Journal of Industrial and Engineering Chemistry 49 (2017) 145–151

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Journal of Industrial and Engineering Chemistry

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Removal of nutrients and COD from wastewater using symbiotic

co-culture of bacterium Pseudomonas putida and immobilized
microalga Chlorella vulgaris
Ghulam Mujtabaa,b , Muhammad Rizwana,c , Kisay Leea,*
a
Department of Environmental Engineering and Energy, Myongji University, Yongin 17058, Republic of Korea
b
Department of Energy and Environment Engineering, Dawood University of Engineering and Technology, Karachi 74800, Pakistan
c
Department of Environmental Sciences, University of Haripur, Haripur 22620, Pakistan

A R T I C L E I N F O A B S T R A C T

Article history:
Received 1 April 2016 Simultaneous removal of nutrients (ammonium and phosphate) and COD was investigated by the co-
Received in revised form 6 December 2016 culture consortium of microalga Chlorella vulgaris and bacterium Pseudomonas putida. The co-culture
Accepted 17 January 2017 system showed higher removal of both nutrients and COD than the each axenic culture, indicating that
Available online 27 January 2017 nutrients uptake capability of C. vulgaris was enhanced in the presence of P. putida. The best performance
in the removal of nitrogen, phosphorus, and COD was obtained through the co-culture with suspended P.
Keywords: putida and immobilized C. vulgaris, demonstrating that the employment of immobilization of one species
Chlorella vulgaris is more synergistic than suspended co-culture system in nutrients removal from wastewater.
Pseudomonas putida
© 2017 The Korean Society of Industrial and Engineering Chemistry. Published by Elsevier B.V. All rights
Symbiotic co-culture
reserved.
Immobilization
Nutrients removal

Introduction accumulating microorganisms need organic carbons to complete

Nitrogen and phosphorus are the main elements for eutrophi-
cation of natural water bodies. Eutrophication became more
common since the mid-20th century and considered as a severe
environmental issue, which can trouble the balance of aquatic
systems. Wastewaters containing nitrogen and phosphorus should
not be discharged directly into water body before adequate
treatment.
Conventional methods include many steps and are expensive
for the removal of nutrients from wastewater. Anaerobic–anoxic–
oxic (A2O) process, the most commonly used biological nutrient
removal (BNR) method, needs at least three bioreactors in series
(such as anaerobic, anoxic, and aerobic) in which working
conditions are contrastingly different and complicated [1].
Nitrogen removal is realized through nitrification and denitrifica-
tion using aerobic and anoxic reactors, respectively. Phosphorus is
removed through the coupling of anaerobic and aerobic reactors.
And, the aerobic reactor serves for the removal of organics.
Availability of COD is necessary for simultaneous removal of
nitrogen and phosphorous because denitrifiers and phosphate-
* Corresponding author. Fax: +82 31 336 6336.
E-mail address: kisay@mju.ac.kr (K. Lee).
reactions [2]. Methanol is usually added in conventional BNR
systems as a supplementary organic carbon source.
In this regard, the removal of nutrients from wastewater by
using the co-culture of bacteria and microalgae is an alternative
and renewable approach. If the growth and maintenance of two
different microorganisms are compatible in a co-culture system,
heterotrophic metabolism of aerobic bacteria and the capabilities
of nutrient uptake and photosynthetic oxygenation of algae can be
mutually symbiotic [3,4]. Photosynthetic microalgae can uptake
nitrogen and phosphorous into their biomass as cell constituents
and release exogenous oxygen to realize most of the aerobic
bacterial requirements. On the other hand, heterotrophic bacteria
can oxidize organic carbon (utilizing O2 from microalgae) and
produce CO2 that can be consumed by microalgae as an
autotrophic carbon source [5]. Sometimes, bacteria can stimulate
algal growth by the release of vitamins and plant hormones [6],
while microalgae can also release organic compounds that can be
used by bacteria as an energy source [7]. Thus, the simultaneous
removal of nitrogen, phosphorous, and organics can be achieved in
a single reactor by selecting appropriate strains of bacteria and
microalgae in co-culture system.
For the purpose of continuous operation of treatment reactor
and miroalgal cells are to be utilized further (e.g., for biofuel
production), the recovery of microalgal cells from the treated

