Bioresource Technology 114 (2012) 26–32

Contents lists available at SciVerse ScienceDirect

Bioresource Technology

journal homepage: www.elsevier .com/locate /bior tech

Immobilization of Chlorella sorokiniana GXNN 01 in alginate for removalof N and P from synthetic wastewater

Kai Liu a, Jian Li a, Hongjin Qiao b,c, Apeng Lin b, Guangce Wang b,⇑a Key Laboratory of Marine Resources and Chemistry, College of Marine Science and Engineering, Tianjin University of Science and Technology, Tianjin 300457, Chinab Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, Nanhai Road 7, Qingdao 266071, Chinac The Graduate School, Chinese Academy of Sciences, Beijing 100049, China

a r t i c l e i n f o

Article history:Received 1 September 2011Received in revised form 31 January 2012Accepted 1 February 2012Available online 16 February 2012

Keywords:ChlorellaImmobilizationPhotosynthesisWastewater treatment

0960-8524/$ - see front matter � 2012 Elsevier Ltd. Ahttp://dx.doi.org/10.1016/j.biortech.2012.02.003

⇑ Corresponding author. Tel.: +86 532 82898574; faE-mail address: gcwang@qdio.ac.cn (G. Wang).

a b s t r a c t

High costs and issues such as a high cell concentrations in effluents are encountered when utilizing mic-roalgae for wastewater treatment. The present study analyzed nitrogen and phosphate removal underautotrophic, heterotrophic, mixotrophic and micro-aerobic conditions by Chlorella sorokiniana GXNN01 immobilized in calcium alginate. The immobilized cells grew as well as free-living cells undermicro-aerobic conditions and better than free-living cells under the other conditions. The immobilizedcells had a higher ammonium removal rate (21.84%, 43.59% and 41.46%) than free living cells (14.35%,38.57% and 40.59%) under autotrophic, heterotrophic, and micro-aerobic conditions, and higher phos-phate removal rate (87.49%, 88.65% and 84.84%) than free living cells (20.21%, 42.27% and 53.52%) underheterotrophic, mixotrophic and micro-aerobic conditions, respectively. The data indicate that immobi-lized Chlorella sorokiniana GXNN 01 is a suitable species for use in wastewater treatment.

� 2012 Elsevier Ltd. All rights reserved.

1. Introduction

Chlorella spp. are a unicellular green microalgae that have a highphotosynthetic efficiency (Zelitch, 1971) and absorb nutrients fromculture media during rapid growth periods (Qiao et al., 2009). SinceChlorella spp. also have a higher capacity than other algae forwastewater nutrient removal, they are regarded as potentiallyuseful as wastewater treatment agents (Gonzalez et al., 1997;Hammouda et al., 1995).

Wastewater treatment by microalgae to remove nitrogen andphosphorus has been studied for almost 50 years (Oswald et al.,1957; Shi et al., 2007), but the presence of free-living microalgaein treated effluents remains a problem (Chevalier and de la Noue,1985). Recent research has therefore focused on the use of immo-bilized microalgae (Jimenez-Perez et al., 2004; Tam and Wong,2000). Alginate has become the most commonly-used polymer inwhich microalgae are entrapped, because of its high diffusivity,low production hazards, low polymer costs, and a simple and fastimmobilization process (Bashan, 1986; De-Bashan and Bashan,2010).

Not all microalgae can thrive in a matrix (Moreno-Garrido et al.,2005; Moreno-Garrido, 2008) possibly because of diminished lightpenetration and nutrient diffusion or chemical interactions thatcause severe stress (De-Bashan and Bashan, 2010). Some

ll rights reserved.

x: +86 532 82880645.

microalgae can damage alginate beads (Moreno-Garrido, 2008).Therefore, it is important to find suitable microalgae species thatcan survive and grow in alginate beads under relatively low illumi-nation and in micro-aerobic or anaerobic conditions.

