Ammonia removal from anaerobic digestion effluent of livestock waste using green alga Scenedesmus sp.

Bioresource Technology 101 (2010) 8649–8657

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Ammonia removal from anaerobic digestion effluent of livestock waste using green alga Scenedesmus sp. Jongmin Park a, Hai-Feng Jin a, Byung-Ran Lim b, Ki-Young Park c, Kisay Lee a,* a

Dept. of Environmental Engineering and Biotechnology, Myongji University, Yongin 449-728, Republic of Korea Environmental Materials Education Center, Seoul National University of Technology, Seoul, Republic of Korea c Dept. of Civil and Environmental System Engineering, Konkuk University, Seoul, Republic of Korea b

a r t i c l e

i n f o

Article history: Received 3 April 2010 Received in revised form 24 June 2010 Accepted 26 June 2010

Keywords: Ammonia removal Microalgae Anaerobic digestion effluent Alkalinity Semi-continuous operation

a b s t r a c t The green alga Scenedesmus was investigated for its ability to remove nitrogen from anaerobic digestion effluent possessing high ammonium content and alkalinity in addition to its growth characteristics. Nitrate and ammonium were indistinguishable as a nitrogen source when the ammonium concentration was at normal cultivation levels. Ammonium up to 100 ppm NH4–N did not inhibit cell growth, but did decrease final cell density by up to 70% at a concentration of 200–500 ppm NH4–N. Inorganic carbon of alkalinity in the form of bicarbonate was consumed rapidly, in turn causing the attenuation of cell growth. Therefore, maintaining a certain level of inorganic carbon is necessary in order to prolong ammonia removal. A moderate degree of aeration was beneficial to ammonia removal, not only due to the stripping of ammonium to ammonia gas but also due to the stripping of oxygen, which is an inhibitor of regular photosynthesis. Magnesium is easily consumed compared to other metallic components and therefore requires periodic supplementation. Maintaining appropriate levels of alkalinity, Mg, aeration along with optimal an initial NH4+/cell ratio were all necessary for long-term semi-continuous ammonium removal and cell growth. Ó 2010 Elsevier Ltd. All rights reserved.

1. Introduction Livestock wastewater contains a high concentration of nitrogen that causes eutrophication when discharged without proper treatment. Livestock waste is often subjected to anaerobic digestion using mesophilic or thermophilic bacteria. The main result of anaerobic digestion is the reduction of organic matter and waste volume through the fermentative degradation of organic constituents. Even so, the levels of nutrients such as ammonia are not completely reduced during anaerobic digestion because the microorganisms employed generally lack sufficient autotrophic metabolism of inorganic nitrogen (Cheng and Liu, 2002; Noike et al., 2004; Ulundag-Demirer et al., 2008). Traditional bacterial nitrification–denitrification can be used to remove ammonia, but it requires the assimilation of extra organic carbon as a carbon source. Another important feature of anaerobic digestion is the 2 high concentration of alkalinity involved (mostly HCO 3 or CO3 , 1000–5000 ppm as CaCO3). High alkalinity also makes it difficult to apply advanced oxidation processes using ozone or peroxides because bicarbonate acts as a radical scavenger (Ma and Graham, 2000; Currie et al., 2003). * Corresponding author. Tel.: +82 31 330 6689; fax: +82 31 336 6336. E-mail address: [email protected] (K. Lee). 0960-8524/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2010.06.142

