J. Phycol. 46, 800–812 (2010)
2010 Phycological Society of America
DOI: 10.1111/j.1529-8817.2010.00862.x
EFFICIENCY OF GROWTH AND NUTRIENT UPTAKE FROM WASTEWATER BY
HETEROTROPHIC, AUTOTROPHIC, AND MIXOTROPHIC CULTIVATION OF
CHLORELLA VULGARIS IMMOBILIZED WITH AZOSPIRILLUM BRASILENSE 1
Octavio Perez-Garcia
Environmental Microbiology Group, The Northwestern Center for Biological Research (CIBNOR), Mar Bermejo 195, Colonia Playa
Palo de Santa Rita, La Paz, B.C.S. 23090, Mexico
Luz E. de-Bashan
Environmental Microbiology Group, The Northwestern Center for Biological Research (CIBNOR), Mar Bermejo 195, Colonia Playa
Palo de Santa Rita, La Paz, B.C.S. 23090, Mexico
The Bashan Foundation, 3740 NW Harrison Blvd., Corvallis, Oregon 97330, USA
Juan-Pablo Hernandez
Environmental Microbiology Group, The Northwestern Center for Biological Research (CIBNOR), Mar Bermejo 195, Colonia Playa
Palo de Santa Rita, La Paz, B.C.S. 23090, Mexico
and Yoav Bashan2
Environmental Microbiology Group, The Northwestern Center for Biological Research (CIBNOR), Mar Bermejo 195, Colonia Playa
Palo de Santa Rita, La Paz, B.C.S. 23090, Mexico
The Bashan Foundation, 3740 NW Harrison Blvd., Corvallis, Oregon 97330, USA
Heterotrophic growth of microalgae presents
significant economic advantages over the more common autotrophic cultivation. The efficiency of
growth and nitrogen, phosphorus, and glucose
uptake from synthetic wastewater was compared
under heterotrophic, autotrophic, and mixotrophic
regimes of Chlorella vulgaris Beij. immobilized in
alginate beads, either alone or with the bacterium
Azospirillum brasilense. Heterotrophic cultivation of
C. vulgaris growing alone was superior to autotrophic cultivation. The added bacteria enhanced growth
only under autotrophic and mixotrophic cultivations. Uptake of ammonium by the culture, yield of
cells per ammonium unit, and total volumetric
productivity of the culture were the highest under
heterotrophic conditions when the microalga grew
without the bacterium. Uptake of phosphate was
higher under autotrophic conditions and similar
under the other two regimes. Positive influence of
the addition of A. brasilense was found only when
light was supplied (autotrophic and mixotrophic),
where affinity to phosphate and yield per phosphate
unit were the highest under heterotrophic conditions. The pH of the culture was significantly
reduced in all regimes where glucose was consumed,
similarly in heterotrophic and mixotrophic cultures.
It was concluded that the heterotrophic regime,
using glucose, is superior to autotrophic and
mixotrophic regimes for the uptake of ammonium
and phosphate. Addition of A. brasilense positively
affects the nutrient uptake only in the two regimes
supplied with light.
Key index words: autotrophic growth; Azospirillum;
Chlorella; heterotrophic growth; immobilization;
microalgae; mixotrophic growth; wastewater treatment
Abbreviations: ANOVA, analysis of variance;
MGPB, microalgae growth-promoting bacteria;
OAB medium, Okon, Albrecht, Burris medium;
SE, standard error; SGM, synthetic wastewater
medium
Autotrophic microalgae Chlorella spp. can grow
heterotrophically (light independent) if supplemented with a preferred carbon source (Chen 1996,
Shi et al. 2000, Lee 2004, Chen and Chen 2006,
Qiao et al. 2009) or mixotrophically, that is, a combination of the two (Kaplan et al. 1986, Ogbonna
et al. 1997, Lee 2004).
Chlorella spp. grown under autotrophic conditions
can remove nitrogen and phosphorous from wastewaters under extremely diverse environmental conditions with efficiency of nitrogen removal generally
higher than that of phosphorus (de la Noüe and de
Pauw 1988, Gonzalez et al. 1997, de-Bashan et al.
2002b, 2008a, Olguı́n 2003, Hernandez et al. 2006);
1
Received 10 July 2009. Accepted 28 January 2010.
Author for correspondence: e-mail [email protected].
2
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H E T E R O T R O P H I C , A U T O T RO PH I C , A N D MI X O T R O PH I C G R O W T H O F I MM O B I L I Z E D MI C R O A L G A E
however, autotrophic tertiary wastewater treatment
by Chlorella spp. poses two inherent major limitations. Wastewater is treated in large volumes in bioreactors where penetration of light into a dense
culture is limited, a fact requiring large exposed surface area of the culture to light (Apt and Behrens
1999, Behrens 2005), and cultivation produces relatively low-cell densities in the wastewater culture
(Lee 2001, Valderrama et al. 2002). Light-independent heterotrophic growth may solve these difficulties (Lee 2001, Chen and Chen 2006). It eliminates
the need for light and may allow cultivation of denser concentrations of the microalgae (Chen 1996,
Ogbonna et al. 1997). Singularly, elimination of
light as a growth factor significantly reduces the
cost of cultivation, the major parameter when large
volumes of wastewater are treated (de la Noüe et al.
