Eutrophication caused by nutrient enrichment in most freshwater, coastal marine and transitional waters has become the tricky issues all over the world since the mid-20th century  . The adverse ecological impacts caused by eutrophication, such as reduction of biodiversity, increment of algae bloom, increased turbidity of the water, decreased crop of aquatic products, economic loss, have made tremendous efforts to control eutrophication   ). Nitrogen and phosphorus have been considered to be the key of eutrophication    . So the reducing the impacts of eutrophication especially nitrogen and phosphorus in water bodies is urgently need   .
Compared to the chemical and physical methods for the treatment of wastewater, biological treatment method is economical, especially bio-treatment with microalgae. There are extensive studies of nutrient removal based on algae growth in municipal   , agricultural   , and industrial wastewaters   . Microalgae have been proved to be as a potential biological treatment material for wastewater   . Tremendous efforts have been put into research of Chlorella or Scenedesmus removing nutrient from different wastewater. It was confirmed that, based on the nitrogen and phosphorous removal efficiencies, there was a range from 8% to 100% and from 30% to 100% in Chlorella and Scenedesmus, respectively. Lau et al. (1996)  found that Chlorella vulgaris can remove 86% inorganic N and 70% inorganic P in wastewater.
However, there is little attention for nitrogen and phosphorous removal in river. Because of industrialization and rapid economic development, rivers are been imposed severe risks. In this study, we applied the isolated microalgal strains Chlorella sp. and Scenedesmus sp. by experiments to river water from Meishe River in Haikou City, Hainan Province. The primary objectives of this study were to test the ability of microalgae removing nutrients in river, to identify the change of algae community. To our opinion, the microalgae can be as a candidate for application of river-water treatment.
2.1. Sample Collection and Experiments Design
The Meishe River with a drainage area of 50.16 km2, originates from the southern of Haikou City, Hainan Province, which flows into the Qiongzhou Strait after 23.86 km. The water quality of river has deteriorated because of discharge of untreated sewage. Although the program for comprehensive management of Meishe River were launched, there are some challenges for improving water quality of river. So we collected the river water for nutrient removal experiments, the experiment used 250ml triangle vase filled with 200ml river water.
The experiments were divided two groups: one was planted with Chlorella sp., the other one was planted with Scenedesmus sp. The cell density of Chlorella sp. was set three level: 2 × 105 cells/ml (C1), 6 × 105 cells/ml (C2), 12 × 105 cells/ml (C3). The cell density of Scenedesmus sp. was set three level: 2 × 105 cells/ml (S1), 4 × 105 cells/ml (S2), 6 × 105 cells/ml (S3). Each experiment had 3 replications. Before each replication poured into algae, Chlorella sp. and Scenedesmus sp. were rinsinged with sterile water for eliminating the effects of nutrient from medium. The experiment lasted for 15 days, TN and TP measurement were conducted every five days. One the fifteenth day water samples were collected for quantitative analysis of algae community.
2.2. Analysis of TN and TP
TN and TP concentration was measured according to Chinese state standard testing methods  .
Removal rates (%) were calculated using relation shown in (1).
where, R is nutrient removal efficiency and Rt and R0 are the nutrient concentration at day t and day 0, respectively.
2.3. Analysis of Algae Community
The water samples were preserved with Lugol’s iodine and sedimented for more than 48 h. The alga density was counted from 0.1 mL of the sediment through a 0.1 mL counting chamber using a microscope at 40 × 10 magnification. Alga taxa was identified to species or varieties according to Hu and Wei (2006)  , and alga biomass was estimated according to the closest geometric shape of each taxa.
Species diversity index was calculated following Shannon-Wiener (H), Simpson indices (P) and Pielou evenness (J) formula.
Ni = number of individuals of a species i, N = total number of individuals.
3.1. The Change of TN and TP Concentration during the Experiment
The TN concentration in river water declined after pouring into Chlorella sp. and Scenedesmus sp., S1 group obtained the higher ability for nitrogen removal on the tenth day, the TN concentration was only 0.81 ± 0.20 mg/l, TN removal rates reached 86%. The TN concentration varied between 11.28 mg/l and 11.83 mg/l in river water at the beginning of experiment.
