1. Assessmentofthe EffectivenessofDifferent FreshwaterMicroalgae Strains for
Phenol Removal
Sulaiman Al-Zuhair1*, Mustafa Nabil1, Yusuf Abdi1, Murad Al Sayyed1 and Hanifa Taher2
1. Chemical and Petroleum Engineering Department, UAE University, 15551 Al Ain, UAE
2. Chemical Engineering Department, Masdar Institute, Abu Dhabi, UAE
Abstract
Phenol is a major constituent in the wastewater of most chemical and petroleum industries. It is
carcinogenic and toxic at relatively low levels, and requires proper treatment before being
discharged. The Biological treatment, using bacteria, has gained a lot of attention as a better
alternative and its effectiveness has been widely proven. However, the produced biomass, i.e.,
bacteria, does not have any obvious value. One the other hand, if phenol can be removed by
microalgae, the produced biomass in this case can be readily used to produce lipids, proteins and
pigments. The former product can be used for biodiesel production and the latter two products have
potential to be used in pharmaceutical and food applications. The ability of different freshwater
strains, namely Chlorella sp., Pseudochlorococcum sp. and an indigenous strain, to grow in water
containing different concentrations of phenol has been tested. The effectiveness of the selected
strains to utilize the phenol as a carbon source and reduce its concentration has also been assessed.
The phenol removal efficiency and cells growth rates were evaluated at different initial phenol
concentrations, in the range of 100-450 ppm. In the tested range of the phenol concentrations,
Pseudochlorococcum sp. has shown to have the highest specific growth and phenol removal rates.
The three strains showed substrate inhibition effect at phenol concentrations above 250 ppm. Three
kinetics models that incorporate substrate inhibition were tested to describe the growth, which show
almost identical fittings.
* Corresponding Author: Email: s.alzuhair@uaeu.ac.ae, Tel: +97137135319

2. Keywords: Microalgae; Phenol; Wastewater treatment; Growth kinetics
1. Introduction
Phenol is considered one of the most hazardous pollutants founds in refinery wastewater, and is
certainly one of the most difficult to remove. Its concentration in the wastewater of petroleum
refinery has been estimated to be 13–88 ppm [1]. It is listed among the priority organic pollutants
by the US Environmental Protection Agency [2]. Phenol may be fatal by ingestion, inhalation, or
skin absorption, since it quickly penetrates the skin and may cause severe irritation to the eyes and
the respiratory tract. Phenol is water soluble and can easily reach water sources downstream from
discharges. Phenol is also considered to be potentially carcinogenic to humans and may be lethal to
fish at concentrations of 5-25 ppm [3]. It is essential, therefore, that phenol concentrations in
refinery effluents be reduced to environmentally acceptable and harmless levels through utilizing
effective and practical treatment methods.
Many processes have been used in the past few years to reduce the concentration of phenol in
petroleum refineries; however, biological treatment has proven to be the most promising and
economical method for the removal of phenol from wastewater [2, 3]. It is believed that this method
leads to a complete removal of phenol and can handle a wide range of concentrations [4]. The
biodegradation of phenols by different types of microbial cultures has attracted the attention of
many researchers in the past two decades. Most studies on phenol degradation have been carried out
with bacteria, mainly from the Pseudomonas genus, due to their ability to degrade organic solvents
in general and its high removal efficiency of phenol in particular [3]. Bacteria from Pseudomonas
genus immobilized in polyvinyl alcohol particles have been successfully used in a bubble column
and a spouted bed bioreactor for the removal of phenols from refinery wastewater [5, 6].
In spite of their high efficiency in degrading phenol, the release of these bacteria could cause
diseases in plants and depletion of fish stocks, and may also have the potential to cause diseases to
humans [7]. Although the use of the bacteria in immobilized form, as in the previously mentioned

3. studies [4, 5], minimizes water contamination by the bacteria, it would be advantageous to use other
types of microorganisms, which are less harmful, if they show comparable performance. In
addition, the grown bacteria do not have any obvious value.
If phenol can be removed from wastewater by microalgae, the produced biomass in this case can be
readily used to produce lipids, proteins and pigment. The former product can be used for biodiesel
production and the latter two products have potential to be used in pharmaceutical and food
applications. In addition, microalgae cultivation does not require the development of agricultural
lands. Furthermore, the use of microalgae in a number of industrial processes has proven to be
cheaper and more environmental friendly than other alternative methods. The ability to carry out a
wide array of bio-physical actives makes it possible for microalgae to be applied in many
biotechnological and environmental processes [8]. In recent years, research on the chemistry of
microalgae has experienced a tremendous increase [9].
Phenol degradation by microalgae have been reported by strains of Chlorella sp., Scenedesmus
obliqus and Spirulina maxima [10], Ochromonas danica [11], Ankistrodesmus braunii and
Scenedesmus quadricauda [12], Chlorella vulgaris [13, 14], Chlorella VT-1 [15], Volvox aureus,
Lyngba lagerlerimi, Nostoc linkia, and Oscillatoria rubescens [16]. However, none of the above
studies have reported algal growth kinetics in phenol [17]. A recent work done by Das et al. [17]
was the first to include such a kinetics study, but was done on a single strain of Chlorella
pyrenoidosa. In addition, of C. pyrenoidosa was inhibited at phenol concentration above 125 ppm,
and hence the work of Das et al. [17] was limited to phenol concentration of 200 ppm. The main
objective of this work is to test the ability of other microalgae strains, namely Chlorella sp.,
Pseudochlorococcum sp. and a locally isolated strain, to grow in wastewater containing phenol and
at the same time reduces its concentration. In addition, the ability to grow at much higher
concentration reaching 450 ppm is also evaluated.
2. Materials and Methods