1992).
Heterotrophic growth of Chlorella spp. has been
known for decades (Droop 1974) and is based on
the addition of glucose, acetate, or glycerol as the
main carbon sources; other carbon sources such as
malate (Qiao et al. 2009) and several more were
also evaluated. In some heterotrophic cultures, the
growth rate of Chlorella, the dry biomass (Ogbonna
et al. 2000, Behrens 2005), ATP generated by the
supplied energy, and the effect on ATP yield (mg of
biomass generated by mg of consumed ATP) are significantly higher than in autotrophic cultures
(Martı́nez and Orús 1991, Chen and Johns 1996,
Shi et al. 2000, Yang et al. 2000) and are mainly
dependent on the species and strain used. Mixotrophic cultivation proved a good strategy to obtain
high biomass and growth rates (Ogawa and Aiba
1981, Lee and Lee 2002) with the extra benefit of
producing photosynthetic metabolites (Chen 1996).
Our working hypothesis for addressing a specific
goal of potential wastewater treatment is that heterotrophic and mixotrophic cultivation of C. vulgaris
are superior to autotrophic cultivation in terms of
growth rate, affinity to the substrates, and the capacity of the developed high populations to uptake
nutrients from wastewater. This has been carried out
by employing a recently proposed technology for
wastewater treatment (de-Bashan et al. 2004) and
plant–bacteria interaction (de-Bashan and Bashan
2008), using a specific microalga immobilized with a
bacterium that promotes growth in microalgae. We
compared the growth rate and population size of
the microalgae, its nitrogen and phosphorus uptake
capacity from synthetic wastewater, and cultural
factors, such as glucose consumption and pH shifts.
This has been carried out under the three types of
cultivation, where the sole difference was the energy
supplied by light or glucose.
MATERIALS AND METHODS
Microorganisms and growth conditions. The unicellular
microalga Chlorella vulgaris (UTEX 2714, Austin, TX) and the
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microalgae growth-promoting bacterium (MGPB) A. brasilense
Cd (DMS 1843, Braunschweig, Germany) were tested. Experiments were carried out under autotrophic, heterotrophic, and
mixotrophic conditions in inverted 1,000 mL conical glass
bioreactors (17 cm tall, 14 cm width at top, and 5 cm at the
bottom), each containing 750 mL synthetic wastewater,
equipped with uplift bottom sterile aeration controlled by a
peristaltic pump providing air at 30 mL airÆmin)1 measured
with a fluxometer (GF-2000, Gilmont Instruments, Barrington,
IL, USA) that pass three microbial filters: one 1 lm pore
bacterial air vent filter (Pall-Gelman Laboratory, Ann Arbor,
MI, USA) and two 0.2 lm sterile filters (Acrodiscs, Pall Corp.,
Ann Arbor, MI, USA). Each bioreactor was equipped with air
outflow protected from contamination by a 0.2 lm filter and a
glass tube exit at the bottom for taking wastewater samples. All
experiments were performed in an environmental chamber
(Biotronette Mark III, Lab-Line Instruments, Melrose Park, IL,
USA) with fluorescent lamps. Heterotrophic cultivation in total
darkness used the same environmental chamber, but the
chamber was sealed. Autotrophic experiments were subjected
to continuous fluorescent light at 90 lmol photonÆm2Æs)1 at
28 ± 1C. Mixotrophic cultures were grown under a 12:12
light:dark (L:D) cycle. All microorganisms were cultivated in
modified, sterile synthetic wastewater medium, hereafter called
SGM (de-Bashan et al. 2002b), where ammonium and phosphate concentrations were adjusted to the concentrations
found in municipal wastewater of the city of La Paz, Baja
California Sur, Mexico (O. Perez-Garcia, Y. Bashan, and M. E.
Puente, unpublished data). The synthetic wastewater at pH 6.7
contained the following ingredients (in mgÆL)1): NaCl, 7; CaCl2,
4; MgSO4Æ7H2O, 2; K2HPO4, 21.7; KH2PO4, 8.5; Na2HPO4, 25;
and NH4Cl, 191. The culture was stirred by continuous bubbling
of sterile air. Heterotrophic and mixotrophic growth media were
supplemented with a d-glucose solution at a concentration of
10 gÆL)1. The glucose stock solution was sterilized by filtration
through two 0.2 lm Whatman cellulose nitrate membranes
(G.E. Healthcare, Piscataway, NJ, USA). The specific growth
medium C30 for Chlorella sp. (Gonzalez et al. 1997), nutrient
broth (Sigma, St. Louis, MO, USA), nitrogen-free OAB medium
specific for Azospirillum sp. (Bashan et al. 1993), and saline
solution (0.85% NaCl) was used to prepare the preinoculum for
the experiments and as controls.
Cell counting and immobilization of microorganisms in alginate.
These procedures were performed according to the description
in de-Bashan et al. (2004). Briefly, axenic cultures (either
C. vulgaris or A. brasilense) were mixed with 2% alginate
solution. Initial cell concentrations among experiments were
similar: 1.0 ± 0.5 · 106 for C. vulgaris and 2.5 ± 0.5 · 105 for
A. brasilense. Beads (2–3 mm in dia.) were automatically
produced in a 2% CaCl2 solidification solution (de-Bashan
and Bashan 2010). To immobilize the two microorganisms in
the same bead, after washing the cultures, each was resuspended in 10 mL 0.85% saline solution and then mixed
together with the alginate. Because immobilization normally
reduces the number of Azospirillum cells in the beads, a second
overnight incubation of the beads in diluted (10% of full
strength) nutrient broth was necessary. Each bioreactor contained 30 g of beads.