Because of the different initial cell density, the variation of TN concentration in Scenedesmus groups and Chlorella groups demonstrated different tendency along with the experiment time. TN concentration declined on the fifth day and the tenth day, then increased on the fifteenth day in the group of S1, S2 and C2. TN concentration had a peak on the tenth day in the group of C1 and C3. However, TN showed a trend of increasing in the group of S3 (Figure 1).
The TP concentration in river water declined after pouring into Chlorella sp. and Scenedesmus sp., C1 group obtained the higher ability for phosphorous removal on the fifth day, the TP concentration was only 0.35 ± 0.08 mg/l, TP
Figure 1. The variation of TN concentration with different initial cell density of Chlorella sp. and Scenedesmus sp.
removal rates reached 95%. The TP concentration varied between 6.61 mg/l and 6.68 mg/l in river water at the beginning of experiment.
Although the different initial cell density, the variation of TP concentration in Scenedesmus groups and Chlorella groups demonstrated same tendency along with the experiment time. TP concentration had a peak on the tenth day in all groups (Figure 2).
3.2. Algae Community Composition
Species of algae in Chlorella group were significantly more than river-water, but those in Scenedesmus group were less than river samples (Table 1). A total of 27 taxa were detected in river samples, including 4 divisions 21 genera. In S group, only 9 taxa were detected, including 3 divisions 6 genera. In C group, a total of 62 taxa were detected, including 4 divisions 36 genera. Comparing to river samples, most algae (e.g. Melosira, Cyclotella, Navicula) disappeared in S group. However, many genera (e.g. Merismopedia, Anabaena, Fragilaria, Carteria, Tetraëdron, Oocystis, Pediastrum, Scenedesmus ) came out in C group.
The total of cell density was higher and Chlorophyta contributed more in Scenedesmus group (Figure 3). Bacillariophyta and Chlorophyta were the dominant groups in Chlorella group, Bacillariophyta contributed more in CK group (Figure 4). Nitzschia was the dominant species in CK, which contributed 54% - 72%, Nitzschia and Chlorella were the dominant species in C group, which contributed 37%, 21%, respectively. Scenedesmus was the dominant species in S group, which contributed 97%.
3.3. Algae Community Diversity Index Change
Compared to CK group, algae community diversity indexes increased in C group and declined in S group. The average Shannon-Wiener, Simpson indices and Pielou evenness increased 37%, 28%, and 18%, respectively in C group. The
Figure 2. The variation of TP concentration with different initial cell density of Chlorella sp. and Scenedesmus sp.
Figure 3. The variation of algae community with different initial cell density of Chlorella sp. and Scenedesmus sp.
Figure 4. The percentage of algae community with different initial cell density of Chlorella sp. and Scenedesmus sp.
average Shannon-Wiener, Simpson indices and Pielou evenness decreased 90%, 90%, and 77%, respectively (Figure 5).
Figure 5. The change of algae community diversity with different initial cell density of Chlorella sp. and Scenedesmus sp.
Table 1. The list of algae in CK, S group and C group.
The success of applying microalgae to remove nitrogen or phosphorus from different wastewater has been demonstrated extensively   ). In our study, it was indicated that the nutrient removal rate from river water was different between Scenedesmus group and Chlorella group, the Scenedesmus sp. had respective advantage in removing nitrogen and Chlorella sp. in removing phosphorous (Figure 1). S1 group had the highest removal rate for TN on the tenth day, this suggests that Scenedesmus sp. with the initial cell density of 2 × 105 cells/ml is likely to have a better TN removal effect. Álvarez-Díaz et al. (2017)  reported that Scenedesmus obliquus achieved higher daily nitrogen removal from wastewater than Chlorella kessleri, Chlorella vulgaris. Compared to free-living cells of Scenedesmus, the chitosan immobilized cells can accomplished a 70% nitrate and 94% phosphate removal within 12 h of incubation  . In the nitrogen/phosphorus ratio of 5:1 - 12:1, 83% - 99% nitrogen and 99% phosphorus could be removed. The cells of Scenedesmus have the benefit of being equipped with spines and bristles, which make them more buoyant, increased nutrient uptake and avoid predation in the water   .
