We studied the removal of chlorophyll-a and eutrophic alga Ankistrodesmus acicularis by mixed and monoculture constructed wetlands of four common wetland macrophytes, Echinodorus berteroi, Hydrocotyle sibthorpioides, Vallisneria natans and Hydrilla verticillata. We also tested the effects of the macrophyte culture solution on the growth of three common eutrophic algae: Chlorella pyrenoidosa, Scnedesmus obliquus and Anabaena flosaquae.
The wetlands had significant effect on the removal of chlorophyll-a and A. acicularis. ANOVA results indicated a significant difference existed among different wetlands (P < 0.01). There was generally no significant difference between the mixed and the monoculture wetlands, while the mixed wetlands generally showed significant difference between them.
Results also indicated that there were significant differences in the effect on the growth of three algae among the culture solutions of different wetlands (p < 0.01). Almost all mixed culture solutions had a significant difference from their monocultures. Similarly, the monoculture treatments generally showed significant differences from their corresponding mixed treatments. However, combinations of different macrophytes generally cannot increase the effect intensity. The results indicated that combinations of macrophytes can produce different effects on alga growth from their monocultures and from their combinations with other macrophytes. Different algae showed different levels of susceptibility to the plant culture solution, and a same culture solution had different effects on different algae, including inhibiting, promoting, or neutral effects.
The eutrophication of lakes and reservoirs has been a severe problem worldwide. Aquatic macrophytes have disappeared from most of the eutrophic water bodies, while alga blooms have occurred more frequently. Lake restoration research has demonstrated that in many cases it is possible to change a ‘turbid water state’ dominated by phytoplankton into an ‘alternative stable state’ with clear water dominated by macrophytes (Moss et al., 1986; Scheffer et al., 1993; Hansson et al., 1998). Aquatic macrophytes may inhibit the growth of algae, control the alga bloom, and improve water self-purification capability. Using macrophytes in water restoration has become a promising and widely used biological control technique (Dai et al., 2012). It has been confirmed that aquatic macrophytes in general inhibit the growth of algae, and several mechanisms contribute simultaneously to their impact. Aquatic macrophytes may compete for nutrients, light and other resources with algae (Ozimek et al., 1990; Moss, 1991), or produce some substances affecting the growth of phytoplankton (Gopal and Goel, 1993).
Research indicated that many submerged macrophytes, such as Myriophyllum spicatum (Gross et al., 1996), Chara globularis and Chara contraria (Mulderij et al., 2003) and Elodea canadensis and Elodea nuttallii (Erhard and Gross, 2006) can inhibit the growth of algae by releasing allelochemicals. Hilt and Gross (2008) divided submerged macrophytes into three types by their ability to inhibit algae: High activity, like Myriophyllum spicatum and Ceratophyllum demersum; moderate activity, like Elodea canadensis and Myriophyllum verticillatum; low activity or no activity, like Stratiotes aloides or Chara aspera. Other wetland macrophytes, even land plants, were also reported producing allelochemicals, which can inhibit phytoplankton growth (Barrett et al., 1996). Recent research reported that emergent plants occupied 32% of the plants which had an inhibitory effect on algae (Hong et al., 2008a,b).
Different aquatic macrophytes show distinct inhibitory effects on the phytoplankton in eutrophic waters (Ruggiero et al., 2003). On a specific alga, different aquatic macrophytes may exhibit different effects, such as positive or negative or neutral, strong or weak (Zhou et al., 2006). On the other hand, a specific aquatic macrophyte may have different effects on different algae (Hilt, 2006; Zhu et al., 2010). Hence, comparing the effects of different aquatic macrophytes on distinct algae has theoretical and practical importance. Most research studying the effect of aquatic macrophytes on algae used a single macrophyte (Mulderij et al., 2005; Zhu et al., 2010). In field conditions, there may be several aquatic macrophytes coexisting in natural water bodies. The coexisting aquatic macrophytes may produce integrated effects on the alga growth, especially the integrated effects of the allelochemicals they produced (Mulderij et al., 2003). The coexisting aquatic macrophytes may also form different plant communities with various dominant species at different stages, which may have different influences on algae (Ruggiero et al., 2003). However, the integrated effects of aquatic macrophytes are unclear when compared with the effects of their mono species. Little is known, especially about the integrated effects of the allelochemicals produced by the coexisting plants on the growth of eutrophic algae (Elakovich and Wooten, 1994). Therefore, studies on the integrated effects of mixed macrophytes on alga growth are urgently needed. This study was aimed at comparing the effects of submerged and emergent macrophytes on alga growth, as well as the effects of mono and mixed culture wetland microcosms.
