Algal–bacterial processes for the treatment of hazardous contaminants: A review
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Transcript of Algal–bacterial processes for the treatment of hazardous contaminants: A review
ARTICLE IN PRESS
Available at www.sciencedirect.com
WAT E R R E S E A R C H 4 0 ( 2 0 0 6 ) 2 7 9 9 – 2 8 1 5
0043-1354/$ - see frodoi:10.1016/j.watres
�Corresponding auE-mail address:
journal homepage: www.elsevier.com/locate/watres
Review
Algal–bacterial processes for the treatment of hazardouscontaminants: A review
Raul Munoza,b, Benoit Guieyssea,�
aDepartment of Biotechnology, Center for Chemistry and Chemical Engineering, Lund University, P.O. Box 124, S-22100 Lund, SwedenbDepartamento de Ingenierıa Quımica y Tecnologıa del Medio Ambiente, Universidad de Valladolid, Paseo del Prado de la Magdalena,
s/n, Valladolid, Spain
a r t i c l e i n f o
Article history:
Received 9 March 2006
Received in revised form
14 June 2006
Accepted 15 June 2006
Keywords:
Heavy metals
Industrial wastewater
Microalgae
Organic pollutants
Photobioreactors
Photosynthesis
nt matter & 2006 Elsevie.2006.06.011
thor. Tel.: +46 46 2224228;[email protected]
A B S T R A C T
Microalgae enhance the removal of nutrients, organic contaminants, heavy metals, and
pathogens from domestic wastewater and furnish an interesting raw material for the
production of high-value chemicals (algae metabolites) or biogas. Photosynthetic oxygen
production also reduces the need for external aeration, which is especially advantageous for
the treatment of hazardous pollutants that must be biodegraded aerobically but might
volatilize during mechanical aeration. Recent studies have therefore shown that when proper
methods for algal selection and cultivation are used, it is possible to use microalgae to
produce the O2 required by acclimatized bacteria to biodegrade hazardous pollutants such as
polycyclic aromatic hydrocarbons, phenolics, and organic solvents. Well-mixed photobior-
eactors with algal biomass recirculation are recommended to protect the microalgae from
effluent toxicity and optimize light utilization efficiency. The optimum biomass concentration
to maintain in the system depends mainly on the light intensity and the reactor
configuration: At low light intensity, the biomass concentration should be optimized to avoid
mutual shading and dark respiration whereas at high light intensity, a high biomass
concentration can be useful to protect microalgae from light inhibition and optimize the light/
dark cycle frequency. Photobioreactors can be designed as open (stabilization ponds or high
rate algal ponds) or enclosed (tubular, flat plate) systems. The latter are generally costly to
construct and operate but more efficient than open systems. The best configuration to select
will depend on factors such as process safety, land cost, and biomass use. Biomass harvest
remains a limitation but recent progresses have been made in the selection of flocculating
strains, the application of bioflocculants, or the use of immobilized biomass systems.
& 2006 Elsevier Ltd. All rights reserved.
Contents
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2800
2. The potential of microalgae for treating hazardous contaminants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2800
2.1. Direct use of algae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2800
2.2. Photosynthetic aeration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2801
r Ltd. All rights reserved.
fax: +46 46 2224713..se (B. Guieysse).
ARTICLE IN PRESS
WAT E R R E S E A R C H 4 0 ( 2 0 0 6 ) 2 7 9 9 – 2 8 1 52800
3. Microbial selection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2802
3.1. Microalgae tolerance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2802
3.2. Microbial interactions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2803
3.3. Microbial growth rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2803
3.4. Microalgae predominance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2804
3.5. Inoculation and selection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2804
4. Photobioreactor design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2804
4.1. Open bioreactors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2804
4.2. Closed photobioreactors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2805
4.3. Mixing. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2805
4.4. Biomass harvesting and biomass retention . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2805
4.5. Biomass concentration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2807
4.6. Surface/volume ratio . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2807
4.7. Hydraulic retention time (HRT) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2808
5. Influence of environmental parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2808
5.1. pH . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2808
5.2. Temperature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2809
5.3. Light supply . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2809
5.4. Dissolved oxygen concentration (DOC) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2809
5.5. Predators. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2809
6. Future prospects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2809
6.1. Potential uses of the algal–bacterial biomass . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2809
6.2. Combining wastewater treatment with CO2 mitigation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2810
7. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2810
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2810
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2811
1. Introduction
Microalgae play an important role during the tertiary treat-
ment of domestic wastewater in maturation ponds or
the treatment of small–middle-scale municipal wastewater
in facultative or aerobic ponds (Aziz and Ng, 1993; Abeliovich,
1986; Mara and Pearson, 1986; Oswald, 1988, 1995).
They enhance the removal of nutrients, heavy metals and
pathogens (Table 1) and furnish O2 to heterotrophic aerobic
bacteria to mineralize organic pollutants, using in turn
the CO2 released from bacterial respiration (Fig. 1). Photo-
synthetic aeration is therefore especially interesting to
reduce operation costs and limit the risks for pollutant
volatilization under mechanical aeration and recent
studies have shown that microalgae can indeed support the
aerobic degradation of various hazardous contaminants
(Munoz et al., 2004; Safonova et al., 2004). Unfortunately,
microalgae are usually quite sensitive towards the hazardous
compounds (Aksmann and Tukaj, 2004; Borde et al., 2003)
and special care must be taken to improve microbial
activity. Hazardous pollutants include a wide range of toxic
and/or persistent substances that can be found in all
environmental compartments. This review will however
focus on the application of algal-based processes for the
detoxification of industrial effluents which biological treat-
ment requires aerobic conditions (biodegradation of recalci-
trant and toxic contaminants) and external oxygen supply
(i.e. highly loaded wastewater). Guidelines for the design,
start-up, and operation of algal–bacterial processes are
provided and discussed, and the areas for further research
are identified.
2. The potential of microalgae for treatinghazardous contaminants
2.1. Direct use of algae
The mechanisms involved in microalgal nutrient removal from
industrial wastewater are similar than that from domestic
wastewater treatment (Table 1). Nutrients are also not con-
sidered as hazardous pollutants and this will not be discussed
further. However, algal-based treatment is especially interest-
ing in the case of N-containing contaminants whose biode-
gradation normally leads to NH4+ or NO3
� release. For instance,
the net amount of NH4+ produced per mole of acetonitrile
biodegraded decreased from 0.74 mol mol�1 in mechanically
aerated batch processes to 0.46 mol mol�1 in photosynthetically
oxygenated batch processes due to algal assimilation (Munoz et
al., 2005a,b). This ratio was further decreased to 0.17 mol mol�1
when the algal–bacterial process was operated in continuous
mode at a HRT of 3.5 d (Munoz et al., 2005a, b).
Heavy metals represent an important group of hazardous
contaminants often found in industrial wastewater (Kratoch-
vil and Volesky, 1998; Volesky, 2001). Microalgae can be
efficiently use to remove these pollutants (Tables 1 and 2)
and a specific metal uptake of 15 mg gBiomass�1 at 99% removal
efficiency has been reported, showing that the process is
competitive compared to other treatment methods (Cani-
zares-Villanueva, 2000). The removal of heavy metals by algae
is therefore well described in the literature and will not be
discussed further in this review (for general reviews, see
Wilde and Benemann, 1993; Perales-Vela et al., 2006).