Many microalgae species such as green algae and cyanobacteria can assimilate nitrogen and phosphate into their biomass as well as inorganic carbon for photosynthesis (Yoshihara et al., 1996; Nagase et al., 2001; Jin et al., 2005). A microalgae system can be employed as an alternative secondary or post-secondary treatment process to remove nutrients from wastewater, especially when a substantial amount of nitrogen remains following the reduction of organic matter by traditional biological methods (Przytocka-Jusiak et al., 1984; Berman and Chava, 1999; Olguin, 2003). Microalgae have been the subject of recent interest due to their ability to increase growth by uptaking various forms of inorganic   nitrogen, including NHþ 4 ; NO3 ; NO2 , or NO etc. (Nolte and Prouix, 1988; Tam and Wong, 1990; Hu et al., 2000; Olguin, 2003; Jin et al., 2008; Park et al., 2009). Microalgae also produce potentially valuable biomass, which can be used as an animal feed additive and biofuel feedstock for biodiesel and hydrogen (Melis and Happe, 2001; Travieso et al., 2006; Tran et al., 2009). It has been recently suggested that a microalgae system can be utilized for the treatment of flue gas through simultaneous CO2 and NOx fixation, followed by the conversion of harvested algal biomass to biofuel (Chisti, 2007; Jin et al., 2005). This study attempted to investigate the characteristics of microalgal growth along with the removal of ammonia from anaerobic digestion effluent containing high levels of ammonium and alkalinity using the green alga Scenedesmus. The effects of ammonium

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2.3. Wastewater

content, alkalinity and aeration on cell growth and ammonia removal are discussed, and a strategy for long-term semi-continuous operation is introduced.

The wastewater used in this study was composed of an anaerobic digestion effluent obtained from a local piggery farm in Hwasung, Korea. In order to prevent interference from other microorganisms, wastewater was filtered through a GF/C filter and then autoclaved. The composition of wastewater before and after this pretreatment is shown in Table 1. After filtration and autoclave, a fraction of SS, COD, TOC and TP was reduced. Meanwhile, SCOD, NH4-N, TN and alkalinity were only slightly changed. Since this study focused ammonia removal and the autotrophic microalgal growth was influenced by inorganic carbon (alkalinity) and nitrogen source (NH4-N or TN), not by organic carbon (COD, SCOD or TOC), it was considered that the obtained results would be applicable to the raw wastewater used in this study. Ammonia is subject to easily evaporate during autoclave if free ammonium is dominant in water body like domestic wastewater. However, the ammonium content is almost invariant in Table 1, because ammonium ions exist in salt forms stably with bicarbonate or carbonate due to high alkalinity in the wastewater used in this study. Cells were initially cultured in Bristol medium, but wastewater was added to the culture suspension when the nitrogen concentration was decreased nearly to zero and the cell density reached a desired value (such as 1.0 g/L). Wastewater was diluted 1/10 upon addition to the culture, making the nitrogen concentration approximately 100 mg/L. Nitrogen removal was performed in batch mode or semi-continuous mode. In semi-continuous mode, new wastewater was added to the culture to increase the nitrogen concentration that had dropped to below 10 ppm back to around 100 ppm. In order to investigate the influence of metallic components on cell growth, boron, iron, manganese, zinc, copper and magnesium were introduced one by one to the ongoing culture when the cell growth rate was markedly attenuated.

2. Methods 2.1. Microalga strain The green alga Scenedesmus accuminatus (KCTC AG10316) was obtained from the Biological Resources Center (BRC) of the Korea Research Institute of Bioscience and Biotechnology (KRIBB). Cells were grown at pH 7.5 in modified Bristol medium (NaNO3, 250 mg/L; K2HPO4, 75 mg/L; KH2PO4, 175 mg/L; CaCl2, 25 mg/L; NaCl, 25 mg/L; H3BO3, 0.2 mg/L; MgSO47H2O, 75 mg/L; FeCl3, 0.3 mg/L; MnSO44H2O, 0.3 mg/L; ZnSO47H2O, 0.2 mg/L; CuSO45H2O, 0.06 mg/L). 250 mg/L of NaNO3 as a nitrogen source is equivalent to 41 mg-N/L. In order to investigate the effect of ammonium, 157 mg/L of NH4Cl (41 mg-N/L) was used instead.

2.2. Cultivation Scenedesmus cells were cultivated in a cylindrical glass reactor with a 1-L working volume. External illuminations were made by fluorescent lamps so that the light intensity at the center of the medium-filled reactor was around 200 lmol/m2/s (Li-250A, Li-COR Lightmeter) with a 12 h:12 h of light:dark cycle.

Table 1 Composition of anaerobic digestion effluent wastewater.