C1 group had the highest removal rate for TP on the fifth day, this suggests that Chlorella sp. with the initial cell density of 2 × 105 cells/ml is likely to have a better TP removal effect (Figure 2) Chlorella is widely used in different type of wastewater treatment such as industrial wastewater, municipal wastewater, swine wastewater   , and it is shown to be effective in removing nitrogen and phosphorus. It is demonstrated that nitrogen and phosphorous removal efficiencies from the growth of Chlorella sp. range from 8% to 100%  , and there exists some differences between different species of Chlorella. Some study confirm that Chlorella vulgaris has higher nutrient removal efficiencies than that of Chlorella kessleri when comparing their performances in artificial medium   . In our study the TP removal efficiency of Chlorella reached 95%, it can be a candidate species for removing the TP in river water. Su et al. (2011)  found that Chlorella pyrenoidosa in soybean processing wastewater obtained the faster removal of nitrogen over phosphorus. The ratio of N/P should be considered in order to ensure the simultaneous utilization of both nitrogen and phosphorus  , an optimal N/P ratio for C. vulgaris was reported to be 7  .
There existed some differences in S group, C group and river sample, algae community composition showed extreme sensitivity to change in the joint of the Scenedesmus or Chlorella. Comparing to river sample, most algae (e.g. Melosira, Cyclotella, Navicula) disappeared in S group. However, many genera (e.g. Merismopedia, Anabaena, Fragilaria, Carteria, Tetraëdron, Oocystis, Pediastrum, Scenedesmus) came out and the diversity of algae increased in C group.
The study tested nutrient removal rates and algal community variation using the isolated microalgal strains Chlorella sp. and Scenedesmus sp. from an urban river water. The results showed:
1) The TN and TP concentration in river water declined after pouring into Chlorella sp. and Scenedesmus sp., the Scenedesmus sp. had respective advantage in removing nitrogen and Chlorella sp. in removing phosphorous. Scenedesmus sp. with the initial cell density of 2 × 105 cells/ml is likely to have a better TN removal effect, TN removal rates reached 86%. Chlorella sp. with the initial cell density of 2 × 105 cells/ml is likely to have a better TP removal effect, TP removal rates reached 95%.
2) Species of algae in Chlorella group were significantly more than river-water, but those in Scenedesmus group were less than river samples (Table 1). A total of 27 taxa were detected in river samples, including 4 divisions 21 genera. In S group, only 9 taxa were detected, including 3 divisions 6 genera. In C group, a total of 62 taxa were detected, including 4 divisions 36 genera. Comparing to river samples, most algae (e.g. Melosira, Cyclotella, Navicula) disappeared in Scenedesmus group. However, many genera (e.g. Merismopedia, Anabaena, Fragilaria, Carteria, Tetraëdron, Oocystis, Pediastrum, Scenedesmus ) came out in Chlorella group.
3) The total of cell density was higher and Chlorophyta contributed more in Scenedesmus group. Bacillariophyta and Chlorophyta were the dominant groups in Chlorella group, Bacillariophyta contributed more in CK group. Nitzschia was the dominant species in CK, which contributed 54% - 72%, Nitzschia and Chlorella were the dominant species in Chlorella group, which contributed 37%, 21%, respectively. Scenedesmus was the dominant species in Scenedesmus group, which contributed 97%.
4) Compared to CK group, algae community diversity indexes increased in Chlorella group and declined in Scenedesmus group. The average Shannon-Wiener, Simpson indices and Pielou evenness increased 37%, 28%, and 18%, respectively in C group. The average Shannon-Wiener, Simpson indices and Pielou evenness decreased 90%, 90%, and 77%, respectively.
In conclusion, this study showed that the Scenedesmus sp. had respective advantage in removing nitrogen and Chlorella sp. in removing phosphorous, the lower diversity and higher dominance of algae can be observed in Scenedesmus group, there existed an opposite tendency in Chlorella group.
The authors gratefully acknowledge anonymous reviewers. This project was supported by the Hainan Provincial Department of Science and Technology (ZDYF2016021).
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