Screening microalgae for some potentially useful ... - Springer Link

Journal of Applied Phycology 16: 309–314, 2004. C 2004 Kluwer Academic Publishers. Printed in the Netherlands.

309

Screening microalgae for some potentially useful agricultural and pharmaceutical secondary metabolites ¨ og1 , W. A. Stirk2,∗ , R. Lenobel3 , M. Banc´ı ˇrová 3 , M. Strnad3 , J. van Staden2 , V. Ord¨ J. Szigeti4 and L. Németh5 1

Department of Plant Physiology and Plant Biotechnology, Faculty of Agricultural and Food Sciences, University of West Hungary, H-9200 Mosonmagyaróvár, Hungary; 2 Research Centre for Plant Growth and Development, University of KwaZulu-Natal Pietermaritzburg, P/Bag X01, Scottsville 3209, South Africa; 3 Laboratory of Growth Regulators, Palacký University & Institute of Experimental Botany AS CR, Slechtitelu˙ 11, 783 71 Olomouc, Czech Republic; 4 Institute of Food Sciences, Faculty of Agricultural and Food Science, University of West Hungary, H-9200 Mosonmagyaróvár, Hungary; 5 Department of Plant Protection, Faculty of Agricultural and Food Sciences, University of West Hungary, H-9200 Mosonmagyaróvár, Hungary ∗ Author for correspondence (e-mail: [email protected]) Received 14 January 2004; revised and accepted 10 May 2004

Key words: anticancer, antimicrobial, Chlorophyta, Cyanobacteria, microalgae Abstract Nearly two hundred microalgal strains (174 Chlorophyta and 23 Cyanobacteria) were screened against some bacteria, filamentous fungi and yeasts using a disc-diffusion type bioassay. From this initial screening, 10 Chlorophyta strains from three genera (Desmococcus, Chlorella and Scenedesmus) were selected because of their high antimicrobial activity. These 10 strains were partially purified and tested using MIC antimicrobial and microtiter IC50 anticancer assays. These preselected algal strains showed a high incidence of antibacterial activity against both Gram-positive (9 out of 10 species) and Gram-negative (7 out of 10 species) bacteria. The extracts were also effective against some tumour cell lines. Abbreviations: cfu, colony forming units; HNCMB, Hungarian National Collection of Medicinal Bacteria (Budapest); MACC, Mosonmagyaróvár Algal Culture Collection; MIC, minimum inhibitory concentration; NCAIM, National Collection of Agricultural and Industrial Microorganisms (Budapest) Introduction Increasing global population and a greater demand for food have led to more intensive farming practices resulting in increased disease (Lewis & Papavizas, 1991). Plant disease epidemics occur in cultivated crops because of monoculture, providing no barrier for the dissemination of the disease agent and a loss of genetic diversity due to selective plant breeding (Scheffer, 1997). There are more than 200 bacteria that infect plants of which almost half belong to the genus Pseudomonas and cause rots, wilts, blights and cankers. Xanthomonas species cause serious leaf blights and

leaf spots. Erwinia species cause blights, wilts and soft rots. Fungi are also very common plant parasites, and most crops are susceptible to one or more fungal diseases (Scheffer, 1997). There are many strategies to control plant disease encompassing breeding resistant varieties and chemical control. The main limitation on plant breeding is the adaptability of pathogens. Agrochemicals (bactericides, fungicides and insecticides) are expensive and cause environmental pollution and pose a health hazard (Lewis & Papavizas, 1991; Scheffer, 1997). One alternative approach is “biocontrol”. This is not a new concept with “suppressive soils,” whose ability

