Desalination and Water Treatment 57 (2016) 29118–29125

[Link] December

doi: 10.1080/19443994.2016.1139101

Isolation and screening of microalgae from agro-industrial wastewater

(POME) for biomass and biodiesel sources

Hesam Kamyaba, Mohd Fadhil Md Dina, Mohanadoss Ponrajb, Ali Keyvanfarb,

Shahabaldin Rezaniaa, Shazwin Mat Taiba, Muhd Zaimi Abd Majidb,*
a
Centre for Environmental Sustainability and Water Security (IPASA), Research Institute for Sustainable Environment, Faculty of
Civil Engineering, Department of Environmental Engineering, Universiti Tteknologi Malaysia (UTM), Skudai, 81310 Johor,
Malaysia, emails: hesam_kamyab@[Link] (H. Kamyab), mfadhil@[Link] (M.F. Md Din), shahab_rezania89@[Link]
(S. Rezania), shazwin@[Link] (S.M. Taib)
b
Construction Research Center (CRC), Faculty of Civil Engineering, Institute for Smart Infrastructure and Innovation Construction
(ISIIC), Universiti Tteknologi Malaysia (UTM), Skudai, 81310 Johor, Malaysia, emails: goldking1977@[Link] (M. Ponraj),
alikeyvanfar@[Link] (A. Keyvanfar), Tel. +60197536100; email: mzaimi@[Link] (M.Z. Abd Majid)
Received 2 September 2015; Accepted 4 December 2015

ABSTRACT

Indigenous microalgae (KR349061) were isolated from the agro-industrial wastewater and
screened in order to determine their potential for biomass and biodiesel sources. Bold’s
Basal Medium was used to isolate the colonies from sample. The nutrient contents were
altered in order to optimize the growth of the microalgae. The isolates were identified based
on the morphology and microscopic appearance of the isolated colonies. It was seen that
the isolates comprised of several common green microalgae and cyanobacteria, among
which the ideal candidate was selected and further investigated. Lipid content was deter-
mined for the strains that showed rapid growth. The results showed that Chlamydomonas
incerta achieved highest lipid content of biomass (42.6%) after 2 d, also highest lipid produc-
tivity of 0.197 mg/L/d was observed when the initial COD concentration was 500 mg/L. C.
incerta also showed higher growth rate and higher amount of lipid was examined for its
potential as sustainable biodiesel feedstock along with its higher biomass yield.

Keywords: Microalgae; Chlamydomonas incerta; POME; Lipid; Biodiesel

1. Introduction Mainly because the use of microalgae to produce bio-

diesel will not compromise the production of products
Microalgae have the potential to be the main
like fodder, food, and others derived from the crops
source of biodiesel, which has the potential to replace
[1,3]. The most important parameters in regulating
fossil fuel [1,2]. It has also been proven to be one of
algal growth by means of photosynthesis are the envi-
the promising resources for producing biodiesel.
ronmental conditions like light intensity, pH, tempera-
ture, turbulence, salinity [4,5], and nutrients [4].
*Corresponding author.

Presented at The 2nd IWA Malaysia Young Water Professionals Conference 2015 (YWP15)
Aspiring Beyond Water Solution, March 17–20, 2015, Langkawi Island, Malaysia

1944-3994/1944-3986 Ó 2016 Balaban Desalination Publications. All rights reserved.