Microalgae can finally biodegrade hazardous organic pollu-
tants and Chlorella, Ankistrodesmus or Scenedesmus species
ARTICLE IN PRESS
Table 1 – Main applications of microalgae during WWT
Application Comment References
BOD removal Microalgae release 1.5–1.92 kg O2 kg�1 of microalgae produced
during photoautotrophic growth and oxygenation rates of
0.48–1.85 kg O2 m�3 d�1 have been reported in pilot-scale ponds
or lab-scale tank photobioreactors treating municipal or
artificially contaminated wastewater
Grobbelaar et al., 1988; Martinez Sancho
et al., 1993; McGriff and McKinney, 1972;
Munoz et al., 2004; Oswald, 1988
Nutrient removal Microalgae assimilate a significant amount of nutrients because
they require high amounts of nitrogen and phosphorous for
proteins (45–60% of microalgae dry weight), nucleic acids and
phospholipids synthesis. Nutrient removal can also be further
increased by NH3 stripping or P precipitation due to the raise in
the pH associated with photosynthesis
Laliberte et al., 1994; Oswald, 2003;
McGriff and McKinney, 1972; Nurdogan
and Oswald, 1995; Vollenweider, 1985
Heavy metal
removal
Photosynthetic microorganisms can accumulate heavy metals
by physical adsorption, ion exchange and chemisorption,
covalent bonding, surface precipitation, redox reactions or
crystallization on the cell surface. Active uptake that often
involves the transport of the metals into the cell interior is often
a defensive tool to avoid poisoning or it serves to accumulate
essential trace elements. Microalgae can also release
extracellular metabolites, which are capable of chelating metal
ions. Finally, the increase in pH associated with microalgae
growth can enhance heavy metal precipitation
Chojnacka et al., 2005 Kaplan et al., 1995;
Kaplan et al., 1987; Rose et al., 1998;
Travieso et al., 1996; Van Hille et al., 1999.
Wilde and Benemann, 1993; Yu and
Wang, 2004
Pathogen removal Microalgae enhance the deactivation of pathogens by raising
the pH value, the temperature and the dissolved oxygen
concentration of the treated effluent
Aiba, 1982; Mallick, 2002; Mezrioui et al.,
1994 ; Robinson, 1998; Schumacher et al.,
2003
Heterotrophic
pollutant removal
Certain green microalgae and cyanobacteria are able to use
toxic recalcitrant compounds as carbon, nitrogen, sulphur or
phosphorous source
Semple et al., 1999; Subaramaniana and
Uma, 1997
Biogas production CH4 production from the anaerobic digestion of algal–bacterial
biomass allows substantial economical savings
Eisenberg et al., 1981; Oswald, 1976
Toxicity
monitoring
Microalgae are used in toxicity tests or in studies of microbial
ecology as they are sensitive indicators of ecological changes
Day et al., 1999
CO2
Microalgalphotosynthesis
O2
Bacterialoxidation
Light
BiomassOrganicmatter
Fig. 1 – Principle of photosynthetic oxygenation in BOD
removal processes.
WAT E R R E S E A R C H 40 (2006) 2799– 2815 2801
have been successfully used for the treatment of olive oil mill
wastewater and paper industry wastewater (Abeliovich and
Weisman, 1978; Narro, 1987; Pinto et al., 2002, 2003; Tarlan
et al., 2002). Lima et al. (2003) reported p-nitrophenol removal
of 50 mg l�1 d�1 by a consortium of Chlorella vulgaris and
Chlorella pyrenoidosa under non-optimized conditions, which
was close to the 100 mg l�1 d�1 achieved with Pseudomonas sp.
by Kulkarni and Chaudhari (2006). However, heterotrophic
microalgae can be out-competed by heterotrophic bacteria in
continuous open systems because microalgae often exhibit
lower specific growth rates than bacteria (Semple et al., 1999;
Lee, 2001). The applicability of pollutant biodegradation by
algae therefore remains uncertain and should be further
investigated.
2.2. Photosynthetic aeration
Mechanical aeration accounts for more than 50% of the total
energy consumption of typical aerobic wastewater treatments
(Tchobanoglous et al., 2003): Hence, microalgae can improve
the energy-efficiency of BOD removal from domestic waste-
water by providing O2 to the heterotrophic aerobic bacteria
(Fig. 1). This synergistic relationship can also be used for the
economical treatment of hazardous contaminants, which is
also safer as there is less risk of pollutant or aerosol release
than during intensive mechanical aeration (Brandi et al., 2000;
Hamoda, 2006). This is especially advantageous knowing that
many recalcitrant and toxic compounds are much easier to
degrade aerobically than anaerobically. For instance, a micro-
algae–bacteria consortium was successfully used for the
degradation of black oil and the detoxification of industrial
wastewater in Russia (Safonova et al., 1999, 2004). Likewise,
Chlorella sorokiniana was able to support the aerobic degrada-
tion of phenanthrene, acetonitrile, phenol, and salicylate by
pollutant-specific bacteria without any external O2 supply
(Borde et al., 2003, Guieysse et al., 2002; Munoz et al., 2005a, b).
Salicylate was thus totally converted under photosynthetic
oxygenation into biomass by the symbiotic consortium
Ralstonia basilensis–C. sorokiniana with an excess of O2 produc-
tion according to the following reactions (Borde et al., 2003):
ARTICLE IN PRESS
Table 2 – Reported studies on heavy metal accumulation by microalgae
Metal Biomass Accumulationcapacity
(mg gBiomass�1 )
Adsorptionremoval rate(mg l�1 d�1)
Experimentalset-up
Reference
Zn Chlorella vulgaris — 114.2 1-l column reactor
with microalgae
immobilized in
k-carrageenan
Travieso et al., 1999
Cr Scenedesmus acutus — 3.5
Cd Chlorella vulgaris — 2.5
Co Scenedesmus
obliquus
0.82 Rotary biofilm
reactor
Travieso et al., 2002
Zn Euglena gracilis 7.5 — 500-ml E-flasks,
free
microorganisms
Fukami et al., 1988
Cd Chlorella
Homosphaera
8.4 1.44 500-ml E-flasks,
free
microorganisms
Zn Chlorella
Homosphaera
15.6 2.67 Costa and Leite
(1990)
Cd Chlorella vulgaris 2.6 — 1-l E-flasks, free
microorganisms
Khoshmanesh
et al. (1996)
Chlorella
pyrenoidosa
2.8 —
Chlamydomonas
reinhardtii
2.3 —
Al Scenedesmus
subspicatus
6.8 — 50-ml
polyethylene-
flasks, free
microorganisms
Schmitt et al., 2001
Cd 7.3 —
Cu 13.2 —
Hg 9.2 —
Cd Chlorella
sorokiniana
192 — Column reactor
with algae
immobilized on a
vegetable sponge
Akhtar et al., 2003
WAT E R R E S E A R C H 4 0 ( 2 0 0 6 ) 2 7 9 9 – 2 8 1 52802
Salicylate mineralisation by R. basilensis (Borde et al., 2003)
C7H6O3 þ 0:396 NO�3 þ 0:396 Hþ þ 4:0795 O2
) 5:02 CO2 þ 1:515 H2Oþ 1:98 CH1:7O0:4N0:2.