http://dx.doi.org/10.1016/j.jiec.2017.01.021
1226-086X/© 2017 The Korean Society of Industrial and Engineering Chemistry. Published by Elsevier B.V. All rights reserved.
146 G. Mujtaba et al. / Journal of Industrial and Engineering Chemistry 49 (2017) 145–151
wastewater is necessary. If cells entrap in an immobilization
matrix, microalgae can easily be separated from bacteria and
harvested from the effluent stream. The main advantages of
immobilization technique also include the prevention of biomass
washout from the process, a greater degree of operational
flexibility, and easy separation [8]. Alginate is a cost-effective
matrix that can easily be used to entrap microbial cells in alginate
beads. Cells were found evenly distributed within each bead, and
cell viability is maintained successfully for an extended time [9].
Algal cells immobilized in alginate beads showed the same
efficiency to remove nitrogen and phosphorous from wastewater
as that by suspended cells.
Green microalga Chlorella vulgaris was selected for the purpose
of nutrients removal and cell immobilization in this study because
it has a fast growth rate and short reproduction time. C. vulgaris is
often used for tertiary treatment of wastewater mainly for the
removal of nitrogen and phosphorus [10]. Heterotrophic aerobic
bacterium Pseudomonas putida is commonly present in activated
sludge process and showing good performance in organics removal
(or BOD removal). It is known that P. putida is co-culturable with C.
vulgaris and can also increase the growth of Chlorella [11].
This study aimed to remove nitrogen, phosphorus, and COD
simultaneously from synthetic wastewater by using the co-culture
system of suspended P. putida and immobilized C. vulgaris in a
single reactor. Alginate beads were used to immobilize C. vulgaris.
It is the first study, to the best of our knowledge, in which the co-
culture of immobilized algae and suspended bacteria is used for
the wastewater treatment. One benefit of this immobilized culture
is the convenience in separating C. vulgaris and P. putida after
treatment. The performance of nutrients and COD removal was
compared in different treatment systems, such as pure P. putida
culture, suspended co-culture, co-immobilized culture, and co-
culture with suspended P. putida and immobilized C. vulgaris. The
change in the cell population of C. vulgaris and P. putida was
compared, and several cycles of repeated treatment were
monitored to test the stability of the co-culture combination with
suspended P. putida and immobilized C. vulgaris in removing
nutrients and COD.
Materials and methods
Microorganisms and cultivation conditions
The freshwater green microalga C. vulgaris AG 30007 was
obtained from Korea Biological Resource Center (Daejeon, Korea).
C. vulgaris was inoculated in a modified BG-11 medium [12] using
250-mL flask and incubated at 25  C and 50 mmol m 2 s 1 of light
intensity (24 h). In order to obtain quicker growth, a bubble-
column photobioreactor [13] was used with the supply of 5% (v/v)
CO2 at 0.1 vvm and 100 mmol m 2 s 1 light using fluorescent tubes
prior to wastewater treatment. The gram-negative aerobic
bacterium P. putida ATCC 17514 was grown in liquid nutrient
broth at 30  C before using for wastewater treatment.
Chlorella was selected because they are widely used for the
studies on the treatment of wastewaters, easily found actually in
municipal wastewater treatment systems, and has been proved to
efficiently assimilate nitrogen and phosphorus from variety
sources of wastewaters. P. putida was selected because it is an
aerobic and culturable bacteria species which is popularly
involved in activated sludge processes for the treatment of
wastewater.
Synthetic municipal wastewater