 H. Kamyab et al. / Desalination and Water Treatment 57 (2016) 29118–29125 29119

The range of optimal conditions as well as the tolera- substrate for the growth of microalgae to produce more
ble range of operating conditions are species specific lipids, which was quantified using Nile red (NR)
and various factors may be interdependent to each method.
other [5]. The potential aspect of microalgae when
compared to other advanced feedstock is that the vari-
able number of species that are available for the pro- 2. Materials and methods
duction of biofuel [6]. Different species of microalgae 2.1. Sample collection
can be selected to optimize its production for making
biofuels. Microalgae, also provide a wide range of Water samples with visible microalgal population
valuable by-products like omega-3 fatty acids, food, were collected from the facultative ponds and algae
animal feed, nutritional compounds, jet fuel, bioetha- ponds located at Kahang, Johor, Malaysia before it is
nol, biodiesel, medicines, fertilizers according to [7]. released into the river. Collections were carried out
Widjaja et al. [8] have reported that many types of for the top and bottom layer of water at each location
microalgal strains capable of producing a higher with the aim of obtaining the dominant microalgal
amount of lipid content have been screened and char- species present in that particular area. Additional sam-
acterized, most of them belonging to the marine ples were obtained from the water bodies specifically
microalgae group. Microalgae produce many different located adjacent to the algal ponds. All the field
types of lipids, hydrocarbons, and other complex oils samples were collected in sterile 50-mL tubes and
which are mostly based on the species specificity [1]. maintained in refrigerator after transferring it to the
Microalgae can utilize organic carbon sources (glucose, laboratory.
acetate, fructose, and others) and inorganic carbon
(CO2) for producing lipid [3]. Whereas, the quality
2.2. Isolation of microalgae
and quantity of lipids produced inside the cell varies
according to the growth conditions like temperature In order to isolate a single microalgal species from
and light intensity or based on the nutrient content the water sample, standard plating method was used
and its concentration such as iron, nitrogen, and phos- in order to separate the mixed algal population. Dif-
phates [9,10]. The analysis of lipid from the sample ferent types of nutrients were used to isolate the colo-
traditionally is based on gravimetric determination nies. The obtained field sample was first serial diluted
and solvent extraction. The further characterization of as to aid the isolation process. Sterile petri plates con-
lipid is performed using conventional techniques such taining approximately 15–20 mL of agar medium was
as the HPLC or GC [11]. Normally, the lipid classes used to plate these diluted samples. One millilitre of
are divided into polar lipids (galactolipids, phospho- the diluted sample was transferred to the media plate
lipids) and neutral lipids (cholesterol, triglycerides). It and spread evenly across the surface. Inoculated
is also known that triglycerides are the main sources plates were placed in a temperature-controlled green-
for the production of biodiesel [3,12]. house (20–25˚C), where the algae were allowed to
More than 30 microalgal strains have been screened grow for 14 d. Grown algal culture was streaked using
for its potential of lipid production by determining the sterile technique onto nutrient agar plates and placed
biomass productivity and lipid content [2,13]. The life back in the greenhouse for isolation. This streaking
cycle assessment on microalgae cultivation has shown method was repeated until pure algal culture was
that most of the energy use and emission of greenhouse obtained. The number of colonies that were trans-
gases are associated with fertilizer (nutrient) produc- ferred from each dilution plate onto other nutrient
tion [14]. However, the culturing of microalgae on a media plate depends on the amount of contamination
larger scale depends on the presence of huge amount and the identification of the present colonies, based on
of nitrogen and other chemical fertilizers, which drives the colony morphology and the microscopic observa-
the practice toward being non-ecofriendly in nature. At tion of each isolate. Following the isolation of individ-
the same time, culturing of microalgae also plays an ual microalgal colonies, each strain was labeled based
important role as for the purification of wastewater on the sampling location and different nutrient
[15,16]. Nowadays, microalgae are getting considerable requirements. The isolated algae were maintained as
attention in terms of feedstock for the production of stock culture and maintained by re-plating each onto
biodiesel. The aim of the research was to isolate and new nutrient media at least once a month or more fre-
cultivate Chlamydomonas incerta from the sample con- quently, depending on the nature of each isolated
taining Palm Oil Mill Effluent (POME) and to assess its strain. The specifications of POME used in this study
impact using different concentrations of POME as a are shown in Table 1.
29120 H. Kamyab et al. / Desalination and Water Treatment 57 (2016) 29118–29125

Table 1
Characteristic of the sampled POME

No Parametera Concentration range (mg/L) Average (mg/L)

1 pH 4.15–4.4.5 4.25
2 COD 1,350–2,120 1,600
3 Soluble COD 20,500–24,500 22,000
4 BOD 300–400 330
5 Total volatile solid 27,300–30,150 28,100
6 Total suspended solid 15,660–23,560 18,900
7 Total phosphorus 200–600 350
8 Total nitrogen 500–800 500
a
All parameters are in units of mg/L except pH.

2.3. Media preparation colonies from each of the taken sample. These nutrient
contents were also modified for optimizing the isola-
Microalgae were isolated from pond and cultured
tion and growth of each colony [18].
in modified Bold’s Basal Medium (BBM) along with
control. This medium was found to be effective for the
growth of isolated microalgal strain. The media solu- 2.4. Morphological identification
tion was sterilized by autoclaving at 121˚C for 15 min
in order to eliminate the possibility of contamination. Microalgal cultures and contaminations were ini-
The pH of the medium was adjusted to 6.8 before tially separated based on the morphological examina-
autoclaving [17]. The microalgal culture was main- tion of colonies on the nutrient agar medium. The
tained in liquid medium and also for its maintenance identification of the isolates was carried out based on
as it is similar to the growth medium (modified BBM). the morphology of the individual cells under micro-
Two millilitre of microalgal culture was inoculated scope. The strains were identified according to the
into sterile conical flasks containing 18 mL of the cul- method used by Wehr and Sheath [19]. Each isolate
ture medium. Now the flasks were incubated in the colony was labeled and photographed at magnifica-
presence of light (100 μmol/s/m2) at room tempera- tions (20×, 40×, 60×) using Canon PAXit microscope
ture. Sterile BBM in presence of different types of equipped (with DXM1200 digital camera and ACT-1
vitamin was used for isolating maximum number of software program). A catalog of these isolates was