C. sorokiniana Photosynthesis
5:02 ðCO2 þ 0:7609 H2Oþ 0:15 NO�3 þ 0:1782 Hþ þ 0:0094 PO3�4 ) CH1:7O0:4N0:15P0:0094 þ 1:4243 O2Þ
C7H6O3 þ 1:149 NO�3 þ 0:047 PO3�4 þ 2:3047 H2Oþ 1:291 Hþ ) 3:070 O2 þ 1:98 CH1:7O0:4N0:2 þ 5:02 CH1:7O0:4N0:15P0:0094
.
CH1.7O0.4N0.2 (Atkinson and Mavituna, 1983) and
CH1.7O0.4N0.15P0.0094 (Oswald, 1988) represent the biomass
compositions of bacteria and algae, respectively.
The same consortium was able to remove sodium salicylate
at a maximum rate of 87mg l�1 h�1 in a continuous enclosed
photobioreactor (Munoz et al., 2004). This corresponded to an
oxygenation capacity of 77mg O2 l�1 h�1 close to that of large-
scale mechanical surface aerators (125 mg O2 l�1 h�1; Boon,
1983). Likewise, 2.3 g acetonitrile l�1 d�1 was removed in a
continuous column photobioreactor inoculated with C. soro-
kiniana and a bacterial consortium, which was comparable
to the 0.91g l�1 d�1 achieved by Dhillon and Shivaraman (1999)
in a continuous 19-l trickling filter bioreactor or the 1.04g l�1 d�1
reported by Manolov et al. (2004) in a 20-l aerobic packed-
bed reactor. These results clearly illustrate the potential
advantages of photobioreactors for the treatment of in-
dustrial wastes. However, new limitations might arise from
the fact that microalgae are generally more sensitive to
hazardous pollutants and grow at slower rates than their
pollutant-degrading bacterial partner. Special care must there-
fore be given in selecting the consortia and supporting
microalgal activity.
3. Microbial selection
3.1. Microalgae tolerance
Microalgae are generally sensitive to toxic pollutants and
are even recommended as test microorganisms for the
ARTICLE IN PRESS
Microalgae Bacteria
- Temperature increase- pH increase - DOC increase - Bactericides
+ Growth promoters + DOC decrease
- Algaecide
+ CO2 consumption+ Extracellular matter
Fig. 2 – Positive (dashed line) and negative (plain line)
interactions between microalgae and bacteria.
WAT E R R E S E A R C H 40 (2006) 2799– 2815 2803
measurement of acute toxicity (OECD201, 1984; Chen and Lin,
2006). Heavy metals are particularly strong inhibitors of
microbial photosynthesis (Clijsters and Vanassche, 1985) that
can also cause morphological changes in the shape and size
of microalgae cells (Pena-Castro et al., 2004; Travieso et al.,
1999). Salicylate removal under photosynthetic oxygenation
by C. sorokiniana was therefore totally inhibited in the
presence of 2 mg Cu2+ l�1 (Munoz et al., 2006a). However, the
system was efficiently protected by pre-treating the effluent
with the algal–bacterial biomass generated during salicylate
degradation (Munoz et al., 2006a). Microalgae are also
sensitive to organic pollutants as Chen and Lin (2006) showed
that in an air-tight environment (i.e. simulating closed
photobioreactors), PCP inhibited Pseudokircheneriella subcapita-
ta (EC50-48 h of 0.004–0.013 mg l�1) more than Daphnia magna
(EC50-48 h of 0.55 mg l�1; Kuhn et al., 1989). Chlorella are more
tolerant with PCP EC50-96 h values ranging from 0.05 (Mostafa
and Helling, 2002) to 3.77 mg l�1 (Iannacone et al., 2001) but
remain more sensitive than activated sludge microflora (IC50
value of 31.2 mg l�1; Chan et al., 1999). Hence, microalgae are
more likely to be inhibited during the treatment of hazardous
compounds than their associated degrading bacteria (which
are normally better equipped to resist their substrate). For
instance, 10 mg phenanthrene l�1 totally inhibited the growth
of C. sorokiniana whereas a phenanthrene-degrading Pseudo-
monas strain used to form the consortium easily biodegraded
this compound at 25 mg l�1 (Borde et al., 2003).
Microalgae are also sensitive to the combined effect of high
NH3 concentrations and high pH values because NH3 un-
couples the electron transport in photosystem II and com-
petes with H2O in the oxidation reactions leading to O2
generation (Azov and Goldman, 1982). For instance, Abelio-
vich and Azov (1976) observed a decline in the efficiency of a
high rate algal pond (HRAP) when NH3 concentrations and pH
were simultaneously above 2 mM and 8, respectively. Like-
wise, Munoz et al. (2005b) reported the complete inhibition of
C. sorokiniana at a total NH3/NH4+ concentration of 15 mM and
pH 8.7 during the photosynthetically oxygenated treatment of
2 g l�1 of acetonitrile in a 50-l column photobioreactor.
The use of NH3-tolerant microalgae can improve the process
stability as Ogbonna et al. (2000a) reported no significant
effect on the growth of C. sorokiniana at 22 mM NH3
whereas Spirulina platensis was nearly completely inhibited
by 11 mM NH3.
Resistant strains can be obtained by genetic manipulation,
cell acclimation to progressively higher pollutant concentra-
tions, or isolation from contaminated sites where indigenous
microorganisms have already been exposed to the target
contaminants (Malik, 2004). For instance, Essam et al. (2006)
isolated a C. vulgaris–Alcaligenes consortium from the treat-
ment plant of a coking factory effluent containing phenolics
that was able to treat simulated wastewater. However, the
algae were still inhibited by un-characterized organic com-
pounds present in the real wastewater. This problem was
solved by pre-treating the effluent (UV irradiation or activated
carbon adsorption). A different approach was used by Munoz
et al. (2003a) to prevent algal inhibition during the treatment
of phenanthrene by a C. sorokiniana–Pseudomonas sp. con-
sortium: the culture was mixed with an immiscible, biocom-
patible organic phase (silicone oil) that was used to lower the
aqueous concentration of phenanthrene (thereby lowering its
toxicity). Thus, the consortium was able to biodegrade
phenanthrene initially supplied at 200–500 mg l�1 at a max-
imum rate of 24.2 mg l�1 h�1 without problems of solvent
emulsification (common under intensive mechanical aera-
tion). For more information about two-liquid-phase systems,
see the reviews of Daugulis (2001) and Deziel et al. (1999).