For cell counting in each experiment, three beads per
bioreactor were solubilized by immersing them in 1 mL 4%
NaHCO3 solution for 30 min. A. brasilense cells were first
stained with fluorescein diacetate (Sigma) described in Chrzanowski et al. (1984) and then directly counted under a
fluorescence microscope (BX41, Olympus, Tokyo, Japan).
Chlorella was counted under LM with a Neubauer hemocytometer (Gonzalez and Bashan 2000) connected to an image
analyzer (Image ProPlus 4.5, Media Cybernetics, Silver Spring,
MD, USA). Growth rate (l) was determined as follows:
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O C T A V I O PE R E Z - G A R C I A E T A L .
l ¼ ðlnNt1 lnNt0 Þ=ðt1 t0 Þ
ð1Þ
where Nt1 is the number of cells at sampling time and Nt0
is the number of cells at the beginning of the experiment
(Oh-Hama and Miyachi 1992).
Analytical methods. Nitrogen and phosphorus were analyzed
from 5 mL samples obtained from the bottom exit of each
bioreactor, using standard water analysis techniques (Eaton
et al. 2005). Orthophosphate was measured by molybdate assay
carried out in an analyzer (Flow Injection Analysis, Lachat
QuikChem series 8000 FIAS+, Loveland, CO, USA) according
to the manufacturer’s manual. Ammonium was measured by
the phenate colorimetric method (Solorzano 1969) and
adapted to the microplate (Versa Max tunable microplate
reader, Molecular Devices, Sunnyvale, CA, USA) (HernándezLópez and Vargas-Albores 2003). Glucose was analyzed by the
glucose oxidase-peroxidase (GOD-PAP) method using a kit
(Randox Laboratories, Crumlin, UK) according to the manufacturer’s manual. The pH was measured by a pH-electrode
(Horiba-twin pH, Irvine, CA, USA).
Experimental design and statistical analysis. The setup of all
experiments was of batch cultures. Each experiment was
performed in triplicate, where one bioreactor served as a
replicate and each experiment was repeated twice. Each
experiment had four regimes: controls (no light, no glucose),
autotrophy (light, no glucose), heterotrophy (no light, glucose
added), and mixotrophy (12:12 photoperiod, glucose added).
Each regime contained five treatments: medium alone, beads
without microorganisms, beads with C. vulgaris, beads with
A. brasilense, and beads with the two microorganisms jointly
immobilized. Three samples were taken for all analyses at each
sampling time. The following variables were analyzed: Volumetric productivity (Qp) was calculated as:
Qp ¼ P1 P0 :
ð2Þ
where P1 and P0 are grams of product (as cells or biomass) in
a defined volume during the time between initial and final
sampling (Cooney 1983). Affinity or specific activity (Sa) of
the microalgal cells to the substrate in a specific interval of
time was calculated as:
Sa ¼ St =Nt
ð3Þ
where St is grams of substrate S consumed or product formed
in a specific time interval t, and Nt is grams or number of catalysts (enzymes or cells) at the end of the time interval t
(Cooney 1983). Yields (YP ⁄ S) (number of cells produced per
mg substrate uptake in 1 L of culture during a defined incubation period) were calculated as:
YP=S ¼ ðP1 P0 Þ=ðS1 S0 Þ
ð4Þ
where P is the number of cells at the end (P1) and at the
beginning (P0) of a defined time interval, and S is the substrate (ammonium, phosphate, or glucose) at the end (S1)
and at the beginning (S0) of this time interval (Bitton 2005).
As data in both repetitions of the each experiment were similar, data were combined for analysis by two-way analysis of variance (ANOVA) and then by Tukey’s post hoc analysis or
Student’s t-test, both at a significance level of P < 0.05, using
Statistica ver. 6.0 software (StatSoft, Tulsa, OK, USA).
RESULTS
Autotrophic, heterotrophic, and mixotrophic growth of
C. vulgaris in synthetic wastewater alone and jointly
immobilized with A. brasilense. In the absence of
light and a carbon source (control treatment), very
slow growth of C. vulgaris occurred (Fig. 1a, lower-
case letter analysis and l calculation) with small, but
significant enhancing effect of immobilization with
the MGPB A. brasilense Cd starting after 3 d of cultivation (Fig. 1a, capital letter analysis).
Application of light significantly enhanced growth
(Fig. 1b, lowercase letter analysis and l calculation),
followed the same trend as joint immobilization, but
started a day earlier (Fig. 1b, capital letter analysis).
Growing alone, C. vulgaris reached the stationary
phase after 3 d, while jointly immobilized with the
MGPB steadily increased population numbers up to
5 d (Fig. 1b, lowercase letter analysis).
Under heterotrophic growth, C. vulgaris grown
alone had slightly larger populations and had
higher growth rates than under autotrophic growth
(compare Fig. 1, b and c), while joint immobilization significantly inhibited microalgal growth
(Fig. 1c, both types of analysis and l calculation).
