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A comparative evaluation of microalgae for the degradation of piggerywastewater under photosynthetic oxygenation

Ignacio de Godos a,b,1, Virginia A. Vargas c,2, Saúl Blanco e,4, María C. García González d,3, Roberto Soto c,2,Pedro A. García-Encina a, Eloy Becares b,1, Raúl Muñoz a,*

a Department of Chemical Engineering and Environmental Technology, University of Valladolid, Paseo del Prado de la Magdalena s/n, 47011 Valladolid, Spainb Department of Biodiversity and Environmental Management, University of León, Campus Vegazana, 24071 León, Spainc Center of Biotechnology, San Simon Mayor University of Cochabamba, Campus Universitario, s/n Cochabamba, Boliviad Institute of Agriculture Technology of Castilla y León (ITACyL), Ctra. Burgos, Km 119, 47071 Valladolid, Spaine Institute of Environmental Sciences, University of León, C/La Serna 58, 24071 León, Spain

a r t i c l e i n f o

 Article history:

Received 26 October 2009

Received in revised form 29 January 2010

Accepted 3 February 2010

Available online 9 March 2010

Keywords:

Bioremediation

Microalgae selection

Nutrients removal

Photosynthetic oxygenation

Piggery wastewater

a b s t r a c t

Two green microalgae (Scenedesmus obliquus   and  Chlorella sorokiniana), one cyanobacterium (Spirulina

 platensis), one euglenophyt (Euglena viridis) and two microalgae consortia were evaluated for their ability

to support carbon, nitrogen and phosphorous removal in symbiosis with activated sludge bacteria during

thebiodegradation of four and eight times diluted piggery wastewater in batch tests. C. sorokiniana and E.

viridis were capable of supporting the biodegradation of four and eight times diluted wastewater. On the

other hand, while S. obliquus and the consortia isolated from a swine manure stabilization pond were only

able to grow in eight times diluted wastewater,  S. platensis  and the consortium isolated from a high rate

algal pond treating swine manure were totally inhibited regardless of the dilution applied. TOC removal

efficiencies (RE) ranging from 42% to 55% and NH4+-RE from 21% to 39% were recorded in the tests exhib-

iting photosynthetic oxygenation. The similar oxygen production rates exhibited by the tested microalgae

under autotrophic conditions (from 116 to 133 mg O2 L 1 d1) suggested that factors other than the pho-

tosynthetic oxygenation potential governed piggery wastewater biodegradation. Microalgal tolerancetowards NH3 was hypothesized as the key selection criterion. Further studies in a continuous algal–bac-

terial photobioreactor inoculated with  C. sorokiniana, S. obliquus  and S. platensis  showed that C. sorokini-

ana, the species showing the highest NH3-tolerance, rapidly outcompeted the rest of the microalgae

during the biodegradation of eight times diluted wastewater, achieving TOC and NH4+-RE comparable

to those recorded in the batch biodegradation tests.

 2010 Elsevier Ltd. All rights reserved.

1. Introduction

Swine manure is considered one of the most polluting agro-

industrial wastewaters worldwide. When not properly managed,

the high organic matter, nitrogen and phosphorous concentrations

present in these wastewaters can cause severe environmental

problems such as eutrophication of water bodies (Carpenter

et al., 1998), groundwater contamination (Krapaca et al., 2002),

air pollution by NH3  volatilization (ApSimon et al., 1987) and soil

degradation due to over-fertilization. In addition, high concentra-

tions of hazardous heavy metals such as Cu+2, Zn+2, and Pb+2 are of-

ten present in piggery wastewaters (de la Torre et al., 2000).

Land application, the traditional piggery wastewater manage-

ment strategy, is nowadays conditioned by the nutrients require-

ments of the crops, the vulnerability of the neighboring

ecosystems and the energy cost derived from its application (Flo-

tats et al., 2009). In areas of intensive farming, piggery wastewater

treatment is often required before discharge into natural water

bodies. In spite of the good performance of activated sludge sys-

tems, their decentralized implementation is often limited by the

high energy requirements and capital costs (Osada et al., 1991).

Likewise, the implementation of anaerobic digestion, despite com-

bining organic matter removal with biogas production, is often re-

stricted by the poor nutrients removal, the need for a complex

process control (temperature, loading rate) and the unfavorable

C/N ratio of piggery wastewaters (Burton and Turner, 2003).

In this context, microalgae-based processes constitute a cost-

effective technology for the degradation of livestock wastewaters

(de Godos et al., 2009a,b; Mulbry et al., 2008). The first microal-

gae-based bioremediation studies were carried out with domestic

0960-8524/$ - see front matter    2010 Elsevier Ltd. All rights reserved.doi:10.1016/j.biortech.2010.02.010

*  Corresponding author. Tel.: +34 983184934; fax: +34 983423013.

E-mail address:   mutora@iq.uva.es (R. Muñoz).1 Tel.: +34 987291568; fax: +34 987291563.2 Tel./fax: +591 4 4542895.3 Tel.: +34 983317388; fax: +34 983414780.4 Tel.: +34 987293136; fax: +34 987291563.

Bioresource Technology 101 (2010) 5150–5158

Contents lists available at  ScienceDirect

Bioresource Technology

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wastewaters 50 years ago in California, at the laboratory of  Oswald

et al. (1957). These preliminary works confirmed that microalgae

can provide the oxygen that heterotrophic bacteria need for the

degradation of the organic matter, while bacteria concomitantly

release the carbon dioxide and the nutrients (N and P) needed by

microalgae during photosynthesis. Recent studies have shown that

microalgae activity is often the limiting component in these sym-

biotic microcosms due to their high sensitivity towards toxicants

(as NH3 in high strength wastewaters) and to the limitations inher-

ent to light supply (Muñoz et al., 2004; González et al., 2008). How-

ever, despite the fact that microalgae play a key role in the

degradation process, little effort has been devoted so far to the

selection of high-performance microalgae and to the evaluation

of the influence of microalgae species on process performance

(Muñoz et al., 2003). In this context, the most important criterion

in the selection of microalgae for wastewater treatment is the

capacity to support high removal rates of carbon and nutrients

(normally associated to high growth rates). Characteristics such

as a facilitated sedimentation and a valuable biomass composition

must be also considered (microalgae from wastewater treatment

can be used as source of protein, biofuels and biofertilizer) (Wilkie

and Mulbry, 2002; An et al., 2003; Mulbry et al., 2005).

