microbial-based assay systems. To aid this, a screening system was necessary which could detect low concentrations of active metabolites, down to 0.1% of the solids in the supernatant (Hanka et al. 1978). As a consequence, an agar diffusion assay was used in conjunction with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), which assisted the visualization of zones of growth inhibition.
(Iceland) was isolated by Dr. I.L. Andrews from a water sample collected by D. Dyer (both of the University of Dundee) from a warm spring in Iceland. Synechocystis sp. (Kenya) and Phormidium sp. (Kenya) were isolated by Dr. I.L. Andrews from field material collected by Dr. W.D. Grant (University of Leicester) from Lake Bogoria, Kenya. Phormidium sp. (Azores) was isolated by Dr. I.L. Andrews from field material collected by Dr. R.J. Sharp (Centre for Applied Microbiology Research, Porton Down) from a hot spring on the island of San Miquel in the Azores. All the cyanobacteria were isolated into axenic culture using phototactic response and antibiotic treatment, according to the procedure of Vaara et aL (1979). Growth Conditions Cultures were grown in 250-ml conical flasks each containing 100ml Medium D which had as its constituents (rag/l): nitrilotriacetic acid, 100; CaSO4. 2H20, 60; MgSO4. 7I-~O, 100; NaC1, 8; KNO3, 103; NaNO3, 689; Na2HPO ¥ 111; FeCI3, 0.2905; M r l S O 4, H20, 2.28; ZnSO4. 71-t20, 0.5; H3BO3, 0.5; CuSO4. 5H20, 0.025; NaMoO 4. 2H20, 0.025; CoC12. 6H20, 0.046; Tris, 605.5; and 0.5 ml conc. H2SO4 in Milli Q deionized and ultrafiltered water (Brock 1978). The cultures were kept at 30°C ( _+1°C) unless specified otherwise, and shaken at 150 rev/min. They were continuously illuminated by cool white fluorescent light which gave an irradiance incident on the surface of the vessels of 2.0 W / m 2.
Isolation of Cyanobacteria Synechococcus PCC 6716 and Mastigocladus laminosus PCC 7521 were from the Pasteur Culture Collection, Paris. Synechococcussp.
Preparation of Material for Initial Bioactivity Screening After 4 weeks of incubation, 100 ml of the cyanobacterial cultures were centrifuged at 19,000 xg for 15 rain and the supernatant freeze-dried, suspended to 2% of the initial volume in Milli Q deionized and ultrafiltered water, and then sterilized by passage through a 0.22-pm pore sterile filter.
The authors am with the Department of Biological Sciences, University of Dundee, Dundee DD1 4HN, UK; fax: 0382 22318, *Corresponding author.
Measurement of Growth/Bioactivity of Phormidium sp. (Azores) Conical flasks, 500 ml, each containing 400 ml of Medium D, were incubated as detailed above. Over 8 weeks, 100-ml samples were
Materials and Methods
© 1994 Rapid Communications of Oxford Ltd
338
WorlaJournalofMicrobiology&Biotechnology,Vo110,1994
Bioactive compounds from cyanobacteria withdrawn from the conical flasks, filtered using glass fibre (grade C; Whatman) and then freeze-dried and prepared as above. The associated cells on the filter paper were freeze-dried and then weighed to plot growth characteristics.
Anti-microbial Assays Using Bacteria Sterile Sensitest agar (Oxoid), 10 ml, at 45 to 47°C, was seeded with 10 pl of an overnight culture of the test organism, gently swirled, poured into a 9-cm diam. Petri dish and allowed to dry in a laminar flow cabinet. Holes, 5 mm in diam., were cut in the agar and 20 lal of the test solution added to each. The plates were held at 4°C for 4 h to allow diffusion of the test material into the agar, then incubated at 37°C for 12h, followed by the application to the surface of the agar of 1 ml of phosphate-buffered saline containing 1 mg 3-(4,5-dimethylthiazoI-2-yl)2,5-diphenyltetrazolium bromide (MTT). After holding at 37°C for a further 15 min clear haloes surrounded by a blue background were seen. [The tetrazolium salt is cleaved by dehydrogenases (Mosmann 1983) to yield a blue formazan product, this indicating zones of growth promotion or growth inhibition of the test microorganism, the diameter of the halo increasing in proportion to growth inhibition.] Anti-microbial Assay using Fungi When using Candida albicans the assay was conducted as in the bacterial assay, except that incubation was extended from 12 to 18 h. When using Cladosporium resinae, sterile water was poured onto the surface of an existing plate culture to collect spores. These were inoculated into 10 ml of sterile Sensitest agar and prepared as previously described. The test material was introduced into 5-mm diam. wells in the agar and the plate incubated for 48 to 72 h, zones of clearing being noted. Cyanobacterial Growth Inhibition The procedure of Bagchi et aI. (1990) was used with minor modifications. Fifteen ml of 1.5% (w/v) agar No. 2 (Lab M, Amersham, UK) was poured into a 9 cm diam. Petri dish and allowed to solidify. Then 0.5ml of a 4-week-old batch culture of cyanobacteria was added to 2.5 ml of 0.8% (w/v) agar No. 2 in Medium D at 45 to 47°C, mixed and poured onto the base layer and the plates incubated at 30°C under cool white fluorescent light until a thick lawn of the test organism had grown. A 5-mm diam. well was then cut in the agar and the test material introduced into the well. The plate was incubated for I week and any growth inhibition noted.