4. 2.1. Strains and culture media
Analytical grade phenol was purchased from BDH Chemicals, UK. All other chemicals were
purchased from Sigma-Aldrich, USA. Freshwater strains, Chlorella sp. and Pseudochlorococcum
sp. were obtained from a local marine research centre in Umm Al-Quwain, UAE. In addition, an
indigenous microalgae strain isolated from Musffah, Abu Dhabi was kindly provided by Prof.
Koroush Salihi, New York University in Abu Dhabi, UAE, which is referred to in this paper as
3112 strain. The strains were grown in modified Bold Bassel medium (3N-BBM) consisting of
(mM): 8.82 NaNO3, 0.17 CaCl2∙2H2O, 0.3 MgSO4∙7H2O, 1.29 KH2PO4, 0.43 K2HPO4, 0.43 NaCl.
The prepared medium, excluding vitamins, was sterilized in an autoclave (Hirayama HV-50, Japan)
at 121 oC for 15 min and cooled to room temperature prior to use. It must be noted that this medium
does not contain a carbon source in order to force the microalgae to use phenol as a carbon source.
2.2. Experimental set-up
The strains were grown in 100 ml Erlenmeyer flasks, which were placed in temperature controlled
shaking water bath (LabTech, DaihanlabTech Co. ltd., Koreas) set at 30 oC. To enhance
heterotrophic growth, and direct the microalgae to utilize the phenol as a sole carbon source, the
cultures were not subjected to excessive lighting, and the lighting was limited to that used to
illuminate the lab. Each flask contained 90 ml of medium containing different concentrations of
phenol in the range of 100-450 ppm. The selected range of phenol concentration was chosen to be
around that found in refinery wastewater. 10 ml of microalgae inoculum, were then added to each
flask to bring the total volume to 100 ml. To account for any abiotic loss of phenol, phenol media
without algal cells was incubated under the same culture conditions, by adding 10 ml distilled
water, instead of microalgae suspension, to prepare control samples. At regular intervals, 2 ml
samples were withdrawn for biomass and phenol concentration determination.
2.3. Analysis

5. The biomass concentration was monitored daily by measuring the optical density at 680 nm
wavelength using UV-spectrophotometer (UV-1800, Shimadzu, Japan). The spectrophotometer was
zeroed using a sample containing the growth medium with no microalgae. The growth was
determined by dividing the optical density at anytime by the initial optical density, measured on day
0. To measure the phenol concentration, the samples were centrifuged centrifugation at 6000 r min-1
for 15 min using multispeed centrifuge (IEC CL31, Thermo Scientific, USA) to remove the
biomass. The supernatant was then collected to measure the phenol concentration using the same
spectrophotometer at 270 nm, which was calibrated using serial dilutions of standard phenol
samples. The concentration of phenol was verified against results found using Chrompack Gas
Chromatograph, Model CP9001. The GC was equipped with capillary column (Stabilwax, 30 m,
0.25 mm ID) and a flame ionization detector (FID), which was set at 250 °C. The temperature
program started at 100 °C and increased at a rate of 20 °C min−1 to 180 °C. The analyses were
conducted in duplicates and the average values are presented with error bars that confirm the
reproducibility of the results.
3. Results and Discussion
3.1. Microalgae growth
The effect of initial phenol concentration on the growth of Chlorella sp., Pseudochlorococcum sp.
and 3112 strains is shown in Fig. 1a, 1b, and 1c, respectively. Values shown in the Figure are
average values for experiments carried in dulicate at same conditions, and the dashed lines are
connection between the experimental data to show the rens. A lag phase of around 4 days was
observed in the growth of 3112 strain, whereas it was less obvious for Chlorella sp. and
Pseudochlorococcum sp. strains. It was also observed that the same lag phase existed regardless of
the initial phenol concentration. This agrees with the results found by Das et al [17], where lag
phase existed when growing C. pyrenoidosa in phenol concentrations higher than 50 ppm.

