ASIA-PACIFIC JOURNAL OF CHEMICAL ENGINEERING

Asia-Pac. J. Chem. Eng. 2014; 9: 368–373

Published online 28 March 2014 in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/apj.1805

Special theme research articles

Dairy effluent treatment and lipids production by Chlorella
pyrenoidosa and Euglena gracilis: study on open and closed
systems
Rajasri Yadavalli,1* C. S. Rao,1 Ramgopal S. Rao1 and Ravichandra Potumarthi2
1
Department of Biotechnology, Sreenidhi Institute of Science and Technology, Hyderabad, India
2
Department of Chemical Engineering, Monash University, Clayton, Australia

Received 31 July 2013; Revised 18 December 2013; Accepted 20 December 2013

ABSTRACT: Organic content and nutrients from dairy effluent by immobilized Chlorella pyrenoidosa and Euglena gracilis
were removed in both open and closed systems. Biochemical constituents viz, carbohydrates, lipids, and proteins were
analyzed to assess the changes in their proximate composition in both the systems for both species. Euglena gracilis has a
high lipid content of 11% at the end of 7 days of cultivation period compared and to C. pyrenoidosa resulted in 7% at the
end of 10 days, respectively. Whereas protein content of Chlorella sp. was found to be higher at 44% compared to 29%
of Euglena sp. A significant increase was also observed in carbohydrates in C. pyrenoidosa at the end of 10 days. Whilst
NH4+-N was completely removed by immobilized Chlorella sp., it was reduced by 96% compared to Euglena sp. Further,
98% PO43 -P removal was achieved with immobilized cultures of both species in two modes of operation. Biological
oxygen demand and chemical oxygen demand were observed in the range of significant decrease (~80%–96%) by this
approach. © 2014 Curtin University of Technology and John Wiley & Sons, Ltd.

KEYWORDS: microalgae; wastewater; bioremediation; photobioreactor; immobilization; lipids; biofuels

