Microalgae could be utilized as an alternative to remove inorganic nutrients from wastewater and makes them a useful phytoremediation tool in wastewater treatment process. Phytoremediation is a process that utilized plant such as microalgae to treat the nutrient pollution in the aquaculture wastewater. Chlorella sp.'s morphology as unicellular microorganism could allow it to absorb nutrient more efficiently as compared to terrestrial plant. Thus, this study was performed to evaluate the performance of Chlorella sp. at different inoculation concentrations to determine its kinetic growth at the same time their nutrient removal efficiency specifically total ammonia nitrogen (TAN) and total phosphorus (TP). Silver Barramundi, Lates calcarifer wastewater was inoculated with microalgae culture with different concentrations of 10%,15%, 20%, 25%, 35% and 40% (v/v) in 14-days batch treatment period at room temperature (25 Â± 2Â°C). Determination of Chlorella sp. growth performance, TAN and TP removal was performed daily based on the APHA's standard method of examination of water and wastewater. In this study, results indicated that the best nutrient removal was in the range of 15 - 20% (v/v) Chlorella sp. inoculation concentrations which yield over 90% nutrient removal with final effluent of 0.08 mg/L TAN and 0.01 mg/L TP. The Chlorella sp. growth kinetics fitted the Monod models and the nutrient removal fitted with the first order kinetics. It could be concluded that Chlorella sp. is a potential candidate to treat aquaculture wastewater and served as green technology to mitigate the greenhouse-related problems.
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Key words: Chlorella sp., Lates calcarifer, batch phytoremediation, total ammonia nitrogen, total phosphorus, Monod Model, First Order Kinetics
Phytoremediation is a biological treatment utilizing any type of plant either terrestrial or marine plant. This type of biological treatment has gained its popularity due to its role in remediating pollution such as heavy metals, high nutrient concentration and also contributed positively to the environmentCho-Ruk et al., 2006Al-Farraj and Al-Wabel, 2007El-Sheekh and Fathy, 2009. Microalgae is recognized as the most promising candidate for bioprocess due to a multiplicity of reactions (Rawat et al., 2011). The use of microalgae is desirable since they are able to serve a multiple role such as bioremediation as well as generating biomass for biofuel production with concomitant carbon sequestration (OlguÄ±ìn, 2003; Mulbry et al., 2008). In addition, wastewater remediation by microalgae is also an eco-friendly process since it does not release any secondary pollution as long as the biomass produced is continuously reused and efficient nutrient recycling is maintained (Muñoz and Guieysse, 2006; Pizarro et al., 2006; MulHYPERLINK "#_ENREF_18"bHYPERLINK "#_ENREF_18"ry et al., 2008).
The release of untreated agricultural, industrial and municipal wastewater poses serious environmental challenges to the receiving water bodies (Arora and SaxeHYPERLINK "#Arora, A. and S. Saxena,2005"nHYPERLINK "#Arora, A. and S. Saxena,2005"a, 2005; De-Bashan and Bashan, 2010). The major effect of releasing wastewater which is rich in organic compounds and inorganic chemicals such as phosphates and nitrates is mainly eutrophication (OlguÄ±ìHYPERLINK "#_ENREF_20"nHYPERLINK "#_ENREF_20", 2003; Pizarro et alHYPERLINK "#_ENREF_22".HYPERLINK "#_ENREF_22", 2006; Mulbry eHYPERLINK "#_ENREF_18"tHYPERLINK "#_ENREF_18" al., 2008; Godos et al., 2009; De-Bashan et al., 2002). According to Rawat et al., HYPERLINK "#_ENREF_23"(HYPERLINK "#_ENREF_23"201HYPERLINK "#_ENREF_23"1), this global problem could be solved by utilizing microalgae whereby the wastewater is utilized as feed for the maintenance of microalgae growth. Throughout the treatment period, there will be concomitant accumulation of biomass for downstream processing enabling the production of biodiesel feedstock (Muñoz and GHYPERLINK "#_ENREF_19"uHYPERLINK "#_ENREF_19"ieysse, 2006; PizaHYPERLINK "#_ENREF_22"rHYPERLINK "#_ENREF_22"ro et al., 2006; Pittman et al., 2011). Cellular nitrogen is mainly used to build proteins, amino acids and nucleic acids while phosphorus is mostly constituent of nucleic acids and phospholipids (Geider and Roche, 2002). These findings has proven that microalgae are able to remove nutrients from wastewater to meet the stringent requirements according to international standards (Rawat eHYPERLINK "#_ENREF_23"tHYPERLINK "#_ENREF_23" al., 2011).