3.2. Microbial interactions
The symbiotic microalgal–bacterial relationship is clear when
microalgae provided the O2 necessary for aerobic bacteria to
biodegrade organic pollutants, consuming in turn the CO2
released from bacterial respiration (Fig. 1). However, micro-
algae and bacteria do not limit their interactions to a simple
CO2/O2 exchange (Fig. 2). Microalgae can have a detrimental
effect on bacterial activity by increasing the pH, the dissolved
oxygen concentration (DOC) or the temperature of the
cultivation broth, or by excreting inhibitory metabolites
(Oswald, 2003; Schumacher et al., 2003). They can however
enhance bacterial activity by releasing extracellular com-
pounds as shown by Wolfaardt et al. (1994) who observed that
diclofop methyl removal by a bacterial consortium increased
up to 36% when actively growing algae or their metabolites
were added to the culture. Similarly, bacterial growth can
enhance microalgal metabolism by releasing growth-promot-
ing factors (Fukami et al., 1997; Gonzalez and Bashan, 2000) or
by reducing O2 concentration in the medium (Mouget et al.,
1995; Paerl and Kellar, 1978). De-Bashan et al. (2002),
for instance, reported that the presence of Azospirillum
brasilense enhanced ammonium and phosphorous removal
by C. vulgaris. Bacteria can also inhibit microalgae by
producing algicidal extracellular metabolites (Fukami et al.,
1997).
3.3. Microbial growth rate
Due to their larger size, microalgae generally grow at slower
rates than heterotrophic bacteria (Fenchel, 1974). In particu-
lar, toluene-degrading Pseudomonas sp. can grow at specific
growth rates of 0.4–0.8 h�1 (Reardon et al., 2000) whereas even
the fast growing Chlorella can hardly grow at rates higher than
0.2 h�1 (Lee, 2001). Hence, pollutant removal is often limited
by O2 production in algal–bacterial systems, which is directly
linked to microalgal activity (Guieysse et al., 2002; Munoz
ARTICLE IN PRESS
Fig. 3 – Aeral view of the Cyanotech Corporation’s
microalgae production facility in Kona, Hawaii. Courtesy of
Cyanotech (USA).
WAT E R R E S E A R C H 4 0 ( 2 0 0 6 ) 2 7 9 9 – 2 8 1 52804
et al., 2004, 2005a, b). Rapidly growing microalgae, which also
exhibit high O2 production rates, should therefore be pre-
ferred as O2 suppliers. Munoz et al. (2003b), for instance,
compared the ability of C. sorokiniana, C. vulgaris, Scenedesmus
obliquus, and Selenastrum capricornutum to support the biode-
gradation of salicylate by a Ralstonia basilensis strain and
showed that C. sorokiniana exhibited the highest specific
growth rates (0.045 h�1) and supported the fastest pollutant
removal rates (18 mg salicylate l�1 h�1). In comparison, Sc.
obliquus exhibited a specific growth rate of 0.013 h�1 and
supported the degradation of 5 mg salicylate l�1 h�1.
3.4. Microalgae predominance
The predominance of slow growing microalgae can be
difficult to maintain in continuous systems due to contam-
ination by small and rapidly growing microalgae (Hoffman,
1998). Closed photobioreactors allow for a better species
control and should therefore be preferred (Tredici, 1999) in
situations where slow growing algae are required (i.e. self-
aggregating microalgae). Attempts to sustain specific micro-
algal populations by manipulating the operational variables
have not always been successful as for instance, Benemann et
al. (1980) failed to maintain Oscillatoria sp. and Micractinium sp.
by microscreening and recirculation of the biomass into the
photobioreactor. However, Wood (1987) successfully estab-
lished the predominance of a Stigeoclonium strain by combin-
ing a short hydraulic retention time (HRT), to wash the freely-
suspended microalgae, with crossflow microscreening of the
target strain. The effluent composition and seasonal environ-
mental conditions also strongly influence microbial predomi-
nance (Fukami et al., 1997; Mara and Pearson, 1986): Euglena
and Chlamydomonas dominate at high organic loads in sewage
treatment while Chlorella and Scenedesmus are the most
abundant species at medium loads (Martinez Sancho et al.,
1993). Euglena and Scenedesmus species also predominate over
Chlorella below 15 1C due to their higher tolerance to low
temperatures (Mara and Pearson, 1986).
3.5. Inoculation and selection
Because microalgae activity and sensitivity usually limit the
removal rate of hazardous pollutants in algal–bacterial
systems, it is important to select fast growing and highly
resistant microalgae. Fortunately, rapidly growing Chlorella
and Scenedesmus sp. naturally dominate most continuous
microalgal-based treatment systems (Garcia et al., 2000a;
Martinez Sancho et al., 1993) and Chlorella species are also
considered as highly resistant microalgae (Palmer 1969;
Munoz et al., 2003b). To start-up facultative (with algae on
the top layer) or maturation (aerobic) ponds for domestic
wastewater treatment, it is therefore sufficient to fill up the
systems with freshwater in order to allow for the develop-
ment of algae and heterotrophic bacteria (UNEP, 2005; Mara
and Pearson, 1998). Raw sewage or activated sludge can be
used when fresh water is not available. A similar strategy is
used for HRAP (water from other ponds can also serve as
inoculum, Tryg Lundquist, Lawrence Berkeley Laboratory,
USA, personal communication) and could be applied for the
treatment of hazardous contaminants. This would permit the
co-selection of the bacteria and algae and ensure that the
microorganisms are compatible with each other. However,
isolation with pre-selected specific strains might be necessary
when, for instance, the target contaminants are too recalci-
trant or too toxic or where there is a need for specific
microalgae (for pigment production, easier harvesting, etc.).
Microbial interaction effects and microbial stability should
then be carefully investigated (Munoz et al., 2003b).
4. Photobioreactor design
As seem above, pollutant removal by algal–bacterial consortia
is often limited by oxygen supply. Hence, without taking into
account any economical consideration, photobioreactors for
the treatment of pollutant-laden effluents and photobioreac-
tors for microalgal mass cultivation (Fig. 3) share the same
basic design criteria: high light utilization efficiency, good
scalability, efficient mixing, control over the reaction condi-
tions, and low hydrodynamic stress on the photosynthetic
cells (Borowitzka, 1999;Lee and Lee, 2003; Pulz, 2001; Tredici,
1999).
4.1. Open bioreactors
Photosynthetic microorganisms can be cultivated in open or
closed reactors (Chaumont, 1993; Molina-Grima, 1999). Typi-
cal aerobic ponds used for WWT are large and shallow open
ditches without internal mixing (Mara and Pearson, 1986,
Racault and Boutin, 2005). They are generally designed upon a
surface-loading criterion such as for instance, 11 m2 per
population equivalent (p.e.) (European Commission, 2001).
These systems, which are not specifically designed to
optimize microalgal activity, were early challenged by Oswald
(1988) who designed HRAPs in order to match algal growth
and O2 production with the BOD of the receiving wastewater.
