Effect of l-glutamicacid on the growth and ammonium removal from ammonium solution and natural wastewater by Chlorella vulgaris

NTM06 m
Masil Khan, Naoto Yoshida
,

Department of Biochemistry and Applied Biosciences, University of Miyazaki, 1-1 Gakuen Kibanadai Nishi, 889-2192 Miyazaki-shi, Miyazaki, Japan Received 7 November 2006; revised 19 December 2006; Accepted 23 December 2006. Available online 26 February 2007.

Abstract
The main objective of this laboratory scale experiment was to study the effect of l-glutamicacid on the growth in media and removal of ammonium from ammonium solution and natural wastewater by Chlorella vulgaris NTM06. It was observed that higher levels (1.0% and 1.5%) of l-glutamicacid compared to control (0% l-glutamicacid) negatively affected growth of C. vulgaris NTM06 and enhanced removal of ammonium from ammonium solution as well as natural wastewater. After 24 h of incubation, 99% of 169.3 mg N/l was removed from ammonium solution by 1.5% l-glutamicacid treated C. vulgairs NTM06 cultures; removal in case of control was 70%. In case of natural wastewaters with initial ammonium concentrations of 1550, 775, 310 and 155 mg N/l, removal after 48 h of incubation were 60%, 88%, 61% and 55% respectively. Ammonium removals from ammonium solutions of pH 4.08.0 were similar, whereas adsorption of ammonium ions on to the surface of dead C. vulgaris NTM06 cells was around 11%. Compared to dark, cultures incubated under the light showed higher initial removal of ammonium, however, after 24 h, differences were not significant. Further research on the role of lglutamicacid in micro-algal treatment of wastewater and its combination with other approaches such as co-immobilization of micro-algae with other organisms, starvation of micro-algal cells and the use of polymers is recommended.

Keywords: Ammonium; Chlorella vulgaris; l-glutamicacid; Wastewater

Article Outline
1. Introduction 2. Methods 2.1. Algal cultures and growth conditions 2.2. Growth measurement 2.3. Sample source 2.4. Ammonium removal by C. vulgaris NTM06 2.5. Dead versus live cells comparison 2.6. Effect of pH on removal of ammonium by C. vulgaris NTM06 2.7. Experimental design and statistical analysis 3. Results and discussion 3.1. Effect of l-glutamic acid on the growth of C. vulgaris NTM06 3.2. Effect of l-glutamic acid levels on removal of ammonium from ammonium solution 3.3. Live and dead C. vulgaris NTM06 cultures comparison 3.4. Light and dark incubation comparison 3.5. pH effect on removal of ammonium by C. vulgaris NTM06 3.6. Total ammonium removal by C. vulgaris NTM06 3.7. Ammonium removal from wastewater Acknowledegments References

1. Introduction
Primary and secondary treatment of wastewater help in settling down of solid materials and removal of organic matter respectively and result in more or less clear wastewater, which can then be recycled as irrigation water or discharged to natural water bodies (de la Noe et al., 1992). However, depending on the source of wastewater, this secondary treated wastewater may sometimes be loaded with inorganic nutrients such as nitrogen and phosphorus, range of salt compounds and heavy metals (Khan et al., 2003). Disposing of such effluent to natural water bodies can cause eutrophication and in case of its use for agriculture or civic amenities (parks etc.), pollution of soil and underground water resources may occur. Due to these potential problems, removal of nutrients (nitrogen and phosphorus), heavy metals and other salts from these primary and secondary