Results and Discussion Table 1 summarizes the results of the initial screening of six thermotolerant/thermophilic cyanobacteria, isolated from a number of locations. Of the cyanobacteria examined, Mastigocladus laminosus PCC 7521 and the Phormidium isolate from the Azores produced extracellular material with the capacity to inhibit the growth of the test microorganisms. Of the two cyanobacteria displaying activity, Phormidium sp. (Azores) was selected for further study as it demonstrated a greater capacity for growth inhibition. Preliminary work associated with Phormidium (Azores) indicated that, although it grew more rapidly at 41°C than at 30°C, the production of the growth-inhibitory material was greatly decreased at the
Table 1, Inhibition of microbial growth supernatants, using the agar diffusion assay. Cyanobacterial source of eupernatant
by
Test organism* Staphylococcus aureus NCIMB 3251
Mastigocladus laminosus PCC 7521 Phormidium sp. (Azores) Phormidium sp. (Kenya) Synechococcus PCC 6716 Synechococcus sp. (Iceland) Synechocystis sp. (Kenya)
cysnobactarlal
++ ++++
Candida albicans 3153 A +++ +++
-
-
-
*The diameter of the inhibition zone was scored + (15 mm). A control, a 50 x concentration of Medium D gave no inhibition (-). NCIMB~National Collection of Industrial and Marine Bacteria, Aberdeen, UK; PCC--Pasteur Culture Collection, Paris, France.
higher temperature. Consequently further incubations of this organism were at 30°C. Table 2 shows an expansion of the microbial screening systems used in Table 1. The extracellular material produced by Phormidium (Azores) displayed a wide range of growth inhibitory activity, affecting Gram-positive and Gram-negative bacteria as well as a yeast and a fungus. Although the growth of a wide range of microbes was inhibited, two of the Gram-negative heterotrophic bacteria were unaffected, this being in keeping with the lack of growth inhibition of the cyanobacteria tested. Figure 1 shows the relationship between the growth of Phormidium (Azores) in batch culture and the production of the growth-inhibitory material, as assayed against Staphylococcus aureus. The results indicate a biphasic pattern of production of the growth-inhibitory material throughout batch culture, unlike the production of many antibiotics which are produced during the stationary phase (Drew & Demain 1977). The detection of extraceUular compounds of cyanobacterial origin which inhibit bacteria or cyanobacteria is not new (Flint & Mooreland 1946; Mason et al. 1982; Flores & Wolk 1986; Cannell et al. 1988). Likewise, products from cyanobacteria that inhibit fungi have been recognized for some time (Welch 1962; Kellam et al. 1988; De Cano et al. 1990; De Mul6 et al. 1991). However, with the exception of one other study (Bloor & England 1989), no cyanobacterium other than Phormidium sp. (Azores) has been isolated with such a broad range of activity against microbes, including Gram-positive and Gramnegative bacteria, a yeast and a fungus. However, it was found that when the extracellular material was tested against other cyanobacteria, no growth inhibition was recorded. The inhibition of growth of most of the diverse group of microbes screened (Table 2) indicates that the Phormidium (Azores) isolate contains an anti-microbial compound with a broad spectrum of activity, a range of more host-specific
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339
S.A. Fish and G.A. Codd Table 2. Anti-microbial activity of supernatant from Phormidlum sp. (Azores), measured using the agar diffusion assay.