INTRODUCTION insoluble fraction or is converted into an activated

Dairy effluents contain high organic load, and unscru-
pulous discharge of these effluents into aquatic bodies
is a matter of serious concern besides deteriorating their
water quality.[1] These effluents contain high biological
oxygen demand (BOD), chemical oxygen demand
(COD), and nutrients such as phosphates, ammonia,
and/or nitrate, which pose a serious threat to the water
quality and cause damage to aquatic life.[2] High
BOD promotes bacterial growth and reduces dissolved
oxygen levels in water, which may prove lethal to
aquatic life. Much alike BOD, a high COD can also
deprive aquatic organisms of oxygen needed for
survival. In addition, removal of high organic content
and nutrients, in particular N and P, is a major
challenge in wastewater treatment, which otherwise
culminates in eutrophication upon their accumulation
in water bodies. Of these, P is particularly difficult to
remove from wastewater. For most commercial
wastewater processing, P is precipitated from the
effluent with the use of chemicals to form a solid
*Correspondence to: Rajasri Yadavalli, Department of Biotechno-
logy, Sreenidhi Institute of Science and Technology, Hyderabad,
India. E-mail: rajasriy@sreenidhi.edu.in, p.rajasri@gmail.com
© 2014 Curtin University of Technology and John Wiley & Sons, Ltd.
Curtin University is a trademark of Curtin University of Technology
sludge by microbial activity.[3] However, P recovered
by these methods is not fully recyclable, and the P
precipitate is either buried in landfill or further treated
to generate sludge fertilizer.
Microalgae are mostly suspension-type
microorganisms and very efficient solar driven cell
factories that have great potential in wastewater
treatment and bioenergy generation.[4,5] Many species
of microalgae effectively grow in wastewaters through
their ability to utilize abundant organic carbon and
inorganic N and P present in it. Use of microalgae in
wastewater treatment has been long promoted.[6,7]
Although application of microalgae in wastewater
treatment industry is still fairly limited, algae are used
throughout the world for wastewater treatment albeit
on a minor scale. Microalgae are efficient in removing
N, P, and toxic metals from wastewater[8,9] and
therefore have potential to play an important role
particularly during the tertiary treatment of
wastewaters. Significant advantage of algal processes
in wastewater treatment over the conventional
chemical-based treatment methods is the potential cost
saving and the lower level technology that is utilized,
therefore making this approach more attractive to
developing countries.
Asia-Pacific Journal of Chemical Engineering
Although conventionally physical, chemical and
biological methods were available for the treatment of
effluents derived from the agro-food industries,[10,11]
algal remediation offers the combined advantages of
treating wastewaters and simultaneously producing
algal biomass, which can further be exploited for
protein complements and food additives for aquacul-
ture, animal and human feed, energies such as biogas
and fuels, and biofertilizer.[12] Moreover, N-rich and
P-rich wastewaters are also viewed as a valuable
substrate for cultivation of microalgae. Chlorella
pyrenoidosa is a common freshwater alga, which
grows in ponds, lakes, and rivers often subjected to
organic pollution. Among the microalgae used to treat
effluents, little information is available on the utility
and efficiency of C. pyrenoidosa and Euglena gracilis
as potential agents for treatment of dairy effluent.
While microalgae can be potentially employed to
treat high organic content, separation of algal biomass
from treated effluent is a key for the success of
wastewater treatment. Efforts have been made to develop
suitable technology for harvesting microalgae.[13–15] Use
of industrial filtration and centrifugation is expensive for
wastewater treatment as they are energy intensive
leading to high cost of operation. In this context,
immobilization of algal cells has been proposed to
overcome harvest problem as well as retaining high-
value algal biomass for further processing.[16,17]
Immobilization has been a proven technology for
microbial confinement for variety of purposes in the area
of industrial biotechnology for enzymes and antibiotics
production and in environmental biotechnology for
wastewater treatment.[18–22]
Research indicates that little work is performed on
immobilized Euglena and Chlorella as potential
candidates for removal of high organic content and
nutrients from dairy effluents. In light of this, we
examined the suitability of dairy effluent as a nutrient
medium for microalgae growth and evaluated the
efficiency of immobilized C. pyrenoidosa and E. gracilis
to reduce/remove some of the organic compounds like
BOD and COD in the dairy effluent in both open and
closed pond systems, and the corresponding results were
presented in this paper.
MATERIALS AND METHODS
Microalgae and culture conditions
Euglena gracilis sp. [National Centre for Industrial
Microorganisms (NCIM): 2790] and C. pyrenoidosa
sp. (NCIM NO: 2738) algae cultures were obtained
from NCIM, Council of Scientific and Industrial
Research National Chemical Laboratories, Pune, India.
Stock cultures of C. pyrenoidosa and E. gracilis sp.
were grown photoautotrophically at 28 °C under
© 2014 Curtin University of Technology and John Wiley & Sons, Ltd.
DAIRY EFFLUENT TREATMENT AND LIPIDS PRODUCTION 369
continuous light illumination in four 100 ml borosil
flasks each having composition (g l 1) in BG11
medium[23] and EG[24] medium, respectively.
Inoculum was prepared by transferring cells from
stock culture and incubated aseptically in a 1000 ml
flask containing 700 ml of respective media under
continuous illumination of 34 μmol m 2 s 1 at 28 °C
for 4 days on an orbital shaker set at 120 rpm. A
4-day-old culture was used as inoculum at 10%
volume for the preparation of immobilized microalgal
beads. Gel entrapment is performed using 2% sodium
alginate and 0.1 M calcium chloride. Average
diameter of the both species algal beads inoculated
was 0.556 mm approx. Untreated dairy effluent was
collected from Vyshnavi dairy, Khammam, Andhra
Pradesh, India.
Photobioreactor operation
Closed system
Approximately 50 g of immobilized beads were
weighed and inoculated in each 16 1 L inverted
polycarbonate bottles set on a wooden rack (Fig. 1(a)
and 1(b)). Artificial air supply was provided through air
pumps at 1 L min 1 for all 16 bottles continuously to
ensure suspension of immobilized beads in dairy water
and thorough contact of beads with the nutrients present
in dairy effluent. A light intensity of 55 μmol m 2 s 1
was maintained at 28 °C for 4 days.
Open pond cultivation
Untreated dairy effluent was transferred to three 40-L
glass tanks used as simulated pond bioreactors for algal
growth with water volume was 10 L in each bioreactor.
Air stones were placed in the middle of each bioreactor
to provide mixing and gas exchange. Air-sparging was
at a rate of 1.5 L min 1 divided among all of the algal
growth containers. The light intensity was determined
using a Lux Meter (LM-52-780). Culture volume was
maintained constant by replenishing dairy effluent to
compensate evaporation losses in open pond system.
Microalgal beads were separated once in every
3 days for C. pyrenoidosa and once in 2 days for E.
gracilis from treated dairy effluent to check algal
biomass growth, which was analyzed for carbohydrate,
protein, and lipid content. During experimental period,
dairy effluent was analyzed for BOD (three at 27 °C),
COD, NH4+-N, phosphates, pH once in every 3 days
for C. pyrenoidosa and once in 2 days for E. gracilis.
Estimation of lipid, carbohydrate, and protein
The carbohydrate content was analyzed based on the
procedure published by Miao and Wu.[25] In short,
0.1 g dried algal pellet was acidified by adding 20 ml
2.5 M HCl. The acidified solution was then hydrolyzed
at 100 °C for 30 min and neutralized to pH 7. The
Asia-Pac. J. Chem. Eng. 2014; 9: 368–373
DOI: 10.1002/apj
370 R. YADAVALLI ET AL. Asia-Pacific Journal of Chemical Engineering

