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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: [email protected] (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.
I.de Godos et al. / Bioresource Technology 101 (2010) 5150–5158 5155
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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