Synthetic municipal wastewater (SMW) was prepared in order
to investigate the removal performance of nitrogen, phosphorous,
and organic carbon. The composition of SMW included: NH4Cl 190,
K2HPO4 40, KH2PO4 15, NaHCO3 275, and glucose 450 mg L 1. The
initial values of inorganic nutrients such as nitrogen and
phosphorous were 50 and 10 mg L 1, respectively, representing
the medium strength municipal wastewater. The initial level of
COD was adjusted at 490 mg L 1 using glucose. The concentrations
of inorganic carbon and alkalinity were measured as 50 and
200 mg L 1, respectively. Prepared SMW was autoclaved at 121  C
to avoid contamination prior to use.
The compositions of municipal wastewater are very diverse
depending upon wastewater sources. The concentrations of
nitrogen, phosphorus, and other components are not always
similar in real municipal wastewaters. The composition of SMW
used in this study was based upon Tchobanoglous and Burton [14],
where the composition of real wastewater generally ranges from
low-strength to high-strength. The medium-strength wastewater
was selected in this study to depict the characteristics of averaged
values of influents in several local municipal wastewater treatment
plants. Glucose is generally employed as an organic carbon in
artificial wastewaters because it is well biodegradable and results
in a stable COD or BOD values. Organic constituents and nutrients
are supposed to be assimilated into the biomass of bacteria and
microalgae.
Cell immobilization
Microorganisms were immobilized using the method de-
scribed by Gonzalez and Bashan [6]. Briefly, 5 mL of the
suspension of C. vulgaris was mixed with 20 mL of sterilized 2%
alginate solution and gently stirred for 15 min. The solution was
dripped from a sterile syringe into a 2% CaCl2 solution with slow
stirring. The beads (4 mm in diameter) formed were left for 1 h at
20  C for curing and then washed in sterile saline solution (0.85% of
NaCl). P. putida was immobilized in the similar manner. For the co-
immobilization of C. vulgaris and P. putida, the same concentration
of each microorganism was mixed, but the volume of each
microbial culture was reduced to 2.5 mL before adding the alginate
solution.
Experimental scheme
After the separate growth of C. vulgaris and P. putida, each was
centrifuged (3000 rpm for 3 min) and then washed three times
with 0.85% sterilized NaCl solution. The initial concentrations of C.
vulgaris and P. putida were determined as 5  106 cells mL 1 and
2.5  106 cells mL 1, respectively. The inoculum ratio of immobi-
lized microalgae to bacteria was 20 (Fig. 2), which was based on
the preliminary study [11] showing that a stable co-culture
consortium and a good nutrient removal performance are
maintained under the conditions with higher microalgae popula-
tion.
Experiments were performed in 250-mL Erlenmeyer flasks
containing 200 mL working volume of wastewater in a shaking
incubator. During the treatment of SMW, the continuous
illumination of 50 mmol m 2 s 1 (24:0 light/dark) was provided
via white fluorescent lamps. The temperature was controlled at
25  C. External aeration or CO2 was not supplied into the reactors;
the mixing was solely driven by flask-shaking. Four different
treatment systems were set up: (i) pure culture of suspended
P. putida, (ii) suspended co-culture of P. putida and C. vulgaris, (iii)
co-immobilized culture, and (iv) co-culture of immobilized
C. vulgaris and suspended P. putida. Experiments have been
performed at least three times for the verification and data are
presented with p-values less than 0.05 through statistical analysis
of variance (ANOVA) in SPSS ver. 22.0.
 G. Mujtaba et al. / Journal of Industrial and Engineering Chemistry 49 (2017) 145–151 147

