TSINGHUA SCIENCE AND TECHNOLOGY ISSNll1007-0214ll04/16llpp391-396 Volume 15, Number 4, August 2010

Removal of Ammonia from Wastewater Effluent by Chlorella Vulgaris*

Jinsoo Kim1, Bala P. Lingaraju1, Rachael Rheaume2, Joo-Youp Lee1,**, Kaniz F. Siddiqui3
1. Department of Chemical and Materials Engineering, University of Cincinnati, Cincinnati, OH 45221, USA; 2. Department of Biomedical Engineering, University of Cincinnati, Cincinnati, OH 45221, USA; 3. The Metropolitan Sewer District of Greater Cincinnati, Cincinnati, OH 45204-2022, USA Abstract: The capability of Chlorella vulgaris to remove nitrogen in the form of ammonia and/or ammonium ions from wastewater effluent in a local wastewater treatment plant (i.e., the Mill Creek Plant in Cincinnati, Ohio, U.S.A.) was studied. The wastewater effluent leaving the plant was found to include high concentrations of nitrogen (7.70.19 mg/L) (ammonia (NH3) and/or ammonium ion (NH4+)) and total inorganic carbon (58.60.28 mg/L) at pH 7, and to be suitable for growing Chlorella vulgaris. When Chlorella vulgaris was cultivated in a batch mode under a closed system, half of the nitrogen concentration was dramatically removed in 48 h after a 24-h lag-phase period. Total inorganic carbon concentration also concomitantly decreased during the rapid growth-phase. The total biomass weight gained during the entire cultivation period balanced out well with the total amount of inorganic carbon and nitrogen removed from the culture medium. These results indicate that wastewater can be synergistically used to polish residual nutrients in wastewater as well as to cultivate microalgae for biofuel production. Key words: microalgae; Chlorella vulgaris; wastewater effluent; nitrogen removal

Introduction
Diminishing petroleum resources along with increased demand for petroleum by emerging economies are driving our society to search for new renewable and carbon neutral liquid transportation fuels. Biofuels such as bioethanol and biodiesel derived from agricultural and forestry residues and oil crops have been considered to be candidates for renewable liquid fuels. Recently, microalgae-derived biodiesel production has received great attention due to their ability to produce substantial amounts of triacylglycerols (e.g., 20%-50% dry cell weight)[1], as a feedstock for biodiesel[2,3], and
Received: 2010-05-19

* Supported in part by the Metropolitan Sewer District of Greater

Cincinnati (MSDGC) under Master Services Agreement (No. 85X10431), Task Order (No. 0210000209), Rachael Rheaume was supported by the Academic Year 2009-10 REU Program funded by the NSF Type 1 STEP Project (No. DUE-0756921)

** To whom correspondence should be addressed.

E-mail: joo.lee@uc.edu; Tel: 1-513-556-0018; Fax: 1-413-566-0018

to utilize a large fraction of solar energy (up to 10%)[4] and carbon dioxide (CO2), and to grow at much faster rates than terrestrial biomass[5]. In addition, sugars generated as metabolites have potential to produce bioalcohols[6-10]. Nitrogen and phosphorous discharged through agricultural, sewage, and industrial effluent are the major contributors to ecological eutrophication[11,12]. In general, treated wastewater still include nitrogen and phosphorous in the form of nitrate, nitrite, ammonia/ammonium, and phosphorus[12]. Microalgae require nitrogen, phosphorous, CO2, and light for their autotrophic growth. It is estimated that there are 210510105 different microalgal strains in nature, but only about 3104 strains have been described[13]. Among the strains, Chlorella vulgaris belongs to a genus of single-celled green algae, and is known as one of the fastest growing microalgae, and typically includes 14%-22% of lipid, 51%-58% of protein, 12%-17% of carbohydrates, and 4%-5% of nucleic acid[14].

