Appl Biochem Biotechnol

DOI 10.1007/s12010-015-1652-9

A Comprehensive Study on Chlorella pyrenoidosa

for Phenol Degradation and its Potential
Applicability as Biodiesel Feedstock and Animal Feed
Bhaskar Das 1 & Tapas K. Mandal 2 & Sanjukta Patra 3

Received: 23 July 2014 / Accepted: 27 April 2015

# Springer Science+Business Media New York 2015

Abstract The present work evaluates the phenol degradative performance of microalgae
Chlorella pyrenoidosa. High-performance liquid chromatography (HPLC) analysis
showed that C. pyrenoidosa degrades phenol completely up to 200 mg/l. It could also
metabolize phenol in refinery wastewater. Biokinetic parameters obtained are the following: growth kinetics, max (media) > max (refinery wastewater), K s (media) <
Ks(refinery wastewater), KI(media) >KI(refinery wastewater); degradation kinetics, qmax
(media)>qmax (refinery wastewater), Ks(media)<Ks(refinery wastewater), KI(media)>
KI(refinery wastewater). The microalgae could cometabolize the alkane components
present in refinery wastewater. Fourier transform infrared (FTIR) fingerprinting of
biomass indicates intercellular phenol uptake and breakdown into its intermediates.
Phenol was metabolized as an organic carbon source leading to higher specific growth
rate of biomass. Phenol degradation pathway was elucidated using HPLC, liquid chromatographymass spectrometry (LC-MS) and ultravioletvisible (UVvisible) spectrophotometry. It involved both ortho- and meta-pathway with prominence of orthopathway. SEM analysis shows that cell membrane gets wrinkled on phenol exposure.
Phenol degradation was growth and photodependent. Infrared analysis shows increased
Electronic supplementary material The online version of this article (doi:10.1007/s12010-015-1652-9)
contains supplementary material, which is available to authorized users.

* Sanjukta Patra
sanjukta@iitg.ernet.in
Bhaskar Das
bhaskar.das@iitg.ernet.in
Tapas K. Mandal
tapasche@iitg.ernet.in
1

Centre for the Environment, Indian Institute of Technology Guwahati, Guwahati 781039, India

Department of Chemical Engineering, Indian Institute of Technology Guwahati, Guwahati 781039,

India

Department of Biotechnology, Indian Institute of Technology Guwahati, Guwahati 781039, India

Appl Biochem Biotechnol

intracellular accumulation of neutral lipids opening possibility for utilization of spent

biomass as biodiesel feedstock. The biomass after lipid extraction could be used as
protein supplement in animal feed owing to enhanced protein content. The phenol
remediation ability coupled with potential applicability of the spent biomass as biofuel
feedstock and animal feed makes it a potential candidate for an environmentally sustainable process.
Keywords Chlorella pyrenoidosa . Phenol degradation . Kinetic parameters . cis . cis-muconic
acid . Ortho-pathway . FTIR fingerprinting . Lipid accumulation . Protein accumulation

