Abstract

A combined high-rate algal pond and submerged macrophyte pond (APMP) reactor was introduced as a novel biotechnique for efficient heavy metals (HMs) removal from wastewater. The role of water temperature, light regime, and N:P mass ratio on algae growth and HMs removal as well as effects of macrophyte species and densities on algae extermination were investigated through batch experiments. Results showed that water temperature significantly affected algae proliferation and HMs removal. Effects of light regime and N:P only showed obvious influences on HMs removal performance at high temperature. HMs removal efficiency reached 75.8% (Cr), 63.6% (Pb), and 61.1% (Zn) at 5-day hydraulic retention time (HRT) in APMP. Positive correlation existed closely between HMs removal and algal growth with long HRT. Algae were strongly inhibited by Ceratophyllum demersum and Vallisneria natans at plant density of 20–30 rhizomes m−2 with effluent algae concentration about 1,000 cells mL−1 at 7-day HRT. Results suggested that the APMP reactor was efficient for HMs removal from wastewater, indicating a possible effective metals removal technique by using APMP.

INTRODUCTION

Heavy metals (HMs) contamination has raised public concern due to its toxicity, wide range of sources, non-biodegradable properties, and accumulation behaviors (Yin et al. 2016). Anthropogenic sources of HMs include mining, electroplating, textile printing, energy and fuel production, and the application of fertilizer to farmlands (Gupta et al. 2014; Kapusta & Sobczyk 2015; Hackbarth et al. 2016). A noticeable concentration of HMs has been detected in municipal sewage. HMs have been suggested to be removed before treated effluents are discharged or reused. In the past few decades, technologies have been developed and applied for HM-contaminated wastewater treatment, such as chemical precipitation, ion-exchange and membrane filtration (Smara et al. 2007; Fu & Wang 2011; Fu et al. 2012). However, these methods involve high investment requirements and cause potential risks through hazardous by-products (Cohen 2006). Limitations include high running costs and unsatisfactory performance of HMs removal especially in the tertiary treatment of wastewater with low HM concentration.

Recently, metal ion removal through biosorption has drawn wide interest among researchers. Biosorption employs an inexhaustible and cost-effective treatment approach with regard to operational complexity, investment costs and low volumes of non-hazardous wastes generation (Muñoz & Guieysse 2006). An abundant source of potential HMs biosorption biomass is algae (Sekomo et al. 2012). It has been reported that algae can efficiently remove HMs. Compared to other methods, algae treatment is competitive in cost, demands of operation and maintenance (Muñoz & Guieysse 2006). Biosorption of metals by algae may be a feasible method for remediating HM-contaminated water (Vijayaraghavan & Balasubramanian 2015). Reports have indicated that numerous species of algae (living or non-living cells) are capable of sequestering significant quantities of toxic metal ions from wastewater (Perales-Vela et al. 2006). Generally, non-living algae species, such as Chlorella vulgaris, are widely adopted in metal ions removal (Kumar et al. 2015). In addition, non-living algal biomass can be used after pretreatment in order to reinforce adsorption performance (Luo et al. 2006). Pretreatments include hardening the algae cell wall, increasing negative charge on cell surface, or increasing the cell surface for adsorption by acid treatment (Muñoz & Guieysse 2006).

To our knowledge, live as well as immobilized microalgae can absorb higher quantity of metals than non-living cells due to the intracellular accumulation. Although algae can grow rapidly in a suitable environment, only limited studies have focused on the use of live microalgae for HMs removal because microalgae are difficult to be harvested. Meanwhile, a surplus of algae in effluent can result in negative impacts on receiving water, such as water-quality deterioration or even inducing eutrophication. Several techniques have now been developed for algae harvesting before discharging; the use of a flocculating agent, centrifugation, chemical cellular interaction, ion binding, etc. are regularly adopted (Kumar et al. 2015). However, these methods are generally less efficient or high-cost demanding.

According to an earlier study (Nakai et al. 1999), the growth of algae could be inhibited by the allelopathic effects from macrophytes. Macrophytes are effective in suppressing photosynthesis of algae and can be used for mitigating algae blooms (Ye et al. 2014). Macrophytes release allelochemicals that can slow down the rate of algae growth. This suggests that macrophytes could be used to exterminate undesirable algae. In addition, macrophytes can utilize their tissues to accumulate and uptake HMs.

Recently, high-rate algal pond (HRAP) biotechnology has received special attention for the advantages that algae can grow on wastewater with low nutritional supply or maintenance and high algae harvesting efficiency (Mehrabadi et al. 2015). Algae growth and pH increase via photosynthesis in HRAPs can enhance HMs precipitation. HRAPs offer a more efficient and economical operation process than conventional wastewater treatment techniques for their ability to remove HMs and tolerate HMs toxicity.

In view of the fact that HRAPs have good performance in HMs removal while macrophytes have the potential of algae inhibition and HMs uptakes, it is possible that the combination of an HRAP and a submerged macrophyte pond (SMP) could form a cost-effective method for HMs removal from wastewater. However, the information on the combination of HRAP and SMP as well as their synergistic effect on HMs removal and algae inhibition is unknown, thus hindering the accurate understanding of HMs removal and algae inhibition in such a combined reactor.

In this paper, we are presenting the results of a study that used a combined algae pond and macrophyte pond (APMP) reactor as a highly efficient treatment technology for HM-contaminated water based on a two-step process: (1) HMs biosorption in HRAP and (2) algae removal by means of inhibition in SMP for further effluent purification from HRAP. The study was focused on the effects of temperature, light regime, and nutrition conditions on algae growth and HMs removal efficiency in HRAP as well as the algae removal efficiency in SMP.