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In previous studies, a variety of wastewaters from urban, industrial, or agricultural sources[15-17] were used to remove nitrogen and phosphorus by using Chlamydomonas reinhardtii[18], Botryococcus braunii[19], Chlorella pyrenoidosa[20], Haematococcus pluvialis[21], Spirulina maxima[22], Spirulina (Arthrospira)[23], Scenedesmus rubescens[24], Scenedesmus abundance[25], Scenedesmus quadricauda[25], Scenedesmus acuminatus, Scenedesmus obliquus[26-28], Scenededesmus dimorphus[29], and Haematococcus plurialis[21]. Chlorella vulgaris has been reported to be able to readily uptake nitrogen from ammonium ion and ammonia and phosphorus through the cell membrane from the wastewater[24,25,29-32]. It was also reported that algaederived biofuels could compensate for environmental burden using wastewater as a source for CO2 and fertilizer according to previous life cycle assessment (LCA) studies[33,34]. The Mill Creek plant located in Cincinnati, Ohio, U.S.A. is a major wastewater treatment facility where ~70% and 30% of the wastewater is typically collected from industrial and domestic sources, respectively. A sample collected from the wastewater effluent contains nitrogen at the concentration level of 7.70.19 mg/L in the form of ammonia (NH3) and/or ammonium (NH4+) with low concentrations (undetectable by ion chromatography) of nitrate, nitrite, and orthophosphate. In this study, the removal of nitrogen (i.e., ammonia (NH3)/ ammonium (NH4+)) by Chlorella vulgaris has been explored for potential wastewater treatment and biofuels production.

was found out to be 253 mg/L. 1.2 Light condition

Fluorescent lamps with 6500 K color temperature similar to natural sunlight were used as the source of light, and the incoming light intensity to beakers was set to 6000 lux (100.8 molm-2s-1) by controlling the distance between the beaker and lamp. The light intensity was measured using a light intensity meter (HQRP digital lux meter, LX1010BS, Osprey-Talon Company), and a 16-h light and 8-h dark cycle was applied to the culture. 1.3 Determination of cell density of Chlorella vulgaris

The cell density of Chlorella vulgaris was determined by measuring the optical density of a 15-mL sample at 682 nm[36] for every 24 h by using UV-vis spectrophotometer (Cary 50, Varian, Inc.). Here, the absorbance of UV spectrophotometer at 682 nm was calibrated by measuring the weight of dried Chlorella vulgaris. Then, the weight of dried biomass was obtained from the prepared calibration curve. 1.4 Determination of nitrate (NO3 ) and phosphate 3 (PO4 ) concentration

1
1.1

Materials and Methods

Culture media

The concentrations of nitrate and phosphate ions were determined with Dionex DX-500 ion chromatography system equipped with IonPac AS14 anion-exchange column. The flow rate of sodium carbonate/sodium bicarbonate effluent (1.8 mmol/L Na2CO3 and 1.7 mmol/L NaHCO3 in 1.0 L of deionized water) was 1.2 mL/min. 1.5 Determination of total inorganic carbon concentration

Wastewater effluent was collected from the Mill Creek wastewater plant in Cincinnati, OH, U.S.A., and was used as a culture medium. The plant monitors the concentrations of ammonia/ammonium, nitrite/nitrate, and total phosphate on a daily basis, and their annual average concentrations were 14.673.65 mg/L, 1.95 1.10 mg/L, and 0.900.31 mg/L, respectively, during 2008 and 2009 (Lachats QuikChem 8500 Series 2 Flow Injection Analysis System). A 30-mL sample of Chlorella vulgaris suspended in shuisheng-4 medium[35] was added to the wastewater effluent. The cell density of Chlorella vulgaris in the wastewater

An acid-base titration method[37] was used to determine the inorganic carbon species in the aqueous phase. This titration method determines total inorganic carbon 2 concentration (carbonate (CO3 ), bicarbonate ( HCO3 ), and aqueous carbon dioxide (CO2 (aq))) using a 0.01 mol/L hydrochloric acid solution as a titrant. 1.6 Determination of nitrogen concentration in the form of ammonia (NH3) and ammonium + ion (NH4 )

Nitrogen concentrations in the form of ammonia and

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ammonium ions present in the wastewater were measured using a commonly used ammonia probe (Model: 9512HPBNWP Orion Thermo Scientific)[38,39]. All ammonium ions were converted into ammonia by raising the pH of the sample solution of the culture medium (i.e., wastewater effluent) above 12 while monitoring its pH (Oakton pH 11 series pH meter), and the resultant ammonia concentration was determined by the ammonia probe. Then the concentration distribution of ammonia and ammonium ions was determined by the equilibrium at the original pH of the sample solution. The standard ammonia solution required for the calibration of the ammonia probe was prepared according to the EPA Method 350.3[40]. 10 mol/L NaOH (reagent grade, Fischer Scientific) was required for raising the pH of the solution above 12. Distilled and deionized water from a MilliQ water purification system was used for preparing the standard and NaOH solutions. The probe was re-calibrated every 5 samples according to the EPA Method 350.3[40] over a range of 1000 mg/L to 1 mg/L of NH4Cl solution. 25 mL of a sample was taken in a container with an equal surface to volume ratio as recommended by the EPA Method 350.3. Then the sample was stirred using a flat micro-magnetic stirrer placed on a magnetic stirrer base (Fischer Scientific magnetic stirrer base). The probe was immersed in the sample at an inclination angle of 45 degrees with continuous stirring. A strong base solution (10 mol/L NaOH) was added to reach a pH higher than 12. A reading was taken after the voltage signal in mV reached a constant value after a 1-min time interval. After each measurement, the probe was washed thoroughly using deionized water and wiped with a soft tissue. When it was not in use, the ammonia probe was stored in a 0.1 mol/L ammonium chloride solution. A 25-mL sample withdrawn from the batch culture at a 24-h interval was used to determine the nitrogen concentration. The sample was filtered out using a syringe filter (0.45 m nominal pore with 25 mm diameter, Whatman filter) in order to avoid potential blockage of the membrane of the ammonia probe. All the measurements were carried out in triplicate to ensure the validity of the data.