310 to reduce disease in susceptible crops is well documented. Natural soil organisms are thought to control soil-borne disease by the production of antibiotics and other inhibitory compounds, as well as by competition, parasitism and predation (Hornby, 1983; Lewis & Papavizas, 1991). Cyanobacteria are common in rice paddies (Whitton, 2000) and their beneficial effect on growth, yield and nitrogen levels has been reported. This was initially attributed to their nitrogen-fixing ability as well as to the production of antibiotics and toxins, organic acids, vitamin B and other hormone-like substances (Pedurand & Reynaud, 1987). These are made available to the crop by exudation, autolysis and microbial decomposition (Boussiba, 1988). Soil microorganisms are also regarded as useful soil conditioners with their polysaccharides aggregating the soil particles to improve soil stability and water-holding capacity (Metting et al., 1990). Although most algalization studies have focussed on the Cyanobacteria because of their nitrogen-fixing ability, microalgae belonging to the Chlorophyta should also be considered. Of practical importance is the fact that these algae generally have a faster growth rate than Cyanobacteria and are easier to culture in mass. Algae produce a number of secondary metabolites as a chemical defence against predation, herbivory and competition for space (De Lara-Isassi et al., 2000; de Nys et al., 1998 ), and some secondary metabolites have been identified. For example, the fatty acid chlorellin was identified as the active antibacterial principal in Chlorella vulgaris in the 1940s (Robles Centeno & Ballentine, 1999). Chlorella vulgaris extracts administered orally to tumour-bearing mice significantly prolonged their survival by enhancing phagocyte production and quality (Justo et al., 2001), and both algae and Cyanobacteria have antiviral activity towards mumps, influenza and the Herpes simplex virus (Huleihel et al., 2001; Schaeffer & Krylov, 2000). Some potential applications to consider for algae are the production of medicinal compounds for the pharmaceutical industry and in the agricultural sector as both biofertilizers and biocontrol agents. It is necessary to screen many algal strains before suitable strains can be selected for either application. The objective of this study was to initially screen many algal strains against phytopathogenic, foodborne and spoilage bacteria, fungi and yeasts to identify strains containing potentially useful secondary metabolites. These few selected algal strains were then further tested against other bacterial strains and for cytotoxic activity with

the idea of finding a higher occurrence of biological activity.

Materials and methods Preliminary screening for biological activity One hundred and ninety seven microalgal strains from 35 genera of Cyanobacteria and Chlorophyta (Table 1) were screened. The algae were obtained from the Mosonmagyaróvár Algal Culture Collection (MACC) and grown in batch culture in the apparatus described ¨ og (1982). Only axenic strains were used. This by Ord¨ was tested by spraying a diluted algal suspension onto the surface of solidified nutrient medium enriched with 0.5% glucose, 0.5% peptone and 1% yeast extract. The absence of bacterial and fungal colonies after 5–7 days incubation period indicated that the culture was axenic. For this screening, the algal cultures were harvested in their early stationary phase of growth, always between 1–2 PM. After centrifugation (Sigma 6K15, Germany) for 15 min at 2150g, the supernatant-free biomass was freeze-dried (Christ Gamma 1–20, Germany) for 22 h at 0.035 mbar and stored at −20 ◦ C until analysed. Algal extracts were made by resuspending the freeze-dried algae in distilled water (10 mg mL−1 ) and ultrasonicated (VirSonic 600, USA) for 2 min. These extracts were tested for biological activity against a number of filamentous fungi, yeasts and bacteria (Table 2) using an agar diffusion method. Agar plates made from specific culture media, depending on the microorganism (Table 2), were inoculated with a standardized quantity of suspension containing 1.5 × 108 cfu mL−1 bacteria, 1.0 × 107 cfu mL−1 yeasts or 1.0 × 105 cfu mL−1 filamentous fungi (conidia). Wells (8 mm diameter) punched in the agar plates were filled with 90 µL of the hydrophilic algal extracts (10 mg mL−1 ). Prediffusion at 4◦ C for 4 h was allowed to improve the sensitivity of the test system. The inhibition or stimulation zones were measured after incubation in aerobic conditions at specific temperatures and culture times (Table 2). For each algal extract and microorganism, there were three replicates per assay (pseudo replicates) and each assay was repeated three times. Ten strains from three Chlorophyta genera were selected, based on their general high inhibitory activity against the various microorganisms. These strains were partially purified and screened using a MIC antimicrobial assay and a microtiter IC50 anticancer assay.