Analyses
(a)
60
Samples of 1 mL of C. vulgaris and P. putida were taken from the
suspended cultures for the determination of cell population and
growth rate. For immobilized samples, five alginate beads were 50

first dissolved in 5 mL of 4% sodium bicarbonate solution [15] in

order to disintegrate the beads before measuring cell density. The 40

NH4 - N (mg L-1)

growth of C. vulgaris was determined by counting the cells using B
Neubauer haemocytometer. The growth of P. putida was assessed M
30 B+M
on nutrient agar plates by counting colonies.
IB+IM
Samples of 10 mL were collected and analyzed for ammonium, B+IM
nitrite, nitrate, phosphate, and COD. The samples were filtered out 20

through Whatman filter paper of 0.45 mm pores. The concen-

trations of NH4+-N, PO43 -P, and COD were measured based upon 10
the salicylate method, molybdovanadate method, and dichromate
digestion method, respectively, using HACH analysis kits and
0
spectrophotometer (HACH, DR 2800). Chromotropic acid method 0 1 2 3 4 5
and diazotization method were used for the measurement of
Time (d)
NO3 -N and NO2 -N, respectively, using the C-MAC kits (C-MAC, (b)
Ltd., Daejon, Korea). 12

Results and discussion

10

Removal of NH4+-N
8
The progress of nitrogen elimination was compared in four PO4 - P (mg L-1)
different treatment systems (Fig. 1a). Only 13% of ammonium was 6
eliminated in the suspended P. putida culture for 2 days and no
further removal was achieved, showing that P. putida alone is not
4 B
able to uptake or oxidize ammonium nitrogen efficiently.
M
Regardless of the state of C. vulgaris, suspended or immobilized, B+M
the addition of C. vulgaris to the culture of P. putida enhanced the 2 IB+IM
removal performance of nitrogen significantly. It indicated that B+IM
microalga C. vulgaris was mainly responsible for the elimination of 0
nitrogen as compared to bacterium P. putida. The uptake rate was 0 1 2 3 4 5
maximum during the first day of treatment (44–54% of total Time (d)
reduction) in all of the algae-employed cultures. The suspended (c)
co-culture eliminated 70% of ammonia for 5 days, which rate was 600
about 5 times higher than that by suspended P. putida alone. When
B
both microorganisms were immobilized, the removal efficiency of M
500
nitrogen was increased to 75%. While, the highest removal of B+M
nitrogen was achieved by the co-culture of immobilized C. vulgaris IB+IM
and suspended P. putida, by which about 85% of ammonia was 400 B+IM
COD (mg L-1)

removed for 5 days.

These results demonstrated that relatively nitrogen can be 300
eliminated from the wastewater more efficiently if microalgae is
entrapped or attached in a matrix. The physical or chemical
200
environment in entrapped or immobilized state seemed to have a
positive influence on microalgal activity regarding nutrient uptake.
However, better results were obtained when the P. putida was kept 100

in suspension (not immobilized) while C. vulgaris was immobi-

lized.
Ammonium is preferred nitrogen compound in its assimilation
by microalgae [16]. Immobilized algae could show more efficient
removal of nitrogen and phosphorous than suspended cells [17]. In
immobilized states, nutrients first adsorb on the surface of the
bead, then penetrate through matrix and are continually sorbed
into cells [18]. This environment may allow cells to face nutrients
in higher concentration and at closer distance. Also the immobili-
zation or entrapment of cells in a small confined space may trigger
interactions resulting in the enhancement of nutrient uptake. It is
known that the nature of alginate gel containing anionic group
(such as carboxyl groups) makes possible for their ionic interaction
with ammonium ions and hence facilitates ammonium removal in
immobilized cell systems [19].
0
0 1 2 3 4 5
Time (d)
Fig. 1. Removal of ammonium, phosphate, and COD by suspended bacteria culture
(B), microalgae (M), suspended co-culture of bacteria and microalgae (B + M),
immobilized co-culture (IB + IM), and the co-culture of suspended bacteria and
immobilized microalgae (B + IM).
de Bashan et al. [20] reported that C. vulgaris co-immobilized
with Azospirillum brasilense (microalgae growth-promoting bacte-
rium) eliminated 99% of ammonium (from 3 mg NH4+ L 1) after
6 days of treatment and 93% removal after 2 days, which is in
agreement with our finding that most of the nitrogen eliminated
within initial few days. Liang et al. [21] obtained 78% of nitrogen
148 G. Mujtaba et al. / Journal of Industrial and Engineering Chemistry 49 (2017) 145–151

removal (from initial 20 mg L 1) after 6 days using co-culture of C.

(a)
vulgaris and Bacillus licheniformis. Co-immobilization of C. vulgaris
and Bacillus pumilus did not increase the nitrogen removal 6.5
capability of the microalgae [22], which implies that some bacteria
may not have positive symbiotic relationship with algae in co-
6.0
immobilized state. No reports were found on the removal of

Microalgae (x10 cells mL )

-1
nutrients by co-culture system in which one member is
immobilized while the other one is suspended in a treatment 5.5

system.

6
In the conventional BNR processes, ammonia is removed from 5.0
wastewater through the oxidation of ammonium to nitrite and
nitrate by aerobic nitrification bacteria [23]. In this study, the
4.5
disappearance of NH4+ did not result in the increase of nitrite or
M
nitrate, showing constantly negligible levels of nitrate and nitrite B+M
through all cultivations (data not shown). This was observed 4.0
IB+IM
similarly in the previous suspended culture experiments [11]. The B+IM
evidence for nitrification was not found, as the concentration of 3.5
nitrate was continuously reduced from its initial value. 0 1 2 3 4 5
Ammonia can also be eliminated through stripping at high pH Time (d)
with a robust aeration. However, pH did not rise enough to support
this phenomenon and the aeration was also not so strong in our (b)
treatment system. Thus, the sole mechanism for the removal of
2.8
ammonia nitrogen in this study was the assimilatory uptake by C.
vulgaris.
2.7
Removal of PO43 -P
Bacteria (×10 cells mL )
-1