In raw wastewater

After pretreatment

8.5 1040 1980 1010 370 1200 1240 140 5990

8.4 negligible 1042 1035 195 1196 1220 75 5562

2.4. Analyses The algal cell density was expressed as dry cell weight (DCW) per liter of culture suspension. The DCW was evaluated by drying cells at 85 oC for 24 h after filtration through a GF/C filter. Other analyses were performed basically according to standard methods (APHA, 1995). HACH DR4000U was used in determination of CODCr, SCODCr, TN and TP. Shimadzu 5000A was used for total

1200

50

1000

40

800 30 600 Cell / NO3-

20

Cell / NH4+

400

Nitrogen (mg/L)

pH SS CODCr SCODCr TOC NH4-N TN TP Alkalinity

Concentration (mg/L)

Dry cell weight (mg/L)

Item

NO3-N NH4-N

10

200

0

0 0

5

10

15

20

Time (day) Fig. 1. Cell growth and nitrogen consumption in the presence of nitrate or ammonium as a nitrogen source.

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normal cultivation levels, resulting in comparable cell growth rates (Fig. 1).

organic carbon (TOC) and inorganic carbon (IC). For NH4-N, HACH DR4000U and Orion electrode were utilized. The Brucine method was used to determine NO3-N. Alkalinity was measured through titrimetric method. Light intensity was analyses using Li-250A light meter (Li-COR).

3.2. Growth inhibition by ammonium Fig. 2 shows the changes in cell density at various initial ammonium concentrations ranging from 100 to 1000 ppm NH4-N. The culture with 100 ppm NH4-N resulted in the best cell growth, which was slightly better than the culture containing 100 ppm NO3-N. The initial growth rates of the culture containing 200– 500 ppm NH4-N were similar to those of 100 ppm NH4-N but leveled off after 7–8 days to a final cell density about 70% as large. This implied that the levels of inhibition were similar from 200 to 500 ppm. The inhibition of cell growth caused by ammonium became severe when the NH4-N content reached 800 ppm, with only 35% of the final cell density attained in the presence of 1000 ppm NH4-N. It is known that microalgae cells are inhibited at high ammonia concentrations, although at low concentrations they can still uptake ammonia (Przytocka-Jusiak, 1976; Azov and Goldman, 1982; Tam and Wong, 1996). Scenedesmus cells used in the current study grew without any sign of inhibition or toxic influence at 100 ppm NH4-N. The final cell mass was reduced by 30% for 200 to 500 ppm NH4-N, although the initial cell growth rate was not affected.

3. Results and discussion 3.1. Preference of nitrogen sources In order to examine which nitrogen source the Scenedesmus culture prefers, cell growth and nitrogen consumption were compared in the presence of 45 mg/L NO3-N or NH4-N. Bristol medium contained either nitrate or ammonium supplied in the form of NaNO3 or NH4Cl, respectively. Fig. 1 shows that the nitrogen concentration profiles were similar regardless of whether nitrate or ammonium was used. This result implies that Scenedesmus cells do not differentiate between nitrate and ammonium as a nitrogen source, and can be utilized for nitrogen removal in the treatment of ammonia-rich wastewater. The uptake of ammonium and nitrate is important in microalgal nitrogen removal because nitrogen often exists as ammonium in wastewater (Table 1), especially for livestock wastewater and anaerobically digested wastewater. In fact, the rate of ammonium uptake is usually inferior to that of nitrate for many microalgae species due to the toxicity of ammonia (Przytocka-Jusiak, 1976; Azov and Goldman, 1982; Tam and Wong, 1996). In addition to nitrate, the Scenedesmus species used in this study was capable of utilizing ammonium when the ammonium concentration was at

3.3. Effect of seeding cell concentration Fig. 3 shows the changes in ammonium content and cell density at different seeding concentration of Scenedesmus at the beginning of cultivation. This set of experiments was performed using diluted real piggery wastewater containing 120 ppm NH4-N. The seeding cell concentration varied from 0.5 g/L to 1.5 g/L DCW. The quantitative values of growth rate and ammonium removal rate were summarized in Table 2. The net cell growth rate for 10 days of cultivation was 45.8 mg/L/d at 0.5 g/L seeding and was increased up to 55.6 mg/L/d at 1.5 g/L seeding (Fig. 3a). In terms of specific growth rate (Fig. 3b), cells grew relatively rapidly and showed the highest specific growth rate at the low seeding concentration of 0.5 g/L. The specific growth rate became smaller as the seeding concentration was increased. Variation in seeding concentration influenced ammonia removal during cell cultivation (Fig. 3c and Table 2). Although the