treated wastewaters are necessary. In many countries it is mandatory to bring level of these contaminants to safe levels before treated wastewater can be discharged to water bodies or recycled. Chemical treatment of wastewater for nutrient removal (tertiary treatment) may be one of the options but this process is in general very expensive and it may at times lead to secondary pollution (de la Noe et al., 1992). In contrast, biological treatment appears to perform well and micro-algal cultures are known to use nitrogen and phosphorus for their growth ( [13] and [Oswald, 1988] ) as well as remove heavy metals ( [Pavasant et al., 2006] and [Yoshida et al., 2006] ). Among all species of micro-algae, Chlorella is common and effective for the immobilization and nutrient removal purposes ( [Lau et al., 1996] and [Tam et al., 1994] ). However, one of the major drawbacks of using Chlorella species or micro-algae for biological wastewater treatment is the slowness of this process. Studies in the recent past have therefore mainly focused on how to enhance the efficiency of biological wastewater treatment systems or nutrient removal by micro-algae. The right choice of micro-algal species (Lau et al., 1996), suitable environmental conditions ( [Gonzalez et al., 1997] and [Puigagut et al., 2005] ), co-immobilization of micro-algae with other microorganisms (de-Bashan et al., 2002), starvation of micro-algal cultures (Hernandez et al., 2006) and use of polymers for immobilization purposes ( [Jimneze-Prez et al., 2004] and [Tam and Wong, 2000] ) have all been reported to be enhancing nutrients removal from wastewater. For example de-Bashan et al. (2002) reported that Chlorella vulgaris and Chlorella sorokiniana in association with Azospirillum brasilense, a plant growth promoting bacteria, increased the growth of biomass and removal of ammonium and phosphorus from synthetic wastewater. Similarly, starvation (3 or 5 days) of C. vulgaris and C. sorokiniana co-immobilized with A. brasilense in alginate beads showed improved nutrients removal (Hernandez et al., 2006). In the same line, the present study investigated the effect of l-glutamicacid on the growth and ammonium removal by C. vulgaris NTM06. It has been reported that plant enzyme glutamine synthetase has high affinity for ammonia and its ability to incorporate ammonia efficiently into organic combination (Oaks, 1994). Furthermore, the role of glutamine synthetase and glutamate synthase in nitrogen metabolism of plants ( [Glass et al., 2002] , [Lea and Miflin, 1974] and [Miflin and Habash, 2002] ) and bacteria (Kanamori et al., 1987) have been emphasized and reported extensively. It was therefore hypothesized that C.

vulgaris NTM06, which is a unicellular plant may also show similar response to l-glutamicacid treatment and might increase ammonium removal from wastewater.

2. Methods
2.1. Algal cultures and growth conditions
Unicellular micro-alga identified as C. vulgaris NTM06 was isolated from soil and maintained on growth media (YPG; defined below) at 4 C in the refrigerator. YPG (agar or broth) contained 2% peptone, 1% yeast extract, 2% glucose and 1.5% agar (in case of agar media only) in distilled water. Concentrations of l-glutamicacid in YPG broth were (w/v) 0.1%, 0.25%, 0.5%, 1.0% and 1.5%. The pH of l-glutamicacid amended broth was readjusted to 6.0 6.2 with the help of NaOH solution. Sterilization was done by autoclaving at 120 C for 20 min. Pre-culture was prepared by inoculating 3 ml of YPG broth with C. vulgaris NTM06 culture and incubated for 4 days at 37 C under continuous light (1000 lux) on a rotary shaker at 120 rpm. For culture, 50 ml of YPG broth (both amended with l-glutamicacid and un-amended) was inoculated with pre-culture at the rate of 1.0% and incubated for 5 days (120 h).

2.2. Growth measurement

Growth measurement based on the number of cells (cells/ml) started at zero time (soon after inoculation) and continued on daily basis until the end of incubation (144 h). Sub-samples were taken aseptically, diluted with distilled water and number of cells in chambers of Haematocytomer was counted under the microscope.

