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ABSTRACT Certain aquatic and/or terrestrial plants have been used as artificial floating island systems in water pollution control. This research was carried out to confirm the release of anticyanobacterial allelochemicals from aquatic and terrestrial plants suitable for artificial floating island systems. A series of cyanobacterial assays using the culture solution extracts of umbrella plant (Cyperus alternifolius) and Canna (Canna generalis) demonstrated the release of anticyanobacterial allelochemicals. GC/MS analysis of the solid extract of C. alternifolius culture solution indicated the existence of 9 phenolic compounds [resorcinol, 3-hydroxy benzoic acid, 4- hydroxy benzoic acid, (4-hydroxyphenyl) acetic acid, vanillic acid, protocatechuic acid, pcoumaric acid, gallic acid, and ferulic acid], and 4 carboxylic compounds (azelaic acid, butanedioic acid, dehydroabietic acid, and malic acid) in which anti-cyanobacterial compounds were involved
Journal of Water and Environment Technology, Vol. 6, No.1, 2008 - 55 - Release of anti-cyanobacterial allelochemicals from aquatic and terrestrial plants applicable for artificial floating islands S. Nakai*, G. Zou**, X. Song**, Q. Pan**, S. Zhou*** and M. Hosomi*** * Graduate School of Engineering, Hiroshima University, 1-4-1 Kagamiyama, Higashi- Hiroshima, Hiroshima 739-8527, Japan ** Shanghai Academy of Agricultural Science, 2901 Beidi Rd, Shanghai 201106,China *** Faculty of Engineering, Tokyo University of Agriculture and Technology, 2-24-16 Naka, Koganei, Tokyo 184-8588, Japan ABSTRACT Certain aquatic and/or terrestrial plants have been used as artificial floating island systems in water pollution control. This research was carried out to confirm the release of anti- cyanobacterial allelochemicals from aquatic and terrestrial plants suitable for artificial floating island systems. A series of cyanobacterial assays using the culture solution extracts of umbrella plant (Cyperus alternifolius) and Canna (Canna generalis) demonstrated the release of anti- cyanobacterial allelochemicals. GC/MS analysis of the solid extract of C. alternifolius culture solution indicated the existence of 9 phenolic compounds [resorcinol, 3-hydroxy benzoic acid, 4- hydroxy benzoic acid, (4-hydroxyphenyl) acetic acid, vanillic acid, protocatechuic acid, p- coumaric acid, gallic acid, and ferulic acid], and 4 carboxylic compounds (azelaic acid, butanedioic acid, dehydroabietic acid, and malic acid) in which anti-cyanobacterial compounds were involved. Keywords: Allelopathy, Canna, carboxylic comp., cyanobacteria, growth inhibition, phenolic comp., umbrella plant. INTRODUCTION In eutrophicated ponds, lakes and dams, occurring cyanobacterial blooms have resulted in serious problems in regard to the effective utilization of water resources, such as fisheries and water-supply reservoirs. In order to avoid the occurrence of blooms by improving water quality, the reduction in nutrient loading is a practical method; however its effectiveness is limited due to the difficulty in controlling non-point sources and/or direct nutrient loading, such as fertilizing soils to maintain vegetation in approximate areas and/or fisheries. Therefore, artificial floating islands with vegetation have been a focus as a means of removing nutrients, and restoring the aquatic ecosystems. The artificial floating islands with vegetation have been applied to many lakes, ponds, and dams (Hoeger, 1988; Nakamura and Shimatani, 1997; Nakamura and Shimatani, 1999; Hayashi et al., 2003, Hirose et al., 2003), and the resultant removal of nutrients such as total nitrogen and phosphorus, as well as reduction in cyanobacterial biomass have been confirmed (Nakamura and Shimatani, 1997; Hayashi et al., 2003). Although light interception and/or nutrients removal causes