This research is motivated by the increasing interest in application of biotechnology and the implementation of environmentally friendly tools in treating wastewater (Yoshimoto et al., 2005; Concas et al., HYPERLINK "#Concas, A., M. Pisu and G. Cao,2010"2HYPERLINK "#Concas, A., M. Pisu and G. Cao,2010"010; Sato et al., 2010). Chlorella sp. was classified as a biological tool in wastewater treatment to reduce the present of nutrient content (Sydney et al., 2010). At the same time, biomass of Chlorella sp. which is the by-product of the treatment process could be marketed as high-value products (Brennan and Owende, 2010). In addition, treatment of phytoremediation could also help in the uptake of various heavy metals and greenhouse gasses present in the wastewater (Davis HYPERLINK "#Davis, T.A., B. Volesky and A. Mucci,2003"eHYPERLINK "#Davis, T.A., B. Volesky and A. Mucci,2003"t al., 2003). This would contribute to overcome environmentally related problem of domestic wastewater reported around the world (Godos et aHYPERLINK "#Godos, I.d., V.A. Vargas, S. Blanco, M.C.G. Gonzalez and R. Soto <I>et al<I/>.,2010."lHYPERLINK "#Godos, I.d., V.A. Vargas, S. Blanco, M.C.G. Gonzalez and R. Soto <I>et al<I/>.,2010."., 2010; Listowski etHYPERLINK "#Listowski, A., H. Ngo, W. Guo, S. Vigneswaran, H. Shin and H. Moon,2011" HYPERLINK "#Listowski, A., H. Ngo, W. Guo, S. Vigneswaran, H. Shin and H. Moon,2011"al., 2011; Rawat et al.HYPERLINK "#_ENREF_23",HYPERLINK "#_ENREF_23" 2011).
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The aim of this study was to determine the possibilities of microalgae Chlorella sp. in treating aquaculture wastewater. This was implemented through monitoring of TP and TAN removal performance in aquaculture wastewater by phytoremediation using Chlorella sp. The most suitable inoculation concentration in batch mode and the growth kinetics of Chlorella sp. throughout the treatment period were investigated.
Materials and methods
The pure strain of green algae Chlorella sp. was obtained from the Institute of Tropical Aquaculture (AKUATROP) of University Malaysia Terengganu on October 2011. It was cultivated for 30 days to produce secondary culture for up-scaling. The medium used for culture was Conway media with autoclaved and filtered seawater. Normal air filtered with 40 Âµm air filter was provided as sterile aeration to prevent any bacterial contamination. Microalgae cultures were maintained at room temperature of about 25Â±2 ËšC under a light intensity of 4100 lux from white fluorescent light for 24 h photoperiod. Maintenance of secondary culture was done for six months throughout the experimental period.
The aquaculture wastewater was supplied from the culture of Lates calcarifer also known as Barramundi or Silver Sea bass as livestock. Lates calcarifer was chosen because of their ability to survive in water at various salinity levels, ranging from 0 ppt. up to 30 ppt.. In this study, 30 fishes were reared in a tank under controlled temperature of 27 Â± 2 Â°C with continuous aeration. The same type of feed was given based on 3% of body weight to control phosphorus and nitrogen content in the effluent. Nutrients content in the effluent were considered maximum on the fourth day of rearing period. At this time, the wastewater produced was channeled for phytoremediation treatment by microalgae.
The treatment of wastewater was conducted in batch mode using 5000 ml flasks. This treatment commenced with the inoculation of Chlorella sp. culture into the wastewater. Six samples with different concentration of Chlorella sp. cultures i.e., 10%, 15%, 20%, 25%, 35%, and 40% (v/v), were examined in this study. Illumination was provided continuously from the top of the flask. The experiments were conducted at controlled room temperature of 25 Â± 2 Â°C for 14 days.
Three parameters of total phosphate (TP), total ammonia nitrogen (TAN) and biomass of Chlorella sp. concentrations were analyzed. The changes of concentrations in the nutrient content and Chlorella sp. biomass were monitored daily for a period of 14 days. Nutrient concentrations (phosphate and nitrogen) were the main concerned for evaluating removal efficiency in accordance to the growth of Chlorella sp. biomass as kinetic study.