These are 2–3 m wide and 0.1-–0.3 m depth shallow open
ponds built in a raceway configuration (Fig. 3), lined with PVC,
clay or asphalt to avoid infiltration and range from 1000 to
5000 m2 in large-scale applications (Abeliovich, 1986; Molina-
Grima, 1999). Under optimal conditions, HRAPs can treat up to
35 g �BOD �m�2 d�1 (175 g BOD �m�3 d�1 in a 0.2 m deep pond)
ARTICLE IN PRESS
WAT E R R E S E A R C H 40 (2006) 2799– 2815 2805
compared to 5–10 g �BOD �m�2�d�1 (5-10 g BOD �m�3 d�1 in a
1 m deep pond) in waste stabilization ponds (Racault and
Boutin, 2005). This superior design also allows for continuous
operation at 2–6 d HRT (Mara and Pearson, 1986) compared to
10–40 d in traditional ponds (Crites and Tchobanoglous, 1998).
However, given the merits of HRAPs, there are nowadays only
a few full-scale systems in operation (De la Noue et al., 1992;
Mara and Pearson, 1986).
4.2. Closed photobioreactors
Enclosed photobioreactors offer higher photosynthetic effi-
ciencies and better control than open systems (less risks of
pollutant volatilization and predation). They can also be built
vertically in order to minimize space requirement (Pulz, 2001;
Tredici, 1999) and minimize water looses by evaporation
which can be very significant in open systems (Pulz, 2001).
Unfortunately, closed systems are also more expensive to
construct (need for transparent materials such as Plexiglas,
glass, PVC, etc.) and difficult to operate and scale up. Enclosed
photobioreactors are often designed as tubular or flat plate
photobioreactors arranged in a horizontal, inclined, vertical
or spiral manner (Fig. 4) (Tredici, 1999). Tubular photobior-
eactors (Fig. 5) are the easiest to scale up by increasing the
length and number of tubes and by the connection of several
units via manifolds (Borowitzka, 1999). They also exhibit
higher light utilization efficiencies than flat plate photobior-
eactors because of the larger reactor surface area per unit of
occupied land (Tredici and Zittelli, 1998). Thus, oxygenation
rates of up to 4.3 kg O2 m�3 d�1 have been achieved in tubular
reactors (Torzillo et al., 2003). This is significantly higher than
the oxygenation rates in ponds and HRAP reported above and
is comparable to the maximum oxygenation capacity of
mechanical surface aerators (3 kg O2 �m�3 d�1, Boon 1983).
Few studies are available on the application of algae for the
treatment of hazardous pollutants and industrial wastes
(Table 3). To the best of our knowledge, only two commercial
Exhaust Air
CO2 enriched air CO2 enriched air
Exh
(A) (B)
Fig. 4 – Schematic representation of (A) a vertical spiral (Biocoil
photobioreactor for mass algal cultivation and fed with air enri
enclosed photobioreactors have so far been tested for waste-
water treatment: the Bio-Fence manifold tubular reactor
(Applied Photosynthetic Limited, Manchester, United King-
dom) with a total volume ranging from 0.050 to 1 m3, and a
helical tubular reactor called Biocoil (Biotechna-Graesser A.P.
Ltd, Australia) with a maximum working volume of 10 m3
(Tredici, 1999). Unfortunately, no data is available on the
removal and oxygenation rates achieved in these systems.
Hence, only few guidelines, mainly based on the oxygenation
capacity (BOD removal capacity) achievable in each config-
uration, can be given to design photobioreactors for large-
scale treatment (Table 4).
4.3. Mixing
Algal ponds are typically operated as plug-flow systems.
However, homogenous conditions in the reactor are prefer-
able during the treatment of toxic effluent as pollutant
dilution lower the risk of microalgae inhibition. Mixing also
limits the formation of anaerobic zones and more generally
reduce any mass transfer limitations (Grobbelaar, 2000). The
device used for mixing should be selected to reduce shear
stress imposed to the microalgal cells (Barbosa et al., 2004;
Mitsuhashi et al., 1995). Gudin and Chaumont (1991) reported
an increase of up to 75% in microalgal productivity when
pumps were replaced by an airlift system to suspend the cells.
Paddle wheels are therefore often used for algal mass
cultivation in open ponds and in HRAP as they provide a cost
efficient gentle mixing.
4.4. Biomass harvesting and biomass retention
Biomass harvesting is necessary to ensure a good effluent
quality (low suspended solids concentration) and prevent cell
washout during continuous operation (Evans and Furlong,
2003; Munoz et al., 2004, 2005; Richmond, 1983). Unfortu-
nately, none of the common industrial approaches (filtration,
CO2 enriched Air
Exhaust Airaust Air
(C)
), (B) an inclined tubular column, and (C) a vertical flat-plate
ched with CO2.
ARTICLE IN PRESS
WAT E R R E S E A R C H 4 0 ( 2 0 0 6 ) 2 7 9 9 – 2 8 1 52806
centrifugation, microstraining, etc.) have been proven to be
economical and suitable for large-scale microalgae removal
(Hoffman, 1998). Wastewater pond effluents are therefore
often characterized by high TSS (Total Suspended Solids)
values, which is especially problematic in the case of
industrial effluents since the biomass might contain heavy
metals or hydrophobic organic compounds.
Microalgal flocculation followed by gravity sedimentation is
the most common harvesting technique during wastewater
treatment because of the large volumes treated and the low
value of the biomass generated (Nurdogan and Oswald, 1996;
Molina-Grima et al., 2003). Unfortunately, this approach is not
always efficient, especially in the case of the small, rapidly
growing Chlorella or Scenedesmus sp. (Garcıa et al., 2000b).
Instead, multicellular cyanobacteria of the genus Spirulina or
Fig. 5 – Outdoor tubular photobioreactor from the Easy
Algaes production facility, Cadiz, Spain. Courtesy of Easy
Algae (Spain).
Table 3 – Organic pollutant removal by algal–bacterial or micro
Compound Experimentalsystem
Microorga
Acetonitrile 600 ml Stirred Tank
Reactor (STR)
C. sorokiniana
consorti
Acetonitrile 50-l column
photobioreactor
C. sorokiniana
consorti
Black oil 5-ml tubes Chorella/Scen
alcanotrophic
Black oil 100 l tank Chorella/Scen
Rhodococcu/Ph
Phenanthrene 2-l STR with silicone oil
at 10%
C. sorokin
Pseudomonas
Phenanthrene 50 ml tubes with
silicone oil at 20%
C. sorokin
Pseudomonas
Phenol 600 ml STR with
NaHCO3 at 8 g l�1
C. vulgaris/A
sp.
Phenol 100 ml E-flasks Anabaena v
Salicylate 600 ml STR C. sorokiniana/
basilen
p-Nitrophenol — C. vulgar
pyrenoid
the self-aggregating Phormidium bohneri have been success-
fully applied in wastewater treatment of farm effluents
(Olguin, 2003). Gutzeit et al. (2005) also recently described a
self-aggregating algal-bacterial process for domestic WWT
where algal-bacterial flocks ranging from 400 to 800mm were
easily removed by gravity. In our laboratory, biomass auto-
flocculation was observed during the continuous degradation
of salicylate supported by a C. sorokiniana when the photo-
bioreactor was operated at high HRT (unpublished data).