0.035
/
0.03 Test Organism
Activity* 0.025
A. Gram-positive heterotrophic bacteria Bacillus cereus NCIMB 3329 Bacillus coagulans NCIMB 9365 Bacillus licheniformis NCIMB 6346 Bacillus sphaericus NCIMB Bacillus subtilis NCIMB 1650
Staphylococcus aureus NCIMB 3251 Streptococcus faeca/is NCIMB 775 Streptococcus viridi NCIMB 8954 B. Gram-negative heterotrophic bacteria Escherichia coli NCIMB 9484 Hafnia alvei NCIMB 6578 Kleb$iel/a pneumoniae NCIMB 8805 Proteus vu/garis NCIMB 67 Proteus morganii NCIMB 232 Pseudomonas aeruginosa NCIMB 8295 Pseudomonas denitdficans NCIMB 9496
+++
.,~
.m
++
o.o15-
++++ ++
0.02
a 0.01
++ ++++
0.005
/
ml
B
m
m
m
+++ +++
a
16
24 32 Time (days)
40
48
56
8
16
24 32 Time ( d a y s )
40
48
56
+ +++ +++ +++ +++
~ 25-
55 ~
20-
~
10-
C. Cyanobacteria
Mastigocladus laminosus PCC 7521 Phormidium sp. (Azores) Phormidium sp. (Kenya) Synechococcus PCC 6716 Synechococcus sp. (Iceland) Synechocystis sp. (Kenya)
i:5 o
D. Yeast/Fungus
Candida albicans 3153 A Cladosporium resinae ATCC 22711
+++ +++
*Inhibition was scored as in Table 1. NCIMB--National Collection of Industrial and Marine Bacteria, Aberdeen, UK; ATCC--Amedcan Type Culture Collection; A--Dundee University Culture Collection, UK.
growth inhibitors, or a combination of the two classes of bioactive products. Indeed, the kinetics of accumulation of extracellular growth-inhibitory material, even when screened against a single test organism, were biphasic (Figure 1B) and further studies (S.A. Fish & G.A. Codd, unpublished work) have revealed the presence of at least two growth-inhibitory products of this cyanobacterium, with M r of approx. 424 and 762. Finally, whilst the biotechnological potential of cyanobacteria is attracting increased attention (Patterson et al. 1991), little work has been done to screen cyanobacteria isolated from extreme environments with regard to their production of bioactive compounds. The results of this work indicate that this group of organisms displays potential in this respect, a potential which warrants further investigation.
340
World Journal of Microbiology & Biotechnology, Vo110,1994
Figure 1. (A) Growth of Phormidium sp. (Azores) in batch culture, determined as an increase in dry weight and (B) detection of bioactive material in the supernatant of the Phormidium cultures when screened against Staphylococcus aureus. Inhibition of S. aureus growth was determined by agar well diffusion assay, each point in (B) representing the mean of three replicate wells each loaded with 20pl of concentrated resuspended freeze-dried supernatant material (see Materials and Methods). Vertical bars represent standard deviations.
Acknowledgements S.A.F. gratefully acknowledges the Science and Engineering Research Council for the award of a Postgraduate Research Studentship.
References Bagchi, S.N., Palod, A. & Chauhan, V.S. 1990 Algicidal properties of a bloom-forming blue-green alga, Oscillatoria sp. Journal of Basic Microbiology 30, 21-29. Bloor, S. & England, R.R. 1989 Antibiotic production by the cyanobacterium Nostoc muscorum. Journal of Applied Phycology 1, 367-372. Brock, T.D. 1978 Thermophflic Microorganisms and Life at High Temperatures. New York: Springer-Verlag.
