Release of anti-cyanobacterial allelochemicals from aquatic and terrestrial plants applicable for artificial floating islands

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

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, (4-4-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,

(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

was inoculated (1 × 104 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

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

Ion source temp 250°C

1 10 100 1000 10000

Control C alternifolius C generalis

4 [cel

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

(BDA), dehydroabietic acid (DHAA), and malic acid (MA) were also detected The structures of these compounds are shown in Fig 4

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Figure 2 - Total ion chromatogram of the solid extract of the C alternifolius culture

solution

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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)

(RES) 3-Hydroxy benzoicacid (3HBA) 4-Hydroxy benzoicacid (4HBA) (4-Hydroxyphenyl)acetic acid (4HPAA)

OH

OH

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CH3 O

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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

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 Since it is unknown whether or not 3HBA, 4HPAA, AA, BDA, DHBA, and MA inhibit growth of M aeruginosa, further research

should be done to confirm their anti-cyanobacterial activity, and their concurrent actions with the 3 anti-cyanobacterial phenolic compounds, as the first step to reveal the

allelopathic effect of C alternifolius on M aeruginosa

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