2.3. Sample source

Low concentration of ammonium is considered to be beneficial to various micro-algae whereas higher concentration of ammonium in wastewater is known to be toxic. The concentration of ammonium in the ammonium solution used in this study was 169.3 mg N/l. (80 mg (NH4)2SO4/100 ml distilled waster). Whereas ammonium concentration in the natural wastewater used in this study was very high (1550 mg N/l) and therefore in addition to concentrated wastewater, diluted forms (2, 5 and 10 times) of this wastewater were also used. pH of the wastewater and ammonium solution was in the range of 8.0 8.3 and 6.06.2 respectively. Reduction of ammonium in the ammonium solution was used as an indicator for selecting an appropriate level of lglutamicacid.

2.4. Ammonium removal by C. vulgaris NTM06

Five days old C. vulgaris NTM06 cultures were harvested by centrifugation at 3000 rpm for 3 min. Supernatant was decanted and cell pellets were washed with saline solution (0.9% NaCl) and then re-suspended in ammonium solution or filtered wastewater and incubated at 37 C under continuous light (1000 lux) except in the light versus dark comparison study where half of the samples were covered with aluminum foils. In case of measuring total potential removal of ammonium ions by C. vulgaris NTM06, ammonium solution was replaced with a new solution after every 24 h of incubation and this process continued until more or less no reduction in ammonium was observed (4 cycles). In case of all incubations, ammonium measurements started at zero time and continued at frequent and regular time intervals untill the end of incubation. The phenate method was used for measuring ammonium concentration in treated ammonium solution/wastewater. In this method phenol and hypochlorite react with ammonium to create blue-colored compound and color intensity is then measured colormetrically. Briefly, 100 L of sub-sample (centrifuged at 12000 rpm) was treated with 2 ml of solution one (g/l of distilled water: 10 g crystal phenol and 0.1 g sodium pentacyanonitrosyl ferrate (III) dihydrate) and 2 ml of solution two (g/l of distilled water; 10 g sodium hydroxide, 53.6 g disodium hydrogenphosphate 12 water, and 10 ml (of 10% available chlorine) sodium hypochlorite solution), mixed and incubated at 37 C in the dark for 30 min. Absorbance was measured at 540 nm using Shimadzu Spectrophotometer (UV-1200).

2.5. Dead versus live cells comparison

In order to investigate that how much ammonium is adsorbed on the surface of C. vulgaris NTM06 cells or disappear due to factors other than taken up by Chlorella cells, comparison of dead versus live cells was carried out. To achieve dead cells, known amount of C. vulgaris NTM06 cultures was placed in boiling water for 10 min and then harvested as mentioned above. For comparison, same amount of C. vulgaris NTM06 cells were harvested alive. Microscopic study of dead and live C. vulgaris cells revealed that dead cells prepared by submerging them in boiling water for 10 min did not cause disintegration of cell wall/structure.

2.6. Effect of pH on removal of ammonium by C. vulgaris NTM06

Effect of pH on removal of ammonium by C. vulgaris NTM06, grown in 1.5% lglutamicacid amended YPG broth, was tested in ammonium solution only. A range of pH values (4.09.0) of ammonium solutions prior to addition of C.

vulgaris NTM06 cells were adjusted with buffers solutions; pH 4.0 and 5.0 with the help of 10 mM sodium acetate buffer, pH 6.0 and 7.0 with the help of 10 mM phosphate buffer and pH 8.0 and 9.0 with the help of TrisHCl buffer.

2.7. Experimental design and statistical analysis

In case of ammonium solution or wastewater, samples with no C. vulgaris NTM06 were defined as positive control (PC) whereas control sample was treated with 0% l-glutamicacid treated C. vulgaris NTM06 cultures. All tests were carried out in triplicate. One-way analysis of variance (ANOVA) was used for analyzing data using SPSS (version 12.0) statistical software. Mean differences were compared using the least significant difference test (LSD) of the same software (SPSS). For graphical presentations the Microsoft Excel 2003 was used.