This work evaluates the performance of two green microalgae

(Chlorella sorokiniana   and  Scenedesmus obliquus), one cyanobacte-

rium (Spirulina platensis), one euglenophyt (Euglena viridis) and

two wild microalgae consortia (isolated from piggery wastewater

treatment ponds) in the biodegradation of piggery wastewater in

symbiosis with an activated sludge bacteria. Each microalga was

first evaluated for its carbon, nitrogen and phosphorous removal

in batch biodegradation tests and its oxygenation capacity under

photoautotrophic conditions. Finally, the dynamics of microalgae

population were assessed in a continuous enclosed 3.5-L photobi-

oreactor inoculated with C. sorokiniana, S. obliquus, and S. platensis

(microalgae species exhibiting a high, moderate, and low tolerance

towards NH3, respectively, according to the result herein obtained)

and activated sludge bacteria.

2. Methods

 2.1. Piggery wastewater 

Piggery wastewater was obtained from the main collector of a

swine manure treatment plant in Hornillos de Eresma (GRUPO

GUASCOR, Valladolid, Spain) and stored at 4 C. Prior to experi-

mentation, swine manure was centrifuged for 10 min at

10000 rpm at 4 C. Therefore, only the soluble fraction of carbon,

nitrogen, phosphorus and heavy metals (Zn+2, Cu+2, As+3, and

Pb+2) was considered in the present study.

 2.2. Microorganism and culture conditions

Chlorella sorokiniana   211/8 k (C. sorokiniana) and   Scenedesmus

obliquus   (S. obliquus) were obtained from the Culture Collection

of Algae and Protozoa of the SAMS Research Services (Argyl, Scot-

land). Euglena viridis  (E. viridis) and  Spirulina platensis  (S. platensis)

were purchased from the Culture Collection of Algae of the Univer-

sity of Goettingen (SAG) (Goettingen, Germany). The microalgae

consortium 1 (composed of the genera   Scenedesmus   74%,   Chla-

mydomonas  16% and   Microspora   7%  Oocystis   1%,  Chlorella   1% and

Nitzschia 1%) was drawn from a 465-L High Rate Algae Pond (HRAP)

treating diluted piggery wastewater at a hydraulic residence time

of 10 days (de Godos et al., 2009b). The microalgae consortium 2

(mainly composed of a Chlorella strain) was obtained from a stabil-

ization pond treating the final effluent of a pig farm in Quillacollo(Bolivia). Swine manure degrading bacteria were obtained from an

activated sludge reactor treating piggery wastewater operated in a

denitrification–nitrification configuration.

Fresh cultures of the axenic microalgae and consortia, prepared

under sterile conditions in 120 mL gas-tight glass serum flasks con-

taining 70 mL of mineral salt medium (Muñoz et al., 2007) and a

20/80 v/v CO2/air atmosphere, were used as inocula. The inoculum

for S. platensis  and E. viridis  was prepared in sterile 250 mL Erlen-

meyer flasks containing 100 mL of the corresponding mineral salt

media recommended by SAG. All inocula were incubated at 25 C

under continuous magnetic stirring at 300 rpm and illumination

at 4500 lux (four fluorescents 40 W Osram L lamps, Germany).

 2.3. Piggery wastewater biodegradation tests under photosynthetic 

oxygenation

Glass bottles of 1250 mL containing 500 mL of diluted centri-

fuged piggery wastewater (1:4 and 1:8 dilutions with tap water)

were inoculated with the target microalga species at initial concen-

trations ranging from4 to 6 mgDW L 1 (Dry Weight) and activated

sludge bacteria at 3 mg DWL 1. The bottles were then flushed with

He in order to establish an initial bioreaction environment totally

deprived from O2, and immediately closed with butyl septa and

sealed with plastic caps. Under these conditions, the biodegrada-

tion of piggery wastewater can only proceed driven by photosyn-

thetic oxygenation. The systems were incubated at 25 C

(temperature controlled by a thermostatic water bath) under con-

tinuous magnetic agitation (300 rpm) and illumination at 4500 lux.

All tests were carried out in duplicate. The systems were allowed

to run until the dissolved total organic carbon (TOC), NH4+ and

pH remained stable during three consecutive days, and at that

point, the pH of one of the duplicates was decreased to 7 via HCl

(37%) addition in order to prevent ammonia-mediated inhibition.

Once the pH of the acidified replicate increased and stabilized

again at previous levels, an additional acidification was carried

out. Control tests in the absence of microalgae and bacteria were

also conducted with four and eight times diluted wastewater in or-

der to account for any potential abiotic wastewater degradation.Liquid samples of 10 mL were periodically drawn and centri-

fuged (5000 rpm during 10 min) for the analysis of TOC, inorganic

carbon (IC), NH4+, NO3

and NO2 concentrations. Culture absor-

bance at 550 nm (OD550) and pH were measured prior to centrifu-

gation. Liquid samples of 10 mL were also drawn at the beginning

and end of each set of tests in order to determine Zn+2, Cu+2, As+3,

Pb+2 and total phosphorous (TP) removal. Liquid samples for heavy

metals determination were stored in pretreated plastic tubes

according to Pott and Mattiasson (2004). In addition, gas samples

of 100 ll were taken using gas-tight syringes (Hamilton Co., Reno,

Nevada) at the end of the experiment in order to determine CO2

and O2, concentration in the headspace of the bottles. The total bio-

mass production was also measured at the end of each experiment.

 2.4. Microalgal oxygenation tests

The oxygenation capacity of the above tested photosynthetic

microorganisms was evaluated under fully autotrophic conditions

in 1250 mL glass bottles containing 500 mL of a sterile mineral salt

medium composed of (mg L 1): NaHCO3, 3402; Na2CO3, 1007;

K2HPO4, 125; NaNO3, 625; K2SO4, 250; NaCl, 250; MgSO4, 50; CaCl2,

10; FeSO4, 2.5; EDTA, 20; ZnSO4, 0.00125; MnSO4, 0.0025; H3BO3,

0.0125; Co(NO3)2, 0.0125; Na2Mo4, 0.0125; and CuSO4,

6.25 106. The systems were flushed with He in order to achieve

an O2-free atmosphere, closed with butyl septa and sealed with

plastic caps. The pH of the cultivation medium was then decreased

to 7 (except in the tests carried out with  S. platensis  (SAG Recom-

mendation)) by injecting 1.1 mL of HCl (37%) and the systems wereallowed to equilibrate for 2 h at 25 C prior to inoculation with the

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target microalgae (initial concentrations ranging from 3 to

6m g DWL  1). The tests were incubated at 25 C under continuous

magnetic agitation and illumination at 300 rpm and 4500 lux,

respectively. Gas samples of 100 lL were periodically taken to re-

cord CO2 and O2 headspace concentrations. In addition, liquid sam-

ples of 10 mL were also periodically drawn to monitor the IC

concentration, pH and culture absorbance at 550 nm.