Chlorella sp. biomass concentration analysis
Analysis of Chlorella sp. biomass concentration was performed simultaneously with nutrient concentrations. Daily samples withdrawn from flasks were centrifuged at 4 Â°C to separate algae biomass from water. Two-hundred-milliliters sample from each flask were poured into the four 50 ml centrifuge tubes and then they were centrifuged at 5000-6000 rpm for about 30 min to separate the Chlorella sp. cells from water. Then, 10 mL sample of algal suspension was again centrifuged at 3000 rpm and the supernatant was discarded. The algae were suspended in 3 mL methanol and heated for about 5 min in a water bath. The samples were cooled to room temperature and then the volume was made up to 5 mL by adding methanol. The Chlorophyll a concentration in the extract was determined using the Equation 1 with the reading of absorption (A) of the pigment extract in a spectrophotometer at the given wavelength, Î» (650nm and 665nm) against a solvent blank (Becker, 2008).
Chlorophyll a (mg/L) = (16.5 Ã- A665) - (8.3 Ã- A650) (1)
TP and TAN concentration analysis
Analysis of TP and TAN concentrations were carried out by using HACH DR2400 kit. A 200 mL samples were daily withdrawn from the flasks. Then, the samples were centrifuged at 5000-6000 rpm to separate algae in order to obtain a clear supernatant. Measurements of TP and TAN were executed in clear supernatant by colorimetric methods i.e., Ascorbic Acid Method and Salicylic Acid Method, respectively. These methods were adapted from Standard Method for Examination of Water and Wastewater (AHYPERLINK "#APHA,2012"PHYPERLINK "#APHA,2012"HA HYPERLINK "#APHA,2012"2HYPERLINK "#APHA,2012"0HYPERLINK "#APHA,2012"1HYPERLINK "#APHA,2012"2). They were equivalent to USEPA Method 365.2 and Standard Method 4500-PE for wastewater.
c. Statistical analysis
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Microalgae cell growth, TP and TAN concentration were recorded in Microsoft Office Excel throughout the experimental period. Graphical analysis was performed using Originlab OriginPro 8.6 whereas statistical analysis involving ANOVA and Tukey's HSD Test was implemented using Minitab 16. Confidence interval of 95% was selected in order to strictly determine the significance of different Chlorella sp. inoculation concentrations on nutrient reduction rate, maximum biomass growth rate, remaining nutrient concentration and treatment period for maximum nutrient reduction.
The comparative studies of the growth performance of Chlorella sp., TAN and TP concentrations between different inoculations were determined as shown in Figure 1. The growth kinetics of Chlorella sp. suited the Monod models in line with the growth phases such as lag, log, stationary and declining phases. Treatment with 15% (v/v) Chlorella sp. had maximum cell concentration of 49.99 mg/L, 51.55 mg/L for 20% (v/v) and 48.36 mg/L for 40% (v/v) inoculation concentration. The TP and TAN reductions followed the First Order Kinetics where removal percentage continued to increase exponentially until reaching an asymptote where the removal percentage was higher than 99% with final nutrient concentration of 0.08 mg/L TAN and 0.01 mg/L TP.
In order to further investigate the effect of different Chlorella sp. inoculation concentrations on TP and TAN removal, correlation and regression analysis were performed specifically on Day 7 of the treatment period since the most obvious reduction occurred at this point. Figure 2 shows the remaining TP and TAN concentrations logarithmically decreased with the increased of Chlorella sp. inoculation concentration. At Day 7 which was the mid-point of treatment period, 0.23 mg/L TP and 0.08 mg/L TAN still remain for 10% (v/v) inoculation as compared to 0.10 mg/L TP and 0.05 mg/L TAN for 40% (v/v) inoculation concentration. Therefore, a higher inoculation concentration of Chlorella sp. would reduce the remaining nutrient concentrations.