3. Results and discussion

Two independent experiments under similar controlled conditions were conducted. Results of both experiments were similar and therefore data of one experiment is presented here. Instead of natural wastewater, ammonium sulphate solution was used in all but the last test where natural wastewater was used. The idea behind this was to avoid possible interference from other inorganic nutrients and indigenous microorganisms and helped us to follow the disappearance of ammonium ions only. Five levels (0.101.5%) of l-glutamicacid were assessed for their effect on the growth rate of C. vulgaris NTM06 in YPG (defined in Methods, Section 2.1) and for choosing appropriate level(s) of l-glutamicacid for ammonium removal. For subsequent tests such as pH effect on the removal of ammonium by C. vulgaris NTM06, live and dead cells comparison, light and dark incubation comparison and ammonium removal from wastewater, Chlorella cultures treated with chosen level of l-glutamicacid (1.5%) were used only.

3.1. Effect of l-glutamicacid on the growth of C. vulgaris NTM06

Fig. 1 shows growth rate of C. vulgaris NTM06 in the YPG media amended with 5 levels (0.1%, 0.25%, 0.5%, 1.0% and 1.5%) of l-glutamicacid. Cells in subsamples were counted once in 24 h with the help of Haematocytometer. Until day 5 (120 h) of incubation, number of cells increased progressively across all treatments with no initial lag phase being observed which might be due to the use of pre-cultures of Chlorella for inoculation. Growth between day 5 and day 6 was less pronounced. Between treatments, zero% and low-levels (0.10.50%)

l-glutamicacid treatments showed higher growth of C. vulgaris NTM06. Whereas higher levels of l-glutamicacid (1.0% and 1.5%) appeared to have slowed down cell multiplications and on day 6 of incubation, compared to control, almost 32% less number of cells per ml were observed in high level l-glutamicacid (1.5%) treatment. This negative effect on the cells growth might be due to high level of acid (l-glutamicacid) in the growth media.

Full-size image (32K) Fig. 1. Effect of different levels of l-glutamicacid (0.11.5%) on the growth of C. vulgaris NTM06 in YPG broth (1% yeast, 2% peptone and 2% glucose) incubated at 37 C under the light (1000 lux) for 160 h.

3.2. Effect of l-glutamicacid levels on removal of ammonium from ammonium solution

The C. vulgaris NTM06 cultures amended with 5 different levels (0.11.5%) of lglutamicacid were used for assessing their effect on ammonium removal, however, for clarity reason and for better understating of graphical illustration, data of only 3 levels (0.1%, 1.0% and 1.5% l-glutamicacid) are presented here. Although reductions in ammonium concentrations were observed in all treatments and at all sampling occasions, differences between control and the first two levels of l-glutamicacid (0.1% and 1.0%) during the first two sampling occasions (2.5 and 6.5 h) were statistically non significant (Fig. 2). In contrast, the 1.5% l-glutamicacid treatment showed more pronounced reductions at all sampling occasions and compared to control and the two other levels of lglutamicacid, differences were always statistically significant (P < 0.05). For example reductions in case of control and the first two treatments were in the range of 2529% at 2.5 h of incubation whereas in case of 1.5% l-glutamicacid treatment it was 45%. Similarly, after 24 h of incubation, 1.5% l-glutamicacid treatment showed almost 99% reductions in ammonium as against 70% in case of the control treatment (Fig. 2).

Full-size image (26K) Fig. 2.

Effect of different levels of l-glutamicacid on removal of ammonium from ammonium solution (169.3 mg N/l) by C. vulgaris NTM06 incubated for 24 h at 37 C (PC; positive control-no C. vulgaris NTM06 cultures, YPG; control-cultures treated with no (0%) l-glutamicacid, YPG + 0.1%; cultures treated with 0.1%, YPG + 1.0%; cultures treated with 1.0% and YPG + 1.5%; cultures treated with 1.5% l-glutamicacid. Points at each sampling occasion associated with different letters differ significantly (<0.05, LSD).