Removal of eutrophic algae by different plant wetlands
Submerged macrophytes Vallisneria natans (V. nata) and Hydrilla verticillata (H. vert), and hygrophytes Echinodorus berteroi (E. bert) and Hydrocotyle sibthorpioides (H. sibt) are widely distributed in China. These plants were chosen to build both the mono and two mixed small wetland systems which consisted of commercially available plastic buckets (upper diameter 23 cm, bottom diameter 19 cm and height 23 cm) and large aperture filter biochemical cotton (model: XY-1038, manufacturer: Foshan Shunde Junyou aouarium accessories plant) as the filling to support the plants. The cotton, with a thickness about 3 cm, was sheared to a diameter appropriate to the bucket's diameter, with two or three small holes for fixing the plants in place. Two sprouts of the same species or two sprouts of two different species (25 cm long, and fresh weight 20 g) were planted in one bucket and the bucket was filled with 5 L of water. Each treatment had three replications and left three unplanted buckets as control buckets. The experimental wetland microcosms were placed in the phytotron, with temperatures 28.0 ± 1.0°C during the daytime (7:00–19:00) and 18.0 ± 1.0°C during night, a relative humidity 70 ± 10%, and an illumination intensity 20,000 lux during the day time. The plants were irrigated with tap water for the first week, and then acclimated with 10% Hoagland nutrient solution for another week before they were fed with the experiment influent. The experiment influent was a 1:1 mixture of Hoagland (100%) nutrient solution and South China Normal University central lake water, which was in the state of eutrophication, and Ankistrodesmus acicularis (A. acic) dominated in the water blooms. The concentrations (mg l−1) of nutrients in the influent were TN (26.3), TP (3.3), CODcr (135.3) and chlorophyll-a (0.3). Water samples of 100 ml were collected at regular intervals from each wetland to examine the concentration of chlorophyll-a and the number of A. acic. Chlorophyll-a concentration was measured using the freezing-thawing method (Lin et al., 2005), and the alga number was tested using the cell-counting method (Zhou and Gao, 2000).
Effect on alga growth of culture solution of different plant wetlands
The aquatic plants were sampled, washed carefully with tap water for 5 min, rinsed with deionized water 3 times to remove adhering epiphyte, zooplankton and other sediments, and then the surface water was blotted with bibulous paper. The cleaned plant samples of 100 g for monoculture, or 50 g each for mixed culture, were cultured in plastic tanks (20 × 30 × 50 cm) containing 3 L of 10% Hoagland nutrient solution, and the tanks were placed in a light incubator with 12 h:12 h cycle of 25°C, 4 200 lux and 22°C, 800 lux. The plant culture solution was sampled after 7 d and filtered through a millipore membrane filter with an average pore diameter of 0.45 μm to remove possible zooplankton, phytoplankton or microorganisms before it served as the algae's cultivation water.
The experimental algae Chlorella pyrenoidosa (C. pyre), Scnedesmus obliquus (S. obli) and Anabaena flosaquae (A. flos) were from the Institute of Hydrobiology of Jinan University. Prior to the laboratory experiments, the algae were incubated in BG-11 media for a short-term (1 week) amplification culture before they were transferred into a revised Hoagland (10%) nutrient solution (Reddy, 1983) for acclimation culture, and then transferred to the revised Hoagland nutrient solution at regular intervals for continuous incubating.
Five milliliter pure solution of each alga species was sampled and put into 200 ml Erlenmeyer flasks filled with 100 ml revised Hoagland nutrient solution, added with 20 ml wetland plant culture water, while the control was added with 20 ml revised Hoagland nutrient solution instead. Each treatment had three replications. These algae were cultured in a light incubator (25°C, 4200 lux, 12 h; 22°C, 800 lux, 12 h). Ten ml solution of algae was sampled to measure the cell densities at the initial day and every 2 days, and ten ml revised Hoagland nutrient solution was subsequently added to keep a stable volume after each sampling. Cell densities of the algae were determined using UV-Vis spectrophotometer (Type: UV-1700; Producer: SHIMADZU international trading Shanghai Co. Ltd.) under 650 nm wavelength to get the OD value. Meanwhile, alga cell number was counted with binocular microscope (Type: XSP-C202; Producer: Chongqing optical instrument factory). Then a standard curve equation could be established from OD650nm (y) and algae cell number (x, ×104), and the alga cell density could be achieved from the equation.