Microalgal autoflocculation can be caused by electrostatic
interactions among the cell walls as a result of Ca/Mg
carbonate or ortophosphate precipitation at high pH (Oswald,
1988). Bioflocculation can occur due to the microalgal release
of long-chain polymers (Garcia et al., 1998). However these
mechanisms are still poorly understood and hard to induce.
The addition of chemical flocculants such as lime, alum or
polyferric sulfate is efficient and reliable but chemical
flocculants remain expensive and increase the effluent
salinity. Instead, chitosan is an edible, economical
(2 US$ �kg�1, 2002) and non-toxic flocculant that is efficient
for the removal of freshwater microalgae (Divakaran and
Sivasankara, 2002). Biomass removal efficiencies of 90% were
thus obtained using 15 mg chitosan � l�1 in our laboratory
during the batch degradation of acetonitrile by an algal-
bacterial consortium (unpublished data). The use of biofloc-
culants from bacteria present within the microcosms is
another very interesting alternative that should be further
investigated (Oh et al., 2001). Finally, recent developments
made in the construction of membrane bioreactors have
made this technology most affordable and increasingly
popular for wastewater treatment (Yang et al., 2006). Such
algal consortia
nisms Removal rate(mg l�1 d�1)
Reference
/bacterial
um
2300 Munoz et al., 2005a
/bacterial
um
432 Munoz et al., 2005b
edesmus/
bacteria
— Safonova et al., 1999
edesmus/
ormidium
5.5 Safonova et al., 2004
iana/
migulae
192 Munoz et al., 2005c
iana/
migulae
576 Munoz et al., 2003a
lcalıgenes 90 Essam et al., 2006
ariabilis 4.4 Hirooka et al., 2003
Ralstonia
sis
2088 Munoz et al., 2004
is/C.
osa
50 Lima et al., 2003
ARTICLE IN PRESS
Table 4 – Comparison of large-scale photobioreactors
Reactor Max.oxygenationcapacity (kgO2 m�3 d�1)a
Lightutilizationefficiency
Scalability Example ofdesign criteria &
featuresb
Reference
WSP 0.01c Very low Easy 11 m2 per
equivalent person,
1 m depth
Racault and
Boutin, 2005
HRAP 0.3–0.38 Low Easy Raceways of 2–3 m
wide and 0.1–0.3 m
deep ponds
Molina-Grima et
al., 1999
Tubular 5.4–6.9 Very high Easy Tubes of 10–100 m
length and 3–6 cm
+
Lee and Low, 1991
Flat plate 6.5–8.3c Very high Difficult Light path 1–5 cm Hu et al., 1996
Tubular (coil) 1.8–2.3c Very high Easy Tube diameter
2–3 cm, cylindrical
structure 8 m
height, 2 m +
Borowitzka, 1999
Vertical
column
3.1–2.4c High Difficult 0.3–0.5 m + and
2–4 m high
columns
Miron et al., 1999
a Except for WSP and the tubular photobioreactor, the oxygenation rates were calculated from reported biomass productivities and conversion
factors of 1.5–1.92 kg O2 kg�1 microalgae.b According to Tredici (1999), Borowitzka (1999), and Janssen et al. (2003).c Based on a pond depth of 1 m, Racault and Boutin (2005).
WAT E R R E S E A R C H 40 (2006) 2799– 2815 2807
bioreactors have been used for the production of algal
pigments (Rossignol et al., 2000) but their potential for algal-
based wastewater treatment must still be proven.
Biomass immobilization is an efficient mean of retaining
biomass during WWT (Nicolella et al., 2000) and microalgae
immobilization in polymeric material such as carrageenan,
chitosan, or alginate has been reported by various authors
(Chevalier and De la Noue, 1985; Lau et al., 1995; Robinson et
al., 1998). However, these matrices are weak and costly, which
has limited their large-scale application (Hoffman, 1998).
Another approach consists on using enclosed photobioreac-
tors where the algal-bacterial microcosm is attached onto the
reactor walls (Munoz et al., 2006b). For instance, Craggs et al.
(1996) successfully operated a shallow open photobioreactor
with the algal-bacterial biomass attached onto the reactor
base for the treatment of agricultural run-off and domestic
wastewater (algal-turf scrubber). Such systems could be
advantageously designed to reduce effluent toxicity (Fig. 6).
4.5. Biomass concentration
Microalgae concentration determines light utilization effi-
ciency (the energy stored as new biomass per unit of light
absorbed, Janseen et al., 2003) in photobioreactors. It there-
fore also controls the oxygenation and pollutant removal
rates achieved in the system. Munoz et al. (2004), for instance,
reported an increase of 44% in salicylate removal when the
biomass concentration was increased from 0.4 to 0.6 g l�1 in a
closed photobioreactor. However, a decrease of 15% on
salicylate removal efficiency was observed when the algal-
bacterial biomass increased from 0.6 g l�1 to 1.3 g l�1. Indeed,
when the biomass concentration reaches a critical value, all
the light provided to the system is used for photosynthesis
and the oxygenation rate reaches a maximum. Increasing the
biomass concentration further only causes mutual shading
and algal dark respiration to occur (Grobbelaar and Soeder,
1985), which reduces the amount of oxygen available to the
bacteria. However, at high light intensities, mutual shading
can be used to increase the frequency of light/dark cycles at
which the cells are exposed in order to optimize the
photosynthetic activity (Hu et al., 1996; Richmond, 2004).
The higher the light intensity, the higher should be the
biomass concentration (Hu et al. 1996). However, the use of
high algal densities to maintain high light/dark frequencies
requires efficient mixing without damaging the cells (Hu et al.
1996). Predicting the optimum biomass concentration under
natural illumination is also very difficult because the light
intensity onsite greatly varies in time.
4.6. Surface/volume ratio
Since the economic cost of artificial lighting is prohibitive,
sunlight must power oxygenation in algal-bacterial photo-
bioreactors. Hence, a crucial design parameter of these
systems is the illuminated surface to volume ratio (Table 4)
that determines the volumetric microalgae growth rate and
therefore the volumetric O2 production and pollutant removal
rates. Oxygenation capacities estimated from outdoors
ARTICLE IN PRESS
Pollutant
O2
Biofilm Bulkliquid
Bulkliquid
Biofilm
Light
Light
(A)
(B)
Fig. 6 – Theoretical dissolved oxygen (dashed line) and
pollutant (plain line) concentration profiles through the
biofilm of (A) a vertical flat photobioreactor with the biofilm
attached on the reactor wall and illuminated from the sides
and (B) a horizontal algal turf reactor with microalgae
attached on the reactor base and illuminated from above. In
the flat reactor, the most active microalgae which are
directly exposed to light are not directly exposed to the
reactor bulk liquid. The concentration of dissolved oxygen is
therefore expected to decrease through the biofilm as a
result of bacterial consumption. At the same time, toxic
pollutants are consumed and their concentration decreases
through the biofilm, which protects the active microalgae
towards pollutant toxicity. In an algal turf reactor, the most
active microalgae which are directly exposed to light are
also directly exposed to the bulk liquid and therefore, to the
highest possible pollutant concentration.
