Wastewater was collected from Whites Creek Wastewater Treatment Plant, Nashville, Tennessee, USA to assess growth and biomass production in the medium. The wastewater was allowed to settle, decanted and stored in plastic containers in the refrigerator at 4˚C until used. Initial levels of N and P in the wastewater effluent were determined as 14 and 6.7 mg/L, using Hach DR 890 colorimeter.

For these experiments, Chlorella vulgaris (ATCC® 9765™) and Scenedesmus obliquus (Ankistrodesmus braunii ATCC® 11457™) were obtained from America type culture collection (ATCC). Pre-inoculation cultures were separately grown in 125 mL Erlenmeyer flasks containing 50 mL modified BG 11 medium supplemented with 10 g/L glucose with agitation at 150 rpm under ambient laboratory conditions in 14:10 hour light-dark cycle for 3 days. The modified BG11 medium contained the following components (g/L): The medium consists of the following components; NaNO₃ (1.5 g∙L⁻¹), K₂HPO₄ 0.04 (g∙L⁻¹), MgSO₄∙7H₂O (0.075 g∙L⁻¹), CaCl₂∙2H₂O (0.036 g∙L⁻¹), citric acid (0.01 g∙L⁻¹), ferric ammonium citrate (0.006 g∙L⁻¹), Na₂EDTA (0.001 g∙L⁻¹), Na₂CO₃ (0.02 g∙L⁻¹) and 1 mL of trace metal solution per liter. The trace metal solution contains a stock of H₃BO₃ (2.86 g∙L⁻¹), MnCl₂∙H₂O (1.81 g∙L⁻¹), ZnSO₄∙H₂O (0.222 g∙L⁻¹), CuSO₄∙5H₂O (0.079 g∙L⁻¹), and Co(NO₃)₂∙6H₂O (0.0494 g∙L⁻¹) [16] . The pH of the medium was adjusted to 7.5 and it was filtered-sterilized using 0.22 µ filter system.

The specific growth rate (µ) was determined as

where N₀ and N represent the optical density at the beginning and any given time t, respectively. A plot of lnN against t yields a straight line, where k was determined through linear regression using the above Equation (1).

The dry weights of the three isolates were determined by filtering 30 mL of the culture suspensions on through 90 mm diameter Glass microfiber (GF/B, Whatman). The filters were oven dried at 90˚C to constant weight in a desiccator. The dry biomass was calculated gravimetrically. The algal biomass was expressed in terms of the dry cell weight (DCW) per liter (g∙L⁻¹).

The overall goal of these investigations is to couple microalgal biomass production to wastewater treatment and to convert biomass produced in the process to precursors of biodiesel. This research recognizes the long-standing knowledge that while production of fuel from microalgae is technically feasible, it is not cost-effective as a standalone process [17] . Rather, investigators have identified wastewater treatment and CO₂ mitigation as feasible and highly desirable processes for coupling microalgal biomass production to environmental protection and mitigation with simultaneous biodiesel production [18] .

Furthermore, research has demonstrated that nitrogen metabolism is a critical aspect in biomass productivity of microalgal lipids. We conducted a preliminary assessment of microalgal biomass production in secondary wastewater from Whites Creek Wastewater Treatment Plant TN and found that it contained levels of N that did not support significant biomass productivity for conversion into biofuel. Accordingly, we focused our attention on finding the levels of nitrogen at which growth and biomass productivities of C. vulgaris and S. obliquuscould be optimized in the wastewater. As reference, we compared biomass productivities in standard defined microalgal medium, BG-11 to those in municipal wastewater. Figure 2 shows a plot of optical densities (OD₆₇₅) over time for C. vulgaris cultivated in BG-11 under varying concentration of N observed over nine days. The plot showed the characteristic sigmoidal growth pattern with different OD optima followed by relatively rapid declines. The 1.0 mg N/L had a highest OD of 9.85 at day 7 compared to 0.50 mg N/L (7.70). There was a decline in OD after 9 days incubation except in 0.25 N/L, which decline at day 8, and increases at day 9. The specific growth rate and biomass productivity of C. vulgaris in BG-11 cultivated in 0.25 mg N/L were 0.28 day⁻¹ and 3.50 g∙L⁻¹ respectively. Increasing levels of N to 0.50 mg/L showed an increase in growth rate and biomass yield to 0.37 day⁻¹ and 4.28 g∙L⁻¹ respectively. A further increase of N to 1 mg/L caused an increase in growth rate to 0.43 day⁻¹. However, at this level of N addition, biomass yield decreased to 3.64 g∙L⁻¹ (Figure 4). A similar study was carried out in 100% piggery wastewater reported that the maximum specific growth rate and biomass productivity of microalga obtained were 0.839 d⁻¹ and 0.681 g∙L⁻¹∙d⁻¹, respectively [21] . Another study showed that in a semi-continuous cultivation of microalgae, the specific growth rate and biomass of Chlorella sp. cultures in the conditions aerated with 2% - 15% CO₂ were 0.58 - 0.66 d⁻¹ and 0.76 - 0.87 g∙L⁻¹, respectively, and the rate of CO₂ reduction was up to 58% [19] . Previous studies have reported at lower nitrogen (<5 mg/L) microalgal lipid content oil can be as high as 40% supporting the fact that biomass productivity is proportional to nitrogen depletion [20] . Our findings also supported previous study that reported the nitrogen sequence of nitrate conversion to nitrite and then ammonium before been incorporated into microalgal metabolic pathway enhance biomass productivity through its nitrogen deficiency [20] .