 2.5. Piggery wastewater biodegradability test 

The biodegradable TOC fraction of the wastewater was evalu-

ated in 250 mL E-flasks containing 100 mL of centrifuged wastewa-

ter and inoculated with 2 mL of the acclimated activated sludge.

The bottles were closed with cotton plugs (allowing air diffusion)

and incubated for 20 days at 25 C under magnetic agitation

(300 rpm). Liquid samples of 5 mL were periodically drawn in or-

der to monitor TOC concentration.

 2.6. Piggery wastewater biodegradation in a continuous algal–

bacterial photobioreactor 

An enclosed jacketed glass tank photobioreactor with a total

working volume of 3.5 L (AFORA, Spain) was used to evaluate the

dynamics of microalgal population during piggery wastewater bio-

degradation. The photobioreactor was illuminated by six fluores-

cent lamps (14 W, PHILIPS, Holland) arranged in a circular

configuration and providing an illuminance of 10,000 lux at the

outer wall of the photobioreactor. The photobioreactor was filled

with 3.5 L of tap water and inoculated with equal concentrations

of  S. platensis,  S. obliquus  and  C. sorokiniana   (1.2 mg DWL 1 each)

and activated sludge bacteria at 0.4 mg DW L 1. Temperature and

magnetic agitation were maintained constant at 25 C (Huber

water bath, Offenburg, Germany) and 300 rpm, respectively. The

photobioreactor was operated during 13 days at a Hydraulic Reten-

tion Time (HRT) of 4.4 days by continuous supply of eight times di-

luted centrifuged piggery wastewater and overflow of the treated

effluent using peristaltic pumps. Polyamide plastic pellets wereadded (4.2 g) in order to avoid microalgal–bacterial biofilm attach-

ment into the inner wall of the photobioreactor.

Samples of 100 mL of the influent and photobioreactor culture

broth were periodically drawn to measure TOC, IC, NH4+, N–

NO2, N–NO3

and volatile suspended solids concentration. In

addition, liquid samples of 10 mL of the culture broth were fixed

with lugol acid at 0.5% and stored at 4 C prior to microalgal cell

counting.

 2.7. Analytical procedures

TOC and IC concentrations were determined using a Shimadzu

TOC-V CSH analyzer (Shimadzu, Japan). N–NH4+ was determined

using an ammonia electrode Orion900/200 (Thermo Electron, Bev-erly, MA, USA). NO3

and NO2 were analyzed via HPLC–IC accord-

ing to Standard Methods (Eaton et al., 2005). Gaseous

concentrations of O2 and CO2, were analyzed using a gas chromato-

graph (Varian CP-3800, Palo Alto, CA, USA) coupled with a thermal

conductivity detector and equipped with a CP-Molsieve 5A

(15 m 0.53 mm,   15lm) and a CP-Pora BOND Q (25 m

0.53 mm,   10lm) columns. Oven was maintained at 40 C for

1.5 min and then heated to 56 C at 10 C min1. Injector and

detector temperatures were 150 C and 175 C, respectively. He-

lium was the carrier gas at 13.7 mL min1.

Aqueous samples for the determination of TP and heavy metals

were digested after acidification (18.6% HNO3) in a microwave

oven (Mars Xpress, CEM, USA). The concentration of TP, Zn+2,

Cu+2

, As+3

, Pb+2

was determined via spectroscopy atomic emission(ICP-AES, Perkin–Elmer, USA).

The pH was measured using a pH probe CRISON micropH 2002

(Crison Instruments, Barcelona, Spain). Biomass concentration was

estimated from culture absorbance measurements at 550 nm

(OD550) using a Spectronic 20Genesys™ spectrophotometer (Spec-

tronic Instruments, USA). In addition, total suspended solids con-

centration was performed according to Standard Methods (Eaton

et al., 2005).

3. Results and discussion

 3.1. Piggery wastewater biodegradation test under photosynthetic 

oxygenation

The experimental results here obtained clearly confirmed that

the species of microalgae supporting process oxygenation signifi-

cantly influenced piggery wastewater biodegradation performance

(Figs. 1 and 2). Thus, whereas   C. sorokiniana   and  E. viridis   were

capable to grow in four and eight times diluted piggery wastewa-

ters, S. obliquus  and consortium 2 were only able to grow in eight

times diluted wastewater. On the other hand, neither  S. platensis

nor consortium 1 exhibited significant growth regardless of the

wastewater dilution applied (data not shown). These findings are

in agreement with previous studies carried out in microalgae-

based sewage treatment processes showing that Euglena and Chlo-

rella  was often dominant at high organic loads while  Scenedesmus

were the most abundant species at medium loads (Martinez San-

cho et al., 1993; González et al., 1997). Palmer (1969) ranked spe-

cies from the genus Euglena, Chlorella  and Scenedesmus  within the

top 10 more resistant microalgae-based on their ability to grow

in organic polluted environments. Likewise, a recent study has re-

ported that a strain of  Euglena exhibited higher growth rates in di-

luted animal waste than Chlorella and Microcystis (cyanobacterium)

strains (Park et al., 2009).

The inhibition of photosynthesis mediated by the high NH3 con-

centrations present in the piggery wastewater were likely the rea-

son underlying the lack of biological activity in   S. platensis   andconsortium 1 cultures, and the inhibition of  Scenedesmus and con-

sortium 2 in four times diluted wastewater. Hence, the high NH3

concentrations resulting from the high pH and NH4+ concentra-

tions present in the piggery wastewater (higher than eight and

300 mg N–NH4+ L 1, respectively, at four times diluted wastewa-

ter) can uncouple the electron transport in photosystem II and

compete with H2O in the oxidation reactions leading to O2 produc-

tion (Azov and Goldman, 1982). Tolerance to NH3 is however spe-

cies dependent. For example, while no significant effect on the

growth of  C. sorokiniana  was observed at 400 mg NH4+ L 1, S. plat-

ensis  was nearly completely inhibited at 200 mg NH4+ L 1 (Ogbon-

na et al., 2000). Likewise,  Gantar et al. (1991)   also reported that

Chlorella   rapidly overcame   S. platensis   in a swine manure batch

degradation tests. At this point, it must be stressed that althoughS. platensis   has been successfully used in continuous HRAP and

batch biodegradation studies, these studies were carried out at sig-

nificantly lower NH4+ concentrations (20 and 80 times diluted pig-

gery wastewater) (Olguín et al., 2001; Cañizares et al., 1994). In

this context, the fact that consortium 1 was collected from a pi-

lot-scale HRAP treating 50 diluted swine manure (effluent

[NH4+] 0 m g L  1) might explain the low NH3-tolerance of this

consortium.