Table 2 Cell growth rate and ammonium removal rate depending upon seeding cell concentrations in Fig. 3. Seeding cell density (g/L)

Net growth rate (mg/L/day)

Specific growth rate (day1)

Ammonium removal rate (mg/L/day)

0.5 0.75 1.0 1.25 1.5

45.8 48.0 49.4 53.8 56.6

0.091 0.064 0.049 0.043 0.038

5.20 5.87 6.14 6.21 6.46

*Specific growth rate is based upon initial seeding cell concentration.

1000 w/o NH4-N 100 ppm 200 ppm 400 ppm 500 ppm 800 ppm 1000 ppm

DCW (mg/L)

800

600

400

200

0 0

2

4

6

8

10

12

14

Time (day) Fig. 2. Influence of ammonium concentration on the growth of Scenedesmus.

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a

2.5

Cell density (g/L)

2.0

1.5

1.0 0.5 g/L 0.75 g/L 1.0 g/L 1.25 g/L 1.5 g/L

0.5

0.0 0

2

4

6

8

10

12

Cultivation time (day)

b

2.4 0.5 g/L 0.75 g/L 1.0 g/L 1.25 g/L 1.5 g/L

Specific cell density (C/C0)

2.2 2.0 1.8 1.6 1.4 1.2 1.0 0.8 0

2

4

6

8

10

12

Cultivation Time (day)

c

1.1 0.5 g/L 0.75 g/L 1.0 g/L 1.25 g/L 1.5 g/L

1.0

NH4-N (C/C0 )

0.9

0.8

0.7

0.6

0.5

0.4 0

2

4

6

8

10

12

Cultivation Time (day) Fig. 3. Influence of seeding cell concentration on (a) net cell growth, (b) specific cell growth and (c) ammonium removal.

culture with the lowest seeding concentration, 0.5 g/L, resulted in the highest ammonia removal rate temporarily during the first

4 days of cultivation, its removal rate was lowered as cultivation time passed and so the overall removal rate became the lowest.

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The ammonium removal rate in cultures of larger seeding size was steadily maintained during 10 days.

a

Table 2 and Fig. 3 showed that the ammonium removal rate had a parallel relationship with the net growth rate. The overall

100 No addition Bicarbonate Carbonate

Inorganic carbon (mg/L)

80

60

40

20

0 0

2

4

6

8

10

Time (day)

b

1.6

Cell density (C/C0)

1.5

1.4

1.3

1.2

1.1 No addition Bicarbonate Carbonate

1.0

0.9 0

2

4

6

8

10

6

8

10

Time (day)

c

1.1

1.0

NH4-N (C/C0)

0.9

0.8

0.7 No addition Bicarbonate Carbonate

0.6

0.5 0

2

4

Time (day) Fig. 4. The intermittent addition of inorganic carbon of alkalinity to Scenedesmus culture. (a) Inorganic carbon, (b) cell growth and (c) NH4-N.

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ammonium removal rate was increased as the seeding concentration was increasing; from 5.20 mg/L/d at 0.5 g/L seeding to 6.46 mg/L/d at 1.5 g/L seeding. This observation indicated that the ammonium removal by the present Scenedesmus species followed the substrate utilization kinetics in a batch reactor where no additional substrate (ammonium as nitrogen source here) is supplied and the product is the increment of biomass itself. Because the final ammonium content reached a similar level after 10 days when the seeding concentration ranged from 1.0 to 1.5 g/L, the 1.0 g/L seeding condition was routinely used thereafter for the long-term removal of nitrogen from wastewater. 3.4. Source of alkalinity Autotrophic algal cells require inorganic carbon for growth; therefore CO2 gas is usually used in many microalgal photobioreactors (Keffer and Kleinheinz, 2002; Yamasaki, 2003). Since no CO2 was supplied to the culture in this study, algal cells were forced to utilize dissolved inorganic carbons in the wastewater. Anaerobically digested wastewater normally contains high level of alkalinity (Hill and Bolte, 2000; Bjornsson et al., 2001) of which major