311 Table 1. Microalgal strains tested in the preliminary screening for biological activity. Genera

No. of strains

Cyanobacteria Anabaena

3

Calothrix

2

Leptolyngbya

6

Lyngbya

2

Microcystis

1

Nostoc

2

Phormidium

3

Pseudanabaena

1

Synechococcus

1

Synechocystis

1

Tolypothrix

1

Genera – 11

Extraction and partial purification of the active compounds

Strains – 23

Chlorophyta Bracteacoccus

2

Chlamydomonas

14

Chlorella

51

Chlorococcum

14

Chlorosarcina

2

Closteriopsis

2

Coccomyxa

2

Coenochloria

9

Desmococcus

11

Klebsormidium

3

Monoraphidium

4

Myrmecia

1

Neochloris

13

Neochlorosarcina

2

Oocystidium

1

Oocystis

7

Poloidion

1

Pseudococcomyxa

2

Scenedesmus

23

Scotiella

2

Scotiellopsis

1

Spongiochloris

2

Tetracystis

4

Westella

1

Genera – 24

Strains – 174

Total Genera – 35

Total Strains – 197

To test for anticancer and antimicrobial activity, 500– 1000 mg of the dried algae was extracted with 30 mL 100% ethanol for 2 h and then centrifuged at 15 000g at 4 ◦ C for 15 min. The pellet was reextracted using the same method. Both extracts were combined and purified using a C18 column to remove any chlorophyll and then dried at 35 ◦ C under vacuum. The dried extracts were dissolved in 200 µL 50% methanol and 800 µL 0.1 M Tris (pH 7.5) and filter sterilised using a 0.2 µm filter. MIC antimicrobial assay The algae were tested against the Gram-positive bacteria strains Enterococcus faecalis 4224, Staphylococcus aureus 3953 and 4223 and the Gram-negative bacteria Pseudomonas aeruginosa 3955 and Escherichia coli 3954, 3988 and 4225 (Czech Collection of Microorganisms, Brno). Bacteria were precultured for 2 h at 37 ◦ C in 5 mL Mueller-Hinton Broth and then diluted with distilled water in a 1:10 ratio. Stock extracts of the algae were prepared in distilled water and added to the sterile microtiter wells in a twofold dilution series. The wells were then inoculated with the bacterium. The cultivating medium for Grampositive bacteria contained 37 g Brian Heart Infusion in 1 L distilled water and the medium for Gram-negative bacteria consisted of 5 g Protose-BE, 17.5 g Casein Acid Hydrolysate and 0.06 g Na2 CO3 in 1 L distilled water. The plates were incubated for 18 h at 37 ◦ C and then examined for bacterial growth. The lowest concentration of each extract dilution series that prevented bacterial growth was considered to be the minimum inhibitory concentration (MIC) of the extract. There were three replicates per assay and each test was repeated at least twice. Anticancer assay Four cancer cell lines of different histogenetic and species origin were used—the MCF7 (human breast adenocarcinoma), CEM (human lymphoblastoid leukaemia), G361 (human malignant melanoma) cancer cell lines and NIH3T3 (mouse fibroblasts) modified normal cell line. These cell lines were grown in DMEM medium (Gibco BRL) supplemented with 10% (v/v) fetal bovine serum and L-glutamine and maintained at 37 ◦ C in a humidified atmosphere with 5% CO2 . Each

312 Table 2. Microorganisms and culture conditions used in the preliminary screening. Incubation conditions Test organism

Culture medium

Time (days)

Temp (◦ C)

Note

Filamentous fungi Alternaria sp. (self isolated)