Bioactive compounds from cyanobacteria Cannell, R.J.P., Kellam, S.J., Owsianka, A.M. & Walker, J.M. 1987 Microalgae and cyanobacteria as a source of glycosidase inhibitors. Journal of General Microbiology 133, 1701-1705. Cannell, R.J.P., Owsianka, A.M. & Walker, J.M. 1988 Results of a large-scale screening programme to detect antibacterial activity from fresh water algae. British PhycologicalJournal 23, 41-44. Carmeli, S., Moore, R.E. & Patterson, G.M.L. 1990 Tantazoles: unusual cytotoxic alkaloids from the blue-green alga Scytonema mirabile.Journal of the American Chemical Society 112, 8195-8197. De Cano, M.M.S., De MulG M.C.Z., De Caire, G.Z. & De Halperin, D.R. 1990 Inhibition of Candida albicans and Staphylococcus aureus by phenolic compounds from the terrestrial cyanobacterium Nostoc muscorum. Journal of Applied Phycology 2, 79-81. De Mul6, M.C.Z., De Caire, G.Z., De Cano, M.S. & De Halperin, D.R. 1991 Bioactive compounds from Nostoc muscorum (cyanobacteria). Cytobios 66, 169-172. Drew, S.W. & Demain, A.L. 1977 Effect of primary metabolites on secondary metabolism. Annual Review of Microbiology 31, 343-356. Flint, L.H. & Moreland, C.F. 1946 Antibiosis in the blue-green algae. American Journal of Botany 33, 218. Flores, E. & Wolk, C.P. 1986 Production, by filamentous, nitrogen-fixing cyanobacteria, of a bacteriocin and of other antibiotics that kill related strains. Archives of Microbiology 145, 215-219. Gustafson, K.R., Cardellina II, J.H., Fuller, R.W., Weislow, O.S., Kiser, R.F., Snader, K.M., Patterson, G.M.L. & Boyd, M.R. 1989 AIDS-Antiviral sulfolipids from cyanobacteria (blue-green algae). Journal of the National Cancer Institute 81, 1254-1258. Hanka, L.J., Kuentzel, S.L., Martin, D.G., Wiley, P.F. & Neff, G.L. 1978 Detection and assay of antitumor antibiotics. Recent Results in Cancer Research 63, 69-76.
Kashiwagi, S., Mynderse, J.S., Moore, R.E. & Norton, T.R. 1980 Antineoplastic evaluation of Pacific basin marine algae. Journal of Pharmaceutical Sciences 69, 735-738. Kellam, S.J., Cannel1, R.J.P., Owsianka, A.M. & Walker, J.M. 1988 Results of a large-scale screening programme to detect antifungal activity from marine and freshwater microalgae in laboratory culture. British Phycological Journal 23, 45-47. Lef6vre, M. 1964 ExtraceUular products of algae. In Algae and Man, ed Jackson, D.F. pp. 337-367. New York: Plenum. Mason, C.P., Edwards, K.R., Carlson, R.E., Pignatello, J., Gleason, F.K. & Wood, J.M. 1982 Isolation of chlorine containing antibiotic from the freshwater cyanobacterium Scytonema hofmanni. Science 215, 400--402. Mosmann, T. 1983 Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. Journal of Immunological Methods 65, 5563. Patterson, G.M.L., Baldwin, C.L., Bolis, C.M., Caplan, F.R., Karnso, H., Larsen, L.K., Levine, I.A., Moore, R.E., Nelson, C.S., Tschappat, K.D., Tuang, G.D., Furusawa, E., Furusawa, S., Norton, T.R. & Raybourne, R.B. 1991 Antineoplastic activity of cultured blue-green algae (cyanophyta) Journal of Phycology 27, 530-536. Reichelt, J.L. & Borowitzka, M.A. 1984 Antimicrobial activity from marine algae: results of a large scale screening programme. Hydrobiologia 116-117, 158-168. Starr, T.J., Dieg, E.F., Church, K.K. & Allen, M.B. 1962 Antibacterial and antiviral activities of algal extracts studied by acridine orange staining. TexasReportson Biologyand Medicine 20, 271-278. Vaara, T., Vaara, M. & Niemela, S. 1979 Two improved methods for obtaining axenic cultures of cyanobacteria. Applied and Environmental Microbiology 38, 1011-1014. Welch, A.M. 1962 Preliminary survey of fungistatic properties of marine algae. Journal of Bacteriology 83, 97-99.
(Received 9 December 1993; accepted 21 December 1993)
World Journal of Microbiology & Biotechnology, Vo110,1994
341
Bioactive compound production by thermophilic and thermotolerant cyanobacteria (blue-green algae) S.A. Fish and G.A. Codd* A thermotolerant species of Phormidium produced extracellular anti-microbial material during batch culture. Although this material was inactive when screened against a number of other cyanobacteria, it inhibited the growth of a wide range of Gram-positive and Gram-negative heterotrophic bacteria, Candida albicans and Cladosporium resinae. Key words: Anti-microbial products, bioactive products, blue-green algae, cyanobacteria, Phormidium.