suppression of cyanobacterial growth (Nakamura and Shimatani, 1999), an improvement of the zooplankton community Address correspondence to Satoshi Nakai, Graduate School of Engineering, Hiroshima University, Email: sn4247621@hiroshima-u.ac.jp Received February 23, 2008, Accepted April 18, 2008. Journal of Water and Environment Technology, Vol. 6, No.1, 2008 - 56 - (Hayashi et al., 2003) and allelopathy (Nakamura and Shimatani, 1999) are also possible causes. Allelopathy is defined as any process involving secondary metabolites produced by plants and other organisms that influence the growth and development of biological and agricultural systems (Marick, 2005). Since certain kinds of macrophytes have been reported to cause allelopathic growth inhibition of cyanobacteria (Barrett et al., 1996; Ridge and Pillinger, 1997; Gross et al., 1994, Nakai et al., 1999, 2006), it is natural to expect that vegetation used in artificial floating islands may also cause allelopathic growth inhibition of cyanobacteria. In fact, some papers reported the existence of anti- cyanobacterial compounds in the plant bodies suitable for artificial floating islands. For example, the anti-cyanobacterial phenylpropanes were identified in the hexane extract of Acorus gramineus (Greca et al., 1989), while in recent Li and Hu (2005) confirmed anti-cyanobacterial activity of 2-methyl acetoacetic acid found in the ethanol extract of Phragmites communis. In addition, Cyperus rotundus is known to contain the anti- cyanobacterial phenolic compounds such as 3,4-dihydroxy benzoic acids (Quayyum et al., 2000; Proestos et al., 2005), though they did not surveyed anti-cyanobacterial activities of the plant extracts. These findings indicates the possible release of anti-cyanobacterial compounds from vegetation in artificial floating islands, and such allelopathic vegetation may be used in artificial floating islands for effective cyanobacterial control, as well as an improvement in water quality. However, the release of anti-cyanobacterial allelochemicals has not been demonstrated. Therefore, this research was carried out to confirm whether or not vegetation used in artificial floating islands releases anti-cyanobacterial allelochemicals and to identify such fascinating compounds. MATERIALS AND METHODS Plants and cyanobacterium Commercially obtained umbrella plant (Cyperus alternifolius) and Canna (Canna generalis) were used for testing, because the artificial floating islands vegetated with these plants applied to Wuli-hu lake in China demonstrated a good performance on water quality improvement (data not published). Prior to the experiments, roots of these plants were carefully washed with tap water to remove soil and debris. C. alternifolius and C. generalis were respectively cultivated in 4 L of tap water for 2 weeks to obtain their exudates. As a cyanobacterium, we used Microcystis aeruginosa (NIES-87), of which clone was obtained from the microbial culture collection of the National Institute for Environmental Studies (NIES). Growth of M. aeruginosa was monitored by using a hemocytometer (Thoma JHS, Hishikaki, Japan). Confirmation of the release of anti-cyanobacterial allelochemicals In order to confirm the release of anti-cyanobacterial allelochemicals, the culture solution of each plant was subjected to a solid extraction procedure followed by a cyanobacterial assay. Briefly, after filtering 2 L of the culture solution through a grass fiber filter (GF/F, Whatman), the filtrate was adjusted to pH2 using 6N HCl, and subsequently passed through a solid phase extraction cartridge (OASIS HLB, Waters). After elution using 5 ml of methanol, 10 µL of the methanol eluent was added to 10 ml of CB medium (Watanabe and Satake, 1991) in a test tube, and after that M. aeruginosa Journal of Water and Environment Technology, Vol. 6, No.1, 2008 - 57 - was inoculated (1 × 10 4 cells/ml), and incubated at 25 ℃ under a light intensity of 4000 lux. As control experiment, tap water was subjected to this solid extraction process, and the resultant tap water extract was assayed. Analysis To identify anti-cyanobacterial allelochemicals released from the tested plant, the solid phase extract of its culture solution was analyzed by gas chromatography/mass spectrometry (GC/MS). After the solid phase was extracted with sodium sulphate anhydrate, 1 ml of the extract was heated at 40 ℃ under a gentle stream of nitrogen for removal of methanol. The resultant residues were treated with 200 µL of N,O- bis(trimethylsilyl) trifluoroacetamide (BSTFA, Tokyo Kasei) for 3 h at 50 ℃ for trimethylsilyl (TMS) derivatization. After removing any excess BSTFA, the samples were dissolved in a small volume of ethyl acetate (40 µL), and subjected to GC/MS analysis in the electron ionization mode (Table 1). Dissolved organic carbon (DOC) concentration of the culture solution was also determined by a total organic carbon analyzer (TOC5000, Shimadzu). Table 1 - Operating conditions for GC/MS. RESULTS AND DISCUSSION Growth inhibition in culture solution Figure 1 compares the growth of M. aeruginosa after 25 d of incubation as affected by the solid extracts of C. alternifolius and C. generalis culture solutions. The growth of M. aeruginosa was inhibited by the solid extracts of both tested plants culture solutions, thereby confirming the release of anti-cyanobacterial allelochemicals from C. alternifolius and C. generalis. Note that the inhibitory effect of the C. alternifolius solid extract is greater than that of C. generalis. C. alternifolius has been used as a plant for water purification (Miyazaki, et al., 1997; Neralla et al., 1999; Kantawanichkul et al., 2001; Hirose et al., 2003). Therefore, we analyzed the solid extract of C. alternifolius culture solution to identify anti-cyanobacterial allelochemicals. GC/MS Hewlett Packard 6890 series/5973 series Column HP5-MS (30.0 m × 250 µm, 0.25 µm) Carrier gas He (99.9999%) Oven temp. 100°C (1 min); 8°C/min to 180°C; 4°C/min to 280°C (10 min) Injection mode Splitless Injection volume 1 µL Injector temp. 250°C Ion source temp. 250°C Journal of Water and Environment Technology, Vol. 6, No.1, 2008 - 58 - 1 10 100 1000 10000 Control C. alternifolius C. generalis Biomass 10 4 [cells/mL] Figure 1 - Inhibitory effects of each culture solution solid phase extract on growth of M. aeruginosa. Bars indicate standard deviation (n=3). Analysis of allelochemicals Although no paper has reported the identified anti-cyanobacterial allelochemicals released from C. alternifolius, certain species of Cyperaceae, i.e. C. rotundus (Proestos et al., 2005; Quayyum et al., 2000; Komai and Ueki, 1981; Komai and Ueki, 1975; Ueki et al., 1974), C. longus (Morikawa et al., 2002), C. esculentus (Parker et al., 2000), C. serotinus (Ueki et al., 1974), and C. conglomeratus (Abdel-Mogib et al., 2000) are known to contain allelopathic phenolic compounds. For example, Quayyum et al. (2000) confirmed growth inhibition of rice (Oryza satives) seedlings by water extracts of C. rotundus and leachate of its leaves, and found the existence of phenolic compounds such as 4-hydroxy benzoic and 3,4-dihydroxy benzoic acids. Additionally, in a recent study, Proestos et al. (2005) investigated the anti-microbial activity of a methanol extract of C. rotundus, and quantified (-) epicatechin, 3,4,5-trihydroxy benzoic and 3-(4-hydroxy-phenyl)-acrylic acids in an acidic hydrolysate of the methanol extract. Since phenolic compounds such as 3,4,5-trihydroxy benzoic and 3,4- dihydroxy benzoic acids are known to have anti-cyanobacterial activities (Saito et al., 1989; Gross et al., 1994, 1996; Nakai et al., 2001, 2006), it was surmised that C. alternifolius also produced such phenolic