a

constituent is bicarbonate HCO 3 ion. The wastewater used in this study was a digestion effluent containing 5900 – 7500 ppm of alkalinity as CaCO3 (Table 1). The cultivation started with a concentration of 1.0 g of cells per L of diluted wastewater, where inorganic carbon ranged from 80 to 90 mg/L as C (equivalent to 650– 750 mg/L of alkalinity as CaCO3). Fig. 4 shows the changes in inorganic carbon (alkalinity), cell density and ammonium concentration during cultivation in the presence of 120 ppm NH4-N. The inorganic carbon, mostly bicarbonate, was consumed rapidly to a level near zero within 2 days (Fig. 4a). Along with the exhaustion of inorganic carbon, the cell growth was attenuated, resulting in an early approach to stationary phase (Fig. 4b). Ammonia removal was also decreased and thus only 13% was removed during 7 days (Fig. 4c). In order to examine the influence of alkalinity, NaHCO3 or Na2CO3 was supplied intermittently as an external source of inorganic carbon (IC) when the level dropped below 5 mg/L on day 2. The level of IC was increased to 85 ppm upon the addition of bicarbonate on day 2, but this was followed by another drop in IC to around 10 mg/L after day 5.5. Fig. 4b shows that the growth pattern until day 3 was similar to that without bicarbonate. However,

1.2 without aeration with aeration

1.0

NH4-N (C/C0)

0.8

0.6

0.4

0.2

0.0 0

2

4

6

8

10

Time (day)

b

1.6

Cell density (C/C0)

1.5

1.4

1.3

1.2

1.1 without aeration with aeration

1.0

0.9 0

2

4

6

8

Time (day) Fig. 5. Influence of aeration on ammonia removal and cell growth.

10

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from day 3 the cell density was steadily increased without attenuation while the ammonium removal rate was maintained highly as the period of 0 – 2 days. The addition of carbonate affected differently compared to bicarbonate addition. Growth rate was not improved by carbonate addition, showing similar growth with no-addition case (Fig. 4b). Ammonium removal rate was a little recovered compared to noaddition case, but its extent was much smaller than that of bicarbonate addition (Fig. 4c). The reason why carbonate addition did not improve growth rate is not clear yet and more investigation is required; however, it is suspected that carbonate addition raised the culture pH up to 10, which did not favor normal cell growth and carbon uptake. The pH value of the culture decreased to 7.5 from the initial value of pH 8.6 upon depletion of inorganic carbon (day 2) and then rose to 8.5 by adding bicarbonate, which means that pH was maintained in the range of 7.5–8.6 during ammonia removal. Meanwhile, in the case carbonate addition, pH increased up to 10.3 which was beyond the range for normal growth although microalgal cells prefer weak alkaline pH for their growth. After carbonate addition (day 2), the carbon uptake rate was actually reduced to nearly half of the bicarbonate addition case (Fig. 4a). The high pH also favors ammonia stripping by shifting

a

equilibrium from ammonium NHþ 4 to ammonia (NH3), which could be a reason that the extent of ammonia removal was much greater than that of cell growth in the case of carbonate addition. These results demonstrate that inorganic carbon in the form of bicarbonate is actively consumed during algal cell growth and ammonium removal, and therefore that maintaining certain level of inorganic carbon is necessary to prolong ammonia removal. The addition of bicarbonate had more positive effects on cultivation than did the addition of carbonate. The bicarbonate ion, which increases the alkalinity for anaerobic digestion, was successfully utilized as a carbon source by autotrophic microalgae grown in ammonium-rich wastewater. Even though the addition of bicarbonate promoted the continuous growth of cells, the growth rate during the second period (days 2 – 5) looked somewhat inferior to the initial activity (days 0 – 2). Firstly, the declining level of inorganic carbon (Fig. 4a) during days 2 – 5 was lower than the rate obtained during days 0 – 2. The cell growth rate during days 2 – 5 was also smaller than that during days 0 – 2, even though the amount of added bicarbonate was identical to the initial level. This difference is perhaps due to the depletion of other nutritional components such as trace metals during the second period (days 2 – 5).