Mixotrophic growth yielded intermediate populations and lower growth rates compared to autotrophic and heterotrophic growth, reached
stationary phase after 2 d for the microalga grown
alone (Fig. 1d, lowercase letter analysis and l calculation), and was declining after 4 d of cultivation,
while joint immobilization prevented population
decline but did not enhance growth further
(Fig. 1d, capital letter analysis).
Analysis of growth showed that 3 d of incubation
is the important time for most cultures, indicating
that heterotrophic growth was similar to autotrophic
growth, while joint immobilization enhanced growth
only under autotrophic conditions, with lesser
enhancement also in the control (no light and no
glucose).
Autotrophic, heterotrophic, and mixotrophic growth of
A. brasilense grown alone in synthetic wastewater and
jointly immobilized with C. vulgaris. No growth of
A. brasilense occurred in wastewater without a carbon
source, with or without C. vulgaris (Fig. 2a). Under
autotrophic conditions, A. brasilense grew only in the
presence of C. vulgaris for 3 d, and then growth
declined (Fig. 2b). Under heterotrophic conditions,
A. brasilense grew for 2 d, but later growth ceased
(Fig. 2c, lowercase letter analysis). When the bacteria were jointly immobilized with C. vulgaris, the
bacteria grew linearly, producing significantly larger
populations (Fig. 2c, capital letter analysis and
linear regression analysis). Under mixotrophic conditions, the bacteria grew for 2 d (jointly immobilized) and 3 d (alone), but then entered the
stationary phase in both treatments (Fig. 2d, lowercase letter analyses). Joint immobilization had, in
general, no effect on the growth of A. brasilense
(Fig. 2d, capital letter analysis).
Uptake of ammonium under autotrophic, heterotrophic,
and mixotrophic growth conditions. Without light and a
carbon source, controls of alginate beads and beads
containing A. brasilense did not remove ammonium
from wastewater similar to wastewater incubated
without any treatment (Fig. 3a, lowercase letter
H E T E R O T R O P H I C , A U T O T RO PH I C , A N D MI X O T R O PH I C G R O W T H O F I MM O B I L I Z E D MI C R O A L G A E
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Fig. 1. Populations of immobilized Chlorella vulgaris alone and jointly immobilized with Azospirillum brasilense growing under autotrophic, heterotrophic, and mixotrophic growth conditions in synthetic wastewater. Values of each curve denoted with a different lowercase
letter differ significantly by two-way analysis of variance (ANOVA) and by Tukey’s post hoc analysis at P < 0.05. Values at each date
denoted with a different capital letter differ significantly by Student’s t-test at P < 0.05. Bars represent standard error of the mean (SE).
Absence of a bar indicates negligible SE. l represents growth rate.
analysis). Under these conditions, C. vulgaris immobilized alone uptakes a small, but statistically significant, amount of ammonium for only 1 d, and later
the uptake ceased (Fig. 3a, lowercase letter analysis).
Jointly immobilized microorganisms could uptake
more ammonium for only 2 d (Fig. 3a, capital letter
analysis) before uptake ceased (Fig. 3a, lowercase
letter analysis). Although the statistical analysis indicated significant ammonium uptake, in several
cases, <20% of ammonium was taken up in the best
cases after 3 d of incubation (Fig. 3e).
Autotrophic, heterotrophic, and mixotrophic
growth conditions did not induce ammonium
uptake in the two control treatments or treatment
with Azospirillum (Fig. 3, b, c, and d, lowercase
letter analyses). Under autotrophic conditions, treatment with immobilized C. vulgaris increased ammonium
uptake,
and
jointly
immobilized
microorganisms enhanced it further for up to 2 d
of incubation (Fig. 3b, capital letter analysis) where
these treatments removed <30% of the ammonium
(Fig. 3e).
Under heterotrophic growth conditions, ammonium uptake was further enhanced by C. vulgaris
immobilized alone for 3 d, reached 44% removal,
and then stabilized (Fig. 3c, lowercase letter analysis, and Fig. 3e). Joint immobilization continuously
took up ammonium for 4 d, but at lower levels,
reaching a maximum uptake of 22% after 3 d of
incubation. Uptake of ammonium under mixotrophic conditions was somewhat similar to uptake
under heterotrophic conditions, where joint immobilization was the more efficient treatment (Fig. 3, d
and e).
At the cellular level, a different ammonium
uptake profile occurred when the affinity of the
cells to the substrate was calculated, and this affinity
varied with time. After 1 d of incubation under heterotrophic conditions, jointly immobilized microorganisms had significantly higher affinity, compared
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Fig. 2. Populations of immobilized Azospirillum brasilense alone and jointly immobilized with Chlorella vulgaris growing under autotrophic, heterotrophic, and mixotrophic growth conditions in synthetic wastewater. Values of each curve denoted with a different lowercase letter differ significantly by two-way analysis of variance (ANOVA) and by Tukey’s post hoc analysis at P < 0.05. Values at each date denoted
with a different capital letter differ significantly by Student’s t-test at P < 0.05. Bars represent standard error of the mean (SE). Absence of
a bar indicates negligible SE. Linear regression under heterotrophic growth is for the joint immobilization treatment.
to all treatments followed by joint immobilization
under mixotrophic conditions (Fig. 3f). Later, even
though joint immobilization was superior, differences with the other treatments were small (Fig. 3f,
days 1 and 2).