2.6
Fig. 1b shows the removal performance of phosphate by
suspended pure culture of P. putida and different co-culture
5

2.5
systems of P. putida and C. vulgaris. Phosphate was not substantially
removed in the pure P. putida culture and the level was even
increased after two days. This increase may happen due to the 2.4
release of some stored phosphates from the bacterial biomass into B
the culture medium. It is known that some bacterial species can B+M
2.3
IB+IM
uptake phosphate beyond the need for balanced growth in aerobic B+IM
environment, converting them into polyphosphates (so-called
2.2
luxury uptake), and release the stored polyphosphates from the 0 1 2 3 4 5
biomass into wastewater medium under anaerobic environment
Time (d)
[24]. In this study, since external aeration was not provided except
O2 evolution due to microalgal photosynthesis, the level of Fig. 2. Changes in the growth of C. vulgaris and P. putida in suspended bacteria
dissolved oxygen (DO) dropped near to zero after 2-d treatment culture (B), microalgae (M), suspended co-culture of bacteria and microalgae
(Fig. 3b) and thus it seems that P. putida released phosphate to the (B + M), immobilized co-culture (IB + IM), and the co-culture of suspended bacteria
medium. and immobilized microalgae (B + IM).

In comparison with pure P. putida culture, the co-culture

systems considerably reduced phosphate concentration. The
existence of C. vulgaris in co-culture systems played a definite
role in the elimination of phosphate. Among three co-culture
systems, the co-culture with immobilized C. vulgaris and
suspended P. putida exhibited the highest removal performance
of 66%. It indicated that microalgae may prefer to confine in an
immobilization matrix rather than in free suspension during the
elimination process. When both microorganisms were co-immo-
bilized in the beads, a little bit lower removal (64%) was obtained
compared to the reactor where immobilized C. vulgaris and
suspended P. putida exist. The removal efficiency was decreased to
60% in the reactor with suspended co-culture of both species (no
immobilization).
All the co-culture systems showed a similar pattern in the
removal of phosphorous. The assimilation rate was maximal
within first day of treatment and then the removal process was
getting slower as mentioned earlier in the removal of nitrogen.
However, the removal rate of phosphorous was relatively low
compared to that of nitrogen. Chevalier and De la Noue [25] also
mentioned that phosphate was not considerably eliminated as
nitrogen. Phosphorous removal by microalgae, compared to
nitrogen, is usually much slower and less efficient [23]. Microalgal
cells tend to saturate with phosphate fast due to the accumulation
of polyphosphates [26].
Phosphate removal process by immobilized Chlorella has been
successful [19]. The co-immobilized culture of C. vulgaris and A.
brasilense removed 75% of phosphorous after 2 days and then
elimination was levelled off [20]. These results suggested that
nutrients uptake rate is fast in the initial treatment time and after
some time removal become stabilized. Liang et al. [21] achieved
92% removal of phosphorous (from initial 4 mg L 1) after 6 days in
the co-culture system of C. vulgaris and B. licheniformis. In this
study, higher concentration of phosphorous (10 mg/L as PO4-P) was
used to simulate the P concentration level in real domestic
wastewater.
Phosphorous can also be removed through precipitation at high
pH of 9–11. However, as pH was not high enough in this study
(Fig. 3a) to trigger precipitation, the uptake of phosphate by C.
vulgaris was the only mechanism for phosphorous removal.
Removal of organic carbon
The removal of organic carbon (or COD removal) by different
treatment systems is shown in Fig. 1c. COD was successfully
 G. Mujtaba et al. / Journal of Industrial and Engineering Chemistry 49 (2017) 145–151 149

More significant amount of COD removal was accomplished in

(a)
the immobilized reactors (Fig. 1c). It seemed that microalgae in
10 beads, compared to suspended state, have more capacity to
consume organic carbons. At the end of treatment, the reactor with
B
M
immobilized C. vulgaris and suspended P. putida resulted in the
9 B+M highest elimination of COD (86%). Co-immobilized treatment
IB+IM system produced nearly similar results by removing 85.5% of COD.
B+IM Compared to these two reactors where C. vulgaris was immobi-
8 lized, the suspended co-culture and individual P. putida culture
exhibited lower reductions, which indicate the importance of
pH