MATERIAL AND METHODS

Experimental setup

One laboratory-scale APMP reactor was built by combining an HRAP and SMP (Figure 1). Inner dimensions (L × W × D) of the HRAP and SMP reactor were 100 cm × 60 cm × 60 cm. Water depth of the HRAP was controlled at 50 cm and the active volume was 300 L. A baffle (60 cm × 2 cm × 60 cm) was vertically positioned in the middle of the HRAP to induce recirculation flow. The recirculating flow was driven using a low-speed agitator (20 rpm) installed on the influent side of the reactor. A light intensity controllable LED tube light was installed on the top of HRAP with the vertical distance of 80 cm above the top surface, and an effluent pipe was installed on the side of the reactor. The influent was fed by a peristaltic pump through a PVC pipe (Ø = 2 cm) installed at the bottom of the HRAP, and an effluent pipe was installed on the side of the reactor. SMP influent was fed by the HRAP effluent using a connecting PVC pipe installed on the side of the SMP. Microcystis aeruginosa provided by the Institute of Hydrobiology, Chinese Academy of Sciences, was introduced as algae species. Before the experiment, M. aeruginosa was preserved and cultivated with BG-11 cultivation solution (Rippka et al. 1979) in a climatic cabinet under the light intensity of 3,000 lx at 30 °C.

Figure 1

Experimental design of the APMP reactor.

Figure 1

Experimental design of the APMP reactor.

The bottom of the SMP reactor was filled with a 10 cm thickness quartz sand layer (Ø = 1.8–2.0 cm), above which was a 5 cm thickness quartz sand layer (Ø = 0.8–1.0 cm). The total effective depth reached 60 cm including the two quartz sand layers. Water level of the HRAP and SMP was equally controlled at 50 cm during the run. An effluent pipe was installed on the side of the SMP. Submerged macrophytes of Ceratophyllum demersum and Vallisneria natans were collected from a local botanical market. Macrophytes with uniform growth and similar biomass were selected. Mean fresh weight of Ceratophyllum demersum and Vallisneria natans were 18 ± 3 g and 34 ± 5 g per plant, respectively. Before being transplanted to the SMP, the plants were propagated for a 3-week acclimation in Hoagland's solution (Hoagland & Arnon 1950).

The APMP reactor was placed on the balcony of the laboratory under a lightproof shelter outside of the laboratory to avoid precipitation and strong solar irradiation. But the reactor still experienced climatic variations in air temperature and humidity. Sampling campaigns in the summer and winter were performed over two 4-month periods (January to April and July to October, 2014). The recorded average seasonal air temperature of the winter and summer campaign was 5 °C and 28 °C while the average relative humidity was 68% and 62% respectively.

The HRAP and SMP were operated in a batch mode. For HMs adsorption, the HRAP reactor was flushed and drained off before adding 250 L artificial HMs wastewater and 50 L algae solution. Initial algae concentration was approximately 3,000 cells mL−1. For the algae inhibition batch experiment in SMP, a culture with algae concentration of 4,800 ± 323 cells mL−1 was used. The macrophyte density was 10, 20, and 30 rhizomes m−2. Plants were cultivated for three complete hydraulic cycles before initiation of the experiment. For the operation of APMP reactor, the mixture of 250 L artificial HMs wastewater and 50 L algae solution was added to HRAP. Initial algae concentration was about 3,000 cells mL−1. Algae were allowed to proliferate for 7 days before running the trail. HRT of 5 days (influent 50 L d−1) was applied. The SMP was fed by receiving HRAP effluent. Effluent was sampled twice per day at 10 a.m. and 10 p.m.

Synthetic HMs wastewater was prepared with tap water. The suggested low HMs concentration artificial wastewater was prepared according to the Wastewater Comprehensive Discharge Standard of China (GB8978-96). The ingredients (mg L−1) included Cr(III), 0.5; Pb(II), 1.0; Zn(II), 2.0 and PO43−, 2.5. In order to investigate the effects of HRT, temperature, light intensity and lighting time, and N:P mass ratio on the algae growth and bioadsorption efficiency, treatments in a 2 × 3 × 2 × 3 factorial design were created (Table 1). N:P was adjusted by adding NaNO3. Synthetic wastewater was stored in a lightproof PVC tank and constantly mixed by a submerged agitator placed on the bottom of the container.

Table 1

Treatments of a 2 × 3 × 2 × 3 factorial design in batch experiments on HRAP reactor: temperature, light regime, N:P mass ratio

Light intensity, lx; lighting time, h Low temperature (5 °C)
 
High temperature (28 °C)
 
N:P = 7:1 N:P = 21:1 N:P = 42:1 N:P = 7:1 N:P = 21:1 N:P = 42:1 
Background (BK) L-BK-7 L-BK-21 L-BK-42 H-BK-7 H-BK-21 H-BK-42 
BK + 3000lx, 12 h L-3k12 h-7 L-3k12 h-21 L-3k12 h-42 H-3k12 h-7 H-3k12 h-21 H-3k12 h-42 
BK + 3000lx, 16 h L-3k16 h-7 L-3k16 h-21 L-3k16 h-42 H-3k16 h-7 H-3k16 h-21 H-3k16 h-42 
BK + 6000lx, 12 h L-6k12 h-7 L-6k12 h-21 L-6k12 h-42 H-6k12 h-7 H-6k12 h-21 H-6k12 h-42 
BK + 6000lx, 16 h L-6k16 h-7 L-6k16 h-21 L-6k16 h-42 H-6k16 h-7 H-6k16 h-21 H-6k16 h-42 
Light intensity, lx; lighting time, h Low temperature (5 °C)
 