Chlorella sorokiniana GXNN 01, isolated from a wastewatertreatment pond sample, grows under autotrophic (AA) conditionsand has the capability to utilize multiple carbon sources duringrapid growth conditions (Qiao et al., 2009). Of particular interestis its ability to assimilate carbon sources under micro-aerobic(MNA) conditions (Qiao et al., 2009). Such conditions are morecost-effective since little or no aeration is required during treat-ment. The present study investigated the feasibility of using C.sorokiniana GXNN 01 in immobilized form for removal of nitrogenand phosphate from a synthetic wastewater under autotrophic(AA), heterotrophic (HA), mixotrophic (MA) and micro-aerobic(MNA) (supplemented with acetate) conditions. The study alsocompared the growth rate and photosynthetic efficiency of free-living cells.

2. Methods

2.1. Microalgae cultivation and immobilization

C. sorokiniana GXNN 01 (Qiao et al., 2009) was maintained inTAP medium (Harris, 1989) without acetate, and provided with amean light intensity of 40 lmol m–2 s–1 under a 12 h:12 hlight:dark (L:D) cycle at 20 ± 2 �C. The medium was changed every2 months.

K. Liu et al. / Bioresource Technology 114 (2012) 26–32 27

Immobilization was carried out as described by Tam and Wong(2000). The algae were cultured in TAP medium (Harris, 1989)without acetate. In the logarithmic growth phase, the algal cellswere harvested by centrifugation at 4468g for 10 min, the cellswere washed three times with deionized water and resuspendedin synthetic wastewater at a concentration of approximately108 cell ml�1. Synthetic wastewater medium was prepared as de-scribed by Perez-Garcia et al. (2010) with some modification andcontained the following (mg l�1): NaCl, 7; CaCl2, 4; MgSO4�7H2O,2; K2HPO4, 21.7; KH2PO4, 8.5; Na2HPO4, 25; NH4Cl, 191; H3BO3,0.57; MnCl2�4H2O, 0.25; ZnSO4�7H2O, 1.1; FeSO4�7H2O, 0.25;CoCl2�6H2O, 0.08; Na2MoO4�2H2O, 0.015, and Na2EDTA, 2.5 at pH6.7. HA, MA and MNA growth media were supplemented withsodium acetate at a concentration of 0.1% (w/v). The ammoniumand phosphate levels of this artificial wastewater were equivalentto those found in municipal wastewater (Perez-Garcia et al., 2010).The suspension was mixed with isopycnic 4% sodium alginatesolution previously autoclaved for 20 min at 121 �C and cooledat room temperature. The alginate–alga mixture was dropped intoa 2% CaCl2 (w/v) solution using a syringe needle connected to aperistaltic pump (BT100-2J; Baoding Longer Precision Pump Co.,Ltd., Baoding, China). Beads of about 3–4 mm diameter wereproduced and allowed to harden for 1 h before being washed threetimes in autoclaved synthetic wastewater to remove any remain-ing CaCl2.

Immobilized algae and free-living algae were cultured in 250-ml flasks filled with 150 ml synthetic wastewater under AA, HA,MA and MNA conditions. The AA, MA and MNA experiments wereilluminated by four fluorescent tubes, giving a mean light intensityof 80 lmol m–2 s–1 under a 12 h:12 h light:dark (L:D) cycle. The HAflasks were covered with a black polypropylene sheet to ensurecultivation in darkness. The MNA flasks were plugged with a sili-cone stopper, in which two syringe needles were inserted (forthe initial purpose of gas exchange) and filled with nitrogen gas,after which the two syringe needles were removed and the stoppersealed with parafilm to ensure air tightness. All flasks were placedin a shaker (DHZ-032LR; Biocolor BioScience and Technology Com-pany, Shanghai, China) at 130 rpm at 30 ± 1 �C. All treatments weredone in triplicate.