Uptake of phosphate under autotrophic, heterotrophic,
and mixotrophic growth conditions. Without light and
a carbon source, all controls and treatments took
up small quantities of phosphate to a level of up
to 15.7% after 3 d of incubation, where the treatments of C. vulgaris immobilized alone removed
the most (Fig. 4, a and e). Although statistical differences among the treatments were detected in
most sampling times (Fig. 4a, capital letter analysis), quantities were small. Under autotrophic,
heterotrophic, and mixotrophic conditions, both
controls and beads with A. brasilense took up meager amounts of phosphate or none at all in some
samplings (Fig. 4, b–d), while C. vulgaris alone
removed up to 20.8% (autotrophic), 17.8% (heterotrophic), and 20.9% (mixotrophic) after 5 d
(Fig. 4c). When jointly immobilized with A. brasilense, uptake increased to 31.5% (autotrophic),
22.6% (heterotrophic), and 24.3% (mixotrophic)
after 5 d of incubation (Fig. 4b). The most rapid
uptake was in the first 2 d under light conditions
(autotrophic and mixotrophic conditions) (Fig. 4,
b and e). Under heterotrophic conditions, similar
quantities of uptake by C. vulgaris alone and jointly
immobilized with A. brasilense occurred for the first
4 d of incubation with an increase in uptake by
C. vulgaris alone after 5 d (Fig. 4, c and e). Similar removal was measured under mixotrophic conditions, where the C. vulgaris immobilized with
A. brasilense performed better and most of the
uptake occurred in the first day (Fig. 4, d and e).
Affinity of individual cells for phosphate after 1 d
followed a similar trend, where jointly immobilized
cells under heterotrophic conditions had the highest affinity, followed by the same treatment under
mixotrophic conditions (Fig. 4f). The superior
affinity of heterotrophic, joint immobilization
H E T E R O T R O P H I C , A U T O T RO PH I C , A N D MI X O T R O PH I C G R O W T H O F I MM O B I L I Z E D MI C R O A L G A E
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Fig. 3. Ammonium uptake (a–d), ammonium consumed (e) on the third day of incubation, and affinity for ammonium (f) of Chlorella vulgaris alone and jointly immobilized with Azospirillum brasilense growing under autotrophic, heterotrophic, and mixotrophic growth
conditions in synthetic wastewater. All statistical analyses were performed using two-way analysis of variance (ANOVA) combined with
Tukey’s post hoc analysis or by Student’s t-test both at P < 0.05 as follows: In (a–d), values on each curve denoted with a different lowercase letter differ significantly by ANOVA. Values at each date denoted with a different capital letter differ significantly by ANOVA. In (e),
columns of C. vulgaris alone or jointly immobilized with A. brasilense separately denoted with a different lowercase letter differ significantly
by ANOVA. Pairs of columns at each treatment type denoted with a different capital letter differ significantly by Student’s t-test. In (f), values corresponding to each day separately denoted with a different capital letter differ significantly with ANOVA. Bars represent standard
error of the mean (SE). Absence of a bar indicates negligible SE.
lasted, at a reduced rate, for an additional day,
diminishing afterward (Fig. 4f).
Consumption of glucose under different conditions of
incubation. Glucose was added only to cultures
growing under heterotrophic and mixotrophic
conditions. Under those conditions, both controls
and beads with A. brasilense did not consume or
remove significant amounts of glucose from the
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O C T A V I O PE R E Z - G A R C I A E T A L .
Fig. 4. Phosphate uptake (a–d), phosphate consumed at the third day of incubation (e), and affinity for phosphate (f) of Chlorella vulgaris alone and jointly immobilized with Azospirillum brasilense growing under autotrophic, heterotrophic, and mixotrophic growth conditions in synthetic wastewater. All statistical analyses used two-way analysis of variance (ANOVA) combined with Tukey’s post hoc analysis or
by Student’s t-test both at P < 0.05 as follows: In (a–d), values on each curves denoted with a different lowercase letter differ significantly
by ANOVA. Values at each date denoted with a different capital letter differ significantly by ANOVA. In (e), columns of C. vulgaris alone
or jointly immobilized with A. brasilense separately denoted with a different lowercase letter differ significantly by ANOVA. Pairs of columns
at each treatment type denoted with a different capital letter differ significantly by Student’s t-test. In (f), values corresponding to each
day separately denoted with a different capital letter differ significantly by ANOVA. Bars represent standard error of the mean (SE).
Absence of a bar indicates negligible SE. Some statistics letters were eliminated for clarity.
medium (Fig. 5, a and b, lowercase letter analysis).
C. vulgaris alone under both growth conditions consumed glucose at the end of the incubation period,
up to 18.6% under heterotrophic and up to 23.7%
under mixotrophic conditions (Fig. 5, a and b).
Joint immobilization enhances consumption of
glucose during the first 2 d, but this level of consumption was not maintained (Fig. 5, a and b).
H E T E R O T R O P H I C , A U T O T RO PH I C , A N D MI X O T R O PH I C G R O W T H O F I MM O B I L I Z E D MI C R O A L G A E
Fig. 5. Glucose uptake of Chlorella vulgaris alone and jointly
immobilized with Azospirillum brasilense growing under heterotrophic and mixotrophic growth conditions in synthetic wastewater.