High temperature (28 °C)
 
N:P = 7:1 N:P = 21:1 N:P = 42:1 N:P = 7:1 N:P = 21:1 N:P = 42:1 
Background (BK) L-BK-7 L-BK-21 L-BK-42 H-BK-7 H-BK-21 H-BK-42 
BK + 3000lx, 12 h L-3k12 h-7 L-3k12 h-21 L-3k12 h-42 H-3k12 h-7 H-3k12 h-21 H-3k12 h-42 
BK + 3000lx, 16 h L-3k16 h-7 L-3k16 h-21 L-3k16 h-42 H-3k16 h-7 H-3k16 h-21 H-3k16 h-42 
BK + 6000lx, 12 h L-6k12 h-7 L-6k12 h-21 L-6k12 h-42 H-6k12 h-7 H-6k12 h-21 H-6k12 h-42 
BK + 6000lx, 16 h L-6k16 h-7 L-6k16 h-21 L-6k16 h-42 H-6k16 h-7 H-6k16 h-21 H-6k16 h-42 

Note: For a treatment name: ‘H’ and ‘L’ represented high and low temperature. ‘BK’ was the background light regime. ‘*k*h’ meant the light intensity with lighting time. The last code in the name was the N:P; e.g. ‘L-3k12 h-7’ means the combination of factors with low temperature of 5 °C, light intensity of 3,000 lx for lighting time of 12 hours, and N:P of 7:1.

Sampling procedure

All samplings were completed in triplicate. Dissolved oxygen (DO), pH and algae concentration in the aqueous solution were measured directly in the reactor during each run. Temperature and DO were measured simultaneously after sampling by a DO meter (H19145, HANNA, Italy). Algae concentration and pH were measured using a multi-parameter water-quality monitoring meter (Manta2.0, Eureka, USA) and a pH meter (H18424, HANNA, Italy). To determine the HMs concentration, 20 mL of aqueous solution was sampled from effluent and stored in a 100-mL flask. After centrifugation (CR22G II, Hitachi), the supernatant fraction was filtered (2.5 μm, Whatman grade 42). Prior to metal content measurement, 5 mL of HNO3 and 5 mL of H2O2 were added into the filtrates then treated via microwave digestion. Biofilm accumulated on the walls of the ponds and the floating algae biomass were harvested and stored in a freezer before HMs analysis. After drying at 105 °C, the mixture of the algae remaining and filter paper was digested with 5 mL of HNO3 by using a microwave technique. Thereafter, 2.0% HNO3 was added to a fixed volume of 25 mL and then the sample filtered. HMs (Cr, Pb and Zn) analysis was conducted by using an inductively coupled plasma mass spectrometer (X Series II, Thermo, USA). Calibration solutions were used to calibrate the instrument response with respect to analyte concentration. Experiments were carried out in triplicate.

Data analysis

The mean algae relative growth rate (RGR, d−1) was calculated according to Equation (1): 
formula
(1)

where Bt1 and Bt0 were the actual and initial biomass density (cell L−1) and t was the cultivation period. The correlation analysis and one-way analysis of variance (ANOVA) were carried out using the SPSS15 package. Two-way ANOVA was used to detect the significance of the effects on RGR, HMs removal efficiencies, and algae removal by submerged macrophyte. The significance level was 5%.

RESULTS AND DISCUSSION

Effects of temperature, light regime, and N:P on RGR of M. aeruginosa

The effects of temperature, light regime, and N:P on M. aeruginosa productivity are shown in Figure 2. Algae RGR was significantly affected by temperature (P < 0.001, Table 2). After 2-day cultivation at high temperature, RGR exceeded 0.6 d−1 (Figure 2(a)). High temperature led to a fast proliferation of M. aeruginosa with appropriate nutrient and irradiation. An algae bloom was observed to occur within a 2-day cultivation. Thereafter, overall RGR declined with HRT and the algae proliferation rate slowed down. RGR decreased most at a long HRT of 6 days to lower than 0.4 d−1 (Figure 2(c)). RGR was negatively inhibited by low temperature. RGR was in general lowered below 0.2 d−1. At low temperature, algae growth was relatively less affected by the variation of HRT (2–6 days) and kept a slow growth rate throughout the entire treatment with the absence of algae bloom.