Phytopathogenic

Potato dextrose agar

4–7

24

Aspergillus wentii NCAIM F.00167


4–7

24

Spoilage

Botrytis cinerea NCAIM F.00744


4–7

24

Phytopathogenic

Fusarium oxysporum NCAIM F.00728


4–7

24

Phytopathogenic

Penicillium expansum NCAIM F.00601


4–7

24

Spoilage

Rhizopus stolonifer NCAIM F.00654


4–7

24

Spoilage

Saccharomyces cerevisiae NCAIM Y.00571

GYP agar

3

26

Useful (bakers) yeast

Zygosaccharomyces bailii NCAIM Y.00734

GYP agar

3

26

Spoilage

Bacillus subtilis NCAIM B.01623

CASO agar

3

26

Foodborne

Bacillus thúringiensis NCAIM B.01286

CASO agar

2

30

Pathogenic for larva of Lepidoptera

Staphylococcus aureus HNCMB 112002

CASO agar

1

37

Foodborne

Erwinia carotovora NCAIM B.01109

CASO Agar

3

26

Phytopathogenic

Escherichia coli HNCMB 35035

CASO Agar

1

37

Foodborne

Pseudomonas syringae pv. syringae NCAIM B.01398

CASO agar

3

26

Phytopathogenic

Salmonella arizonae HNCMB 42021

CASO agar

1

37

Foodborne

Xanthomonas campestris pv. campestris NCAIM B.01224

CASO agar

3

26

Phytopathogenic

Yeasts

Bacteria (Gram-positive)

Bacteria (Gram-negative)

well of a 96-well plate was seeded with 104 cells, allowed to stabilise for at least 2 h after which 20 µL of the algal extracts was added at six concentrations ranging from 5 to 500 µL. The plates were incubated at 37 ◦ C and 5% CO2 for 3 days after which Calcein AM solution (Molecular Probes) was added. After 1 h, the fluorescence of the viable cells was quantified using Fluoroskan Ascent (Labsystems). The assay was done in triplicate. The extract concentration lethal to 50% of the tumour cells (IC50 ) was calculated from the dose response curves obtained.

gal strains inhibited the Gram-positive bacteria Staphylococcus aureus. Desmococcus olivaceus (MACC 343) stimulated the growth of the Gram-positive bacteria Bacillis thúringiensis. Scenedesmus sp. (MACC 540) inhibited the fungi Alternaria sp. MIC antimicrobial assay

Results

As hoped, the preselected algal strains showed a high incidence of antibacterial activity against both the Gram-positive (9 out of the 10 species) and the Gramnegative (7 out of the 10 species) bacterial strains tested (Table 4). There was variation within species with different strains showing differences in biological activity.

Preliminary screening for biological activity

Anticancer assay

Ten strains from three Chlorophyta genera (Desmococcus, Chlorella and Scenedesmus) were selected for the high antimicrobial activity of their aqueous extracts against the various microorganisms (Table 3). Eight al-

The algal extracts inhibited the four tumour cell lines (Table 5). Growth of normal mouse fibroblasts ( NIH3T3) was inhibited but at least at 10× higher algal concentrations. The algal extracts were the most

313 Table 3. Results of the initial screening disc-diffusion assays against various microorganisms. Genera and species (MACC)

Microorganisms

Inhibition zone (mm)

Desmococcus olivaceus (324)

Staphylococcus aureus

6.7 ± 0.3


Pseudomonas syringiae

5.3 ± 1.1

Stimulation zone (mm)

16.7 ± 3.8

Bacillus thúringiensis Chlorella minutissima (357)


6.3 ± 1.1

Chlorella minutissima (360)


6.2 ± 0.1



8.0 ± 0.0

Chlorella sp. (313)


6.5 ± 0.3

Chlorella sp. (381)


5.7 ± 0.9

Chlorella sp. (458)


7.7 ± 0.8

Scenedesmus sp. (469)



Alternaria sp.