6. (a)
(b)
(c)
Figure 1: Biomass growth profile of (a) Chlorella sp., (b) Pseudochlorococcum sp. and (c) 3112
strains at various initial phenol concentrations
0
3
6
9
12
0 2 4 6 8 10
100ppm
150ppm
200ppm
250ppm
300 ppmBiomassconcentrationover
initialconcentration
Time, Days
0
3
6
9
12
15
0 2 4 6
100ppm
150ppm
200ppm
250ppm
300 ppm
Biomassconcentrationover
initialconcentration
Time, Days
0
4
8
12
16
20
0 2 4 6 8
100ppm
150ppm
200ppm
250ppm
300 ppm
Biomassconcentrationover
initialconcentration
Time, Days

7. From the growth curves, the specific growth rates were determined for the three strains as shown in
Fig. 2. Assuming the maintenance requirement for the limiting substrate to be small enough to be
neglected, the specific growth rates, shown in Fig 2, were calculated from the slope of ln (X/Xo)
versus time line, in the exponential growth region, where X and Xo are the biomass concentration at
any time and at initial time, respectively.
Figure 2: Specific growth rate of Chlorella sp., Pseudochlorococcum sp. and 3112 strains at various
initial phenol concentrations
The specific growth rate was found to increase with increase in phenol concentration until the
highest value was reached at phenol concentration of 250 ppm. However, the growth rate was found
to decline with increase in phenol concentration beyond 250 ppm, suggesting substrate inhibition
effect of phenol. Similar trend was also observed using C. pyrenoidosa [17], but the highest specific
rate was reached at 125 ppm. The highest growth rates of tested strains were 0.25, 0.49 and 0.59
day-1 for Chlorella sp., Pseudochlorococcum sp. and 3112 strains, respectively. These values are
slightly lower than that found for C. pyrenoidosa grown in phenolic media, which was found to be
0.65 day-1 [17]. The lower growth rate found in this work, compared to that obtained by Das et al
[17], could be due to the alternate strong illumination that C. pyrenoidosa was subjected to. As
mentioned earlier, the cultures were not subjected to excessive lighting, to enhance heterotrophic
growth, and direct the microalgae to utilize the phenol as a sole carbon source. It was interesting to
0
0.1
0.2
0.3
0.4
0.5
0.6
0 50 100 150 200 250 300 350 400 450
Chlorella
Psuedo
3112
SpecificGrowthrate,m(day-1)
Initial Phenol Concentration, ppm

8. notice that, although the microalgae growth rate dropped with at phenol concentration higher than
250 ppm. Yet, they were able to survive up to a concentration of as high as 450 ppm.
3.2. Phenol degradation
To determine the effectiveness of the tested strain for phenol degradation, the residual phenol was
estimated at different times. The effect of initial phenol concentration on the drop of phenol using
Chlorella sp., Pseudochlorococcum sp. and 3112 strains is shown in Fig. 3a, 3b, and 3c,
respectively. The results show that the fastest phenol concentration drop was recorded using
Pseudochlorococcum sp. To cancel out the effect of any abiotic factors in phenol removal, the loss
of phenol from culture media without microalgae was determined, and a 1 % abiotic loss of phenol
was found within 4 days. This proves that the microalgae cells were solely responsible for phenol
removal from the sample. Utilization of phenol as an organic carbon source by algae has also been
reported by Semple and Cain [11]. They suggested that phenol can be metabolized into organic end
products like pyruvate, which can contribute to biomass growth.
The effectiveness of phenol removal at each initial phenol concentration may not be clearly evident
from the results shown in Fig 3, as it shows the concentration of remaining phenol over initial
phenol concentration that differs from one test to another. Therefore, the rate of the drop of phenol
concentration with time was determined, and shown in Fig. 4, for the three tested strains. Since we
are only interested in the initial drop rate, the measurement of phenol concentration was limited to
the first three days. As expected, up to an initial phenol concentration of 250 ppm, the phenol drop
rate increased with the increase in initial phenol concentration, which corresponds well with the
growth results shown in Fig. 2. However, for Chlorella sp., the maximum drop was observed at
initial phenol concentration of 200 ppm. The results found in this work agree with those found
using C. pyrenoidosa [17]. However, in the latter, the substrate inhibition was encountered at 125
ppm.