For cell counts in alginate beads, ten beads were taken fromeach flask and solubilized by immersion in 1 ml of a 4% NaHCO3

solution for 30 min (Perez-Garcia et al., 2010). Cell number wasdetermined on a daily basis, using a haemocytometer (Hengtai,Yancheng City, China) under a microscope (Eclipse E50i; Nikon,Tokyo, Japan). Free-living algal growth was determined daily byOD750 measurements using a spectrophotometer (ShimadzuUV-1800, Japan). A relationship between cell number and opticaldensity (OD) was determined according to the equation:

y ¼ 2� 107x� 36764;R2 ¼ 0:9986 ðP < 0:05Þ ð1Þ

where y is the number of cells in 1 ml wastewater, and x is the valueof OD750.

2.2. Determination of PSII activity

Non-invasive chlorophyll fluorescence of PSII measurementswere taken using the pulse-amplitude-modulated method on anImaging-PAM system (Heinz Walz, Effeltrich, Germany) connectedto a PC with WinControl software. For all experiments, three beadswere removed from flasks containing immobilized cells and 1-mlsamples were taken from flasks containing free-living cells.Individual beads together with 100 ll synthetic wastewater or a200-ll sample of free-living cells were dispensed into 96-wellplates, adapted to the dark for 15 min, and minimum (F0) and

maximum (Fm) fluorescence were immediately determined usinga saturation pulse for calculation of the maximum Photosystem II(PSII) quantum yield (Fv/Fm):

Fv=Fm ¼ ðFm � F0Þ=Fm ð2Þ

where Fm is maximum fluorescence yield and F0 is minimal fluores-cence yield resulting in the variable fluorescence Fv.

Actinic light with photosynthetically active radiation (PAR) of35 lmol m–2 s–1 was turned onto initiate algal photosynthesisand the maximum fluorescence yield for illuminated samples(F 0m) was recorded. The effective PSII quantum yield was calculatedas follows:

YðIIÞ ¼ ðF 0m � FÞ=F 0m: ð3Þ

All determinations were carried out in darkness.

2.3. Determination of ammonium and phosphorus concentrations

Five-milliliters of samples were removed from cultures andammonium and phosphate concentrations were determined usingNessler’s reagent (Koch and McMeekin, 1924) and the molybde-num blue spectrophotometric method (Ma and Xu, 1998),respectively.

2.4. Experimental design and statistical analysis

Four cultivation conditions were set up (MA, HA, MNA and AA)and for each condition, controls (beads without cells), cultureswith immobilized cells, and cultures with free-living cells wereestablished. Mean and standard deviation values of the triplicatesfor each treatment were calculated. The removal of nutrients at aparticular treatment time was evaluated using parametric one-way analysis of variance (ANOVA).

3. Results and discussion

3.1. Growth of immobilized and free-living cells in syntheticwastewater under different conditions

Growth curves were determined for immobilized and free-living cells (Fig. 1). Very slow growth of immobilized microalgaeoccurred under AA conditions during days 0–3 (Fig. 1a), but growthincreased slightly after 3 d and by day 4, the population hadreached a concentration of 1.85 � 109 cells�flask�1. Under MA andHA conditions, the cells grew logarithmically for 2 d and reaching4.00 � 109 and 3.63 � 109 cells�flask�1, respectively after 3 d.Thereafter, the population declined slightly.

Although population growth of MNA cells was slightly slowerduring day 1–3 than that of HA and MA cells, the cell count reached3.78 � 109 cells�flask�1 and stationary phase was observed after4 d. The cell density of C. sorokiniana GXNN 01 in alginate beadswas estimated to be 6.66 � 106, 6.05 � 106, 5.13 � 106 and2.15 � 106 cells�bead�1 under MA, HA, MNA and AA conditions,respectively, after 3 d of incubation (P < 0.05) (Fig. 1c).

A similar trend was noted in the growth of free-living microal-gae (Fig. 1a, b). Growth of free-living cells was slowest under AAconditions; no logarithmic growth was observed, and the popula-tion reached only 1.11 � 109 cells�flask�1 after 5 d. Rapid cellgrowth did occur under MA, HA and MNA conditions (Fig. 1b).After a 2-d logarithmic growth period, the cells entered stationaryphase and the population reached 3.45 � 109(MA), 3.10 � 109(HA),and 3.43 � 109(MNA) cells�ml�1. There were similarities betweenMA and MNA cell growth patterns, whereas a slightly lower bio-mass was recorded under heterotrophic (HA) conditions (P < 0.05).