WAT E R R E S E A R C H 4 0 ( 2 0 0 6 ) 2 7 9 9 – 2 8 1 52808
photobioreactors suggest that horizontal or inclined tubular
and flat plate photobioreactors are the most efficient config-
urations for wastewater bioremediation due to their high
illuminated surface to volume ratio (Lee, 2001). However, the
optimum surface/volume ratio is also dependant on factors
such as the biomass concentration established (or main-
tained) in the system, the hydrodynamic regime, and the
impinging light intensity (Molina-Grima et al., 1999).
High illuminated surface/volume ratio generally means
high land requirement, especially when open reactors are
used. For this reasons, waste stabilization ponds are generally
recommended for small-scale decentralized applications
(Crites and Tchobanoglous, 1998) where the expenses for
land are balanced out by lower costs of the initial investment,
operation and management, and water collection. There is
however no clear definition on the size range of decentralized
or small wastewater treatment plant as Crites and Tchoba-
noglous (1998) define them as facilities handling less than
approx. 3800 m3 of wastewater d�1 (approx. 32 000 p.e. using a
equivalent of 120 l p.e.�1 d�1) whereas the European Commis-
sion (2001) classify small and medium sized communities as
500–5000 p.e. Based on the same criteria, algal–bacterial
processes should be suitable for treating up 60–4000 m3
wastewater d�1 at loads of 30–1800 kg d�1, depending on the
local land value. Essam et al. (2006), for instance, reported
complete phenol removal from a synthetic coking wastewater
at 6 d HRT in an algal–bacterial photobioreactor. Thus, the
full-scale treatment of 300 m3 wastewater d�1 should then be
achieved in a 6000 m2�0.3 m HRAP, the size of a 550 p.e. pond
treatment. Unfortunately, not enough current data are avail-
able on the specific use of algal–bacterial processes for
industrial waste treatment to better predict their applicability.
Small-scale decentralized wastewater treatment could also
allow water reuse onsite and reduce the need for transporta-
tion of hazardous wastes.
4.7. Hydraulic retention time (HRT)
HRAP are traditionally operated at 2–6 d HRT (Mara and
Pearson, 1986) and similar values have been reported in
enclosed photobioreactors (Essam et al., 2006; Munoz et al.,
2005a). Munoz et al. (2004), for instance, reported complete
salicylate removal and high DOCs at 2.7 HRT. When the HRT
was decreased from 2.7 to 1.7 d, both the removal efficiency
and the DOC decreased from 18 to approx. 0.5 mg l�1,
indicating that the process became limited by O2 supply and
therefore by the algal activity. Complete pollutant removal
was however achieved at 0.9 d HRT by sedimentation and
recirculation of a portion of the biomass produced (Munoz
et al., 2004).
5. Influence of environmental parameters
5.1. pH
Microalgal CO2 uptake can cause the pH to rise to 10–11 in
HRAPs and high pH values (up to 9) were also recorded during
salicylate biodegradation by an algal–bacterial consortium in
an enclosed photobioreactor (Munoz et al., 2003b). This
increase, which is beneficial for the disinfection of pathogens,
can also cause a decrease in the pollutant removal efficiency
(Oswald, 1988; Schumacher et al., 2003) as complete bacterial
inhibition at pH above 10 is commonly observed in stabiliza-
tion ponds (Mara and Pearson 1986; Oswald 1988). It is
however difficult to dissociate the direct effects of pH on
microbial growth from collateral effects such as modifications
in the CO2/HCO3�/CO3
�2 and NH3/NH4+ equilibria or in phos-
phorus and heavy metal availability (Laliberte et al., 1994).
The pH also influences N and P removal via NH3 volatilization
and orthophosphate precipitation at a high pH (9–11) (Craggs
et al., 1996; Garcia et al., 2000b; Nurdogan and Oswald, 1995).
Fortunately, it is relatively easy to control the pH in biological
systems.
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WAT E R R E S E A R C H 40 (2006) 2799– 2815 2809
5.2. Temperature
The efficiency of microalgae-based treatments normally
decreases at low temperatures (Abeliovich, 1986). Munoz
et al. (2004) observed that the removal efficiency doubled
when the temperature increased from 25 to 30 1C using a
symbiotic microcosm formed by a C. sorokiniana and a R.
basilensis strain (the activities of both microorganisms in-
creased with the temperature in the tested range). However,
Chevalier et al. (2002) demonstrated that a cold-adapted
cyanobacteria strain was suitable for nutrient removal at
average temperature of 15 1C. Likewise, Gronlund (2004)
described a pilot-scale HRAP capable to support 90% BOD
removal at 2.5 d HRT at temperature below 10 1C and light
intensity below 200mE m�2s�1 (Swedish subartic region,
latitude 631N). These studies therefore show the wastewater
treatment with cold-adapted photosynthetic strains in opti-
mized bioreactors is possible despite the decrease in biologi-
cal activity with temperature inherent to any biological
methods.
Excessive temperature at high light intensities and high
biomass concentrations can also arise from the fact algae
convert a large fraction of the sunlight into heat (Abeliovich,
1986). Temperature control by external heat exchanger or
water spray have been proposed to ensure a stable microalgal
population but their costs remain often prohibitive, even for
high-quality algal mass cultivation (Tredici, 1999). An alter-
native to temperature control is the combination of micro-
algal strains with similar characteristics (in terms of O2
supply, inhibition and harvesting) but with different optimum
growth temperatures (Morita et al., 2001).
5.3. Light supply
Sunlight intensity greatly varies during the day and during
the year. Algal activity increases with light intensity up to
200–400mE m�2 s�1, where the photosynthetic apparatus be-
comes saturated, to decrease at higher light intensities
(Ogbonna and Tanaka, 2000b; Sorokin and Krauss, 1958).
Photoinhibition has therefore been observed during the
central hours of a sunny day when irradiance can reach up
to 4000mE m�2 s�1 (Rebolloso Fuentes et al., 1999). It is more
likely to occur at low microalgal concentration, such as during
start-up (Goksan et al., 2003), because the light intensity to
which microalgae are actually exposed is not reduced by
mutual shading (Evers, 1991; Contreras-Flores et al., 2003;
Richmond, 2000). Careful photobioreactor designing can also
avoid excessive damage of the photosynthetic apparatus
by distributing the light irradiating a certain land area
onto a larger surface (Torzillo et al., 2003). Reducing the
size of the antenna of photosynthetic cells using molecular
tools reduces light adsorption and usually allows higher
photosynthesis rates under high light intensities (Melis et al.,
1999).
