Allelopathic Effects of Eichhornia crassipes on the ...

Journal of Agricultural Science and Technology A 2 (2012) 1400-1406 Earlier title: Journal of Agricultural Science and Technology, ISSN 1939-1250

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Allelopathic Effects of Eichhornia crassipes on the Growth of Microcystis aeruginosa Xiaoxia Wu, Zhenye Zhang, Dingli Chen, Junsong Zhang, Wenbing Yang and Yingen Jin College of Bioscience and Biotechnology, Yangzhou University, Yangzhou 225009, China Received: September 10, 2012 / Published: December 20, 2012. Abstract: Algal blooms caused by eutrophication in fresh water are one of the major environmental problems in the world. Using biological methods to control algal growth, especially based on allelopathic inhibitory effects of aquatic macrophytes on phytoplankton growth, have been received world-wide attention. In this study, the allelopathic activity of the invasive macrophyte, Eichhornia crassipes (water hyacinth), on blue-green algae, Microcystis aeruginosa (PCC7806) was investigated using coexistence assay. Our results showed that water hyacinth had disparate effects on the growth of M. aeruginosa (PCC7806) under different initial algal densities. Under lower initial algal density (OD650 = 0.10 and OD650 = 0.05), the algal growth was significantly inhibited by water hyacinth (inhibition ratio was 95.6% and 97.3%, respectively). While it was stimulated at higher initial algal densities (OD650 = 0.20). Water hyacinth inhibited the growth of algae mainly through its root system. Culture water from water hyacinth and aqueous methanol extracts from dry roots samples also showed inhibition effects on algal growth. The inhibition effects increased as the increase of crude extract concentration, suggest that water hyacinth may excrete inhibitory substances from root system and show allelopathic inhibitory potential to the growth of M. aeruginosa. Key words: Eichhornia crassipes, allelopathic effects, water blooms, Microcystis aeruginosa.

1. Introduction Microcystis aeruginosa is one kind of main algae causing water blooms. The problem of how effectively inhibit the growth of M. aeruginosa and control the water blooms has caused environmentalist’s attention. Common used methods were designed on the basis of the physical, chemical and biological aspects. Now, the more effective methods used mainly based on the use of chemicals such as bluestone, potassium permanganate, a practice which is becoming increasingly controversial as public concern for environmental matters increases. Thus, there is a need for an algal control method that is environmentally acceptable, inexpensive, selective and has a long-term effect [1]. In the last three decades, a special interest in the world was aroused by the potential of using the biological methods in wastewater treatment, whose Corresponding author: Yingen Jin, professor, research field: plant ecophysiology. E-mail: [email protected].

application as of natural procedures of tertiary processing of effluents provided the effluents of required quality in an economically acceptable way in the technically simple structures [2]. Allelopathy is a widely existing natural phenomenon. It is defined as the direct or indirect harmful or beneficial effects of one plant on another through the production of chemical compounds that escape into the environment [3]. In aquatic plants, all primary producing organisms of aquatic plants are capable of producing and releasing allelopathic active compounds [4]. Zhou et al. [5] reviewed that macro aquatic plants, such as bulrush (Typha augustifolia), fanwort (Cabomba caroliniana), water milfoil (Myriophyllum spicatum), hornwort (Ceratophyllum demersum), Chinese water chestnut (Eleocharis tuberose), pondweed (Potamogeton pectinatus), water hyacinth (Eichhornia crassipes) and duckweed (Lemna minor), could produce allelochemicals and inhibit different kinds of algae. Some algicide-like