Cyanobacteria have long been recognized to be capable of producing bioactive compounds (Lef6vre 1964). Recently, compounds from cyanobacteria have been isolated which display inhibitory effects on bacterial growth (Reichelt & Borowitzka 1984; Cannell et al. 1988), fungal growth (Kellam et al. 1988; De Mul6 et al. 1991), cancer cells (Kashiwagi et al. 1980; Carmeli et al, 1990), viruses (Starr et al. 1962; Gustafson et al. 1989) and enzymes (Cannell et al. 1987). Many mesophilic cyanobacteria have been screened for the above activities but before the present study, thermotolerant or thermophilic species had not been specifically targeted. The intention of the present investigation was to examine the production of bioactive compounds by cyanobacteria from thermal environments using microbial-based assay systems. To aid this, a screening system was necessary which could detect low concentrations of active metabolites, down to 0.1% of the solids in the supernatant (Hanka et al. 1978). As a consequence, an agar diffusion assay was used in conjunction with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), which assisted the visualization of zones of growth inhibition.
(Iceland) was isolated by Dr. I.L. Andrews from a water sample collected by D. Dyer (both of the University of Dundee) from a warm spring in Iceland. Synechocystis sp. (Kenya) and Phormidium sp. (Kenya) were isolated by Dr. I.L. Andrews from field material collected by Dr. W.D. Grant (University of Leicester) from Lake Bogoria, Kenya. Phormidium sp. (Azores) was isolated by Dr. I.L. Andrews from field material collected by Dr. R.J. Sharp (Centre for Applied Microbiology Research, Porton Down) from a hot spring on the island of San Miquel in the Azores. All the cyanobacteria were isolated into axenic culture using phototactic response and antibiotic treatment, according to the procedure of Vaara et aL (1979). Growth Conditions Cultures were grown in 250-ml conical flasks each containing 100ml Medium D which had as its constituents (rag/l): nitrilotriacetic acid, 100; CaSO4. 2H20, 60; MgSO4. 7I-~O, 100; NaC1, 8; KNO3, 103; NaNO3, 689; Na2HPO ¥ 111; FeCI3, 0.2905; M r l S O 4, H20, 2.28; ZnSO4. 71-t20, 0.5; H3BO3, 0.5; CuSO4. 5H20, 0.025; NaMoO 4. 2H20, 0.025; CoC12. 6H20, 0.046; Tris, 605.5; and 0.5 ml conc. H2SO4 in Milli Q deionized and ultrafiltered water (Brock 1978). The cultures were kept at 30°C ( _+1°C) unless specified otherwise, and shaken at 150 rev/min. They were continuously illuminated by cool white fluorescent light which gave an irradiance incident on the surface of the vessels of 2.0 W / m 2.
Isolation of Cyanobacteria Synechococcus PCC 6716 and Mastigocladus laminosus PCC 7521 were from the Pasteur Culture Collection, Paris. Synechococcussp.
Preparation of Material for Initial Bioactivity Screening After 4 weeks of incubation, 100 ml of the cyanobacterial cultures were centrifuged at 19,000 xg for 15 rain and the supernatant freeze-dried, suspended to 2% of the initial volume in Milli Q deionized and ultrafiltered water, and then sterilized by passage through a 0.22-pm pore sterile filter.
The authors am with the Department of Biological Sciences, University of Dundee, Dundee DD1 4HN, UK; fax: 0382 22318, *Corresponding author.
Measurement of Growth/Bioactivity of Phormidium sp. (Azores) Conical flasks, 500 ml, each containing 400 ml of Medium D, were incubated as detailed above. Over 8 weeks, 100-ml samples were
Materials and Methods
© 1994 Rapid Communications of Oxford Ltd
338
WorlaJournalofMicrobiology&Biotechnology,Vo110,1994
Bioactive compounds from cyanobacteria withdrawn from the conical flasks, filtered using glass fibre (grade C; Whatman) and then freeze-dried and prepared as above. The associated cells on the filter paper were freeze-dried and then weighed to plot growth characteristics.