Introduction
Phenol widely used in industrial processes of petroleum refineries, resin plants, coking
operations, etc. are released in wastewaters. The phenol concentration in the wastewater
of petroleum refinery have been estimated to be 1388 mg/l [14], 180 mg/l in coke
wastewater from a steel facility [5], and 70 mg/l in resin industry wastewater [6]. Phenol
is water soluble, so it can easily reach water sources downstream from discharges
causing harmful effects to aquatic flora, fauna, and humans. Phenol released into the
aquatic ecosystems is biodegraded by the naturally occurring microflora as bacteria,
fungi, as well as algae. However, the studies related to phenol biodegradation by algae
are much less than that available concerning bacteria and fungi. Phenol degradation by
microalgae have been reported by strains of Chlorella sp., Scenedesmus obliqus and
Spirulina maxima [7], Ochromonas danica [8], Ankistrodesmus braunii and Scenedesmus
quadricauda [9], Chlorella vulgaris [10, 11], Chlorella VT-1 [10], Volvox aureus, Lyngba
lagerlerimi, Nostoc linkia, and Oscillatoria rubescens [11]. However, none of the above
studies have reported algal growth and degradation kinetics in phenol. Knowledge of
growth and substrate utilization kinetics is essential to better understand the role played
by microalgae during the natural biodegradation process in phenol-polluted aquatic
ecosystems. The microbial phenol mineralization capability is completely dependent on
activity of metabolic pathway involving a cascade of phenol metabolizing enzymes. The
metabolic pathways involved in phenol biodegradation have been well studied in bacteria
[1214] and fungi [1517]. The only complete pathway of algal phenol mineralization
has been reported in batch cultures of achlorophyllus algae O. danica [8]. Recently,
phenol oxidation to catechol by different green algal species as V. aureus, N. linkia and
O. rubescens [11] have been reported. Oxidation of catechol by C. vulgaris and V. aureus
have also been reported; however, the product of oxidation was not determined [11]. The
present research work provides evidence that green algae possess an enzymatic mechanism for phenol removal as in other microbial systems. Thus, the metabolic mechanism
of phenol mineralization for green unicellular algae abundantly found in aquatic water
bodies deserves adequate attention. To fulfill the lacuna of literature, we have undertaken
this work with multi-fold objectives: (a) to evaluate the biomass growth and phenol
degradation performance in nutrient media and refinery wastewater by kinetic modeling,
(b) to understand the enzymatic mechanism and pathway elucidation, and (c) to evaluate
the biochemical parameters of the algal biomass for further applications. Chlorella
pyrenoidosa (NCIM 2738) was chosen as the model organism since it is one of the most
predominant green microalgae in aquatic ecosystems and water treatment plants.

Appl Biochem Biotechnol

Materials and Methods

Chemicals
MgSO 4 ,CaCl 2, K 2 HPO 4, FeSO 4, Na 2 EDTA, H 3 BO 3, MnCl2 .4H 2 O, ZnSO 4.7H 2O,
Na2MoO4.2H2O, CuSO4.5H2O, and phenol were of analytical grade obtained from
Merck, India. Catechol and cis,cis-muconic acid standards were of high-performance
liquid chromatography (HPLC) grade obtained from Sigma-Aldrich, India. Fourier
transform infrared (FTIR) grade KBR was obtained from Spectrochem, India. Glutaraldehyde solution, 25 % for electron microscopy, was obtained from Himedia,
India.

Microorganism and its Culture Condition

Axenic culture of C. pyrenoidosa (NCIM 2738) obtained from NCIM Pune was cultured
in Fogs medium (pH 7.5) containing 0.2 g/l MgSO4, 0.2 g/l K2HPO4, 0.1 g/l CaCl2.H20,
1 ml/l micronutrient solution, and 5 ml/l Fe-EDTA solution. The micronutrient solution
is composed of 2.86 mg H3BO3, 181.0 mg MnCl2.4H20, 22.0 mg ZnSO4.7H2O, 39 mg
Na2MoO4.2H2O, and 8 mg CuSO4.5H2O dissolved in a final volume of 100 ml distilled
water. Cultures were maintained on an orbital shaker operated at 140 rpm with illumination of 3500 lx for a photoperiod of 14 h light/10 h dark in order to simulate the
natural light/dark cycle. For growth of microorganisms in refinery wastewater, samples
were collected in sterile sample bottles from Indian petroleum refinery and then
transported in ice packs to the laboratory. The oil components in refinery wastewater
were determined by China National Standard Method [18]. The amount of phenol was
determined in 0.2-m filtered water sample by HPLC as described below. The pH of
refinery wastewater was not adjusted for the degradation experiments. All other growth
and culture conditions were similar as mentioned above.