Allelopathic Effects of Eichhornia crassipes on the Growth of Microcystis aeruginosa

substances have been extracted, and showed very good potential in the control of algal blooms. Water hyacinth is one kind of invasive, free-floating aquatic macrophytes. Because of its rapid multiplication rate [6], it has been considered the world’s worst aquatic weeds [2]. On the other hand, water hyacinth has also been studied as one kind of very promising plants with tremendous application in wastewater treatment [7-9], especially in metal-polluted water [10-13]. In addition, more scientists have focused on its allelopathic effects on algae [14-17]. Allelopathic experiments indicated that water hyacinth has great allelopathic effects on green algal growth in eutrophic freshwater. Control of water hyacinth can frequently leads to outbreaks of blue-green algae [18]. The controlled culture of water hyacinth in shallow basins can effectively removed algae and reduced other parameters concerned with water quality such as biochemical oxygen demand (BOD), chemical oxygen demand (COD) and total suspended solids (TSS) [9]. Sharma et al. [19] demonstrated that decaying water hyacinth litter could release some kinds of chemical substances, which caused an acute toxicity to growth of Scenedesmus obliquus. Some algicide-like substances extracted from water hyacinth have shown good effects in the control of algae [20].

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organic materials, and planted in the outdoor cement pool (150 cm  100 cm  50 cm) for acclimatization culture. The cyanobacteria species M. aeruginosa PCC7806 (unicellular algae, kindly provided by Professor Jian hong Li, Department of Biology, Nanjing Normal University) were inoculated and cultured under controlled condition. 2.2 Cyanobacteria Cultivation and Growth Status Determination

aeruginosa, the major algae causing freshwater bloom.

M. aeruginosa were cultured in BG-11 medium to logarithmic growth period and subcultured every 15 days. Culture conditions were controlled at 25 ± 1 °C under cool-white fluorescent light (25 ± 5 µmol m-2 s-1) and photoperiod 12L:12D, all the flasks were shaken four times every day. Growth condition of M. aeruginosa was determined by three methods according to different culture system. First method was the measurement of optical density (i.e., turbidity) at 650 nm on a 722 type spectrophotometer (UV755B, Shanghai, China). Secondly, the chlorophyll content of algae was estimated by methanol extraction, and thirdly, numeration of cell density was measured by a haemocytometer cell under light microscope (×1,000). Our preliminary experiments had shown that above three parameters had very high positive correlations with each other and got very similar results on the growth of test algae (P < 0.05). So, we can select different method to determine the growth status of algae according to different algae culture condition.

The aim of this study was to describe the effect of water

2.3 Experiments Using Fresh Samples of Water Hyacinth

However, researches on allelopathy of water hyacinth on algae have got many conclusive results, most of these researches are mainly focused on green algae control. Little attention was concerned with M.

hyacinth on the growth of cyanobacteria M. aeruginosa. To investigate possible interactions between two organisms, simultaneous culture was carried.

2. Materials and Methods 2.1 Materials Water hyacinth, collected from a small river in suburb of Suzhou City, Jiangsu Province, were washed to remove surface-deposited inorganic and

To test the algicidal activity of water hyacinth on M. aeruginosa, different initial concentration (OD650 = 0.05, 0.1, 0.2, respectively) of algae suspension were co-cultured with above mentioned acclimatized cultured water hyacinths (17 ± 2 g fresh weight) in 3 L beaker containing 2 L BG-11 medium. M. aeruginosa (OD650 = 0.05, 0.1, 0.2, respectively) growing in BG-11 medium was prepared as control. The experimental beakers were stuffed with sterile silica