5.0 ± 1.3 10.0 ± 0.0

Note. Only results where inhibition or stimulation of microbial activity was achieved are shown. The results are presented as mean ± SE from three assays each with three replicates. Table 4. Antimicrobial MIC concentrations (mg mL−1 ) of algal extracts. Gram-positive bacteria

Gram-negative bacteria

Genera and species (MACC)

Sa3953

Sa4223

Ef4224

Ec4225

Ec3988

Ec3954

Pa3955


0.015

0.008

0.008

>0.120

>0.120

>0.120

>0.120


0.003

0.003

0.003

0.115

0.058

0.115

0.058


0.012

0.012

0.018

>0.118

>0.118

0.118

0.059


0.006

0.007

0.012

0.118

0.059

0.118

0.059


0.001

0.003

0.003

0.115

0.058

0.115

0.058

Chlorella sp. (313)

0.063

>0.125

>0.125

0.125

0.125

0.125

0.125

Chlorella sp. (381)

0.002

0.002

0.002

>0.125

0.013

0.013

>0.125

Chlorella sp. (458)

0.012

0.006

0.006

0.120

0.060

0.120

0.060


0.028

0.056

0.056

0.113

0.056

0.113

0.056


>0.115

>0.115

0.115

>0.115

0.115

>0.115

0.115

Note. Sa = Staphylococcus aureus, Ef = Enterococcus faecalis, Ec = Escherichia coli, Pa = Pseudomonas aeruginosa.

effective against the cell line with mutations in the cell cycle associated with proteins ( CEM). Discussion The initial screening identified 10 algal strains whose water extracts had antimicrobial activity. When ethanolic extracts of these 10 Chlorophyta strains were tested against different bacterial strains, high levels of activity were noted for almost all of the species. Particularly encouraging was the inhibitory (bacteriostatic) activity against the Gram-negative bacteria. Generally antibiotics are less effective against Gram-negative bacteria because of their more complex multilayered cell wall structure with additional lipopolysaccharides on

the outer cell surface, which makes it more difficult for the active compound to penetrate (Rang & Dale, 1987). This is an important consideration as most of the bacterial diseases affecting crops are due to Gram-negative bacteria (Scheffer, 1997). However, this potential ability of algae to inhibit certain soil-borne diseases needs further investigation before they can be accepted as “biocontrol agents” for agriculture. A further advantage of these 10 algal strains is that they ¨ og et al., contain many endogenous cytokinins (Ord¨ 2004) that are known to be beneficial to crop growth and yield (Metting et al., 1990). These 10 preselected strains could also potentially be effective against tumours with various alterations of tumour suppressor genes such as p53 and pRb.

314 Table 5. IC50 inhibitory concentrations (mg well−1 ) of the algal strains tested against four cancer cell lines. Cell lines Genera and species (MACC)

MCF7

CEM

G361

NIH3T3


1.27

0.64

1.45

>2.0


0.26

0.20

1.21

1.52


0.95

0.32

1.25

>2.0


0.56

0.26

1.37

1.54


0.20

0.22

1.15

1.31

Chlorella sp. (313)

0.93

0.61

1.65

>2.0

Chlorella sp. (381)

0.21

0.15

1.03

1.15

Chlorella sp. (458)

>1.0

0.60

1.45

>2.0


0.07

0.06

0.17

1.41


0.29

0.25

1.36

1.58

Strain selection is important, as biological activity varied between strains of the same species tested in this study. It is uncertain if this variation was due to inherent strain differences or due to different physiological states of the cultures when harvested although the cultures were harvested in their early stationary phase of growth to minimize some of this variation. Growth conditions do affect biological activity, as there are optimal conditions for the synthesis of secondary metabolites. For example, various irradiance levels resulted in different degrees of antimicrobial activity in the red macroalgae Spyridia filamentous (Robles Centeno & Ballentine, 1999). The conditions under which the various secondary metabolites are produced by the algae must still be determined in order to maximise the production of the useful secondary metabolites. The importance of being able to easily grow and manipulate culture conditions of suitable axenic strains of algae must be emphasised in this regard. These results show that some microalgae do contain useful secondary metabolites and as they are relatively easily grown in mass culture, they provide a source of biological material with potential application in both agricultural and pharmaceutical industries. The ultimate goal is to develop suitable algal strains that improve crop growth and control disease in a costeffective, environmental-friendly manner. This study has identified some strains on which more detailed studies should concentrate. Acknowledgements This study was made in the framework of the intergovernmental South African–Hungarian scientific and