Biomass Growth, Phenol Degradation Analysis, and Kinetic Modeling

The phenol biodegradation capabilities of the microalgae were analyzed in the concentration range of 25200 mg/l. The lowest phenol concentration was chosen to be 25 mg/l
as it is lethal to aquatic organisms like fishes [19]. The upper level of phenol concentration was chosen to be 200 mg/l, keeping in mind the phenol concentration found in
refinery wastewater [14]. A control media without phenol was maintained with all other
conditions similar. To account for any abiotic loss of phenol, phenol media without algal
cells was incubated under the same culture conditions. To determine photodependency of
phenol metabolism, a similar experiment was carried out in the dark. Samples were
collected at regular intervals of 24 h, and growth analysis was carried out by dry weight
analysis. For measurement of residual phenol concentration, the samples were filtered
using a 0.2-m membrane filter. The filtrate was quantified for phenol concentration by
HPLC (Prostar, Varian, USA) equipped with an ultravioletvisible (UVvisible) detector
operating at 270 nm and a C-18 column. HPLC analysis was performed using mobile
phase of acetonitrile/water (70:30) at a flow rate of 0.5 ml/min. To verify differences in
total chlorophyll concentration between phenol-degrading and control biomass,

Appl Biochem Biotechnol

extraction was performed as per Cuaresma et al. [20]. The total chlorophyll in extracts
was determined following the modified Arnons equation [21] as follows:
Chlb 16:72A665 9:16A652 dilution factor mg=l
Chla 34:9A652 15:28A665 dilution factor mg=l
Chltot
Chla
Chlb mg=l
All experiments were carried in triplicates, and the mean values and standard error were
calculated using Origin Pro8 and reported in the respective plots
The biomass growth at various initial concentrations of phenol was utilized for calculating
the specific growth rates, (day1) according to the following equations:

InN 2 InN 1
t1 t1

where N1 (mg/l) and N2 (mg/l) are biomass growth at time t1 (day) and t2 (day) [22].
The growth kinetics of C. pyrenoidosa in phenol was studied. The experimental data were
analyzed with several available growth kinetic models like Haldane [23], Yano [24], Webb
[25], Aiba [26], and Edward [27] to select a suitable kinetic model that can represent growth
pattern of C. pyrenoidosa. From the experimental data of specific growth rate (, day1) with
respect to various initial concentration of phenol (S0), the model equations were solved using
nonlinear regression method and the values of kinetic parameters of different models were
determined based on highest regression coefficient and least standard deviation value.
The experimental data on the substrate degradation were utilized for calculating the specific
degradation rate, q (day1) according to the following equations:
q

1 dS o
x dt

where x and S0 are biomass (mg/l) and phenol concentration (mg/l ) at time t (day) [28].
The phenol degradation kinetics of C. pyrenoidosa was studied, and the experimental data
were analyzed with several available degradation kinetic models that can represent present
experimental data. The model equations were solved to determine the degradation kinetic
parameters using present experimental values of q (day1) for various phenol concentrations
(So) [29].
For growth and degradation kinetics study in refinery wastewater log phase C. pyrenoidosa
cells at the concentration of 220 mg/l was inoculated to refinery wastewater (0.2 m filtered).
Biomass growth was determined by dry cell weight analysis. The changes in the nature of
other oil components of the wastewater were characterized after treating the sample with the
microalgae. The specific growth and degradation rates were calculated according to Eqs. 1 and
2 as mentioned above. Experimental growth and degradation kinetic data were fitted to the
kinetic models, and the biokinetic parameters were estimated.

Analysis of Effect of Phenol on Cell Surface Morphology

The effect of phenol on the cells of C. pyrenoidosa was studied by imaging with scanning
electron microscope (1430vp, Leo, Germany). The surface of the algal biomass treated with
200 mg/l phenol for a period of 48 h were observed by SEM and also compared with control
cells. Sample preparation was carried out as per protocol given by Sadiq et al. [30].