Values of each curve denoted with a different lowercase letter differ significantly by two-way analysis of variance (ANOVA) and by
Tukey’s post hoc analysis. Values at each date denoted with a different capital letter differ significantly by ANOVA, all at P < 0.05.
Bars represent standard error of the mean (SE).
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analysis). Jointly immobilized microalga and bacteria
under autotrophic and heterotrophic conditions
produced significantly fewer cells for the quantity of
ammonium consumed, where no effect occurred
under mixotrophic and control conditions (Fig. 7a,
capital letter analysis).
C. vulgaris immobilized alone under heterotrophic conditions had the highest yield for phosphate
consumed, and the autotrophic and mixotrophic
growth was less (Fig. 7b, lowercase letter analysis).
Immobilization of the bacterium reduced the yield
(heterotrophic condition) or had no effect (autotrophic and mixotrophic conditions). Only under
the conditions of the control did joint immobilization have a significant effect on using phosphate
(Fig. 7b, capital letter analysis).
C. vulgaris immobilized alone under heterotrophic
conditions had the highest yield per glucose consumed; mixotrophic growth was less efficient (Fig. 7c,
lowercase letter analysis). Immobilization under
heterotrophic conditions had a negative impact on
glucose yield and had no effect under mixotrophic
conditions (Fig. 7c, capital letter analysis).
Volumetric productivity per liter of culture of the
three systems indicates that microalga immobilized
alone under heterotrophic conditions had the best
performance, followed by autotrophic and the mixotrophic (Fig. 7d, lowercase letter analysis). Joint
immobilization with the bacterium enhanced total
volumetric productivity under autotrophic and
mixotrophic conditions, but not under heterotrophic conditions (Fig. 7d, capital letter analysis);
under autotrophic conditions, p volumetric productivity of jointly immobilized cultures was as high as
under heterotrophic cultures solely of C. vulgaris.
DISCUSSION
Changes in the pH under different conditions of
incubation. In the absence of light and a carbon
source, the pH did not change with time for any
treatment (Fig. 6a). Under autotrophic conditions,
C. vulgaris alone or jointly immobilized with A. brasilense significantly and similarly reduced the pH to
<5 (Fig. 6b). Under heterotrophic conditions, significant reduction in the pH of both treatments was
recorded (Fig. 6c, lowercase letter analysis), where
joint immobilization reduced it faster in the first
3 d (Fig. 6c, capital letter analysis), but the level was
similar to C. vulgaris alone afterward. Under mixotrophic conditions, similar phenomena occurred,
with the single difference that C. vulgaris alone lowered the pH faster than a culture with jointly immobilized microorganisms (Fig. 6d).
Productivity of the three incubation regimes. The
number of cells grown per mg of ammonium per
liter of culture after 3 d of incubation was the highest under autotrophic growth; the other treatments
had no significant effect (Fig. 7a, lowercase letter
Most applications of microalgae use light because
microalgae are very efficient solar energy converters,
and they can produce a great variety of useful
metabolites autotrophically (Lebeau and Robert
2006). Yet, light-independent heterotrophic growth,
wherever possible, has significant economic advantage over light-dependent growth in mass-producing
microalgae (Chaumont 1993, Borowitzka 1999). So
far, because of the high volume of wastewater, autotrophic tertiary nutrient removal is uncommon (deBashan and Bashan 2004, Muñoz and Guieysse
2006).
Despite the economic incentive, heterotrophic
growth in wastewater treatment has inherent major
limitations. Those included (a) low tolerance to
high carbon concentrations; (b) competition with
other bacteria and yeasts that have enhanced growth
in heterotrophic, nonaxenic wastewater and that
have shorter generation times than the microalgae;
(c) not all species of microalgae can grow without
light (Lee 2001); and (d) some organic compounds
inhibit microalgal growth (Chen 1996). As an initial
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O C T A V I O PE R E Z - G A R C I A E T A L .
Fig. 6. Changes in the pH during cultivation of Chlorella vulgaris alone and jointly immobilized with Azospirillum brasilense growing
under autotrophic, heterotrophic, and mixotrophic growth conditions in synthetic wastewater. Values of each curve denoted with a different lowercase letter differ significantly by two-way analysis of variance (ANOVA) and by Tukey’s post hoc analysis at P < 0.05. Values at
each date denoted with a different capital letter differ significantly by ANOVA, all at P < 0.05. Bars represent standard error of the mean
(SE). Absence of a bar indicates negligible SE.
step for evaluating the potential of different cultivation systems for wastewater treatment, this study
used a defined synthetic wastewater in small laboratory bioreactors and a common strain of C. vulgaris,
previously shown to be an efficient wastewater treatment agent (de-Bashan et al. 2002b, 2004) and
capable of growing heterotrophically on municipal
wastewater supplemented either with glucose or Naacetate (O. Perez-Garcia, Y. Bashan, M. E. Puente,
unpublished data). Glucose is known to support
heterotrophic growth of several Chlorella sp., including C. vulgaris (Lee 2004).