While TOC was rapidly removed in tests supplied with actively

growing microalgae (Figs. 1 and 2), no TOC removal was recorded

neither in the tests inoculated with S. platensis or consortium 1 nor

in oxygen-deprived control tests (data not shown). Biological oxi-

dation supported by microalgal photosynthesis can be thus consid-

ered as the main TOC-RE mechanism in enclosed algal–bacterialsystems. Maximum TOC degradation rates (estimated from the

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slope of the TOC concentration vs. time curves during the initial

stages of the biodegradation process) of 52 ± 5, 48 ± 3, 66 ± 4, and

5 6 ± 3 m g C L  1 d1 were recorded in the tests supplied with eight

times diluted wastewater and inoculated with E. viridis, S. obliquus,

C. sorokiniana, and consortium 2, respectively (Figs. 1 and 2a and

b). In tests supplied with four times diluted wastewater,  E. viridis

supported the maximum TOC degradation rates (111 ± 10 mg C

L 1 d1), whereas these rates decreased to 89 ± 13 mgC L 1 d1

in C. sorokiniana tests. These values were lower that those obtained

by González et al. (2008) (127 ± 26 and 98 ± 8 mg C L  1 d1 for four

and eight times diluted wastewaters, respectively) under similar

experimental conditions using   C. sorokiniana   as photosynthetic

oxygenating microorganism. In this context, the characteristics of piggery wastewater (i.e. fraction of readily biodegradable organic

carbon) are known to significantly impact the kinetics of TOC re-

moval and make inter-studies comparison a rather unfruitful task

(González et al., 2008). Farm swine manure management practices

(shed cleansing, waste storage conditions) and pig nutrition usu-

ally account for the different TOC biodegradability observed, which

might range from 0% to 80% (Boursier et al., 2005; González et al.,

2008). The rates of TOC removal gradually levelled off and stabi-

lized by the 8–9 days of cultivation. Removal efficiencies (RE) of 

55 ± 5%, 42 ± 4%, 42 ± 1% and 46 ± 1% were recorded in tests sup-

plied with eight times diluted wastewater and inoculated with  E.

viridis,  S. obliquus,  C. sorokiniana, and consortium 2, respectively,

prior to acidification of one of the duplicates (Table 1). Likewise,

TOC-RE of 51 ± 3% and 47 ± 8% were achieved in the systems sup-plied with four times diluted wastewater and inoculated with  E.

0

200

400

600

0 100 200 300 400 500

   I   C   (  m  g   L  -   1   )

Time (h)

d

0

200

400

600

0 100 200 300 400 500

   T   O   C   (  m  g   L  -   1   )

a

0

200

400

600

0 100 200 300 400 500

   T   O   C   (  m  g   L  -   1   )

b

0

200

400

600

0 100 200 300 400 500

   I   C   (  m  g   L  -   1   )

Time (h)

c

Fig. 1.  Time course of TOC and IC concentrations in enclosed algal–bacterial systems inoculated with  E. viridis (a and c, respectively) and  S. obliquus (b and d, respectively)

during the biodegradation of piggery wastewater diluted four times (diamonds) and eight times (squares). The acidified duplicate after TOC stabilization at each dilution isrepresented with open symbols (e andh, respectively). Vertical dashed lines illustrate the time of culture medium acidification.

0

200

400

600

0 100 200 300 400 500

   T   O   C   (  m  g   L  -   1   )

0

200

400

600

0 100 200 300 400 500

   T   O   C   (  m  g   L  -   1   )

a

0

200

400

600

0 100 200 300 400 500

   I   C   (  m  g   L  -   1   )

Time (h)

c

0

200

400

600

0 100 200 300 400 500

   I   C   (  m  g   L  -   1   )

T ime (h)

d

b

Fig. 2.   Time course of TOC and IC concentrations in enclosed algal–bacterial systems inoculated with   C. sorokiniana  (a and c, respectively) and consortium 2 (b and d,

respectively) during the biodegradation of piggery wastewater diluted four times (diamonds) and eight times (squares). The acidified duplicate after TOCstabilization at each

dilution is represented with open symbols (e andh, respectively). Vertical dashed lines illustrate the time of culture medium acidification.

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viridis   and   C. sorokiniana, correspondingly. These results are in

agreement with the TOC biodegradable fraction of piggery waste-

water herein employed (54 ± 2%) and determined independently

in biodegradability tests inoculated with activated sludge bacteria

and aerated mechanically during 20 days.

Piggery wastewater biodegradation under photosynthetic oxy-

genation resulted in gradual pH increases as a result of inorganic

carbon consumption and the release of basic bioreaction metabo-

lites (González et al., 2008) (Fig. 4). This increase in pH was moresevere in the tests performed with 1:8 dilution, which might be

due to the lower buffer capacity of this cultivation medium and

the higher photosynthetic activity as a result of a lower NH3  inhi-

bition.   C. sorokiniana, consortium 2,   E. viridis, and   S. obliquus

achieved maximum pH values of 10.1 ± 0.2, 9.9 ± 0.2, 9.6 ± 0.1

and 8.6 ± 0.1, respectively, prior to acidification. In the tests sup-

plied with four times diluted wastewater, the increase in pH was

notably lower: 9.5 ± 0.1 and 8.9 ± 0.1 for C. sorokiniana  and E. viri-

dis, respectively. Following the stabilization of TOC concentrations,

the pH of one of the duplicate systems was intentionally decreased

to 7 via HCl addition in order to reduce NH3-mediated toxicity and

to increase fraction of IC available for microalgal growth. In this

context, biomass concentration and IC consumption rapidly re-

sumed in the acidified systems, which clearly indicates that mediaacidification resulted in an increase in CO2 availability for microal-

gal growth. In this regard, the high pH derived from microalgae-

based piggery wastewater treatment (Fig. 3) mediated an increase

in carbonate and bicarbonate concentrations (non-bioavailable

forms of IC for many species of photosynthetic microorganisms)