Fig. 1. Populations of immobilized (a) and free-living cells (b) per flask (containing 150 ml synthetic wastewater) under MA, HA, MNA and AA conditions, with cellconcentrations measured as ‘per bead’ under different conditions (c). Growth rates on day 3 (d). (Values = average ± SD).

28 K. Liu et al. / Bioresource Technology 114 (2012) 26–32

The addition of acetate significantly enhanced growth of immo-bilized and free-living cells. Immobilized cells grew more slowlythan free-living cells on day 1, but their growth rate subsequentlysurpassed that of free-living cells, resulting in a high maximumbiomass of immobilized cells (Fig. 1a, b). The growth rates ofimmobilized cells (l) was faster than that of free-living cells(P < 0.05) during the first 3 d under MA, HA and AA conditions,with the exception of cells grown under MNA conditions(Fig. 1d). The overall growth rate (l) of immobilized microalgaewas therefore higher than that of free-living microalgae after 4 d.

When C. sorokiniana GXNN 01 was cultured using acetate as acarbon source (i.e., C. sorokiniana GXNN 01 kept under MA andHA conditions), the growth rates increased significantly (Fig. 1d).Furthermore, C. sorokiniana GXNN 01 even exhibited superiorgrowth when kept under MNA conditions (Fig. 1). This indicatesthat the treatment of wastewater by C. sorokiniana GXNN 01 underHA and MNA conditions has significant advantages because (1)microalgae have a relatively higher growth rate and higher celldensities under HA and MNA conditions than under AA conditions,as indicated by the positive correlation with nutrient uptake ratesfrom wastewater and (2) HA growth occurred without light andMNA growth without aeration, conditions that would be beneficialfor cultivation of microalgae in high-volume wastewater, wherelight and aeration were limited. C. sorokiniana GXNN 01 canremove nutrients under HA (without light) and MNA (withoutaeration) conditions, which would reduce the cost of wastewatertreatment.

3.2. PSII activity of immobilized and free microalgae under differentgrowth conditions

PSII activity can be represented by maximum quantum yield(Fv/Fm), and effective PSII quantum yield (Y(II)). The Fv/Fm ratio isa sensitive means of assessing photosynthetic activity (Forsteret al., 1997; Herrmann et al., 1996). For immobilized cells, valuesof Fv/Fm reflect the quantum yield of PSII photochemistry whenall PSII reaction centers are open. This value was the highest underAA conditions during 5 d of treatment; it increased in the first 2 dand then stabilized (0.58 ± 0.008). Under MA and HA conditions,the values of Fv/Fm decreased slowly and stabilized at 0.41 ±0.008 and 0.32 ± 0.006, respectively. Under MNA conditions, Fv/Fm

values increased slightly on the first day and then decreased to0.37 ± 0.002 after 5 d of treatment (Fig. 2a).

For free-living cells, the highest values of Fv/Fm occurred underAA conditions throughout the experimental period and increasedto 0.63 ± 0.004 at day 4. The values under MA, HA and MNA growthconditions were initially similar to those under AA conditions butdecreased slightly to 0.21 ± 0.020, 0.32 ± 0.030 and 0.34 ± 0.049(Fig. 2b).

Likewise, Y(II) ðyield ¼ F 0v=F 0mÞ of immobilized cells and free-living cells with PAR of 35 lmol m–2 s–1 were consistently higherunder AA conditions than under other conditions. For immobilizedmicroalgae (Fig. 2c) under MNA conditions, the value of Y(II) rap-idly increased after the first 1-d adaptive phase, similar to resultswhen kept under AA conditions, but then decreased to

Fig. 2. The values of the maximum PSII quantum yield (Fv/Fm) (a, b) and the effective PSII quantum yield (Y(II)) (c, d) of both immobilized microalgae (a, c) and free-livingmicroalgae (b, d) under MA, HA, MNA and AA conditions in synthetic wastewater (values = average ± SD).