In terms of number of cells per unit volume, this increased removal of ammonium by 1.5% l-glutamicacid treatment compared to control was in fact done by 19% less number of cells; 5.4 108 cells/ml in the 1.5% l-glutamicacid treatment and 6.6 108 cells/ml in the control. This would mean that per unit reduction of ammonium was 70% more in the 1.5% l-glutamicacid treated cells than in the cells with no l-glutamicacid. This highly significant removal of ammonium by 1.5% l-glutamicacid treated cultures might be due to increased level of enzymes in treated Chlorella cells capable of assimilating increased amount of ammonium. It has been reported that enzymes glutamine synthetase and glutamate synthase are known to be mainly responsible for ammonium assimilation in plant and bacteria ( [Kanamori et al., 1987] , [Glass et al., 2002] , [Miflin and Habash, 2002] and [Oaks, 1994] ) and treating of C. vulgaris NTM06 with l-glutamic in the present study might have increased activities of these enzymes (Ahmad and Hellebust, 1990). Although reports regarding response of enzymes to l-glutamicacid treatment of micro-algae are limited, Rigano et al. (1974) had, however, reported that Cyanidium caldarium grown on glutamate showed high nitrate reductase activity. It would have been worth assessing responses of enzymes activities particularly those involved in ammonium assimilation (e.g. glutamine synthetase and glutamate synthase) to l-glutamicacid treatment but because of the lack of time and resources availability it was not possible.

3.3. Live and dead C. vulgaris NTM06 cultures comparison

Adsorption of ammonium to cell surfaces or volatilization of ammonium could be some of the factors, which might have also contributed to the loss of ammonium from ammonium solution during the course of incubation but reduction of ammonium in case of dead C. vulgaris cells was around 11% at 2.5 h of incubation (Fig. 3) and unlike the live cells no further significant changes were observed at the other two sampling occasions (6.5 and 24 h). Whereas volatilization of ammonium can be discounted because the initial pH of the

solution was in the range of 6.06.2 and concentration of ammonium in case of positive control (PC) remained almost unchanged during the course of incubation.

Full-size image (18K) Fig. 3. Removal of ammonium from ammonium solution (169.3 mg N/l) by live and dead cells of C. vulgaris NTM06 treated with 1.5% l-glutamicacid and incubated for 24 h at 37 C (PC; positive control-no C. vulgaris NTM06). Points at each sampling occasion associated with different letters differ significantly (<0.05, LSD).

It is also evident from data of dead Chlorella cells that adsorption on to cell surfaces happened in the initial hours (2.5 h) of incubation only whereas live cells showed continuous uptake of ammonium. This suggests that live Chlorella cells were metabolically active through out the incubation time.

3.4. Light and dark incubation comparison

Fig. 4 shows comparison of ammonium removal by C. vulgaris NTM06 incubated under the light and in the dark. Differences in ammonium reduction between the light and dark incubations were significant (P < 0.001) at 2.5 h and 6.5 h with the highest values (28% and 45% for 2.5 and 6.5 h respectively) being associated with the light compared to the dark incubated samples (21% and 33% for 2.5 and 6.5 h respectively). However, measurements at 24 h showed almost similar values (99.6% and 98.0% for the light and dark incubated samples respectively).

Full-size image (21K) Fig. 4. Removal of ammonium from ammonium solution (169.3 mg N/l) by C. vulgaris NTM06 cultures treated with 1.5% l-glutamicacid and incubated under the light (1000 lux) and in the dark for 24 h at 37 C (PC; positive control-no C. vulgaris NTM06). Points at each sampling occasion associated with different letters differ significantly (<0.05, LSD).