Heterotrophic cultivation of C. vulgaris was superior to autotrophic and mixotrophic growth,
although each cultivation scheme has its advantages
under specific conditions, depending on the end
goal of cultivation: biomass or nutrient uptake. The
highest ammonium uptake (36%) occurred in heterotrophic cultivation when the culture was loaded
with a high concentration of ammonium, higher
than under autotrophic cultivation. This uptake by
the entire heterotrophic culture contrasts with the
superiority of autotrophic cultivation in terms of the
amount of growing cells, biomass produced, growth
rate, and the yield of cells per quantity of ammonium consumed. Although even higher percentage
uptakes of ammonium were previously recorded
using this immobilization system and this strain
under autotrophic conditions (de-Bashan et al.
2002b, 2004), those were performed in a far lower
initial ammonium concentration (<10 vs. 50 mgÆL)1
in this study). Consequently, in a total quantity of
ammonium uptake, the heterotrophic regime is
superior to the recorded level.
A plausible explanation for these results might be
found in a combination of high affinity of cells for
ammonium, and their high growth rate in heterotrophic cultivation of C. vulgaris, used alone, was far
superior to other treatments and culture conditions.
It appears that under heterotrophic conditions, cells
that are growing alone uptake large quantities of
ammonium and use it for cell multiplication. However, when these cells, jointly immobilized with
A. brasilense, are used for ammonium uptake, it is
not used for multiplication, but possibly stored, as
in cell cultures of higher plants growing in the dark
H E T E R O T R O P H I C , A U T O T RO PH I C , A N D MI X O T R O PH I C G R O W T H O F I MM O B I L I Z E D MI C R O A L G A E
809
Fig. 7. (a–c) Yield (number of cells produced per mg substrate uptake in 1 L of culture during the first 3 d of incubation) of Chlorella vulgaris alone and jointly immobilized with Azospirillum brasilense growing under autotrophic, heterotrophic, and mixotrophic growth
conditions in synthetic wastewater. (a) Ammonium, (b) phosphate, (c) glucose, and (d) volumetric productivity of all immobilized and
jointly immobilized systems. In each panel, columns of C. vulgaris alone or jointly immobilized with A. brasilense separately denoted with a
different lowercase letter differ significantly by two-way analysis of variance (ANOVA) and by Tukey’s post hoc analysis. Pairs of columns of
each treatment type denoted with a different capital letter differ significantly by Student’s t-test, all at P < 0.05.
and using glucose nutrition (Buchanan et al. 2000).
The reason that heterotrophic cultures of C. vulgaris
growing alone uptake more ammonium than cultures that are jointly immobilized with A. brasilense is
because of the higher growth rate. Consequently,
larger populations form, and this is not related to
the high affinity of the cells for ammonium. Under
mixotrophic conditions, cultures of C. vulgaris
(alone or with A. brasilense) take up more ammonium than cultures under autotrophic conditions
because of higher affinity for ammonium under
these conditions. Higher affinity for ammonium in
heterotrophic and mixotrophic cultures occurs
because more ATP and NAD(P)H for metabolic
processes is available, which is unrelated to autotrophic carbon assimilation (Calvin cycle) (Yang
et al. 2000), such as for primary nitrogen assimilation by the enzymes of the nitrogen cycle in Chlorella spp. The difference between the Sa values for
nitrogen and phosphorus (discussed later) might be
explained because during the first day of sampling
the largest uptake of both nutrients occurred with
the smallest population of microalgae, compared to
later samplings, where the population was larger but
removal of the nutrients did not significantly
increase or increased only to a small extent.
The presence of the MGPB A. brasilense enhanced
ammonium and phosphate uptake by the microalgae when light was provided under autotrophic and
mixotrophic conditions (de-Bashan et al. 2002b,
2004, 2008c), but not under heterotrophic conditions. This occurred because the ‘‘helper’’ bacterium enhanced, in general, the growth rate of the
microalgae (Gonzalez and Bashan 2000, de-Bashan
et al. 2008a, this study) by affecting photosynthesis
patterns. The specific role of Azospirillum had been
shown by producing phytohormones and affecting several cellular metabolisms of the microalga
(de-Bashan et al. 2002a, 2008a). So far, previous
studies have shown that its role in removing nutrients is minimal (de-Bashan et al. 2002b, 2004).
Furthermore, the two microorganisms significantly
differ in individual cell size and their final population size in culture. We showed that microalgal populations are at least one order of magnitude larger
than those of the MGPB. While these microalgal
810
O C T A V I O PE R E Z - G A R C I A E T A L .
populations are common for this species (Gonzalez
and Bashan 2000), the levels of the bacterial population for this bacterial species are small (Bashan
et al. 2004). This happened because the medium
suitable for the microalga is less suitable for the
growth of A. brasilense. Although A. brasilense can
use ammonium for growth (Van Dommelen et al.
1997), the relatively small populations of A. brasilense in a jointly immobilized system removed only
minute amounts of nitrogen and phosphate from
wastewater (de-Bashan et al. 2002b, this study) or
may live also on exudates from the microalgae
(Watanabe et al. 2006).
Under heterotrophic cultivation, joint immobilization significantly increased the affinity of C. vulgaris to ammonium and phosphate, compared to
other treatments, but did not increase the population. The yield, a value composed of the number of
cells per substrate (either ammonium or phosphate), is very low. All of these findings indicate
that the cells are metabolically active but do not use
these nutrients for multiplication. Our experimental
evidence supports the claim by de-Bashan et al.