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gel-stopper and cultured under the conditions as described above for seven days. Experiments were repeated twice and every treatment had three replications. The effect of water hyacinth roots on algal growth was also determined by co-culturing of M. aeruginosa with roots of water hyacinths (11 ± 2 g fresh weight). During the experiments, 1 mL cultural solution was collected from each beaker every day to determine the growth status of M. aeruginosa by spectrophotometry. 2.4 Experiments Using Dried Roots Powder of Water Hyacinth Water hyacinths in vigorous vegetative growth period were harvest, shade dried, chopped into small pieces and grounded to powder. Different concentration of root powder (0.0 g L-1, 0.8 g L-1, 4 g L-1, 20 g L-1) was added to algal culture system and cultured for seven days. Density changes of algae samples were determined every day at a fixed time under a microscope. 2.5 Experiments Using Culture Water from Water Hyacinth To collect the cultured water of water hyacinth, 100 g fresh weight of water hyacinth were cultured in 10 L Hogland solution (0.1) for seven days and then filtered (Whatman GF/F, Ø 47 mm) in dim light to remove residues and microorganisms. M. aeruginosa were cultured for seven days in BG-11 medium prepared with different concentration (100%, 50%, 25%, 0%) of water hyacinth culture water. Density changes of M. aeruginosa were determined every day at a fixed time under microscope. The inhibition ratio (IR) was calculated as the following: IR (100%) = (1  Nt/Nc)  100 Nt and Nc indicate the OD650 value of M. aeruginosa of the treatment and control at different days, respectively. 2.6 Experiments Using Methanol Extracts of Dried Roots Powder of Water Hyacinth Water hyacinth roots were carefully rinsed with tap

water and air dried, then chopped and extracted for 24 h with 1 mL methanol per 40 mg plant dry mass. Extraction was repeated three times. The extraction was evaporated under vacuum to dry and stored at -20 °C. Solvent controls were performed using the same procedure without adding plant material. For analyzing the effects of methanol extract from water hyacinth roots, an appropriate aliquot of the extracts was measured. In each of the required number of 100 mL Erlenmeyer flasks containing 1 mL of the stock liquid of methanol extracts. The final volume in each flask was made up to 40 mL from BG-11 medium to obtain 1.0, 1.5, 2.0 mg mL-1 concentrations of methanol extracts. Each test flask was inoculated with 1 mL algal cell suspension and keep an initial cell density of about 105 cells mL-1. A set of 12 test flasks was used for each concentration of the extracts. The control sets, i.e., with no extracts, algae was cultured in sterile BG-11 medium and treated in the same way as the test preparation. The test and control preparations were incubated under controlled condition and shaken twice a day. The growth status of algae in the test was measured every 24 h for seven days incubation periods. Inhibition effects were determined by the paper disk-agar plate method [21] and spectrophotometric method.

3. Results and Discussion 3.1 Effects of Fresh Water Hyacinth on the Growth of M. aeruginosa In order to test the effects of water hyacinth on the growth of M. aeruginosa, different initial densities of M. aeruginosa were incubated with or without entire water hyacinths for seven days, respectively, and the inhibitory activity of water hyacinths were determined by the changes of algal concentration (OD650 values). Our results showed that water hyacinth affected the growth of M. aeruginosa in a concentration dependent way (Fig. 1). Compared to the corresponding control, water hyacinth showed no inhibitory effects on M. aeruginosa at higher initial algal density (IAD) (Fig.


M. aeruginosa concentration (OD650)

1A, OD650 = 0.2), whereas the inhibitory effects were observed at lower IADs (Figs. 1B and C) , and the IRs of water hyacinth on the growth of algae were up to 95.6% and 97.3%, respectively after seven days co-cultivation. In Lake Taihu (China), the engineered use of water hyacinths has proved to be one of the most efficient approaches to reducing the nutrient load and restricting blue-green algal blooms [22]. According to Evans [23], the inhibitory effects of water hyacinth may be due to its allelopathic mechanisms or due to its effective competitive potential to algae. Kim [24] also suggested that reducing algal concentration in adsorptive capacity of water hyacinth. It is true that the plant roots may provide a large surface area for microbial growth and allow for biofilm formation [25]. In contrast, Sun et al. [26] found that water hyacinth was still shown significant inhibition effects on algal growth excluded from the factors of compete for light and nutrient between water hyacinth and algae. 3.2 Water Hyacinth Inhibited the Growth of M. aeruginosa Mainly through Its Root System According to Sharma et al. [19], leachate of the leaf and root litters of water hyacinths showed very similar effects on growth of algae. In contrast, Sun et al. [14] believed that mainly roots of water hyacinths showed allelopathic inhibitory activities on algae. In order to tracing algicidal active parts of water hyacinth, water hyacinth roots were isolated from whole plants and determined its algicidal effects by co-culture method. M. aeruginosa cultured with or without entire plants of water hyacinths were used as control. The results of OD650 values (Fig. 2) indicated that roots of water hyacinth alone also showed the similar algicidal activity as whole plants did. Paired t-test analysis showed no statistical significance between them (P > 0.05), and both showed very significant difference compared to M. aeruginosa control without water hyacinths (P < 0.01).