9. (a)
(b)
(c)
Figure 3: Phenol concentration profile of (a) Chlorella sp., (b) Pseudochlorococcum sp. and (c)
3112 strains at various initial phenol concentrations
0.1
0.3
0.5
0.7
0.9
1.1
0 1 2 3
Blank
100 ppm
150 ppm
200 ppm
250 ppm
300 ppm
Phenolconcentrationover
initialcconcentration
Time, Days
0
0.2
0.4
0.6
0.8
1
0 1 2 3
Blank
100 ppm
150 ppm
200 ppm
250 ppm
300 ppm
Phenolconcentrationover
initialcconcentration
Time, Days
0.23
0.43
0.63
0.83
1.03
0 1 2 3 4
Blank
100 ppm
150 ppm
200 ppm
250 ppm
300 ppm
Phenolconcentrationover
initialcconcentration
Time, Days

10. Figure 4: Phenol drop rate using Chlorella sp., Pseudochlorococcum sp. and 3112 strains at various
initial phenol concentrations
3.3. Kinetics of cells growth
Three growth models were used to describe the specific growth rate of the tested strains. The
selected models are those that incorporate limiting substrate-inhibition kinetics, namely Haldane
(Eq.1), Aiba (Eq.2) and Andrews (Eq.3)
i
2
iiS
im
KSSK
Sμ
μ

 (1)
 i
iS
im
KSexp
SK
Sμ
μ 

 (2)
     iiS
m
KS11SK
μ
μ

 (3)
Where, mm is the maximum growth rate, Si is the initial substrate concentration, Ks is the substrate
constant and Ki is the inhibition constant. All the models were fitted to the experimental data shown
in Fig. 2. The estimated values for the model kinetic parameters as returned by the fitting non-linear
regression algorithm, using Excel solver with an objective function (O.F.) given by Eq. (4), are
shown in Table 1.
0
30
60
90
120
150
0 100 200 300 400
Chlorella
Psuedo
3112
RatepfPhenoldrop,(ppm/day)
Initial Phenol Concentration, ppm

11. Where, μexp and μpred are the experimental and model predicted specific growth rates, respectively,
and n is the number of points used.
Table 1: Estimated value of microalgae growth kinetic parameters in phenol-containing media
Strain mm (day-1) KS ( ppm) Ki ( ppm)
Haldane Model
Chlorella 0.43 145.58 177.51
Pseudochlorococcum sp 0.88 45.17 142.64
3112 0.96 135.46 157.32
C. pyrenoidosa [17] 0.55 89.99 100.24
Aiba Model
Chlorella 0.45 85.24 291.97
Pseudochlorococcum sp 1.32 109.46 86.23
3112 1.56 85.23 291.98
C. pyrenoidosa [17] 0.71 58.13 200.40
Andrews Model
Chlorella 0.63 150.79.39 141.82
Pseudochlorococcum sp 1.74 70.90 70.92
3112 2.64 355.10 45.15
It is clear that the three models fittings are very close. The determined values of the model
parameters were found to be close to those found using C. pyrenoidosa [17]. It was not possible to
compare the results with other studies done on microalgae growth in phenolic media, because as
mentioned earlier, they did not include kinetics studies. Graphical outputs showing the fits of the
experimental data by the models are shown in Figs. 5a, 5b and 5c, for Chlorella sp.,
Pseudochlorococcum sp. and 3112 strains, respectively.
O.F. = ∑(μexp − μpred.)
2
n
i
( 4)

12. (a)
(b)
(c)
Figure 5: Growth kinetics model predictions of the specific growth rate of (a) Chlorella sp., (b)
Pseudochlorococcum sp. and (c) 3112 strains at various initial phenol concentrations
0
0.1
0.2
0.3
0.4
0 100 200 300 400 500
Exp. data
Haldane
Aiba
Andrews
SpecificGrowthrate,m(day-1)
Initial Phenol Concentration, ppm
0
0.1
0.2
0.3
0.4
0.5
0.6
0 100 200 300 400 500
Exp.data
Haldane
Aiba
Andrews
SpecificGrowthrate,m(day-1)
Initial Phenol Concentration, ppm
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0 100 200 300 400 500
Exp. data
Haldane
Aiba
Andrews
SpecificGrowthrate,m(day-1)
Initial Phenol Concentration, ppm

13. Conclusion
The effect of initial phenol concentration on the growth of Chlorella sp., Pseudochlorococcum sp.
and 3112 strains has been tested. It was found that the specific growth rates of the
Pseudochlorococcum sp. and 3112 strains increase with the increase in phenol concentration up to
an optimum concentration of 250 ppm, and then start to drop. Whereas, the maximum drop rate for
Chlorella sp. was observed at 200 ppm. Three kinetics models were used to describe growth rate,
and their fittings were very close. The effect of initial phenol concentration on the drop of phenol
was also tested. The fastest phenol concentration drop was recorded using Pseudochlorococcum sp.
Up to an initial phenol concentration of 250 ppm, the phenol drop rate increased with the increase
in initial phenol concentration, and a drop in phenol degradation rate was observed beyond that
concentration.
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