(a) (b)

40 X

(b) (b)

10 X 20 X

Fig. 1. (a) Isolation of microalgae from POME (b) microscopic observation of C. incerta under light microscope.
 H. Kamyab et al. / Desalination and Water Treatment 57 (2016) 29118–29125 29121

3. Results and discussion

3.1. Isolation and identification
The samples obtained from the algal ponds located
in Kahang, Johor, Malaysia were cultured in wastewa-
ter containing POME and allowed to grow by expos-
ing it to direct sunlight. The characteristics and
morphological features of the isolates showed similar-
ity with C. incerta. The individual cells of the colony
were found to be in the range of 10 μm, greenish, uni-
cellular, and spherical in shape (Fig. 1).

Fig. 2. C. incerta cultivation under different COD 3.2. Selection of suitable microalgal concentration
concentrations.
Notes: a = 0 mg/L, b = 250 mg/L, c = 500 mg/L, and C. incerta during its exponential period was
d = 1,000 mg/L. inoculated (2%, v/v) in the liquid medium.

established for future reference. Photographic compar-

1.60
ison of the original isolates along with the stock cul-
tures was performed periodically as to ensure that 1.40
contamination had not occurred.
1.20

1.00
Ln(X /X 0)

2.5. Cultivation and harvesting 0 (mg/L)

0.80
250 (mg/L)
Preliminary study was done through the
0.60 500 (mg/L)
cultivation of microalgae C. incerta in POME with dif-
1000 (mg/L)
ferent concentrations at 0, 250, 500, and 1,000 (mg 0.40

COD/L).The sample containing microalgal culture 0.20

was monitored every day for its growth rate by mea-
0.00
suring the optical density at 620 nm. The cultures 0 1 2 6 10 14 16 20 28
were aerated using air pump and the sterile-specified Time (days)
media was added to each culture at the end of each
week. Constant mixing of the algal culture in 2-L flask Fig. 3. Growth rate of C. incerta under different initial
was provided by means of aeration. The temperature substrate concentrations of POME.
of the algal culture in the flask was found to be
between 24 and 33˚C. Harvesting of the microalgae
1.8 0.5
was carried out using the centrifugation process [16].
Specific growth rate
1.6 0.45
Growth yield 0.4
Specific growth rate (1/h)

2.6. NR staining for the determination of lipid 1.4

Growth yield (g/g)

0.35
1.2
One millilitre of culture was taken and centrifuged
0.3
at 2,000 rpm for 2 min [20]. Now, the supernatant was 1
0.25
discarded and the pellet obtained was washed using 0.8
phosphate buffer at pH 7.0. Further, 1 mL of phos- 0.2
0.6
phate buffer was added to the washed pellet in order 0.15
to be used as stock solution. After that, 1 mL of the 0.4
0.1
cell suspension diluted to 100 × 2.8 mL of the diluted 0.2 0.05
cell suspension was transferred to a 5-mL cuvette.
1.8 μL of NR solution was added to the sample and it 0
0 200 400 600 800 1000 1200
0

was vortexed well for 5 min. Now it was left under Substrate concentration (mg/L)
dark condition for 20 min before taking the reading
of fluorescence intensity with chemiluminescence Fig. 4. Effect of COD concentration on the growth of C.
spectrophotometer [12]. incerta.
29122 H. Kamyab et al. / Desalination and Water Treatment 57 (2016) 29118–29125

(a) (b)

(c)

Fig. 5. Lipid productivity and lipid content (% of biomass) of C. incerta under different COD concentrations. (a) POME
COD of 250 mg/L, (b) POME COD of 500 mg/L, and (c) POME COD of 1,000 mg/L.

The cultivation of C. incerta was initially carried out in illumination with the intensity of 1,033 Lux or
a 250-mL conical flask containing 100 mL of liquid ±14 μmol/m2/s for 28 d. After that 1 mL of chloram-
medium. POME used during the batch experiment phenicol (30 μg/mL) was added to each medium con-
was previously allowed to settle for 1 h and further it sisting of POME as to ensure that the culture will be
was diluted with BBM at the concentration of (250, free from contamination.
500, and 1,000 mg/L), also in the absence of POME to
serve as control for the experiment (0 mg/L), as
3.3. Optimization of microalgal growth rate using POME
shown in Fig. 2. The substrate concentration was
chosen based on the similar type of research con- It is seen from Fig. 3, cells obtained higher growth
ducted in the previous studies [4,21]. C. incerta was rate in presence of 250 mg/L POME, lower growth
grown at room temperature under continuous rate was observed with 1,000 mg/L of POME and
 H. Kamyab et al. / Desalination and Water Treatment 57 (2016) 29118–29125 29123

intermediate growth rate was found in presence of 0.18 0.35

0.16
500 mg/L POME. Variation in Napierian logarithm of y = 0.0021x- 0.0313 0.3

Biomass Productivity (g/L.d)