Appl Biochem Biotechnol

Whole Cell Finger Printing of Biomass

The whole cell fingerprint of the biomass was analyzed by FTIR with two objectives: (a) to
determine whether phenol is bioaccumulated in the cell or biodegraded and (b) to analyze the
biomolecular changes in the algal cell during the phenol removal process. Algal cells actively
degrading 125 mg/l phenol were taken for this study. The harvested biomass was washed and
dried in vacuum desiccator. One volume of dried algal sample was blended with 100 volume
of dried KBr powder and pressed into tablets before measurement. The spectral acquisition
was performed using a FTIR spectrometer (IR Affinity, Shimadzu, Japan) by means of 500
scans with 4 cm1 of spectral resolution over the wave number range of 4004000 cm1. The
spectrum was submitted to a 15-point smoothing filter for noise reduction. The characteristic
peak areas were obtained using IR Solution FTIR software (Shimadzu, Japan).

Characterization of the Phenol Degradation Pathway

Log phase algal biomass growing in Fogs media supplemented with 125 mg/l phenol was
harvested, washed thrice, and grounded in potassium phosphate buffer solution (50 mM) using
a mortarpestle in an ice bath. The extract was centrifuged at 10,000 rpm for 10 min at 4 C,
and the supernatant obtained was used for enzyme activity assays. Total protein in the
supernatant was determined as per procedure given by Lowry et al. [31].
Phenol hydroxylase activity was analyzed in a assay mixture containing 50 mM potassium
phosphate buffer solution (pH 7.2), 88 g protein, 1.5 M phenol, and 1.5 M NADPH. Heatinactivated enzyme extract served as control. The incubation was stopped at equal intervals
with 20 l of 0.6 M perchloric acid. Samples were analyzed for phenol utilization and
concomitant accumulation of the reaction product catechol by HPLC.
The ortho-cleavage of catechol (hydroxylation product of phenol) to cis,cis-muconic acid is
carried out by catechol-1,2-dioxygenase. Catechol-1,2-dioxygenase activity was analyzed in a
reaction mixture containing 50 mM potassium phosphate buffer pH 7.2, 88 g protein, and
1.5 M catechol. Catechol cleavage to cis,cis-muconic acid was analyzed by HPLC. Catechol2,3-dioxygenase is responsible for meta cleavage of catechol to 2-hydroxymuconic semialdehyde (2-HMS). Catechol-2,3-dioxygenase activity was determined by increase in absorbance
at 375 nm due to accumulation of the reaction product 2-HMS (E375 =14,700 mol L1 cm1).
The breakdown products of cis,cis-muconic acid and 2-HMS were identified by
electrospray ionization liquid chromatographymass spectrometry (LC-MS) (Make: Agilent,
Model: Infinity LC system) in negative charge mode. To characterize the metabolites, the
catechol dioxygenase assay was carried out as mentioned above. The LC-MS was operated
using acetonitrile/water (60:40) mixture as solvent at a flow rate of 0.5 ml/min with detector at
270 nm. The m/z signals corresponding to the metabolites were identified using the Tandem
Mass Spectrum database (open sourceCentral Drug Research Institute, India).

Neutral Lipid Analysis

The success of microalgal system to serve as an efficient biodiesel feedstock depends on its
high neutral lipid productivity [32]. To determine neutral lipid accumulation, algal cells were
stained with a microwave-assisted Nile red staining method as per Chen et al. [33]. For
staining, the cell density was chosen to provide 0.06 (OD750) as optimized for the staining
protocol by Chen et al. [33]. Fluorescence from stained algal cells were measured on a

Appl Biochem Biotechnol

multimode microplate reader (Infinity, Tecan, Switzerland) at excitation and emission wavelengths at 490 and 580 nm, respectively. The excitation and emission standards were determined based on pre-scan characteristics of neutral lipid standard, triloein (Himedia, India). A
standard curve of triloein (R2 =0.99) in the concentration range of 5100 g/ml was used for
quantification of neutral lipids. The Nile red stained cells were also observed under a
fluorescence microscope (CX41, Olympus, Japan) as per Greenspan et al. [34] and imaged
at 100 magnification.