Tischner and Httermann (1980) reported that exposure to light of C. sorokiniana showed significant increase in the activity of glutamine synthetase, an enzyme catalyzing the assimilation of ammonium. Our results of initial hours of incubation (2.5 and 6.5 h) support this finding. However, no significant differences between the light and dark incubations after 24 h suggest that reduction of ammonium in the dark might be due to adsorption on to cell surfaces only. But our findings of live and dead cells comparison study showed that only around 11% of ammonium could be adsorbed on to dead cells surfaces. In the absence of light (no photosynthesis) and with limited adsorption of ammonium (up to 11%) on to Chlorella cells surfaces, the disappearance of ammonium from acidic medium (pH 6.06.2, no volatilization) in the dark is hard to be explained on the basis of data generated in this study and further study for knowing the exact mechanism of ammonium uptake by Chlorella is recommended.

3.5. pH effect on removal of ammonium by C. vulgaris NTM06

Six pH levels of ammonium solution ranging from 4.0 to 9.0 were adjusted (prior to the addition of C. vulgaris NTM06 cultures) with the help of buffers. Details are mentioned in the methods section (Section 2.6). The pH treatments ranging from 4.0 to 8.0 showed a similar trend on all sampling occasions (Fig. 5). In contrast ammonium reductions in case of pH 9.0 during the first two sampling occasions (2.5 and 6.5 h) were significantly higher. For example at 2.5 h, pH 4.08.0 treatments showed reductions in the range of 1317%, whereas in the case of pH 9.0 reduction in ammonium for the same period was 41%. However, after 24 h of incubation this trend reversed with higher reductions (8890%) being associated with pH 4.08.0 treatments; pH 9.0 treatment showed 59% reduction at this point (Fig. 5).

Full-size image (25K) Fig. 5. Effect of different levels of pH (4.09.0) on the removal of ammonium from ammonium solution (169.3 mg N/l) by 1.5% l-glutamicacid treated C. vulgaris NTM06 incubated for 24 h at 37 C (PC; positive control-no C. vulgaris NTM06). Points at each sampling occasion associated with different letters differ significantly (<0.05, LSD).

Our data suggest that C. vulgaris NTM06 might remove similar amount of ammonium under a wide range of pH; both acidic and alkaline conditions (up to pH 8.0). Yan et al. (1996) also reported similar findings but for orthophosphate removal from artificial wastewater and C. vulgaris were immobilized in calcium alginate beads. In case of pH 9.0, although initial reduction in case of pH 9.0 was high, reduction after 24 h of incubation suggests that removal of ammonium by C. vulgaris NTM06 might be less in the highly alkaline media.

3.6. Total ammonium removal by C. vulgaris NTM06

A total of 4 cycles of 24 h each were employed and a new ammonium solution (N solution; 169.3 mg/l) was introduced to the same C. vulgaris NTM06 cultures at the end of each cycle. As expected, ammonium removal was almost 100% at the end of cycle one (data not shown). Subsequent cycles showed less progressive reductions and between cycle 3 and 4 ammonium reduction was almost negligible. A total of 229 mg N/l was removed over the course of 4 cycles.

3.7. Ammonium removal from wastewater

Compared to ammonium solution (169.3 mg N/l), concentration of ammonium in wastewater was very high (1550 mg N/l) and therefore along with concentrated wastewater, diluted forms of this wastewater sample (775, 310 and 155 N/l) were also included for assessing ammonium removal by C. vulgaris NTM06. This also gave us the opportunity to assess reduction across a wide range of ammonium concentrations. The concentrated wastewater with no C. vulgaris cultures was used as positive control. Unlike other tests incubation in this instance continued for 48 h. Fig. 6 shows reduction (%) of ammonium across all sampling occasions and for all treatments (the concentrated, and its diluted forms). At 2.5 h of incubation, ammonium reductions in case of all treatments were in the range of 16.6 21.4% and were statistically non significant. However, at the other three sampling points (6.5, 24 and 48 h), highly significant differences (<0.000) among all treatments were observed. The treatment with initial ammonium concentration of 775 mg N/l (2 times diluted wastewater) showed the highest % reductions, which were 35%, 73% and 88% for 6.5, 24 and 48 h incubations respectively. Percent reductions in case of concentrated (1550 mg N/l) and 310 mg N/l (5 times diluted wastewater) treatments were intermediate whereas the lowest values for the last two sampling points were associated with the 155 mg N/l (10 times diluted wastewater) treatment. Reductions in case of positive control (no C. vulgaris) during the same time period were in the range of 0.26.0% (Fig. 6).