K. Liu et al. / Bioresource Technology 114 (2012) 26–32 29

0.15 ± 0.008 at day 2 and stabilized. After 1 d, values increased to0.20 ± 0.014 but the values of Y(II) decreased to 0.09 ± 0.013 underMA conditions. Under HA conditions, the values slowly increasedin the first 3 d and then decreased slightly (0.13 ± 0.044).

In free microalgae, the values of Y(II) under MA and HA condi-tions rapidly declined at day 2 (0.058 ± 0.002 and 0.067 ± 0.015)but decreased slowly under MA conditions (0.02 ± 0.005) andincreased slowly under HA conditions (0.18 ± 0.024) during days3–5 (Fig. 2d). Under MNA conditions, the values increased afterthe first day (0.26 ± 0.014) to a level slightly higher than thosemeasured under AA conditions, then decreased to 0.10 ± 0.030 onday 2, after which the system stabilized.

Immobilized cells of C. sorokiniana GXNN 01 had a 1-d lag per-iod that was not observed in free-living cells, which is consistentwith previous reports (Moreno-Garrido, 2008; Vílchez et al.,1997). The values for both Fv/Fm and Y(II) in the immobilized cellswere slightly lower than those of free-living cells at the beginningof incubation (0 d) (Fig. 2), indicating that immobilized cells werestressed and that photosynthetic activity in cells was slightlyinhibited, since Fv/Fm represents the maximum photosyntheticcapability of the plant under investigation and can decrease whenplants on stress in the surrounding environment (Lin et al., 2009).Thus, the immobilized cells had a lag period for 1 d (i.e., these cellswere in a recovery state), after which growth of the immobilizedcells rapidly increased and their final biomass became higher thanthat of free-living cells (Fig. 1). This observation is consistent withresults from previous studies (Lau et al., 1998; Mallick, 2002).

The biomass of C. sorokiniana GXNN 01 was higher under MA,HA and MNA conditions than under AA conditions for both immo-bilized and free-living cells (Fig. 1). In contrast, results also

indicated higher values for Fv/Fm and Y(II) under AA conditionsthan under MA, HA and MNA conditions, for both immobilizedand free-living cells (Fig. 2). Thus it was inferred that biomassaccumulated through utilization of acetate as a carbon source,not by photosynthesis. The values of Fv/Fm under AA conditionswere obviously higher than those under MA, HA and MNA condi-tions (Fig. 2), indicating that acetate addition slightly inhibitedphotosynthetic activity. Pringsheim and Wiessner (1960) reportedthat acetate caused abnormal photosynthesis in Chlamydobotrysstrains and slightly reduced photosynthesis in Chlorella vulgaris.Moreover, the results also indicated that the alginate matrix mighthave reduced the inhibition of photosynthetic activity of immobi-lized cells by acetate.

Therefore, Chlorella sp. has the capacity, when cultivated undercertain trophic conditions and in particular wastewaters, to utilizeorganic carbons, such as acetate, to accumulate biomass.

3.3. Removal of ammonium and phosphorus from syntheticwastewater under different conditions

In immobilized cells, the controls (blank beads without algae)were almost ineffectual in removing ammonium from wastewater.Under different growth conditions, residual ammonium concentra-tions in sewage decreased rapidly in the first 3 d and then leveledoff in the last 2 d. Under AA conditions, only 21.84% of the ammo-nium was eliminated (from 41.87 to 32.73 mg l�1) during a 5-dtreatment, whereas 47.17% of the ammonium was eliminated(from 40.99 to 21.82 mg l�1) under MA conditions compared to43.59% and 41.46% under HA and MNA conditions (from 41.52 to

Fig. 3. Removal of ammonium by immobilized microalgae (a) and free-living microalgae (b) and removal rates of ammonium (c) under MA, HA, MNA and AA conditions in thesynthetic wastewater (values = average ± SD).