2.6

(I)

2.4

(II)

(III)

(IV)

(V)

Cell density (g/L)

2.2 2.0 1.8 1.6 1.4 B F N Z C M

1.2

Mg

1.0

Mg 0.8 0

5

10

15

20

25

20

25

30

Time (day)

b

120

100

NH4-N (ppm)

80

60

40

20

0 0

5

10

15

30

Time (day) Fig. 6. Profiles of cell density and ammonium concentration in semi-continuous treatment of wastewater using Scenedesmus cells. B = H3BO3; F = FeCl3; N = MnSO4; Z = ZnSO4; C = CuSO4; M = MgSO4.

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3.5. Influence of aeration In order to determine the influence of aeration on ammonium removal and cell growth, sterilized air was supplied at a rate of 0.75 vvm (volume to volume per minute). The algal culture was controlled at pH 7.5. As seen in Fig. 5, aeration provided significant benefits to ammonium removal while simultaneously increasing algal biomass; Ammonium removal rate was 6.3 mg/L/day without aeration and 18.4 mg/L/day with aeration. Net cell growth rate was 66.8 mg/L/day without aeration and enhanced to 118 mg/L/day with aeration. This increase in NH4-N removal rate is probably due to the stripping effect promoted by aeration. It is well known that aqueous ammonia can be stripped away from water by aeration, a process highly favored in alkaline pH above 9.5. Nevertheless, ammonia stripping could not be the only cause of ammonium reduction in this study. Besides ammonia, dissolved oxygen can be stripped away from water, an important notion in treating wastewater with photosynthetic microalgae. Molecular oxygen normally evolves as a consequence of photosynthesis while dissolved oxygen inhibits regular photosynthesis and carbon fixation (Merrett and Armitage, 1982; Markez et al., 1995). The concentration of dissolved oxygen becomes higher in un-aerated algal culture compared to the aerated one, creating an unfavorable situation for algal cell growth and algal ammonia uptake. This speculation is supported by the comparison of dissolved oxygen (DO) values and the enhanced cell density produced in aerated culture (Fig. 5b). The rate of oxygen evolution by microalgal photosynthesis and the resulting DO increase in the culture is very fast, and so it is known that the DO value can exceed the ambient saturation value (Rubio et al., 1999; Miron et al., 2002). According to separated experiments, the DO value in the culture was 0.76 mg/L initially and it was maintained without change if light was not supplied. However, once light illuminated, DO increased so rapidly that it exceeded 6.2 mg/L within 18 min and 10 mg/L within 35 min. The value 6.2 mg/L was the saturated DO value that was obtained by continuous bubbling with air at ambient condition through the culture containing Scenedesmus cells. DO value could rise even higher than 2.5-folds of saturation value after 60 min if inorganic carbon was sufficient. Meanwhile, DO value was kept constant around 6.2 mg/L when aeration was carried out. Therefore, the use of appropriate aeration was beneficial to enhance microalgal ammonium uptake by preventing DO level from going up as well as to strip ammonia out of the wastewater. Aeration using inert gases like nitrogen or argon, instead of cheap air, would achieve further lowered DO value. 3.6. Semi-continuous treatment Semi-continuous type ammonia removal was performed with repeated cell withdrawal and wastewater supplement. When the ammonium level was dropped to below 10 ppm, a fraction approximately 1/10 of the culture broth and cells was withdrawn. New wastewater was then supplied so the culture could be adjusted to 90–100 ppm NHþ 4 , 90 ppm alkalinity and 1 g/L of cell mass (Fig. 6). Initially, cells harvested from a stock culture were seeded to a concentration of 1 g/L. The first cycle lasted 5 days during which the cell mass more than doubled and 89% of ammonium was successfully removed. In the second cycle, however, the cell growth rate was decreased to half and the extent of ammonium removal was also reduced. Therefore, fresh wastewater was supplied at day 11, but no sign of performance recovery was observed. Following this, metallic components (B, Fe, Mn, Zn, Cu or Mg) were added individually with 1-day intervals to the culture medium. Fig. 6 shows that none of the elements supplied to the culture produced