The experimental data were processed using Microsoft Excel 2007 and performed using SPSS 19.0. The least significant difference at 5% level and 1% level were used for ANOVA. Correlation between different wetlands was analyzed using correlation analysis. Multiple comparison testing was performed to analyze the significant difference between different wetlands.
Effect of different plant wetlands on chlorophyll-a concentration
Each wetland showed similar chlorophyll-a (chl-a) concentration to the control on the first day, while each had significantly lower concentration than the control on the fourth day (Figure 1). After that, the wetlands showed significantly lower concentration than the control except V. nata wetland and V. nata + H. vert wetland. There was great difference in chl-a concentration between the ten treatments, E. bert, H. vert, E. bert + H.vert and H. sibt + H. vert wetlands had the lowest chl-a concentration, while V. nata wetland had the highest value. The monoculture and mixed culture wetlands had no significant difference, while the wetlands containing V. nata generally had higher chl-a concentration. H. sibt + H. vert wetlands showed a significantly lower chl-a concentration than all other mixed wetlands (P < 0.01).
Effect of different plant wetlands on A. acic growth
The density of A. acic decreased dramatically since the fourth day, especially in H. vert wetland and H. vert + E. bert wetland. The alga densities in V. nata, H. vert, E. bert + H. vert and V. nata + H. vert wetlands were significantly lower than that in the control (Figure 2). Then the alga densities in V. nata, E. bert + H. sibt and H. sibt + H. vert wetlands were significantly lower than that in the control on the seventh and tenth day. Until the fourteenth day all wetlands except V. nata + H. vert wetland had significantly lower alga concentrations than the control. There was no significant difference in A. acic density among different monoculture wetlands, and between mono and mixed wetlands, whereas A. acic density in V. nata + H. vert mixed wetland was generally significantly higher than those in the monoculture wetlands of the two species. Monocultures of E. bert and V. nata had significantly higher alga densities than any of their mixed wetlands.
Wetlands of H. sibt, H. vert, H. sibt + H. vert, E. bert + H. sibt, and E. bert + H. vert displayed higher A. acic removal rates than other wetlands (Figure 3).
Effect of different culture solutions on eutrophic alga growth
When co-cultured for one week, most of the culture solution treatments showed a lower C. pyre density than the control (Figure 4). The treatments of H. vert, H. sibt, E. bert, E. bert + V. nata and E. bert + H. sibt showed lower alga densities. On the fifteenth day, all the treatments except E. bert + H. vert showed significantly lower alga densities than the control. In general, V. nata + H. vert and E. bert + V. nata treatments showed the strongest alga inhibition, followed by H. sibt, E. bert, H. vert and H. sibt + V. nata treatments.
Among the monoculture solution treatments, H. vert showed the strongest inhibition while V. nata exhibited the weakest inhibition on C. pyre growth, with V. nata culture solution treatment having significantly higher alga density than the other three treatments (P < 0.05).
The mixed culture solution treatments of E. bert + V. nata and E. bert + H. sibt had significantly lower alga densities than the control after 1 week (Figure 4). All the treatments except E. bert + H. vert treatment had significantly lower alga densities than the control after 15 days. Among the treatments, V. nata + H. vert and E. bert + V. nata exhibited a stronger inhibition than other mix treatments, and H. sibt + V. nata and E. bert + H. sibt had stronger inhibition than H. vert + H. sibt.
The culture solution treatment of H. vert had a significantly lower alga density than any mixed culture solution treatments of the species.
Most plant culture solution treatments had no significant inhibition on the growth of S. obli (Figure 4). Among the monoculture solution treatments, only V. nata (since the fifth day) and H. vert (since the eleventh day) treatments showed a significantly lower S. obli growth than the control. V. nata showed a significantly stronger inhibition on S. obli growth than E. bert, H. vert and H. sibt (P < 0.01). In the mixed culture solution treatments, E. bert + H. sibt and H. sibt + V. nata had significantly lower S. obli densities than the control. E. bert + H. sibt had a significantly lower S. obli density than the other mixed treatments except H. sibt + V. nata treatment (P < 0.01). H. sibt + V. nata had a significantly lower S. obli density than H. sibt + H. vert, V. nata + H. vert and E. bert + H. vert (P < 0.01). Contrarily, E. bert, H. sibt, E. bert + H. vert and H. sibt + H. vert showed significantly higher S. obli densities than the control (P < 0.05). ANOVA indicated that V. nata had a significantly lower alga density than any mixed culture treatments with this species.