due to a shift in the acid–base equilibrium. The results also showed

that most of the biodegradable organic carbon was already de-

pleted prior to acidification and therefore no enhancement in

TOC removal derived from the mitigation of NH3 inhibition. Hence,

no significant differences were found when the performance of the

acidified and non-acidified replicates of  C. sorokiniana and consor-

tium 2 was evaluated (Fig. 2a and b). TOC concentrations slightly

increased following acidification both in four and eight times di-

luted  E. viridis   tests (Fig. 1a). This increase in TOC concentration

suggest either the excretion of significant amounts of extracellular

organic matter (EOM) or an intensive biomass hydrolysis at the last

stages of the cultivation period. The former hypothesis is sup-

ported by previous investigations that observed the excretion of 

EOM concentrations of up to 80 mg L 1 in some microalgal cul-

tures, this excretion (mainly composed of polysaccharides) being

higher the larger the microalgae age was (Hoyer et al., 1985; Hen-

derson et al., 2008). Unexpectedly, a significant difference in TOC

concentration between acidified and non-acidified systems wasfound in the tests supplied with  S. obliquus. These tests were the

first ones to be carried out and were therefore performed with

fresh swine manure (prior to storage at 4 C). This suggests the

presence of a higher fraction of easily biodegradable TOC since it

has been observed that swine manure gradually stabilizes even

at 4 C. At this point it must be highlighted that some experimental

error due to the temporal variability of swine manure properties

must be allowed in research carried out with real piggery waste-

waters, since even tests carried with fresh wastewater will present

gross variation from day to day (de Godos et al., 2009a). In addi-

tion, this apparent mismatch between the data observed for TOC

evolution in S. obliquus  and the rests of the microalgae tested high-

lights the complex nature of the processes underlying piggery

wastewater biodegradation and the need for further researcher inthe potential excretion of EOM, since this issue can significantly

impact wastewater treatment performance (Henderson et al.,

2008).

 3.2. Microalgae oxygenation test 

The evaluation of the oxygenation capacity of the tested micro-

algae revealed that C. sorokiniana,  S. obliquus  and  S. platensis, and

the two consortia exhibited comparable oxygenation capacities

(ranging from 116 ± 27 for consortium 1 to 133 ± 9 mg O2 L 1 d1

for consortium 2, respectively) (Table 1). These oxygenation rates

were estimated from the slope of the oxygen concentration vs.

time curves during the exponential phase of microalgal growth

(Fig. 3a and b). Based on these similarities in microalgal oxygena-

 Table 1

Influence of microalgae species on the potential oxygen supply under autotrophic growth, maximum TOC degradation rates, TOC and NH4+ removal efficiencies and pH reached

prior to media acidification in the biodegradation tests conducted under photosynthetic oxygenation.

Oxygenation capacity (mg O2 L 1 d1) Maximum TOC degradation rate (mg C L 1 d1) TOC-RE (%) NH4+-RE (%) Maximum pH

1:4 1:8 1:4 1:8 1:4 1:8 1:4 1:8

E. viridis   * 111 ± 10 52 ± 5 51 ± 3 55 ± 5 34 ± 0 39 ± 3 8.9 ± 0.1 9.6 ± 0.1

C. sorokiniana   131 ± 1 89 ± 13 66 ± 4 47 ± 8 42 ± 4 21 ± 4 25 ± 13 9.5 ± 0.1 10.1 ± 0.2

S. obliquus   125 ± 8 – 48 ± 3 – 42 ± 1 – 36 ± 2 – 8.6 ± 0.1

S. platensis   128 ± 10 – – – – – – – –

Consortium 1 116 ± 27 – – – – – – – –

Consortium 2 133 ± 9 – 56 ± 3 – 46 ± 1 – 36 ± 3 – 9.9 ± 0.2

* Non cultivate in CO2  as the sole carbon source in the microalgae oxygenation tests.

0

200

400

600

0 100 200 300 400

   O   2

   (  m  g   )

Time (h)

0

200

400

600

0 100 200 300 400

   O   2   (  m  g   )

Time (h)

a b

Fig. 3.  Time course of O2 produced by  S. obliquus  (d), C. sorokiniana  (), consortium 1 (N) (a),  S. platensis  (j) and consortium 2 () (b) under autotrophic conditions.

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tion capacity and the significantly different TOC oxidation rates re-

corded in the biodegradation tests, one can assume that biological

carbon oxidation process is determined by factors others than the

oxygenation capacity of the microalgae selected. The different tol-

erance of microalgae towards the toxic effects of NH3,   which di-

rectly influence microalgae growth, might explain this apparent

mismatch between microalgae oxygenation rates and TOC removal

rates. The oxygenation capacity of  E. viridis  could not be assessed

under the fully autotrophic cultivation conditions used in the oxy-genation tests since the growth of this particular euglenophyt

seems to require an organic carbon source (SAG curator, personal

communication).

 3.3. Nitrogen and phosphorous removal

Based on the low NH4+ removal efficiencies recorded (25–39%)

and the absence of NO3 and NO2

, nitrogen assimilation into bio-

mass was likely the only NH4+ removal mechanism present during

the initial stages of piggery wastewater biodegradation (Fig. 5). To-

tal nitrogen analyses (Shimadzu TNM-1, Tokyo, Japan) of the cen-

trifuged piggery wastewater revealed that 84% of the nitrogen

present was in the form of NH4+. At 1:8 dilution, NH4+-RE priorto acidification of 39 ± 3%, 36 ± 2%, 25 ± 13% and 36 ± 3% were re-

corded in tests inoculated with  E. viridis, S. obliquus, C. sorokiniana

and consortium 2, respectively (Table 1). NH4+-REs of 34 ± 0% and

6

7

8

9

10

11

0 100 200 300 400 500

  p   H

a

6

7

8

9

10

11

0 100 200 300 400 500

  p   H

b

6

7

8

9

10

11

0 100 200 300 400 500

  p   H

T ime (h)

6

7

8

9

10

11

0 100 200 300 400 500

  p   H

Time (h)

c d

Fig. 4.   Time course of pH in enclosed algal–bacterial systems inoculated with E. viridis (a), S. obliquus (b), C. sorokiniana (c) and consortium 2 (d) during the biodegradation of 

piggery wastewater diluted four times (diamonds) and eight times (squares). The acidified duplicate after pH stabilization at each dilution is represented with open symbols

(e andh, respectively). Vertical dashed lines illustrate the time of culture medium acidification.