30 K. Liu et al. / Bioresource Technology 114 (2012) 26–32

23.58 mg l�1 and from 41.51 to 24.28 mg l�1), respectively (Fig. 3a,c).

In wastewater containing free microalgae, ammonium concen-trations declined rapidly in the first 3 d and then declined at aslower rate after day 3–5 (Fig. 3b). Removal rates of ammoniumby free microalgae were 48.12 (MA), 38.57 (HA), 40.59 (MNA),and 14.35% (AA) (from 38.70 to 20.59 mg l�1, 38.70 to 23.76 mg l�1,38.53 to 23.05 mg l�1 and 39.06 to 33.61 mg l�1, respectively)(Fig. 3b, c).

The above results indicate that acetate addition resulted in theremoval of more ammonium from the synthetic wastewater. In

Fig. 4. Removal of phosphate by immobilized microalgae (a) and free-living microalgaesynthetic wastewater (values + average ± SD).

contrast, under AA, HA and MNA conditions, the removal rates ofthe ammonium by immobilized cells were slightly higher thanthose by free-living cells, but similar rates for immobilized andfree-living cells were measured under MA conditions. The removalof ammonium in immobilized cells was almost complete after thefirst 3 d, but in the case of free-living cells, continued for the whole5-d period.

The reduction of phosphate concentrations in wastewaters oc-curred in a similar way to that observed for ammonium. Phos-phates declined under different conditions during the 5-dtreatment (Fig. 3a, b), and controls (blank alginate beads without

(b) and removal rates of phosphate (c), under MA, HA, MNA and AA conditions in

Fig. 5. Population levels of leaked microalgae from alginate beads into wastewater (a) and rate of leakage (b) under MA, HA, MNA and AA conditions in the syntheticwastewater. (Values are average ± SD).

Table 1Changes in pH in wastewater, after treatment using immobilized cells under different conditions.

0 d 1 d 2 d 3 d 4 d 5 d

MA 6.98 ± 0.04 8.34 ± 0.02 8.50 ± 0.05 8.69 ± 0.03 8.70 ± 0.02 8.75 ± 0.02HA 6.99 ± 0.08 8.35 ± 0.02 8.53 ± 0.06 8.83 ± 0.12 8.91 ± 0.19 8.95 ± 0.05MNA 6.96 ± 0.02 8.02 ± 0.05 8.81 ± 0.01 8.38 ± 0.06 8.44 ± 0.12 8.48 ± 0.05AA 6.95 ± 0.03 6.87 ± 0.03 6.33 ± 0.07 5.85 ± 0.04 5.29 ± 0.04 4.75 ± 0.19

K. Liu et al. / Bioresource Technology 114 (2012) 26–32 31

cells) were almost ineffectual in removing phosphates fromwastewater.

In the case of immobilized cells maintained under MA, HA andMNA conditions, phosphate concentrations declined rapidly in thefirst 2 d (from 10.13 to 1.82 mg l�1, from 10.26 to 2.30 mg l�1 andfrom 10.26 to 2.30 mg l�1, respectively) and then declined slowly(Fig. 4a). A minor reduction in phosphates (from 11.21 to9.45 mg l�1) was noted under AA conditions. Removal rates byimmobilized microalgae were 88.65 (MA), 87.49 (HA), 84.84(MNA), and 17.63% (AA) (Fig. 4c).

Free-living cells showed lower removal rates of phosphate thanimmobilized cells (Fig. 4c). Under MA conditions, free microalgaerapidly removed phosphate in the first 3 d (from 11.34 to7.02 mg l�1), whereas under MNA conditions, this continued tothe fourth day (from 11.48 to 6.14 mg l�1). Under HA conditions,reduction of phosphate was fast during the first day and then lev-eled off (from 12.02 to 9.72 mg l�1). Under AA conditions, phos-phate concentrations declined slowly during the whole treatmentperiod (from 12.36 to 10.26 mg l�1) (Fig. 4b). The removal ratesof phosphate by free-living microalgae were 42.27 (MA), 20.21(HA), 53.52 (MNA) and 18.04% (AA) (Fig. 4c).