Table 2

Effects of temperature, light regime and N:P on HMs removal at 6-day HRT

Treatments RGR, mg L−1 d−1 Cr, % Pb, % Zn, % Treatments RGR, mg L−1 d−1 Cr, % Pb, % Zn, % 
L-BK-7 0.059 ± 0.022 19.34 ± 4.33 16.55 ± 3.65 18.11 ± 4.55 H-BK-7 0.210 ± 0.031 40.11 ± 5.44 52.58 ± 6.55 40.82 ± 7.64 
L-3k12 h-7 0.060 ± 0.037 18.15 ± 3.28 18.68 ± 4.28 22.57 ± 5.86 H-3k12 h-7 0.362 ± 0.024 50.49 ± 4.32 58.19 ± 8.46 58.41 ± 4.11 
L-3k16 h-7 0.071 ± 0.027 23.84 ± 5.96 18.49 ± 5.75 21.72 ± 4.33 H-3k16 h-7 0.352 ± 0.040 53.06 ± 4.61 67.70 ± 9.29 65.11 ± 7.22 
L-6k12 h-7 0.070 ± 0.047 25.73 ± 4.57 11.20 ± 6.49 17.21 ± 3.72 H-6k12 h-7 0.278 ± 0.022 43.01 ± 5.83 51.66 ± 5.54 50.80 ± 6.30 
L-6k16 h-7 0.111 ± 0.026 27.96 ± 3.18 15.78 ± 4.17 25.20 ± 5.26 H-6k16 h-7 0.246 ± 0.069 42.70 ± 3.38 63.78 ± 5.93 44.32 ± 4.54 
L-BK-21 0.121 ± 0.025 30.02 ± 4.33 37.22 ± 5.03 33.18 ± 6.30 H-BK-21 0.271 ± 0.035 42.34 ± 6.87 55.11 ± 7.55 51.21 ± 7.98 
L-3k12 h-21 0.132 ± 0.044 31.22 ± 6.10 39.59 ± 5.89 36.13 ± 4.74 H-3k12 h-21 0.367 ± 0.033 54.14 ± 5.18 85.97 ± 7.28 64.87 ± 8.47 
L-3k16 h-21 0.151 ± 0.036 33.36 ± 3.18 54.56 ± 3.36 36.39 ± 3.02 H-3k16 h-21 0.368 ± 0.042 56.70 ± 6.87 81.42 ± 6.14 63.67 ± 8.81 
L-6k12 h-21 0.169 ± 0.030 39.06 ± 5.08 54.81 ± 3.28 40.87 ± 6.41 H-6k12 h-21 0.293 ± 0.023 44.75 ± 5.31 59.85 ± 5.78 49.47 ± 10.60 
L-6k16 h-21 0.120 ± 0.038 36.69 ± 6.17 61.24 ± 4.81 45.43 ± 7.34 H-6k16 h-21 0.305 ± 0.025 46.27 ± 6.91 71.21 ± 4.39 52.48 ± 4.45 
L-BK-42 0.135 ± 0.035 27.66 ± 6.43 23.56 ± 4.19 26.55 ± 5.68 H-BK-42 0.310 ± 0.034 46.91 ± 6.78 56.87 ± 7.66 49.87 ± 8.54 
L-3k12 h-42 0.170 ± 0.032 28.17 ± 4.17 34.51 ± 5.51 27.02 ± 3.85 H-3k12 h-42 0.378 ± 0.035 60.87 ± 8.26 81.95 ± 6.26 71.49 ± 12.95 
L-3k16 h-42 0.129 ± 0.023 29.92 ± 3.57 24.49 ± 3.45 37.23 ± 3.89 H-3k16 h-42 0.388 ± 0.025 71.26 ± 8.33 84.13 ± 8.39 75.37 ± 6.05 
L-6k12 h-42 0.151 ± 0.025 34.37 ± 4.18 37.41 ± 8.74 35.31 ± 4.27 H-6k12 h-42 0.321 ± 0.033 50.99 ± 6.21 60.61 ± 8.43 55.68 ± 5.12 
L-6k16 h-42 0.151 ± 0.030 35.83 ± 5.45 53.39 ± 4.00 38.53 ± 4.99 H-6k16 h-42 0.322 ± 0.028 50.58 ± 7.82 65.31 ± 10.63 51.87 ± 4.51 
Significance 
 Tem *** *** *** *** LI × LT NS NS NS 
 LI *** ** *** LI × N:P NS NS NS NS 
 LT NS NS *** NS LT × N:P NS NS NS NS 
 N:P *** *** *** *** Tem × LI × LT NS NS NS NS 
 Tem × LI *** *** *** *** Tem × LI × N:P NS NS *** NS 
 Tem × LT NS NS NS NS Tem × LT × N:P NS NS NS NS 
 Tem × N:P *** ** *** *** LI × LT × N:P NS NS NS NS 
 Tem × LI × LT × N:P NS NS ** NS      
Treatments RGR, mg L−1 d−1 Cr, % Pb, % Zn, % Treatments RGR, mg L−1 d−1 Cr, % Pb, % Zn, % 
L-BK-7 0.059 ± 0.022 19.34 ± 4.33 16.55 ± 3.65 18.11 ± 4.55 H-BK-7 0.210 ± 0.031 40.11 ± 5.44 52.58 ± 6.55 40.82 ± 7.64 
L-3k12 h-7 0.060 ± 0.037 18.15 ± 3.28 18.68 ± 4.28 22.57 ± 5.86 H-3k12 h-7 0.362 ± 0.024 50.49 ± 4.32 58.19 ± 8.46 58.41 ± 4.11 
L-3k16 h-7 0.071 ± 0.027 23.84 ± 5.96 18.49 ± 5.75 21.72 ± 4.33 H-3k16 h-7 0.352 ± 0.040 53.06 ± 4.61 67.70 ± 9.29 65.11 ± 7.22 
L-6k12 h-7 0.070 ± 0.047 25.73 ± 4.57 11.20 ± 6.49 17.21 ± 3.72 H-6k12 h-7 0.278 ± 0.022 43.01 ± 5.83 51.66 ± 5.54 50.80 ± 6.30 
L-6k16 h-7 0.111 ± 0.026 27.96 ± 3.18 15.78 ± 4.17 25.20 ± 5.26 H-6k16 h-7 0.246 ± 0.069 42.70 ± 3.38 63.78 ± 5.93 44.32 ± 4.54 
L-BK-21 0.121 ± 0.025 30.02 ± 4.33 37.22 ± 5.03 33.18 ± 6.30 H-BK-21 0.271 ± 0.035 42.34 ± 6.87 55.11 ± 7.55 51.21 ± 7.98 
L-3k12 h-21 0.132 ± 0.044 31.22 ± 6.10 39.59 ± 5.89 36.13 ± 4.74 H-3k12 h-21 0.367 ± 0.033 54.14 ± 5.18 85.97 ± 7.28 64.87 ± 8.47 
L-3k16 h-21 0.151 ± 0.036 33.36 ± 3.18 54.56 ± 3.36 36.39 ± 3.02 H-3k16 h-21 0.368 ± 0.042 56.70 ± 6.87 81.42 ± 6.14 63.67 ± 8.81 
L-6k12 h-21 0.169 ± 0.030 39.06 ± 5.08 54.81 ± 3.28 40.87 ± 6.41 H-6k12 h-21 0.293 ± 0.023 44.75 ± 5.31 59.85 ± 5.78 49.47 ± 10.60 
L-6k16 h-21 0.120 ± 0.038 36.69 ± 6.17 61.24 ± 4.81 45.43 ± 7.34 H-6k16 h-21 0.305 ± 0.025 46.27 ± 6.91 71.21 ± 4.39 52.48 ± 4.45 
L-BK-42 0.135 ± 0.035 27.66 ± 6.43 23.56 ± 4.19 26.55 ± 5.68 H-BK-42 0.310 ± 0.034 46.91 ± 6.78 56.87 ± 7.66 49.87 ± 8.54 
L-3k12 h-42 0.170 ± 0.032 28.17 ± 4.17 34.51 ± 5.51 27.02 ± 3.85 H-3k12 h-42 0.378 ± 0.035 60.87 ± 8.26 81.95 ± 6.26 71.49 ± 12.95 
L-3k16 h-42 0.129 ± 0.023 29.92 ± 3.57 24.49 ± 3.45 37.23 ± 3.89 H-3k16 h-42 0.388 ± 0.025 71.26 ± 8.33 84.13 ± 8.39 75.37 ± 6.05 
L-6k12 h-42 0.151 ± 0.025 34.37 ± 4.18 37.41 ± 8.74 35.31 ± 4.27 H-6k12 h-42 0.321 ± 0.033 50.99 ± 6.21 60.61 ± 8.43 55.68 ± 5.12 
L-6k16 h-42 0.151 ± 0.030 35.83 ± 5.45 53.39 ± 4.00 38.53 ± 4.99 H-6k16 h-42 0.322 ± 0.028 50.58 ± 7.82 65.31 ± 10.63 51.87 ± 4.51 
Significance 
 Tem *** *** *** *** LI × LT NS NS NS 
 LI *** ** *** LI × N:P NS NS NS NS 
 LT NS NS *** NS LT × N:P NS NS NS NS 
 N:P *** *** *** *** Tem × LI × LT NS NS NS NS 
 Tem × LI *** *** *** *** Tem × LI × N:P NS NS *** NS 
 Tem × LT NS NS NS NS Tem × LT × N:P NS NS NS NS 
 Tem × N:P *** ** *** *** LI × LT × N:P NS NS NS NS 
 Tem × LI × LT × N:P NS NS ** NS      