Most of the plant culture solutions showed a significant inhibition on A. flos growth (Figure 4). Treatments of E. bert, E. bert + H. vert and E. bert + H. sibt showed significantly lower A. flos densities than the control treatment after 7 days, and all treatments except H. sibt + H. vert and H. sibt + V. nata showed significantly lower alga densities than the control on the fifteenth day. Among the monoculture solution treatments, treatment of E. bert had a significant stronger inhibition on A. flos growth than H. sibt and H. vert treatments (P < 0.01). Among the mixed culture solution treatments, treatments of E. bert + H. vert and E. bert + H. sibt had a significantly stronger inhibition on A. flos growth than the other mixed treatments, and treatment of E. bert + V. nata also had a significantly stronger inhibition on the alga growth than H. sibt + V. nata and H. sibt + H. vert treatments (P < 0.01). H. sibt, H. sibt + H. vert and H. sibt + V. nata had significant higher alga densities than the control.
In general, the effects of the plant culture solutions on the growth of three eutrophic algae included inhibition, promotion, or no significant effect (Table 1). The three algae had different levels of sensitivity to the culture solutions, with C. pyre being the most sensitive. The same plant culture solution might have distinct effects on different algae: it may have an inhibition effect on one alga while having a promotion effect on another, or no significant effect on a third alga. Macrophyte combinations generally cannot increase the effect strength comparing their monoculture, but can produce different effect direction (positive, negative, or neutral) from their mono cultures, and from their combitions with other macrophytes.
Discussion and conclusions
Wetland macrophytes are generally diverse in natural communities. They may cause a mutual influence on each other and on eutrophic algae (Ellen et al., 2002; Korner and Nicklisch, 2002; Lürling et al., 2006), and there may be synergistic, antagonistic and adduction effects (Hairston et al., 2005; Mougi and Iwasa 2010, 2011a,b). It is still inconclusive whether mono or mixed macrophytes community can exert stronger effect on eutrophic algae (Wu et al., 2007; Li et al., 2010; Tanner and Headley, 2011), which might depend on the interaction of the coexisting macrophytes and the sensitivity of the algae. In the mixed culture wetlands of this study the macrophytes may also interact with each other and exert an integrated effect on the alga growth. For example, macrophytes combinations can produce different effects on alga growth from their monocultures and from their combinations with other macrophytes. The results of this study indicated that a mixed culture generally cannot increase the affecting intensity. Hence, the integration effect of different macrophytes on phytoplankton should be emphasized during the application of macrophytes in controlling eutrophic algae, and needs further study.
The most probable mechanism of macrophytes influencing other plants or algae was that the macrophytes could release allelochemicals to affect the life activities of the receptor plants or algae, such as cell growth, differentiation, photosynthesis and, respiration (Zhu et al., 2010). Shao et al. (2013) reported that the allelochemicals can inhibit, promote, or have no effect on receptor organisms. Chen and Guo (2014) indicated that allelopathic effects should include not only inhibitory but also stimulatory effects. Research also indicated that the possible mechanisms of allelopathic interaction might relate to the specific species of allelochemicals (Nakai et al., 2000; Elisabeth et al., 2003). Other possible mechanisms might relate to the allelochemical concentration and the sensitivity to allelochemicals of specific species. Most allelopathic effects relate their concentrations, with low concentration producing a promoting effect and high concentration expressing an inhibitory effect (Choi et al., 2002). An allelochemical concentration might show an inhibitory effect on a sensitive alga, while showing no significant effect, or even a promoting effect on a less sensitive alga. The results of our study also showed that different macrophytes could result in inhibition, promotion, or no effect on different alga. A same macrophyte could exert an inhibition on one alga, but produce a promotion effect, or no significant effect on other algae. In addition, the present study indicated that different culture solutions had different affecting times. Inhibition of some macrophytes worked at the initial 2–3 days, while other macrophytes needed a longer time to show their inhibitory effect. These results can help better formulate hypotheses on the effect mechanisms, including potential alleopathy. However, we have not directly measured any allelophic chemicals in the systems which needs to be tested later.
Brammer and Wetzel (1984) reported that the availability of potassium or sodium rather than the presence of allelochemicals from Stratiotes appeared to be the most influential growth-limiting factor for algae or whole phytoplankton. Besides, the co-precipitation of phosphates with calcium was considered an important mechanism that inhibits the growth of algae (Brammer, 1979; Brammer and Wetzel, 1984). Morris et al. (2009) indicated that inorganic elements might also play an allelopathic role. This needs further study to determine whether the inorganic elements of the nutrient solution used in our experiment affected the interaction of the macrophytes and the eutrophic algae.