Lipid Productivity (g/L.d)

Specific Growth Rate (d¯¹)

0.14 R² = 0.9421
(X´/X´0) ratio with the operation time for differential 0.12
0.25

substrate concentrations is also shown in Fig. 3. X´ 0.1

y = 0.0045x- 0.1392
R² = 0.9606
0.2
Biomass Productivity

represents the growth of microalgal concentration 0.08

y = -0.0015x + 0.1489
0.15
(g/L.d)
Lipid productivity
(g/L.d)
(absorbance) during the experiment and X´0 repre- 0.06 R² = 0.954
0.1 Specific Growth rate
0.04 (1/d)
sents the value of growth concentration (absorbance) 0.02
0.05 Linear (Biomass
Productivity (g/L.d))
at the beginning of the experiments It also clearly indi- 0 0 Linear (Lipid
productivity (g/L.d))
cates that 250 mg/L is the most suitable substrate con- 0 20 40 60 80 100 120
Linear (Specific
Organic Loading Rate (kg COD/m³.d) Growth rate (1/d))
centration needed for the growth of C. incerta growth,
when compared to other concentrations, except for the
substrate concentration of 0 mg/L. Fig. 6. Specific growth rate (μ), biomass productivity
(Pbiomass), and lipid productivity (PLipid) of C. incerta using
different organic loading rates in diluted POME.
3.4. Effect of POME on microalgal growth rate
3.7. Effect of various concentrations of POME on cell
The results obtained from Fig. 4 show that specific
density and lipid content of C. incerta
growth rate was found to decrease along with the
increasing substrate concentration. This indicates that Four different concentrations of POME were tested:
substrates can serve as inhibitors at higher concentra- 0, 250, 500, and 1,000 mg/L. The readings were taken
tion. The presence of organic substances may directly for lipid content and cell density (OD) at 620 nm.
turn out to be the essential organic nutrient or can act Whereas, the lipid content was analyzed using NR
as the accessory growth factor [22]. Growth was found method.
to be slower with apparent COD inhibition at the con- It is seen from Fig. 7, that lowest lipid content was
centration above 500 mg/L, which greatly affected the observed when the cells were cultivated in 500 mg/L
efficiency of biomass production. of POME concentration. It can be concluded that
microalgal cells do not make lipid as a storage pro-
duct to survive. Instead, they can use these products
3.5. Lipid production as a result of protein biosynthesis for energy storage
The formation of lipid content is dependent on [3]. Therefore, the lipid content was found to be
several factors like pH, substrate composition, light decreased.
intensity according to the findings of [9,23,24]. In this Almost all of the microalgal cells showed decrease
study, in considering the different factors used for the in the production of lipid from the initial day to until
production of lipid, C. incerta achieved highest lipid the 15th day. It was noticed that nutrient starvation
content (42.6%) of biomass after 2 d, also highest lipid increased the lipid content. However, the decrease in
productivity of 0.197 mg/L/d was obtained when the lipid content may be also due to the presence of more
initial COD concentration was 500 mg/L (Fig. 5). dead cells because the NR method used was unable to
stain the cells. Therefore, lower observation of lipid
content shows that the accuracy of the method can be
3.6. Effect of organic loading rate on biomass and lipid affected due to the viability of cell [11]. Also, different
production using C. incerta in POME sizes and shapes of cells contribute for the lower pro-
duction of lipid [20]. The increase in biomass concen-
The growth of C. incerta using POME was carried
tration does not always increase the storage of lipid
out using different organic loading rates ranging from
36 to 96 kg COD/m3 d for 14 d. The obtained values
were a result of regulating the influent flow rate. The 0 mg/L

biomass productivity and specific growth rate in all of 250 mg/L

4.500
Absorbance, 620 nm

the diluted POME wastewater sample was found to 4.000

3.500 500 mg/L
3.000
increase along with the increasing organic loading rate 2.500
2.000
as shown in Fig. 6. It also shows that decrease in the 1.500
1.000
0.500
lipid productivity of C. incerta at the end of the experi- 0.000

ment was obtained with the organic loading rate 0 5 10 15 20

Time (Days)
(R2 = 0.954), which in turn provides a good correlation
between the biomass production (R2 = 0.9421) and
Fig. 7. Lipid content using different concentrations of
lipid production. POME.
29124 H. Kamyab et al. / Desalination and Water Treatment 57 (2016) 29118–29125

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