3.4. Leakage of immobilized microalgae under different growthconditions

With increasing treatment time, small quantities of algae cellsescaped from alginate beads and multiplied under MA, HA andMNA conditions. While very little cell leakage was observed underAA conditions (Fig. 5), the leakage rate from day 3–5 (algal cells inwastewater/total immobilized algal cells) were 1.16–3.80% (MA),1.37–4.93% (HA), 4.11–4.56% (MNA) and 0–0.09% (AA).

Growth trends in both immobilized, and free-living, cells werepositively correlated with the rates of ammonium removal fromsynthetic wastewaters, as measured in these cells (Fig. 1 and

Fig. 3). These removal rates were slightly higher in immobilizedcells than in free-living cells (Fig. 3c). In the case of phosphate re-moval, however, the rates in immobilized cells (>80%) were statis-tically higher than those measured in free-living cells (<50%) underMA, HA and MNA conditions (Fig. 4), possibly due calcium ions inthe alginate matrix and phosphate in wastewater causing precipi-tation of phosphate as calcium phosphate, at pH P 8 (Megharajet al., 1992; Tam and Wong, 2000). In the present study, the pHvalues of immobilized cell flasks were >8.0 under MA, HA andMNA conditions (Table 1), indicating conditions favoring signifi-cant phosphate precipitation. The leakage of microalgae from thealginate beads also provides an indication of phosphate precipita-tion (Fig. 5). Therefore, chemical precipitation using iron, alum, orlime (De-Bashan and Bashan, 2010) should be used to initially re-move phosphate from wastewater, after which immobilized cellscan be used to remove nutrients from wastewaters effluents.

4. Conclusions

The present study demonstrates that immobilized C. sorokinianaGXNN 01 is likely a useful organism for wastewater treatment,since it grew well under a variety of conditions, especially underMNA conditions, and had a higher rate of removal of ammoniumand phosphate from synthetic wastewater than was the case forfree-living cells. In addition to the growth of microalgal cells, theprecipitation of phosphate as calcium phosphate also contributedto the removal of phosphate, which resulted in slight leakage ofcells from alginate beads. Research with real wastewaters stillneeds to confirm the utility of this system in practice.

Acknowledgements

The work was supported by the National Natural ScienceFoundation of China [30830015, 30970302], Basic Research Project

32 K. Liu et al. / Bioresource Technology 114 (2012) 26–32

of the Ministry of Science and Technology in China[SQ2012FY4910019-1], the key project of Science and Technologyfor Supporting Tianjin Development [10ZCKFSH00700], the Inno-vative Foundation of Chinese Academy of Sciences [Y12336102L],the Project Supported by the Natural Science Foundation of Tianjin[2011].

References

Bashan, Y., 1986. Alginate beads as synthetic inoculant carriers for slow release ofbacteria that affect plant growth. Appl. Environ. Microbiol. 51, 1089–1098.

Chevalier, P., de la Noue, J., 1985. Wastewater nutrient removal with microalgaeimmobilized in carrageenan. Enzyme Microb. Technol. 7, 621–624.

De-Bashan, L.E.B., Bashan, Y., 2010. Immobilized microalgae for removingpollutants: review of practical aspects. Bioresour. Technol. 101, 1611–1627.

Forster, R., Haker, A., Schubert, H., 1997. Wavelength dependence of photoinhibitionin the green alga Chlorella vulgaris. Photosynthetica 33, 541–552.

Gonzalez, L.E., Canizares, R.O., Baena, S., 1997. Efficiency of ammonia andphosphorus removal from a Colombian agroindustrial wastewater by themicroalgae Chlorella vulgaris and Scenedesmus dimorphus. Bioresour. Technol.60, 259–262.

Hammouda, O., Gaber, A., Abdelraouf, N., 1995. Microalgae and wastewatertreatment. Ecotoxicol. Environ. Saf. 31, 205–210.