0

100

200

300

400

0 100 200 300 400 500

   N  -   N   H

   4   +   (  m  g   L  -   1   )

a

0

100

200

300

400

0 100 200 300 400 500

   N  -   N   H   4

   +   (  m  g   L  -   1   )

0

100

200

300

400

0 100 200 300 400 500

   N  -   N   H   4

   +   (  m  g   L  -   1   )

Time (h)

0

100

200

300

400

0 100 200 300 400 500

   N  -   N   H   4

   +   (  m  g   L  -   1   )

Time (h)

b

c d

Fig. 5.   Time course of N–NH4+ in enclosed algal–bacterial systems inoculated with  E. viridis   (a),   S. obliquus   (b),   C. sorokiniana   (c) and consortium 2 (d) during the

biodegradation of piggery wastewater diluted four times (diamonds) and eight times (squares). The acidified duplicate after pH stabilization at each dilution is representedwith open symbols (e andh, respectively). Vertical dashed lines illustrate the time of culture medium acidification.

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21 ± 4% were recorded at day 8 of experimentation in E. viridis and

C sorokiniana   in four times diluted tests. On the other hand, the

occurrence of NH4+ nitrification prior to acidification was ruled

out based on the absence of NO3 and NO2

and the high pH values

present in the cultivation medium. S. obliquus was the only micro-

algae supporting NH4+ nitrification following the 2nd acidification.

Thus, 36 and 8 mg/L of N–NO2 and N–NO3

(accounting for 33% if 

the initial nitrogen present in the test) and a decrease in the culti-

vation pH down to 6.3 were detected at the end of the acidified  S.

obliquus test supplied with eight times diluted wastewater (Fig. 4),

which confirmed the presence of a nitrifying population in the acti-

vate sludge used as inoculum. Unexpectedly, none of the other

microalgae supported ammonium nitrification despite the occur-

rence of N–NH4+, high oxygen concentrations in the headspace

and nitrifying bacteria. The fact that nitrification was only ob-

served in the acidified test containing the less active microalgae

(in terms of pH increase and biomass growth rate) suggest the

occurrence of CO2  limiting conditions for nitrifying growth in the

test carried out with  C. sorokiniana,   E. viridis, and consortium 2.

This limitation in the CO2 available for nitrifiers growth was likely

the result of the prevalence of high pH values and the intense com-

petition between nitrifiers and photosynthetic microorganism. In

our particular case, although nitrification and microalgae growth

can coexist during the biological degradation of piggery wastewa-

ters (de Godos et al., 2009a,b; González et al., 2008), the compe-

tence for IC between both autotrophic groups was likely unequal

due to the large populationof microalgae present in the cultivation

prior to media acidification (Wolf et al., 2006). Likewise, Wett and

Rauch (2003) reported limitations in IC in the nitrification process

of an activated sludge systems treating wastewaters with high

ammonia concentrations. This hypothesis must be however con-

firmed with further experiments specifically devoted to this issue.

Initial concentrations of 19.4 ± 0.8 and 11 ± 2.3 mg P L 1 were

detected in systems supplied the four and eight times diluted

wastewater, correspondingly. C. sorokiniana, S. obliquus  and E. viri-

dis supported TP-RE ranging from 20% to 65% in eight times diluted

wastewaters, while these removals increased up to 45–60% in fourtimes diluted wastewater (Table 2). In this context,  Powell et al.

(2009) recently reported a luxury P uptake at high phosphate con-

centrations in a mixed microalgal consortium dominated by Scene-

desmus. These authors observed up to three times higher

microalgal acid soluble polyphosphate content when phosphate

aqueous concentration increased from 5 to 15 mg P L 1. The max-

imum PO4+-RE corresponded to the acidified S. obliquus  test (65%)

and the lowest values to consortium 2 (7–13%). No clear correla-

tion between media acidification and P-RE could be drawn from

the data herein obtained, with acidified and non-acidified systems

exhibiting comparable removals (except for S. obliquus). This high-

lights the high complexity of P removal mechanisms in microal-

gae-based systems. P removal in algal–bacterial processes involve

from P precipitation (at high pH values) to microbial mediated

assimilation in the form of biomass and intracellular polyphos-

phate compounds and is highly sensitivity to variations in PO43

concentration, light intensity and temperature (Nurdogan and Os-

wald, 1995; Powell et al., 2009).

The final oxygen headspace concentrations in acidified systems

were always substantially higher (2.6 times) than those mea-

sured in non-acidified systems regardless of the microalgae and

piggery wastewater dilution. For example, O2

 headspace concen-

trations of 388 and 266 mg L 1 were measured in eight times di-

luted acidified duplicates of    E. viridis   and   C. sorokiniana,

respectively, compared to 179 and 174 mg L 1 in non-acidified sys-

tems. Likewise, higher final biomass concentrations were found in

acidified systems (940 vs. 666 mg DW L 1 in C. sorokiniana  tests).

These findings confirm the key role of pH/NH3-mediated inhibition

on the bioavailability of IC and therefore photosynthetic biomass

production.

Although important concentrations of heavy metals have been

observed in livestock wastewaters (de la Torre et al., 2000), the

concentrations of Zn+2, Cu+2, As+3 and Pb+2 recorded in this study

were below the quantification limits of the analytical procedures

(Zn+2 0.1, Cu+2 0.15, As+3 0.65 and Pb+2 0.30mg L 1).

 3.4. Piggery wastewater biodegradation in a continuous algal–

bacterial photobioreactor 

Piggery wastewater biodegradation in the continuous photobi-

oreactor was characterized by a rapid microalgal–bacterial growth,

which finally stabilized by day 6 at 237 ± 31 mg DW L 1. pH also

 Table 2

Influence of microalgae species on total phosphorous removal.

TP–RE (%)

1:4 1:8

A N A N

E. viridis   53 60 31 28

C. sorokiniana   54 45 23 20

S. obliquus   – – 65 27

S. platensis   – – – –

Consortium 1 – – – –

Consortium 2 – – 13 7

A: acidified duplicate in the biodegradation tests.

N: non-acidified duplicate in the biodegradation tests.–: no biodegradation.

0

5

10

15

0

100

200

300

0 100 200 300 400

   D   O   C   (  m  g   L  -   1   )

   T   O   C   (  m  g   L  -   1   )

b

0

2

4

6

8

10

0

50

100

150

200

0 100 200 300 400

  p   H

   N  -   N   H   4

   +   (  m  g   L  -   1   )

Time (h)

c

0

100

200

300

400

500

0

25

50

75

100

0 100 200 300 400

   S   S   T

   (  m  g   L  -   1   )

   N   º

  c  e   l   l  s   (   1   0   6   )

a

Fig. 6.  Population dynamics of  C. sorokiniana (N), S. obliquus (d) and S. platensis ()

and total suspended solids (—) (a), time course of inlet (j), outlet (), TOC

concentration and dissolved oxygen concentration (—) (b), and time course of inlet

(h), outlet (e) N–NH4+ concentrations and pH () in a continuous algal–bacterialphotobioreactor treating eight times diluted piggery wastewater.