using immobilized algae in the treatment. The suspended co-

7 culture achieved 78% of COD removal after 5 days of treatment,
which is still high as compared to that by pure P. putida culture. In
the pure P. putida culture, the removal efficiency was only 72% after
6
4 days. The possible reason for low COD removal rate in the P.
putida culture would be non-availability of sufficient oxygen
(Fig. 3b) for further mineralization of organic carbons as their
5
0 1 2 3 4 5
photosynthetic partner (C. vulgaris) was absent. In a study of Su
et al. [29], considerably more COD was removed in the reactor
Time (d)
employing activated sludge and microalgae compared to the
reactor with activated sludge only, indicating the collaboration
between algae and bacterial sludge.
As conclusions from Fig. 1, the simultaneous removal of
(b) nitrogen, phosphorous, and organic carbon was achieved in the
8
co-culture systems, showing the symbiotic benefits between C.
vulgaris and P. putida. Microalgae, through photosynthesis,
B
generate O2 that can be utilized by the bacteria as an electron
M
B+M
acceptor. On the other hand, through oxidation, bacteria are
6 IB+IM capable to produce CO2 that can be consumed as a carbon source by
B+IM microalgae. By comparing the removal performance of various co-
culture combinations with Chlorella alone (M) and P. putida alone
DO (mg L )
-1

(B) in Fig. 1, the synergistic effect on nutrients and COD removal in

4
co-culture systems is actually evidenced and the effect is enhanced
especially when Chlorella is immobilized (B + IM).

2 Changes in cell population

The change in the growth of microalga C. vulgaris during the

0 concentration of C. vulgaris reduced in the initial and then
0 1 2 3 4 5
Time (d)
Fig. 3. Changes in the concentrations of pH and DO in suspended bacteria culture
(B), microalgae (M), suspended co-culture of bacteria and microalgae (B + M),
immobilized co-culture (IB + IM), and the co-culture of suspended bacteria and
immobilized microalgae (B + IM).
reduced in all the systems indicating that both of C. vulgaris and P.
putida were capable to utilize the organic carbon for their
metabolisms. To acquire the carbon and energy, P. putida
mineralize organic carbons by consuming the dissolved oxygen
in wastewater (heterotrophic metabolism). This is the common
mechanism for COD reduction in aerobic wastewater treatment
systems using bacteria. C. vulgaris also can reduce COD levels
through mixotrophic metabolism as both light and organic carbon
were present. Generally, microalgae use photoautotrophic metab-
olism by utilizing light and CO2 (or inorganic carbon) as energy and
carbon sources. However, many algal species, including Chlorella,
can assimilate organic compounds (such as well-reduced organic
compounds like glucose) as a carbon source under the light [3,27].
Abeliovich and Weisman [28] also reported that organic carbons,
through mixotrophic or heterotrophic metabolism, were mutually
consumed by algae and bacteria in the algal-bacterial treatment
system. The present C. vulgaris strain is capable of consuming
organic carbon source (glucose) through mixotrophic metabolism.
treatment was explored in different co-culture systems. Cell
increased continuously (Fig. 2a) before the final day of treatment
where further growth did not occur. The reason of temporary
reduction in cell growth at the start is may be because C. vulgaris
need some adaption time in the new wastewater medium and in
the new co-culture environment. After adapting to new environ-
ment, C. vulgaris started growing actively. Cell concentrations
increased in all the cases from their initial ones. C. vulgaris grew
relatively well when it was immobilized. The highest growth rate
was achieved when both C. vulgaris and P. putida were immobilized
in the co-culture system. Almost similar results were obtained
when C. vulgaris was immobilized and P. putida was suspended. The
lowest growth rate was obtained in the suspended co-culture
system, which indicates the importance of growing microalgae in
immobilized state. No leakage of algal cells into the culture
medium was detected and the stability of alginate beads was
maintained during the cultivation.
It was reported that the growth of C. vulgaris is not reduced in
the gel matrix of alginate or carrageenan, and that chlorophyll
content was found even higher in the immobilized cells than that
in free cells [19]. The viability and catalytic performance were
maintained in the immobilized state as free cells [30]. Co-
immobilization of C. vulgaris and A. brasilense in alginate beads
significantly promoted the growth of microalgae [6,20]. Some
other bacterial strains (for example, B. licheniformis and B. pumilus)
also enhanced the growth of C. vulgaris [21,22]. However, such
synergistic interaction is not general in all co-culture situations.
150 G. Mujtaba et al. / Journal of Industrial and Engineering Chemistry 49 (2017) 145–151