plant contained large amounts of total inorganic carbon (58.60.28 mg/L) and nitrogen (7.70.19 mg/L) in the + form of ammonia (NH3)/ammonium (NH4 ) with the small amounts of nitrate (NO3 ) and nitrite (NO2 ) (1.951.10 mg/L on annual average) and phosphate 3 (PO4 ) (0.900.31 mg/L on annual average). In addition, the initial pH of the wastewater effluent was almost constant at pH 7. Chlorella vulgaris can grow using carbon dioxide and light, known as the photosynthetic process: 6CO 2 + 12H 2 O + light (energy) C6 H12 O6 + 6O2 + 6H 2 O. Microalgae can utilize bi carbonate ions (HCO3 ) as a carbon source for photosynthesis with the help of an enzyme called carbonic anhydrases (CA) as well as carbon dioxide. In addition, inorganic nutrients, especially nitrogen and phosphate are required for their growth. The growth rate of Chlorella vulgaris and the uptake rate of total inorganic carbon are shown in Fig. 1. The lag-phase period for adaptation to the wastewater condition was found to be short (24 h), and total inorganic carbon was not reduced during the period. During the growth phase, the cell density of Chlorella vulgaris dramatically increased, and the total inorganic carbon concentration sharply decreased until 96 h, indicating active photosynthetic reaction. The pH of the wastewater continued to increase from the lag phase through the growth phase as shown in Fig. 2. Microalgae are known to produce hydroxyl ions (OH) when a bicarbonate ion is consumed for photosynthesis within the algal cell by following the reaction: HCO3 CO 2 +

OH [41-43]. The pH started to level off as the uptake rates of total inorganic carbon and nitrogen were discontinued after 144 h.

Results and Discussion

Fig. 1 Growth rate of Chlorella vulgaris and uptake rate of total inorganic carbon

The wastewater effluent collected from the Mill Creek

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Fig. 2 Total inorganic carbon uptake and pH increase

that the weight gained by biomass is almost comparable to the amount of inorganic carbon and nitrogen removed from wastewater. The experimental results indicated that Chlorella vulgaris has potential to remove nitrogen (i.e., ammonia and ammonium ion) at a reasonable uptake rate from wastewater while being cultivated using wastewater effluent. As a result of consuming bicarbonate ions, the pH of the culture medium increased. This can open up an opportunity to utilize wastewater in order to cultivate microalgae for the dual purpose of removing nutrients and producing biofuels.
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The removal rate of nitrogen (ammonia (NH3)/ammonium ion (NH4+)) is shown in Fig. 3. During the lag phase, the nitrogen removal efficiency varied little from 7.60.24 mg/L to 7.70.19 mg/L matched with no growth of the Chlorella vulgaris or uptake of total inorganic carbon. As with the uptake of the inorganic carbon, the nitrogen concentration did not decrease during the initial lag phase, but then sharply decreased from 7.60.24 mg/L to 3.40.17 mg/L at the same time as the decrease in total inorganic carbon concentration during the rapid growth-phase (i.e., 24 to 96 h). It is interesting to note that the total biomass weight gained during the entire cultivation period is well balanced by the total amount of inorganic carbon and nitrogen removed from the culture medium as clearly shown in Fig. 1.

Fig. 3 Removal of nitrogen in the form of NH3/NH4 from wastewater effluent by Chlorella vulgaris

Conclusions

In this study, Chlorella vulgaris was used to remove nitrogen in the form of ammonia and ammonium ions from the wastewater effluent collected from the Mill Creek plant in Cincinnati, OH, U.S.A. The mass balances taken for inorganic carbon and nitrogen show

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