Harris, E.H., 1989. The Chlamydomonas Sourcebook: A Comprehensive Guide ToBiology And Laboratory Use. Academic Press, San Diego.

Herrmann, H., H der, D.P., K fferlein, M., Seidlitz, H., Ghetti, F., 1996. Effects of UVradiation on photosynthesis of phytoplankton exposed to solar simulator light.J. Photochem. Photobiol. B 34, 21–28.

Jimenez-Perez, M., Sanchez-Castillo, P., Romera, O., Fernandez-Moreno, D., Perez-Martinez, C., 2004. Growth and nutrient removal in free and immobilizedplanktonic green algae isolated from pig manure. Enzyme Microb. Technol. 34,392–398.

Koch, F.C., McMeekin, T.L., 1924. A new direct Nesslerization micro-kjeldahl methodand a modification of the Nessler-folin reagent for ammonia. J. Am. Chem. Soc.46, 2066–2069.

Lau, P., Tam, N., Wong, Y., 1998. Effect of carrageenan immobilization on thephysiological activities of Chlorella vulgaris. Bioresour. Technol. 63, 115–121.

Lin, A.P., Wang, G.C., Yang, F., Pan, G.H., 2009. Photosynthetic parameters of sexuallydifferent parts of Porphyra katadai var. hemiphylla (Bangiales, Rhodophyta)during dehydration and re-hydration. Planta 229, 803–810.

Ma, Y.A., Xu, H.Z. et al., 1998. The Specification for Marine Monitoring – Part 4,Seawater Analysis (GB17378.4) vol. 4. China standard press, Beijing.

Mallick, N., 2002. Biotechnological potential of immobilized algae for wastewater N,P and metal removal: a review. Biometals 15, 377–390.

Megharaj, M., Pearson, H., Venkateswarlu, K., 1992. Removal of nitrogen andphosphorus by immobilized cells of Chlorella vulgaris and Scenedesmus bijugatusisolated from soil. Enzyme Microb. Technol. 14, 656–658.

Moreno-Garrido, I., 2008. Microalgae immobilization: current techniques and uses.Bioresour. Technol. 99, 3949–3964.

Moreno-Garrido, I., Campana, O., Lubian, L., Blasco, J., 2005. Calcium alginateimmobilized marine microalgae: experiments on growth and short-term heavymetal accumulation. Mar. Pollut. Bull. 51, 823–829.

Oswald, W., Gotaas, H., Golueke, C., Kellen, W., Gloyna, E., Hermann, E., 1957. Algaein waste treatment. Sew. Ind. Wastes 29, 437–457.

Perez-Garcia, O., de-Bashan, L.E., Hernandez, J.P., Bashan, Y., 2010. Efficiency ofgrowth and nutrient uptake from wastewater by heterotrophic, autotrophic,and mixtrophic cultivation of Chlorella vulgaris immobilized with AzospirillumBrasilense. J. Phycol. 46, 800–812.

Pringsheim, E., Wiessner, W., 1960. Photo-assimilation of acetate by greenorganisms. Nature 188, 919–921.

Qiao, H., Wang, G., Zhang, X., 2009. Isolation and characterization of Chlorellasorokiniana GXNN01 (Chlorophyta) with the properties of heterotrophic andmicroaerobic growth. J. Phycol. 45, 1153–1162.

Shi, J., Podola, B., Melkonian, M., 2007. Removal of nitrogen and phosphorus fromwastewater using microalgae immobilized on twin layers: an experimentalstudy. J. Appl. Phycol. 19, 417–423.

Tam, N.F.Y., Wong, Y.S., 2000. Effect of immobilized microalgal bead concentrationson wastewater nutrient removal. Environ. Pollut. 107, 145–151.

Vílchez, M.J., Vigara, J., Garbayo, I., Vílchez, C., 1997. Electron microscopic studies onimmobilized growing Chlamydomonas reinhardtii cells. Enzyme Microb.Technol. 21, 45–47.

Zelitch, I., 1971. Photosynthesis, Photorespiration and Plant Productivity. AcademicPress, New York.