Figure 3 shows the correlation between treatment periods required to achieve the maximum reduction and different inoculation concentrations of Chlorella sp. The increased of Chlorella sp. inoculation concentration contributed to a shorter treatment period required to reach the maximum TP and TAN removal. A very strong negatively linear relationship with slope of -0.1335 and adjusted R2 of 0.9057 was established for TP, whereas a similar pattern of strong relationship with slope of -0.0832 and adjusted R2 of 0.8713 for TAN removal with the increase of Chlorella sp. inoculation concentration. The higher inoculation concentration contributed to a higher readily available biomass which produces higher initial nutrient reduction. After that, Chlorella sp. would undergo subsequent mitotic cell division of growth to increase its biomass concentration by utilizing the remaining nutrients exist in the wastewater (De-Bashan and BashHYPERLINK "#_ENREF_11"aHYPERLINK "#_ENREF_11"n, 2010).
Batch phytoremediation on aquaculture wastewater utilizing Chlorella sp. was successfully performed. During the study, the concentrations of Chlorella sp. used for phytoremediation was varied at 10%, 15%, 20%, 25%, 35% and 40% (v/v). All samples were treated for a period of 14 days after Chlorella sp. inoculation. Different concentrations of Chlorella sp. have shown a quite similar growth pattern and suited the Monod Model. As shown in Table 1, the maximum growth rate constant and half saturation constant of TP show reduction with the increase of inoculation concentrations. For 10% (v/v), the half saturation constant, Ks was 0.354 mg/L and decreasing to 0.164 mg/L at 40% (v/v) Chlorella sp.. However, there was no clear trend between kinetics coefficients of TAN and different Chlorella sp. inoculation concentrations.
Total phosphorus and total ammonia nitrogen removal
The initial concentrations of TP for all treatments were maintained at 2.50Â±0.05 mg/L orthophosphate, POâ‚„Â³--P in order to determine the percentage of TP removal for a period of 14 days. The reading for TP concentration was taken daily to observe its reduction in the effluent. TP concentration in each treatment decreased sharply at early period of Chlorella sp. growth due to the rapid increase of Chlorella sp. biomass as indicated in log phase. All samples achieved the high percentage of TP removal, 99.6% which indicate that the effluent contain 0.01 mg/L PO43- except for sample of 10% (v/v) Chlorella sp.. The concentration of 0.01mg/L PO43- in effluent is being considered as a safe level for effluent to be released to water body and being used for water recycled in aquaculture. The earliest sample that achieved the maximum removal percentage of more than 99% of TP is the sample with 35% (v/v) Chlorella sp., followed by the samples with 20% and 40% (v/v) Chlorella sp.. Next is the sample with 25% (v/v) Chlorella sp. and the last one is the sample with 15% (v/v) of Chlorella sp.. As shown in Table 3, the initial TAN concentrations for all 6 aquaculture wastewater samples were approximately 0.80Â±0.05 mg/L before being inoculated with Chlorella sp. Once the treatment of phytoremediation completed, the final TAN concentration with 10% (v/v) Chlorella sp. was reduced to 0.12mg/L. Therefore, the optimum percentage of microalgae for phytoremediation at specified treatment period was found to be in the range of 15 - 25% (v/v) Chlorella sp.
Figure 4 shows the maximum growth rate constant of Chlorella sp. analyzed using Monod Model and reduction rate analyzed using First Order Kinetics with regard to TAN and TP. The characteristics of log phase were accurately quantified in accordance to the First Order Kinetics. The highest maximum growth rate constant was at 20% (v/v) inoculation concentration for TP whereas for TAN at 35% (v/v). However, the highest cumulative nutrient reduction rate was at 15% (v/v) slightly decreased as inoculation concentration increased to 25% (v/v). Reduction rate of TP was recorded as the highest within the range of 15 - 35% (v/v) whereas for TAN at the range of 10 - 20 % (v/v) inoculation concentrations. Thus, the most suitable concentration of Chlorella sp. inoculation that produced the optimum reduction for both nutrients was determined in the range of 15 - 20% (v/v).