A

B

C

0

1 2 3 4 5 6 Time of incubation (Days)

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Fig. 1 Effects of water hyacinth (whole plant) on growth of M. aeruginosa. The growth of M. aeruginosa co-cultured with water hyacinth plants showed initial concentration dependence. The vertical bars represent the standard error. a, initial OD650 of M. aeruginosa was 0.20; b, initial OD650 was 0.10; c, initial OD650 was 0.05. M. aeruginosa concentration (OD650)

water hyacinths ponds may be due to strong

without water hyacinth with water hyacinth

.35 .30 .25 .20 .15 .10 .05 0.00 .18 .16 .14 .12 .10 .08 .06 .04 .02 0.00 .12 .10 .08 .06 .04 .02 0.00

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Control Control * Only roots

.30 .25 .20 .15 .10 .05 0.00 0

1 2 3 4 5 6 7 8 Time of incubation (days) Fig. 2 Effects of water hyacinth roots on growth of M. aeruginosa. Control* and Control stands for M. aeruginosa cultured with and without entire plants of water hyacinths respectively.

Different dried root samples were also used to further identify its algal inhibitory activity. Considering the influence of root powder on absorbance, changes of algae density during experiments were determined by hemacytometer. Compared to control, root powders of water hyacinth could obviously inhibit the growth of M. aeruginosa cells (Fig. 3). At the eighth day after treatments, cell growth of M. aeruginosa under 0.8 g L-1, 4.0 g L-1 and 20.0 g L-1 was reduced to 64.47%, 22.52% and 0.7% of control, respectively. Our results suggest that water hyacinth inhibit the growth of


cell density 4 -1 (×5×10 cells.ml )

1404 700 600 500 400 300 200 100 0

Control Dry roots 0.8 g/L Dry roots 4.0 g/L Dry roots 20.0 g/L

0

1 2 3 4 5 6 7 Time of incubation (days)

8

Fig. 3 Effects of root powder of water hyacinth on growth of algae. A

M. aeruginosa mainly through its root system, and the inhibitory effects also showed the concentration dependant. 3.3 Effect of Culture Water from Water Hyacinth on the Growth of M. aeruginosa

Fig. 4

B Effects of culture water from water hyacinth on

algae growth. A: Inhibitory ratio (IR) of Microcystis aeruginosa at different concentrations of water hyacinth culture water. B: 1. control group’s algae liquor at the 4th day; 2. 100% group algae liquor at the 4th day; 3. control groups algae liquor at the 7th day; 4. 100% group algae liquor at the 7th day). .25 M. aeruginosa concentration (OD650)

An allelopathic effect of culture water from water hyacinth on growth of M. aeruginosa was investigated. The results showed that growth of M. aeruginosa was inhibited by culture water (Fig. 4A). Increasing percentage of culture water in medium caused increase of IR on growth of M. aeruginosa. The strongest inhibition was observed when concentration of culture water up to 100%. Compared with the control, the IR of 100% treatment on M. aeruginosa arrived 94.9% at the 7th day after treatment. By significance analyses, the difference of M. aeruginosa density was remarkable (P = 0.012 < 0.05) between control and 100% culture water treatment. For continuous addition, the results indicated that the growth of M. aeruginosa was inhibited from the second day after treatment and then the growth curve of M. aeruginosa dropped rapidly. Cells of M. aeruginosa began to be pale and then died on the 4th day. On the seventh day, the M. aeruginosa cells decomposed gradually and died out (Fig. 4B). In order to further investigate the nature of inhibited substance in culture water, the culture water was heated at 105 °C for 15 min and then cultured algae with BG-11 medium prepared by aforesaid 100% culture water instead of distilled water. In Fig. 5, high temperature treated group shown no significant inhibited effect on algae growth compared to control with BG-11 medium cultured algae, while room