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increased from 8.1 to steady state values of 9.3 ± 0.3 (Fig 6). S. obli-

quus was the dominant species within the first hours of operation,

reaching its maximum concentration by the 3rd day of cultivation

(55 106 cells L 1). However, the number of cells of this green

microalga rapidly decreased with a concomitant increase in the

number of  C. sorokiniana (Fig. 6a). C. sorokiniana finally outcompet-

ed S. obliquus  by the 6th day of cultivation achieving a number of 

cells of approximately 85 106 cells L 1). No significant number

of cells of  S. platensis was detected during piggery wastewater bio-

degradation, which confirmed the high sensitivity of this cyano-

bacterial species. The dynamics of these three microalgae species

were in agreement with the results obtained in the previous batch

degradation processes: a high NH3-tolerance of   C. sorokiniana, a

moderate tolerance towards NH3 of S. obliquus, and a low tolerance

of  S. platensis. Thus, the low TOC and NH4+ concentrations present

during process start-up (since the photobioreactor was initially

filled with tap water) promoted the initial growth of  S. obliquus,

population which gradually declined with the increase in TOC

and NH4+ concentrations as a result of piggery wastewater input

(Fig 6b and c). Likewise, the gradual increase in N–NH4+ triggered

the proliferation of the highly NH3-tolerant   C. sorokiniana.   The

TOC-RE and NH4+-RE achieved during the steady state operation

of the photobioreactor were similar to those obtained in the batch

biodegradation tests (58 ± 18% and 37 ± 8%, respectively) (Fig. 6b

and c). These results confirmed the validity of the batch biodegra-

dation tests as a tool to select high-performance microalgae for the

continuous biodegradation of piggery wastewater. In addition, the

high steady state dissolved oxygen concentrations recorded in the

microalgal–bacterial cultivation medium (10.6 ± 1.2 mg O2 L 1)

can be considered as an evidence of the complete depletion of 

the biodegradable fraction of the TOC present in the piggery waste-

water (Muñoz et al., 2004). Nitrogen assimilation into algal–bacte-

rial biomass was likely the only NH4+ removal mechanisms, since

neither NO3 nor NO2

were detected into bioreactor medium.

4. Conclusions

This study assessed the ability of two green microalgae, one cya-

nobacterium, oneeuglenophytand twowildmicroalgaeconsortia to

photosynthetically support carbon, nitrogen and phosphorous re-

moval fromdiluted piggery wastewaters. Theresultsfrom thebatch

biodegradation tests, the batch oxygenation tests and the continu-

ous piggery wastewater biodegradation operation confirmed that

tolerance towards ammonia was the most important criterion for

microalgae selection. C. sorokiniana and E. viridis species supported

the highest TOC and NH4+ removal rates which agrees with previous

studies ranking Euglena and Chlorella species among the top10 best

performingmicroalgae-based on their occurrence in highlypolluted

environments. Surprisingly microalgae isolated from polluted envi-

ronments exhibited a poorestperformance than well knownorganic

pollution-resistant microalgae from culture collection. Besides, itwas shown that NH3   inhibition could eventually determine the

dynamics of microalgal population during continuous piggery

wastewater biodegradation. Comparable TOC and NH4+ removal

efficiencies were observed in batch and continuous biodegradation

studies (47–58% and 31–37%, respectively), which agreed with the

maximum TOC biodegradable fraction (54%). In addition, nitrifica-

tion inhibition due to an intense competition between nitrifiers

and microalgae for CO2 was hypothesized based on batch biodegra-

dation test. However, further experiments addressing to this issue

must be performed to confirm this hypothesis.

 Acknowledgements

This research was supported by the Autonomous Governmentof Castilla y León through the Institute of Agriculture Technology

(ITACYL project VA13-C3-1) and the program of Excellence for Re-

search Groups (GR76), the Spanish Ministry of Education and Sci-

ence (RYC-2007-01667 contract and projects CTC2007-64324;

CONSOLIDER-INGENIO 2010 CSD 2007-00055) and the Spanish

International Cooperation Agency (A/016603/08 Project). Araceli

Crespo, Javier Iglesias, Sara Santamarta and the Laboratory of the

Instrumental Techniques of the University of Leon (LTI-ULE) are

gratefully acknowledged.

References

An, J.Y., Sim, S.J., Lee, J.S., Kim, B.W., 2003. Hydrocarbon production from secondary

treated piggery wastewater by the green algae   Botryococcus braunii. J. Appl.

Phycol. 15, 185–191.

ApSimon, H.M., Kruse, M., Bell, J.N.B., 1987. Ammonia emissions and their role in

acid deposition. Atmos. Environ. 21, 1939–1946.

Azov, Y., Goldman, J.C., 1982. Free ammonia inhibition of algal photosynthesis in

intensive cultures. Appl. Environ. Microbiol. 43, 735–739.

Boursier, H., Béline, F., Paul, E., 2005. Piggery wastewater characterization for

biological nitrogen removal process design. Bioresour. Technol. 96, 351–358.

Burton, C.H., Turner, C., 2003. Manure Management. Treatment Strategies for

Sustainable Agriculture, second ed. Silsoe Research Institute, Bedford, United

Kingdom.

Cañizares, R.O., Rivas, L., Montes, C., Dominguez, A.R., 1994. Aerated swine

wastewater treatment with K-carrageenan immobilised   Spirulina maxima.

Bioresour. Technol. 47 (1), 89–91.Carpenter, S.R., Caraco, N.F., Correll, D.L., Howarth, R.W., Sharpley, A.N., Smith, V.H.,

1998. Nonpoint pollution of surface waters with phosphorus and nitrogen. Ecol.

Appl. 8, 559–568.

de Godos, I., González, C., García-Encina, P., Bécares, E., Muñoz, R., 2009a.