Results and Discussion

Biomass Growth and Phenol Degradation
The effect of phenol concentration on biomass growth profile and experimental specific
growth rate of C. pyrenoidosa have been shown in Fig. 1a and b, respectively. The growth
curves show that there is no lag phase in control and low concentration of phenol (25 mg/l)
cultures. However, lag phase is observed from 50 mg/l phenol, and the phenol concentrations
preceding it as seen from Fig. 1a. Lag phase is followed by exponential growth phase, which is
simultaneously followed by phenol utilization by the biomass. After the exponential growth
period, phenol is depleted, and the microalgae enters the stationary phase. The biomass growth
even when phenol is depleted may be explained by biotransformation of phenol into its
metabolic intermediates, which serves as growth substrates until fully utilized [35]. Li et al.
[35] worked on phenol degradation by Pseudomonas putida LY1 and observed similar results
of appearance of lag phase with increased phenol concentration, simultaneous phenol transformation in the exponential phase, appearance of stationary phase concomitant with phenol
depletion, and increase in biomass even after complete phenol utilization. From the growth
curve, the specific growth rate of 0.16 day1 of control culture was found to be comparatively
lower as compared to specific growth rate achieved in presence of phenol (Fig. 1b). This
higher specific growth rate is probably because of phenol utilization as an organic carbon
source by C. pyrenoidosa. The specific growth rate was found to increase with increase in
substrate concentration until the highest value of 0.65 day1 was attained at phenol concentration of 125 mg/l (Fig. 1b). The highest total chlorophyll content of 27.03 mg/l in 125 mg/l
phenol cultures is 26.07 % higher than that of 21.44 mg/l total chlorophyll in control cultures
further supporting the high biomass growth rate in phenol (Supplementary Fig. 1a and b).
However, the growth rate was found to decline with increase in phenol concentration beyond
125 mg/l, suggesting growth inhibition effect of phenol. Utilization of phenol as an organic
carbon source by algae has also been reported by Semple and Cain [8] and Lika and Papdakis
[36]. They suggested that phenol can be metabolized into organic end products like pyruvate
and CO2, which can contribute to biomass growth.
To determine the phenol degradation profile, the residual phenol was estimated by HPLC
with a retention time of 19.4 min. HPLC data shows complete phenol degradation as shown in
Fig. 2a. The specific degradation rate increases with phenol concentration until a maximum
rate of 0.29 day1, which was achieved at 125 mg/l phenol (Fig. 2b.). It is due to the highest
specific growth rate of the microalgae at 125 mg/l phenol. Beyond this phenol concentration
(125 mg/l), progressive decrease in specific degradation rate could be well explained by
inhibited biomass growth. Mathur and Mazumdar [37] observed similar phenomena during
phenol degradation by P. putida. They reported that both specific growth and degradation rate

Appl Biochem Biotechnol

Fig. 1 a Biomass growth profile of Chlorella pyrenoidosa in various initial phenol concentrations. b Variation in
specific growth rate of Chlorella pyrenoidosa in various initial phenol concentrations