Anti-microbial Assays Using Bacteria Sterile Sensitest agar (Oxoid), 10 ml, at 45 to 47°C, was seeded with 10 pl of an overnight culture of the test organism, gently swirled, poured into a 9-cm diam. Petri dish and allowed to dry in a laminar flow cabinet. Holes, 5 mm in diam., were cut in the agar and 20 lal of the test solution added to each. The plates were held at 4°C for 4 h to allow diffusion of the test material into the agar, then incubated at 37°C for 12h, followed by the application to the surface of the agar of 1 ml of phosphate-buffered saline containing 1 mg 3-(4,5-dimethylthiazoI-2-yl)2,5-diphenyltetrazolium bromide (MTT). After holding at 37°C for a further 15 min clear haloes surrounded by a blue background were seen. [The tetrazolium salt is cleaved by dehydrogenases (Mosmann 1983) to yield a blue formazan product, this indicating zones of growth promotion or growth inhibition of the test microorganism, the diameter of the halo increasing in proportion to growth inhibition.] Anti-microbial Assay using Fungi When using Candida albicans the assay was conducted as in the bacterial assay, except that incubation was extended from 12 to 18 h. When using Cladosporium resinae, sterile water was poured onto the surface of an existing plate culture to collect spores. These were inoculated into 10 ml of sterile Sensitest agar and prepared as previously described. The test material was introduced into 5-mm diam. wells in the agar and the plate incubated for 48 to 72 h, zones of clearing being noted. Cyanobacterial Growth Inhibition The procedure of Bagchi et aI. (1990) was used with minor modifications. Fifteen ml of 1.5% (w/v) agar No. 2 (Lab M, Amersham, UK) was poured into a 9 cm diam. Petri dish and allowed to solidify. Then 0.5ml of a 4-week-old batch culture of cyanobacteria was added to 2.5 ml of 0.8% (w/v) agar No. 2 in Medium D at 45 to 47°C, mixed and poured onto the base layer and the plates incubated at 30°C under cool white fluorescent light until a thick lawn of the test organism had grown. A 5-mm diam. well was then cut in the agar and the test material introduced into the well. The plate was incubated for I week and any growth inhibition noted.
Results and Discussion Table 1 summarizes the results of the initial screening of six thermotolerant/thermophilic cyanobacteria, isolated from a number of locations. Of the cyanobacteria examined, Mastigocladus laminosus PCC 7521 and the Phormidium isolate from the Azores produced extracellular material with the capacity to inhibit the growth of the test microorganisms. Of the two cyanobacteria displaying activity, Phormidium sp. (Azores) was selected for further study as it demonstrated a greater capacity for growth inhibition. Preliminary work associated with Phormidium (Azores) indicated that, although it grew more rapidly at 41°C than at 30°C, the production of the growth-inhibitory material was greatly decreased at the
Table 1, Inhibition of microbial growth supernatants, using the agar diffusion assay. Cyanobacterial source of eupernatant
by
Test organism* Staphylococcus aureus NCIMB 3251
Mastigocladus laminosus PCC 7521 Phormidium sp. (Azores) Phormidium sp. (Kenya) Synechococcus PCC 6716 Synechococcus sp. (Iceland) Synechocystis sp. (Kenya)
cysnobactarlal
++ ++++
Candida albicans 3153 A +++ +++
-
-
-
*The diameter of the inhibition zone was scored + (15 mm). A control, a 50 x concentration of Medium D gave no inhibition (-). NCIMB~National Collection of Industrial and Marine Bacteria, Aberdeen, UK; PCC--Pasteur Culture Collection, Paris, France.
higher temperature. Consequently further incubations of this organism were at 30°C. Table 2 shows an expansion of the microbial screening systems used in Table 1. The extracellular material produced by Phormidium (Azores) displayed a wide range of growth inhibitory activity, affecting Gram-positive and Gram-negative bacteria as well as a yeast and a fungus. Although the growth of a wide range of microbes was inhibited, two of the Gram-negative heterotrophic bacteria were unaffected, this being in keeping with the lack of growth inhibition of the cyanobacteria tested. Figure 1 shows the relationship between the growth of Phormidium (Azores) in batch culture and the production of the growth-inhibitory material, as assayed against Staphylococcus aureus. The results indicate a biphasic pattern of production of the growth-inhibitory material throughout batch culture, unlike the production of many antibiotics which are produced during the stationary phase (Drew & Demain 1977). The detection of extraceUular compounds of cyanobacterial origin which inhibit bacteria or cyanobacteria is not new (Flint & Mooreland 1946; Mason et al. 1982; Flores & Wolk 1986; Cannell et al. 1988). Likewise, products from cyanobacteria that inhibit fungi have been recognized for some time (Welch 1962; Kellam et al. 1988; De Cano et al. 1990; De Mul6 et al. 1991). However, with the exception of one other study (Bloor & England 1989), no cyanobacterium other than Phormidium sp. (Azores) has been isolated with such a broad range of activity against microbes, including Gram-positive and Gramnegative bacteria, a yeast and a fungus. However, it was found that when the extracellular material was tested against other cyanobacteria, no growth inhibition was recorded. The inhibition of growth of most of the diverse group of microbes screened (Table 2) indicates that the Phormidium (Azores) isolate contains an anti-microbial compound with a broad spectrum of activity, a range of more host-specific
WorldJournalofMicrobiology&Biotechnology,Vo110,1994
339
S.A. Fish and G.A. Codd Table 2. Anti-microbial activity of supernatant from Phormidlum sp. (Azores), measured using the agar diffusion assay.