Note: Values are presented as mean ± standard error (n = 4). Effects of factors according to two-way ANOVA: NS, non-significant effect; *p < 0.05;**p < 0.01;***p < 0.001. Multiplication sign (×) represents the interaction between or among factors. For a treatment name: ‘H’ and ‘L’ represented high and low temperature. ‘BK’ was the background light regime. ‘*k*h’ meant the light intensity with lighting time. The last code in the name was the N:P; e.g. configuration ‘L-3k12 h-7’ means the combination of factors with low temperature of 5 °C, light intensity of 3,000 lx and lighting time of 12 hours, and N:P of 7:1. ‘Tem’ was the temperature, ‘LI’ was the light intensity and ‘LT’ was the lighting time.

Figure 2

Effects of temperature, light regime, and N:P on algae RGR.

Figure 2

Effects of temperature, light regime, and N:P on algae RGR.

Table 2 indicates that the increase in N:P resulted in higher RGR for all treatments. The interaction effect of temperature and N:P on RGR was significant (P < 0.01). The nutrient effect was notable at higher temperature. The magnitude of M. aeruginosa bloom was influenced by both the concentrations of nitrogen and phosphorus and the ratio of their occurrence. A previous study suggested that N:P between 5.5:1 and 17.4:1 was the optimal range (Klausmeier et al. 2004). Otherwise, low N:P (<5.5:1) was not conducive to algae growth due to the limitation of nitrogen, and the high N:P (>30:1) implied the limitation of phosphorus (Zhang & Hu 2011). In this study, N:P between 7:1 and 21:1, close to the optimal range, was met. In general, the N:P was proportional to the RGR in HRAP and consistent with the trophic level of the cultures.

Algae growth was closely related to autologous photosynthesis through illumination. Statistical analysis indicated the significant effects of light intensity on algae growth, but the lighting time was not a significant factor (P > 0.05, Table 2). RGR was steady and slightly increased when light intensity and lighting time increased to 3,000 lx for 12 h and 3,000 lx for 16 h, whereas RGR was negatively affected by light regime of 6,000 lx for 12 h and 6,000 lx for 16 h (Figure 2). The results indicated that over-irradiation inhibited algae growth and 3,000 lx was a suitable light intensity level in this study. In addition, a significant interaction effect between temperature and light intensity was found (P < 0.05). It was observed that light regime effect was more pronounced at high temperature. However, in combination with the increase of HRT of 6 days, the variation of light regime did not display significant effects on the various RGR investigated. The role of light regime was found most obvious during the algae bloom period. At low temperature, light intensity and nutrient had no prominent effects on algae growth and RGR kept below 0.2 d−1.