increases with increase in phenol concentration until a maximum value of 100 mg/l. They also
reported that both growth and degradation rate declines due to substrate inhibition of phenol
concentration beyond 100 mg/l.
To negate the effect of any abiotic factors in phenol removal, the loss of phenol from culture
media without C. pyrenoidosa was determined, and a 1 % abiotic loss of phenol was found
within 4 days as compared to complete removal of phenol in C. pyrenoidosa inoculated
cultures. This proves that C. pyrenoidosa is solely responsible for phenol removal from the
sample. To determine if the process of phenol degradation in C. pyrenoidosa is
photodependent, microalgal cells were incubated in phenol under dark condition. During this
process, the biomass growth and phenol degradation have been found to be negligible as
compared to that in light/dark cycle (Supp. Fig. 2). C. pyrenoidosa could metabolize only 7 %
phenol in 3 days under dark condition, which is almost negligible as compared to light/dark
cycle (81.56 % phenol removed within 3 days). It resembles the observation of Papazi and
Kotzabasis [38]. They reported that phenol biodegradation is a photoregulated response in the
algae Scenedesmus obliquus. They showed that phenol biodegradation by S. obliquus was

Fig. 2 a Phenol degradation profile of Chlorella pyrenoidosa. b Specific degradation rate of C. pyrenoidosa for
different phenol concentrations

Appl Biochem Biotechnol

reduced to 5 % in dark. Scraag [10] reported that there is no growth as well as phenol
degradation by C. vulgaris under dark condition, which is in accord with the present observations. Attempts were also made to understand the dynamics of biomass growth and phenol
degradation of C. pyrenoidosa in refinery wastewater. The refinery wastewater was quantified
to contain 23.33 mg/l phenol. The pH of the refinery wastewater was noted as 7.9. Intending to
understand the natural growth and phenol degradation profile of C. pyrenoidosa, the experiment in refinery wastewater was carried out without attempting to meet the nutritional
requirement of the microalgae and maintaining the actual pH of the refinery wastewater.
Figure 3a shows the growth and phenol degradation ability of the microalgae in refinery
wastewater. The biomass growth profile indicates an initial lag phase of 2 days unlike that in
25 mg/l phenol supplemented Fogs media where no lag phase was observed. After the lag
phase, the biomass grows exponentially on the fourth day and then enters the stationary phase
on the 5th day with a final biomass of 339 mg/l. There is no phenol removal by C. pyrenoidosa
when it is in the lag phase of growth (till second day). After the second day, the microalgae
uptakes phenol, which is coincident with exponential growth of the algal biomass.
C. pyrenoidosa mineralized 38.32 % of phenol in refinery wastewater by 7 days unlike
complete mineralization of 25 mg/l phenol concentration by third day in Fogs media. Agarry
et al. [39] reported inhibition of complete mineralization of 30 mg/l phenol in refinery
wastewater by Pseudomonas aeruginosa and Pseudomonas fluorescens, which correlates well
with present finding. P. aeruginosa mineralized 94.5 %, while P. fluorescens mineralized
69.4 % of initial phenol concentration. However, contrary to the present work, they added
mineral salt medium to refinery wastewater to meet the nutritional requirement of the
microorganism for proper growth. Refinery wastewater may contain other constituents that
may prove inhibitory to the phenol degradation potential of the microorganisms [39]. Characterization of the nature of oil present in refinery wastewater by UV-spectophotometry shown
in Fig. 3b indicates that the refinery wastewater before treatment (day 0) consists of both
alkane (absorbance at 215230 nm) and aromatic compounds (absorbance at 250260 nm).
Since the peak of maximum absorbance is around 215 nm, the nature of oil in refinery
wastewater is clean oil. Dotted line (Fig. 3b) represents the oil component characteristics in
wastewater after treatment with C. pyrenoidosa for 8 days, indicating the degradation of both
alkanes and aromatic compounds. This adds to the potential of the algal candidate.
Cometabolism of other substrates along with phenol may have slowed the phenol degradation
rate.