Full-size image (24K) Fig. 6. Reduction (%) of ammonium in wastewaters treated with 1.5% l-glutamicacid treated C. vulgaris NTM06 and incubated for 48 h at 37 C (PC: positive controlno C. vulgaris NTM06, WW-1550: wastewater with 1550 mg N/l, WW-775: wastewater with 775 mg N/l, WW-310: wastewater with 310 mg N/l, WW-155: wastewater with 155 mg N/l. Points at each sampling occasion associated with different letters differ significantly (<0.05, LSD).

Although the % reduction of ammonium in case of 2 times diluted wastewater (775 mg treatment was highest, net removal of ammonium (mg/l) increased as the concentration of ammonium in wastewater increased. For example after 48 h of incubation, the highest net removal of ammonium (940 mg N/N/l) was observed in the highly concentrated wastewater (1550 mg N/l) and the lowest (85 mg N/l) being recorded in the low concentrated wastewater treatment (155 mg N/l). These findings suggest that the rate of ammonium removal by Chlorella might be determined by the initial concentration of ammonium in the medium. This can further be supported by our findings of total removal of ammonium from ammonium sulphate solution (169.3 mg N/l, Section 3.6, 4 cycles). Where total removal of ammonium over a period of 96 h by almost similar number of Chlorella cells was only 229 mg N/l; much less than in case of concentrated (1550 mg N/l) and its 2 times diluted (775 mg N/l) wastewater treatments. Similar results but for nitrate uptake by C. vulgaris were also reported by Jeanfils et al. (1993). de-Bashan et al. (2002) observed that after 48 h of incubation, C. vulgaris in association with plant growth promoting bacteria Azospirillum brasilense did not result in additional removal of ammonium ions. Our results of the highly concentrated wastewater treatment only support these findings (de-Bashan et al., 2002) as no reduction in ammonium was observed after 24 h of incubation (Fig. 6). In contrast, diluted wastewater treatments (less concentrated) showed further reductions. This suggests that the inherent capacity of C. vulgaris to absorb ammonium might reach saturation at some point and operators of biological wastewater treatment facilities need to be aware of it. It may be noted that although the % removal of ammonium from wastewater in the present study compared to previous studies was not 100% but the net

removal of ammonium (mg/l) was very high. Tam and Wong (2000) reported that C. vulgaris entrapped in calcium alginate beads (12 beads/ml) removed 100% of ammonium ions after 24 h of incubation. The initial concentration in their study was, however, only 30 mg N/l; much less than even the low wastewater treatment (155 mg N/l) of the present study. Results by other authors such as ( [de-Bashan et al., 2002] , [de-Bashan et al., 2004] and [Hernandez et al., 2006] ) also showed almost 100% removal of ammonium for the same specie but initial concentration of ammonium in wastewater treated were always lower than ours and cells were either co-immobilized with alginate beads or starved. Based on the findings of this study it can be concluded that treating C. vulgaris NTM06 with l-glutamicacid can increase removal of ammonium from wastewater and therefore further research is warranted in this regard. The feasibility of combining l-glutamicacid treatment of Chlorella species with other approaches such as co-immobilization with plant growth promoting bacteria (de-Bashan et al., 2004), starvation of Chlorella cells (Hernandez et al., 2006) and or the use of polymers ( [Canizares et al., 1994] and [Jimneze-Prez et al., 2004] ) for example is also recommended.

Acknowledegments
The authors thank the Japan Society for the Promotion of Science (JSPS) for the award of JSPS Postdoctoral Fellowship to the first author and the financial support termed Grants-in-Aid for this research.

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