Control High temperture Room temperture

.20

.15

.10

.05

0.00 0

2 4 Time of incubation (days)

6

8

Fig. 5 Influence of cultured water of E. crassipes treated by high temperature on the growth of algae.

temperature group shown significant inhibited effect. 3.4 Allelopathic Activity of Water Hyacinth Extracts Allelopathic effects of methanol extracts was tested by paper disk-agar plate method [27]. The results showed that clear zones around filter piece soaked with methanol extracts compared with control, which meant that M. aeruginosa was strongly inhibited by the methanol extracts (Fig. 6A). Concentration test results (Fig. 6B) by liquid culture assay showed that


-1

Chlorophyll a ( mg.L )

.5

A

B

Control 1.0 mg/ml 1.5 mg/ml 2.0 mg/ml

.4

.3

.2

.1

0.0 0

2

4

6

8

10

Time of incubation (days)

Fig. 6 Effect of methanol extract of water hyacinth on growth of M. aeruginosa. A graph showed algae inhibited ring of the M. aeruginosa grown in agar plate contain methanol extract of water hyacinth (below) compared with control (upper). B graph showed Chl a content of the M. aeruginosa. grew in BG-11 medium with different concentration of methanol extracts

M. aeruginosa was much more sensitive and strongly inhibited by 1.5 mg mL-1 and 2.0 mg mL-1 methanol extracts. The strongest inhibition was observed when the concentration of methanol extract was up to 2.0 mg mL-1. These results suggested that water hyacinth might contain growth inhibitory substances and possess allelopathic potential. Growth inhibition of the green algal caused by exudates from water hyacinth was already reported by many researches [14-17, 28-30]. Our results expand the so far scarce studies on allelopathy of water hyacinth towards ecologically main kinds of bloom-water algae, M. aeruginosa. We showed that extracts from water hyacinth reduced the growth rate of M. aeruginosa. The effect increased when increasing the concentration of extract, which also meant water hyacinth might excrete growth inhibitory substances and shown allelopathic potential to blue green algae, M. aeruginosa PCC7806. In contrast to our hypothesis, Shanab et al. observed no strong inhibited effects on two test species of cyanobacteria of methanol extract from water hyacinth [17]. Whereas Liu et al. tested allelopathic effects of five allelochemicals found in roots of water hyacinth on M. aeruginosa and founded that allelopathic compounds from water hyacinths could inhibit algal growth at higher concentration [31].

4. Conclusions In recent decades, water hyacinth has no longer be

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thought as aquatic weeds, but have been found to have potential for use in phytoremediation, paper, organic fertilizer, biogas production, human food, fiber, and animal fodder [2]. Water hyacinth has already proved as a very promising plant in wastewater treatment [32]. It can be used as high effective biosorption [33] and successfully used for fast removal of metals in the initial stage of water body remediation [34]. The obtained results showed that the algae growth was inhibited by water hyacinth at low IADs, but stimulated at high IADs. Not only dry sample of water hyacinth but also water culture filtrate had inhibitory effects on M. aeruginosa growth, so we concluded that water hyacinth especially its roots system could exude allelopathic compounds and shown allelopathic inhibition to the growth of blue green algae, M. aeruginosa.

Acknowledgments The authors thank Ding H.D and Chen G. for their earlier review of the manuscript. This work was supported by grants from the Natural Science Foundation of Jiangsu Province (BK2009188) and the Natural Science Foundation of Yangzhou University. (2011CXJ075)

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