(2005, 2008b) that the effect of the ‘‘helper’’ bacte-
rium is to increase ammonium consumption of each
cell and not that the elimination of ammonium is a
result of increase in the size of the population.
Removal of phosphate is far less affected by the
different treatments, where autotrophic cultivation
was the best one and adding the MGPB into autotrophic, heterotrophic, and mixotrophic conditions
enhanced this ability, similar to previous studies on
that topic (Hernandez et al. 2006). Differences in
rates of removal between the regimes are not a consequence of the affinity of the cells for phosphate.
In spite of the higher affinity in mixotrophic and
heterotrophic cultures for phosphate, removal in
autotrophic cultures was significantly superior. Superiority of C. vulgaris jointly immobilized with A. brasilense under autotrophic conditions could be
explained by accumulation of phosphorus as
polyphosphates (Hernandez et al. 2006) and ⁄ or a
higher efficiency in metabolizing inorganic phosphorus to ATP via photo and oxidative phosphorylation (Yang et al. 2000).
Regardless of the cultivation conditions and
because of the use of ammonium as the sole nitrogen source, the pH of the culture always declined to
Table 1. General analysis of the best growth condition (autotrophic, heterotrophic, mixotrophic) and immobilization type
(Chlorella alone or immobilized with Azospirillum brasilense) for each parameter tested.
Parameter
Chlorella growth
Azospirillum growth
Ammonium consumption
Affinity for ammonium
Phosphate consumption
Affinity for phosphate
Glucose consumption
Ammonium yield
Phosphate yield
Glucose yield
Volumetric productivity
Per specific growth conditions
Control – jointly immobilized
Autotrophic – jointly immobilized
Heterotrophic – Chlorella alone
Mixotrophic – jointly immobilized
Control – similar
Autotrophic – jointly immobilized
Heterotrophic – jointly immobilized
Mixotrophic – similar
Control – jointly immobilized
Autotrophic – jointly immobilized
Heterotrophic – Chlorella alone
Mixotrophic – jointly immobilized
Not applicable
Control – Chlorella alone
Autotrophic – jointly immobilized
Heterotrophic – jointly immobilized
Mixotrophic – similar
Not applicable
Heterotrophic – jointly immobilized
Mixotrophic – jointly immobilized
Control – similar
Autotrophic – Chlorella alone
Heterotrophic – Chlorella alone
Mixotrophic – similar
Control – jointly immobilized
Autotrophic – similar
Heterotrophic – Chlorella alone
Mixotrophic – similar
Heterotrophic – Chlorella alone
Mixotrophic – similar
Control – jointly immobilized
Autotrophic – jointly immobilized
Heterotrophic – Chlorella alone
Mixotrophic – jointly immobilized
Similar: treatment of Chlorella alone = treatment of jointly immobilized with A. brasilense.
Comparison among all growth conditions
Heterotrophic – Chlorella alone
Heterotrophic – jointly immobilized
Heterotrophic – Chlorella alone
Heterotrophic – jointly immobilized
Autotrophic – jointly immobilized
Heterotrophic – jointly immobilized
Similar
Autotrophic – Chlorella alone
Heterotrophic – Chlorella alone
Heterotrophic – Chlorella alone
Autotrophic – jointly immobilized
Heterotrophic – Chlorella alone
H E T E R O T R O P H I C , A U T O T RO PH I C , A N D MI X O T R O PH I C G R O W T H O F I MM O B I L I Z E D MI C R O A L G A E
low values because consumption of ammonium
releases protons and the decreased pH impairs cultivation (Kaplan et al. 1986, Grobbelaar 2004, this
study). When C. vulgaris grew on high concentrations of ammonium, the pH of the culture dropped
as long as ammonium was present, even if the pH
was adjusted daily (Tam and Wang 1996). All these
factors imply that, in these cultures, there is no loss
of ammonium via volatilization, because ammonium
is converted to volatile ammonia only at pH > 8.5.
Joint immobilization with A. brasilense reduced the
negative effects of pH shifts, but at higher pH levels
than the ones present in this study (de-Bashan et al.
2005).
A general evaluation of the various growing
regimes and immobilization parameters explored in
this study (Table 1) showed that heterotrophic cultivation of C. vulgaris yielded the highest uptake of
ammonium from synthetic wastewater compared to
autotrophic and mixotrophic growth. Heterotrophic
cultivation did not enhance uptake of phosphate,
compared to the other treatments. Adding the MGPB
to the culture had positive effects only for autotrophic cultivations (high ammonium and phosphate
removal and high cell populations of C. vulgaris), but
not for heterotrophic and mixotrophic cultures.
We thank the following personnel of CIBNOR: Iban Murillo
(phosphate analysis), Jorge Cobos (bioreactor manufacturing), Roberto Hernandez (glucose analysis), and Esther
Puente (advice concerning microbiological techniques). This
study was mainly supported by Consejo Nacional de Ciencia y
Tecnologı́a (CONACYT contract 23917), Secretaria de Medio
Ambiente y Recursos Naturales (SEMARNAT contract 23510),
and time for writing by the Bashan Foundation, USA.
O. P.-G. is a recipient of a fellowship from CONACYT (contract 207021) with additional funding from SEMARNAT and
the Bashan Foundation.
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