As mentioned previously, temperature was a limitation and prerequisite factor for algae growth. Temperature not only affected cellular chemical composition but also influenced nutrient uptake and algae photosynthesis (Singh & Singh 2015). Therefore, the effect of temperature on the algae growth was much more significant than other restricting factors in this study. At proper temperature, the effects of light regime and nutrients were remarkable. In these treatments, HRT of 6 days presented the largest algae proliferation reaching cell concentration of 30,828 ± 255 cell L−1, while the treatment for 2-day HRT showed a fast algae bloom in the first phase at high temperature. Then, the algae cell amount increased to the maximum (12,367 ± 659 cell L−1) but the growing rate decelerated in the later phase.

Removal of Cr, Pb, and Zn in HRAP

Table 2 gives an overview of HRAP batch run results of the effects of temperature, light regime and N:P on HMs removal at 6-day HRT. In general, Cr, Pb, and Zn showed a similar pattern in their removal rate. Maximum removal rate varied from 18.15% to 60.87% for Cr, 11.20% to 85.97% for Pb, and 17.21% to 64.80% for Zn, respectively. Significant differences were found among HMs removal (P < 0.05). The following order was observed for HMs removal under all conditions: Pb > Zn > Cr. HMs removal increased with RGR and the removal rate was closely correlated to RGR at 6-day HRT (Table 2). ANOVA results showed significant effects of temperature, light intensity, and N:P on HMs removal. The interaction between temperature and light intensity and the interaction between temperature and N:P on HMs removal were statistically significant (Table 2). High temperature presented higher HMs removal than low temperature. This effect of temperature on HMs removal was identical to that on RGR. Nutrient condition affected the HMs removal rate. Higher N:P resulted in a greater HMs removal rate (P < 0.05). Moreover, an intense light irradiation also enhanced HMs removal, but lighting time showed no significant effects on removal rate of HMs except Pb.

Generally, HMs uptake quantity by live algae was larger than by dead biomass especially in the low concentration of HMs. It was reported that the quantity of Cr uptake by live algae Spirulina spp. was up to 304 mg g−1, nearly twice larger than that of dead algae (Doshi et al. 2007). Arunakumara et al. (2008) and Sandau et al. (1996) indicated that live Spirulina platensis presented Pb uptake capacity of 188 mg g−1 in contrast to that of dead algae of 16.97 mg g−1. Live species, such as Isochrysis galbana, Planothidium lanceolatum and Scenedesmus subspicatus, also showed a more efficient Zn removal than other dead algae biomass (Sandau et al. 1996; Sbihi et al. 2012).

Effects of HRT on the HMs removal are illustrated in Figure 3. The removal efficiency increased with HRT. The highest removal was observed at 6-day HRT. As known, metal accumulative bioprocesses are based on the extent of metabolic dependence (Gadd 1990). HMs removal from culture solution by algae involved two major mechanisms: active uptake and passive uptake (Dhabab 2011). It should be noted that the HMs removal and RGR were not closely related at the short HRT of 2 days indicating that the intracellular accumulation was relatively a major process during the algae blooming period. HMs ions were actively transported into cells across the cell membrane, which required bioactivity through the cell metabolic cycle (Cossich et al. 2002). Therefore, algae bioactivity determined whether the bioabsorption efficiency was significantly affected by temperature, light regime, and nutrients. Either at low temperature or irradiation, weak algae bioactivity probably caused low bioaccumulation. Consequently, the correlation between HMs removal and RGR was not significant. However, the higher correlation between HMs removal and RGR was found at 4–6 day HRT. In the later stage of algae growth, cell metabolic cycle dependent biosorption or passive uptake was instead a dominant process (Malik 2004). HMs ions were entrapped in the cellular structure and biosorbed onto the cellular binding sites (Malik 2004). At 6-day HRT, both absorption and adsorption capacity reached the maximum. Therefore, the removal efficiency was highly correlated to the concentration of algae in cultures and R2 was up to 0.91 (Cr), 0.77 (Pb), and 0.89 (Zn), respectively.

Figure 3

HMs removal under various algae RGR.

Figure 3

HMs removal under various algae RGR.

Algae removal in SMP

In SMP batch experiments, algae were strongly inhibited by C. demersum and V. natans (Figure 4). The effect of inhibition on algae was closely related to species and plant density. The variation trend of algae concentration showed that culture water treatment with C. demersum and V. natans exhibited an obvious inhibition on M. aeruginosa, with the algae cell concentration significantly lower than the control (P < 0.05). A higher plant density exhibited stronger inhibition effect on algae growth. The inhibition effect of C. demersum at plant density of 10 and 20 rhizomes m−2 was significantly higher than that of V. natans. In the presence of V. natans at the plant density of 10 rhizomes m−2, the algae concentration increased with HRT, indicating a weak inhibition. The reduction of algae at different plant density of 20 and 30 rhizomes m−2 with C. demersum showed insignificant difference (P > 0.05). At the plant density of 30 rhizomes m−2, both species presented high algae removal. The removal rate in V. natans planted SMP was 65% and 64% for C. demersum planted SMP.

Figure 4

Algae inhibition in SMP with different species and plant densities.

Figure 4

Algae inhibition in SMP with different species and plant densities.