any positive effect, except for Mg which increased the cell density. Therefore, Mg was supplied repeatedly at the beginning of the 4th and 5th cycles and caused normal cell growth and ammonium removal just like that observed in the 1st cycle. The averaged values of cell growth rate and ammonia removal rate in the 1st, 4th and 5th cycles were 213 and 19.2 mg/L/day, respectively. It is speculated that the other metallic elements tested besides Mg were not limiting because they were supplied in sufficient amounts when new wastewater was added. Mg is an important component of photosynthetic pigments in many algal species (Finkel and Appleman, 1963; Walker, 1994). Therefore, it can be concluded that the addition of Mg along with the maintenance of alkalinity and optimal initial NHþ 4 /cell ratio are critical for stable long-term ammonia removal during semi-continuous treatment using microalgae.

4. Conclusions The bicarbonate ion, which is constituent of the alkalinity for anaerobic digestion, was actively consumed as a carbon source by autotrophic microalgae grown in ammonium-rich wastewater. Therefore, maintaining a certain level of inorganic carbon is necessary to prolong ammonia removal. In a long-term semi-continuous process, Mg is easily consumed compared to other metallic components and thus requires periodic supplementation. The concerted maintenance of alkalinity, Mg levels, optimal initial NHþ 4 /cell ratio and moderate aeration is required in order to achieve stable longterm ammonia removal in a semi-continuous process using microalgae. Acknowledgements This study was financially supported by Korea Research Foundation Grant funded by the Korean Government (MOEHRD), KRF2006-D00131. Jongmin Park is also thankful to scholarships from the BK21 Program of the Ministry of Education, Korea. References APHA, 1995. Standard Methods for the Examination of Water and Wastewater, 19th ed., APHA, Washington, DC. Azov, Y., Goldman, J.C., 1982. Free ammonia inhibition of algal photosynthesis in intensive cultures. Applied and Environmental Microbiology 43, 735–739. Berman, T., Chava, S., 1999. Algal growth on organic compounds as nitrogen sources. Journal of Plankton Research 21, 1423–1437. Bjornsson, L., Murto, M., Jantsch, T.G., Mattiasson, B., 2001. Evaluation of new methods for the monitoring of alkalinity, dissolved hydrogen and the microbial community in anaerobic digestion. Water Research 35, 2833–2840. Cheng, J., Liu, B., 2002. Swine wastewater treatment in anaerobic digesters with floating medium. Transactions of the American Society of Agriculture and Biological Engineers 45, 799–805. Chisti, Y., 2007. Biodiesel from microalgae. Biotechnology Advances 25, 294–306. Currie, M., Graham, N., Hall, T., Lambert, S., 2003. The effect of bicarbonate on ozone-enhanced particle removal in water treatment. Ozone Science and Engineering 25, 285–293. Finkel, B.J., Appleman, D., 1963. The effect of magnesium concentration on growth of Chlorella. Plant Physiology 28, 664–673. Hill, D.T., Bolte, J.P., 2000. Methane production from low solid concentration liquid swine waste using conventional anaerobic fermentation. Bioresource Technology 74, 241–247. Hu, Q., Westerhoff, P., Vermaas, W., 2000. Removal of nitrate from groundwater by cyanobacteria: quantitative assessment of factors influencing nitrate uptake. Applied and Environmental Microbiology 66, 133–139. Jin, H.F., Santiago, D., Park, J., Lee, K., 2008. Enhancement of nitric oxide solubility using Fe(II)EDTA and its removal by green algae Scenedesmus sp. Biotechnology and Bioprocess Engineering 13, 48–52. Jin, Y., Veiga, M.C., Kennes, C., 2005. Bioprocesses for the removal of nitrogen oxides from polluted air. Journal of Chemical Technology and Biotechnology 80, 483– 494. Keffer, J.E., Kleinheinz, G.T., 2002. Use of Chlorella vulgaris for CO2 mitigation in a photobioreactor. Journal of Industrial Microbiology and Biotechnology 29, 275– 280.

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