Growth and Degradation Kinetic Modeling: Biokinetic Parameter Evaluation

and Performance Assessment
The behavior of biodegradation rate is a strong function of biomass growth rate. Any growth
medium where the microbial population can double itself faster will potentially result in higher
biodegradation rate [39]. Understanding of a microorganisms degradation and growth kinetics
will bring out its potential for phenol biodegradation [40]. Thus, an attempt has been made to
find out the mathematical relationship between (a) growth rate of biomass and substrate
concentration and (b) phenol degradation rate and its initial concentration. The results obtained
by solving the various growth kinetic models have been tabulated in Table 1. From this table, it
is clear that Yano model yielded comparatively high R2 value and least SD value confirming
that Yano model best fitted the experimental data. A comparative plot of experimental and
model predicted specific growth rates has been shown in Fig. 4a. The value of KS (half

Appl Biochem Biotechnol

Fig. 3 a Biomass growth and phenol degradation by C. pyrenoidosa in refinery wastewater. b Oil characteristics
of refinery wastewater before (day 0) and after treatment (day 8) with C. pyrenoidosa

saturation coefficient) indicates the affinity of the microorganism to the substrate. The value of
KI (substrate inhibition constant) signifies the degree of resistance of microorganism to the
toxic effect of the substrate [41].
The substrate consumption rate is the most important parameter for denoting microbial
degradative performance [42]. Initial phenol concentration has strong influence on specific
degradation rate making kinetic analysis of substrate consumption essential [39]. The value of
kinetic parameters obtained by solving the various degradation kinetic models has been shown
in Table 2. The specific degradation rate predicted by the various kinetic models at different
initial concentration of phenol has also been plotted graphically in Fig. 4b. Table 2 indicates
that Yano model yielded the highest correlation coefficient (R2) and the least standard
deviation (SD) among all other models and thus best fitted the experimental data.
On the basis of the encouraging results in cultured condition, we attempted to verify the
applicability of C. pyrenoidosa for phenol removal from refinery wastewater. The kinetic
parameters obtained for various growth kinetic models have been shown in Table 3. Table 3
shows that Haldane model yielded the highest correlation coefficient (R2) and the least
standard deviation (SD) and thus best fitted the experimental data. Table 4 describes the
kinetic parameters of degradation kinetic models, and the Haldane model shows both highest
correlation coefficient (R2) and least standard deviation (SD). Thus, the Haldane model
represents appropriately the phenol degradation behavior of the microalgae in refinery wastewater used in the present study.

Table 1 Estimated value of growth kinetic parameters of C. pyrenoidosa (NCIM 2738) in phenol-containing
nutrient media
Model

max (day1)

Ks (mg/l)

KI (mg/l)

Haldane

5.572

444.1

24.46

Yano

4.344

410.5

214.5

32.26
519.7

Webb

5.394

480.74

15.44

Aiba
Edward

7.15
28.1

472.5
111.5

132.7
105.3

K (mg/l)

R2

SD

0.94

0.049

0.97

0.035

0.92

0.055

0.95
0.95

0.045
0.044

Appl Biochem Biotechnol

Fig. 4 a Growth kinetic model fitted to experimental batch growth data of C. pyrenoidosa. b Degradation kinetic
model fitted to experimental batch degradation data of C. pyrenoidosa

Comparison of the biokinetic parameters may give the indication of how C. pyrenoidosa
behaved under two significantly different culture conditions of nutrient sufficient media and
refinery wastewater. For this reason, a comparison was made between the respective biokinetic
parameters of best fit kinetic models representing the biomass growth and phenol degradation
behavior. Kinetic modeling of the experimental biomass growth data suggests that max
(0.017 day1) and KI (10.46 mg/l) is lower in refinery wastewater (first row in Table 3) as
compared to that in nutrient media (second row in Table 2). The Ks value (600.1 mg/l) in
refinery wastewater (first row in Table 3) is higher than that in nutrient media (second row in
Table 2). While degradation kinetic modeling shows lowered qmax (0.012 day1) and KI
(53.24 mg/l) values in refinery wastewater (first row in Table 4) as compared to that in nutrient
media (second row in Table 2). The Ks value (300.99 mg/l) in refinery wastewater (third
column of first row in Table 4) is higher compared to that in nutrient media (thirrd column of
second row in Table 2). Lower max values in refinery wastewater is probably due to the lack
of optimal nutrient factors for growth as well as other growth inhibitory constituents, which
may be present in refinery wastewater. Maximum degradation rate (qmax) is also lower in
refinery wastewater due to the lower specific growth rate as mentioned above. Therefore,
efficient phenol utilization is less in refinery wastewater as compared to that in nutrient media
containing optimal biomass growth conditions. Secondly, cometabolism of alkanes in refinery
wastewater along with phenol (aromatic) as discussed in Biomass growth and phenol
degradation may lead to decrease in phenol degradation rate (qmax). Ks being inversely related
to affinity of microbial system for substrate, a higher Ks value indicates its lower affinity to the
Table 2 Estimated value of degradation kinetic parameters during phenol biodegradation by C. pyrenoidosa
(NCIM 2738) in phenol-containing nutrient media
Model