Figure 4 shows that the algae reduction was gradually enhanced with HRT. After 7-day HRT at the plant density of 30 rhizomes m−2, effluent algae concentration supplied to the experimental set-up was the lowest. M. aeruginosa was strongly suppressed by C. demersum and V. natans in this study and there was a clear plant density dependent pattern, indicating a deactivation in algae physiology, with regard to the antagonistic relationship between algae and macrophytes. Algae growth was inhibited by competition for available nutrients and light between algae and macrophytes (Hasler & Jones 1949), while algae inhibition could be caused by the release of allelochemicals to culture (Ye et al. 2014). Previous studies also indicated the potential application of submerged macrophytes in the removal of undesirable algae (Tang & Gobler 2011; Wang et al. 2012; Wang et al. 2014). The findings strongly suggested that C. demersum and V. natans could be used to reduce M. aeruginosa after HMs biosorption in an HRAP at the plant density of 20–30 rhizomes m−2.

HMs removal in batch operated APMP reactor

The removal of HMs in the APMP reactor is shown in Figure 5. The performance was good but the removal rate was usually lower than for individual batch experiments in HRAP and SMP reactors. Removal of Cr, Pb, and Zn showed similar patterns. During the algae proliferation period, no influent was fed and the HMs removal efficiency increased rapidly. After 5 days, HMs removal reached the highest values of 75.8% (Cr), 63.6% (Pb), and 61.1% (Zn). The treatment period in HRAP showed a slightly lower, but steady, HMs removal efficiency than did the proliferation period. SMP effluent presented a removal efficiency that was approximately 10 percentage points higher, compared with the HRP effluent. Moreover, no significant diurnal removal difference was found both in HRAP and SMP.

Figure 5

HMs removal in APMP reactor. APMP was operated during summer campaign with the C. demersum density of 20 rhizomes m−2; D, day; N, night.

Figure 5

HMs removal in APMP reactor. APMP was operated during summer campaign with the C. demersum density of 20 rhizomes m−2; D, day; N, night.

Figure 6

pH and DO profiles in HRAP and SMP for day/night time during operation on APMP reactor; D, day; N, night.

Figure 6

pH and DO profiles in HRAP and SMP for day/night time during operation on APMP reactor; D, day; N, night.

As shown in Figure 6, pH varied from 2.5 to 7.0 in the APMP reactor. The pH during the treatment period was found to be higher than during the proliferation period in HRAP. In the proliferation period, no obvious pH variation was observed. Thereafter, pH gradually changed in a sigmoid-like pattern with the rise of algae cell concentration. Due to the carbon dioxide consumption through algae photosynthetic activity, pH increased obviously in the daytime. In return, carbon dioxide was released back to the culture again through algae respiration at night, which resulted in pH decrease. In SMP, pH in daytime showed a decreasing trend because the inhibition of macrophytes led to a reduced algae photosynthetic activity during the lighting period.

It was reported that higher HMs removal was reached at pH of approximately 5. At low pH, functional groups on the algal surface are associated with H+ ions, thus hampering the positively charged metal ions from binding (Monteiro et al. 2012). Higher pH could facilitate metal uptake since the cell surfaces were negatively charged. With pH increase from 1.0 to 7.0, HMs removal could be largely enhanced (Bishnoi et al. 2004; Brinza et al. 2007). When the pH was above 6.5, HMs tended to precipitate as hydroxides; thus only a low amount of HMs remaining in solution could complex with ligands on the algae surface. Contributed by the algae photosynthesis, DO in effluent was higher than influent. DO showed a similar changing pattern as pH, and ranged from 7 mg L−1 to 9 mg L−1.

The accumulation of HMs in the operation of APMP reactor was calculated by summing HMs removal through algae biosorption in HRAP and SMP, and the precipitation in water phase. Figure 7 shows the partition of HMs removal during the entire period, i.e. from the start-up phase to the end of the treatment. The removal partitioning showed that HMs removed by algae in HRAP accounted for about 50% of the total. It was notable that HMs removal in SMP was respectively 13.67% (Cr), 19.53% (Pb), and 16.38% (Zn). A study by Wang & Chen (2010) indicated that dead algae probably had the stronger surface adsorptive reactivity. Therefore, dead algae biomass produced in SMP could further reduce HMs in HRAP by surface adsorption after bioabsorption. As mentioned above, HMs tended to precipitate as hydroxides at pH above 6.5. It inferred that HMs removal occurred via precipitation in HRAP as pH exceeded 6.5 during daytime. The HMs removal by precipitation accounted for 13.75% (Cr), 11.11% (Pb), and 4.92% (Zn) of the total influent. As it was difficult to determine the HMs quantity in the form of precipitation through chemical analysis, the HMs precipitation amount was calculated by mass balance. In addition, HMs adsorbed by the reactor surfaces and the quartz sand in the SMP could also contribute to the total removal. Such removal via abiotic ways was not obvious considering that HMs adsorptive equilibrium had been reached in the warming-up operation before treatment.

Figure 7

Average HMs removal partitioning in APMP reactor.

Figure 7

Average HMs removal partitioning in APMP reactor.

CONCLUSION

In this study, a novel combined algae pond and macrophyte pond reactor, APMP, was proposed for efficient treatment of HM-contaminated water. The results indicated that the APMP reactor was applicable for Zn, Cr, and Pb removal from wastewater. Temperature, as a key factor, yielded significant difference in algae growth and HMs biosorption. The effect of light regime and nutrients at low temperature was not significant. C. demersum and V. natans at the plant density of 20–30 rhizomes m−2 presented high removal of algae. Effluent algae concentration of SMP was about 1,000 cells mL−1. Treatment in HRAP showed good performance. HMs precipitation was probably caused by pH variation above 6.5 during the daytime. Dead algae biomass in SMP further contributed to HMs removal through algae surface adsorption. The study may provide an effective option for metals removal in water.