Periodical absence of light (or periods of low light intensity)
causes a halt (or sever reduction) of photosynthesis, which
generally leads to the occurrence of anaerobic conditions in
the reactor. However, photosynthesis and pollutant removal
normally resume once light is available again. Waste stabili-
zation ponds are therefore designed to cope with natural
diurnal or seasonal light intensity fluctuations by, for
instance, increasing the HRT in the system (Tadesse et al.,
2004). High HRT, or the use of storage tanks during period
of low light intensities, are also important to avoid increases
of toxic pollutant concentrations and inhibition. In a pilot-
scale closed photobioreactor inoculated with a C. sorokinia-
na–Comamonas sp. consortium, oxygen production and acet-
onitrile removal dropped when illumination was stopped for
10 h but it quickly recovered each time illumination was
resumed (Munoz et al., 2005b). Wastewater storage during
nighttime should therefore no affect the overall process
efficiency.
5.4. Dissolved oxygen concentration (DOC)
High DOC levels can generate photo-oxidative damage on
microalgal cells and therefore decrease treatment efficiency
(Oswald, 1988; Suh and Lee, 2003). For instance, Matsumoto
et al. (1996) reported a 98% decrease in the photosynthetic O2
production rate when the DOC increased from 0 to 29 mg l�1
(E350%). O2 supersaturation in enclosed photobioreactors
designed for mass algal cultivation can reach up to 400%,
which severely inhibits microalgal growth (Lee and Lee, 2003).
Fortunately, O2 supersaturation does not constitute a
severe problem in biodegradation processes due to the
continuous O2 consumption by heterotrophic bacteria. For
instance, the DOC was always very low (E0 mg l�1) during the
biodegradation of acetonitrile and salicylate in the batch
mode when the pollutants were present and being degraded.
However, it also always rapidly increased after complete
pollutant depletion (Guieysse et al., 2002; Munoz et al., 2005a).
High O2 concentrations are therefore a good indication of
complete pollutant depletion in continuous processes (Munoz
et al., 2004). Further research should be conducted to
investigate if the DOC can be used for process control to
optimize, for instance, the biomass concentration in the
system.
5.5. Predators
Infections by parasitic fungi like Chytridium sp. or the
development of food chains in the photobioreactor can
cause unexpected process failure (Abeliovich and Dikbuck,
1977). Fortunately, these potential problems can easily
be avoided by daily operating the process at low O2
levels for a short period of time (1 h) in order to
suppress the growth of higher aerobic organisms (Abeliovich,
1986).
6. Future prospects
6.1. Potential uses of the algal–bacterial biomass
Algal–bacterial biomass can be used for various purposes
(Table 5). However, biomass produced from wastewater will
seldom be suitable for the production of food or even high
value chemicals due to high-quality requirements and public
acceptance. Likewise, fertilization should only be conducted if
the biomass does not contains heavy metals or recalcitrant
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Table 5 – Potential uses of algal–bacterial biomass
Algae application Examples/comments References
Human food source Drinks, noodles, health products Liang et al., 2004
Animal feed Tetraselmis sp., Spirulina sp. and Chaetoceros sp. are currently employed as
food source for shrimps or salmonids production
Borowitzka, 1997; Day et al.,
1999
High-value
biomolecules
Astaxanthin, ascorbic acid, b-carotene, glycerol, or poly-b-
hydroxybutyrate
Ghirardi et al., 2000; Pulz
and Gross, 2004; Tsygankov,
2001
Fertilizer Because algae contain large amounts of nitrogen and phosphorus,
algal–bacterial biomass from wastewater treatment represents an
interesting inexpensive fertilizer. Two million hectares for rice
cultivation were thus fertilized in India in 1977
Oswald and Benemann,
1977
Biogas production Anaerobic digestion of biomass into CH4 and CO2 Munoz et al., 2005a
Biofuels Liquid fuels can be produced from the thermochemical liquefaction or
pyrolysis of microalgae. Certain microalgae also have the capability to
accumulate oils in their cells.
Sawayama et al., 1999
WAT E R R E S E A R C H 4 0 ( 2 0 0 6 ) 2 7 9 9 – 2 8 1 52810
compounds (which are often found in industrial effluents).
Hence, the best option remains to use the algal–bacterial
biomass for energy production by anaerobic digestion into
biogas. Due to CO2 fixation by the algae, all the organic matter
biodegraded is converted into biomass under photosyntheti-
cally oxygenated treatment. This represents a considerable
gain in the carbon available for CH4 production compared to
classical aerated processes where approx. 50% of the original
carbon is lost as CO2 released in the atmosphere. For instance,
Munoz et al. (2005a) reported more than a 100% gain in CH4
production when algal–bacterial biomass produced from
acetonitrile treatment was used instead of bacterial biomass
alone. Furthermore, the biogas produced from the digester
can also be sparged and treated in the algal pond to convert
the CO2 anaerobically produced (biogas usually contains
approx, 60% CH4 and 40% CO2) into algal biomass (Eisenberg
et al., 1981; Mandeno et al., 2005). This further improves the
overall C-mitigation and energy-recovery efficiency of the
system.
6.2. Combining wastewater treatment with CO2
mitigation
As algal cultures require large amounts of nutrients, it can be
very advantageous to combine CO2 fixation from gaseous
streams (i.e. combustion systems) with wastewater treatment
(Nakamura, 2003). This could also help removing hazardous
combustion products such as NOx and SOx (Nagase et al.,
1998, 2001). Thus, 26.0 g CO2 m�3 h�1 was fixed and 0.92 g
NH3 �m�3 h�1 was removed when flue gas and wastewater
from a steel making plant were simultaneously treated (Yun
et al., 1997). Benemann et al. (2003) concluded that productiv-
ities near the theoretical maximum, high-energy prices,
and greenhouse gas abatement credits would however be
required to make this process economically realistic. With
current oil prices and the increase pressure to reduce CO2
emissions and dependence to fossil fuels, this might already
be happening.
7. Conclusions
Algal–bacterial systems are efficient for the treatment of
hazardous pollutants but remains limited by the difficulty of
harvesting the biomass formed, the high land requirement of
open systems, or the high construction costs of enclosed
photobioreactors. Hence, suitable applications will be found
when the effluents to be treated contain hazardous volatile
pollutants, where combined removal capacities (organic
pollutants/nutrients/heavy metals) are desired, or when the
biomass produced can be commercialized. In such cases, the
additional costs brought about by land use, reactor construc-
tion and biomass harvesting will be justified by the gains in
safety and energy savings achieved.
Before algal–bacterial processes can widely be implemented
for the treatment of industrial wastes, more research is still
needed to (1) select ‘‘extreme’’ algal strains capable to grow
under wider and more extreme conditions of light, pH,
pollutant concentrations, etc.; (2) understand and control
the mechanisms of autoflocculation and bioflocculation to
improve harvesting and biomass control; (3) scale-up and
model photobioreactors to provide better design guidelines;
and (4) develop new treatment methods such as membrane
photobioreactors or combined physical–biological processes
to improve biomass control and protect algae against
inhibitory effects.
Acknowledgements
This work is dedicated to Professor William J. Oswald
(1919–2005) who was a pioneer in the development of algae-
based wastewater treatment. The financial support from SIDA
(The Swedish International Development Cooperation
Agency, projects SWE-2002-205 and SWE-2005-439) and the
Spanish Ministry for Science and Education (Juan De La Cierva
Program, JCI-2005-1881-5 Contract) are gratefully acknowl-
edged.
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