compounds, and released them to inhibit the cyanobacterial growth. Figure 2 shows a total ion chromatogram of the solid extract of the C. alternifolius culture solution, where many compounds appear. No compound was detected after 30 min (data not shown). For identification, the mass spectra patterns of the respective peaks were compared with patterns stored in the mass spectral library ver. 2 of the U.S. National Institute of Standards and Technology, as the examples are shown in Fig. 3. The fragment patterns of peaks E and F respectively agreed with that of vanillic and protocatechuic acids-TMS esters. Finally, we identified 9 phenolic compounds, A) resorcinol (RES), B) 3-hydroxy benzoic acid (3HBA), C) 4-hydroxy benzoic acid (4HBA), D) (4-hydroxyphenyl)acetic acid (4HPAA), E) vanillic acid (VA), F) protocatechuic acid (PCA), G) p-coumaric acid (CA), H) gallic acid (GA), I) ferulic acid (FA), while the 4 carboxylic compounds, azelaic acid (AA), butanedioic acid Journal of Water and Environment Technology, Vol. 6, No.1, 2008 - 59 - (BDA), dehydroabietic acid (DHAA), and malic acid (MA) were also detected. The structures of these compounds are shown in Fig. 4. 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 2000000 4000000 6000000 8000000 1e+07 1.2e+07 1.4e+07 1.6e+07 Time--> Abundance TIC: SHURO.D 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 2000000 4000000 6000000 8000000 1e+07 1.2e+07 1.4e+07 1.6e+07 Time--> Abundance TIC: SHURO.D A B C D E F H I G Figure 2 - Total ion chromatogram of the solid extract of the C. alternifolius culture solution. 50 100 150 200 250 300 350 400 450 500 0 1000 2000 3000 4000 5000 6000 7000 8000 9000 m/z--> Abundance Scan 1073 (13.508 min): SHURO.D (-) 297 267 223 73 193 126 163 361 469 331 399 439 50 100 150 200 250 300 350 400 450 500 0 1000 2000 3000 4000 5000 6000 7000 8000 9000 m/z--> Abundance #128375: Benzoic acid, 3-methoxy-4-[(trimethylsilyl)oxy]-, t 297 73 267 223 126 193 43 163 50 100 150 200 250 300 350 400 450 500 0 1000 2000 3000 4000 5000 6000 7000 8000 9000 370 193 73 311 281 223 147 117 339 251 437 403 465495 50 100 150 200 250 300 350 400 450 500 0 1000 2000 3000 4000 5000 6000 7000 8000 9000 193 73 370 311 45 281 223 165 137 341105 m/z--> Abundance Scan 1201 (14.513 min): SHURO.D m/z--> Abundance #125879: Benzoic acid, 3,4-bis[(trimethylsilyl)oxy]-, trimet 50 100 150 200 250 300 350 400 450 500 0 1000 2000 3000 4000 5000 6000 7000 8000 9000 m/z--> Abundance Scan 1073 (13.508 min): SHURO.D (-) 297 267 223 73 193 126 163 361 469 331 399 439 50 100 150 200 250 300 350 400 450 500 0 1000 2000 3000 4000 5000 6000 7000 8000 9000 m/z--> Abundance #128375: Benzoic acid, 3-methoxy-4-[(trimethylsilyl)oxy]-, t 297 73 267 223 126 193 43 163 50 100 150 200 250 300 350 400 450 500 0 1000 2000 3000 4000 5000 6000 7000 8000 9000 m/z--> Abundance Scan 1073 (13.508 min): SHURO.D (-) 297 267 223 73 193 126 163 361 469 331 399 439 50 100 150 200 250 300 350 400 450 500 0 1000 2000 3000 4000 5000 6000 7000 8000 9000 m/z--> Abundance #128375: Benzoic acid, 3-methoxy-4-[(trimethylsilyl)oxy]-, t 297 73 267 223 126 193 43 163 50 100 150 200 250 300 350 400 450 500 0 1000 2000 3000 4000 5000 6000 7000 8000 9000 370 193 73 311 281 223 147 117 339 251 437 403 465495 50 100 150 200 250 300 350 400 450 500 0 1000 2000 3000 4000 5000 6000 7000 8000 9000 193 73 370 311 45 281 223 165 137 341105 m/z--> Abundance Scan 1201 (14.513 min): SHURO.D m/z--> Abundance #125879: Benzoic acid, 3,4-bis[(trimethylsilyl)oxy]-, trimet (a) (b) (c) (d) Figure 3 Comparison of fragment pattern of the peak E (a) at 13.508 min with that of vanillic acid-TMS ester (b) and fragment pattern of the peak F (c) at 14.513 min with that of protocatechuic acid-TMS ester (d). Journal of Water and Environment Technology, Vol. 6, No.1, 2008 - 60 - Resorcinol (RES) 3-Hydroxy benzoic acid (3HBA) 