The isolate C. vulgaris grown in wastewater containing different levels of 0.25, 0.50 and 1.0 mg N/L were able to survive in the wastewater used. The main reasons for coupling microalga biodiesel production with waste- water treatment are to reduce the dependence on freshwater, minimize the cost of nutrients used in algal cultivation and to eliminate N and phosphorus from wastewater [22] . The specific growth rates were 0.44, 0.32, and 0.32 day⁻¹, respectively for cultures fortified with 0.25, 0.50 and 1.0 mg N/L, (Figure 1). Their corresponding biomass productivities were 4.96, 2.19, and 2.31 g∙L⁻¹, respectively (Figure 4). The growth rates of microalgae are often lower in various industrial wastewaters due to low nitrate and phosphate and high toxin levels [8] . The fortified wastewater used in this study was able to support the microalgal growth. Interestingly, the addition of 0.25 mg N/L to wastewater of C. vulgaris showed the highest OD (8.99 as shown in Figure 2) at day 8 with a biomass

yield of 4.96 g∙L⁻¹. This indicated that the organism performed better at 0.25 mg N/L than wastewater supplemented with 0.50 and 1.0 mg N/L. It demonstrated that the biomass productivity increased with the decreased concentration of nitrogen. Since, the main reason for combining microalga biomass production with wastewater treatment is to reduce the dependence on freshwater and minimize the cost of nutrients used in algal cultivation. A similar study conducted using Chlamydomonas obtained the maximal biomass of 1.5 g∙L⁻¹ after 5 days of cultivation [22] .

In this study, S. obliquus was also evaluated under the same conditions described for C. vulgaris. The optical density (Figure 3) monitored over a period of 9 days showed a similar pattern to the biomass production. The OD of 0.25 mg N/L reached the highest OD (6.74) at day 9. Scenedesmus obliquus grown in BG-11 with N additions of 0.25, 0.50 and 1.0 mg/L were 0.59, 0.73 and 0.71 day⁻¹, respectively (Figure 1). BG-11medium containing 0.50 mg N/L had a higher growth rate of 0.73 day⁻¹. Correspondingly, to different growth rates observed in BG-11, biomass productivities by S. obliquus were 2.15, 3.10 and 3.37 g∙L⁻¹, respectively (Figure 4).

As shown in Figure 1 and Figure 3, the specific growth rate of 0.25, 0.50, 1.0 mg N/L W, were 0.56, 0.47, and 0.55, respectively. The corresponding biomass productivities were 1.05 ± 0.0.16, 1.18 ± 0.08 and 1.27 ± 0.06 g∙L⁻¹ respectively. However, this is in contrast to higher specific growth rate and biomass yield observed in wastewater for C. vulgaris. Among the six microalga isolates investigated during a similar study, the average specific dry biomasses of 0.53 and 0.49 g/dwt/L were found for S. obliquus, and C. vulgaris, respectively, after 20 days of cultivation in piggery wastewater [23] . In another study that evaluated the prospect of using microalgae S. obliquus for brewery wastewater treatment and biomass production. After 9 days, a maximum of 0.9 g∙L⁻¹ was observed at 12 h period of daily light exposure in aerated culture [24] .

Comparing the graph of Figure 4, the biomass productivity of C. vulgaris was generally higher than the productivity of S. obliquus under varying conditions of the test media. The biomass productivity of C. vulgaris was significantly higher than S. obliquus in 0.25 N/L BG-11, 0.50 N/L BG-11, 0.25 mg N/L W, 0.50 mg N/L W, and 1.0 mg N/L W, respectively at p < 0.05. The biomass productivity of C. vulgaris in 0.25 mg N/L W was significantly

higher than the production in 0.50 mg N/L W and 1 mg N/L W. A higher significant productivity of S. obliquus was observed in 0.50 mg N/L BG 11 as compared with 0.25 mg N/L W, 0.50 mg N/L W, and 1 mg N/L W. The biomass yield increased as the N concentration was increased from 0.25 mg to 0.50 mg N/L, a similar study conducted showed the highest biomass obtained was 10.04 g∙L⁻¹ with biomass productivity of 1.673 g/L d was obtained using 1.0 g∙L⁻¹ of NaNO₃ as N source and 40 g∙L⁻¹ glucose, while a biomass of 7.09, 8.55 and 9.45 g∙L⁻¹ with biomass productivity of 1.182, 1.425 and 1.575 g/L d were found at 20, 30 and 50 g∙L⁻¹ glucose, respectively [25] . In another study, different pretreatment options test and filtration (by 0.2 μM) resulted in the highest biomass and lipid productivity [26] . The effect of N concentration on of Chlorella sp. showed that lower N supplement 0.50 mg N/L BG 11 and 0.25 mg N/L W, increased the biomass yield to 4.28 ± 0.22 and 4.96 ± 0.17 g∙L⁻¹, respectively, when compared to 1mg N/L BG 11 (3.64 ± 0.68 g∙L⁻¹). The results obtained in this research have similar trend with the result that showed excess N source reduces productivity of C. vulgaris [27] . The highest biomass productivity was obtained at 10 mM sodium nitrate, with a biomass concentration of 3.2 g∙L⁻¹ and a biomass productivity of 0.63 g⁻¹∙day⁻¹ [28] .