Figure 1. (a) Closed system and (b) open pond system for treatment of dairy effluent. This
figure is available in colour online at www.apjChemEng.com.

volume was adjusted to 100 ml. The filtered sample

was subjected to 3,5-dinitrosalicylic acid assay.[26]
Lipid extraction was performed in a way that was
similar to the procedures reported by previous
studies.[27,28] Briefly, 1.0 g dried cells were ground in
a mortar and pestle. The dried powder is then
transferred to a 50 ml glass centrifuge tube. Chloroform
was then added to the tube to make the chloroform/
methanol (2 : 1, v/v). The tube was vortexed for 5 min
and was allowed to stand for 24 h. After that, the tube
was centrifuged at 4000 g for 15 min to remove the algal
solids. The supernatant was collected, and the solvent
was vaporized using Rotovap at 65 °C. Oil left in the flask
without solvent was weighed to calculate lipid content.
Protein estimation was performed using Lowry’s
method.[29] Briefly, 0.1 g algal powder was taken, 2 ml Figure 2. General process flowdiagram of dairy treatment
of 5% trichloro acetic acid was added, and the mixture using microalgae. This figure is available in colour online
was centrifuged at 2000 rpm for 10 min. Solution at www.apjChemEng.com.
containing pellet was dissolved in 1.5 ml of 0.1 N NaOH.
Around 0.2 ml of the aforementioned prepared solution
was taken into 20 ml test tube, and 5 ml of alkaline copper be stable in acidic pH ranges up to 4 days (5.5–6.5).[30]
solution was added. After incubation at room temperature Hence, the bead stability was ensured throughout the
for 15 min, 0.5 ml of 1 : 2 diluted Folin–Ciocalteu reagent experiment. Fully bulged algal beads of diameter
was added. Again, the mixture was incubated for 30 min, 1.078 mm and 1.081 mm of C. pyrenoidosa and E.
and Optical Density (OD) values were taken at 540 nm. A gracilis, respectively, were analyzed for lipid, carbo-
general process flow diagram was represented for the hydrate, and protein content (Figs 5 and 6).
treatment of dairy effluents using microalgae in Fig 2.
Biochemical composition analysis