ACKNOWLEDGEMENTS

This study was supported by Fundamental Research Funds for the Central Universities-DHU Distinguished Young Professor Program (Grant No. 18D111310) and the National Nature Science Foundation of China (Grant Nos. 51809162 and 51679041).

REFERENCES

REFERENCES
Bishnoi
N. R.
,
Pant
A.
&
Garima
P.
2004
Biosorption of copper from aqueous solution using algal biomass
.
J. Sci. Ind. Res.
63
,
813
816
.
Brinza
L.
,
Dring
M. J.
&
Gavrilescu
M.
2007
Marine micro and macro algal species as biosorbents for heavy metals
.
Environ. Eng. Manag. J.
6
(
3
),
237
251
.
Cossich
E. S.
,
Tavares
C. R. G.
&
Ravagnani
T. M. K.
2002
Biosorption of chromium(III) by Sargassum sp. biomass
.
Electron. J. Biotechnol.
5
(
2
),
133
140
.
Dhabab
J. M.
2011
Removal of Fe(II), Cu(II), Zn(II), and Pb(II) ions from aqueous solutions by duckweed
.
J. Oceanogr. Mar. Sci.
2
,
17
22
.
Fu
F.
&
Wang
Q.
2011
Removal of heavy metal ions from wastewaters: a review
.
J. Environ. Manage.
92
(
3
),
407
418
.
Gupta
D. K.
,
Chatterjee
S.
,
Datta
S.
,
Veer
V.
&
Walther
C.
2014
Role of phosphate fertilizers in heavy metal uptake and detoxification of toxic metals
.
Chemosphere
108
,
134
144
.
Hackbarth
F. V.
,
Maass
D.
,
de Souza
A. A. U.
,
Vilar
V. J. P.
&
de Souza
S. M. A. G. U.
2016
Removal of hexavalent chromium from electroplating wastewaters using marine macroalga Pelvetia canaliculata as natural electron donor
.
Chem. Eng. J.
290
,
477
489
.
Hoagland
D. R.
&
Arnon
D. I.
1950
The water-culture method for growing plants without soil
.
Circ. Calif. Agr. Exp. Sta.
347
,
1
39
.
Klausmeier
C. A.
,
Litchman
E.
,
Daufresne
T.
&
Levin
S. A.
2004
Optimal nitrogen-to-phosphorus stoichiometry of phytoplankton
.
Nature
429
,
171
174
.
Kumar
K. S.
,
Dahms
H. U.
,
Won
E. J.
,
Lee
J. S.
&
Shin
K. H.
2015
Microalgae – a promising tool for heavy metal remediation
.
Ecotoxicol. Environ. Safe
113
,
329
352
.
Mehrabadi
A.
,
Craggs
R.
&
Farid
M. M.
2015
Wastewater treatment high rate algal ponds (WWT HRAP) for low-cost biofuel production
.
Bioresour. Technol.
184
,
202
214
.
Monteiro
C. M.
,
Castro
P. M. L.
&
Malcata
F. X.
2012
Metal uptake by microalgae: underlying mechanisms and practical applications
.
Biotechnol. Prog.
28
(
2
),
299
311
.
Nakai
S.
,
Inoue
Y.
,
Hosomi
M.
&
Murakami
A.
1999
Growth inhibition of blue–green algae by allelopathic effects of macrophytes
.
Water Sci. Technol.
39
(
8
),
47
53
.
Perales-Vela
H. V.
,
Pena-Castro
J. M.
&
Canizares-Villanueva
R. O.
2006
Heavy metal detoxification in eukaryotic microalgae
.
Chemosphere
64
,
1
10
.
Rippka
R.
,
Deruelles
J.
,
Waterbury
J. B.
,
Herdman
M.
&
Stanier
R. Y.
1979
Generic assignments, strain histories and properties of pure cultures of cyanobacteria
.
Journal of General Microbiology
111
,
1
61
.
Sandau
E. E.
,
Sandau
P.
&
Pulz
O.
1996
Heavy metal sorption by microalgae
.
Acta Biotechnol.
16
(
4
),
227
235
.
Sbihi
K.
,
Cherifi
O.
,
El Gharmali
A.
,
Oudra
B.
&
Aziz
F.
2012
Accumulation and toxicological effects of cadmium, copper and zinc on the growth and photosynthesis of the freshwater diatom Planothidium lanceolatum (Brébisson) Lange-Bertalot: a laboratory study
.
J. Mater. Environ. Sci.
3
(
3
),
497
506
.
Sekomo
C. B.
,
Rousseau
D. P. L.
,
Saleh
S. A.
&
Lens
P. N. L.
2012
Heavy metal removal in duckweed and algae ponds as a polishing step for textile wastewater treatment
.
Ecol. Eng.
44
,
102
110
.
Singh
S. P.
&
Singh
P.
2015
Effect of temperature and light on the growth of algae species: a review
.
Renew. Sust. Energ. Rev.
50
,
431
444
.
Smara
A.
,
Delimi
R.
,
Chainet
E.
&
Sandeaux
J.
2007
Removal of heavy metals from diluted mixtures by a hybrid ion-exchange/electrodialysis process
.
Sep. Purif. Technol.
57
,
103
110
.
Wang
J.
&
Chen
C.
2010
Research advances in heavy metal removal by biosorption
.
Acta Scientiae Circumstantiae
30
(
4
),
673
701
.
Wang
Z.
,
Yao
L.
,
Liu
G.
&
Liu
W.
2014
Heavy metals in water, sediments and submerged macrophytes in ponds around the Dianchi Lake, China
.
Ecotoxicol. Environ. Safe
107
(
9
),
200
206
.