Simultaneous nitrification–denitrification, phosphorous and carbon removal

during pre-treated swine slurry degradation in a tubular biofilm

photobioreactor. Appl. Microbiol. Biotechnol. 82 (1), 187–194.

de Godos, I., Blanco, S., García-Encina, P., Bécares, E., Muñoz, R., 2009b. Long-term

operation of high rate algal ponds for the bioremediation of piggery

wastewaters at high loading rates. Bioresour. Technol. 100, 4332–4339.

de la Torre, A.I., Jimenez, J.A., Carballo, M., 2000. Ecotoxicological evaluation of pig

slurry. Chemosphere 41 (10), 1629–1635.

Eaton, A.D., Clesceri, L.S., Greenberg, A.E., 2005. Standard Methods for the

Examination of Water and Wastewater, 21st ed. American Public Health

Association/American Water Works Association/Water Environment

Federation, Washington, DC, USA.

Flotats, X., Bonmatí, A., Fernández, B., Magrí, A., 2009. Manure treatment

technologies: on-farm versus centralized strategies. NE Spain as case study.

Bioresour. Technol. 100, 5519–5526.

Gantar, M.Z., Obreht, Z., Dalmacija, B., 1991. Nutrient removal and algal succession

during the growth of  Spirulina platensis  and Scenedesmus quadricauda  on swine

wastewater. Bioresour. Technol. 37, 167–171.

González, L.E., Canizares, R.O., Baena, S., 1997. Efficiency of ammonia and

phosphorus removal from a Colombian agroindustrial wastewater by the

microalgae Chlorella vulgaris and Scenedesmus dimorphus. Bioresour. Technol. 60

(3), 259–262.

González, C., Marciniak, J., Villaverde, S., García-Encina, P.A., Muñoz, R., 2008.

Microalgal-based processes for the degradation of pre-treated piggery

wastewaters. Appl. Microbiol. Biotechnol. 80, 891–898.

Henderson, R.K., Baker, A., Parsons, S.A., Jefferson, B., 2008. Characterisation of 

algogenic organic matter extracted from cyanobacteria, green algae and

diatoms. Water Res. 42, 3435–3445.

Hoyer, O., Lusse, B., Bernhardt, H., 1985. Isolation and characterisation of 

extracellular organic matter (EOM) from algae. Z. Wasser Abwasser Forsch.

18, 76–90.

Krapaca, I.G., Deya, W.S., Roya, W.R., Smythb, C.A., Stormentc, E., Sargenta, S.L., 2002.

Impacts of swine manure pits on groundwater quality. Environ. Pollut. 120 (2),475–492.

Martinez Sancho, M.E., Jimenez Castillo, J.M., Espinola Lozano, J.B., El Yousfi, F.,

1993. Sistemas algas–bacterias para tratamiento de residuos líquidos. Ing.

Quím. 25, 131–135.

Mulbry, W., Kebede-Westhead, E., Pizarro, C., Sikora, L., 2005. Recycling of manure

nutrients: use of algal biomass from dairy manure treatment as a slow release

fertilizer. Bioresour. Technol. 96, 451–458.

Mulbry, W., Kondrad, S., Pizarro, C., Kebede-Westhead, E., 2008. Treatment of dairy

manure effluent using freshwater microalgae: algal productivity and recovery

manure nutrients using pilot-scale algal turf scrubbers. Bioresour. Technol. 99

(17), 8137–8142.

Muñoz, R., Köllner, C., Guieysse, B., Mattiasson, B., 2003. Salicylate biodegradation

by various algal–bacterial consortia under photosynthetic oxygenation.

Biotechnol. Lett. 25 (22), 1905–1911.

Muñoz, R., Köllner, C., Guieysse, B., Mattiasson, B., 2004. Photosynthetically

oxygenated salicylate biodegradation in a continuous stirred tank

photobioreactor. Biotechnol. Bioeng. 87 (6), 797–803.

Muñoz, R., Díaz, L.F., Bordel, S., Villaverde, S., 2007. Inhibitory effects of catechol

accumulation on benzene biodegradation in   Pseudomonas putida   F1 cultures.Chemosphere 64, 244–252.

I.de Godos et al. / Bioresource Technology 101 (2010) 5150–5158   5157

8/13/2019 40830_Bioremediacion

http://slidepdf.com/reader/full/40830bioremediacion 9/9

Nurdogan, Y., Oswald, W.J., 1995. Enhanced nutrient removal in high rate algae

ponds. Water Sci. Technol. 31, 33–43.

Ogbonna, J.C., Yoshizowa, H., Tanaka, H., 2000. Treatment of a high strength organic

wastewater by a mixed culture of photosynthetic microorganisms. J. Appl.

Phycol. 12 (3–5), 277–284.

Olguín, E.J., Galicia, S., Angulo-Guerrero, O., Hernández, E., 2001. The effect of low

light flux and nitrogen deficiency on the chemical composition of  Spirulina  sp.

( Arthrospira) grown on digested pig waste. Bioresour. Technol. 77 (1), 19–24.

Osada, T., Haga, K., Harada, Y., 1991. Removal of nitrogen and phosphorous from

swine wastewater by the activated sludge units with the intermittent aeration

process. Water Res. 25 (11), 1377–1388.Oswald, W.J., Gotaas, H.B., Golueke, C.G., 1957. Algae in wastewater treatment.

Sewage Ind. Wastes 29 (4), 437–455.

Palmer, C.M., 1969. A composite ranting of algae tolerating organic pollution. J.

Phycol. 5, 78–82.

Park, K.Y., Lim, B.R., Lee, K., 2009. Growth of microalgae in diluted process water of 

the animal wastewater treatment plant. Water Sci. Technol. 59 (11), 2111–

2116.

Pott,B., Mattiasson, B.,2004. Separation of heavy metals from watersolutions at the

laboratory scale. Biotechnol. Lett. 26, 451–456.

Powell, N., Shilton, A., Chisti, Y., Pratt, S., 2009. Towards a luxury uptake process via

microalgae – defining the polyphosphate dynamics. Water Res. 43, 4207–4213.

SAG. Available from:  <http://www.epsag.uni-goettingen.de/>.

Wett, B., Rauch, W., 2003. The role of inorganic carbon limitation in biological

nitrogen removal of extremely ammonia concentrated wastewater. Water Res.

37, 1100–1110.Wilkie, A.C., Mulbry, W.W., 2002. Recovery of dairy manure nutrients by benthic

fresh microalgae. Bioresour. Technol. 84, 81–91.

Wolf, G., Picioreanu, C., van Loodrecht, Mark C.M., 2006. Kinetic modeling of 

phototrophic biofilms: the PHOBIA model. Biotechnol. Bioeng. 97, 1064–1079.

5158   I.de Godos et al. / Bioresource Technology 101 (2010) 5150–5158