The use of several species of microalgae as a tertiary wastewater treatment was proposed over a decade ago, and various potential treatments continue to be evaluated today (DHYPERLINK "#_ENREF_12"eHYPERLINK "#_ENREF_12"-Bashan and Bashan, 2010). The underlying assumption is that the microalgae will transform some of the contaminants to non-hazardous materials and then the treated water can be reused or safely discharged (Borowitzka and BorowitHYPERLINK "#Borowitzka, M.A. and L.J. Borowitzka,1988"zHYPERLINK "#Borowitzka, M.A. and L.J. Borowitzka,1988"ka, 1988). In this study, once the TP and TAN concentrations mostly depleted, Chlorella sp. growth began to decline. This was also supported by Droop-based model suggested by Bougaran et al. (2010). The interaction of TP and TAN concentrations on the growth of microalgae should be considered at the acquisition level rather than at the assembly level. Nutrient present in the wastewater (extracellular region) affect the Chlorella sp. growth more than those in the intracellular region. Thus, this phenomenon indicated that the present of these nutrients in wastewater contributed significantly on the growth of Chlorella sp..
Based on the previous study, microalgae can be effectively used to remove high amount of nutrient because they require large quantity of nitrogen and phosphorus for protein (45 - 60% of microalgae dry weight), nucleic acid and phospholipids synthesis (Rawat et alHYPERLINK "#_ENREF_23".HYPERLINK "#_ENREF_23", 2011).In this study, the concentrations of Chlorella sp. used for phytoremediation was varied at 10%, 15%, 20%, 25%, 35% and 40% (v/v). Different concentrations of Chlorella sp. have shown a quite similar growth pattern and suited the Monod Model which consists of lag phase, log phase, stationary phase and death phase. After the inoculation, reduction of microalgae growth occurred within a short period of about 2 days known as lag phase. In this phase, the reduction of growth occurred because microalgae were still adapting with the new environment (El-SheHYPERLINK "#_ENREF_13"eHYPERLINK "#_ENREF_13"kh and Fathy, 2009). When microalgae culture inoculated into the aquaculture wastewater, other pollutants present may also contribute to the reduction in its growth. The microalgae growth rate rebounded back once they were successfully adapted to the new environment. This stage was known as log phase. This is indicated by the rapid increase in the biomass as depicted with exponential growth of microalgae. Thus, microalgae utilized the TP and TAN that present in aquaculture wastewater as source of food contributing to the reduction of nutrient.
From nutrients removals aspect, TP in the form of orthophosphate was absorbed by Chlorella sp. and being stored as polyphosphates within the cells (RawHYPERLINK "#_ENREF_23"aHYPERLINK "#_ENREF_23"t et al., 2011). Under subsequent aerobic condition, the orthophosphate is oxidized to produce energy and re-accumulation of phosphate into polyphosphate. The energy generated in this transformation process was used by Chlorella sp. for its growth. However, phosphorus concentration above 6 mg/L will lead to explosive growth of algae. It has become a global problem where ponds and lakes gradually turn into marshes. Algae are considered to be a biological means of phosphorus removal which helps in detecting a potential eutrophication. Since phosphorus is ecologically significant in algal productivity, its removal from aquatic bodies is essential to prevent the occurrence of eutrophication problems.
During the experiment, it was found that microalgae started to degrade themselves after all substrate had been used. When degradation of microalgae occurred, the microalgae would release the nutrients that it had absorbed. Due to this phenomenon, the removal percentage of TAN had decreased after achieving its maximum value. The determination of proper inoculation concentration of Chlorella sp. is crucially important for the implementation on the real scale aquaculture wastewater treatment.
The final TAN concentration has been reduced to 0.08 mg/L after undergoing the full treatment period. In the real application of phytoremediation, this value would represent the actual concentration of TAN before it is disposed into the water body such as river or sea. As recommended by the USEPA Standard, effluent TAN concentration released must be less than 0.2 mg/L to be considered as environmentally safe since the effluent discharged did not exert significant impact on the flora and fauna. Thus, this indicated that the phytoremediation treatment of aquaculture wastewater was an excellent method in removing nutrient especially TAN and TP.
It was shown that the used of microalgae Chlorella sp. in reducing harmful nutrients in the aquaculture wastewater was suitable and effective. Based on the kinetic coefficients using Monod Models and First Order Kinetics on the removal of both TAN and TP, the optimum concentration of Chlorella sp. biomass occurred in the range of 15 - 20% (v/v) that contributed over 99% nutrient removal with final effluent of 0.08 mg/L and 0.01mg/L, respectively. Microalgae Chlorella sp. did exhibit a normal growth patterns with clear distinction of growth phases. Aquaculture wastewater treatment using microalgae Chlorella sp. also known as phytoremediation could be considered as a novel innovation of wastewater treatment technology.