0.035
/
0.03 Test Organism
Activity* 0.025
A. Gram-positive heterotrophic bacteria Bacillus cereus NCIMB 3329 Bacillus coagulans NCIMB 9365 Bacillus licheniformis NCIMB 6346 Bacillus sphaericus NCIMB Bacillus subtilis NCIMB 1650
Staphylococcus aureus NCIMB 3251 Streptococcus faeca/is NCIMB 775 Streptococcus viridi NCIMB 8954 B. Gram-negative heterotrophic bacteria Escherichia coli NCIMB 9484 Hafnia alvei NCIMB 6578 Kleb$iel/a pneumoniae NCIMB 8805 Proteus vu/garis NCIMB 67 Proteus morganii NCIMB 232 Pseudomonas aeruginosa NCIMB 8295 Pseudomonas denitdficans NCIMB 9496
+++
.,~
.m
++
o.o15-
++++ ++
0.02
a 0.01
++ ++++
0.005
/
ml
B
m
m
m
+++ +++
a
16
24 32 Time (days)
40
48
56
8
16
24 32 Time ( d a y s )
40
48
56
+ +++ +++ +++ +++
~ 25-
55 ~
20-
~
10-
C. Cyanobacteria
Mastigocladus laminosus PCC 7521 Phormidium sp. (Azores) Phormidium sp. (Kenya) Synechococcus PCC 6716 Synechococcus sp. (Iceland) Synechocystis sp. (Kenya)
i:5 o
D. Yeast/Fungus
Candida albicans 3153 A Cladosporium resinae ATCC 22711
+++ +++
*Inhibition was scored as in Table 1. NCIMB--National Collection of Industrial and Marine Bacteria, Aberdeen, UK; ATCC--Amedcan Type Culture Collection; A--Dundee University Culture Collection, UK.
growth inhibitors, or a combination of the two classes of bioactive products. Indeed, the kinetics of accumulation of extracellular growth-inhibitory material, even when screened against a single test organism, were biphasic (Figure 1B) and further studies (S.A. Fish & G.A. Codd, unpublished work) have revealed the presence of at least two growth-inhibitory products of this cyanobacterium, with M r of approx. 424 and 762. Finally, whilst the biotechnological potential of cyanobacteria is attracting increased attention (Patterson et al. 1991), little work has been done to screen cyanobacteria isolated from extreme environments with regard to their production of bioactive compounds. The results of this work indicate that this group of organisms displays potential in this respect, a potential which warrants further investigation.
340
World Journal of Microbiology & Biotechnology, Vo110,1994
Figure 1. (A) Growth of Phormidium sp. (Azores) in batch culture, determined as an increase in dry weight and (B) detection of bioactive material in the supernatant of the Phormidium cultures when screened against Staphylococcus aureus. Inhibition of S. aureus growth was determined by agar well diffusion assay, each point in (B) representing the mean of three replicate wells each loaded with 20pl of concentrated resuspended freeze-dried supernatant material (see Materials and Methods). Vertical bars represent standard deviations.
Acknowledgements S.A.F. gratefully acknowledges the Science and Engineering Research Council for the award of a Postgraduate Research Studentship.
References Bagchi, S.N., Palod, A. & Chauhan, V.S. 1990 Algicidal properties of a bloom-forming blue-green alga, Oscillatoria sp. Journal of Basic Microbiology 30, 21-29. Bloor, S. & England, R.R. 1989 Antibiotic production by the cyanobacterium Nostoc muscorum. Journal of Applied Phycology 1, 367-372. Brock, T.D. 1978 Thermophflic Microorganisms and Life at High Temperatures. New York: Springer-Verlag.
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(Received 9 December 1993; accepted 21 December 1993)
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