technical cooperation programme that had been established between the International Deputy State Secretariat of the Ministry for Education of Hungary and the National Research Foundation of South Africa. This work was also supported by a grant of the Czech Ministry of Education No. MSM 153100008 and the Grant Agency of the Czech Republic No. 522/03/0323. The International Foundation for Science (IFS) in Stockholm, Sweden is also thanked for financial assistance. References Boussiba S (1988) Anabaena azollae as a nitrogen biofertilizer. In Stadler T, Mollion J, Verdus M-C, Karamanos Y, Morvan H, Christiaen D (eds), Algal Biotechnology, Elsevier Applied Sciences, England, pp. 169–178. ´ De Lara-Isassi G, Alvarez-Hern´ andez S, Collado-Vides L (2000) Ichtyotoxic activity of extracts from Mexico marine macroalgae. J. Appl. Phycol. 12: 45–52. De Nys R, Dworjanyn SA, Steinberg PD (1998) A new method for determining surface concentrations of marine natural products on seaweeds. Marine Ecol. Prog. Ser. 162: 79–87. Hornby D (1983) Suppressive soils. Ann. Rev. Phytopath. 21: 65–85. Huleihel M, Ishanu V, Tal J, Arad S (2001) Antiviral effect of red microalgal polysaccharides on Herpes simplex and Varicella zoster viruses. J. Appl. Phycol. 13: 127–134. Justo GZ, Silva MR, Queiroz MLS (2001) Effects of the green algae Chlorella vulgaris on the response of the host hematopoietic system to intraperitoneal ehrlich ascites tumour transplantation in mice. Immunopharm. Immunotoxicol. 23: 119–131. Lewis JA, Papavizas GC (1991) Biocontrol of plant diseases: The approach of tomorrow. Crop Protect. 10: 95–105. Metting B, Zimmerman WJ, Crouch IJ, van Staden J (1990) Agronomic use of seaweeds and microalgae. In Akatsuka I (ed), Intoduction to Applied Phycology, SPB Academic Publishing, The Hague, The Netherlands, pp. 589–627. ¨ og V (1982) Apparatus for laboratory algal bioassay. Int. Rev. Ord¨ Ges. Hydrobiol. 67: 127–136. ¨ og V, Stirk WA, van Staden J, Novák O, Strnad M (2004) EnOrd¨ dogenous cytokinins in microalgae (Chlorophyta). J. Phycol. 40: 88–95. Pedurand P, Reynaud PA (1987) Do cyanobacteria enhance germination and growth of rice? Plant Soil 101: 235–240. Rang HP, Dale MM (1987). Pharmacology. Churchill Livingstone, Edinburgh, UK. Robles Centeno P, Ballentine DL (1999) Effects of culture conditions on production of antibiotically active metabolites by the marine alga Spyridia filamentosa (Ceramiaceae, Rhodophyta), I. Light. J. Appl. Phycol. 11: 217–224. Shaeffer DJ, Krylov V (2000) Anti-HIV activity of extracts and compounds from algae and cyanobacteria. Ecotoxic. Environ. Safety 45: 208–227. Scheffer RP (1997) The Nature of Disease in Plants. Cambridge University Press, Cambridge, UK. Whitton BA (2000) Soils and rice-fields. In Whitton BA, Potts M (eds), The Ecology of Cyanobacteria, Kluwer Academic, Dordrecht, pp. 233–255.