RESULTS AND DISCUSSION Chlorella pyrenoidosa

Biochemical analysis of algal biomass
Growth of both algae cultures in dairy effluent was
determined in closed and open systems, and time
maxima were found to be 10 days and 7 days for C.
pyrenoidosa and Euglena, respectively (Fig. 3a and
3b). Subsequently, algal beads were separated from dairy
water effluent using nylon filter cloth (Fig. 4). Earlier
studies mentioned that even at high concentrations (up
to 900 mg L 1) of phosphates, immobilized beads tend to
© 2014 Curtin University of Technology and John Wiley & Sons, Ltd.
Lipid content increased to 6% after 10 days of
cultivation of C. pyrenoidosa in closed system.
Carbohydrate was 14% on the 3rd day and increased
by 100% on the 10th day. Protein content was highest
of all the biochemical compositions analyzed in closed
system for C. pyrenoidosa, which increased from 19%
to 44% by the 10th day. Whereas lipid content
increased from 2% to 7% and carbohydrate increased
up to 28% after last day of cultivation in open pond
system for the growth of C. pyrenoidosa. The increase
of protein content, however, was similar to that of
Asia-Pac. J. Chem. Eng. 2014; 9: 368–373
DOI: 10.1002/apj
Asia-Pacific Journal of Chemical Engineering DAIRY EFFLUENT TREATMENT AND LIPIDS PRODUCTION 371

Figure 3. Growth curves of Chlorella pyreniodosa and Euglena gracilis in dairy

effluent in (a) closed system and (b) open pond system. This figure is available in
colour online at www.apjChemEng.com.

Figure 4. Wet algal beads of (a) Chlorella pyrenoidosa and (b) Euglena gracilis (sample and
control) separated after cultivation period. This figure is available in colour online at www.
apjChemEng.com.

Figure 5. Chlorella pyrenoidosa biochemical composition
in (a) closed system and (b) open system during cultivation
time. This figure is available in colour online at www.
apjChemEng.com.
© 2014 Curtin University of Technology and John Wiley & Sons, Ltd.
Figure 6. Euglena gracilis biochemical composition in
(a) closed system and (b) open system during
cultivation time. This figure is available in colour
online at www.apjChemEng.com.
Asia-Pac. J. Chem. Eng. 2014; 9: 368–373
DOI: 10.1002/apj
372 R. YADAVALLI ET AL. Asia-Pacific Journal of Chemical Engineering