For instance, no positive influence on the growth of microalgae was

(a) 60
appeared in an agro-industrial wastewater pond where a naturally
I II III
occurring C. vulgaris was closely associated with the nitrogen-
fixing bacterium Phyllobacterium myrsinacearum [31]. 50
From the growth of Chlorella in Fig. 2a and corresponding
phosphate removal in Fig. 1b, it is noted that the phosphate 40

NH4 - N (mg L-1)

removal rate is pretty high at the beginning of the culture (for 1-d
period) even though microalgae population is not actively 30
increasing. It is suspected that the phosphate removal during this
early period is due to luxury uptake mechanisms of phosphate by
20
microalgae [32], although the polyphosphate accumulation was
not examined yet. Luxury uptake is the phenomenon of taking up
phosphorous by microorganisms more than is necessary for 10

growth. This additional phosphorus uptake is stored as poly-

phosphate which can then be used as an internal P source when the 0
external P concentration is not sufficient. 0 2 4 6 8 10 12 14 16
Fig. 2b shows the change in bacterium P. putida propagation in Time (d)
different treatment systems. Similar to the growth pattern of C.
vulgaris, the concentrations of P. putida were reduced at first. P. (b) 12
putida may also need some adaptation time in the new I II III
environment, and after adjusting in the new conditions, P. putida 10
gradually started to grow likewise C. vulgaris. The highest
concentration was achieved in the co-culture of immobilized C. 8

PO4 - P (mg L-1)

vulgaris and suspended P. putida. When C. vulgaris and P. putida
were co-immobilized or both suspended, the populations of P.
6
putida became lower than those of pure P. putida culture and the
co-culture of immobilized C. vulgaris and suspended P. putida. In
the pure P. putida culture, the growth was not increased further 4
after 4 days because of no DO was available in the culture medium
(Fig. 3b) to degrade the organic carbon. 2

Changes in pH and DO 0
0 2 4 6 8 10 12 14 16
Fig. 3 shows the changes in pH and DO during the treatment Time (d)
with pure P. putida culture and different co-culture systems. The
pH dropped from the initial value in all the treatment systems (c) 600
(Fig. 3a). This decrease is may be due to the formation of some I II III
organic acids by bacteria (during the degradation of organic
500
carbon) and due to releasing of H+ ions by microalgae (during the
consumption of NH4+) [33]. Larger pH drop in the co-culture
systems would be because both phenomena occurred simulta- 400
COD (mg L-1)

neously in these treatment systems. In comparison of different co-

culture systems, the reductions in pH were clearly corresponded to 300
the reductions in ammonium ions (Fig. 1a); more pH dropped as
more the ammonium ions were eliminated by C. vulgaris. After 200
certain time of treatment, pH did not drop further when microalgal
uptake of nitrogen became stable.
100
Fig. 3b shows the change in DO. All the treatment systems
displayed the decrease in DO concentrations from the initial
0
3 mg L 1. DO was completely diminished in the pure P. putida
0 2 4 6 8 10 12 14 16
culture and the level was reduced to almost zero after 3 days. It is
noted that no external aeration was supplied in these experiments. Time (d)

The DO levels were significantly higher in the co-culture systems Fig. 4. Removal of ammonia, phosphate, and COD by the co-culture of suspended P.
than in pure P. putida culture, due to the presence of photosyn- putida and immobilized C. vulgaris during three repeated operations.
thetic microalga C. vulgaris.

Repeated batch treatment

The stability of co-culture system with suspended P. putida and
immobilized C. vulgaris was investigated by repeated batch
operation for three cycles. After the completion of one batch
operation, fresh wastewater was replenished after discarding the
spent medium through centrifugation. The stability of the co-
culture was confirmed as nutrients and COD were continuously
reduced during 3 cycles of repeated batch operation (Fig. 4). The
co-culture exhibited the removal efficiencies of 78–85% for
nitrogen (Fig. 4a), 54–65% for phosphorous (Fig. 4b), and about
86% for COD (Fig. 4c). The removal patterns of nitrogen,
phosphorous, and COD were almost unchanged as treatment
was repeated, which indicated that the co-culture consortium of P.
putida or C. vulgaris was stable enough to expand this system to a
continuous culture and to a practical scale.
In real wastewater, it is expected that the characteristics of N
and P removal would be similar because C. vulgaris are able to
 G. Mujtaba et al. / Journal of Industrial and Engineering Chemistry 49 (2017) 145–151 151

uptake ammonia, nitrate and some organic nitrogen. However, it is References

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