4-Hydroxy benzoic acid (4HBA) (4-Hydroxyphenyl) acetic acid (4HPAA) OH OH Vanillic acid (VA) COOH OH COOH OH COOH OH OCH 3 OH CH 2 COOH COOH OH OH OH COOH COOH OH OHHO OH COOH OCH 3 Protocatechuic acid (PCA) Gallic acid (GA)p-Coumaric acid (CA) Ferulic acid (FA) CH 3 CH 3 O OH Dehydroabietic acid (DHAA) OH O HO O OH Malic acid (MA) O OH O HO Butanedioic acid (BDA) OOH O HO Azelaic acid (AA) Figure 4 - Structures of phenolic and carboxylic compounds identified in the C. alternifolius culture solution. Note that 4HBA, VA, PCA, CA, GA, FA were the phenolic compounds detected in the hydrolyzed extracts of C. rotundus (Proestos et al., 2005; Komai and Ueki, 1975), whereas RES, 3HBA, 4HPAA, AA, BDA, and MA were newly found. The existence of DHAA, 4HBA, and CA in C. rotundus was confirmed by GC/MS analysis of its water extract (Quayyum et al., 2000). In our previous studies, anti-cyanobacterial effects of VA, PCA, and GA were confirmed by a series of assays using M. aeruginosa, while RES, CA, and FA did not show any effect (Nakai et al., 2001). The concentration at which the three anti- cyanobacterial phenolic compounds respectively inhibited the normal growth of M. aeruginosa by 50% of the control, denoted as EC50, was > 10 mg/L for VA, 3 mg/L for PCA, and 1.0 mg/L for GA. This indicates that VA, PCA, and GA can contribute to the allelopathic effect of C. alternifolius on M. aeruginosa; however, these compounds may not account for the allelopathic effect. In fact, a number of other organic compounds were detected in the solid extract in addition to these anti-cyanobacterial compounds (Fig. 2), though the DOC concentration of the C. alternifolius culture solution was 2.5 mg/L. This suggests that the amounts of the 3 anti-cyanobacterial phenolic compounds in the culture solution might be not enough to cause growth inhibition of M. aeruginosa. As for the newly found 3HBA, 4HPAA, AA, BDA, DHBA and MA, their anti- cyanobacterial activities are unknown, and the possibility still remains that these compounds together with the 3 anti-cyanobacterial phenolics cause the observed allelopathic growth inhibition of M. aeruginosa by C. alternifolius. In the future, their anti-cyanobacterial effect should be evaluated as a first step to understand the mechanisms of the allelopathic effect by C. alternifolius on M. aeruginosa. Towards Journal of Water and Environment Technology, Vol. 6, No.1, 2008 - 61 - revealing the allelochemicals responsible for the allelopathy caused by C. alternifolius, essential information may be the actual concentrations of the candidates in its culture solution and their concurrent action on the growth inhibition of M. aeruginosa. CONCLUSIONS The solid extracts of C. alternifolius and C. generalis culture solutions respectively demonstrated the growth inhibition of M. aeruginosa, thereby confirming the release of anti-cyanobacterial compounds from these plants. GC/MS analyses of the solid extract of C. alternifolius culture solution indicated the existence of 9 phenolic compounds (RES, 3HBA, 4HBA, 4HPAA, VA, PCA, CA, GA, and FA) and 4 carboxylic compounds (AA, BDA, DHAA, and MA). Although the 3 anti-cyanobacterial phenolic compounds (VA, PCA, and GA) could contribute to the allelopathic effect of C. alternifolius, their apparent amounts in the culture solution might not be sufficient to cause growth inhibition of M. aeruginosa. 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Microalgae and Protozoa, National Institute for Environmental StuJdies Environmental Agency, Japan, Tsukuba Japan. . 1000 2000 3000 4000 5000 60 00 7000 8000 9000 m/z--> Abundance Scan 1073 (13.508 min): SHURO.D (-) 297 267 223 73 193 1 26 163 361 469 331 399 439 50 100. 1000 2000 3000 4000 5000 60 00 7000 8000 9000 m/z--> Abundance Scan 1073 (13.508 min): SHURO.D (-) 297 267 223 73 193 1 26 163 361 469 331 399 439 50 100