closed system, i.e. by 43% (Fig. 5). High protein
content of algal biomass reported in present study in
both closed and open pond systems was supported by
Rodulfo in his earlier studies.[31]
Euglena gracilis
Overall, lipid production of 11% was relatively higher
in E. gracilis compared to C. pyrenoidosa, which was
in support with earlier studies[32,33] while protein
content of Chlorella sp was found to be high of 44%
in both the systems (Fig. 6). High protein content of
algal biomass reported in present study in both closed
and open pond systems was in accordance with earlier
studies.[25] Carbohydrate content was also more when
compared to C. pyrenoidosa, but protein content was
slightly less (~32%) in E. gracilis when compared with
that of C. pyrenoidosa.
Dairy water analysis
Nitrogen and phosphate removal
Phosphates are reduced by 99% and 97% in closed
system after algal bead treatment for C. pyrenoidosa
and E. gracilis, respectively (Tables 1 and 2). Whereas
reduction in phosphates was 96% in open pond system
for both species, which is less when compared to
closed system. NH4+-N was almost completely reduced
in both closed and open systems for C. pyrenoidosa
whereas reduction with E. gracilis for closed and open
pond systems is 97% and 95%, respectively. However,
on a whole, there was a significant decrease of N and P,
which are under acceptable limits by Andhra Pradesh
Pollution Control Board, may be because of algal
uptake and adsorption on alginate gels that were found
to be major processes involved in the removal of N and
phosphate in present study. Previous studies have also
reported that air-stripping of ammonia is a possible
mechanism for N removal in an intensively aerated
microalgal system with alkaline pH resulting from
photosynthetic activity and aeration.[34,35]
Effect on pH
Untreated dairy effluent has an acidic pH value initially,
but after algal beads treatment with both the species, the
treated effluent is shifted to alkaline pH range. NH4+-N
could be lost via ammonia volatilization while PO43 -P
was removed by chemical precipitation, because of
which alkaline pH was recorded in the treatment process.
BOD and COD removal
Wastewater of dairy industry contains large quantities
of milk constituents such as casein, lactose, fat, and
inorganic salts besides detergents and sanitizers used
for washing. All these components contribute largely
toward their high biochemical oxygen demand. High
BOD and COD values lead to the deprival of oxygen
for aquatic life in water. BOD and COD have decreased
up to 92% and 86% (approximately), respectively, after
algal bead treatment with C. pyrenoidosa in both types of
operating systems (Table 1). Whereas with Euglena algal
bead treatment, the BOD and COD reduced by 96% and
80% (approximately) in both closed and open pond
systems (Table 2). Previous work also showed that algal
uptake had little effect on the removal of COD.[3]

Table 1. Water analysis of dairy effluent with Chlorella pyrenoidosa in closed and open systems during cultivation
period.

Type of system Operation (days) BOD (mg L 1) COD (mg L 1) NH4+-N (mg L 1 1
) Phosphates pH
Control (untreated) 1200 2900 35 30 6.5
Closed system 3rd day 600 1500 15 18 8.01
6th day 246 1100 9 8 8.01
10th day 101 400 0.64 0.87 8
Open system 3rd day 550 1400 15 20 8.01
6th day 232 1000 9 10 8.01
10th day 97 350 0.64 0.94 8

BOD, biological oxygen demand; COD, chemical oxygen demand.

Table 2. Water analysis of dairy effluent with Euglena gracilis in closed and open systems during cultivation
period.

Type of system Operation (days) BOD (mg L 1) COD (mg L 1) NH4+-N (mg L 1) Phosphates pH
Control (untreated) 1200 2900 35 30 6.5
Closed system 3rd day 314 1200 1.9 1.23 8.23
7th day 45 700 0.89 1.01 8.76
Open system 3rd day 225 1100 2.2 1.8 8.2
7th day 42 448 1.6 1.22 7.6

BOD, biological oxygen demand; COD, chemical oxygen demand.

© 2014 Curtin University of Technology and John Wiley & Sons, Ltd. Asia-Pac. J. Chem. Eng. 2014; 9: 368–373
DOI: 10.1002/apj
Asia-Pacific Journal of Chemical Engineering
CONCLUSIONS
In the present study, we conclude that both the
microalgae demonstrated similar efficiency in treating
BOD, COD, and nutrients present in the dairy effluent.
While E. gracilis showed high total lipid content,
which may be further used for biodiesel production,
high protein content observed in C. pyrenoidosa can
be further used as animal feed upon harvesting.
Moreover, the mode of operation has little effect on
the efficiency of treatment with more or less similar
results in both closed and open systems. This study
demonstrates that microalgae can be potentially
employed for simultaneous treatment of dairy effluent
along with the production of value added products for
commercialization.
Acknowledgements
Author, Rajasri Y. is thankful to the management of
Sreenidhi Institute of Science and Technology (SNIST)
for their financial support to carry out current work
through in-house project fund. Thanks to Mr. H. R.
V. N. Gowtham Rao and G. Praveen Kumar for their
help during the project. Special thanks to Vyshanavi
Dairy Pvt Ltd., Khammam, Andhra Pradesh, India for
providing dairy effluent for conducting experiments.
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