qmax (day1)

Ks (mg/l)

KI (mg/l)

Haldane

0.55

89.99

100.24

0.73

0.05

Yano

0.76

170.60

250.6

86.54

0.81

0.04

K (mg/l)

R2

SD

Webb

0.30

77.93

100.9

350.06

0.70

0.07

Aiba
Edward

0.71
2.78

58.13
77.94

200.40
100.9

0.65
0.76

0.08
0.06

Appl Biochem Biotechnol

Table 3 Estimated growth kinetic parameters of Chlorella pyrenoidosa (NCIM 2738) in refinery wastewater
Model

max (day1)

Ks (mg/l)

KI (mg/l)

K (mg/l)

R2

SD

Haldane

0.017

600.1

10.46

0.96

0.025

Yano

4.344

600.5

150.5

10.26

0.96

0.412

Webb

0.356

580.7

5.44

530.7

0.82

0.113

Aiba
Edward

0.019
0.03

572.5
50.94

70.7
70.9

0.84
0.34

0.023
0.05

substrate [43]. Higher Ks value suggests a decreased affinity for phenol of C. pyrenoidosa in
refinery wastewater compared to that in nutrient media. Higher Ks value in refinery wastewater
explains inhibition of complete phenol mineralization unlike complete mineralization in
media. C. pyrenoidosa utilizes phenol less efficiently in refinery wastewater due to decreased
affinity for phenol. KI value is involved in quantification of the effect of toxicity of a
compound during the biodegradation process. A higher KI value implies less sensitivity of
the microbe to substrate inhibition. Lower KI value in refinery wastewater suggests high
sensitivity of C. pyrenoidosa to toxic effect of phenol compared to that in nutrient media.
This can be understood from the fact that optimal growth conditions in media helps the
microalgae counter the inhibitory effect of phenol in a better way. On the other hand, lack of
optimal biomass growth factors and possible presence of other additional inhibitory constituents in refinery wastewater compromises the ability of C.pyrenoidosa to resist the inhibitory
effect of phenol.

Characteristics of Cell Surface Morphology on Phenol Treatment

The SEM micrograph (Supp. Fig. 3a and b.) shows that phenol exposure affects the membrane
morphology of cells. When cells were exposed to 200 mg/l phenol for 48 h, the cell surface
was found to be wrinkled. Accumulation of phenol in the hydrophobic part of the membrane
leads to disturbance in the interactions between the acyl chain of phospholipids. This causes
modification of membrane fluidity and may lead to swelling of the bilayer [44].

FTIR Fingerprinting Analysis

The FTIR spectrum (Supp. Fig. 4a) depicts the whole-cell fingerprint of the biochemical
changes in response to phenol. Adsorption at 34443419 cm1 is due to stretching of OH
groups of alcohol, phenol, or carboxyl OH and hydrogen vibration of the amide NH [45].
Table 4 Estimated degradation kinetic parameters for Chlorella pyrenoidosa (NCIM 2738) during phenol
degradation in refinery wastewater
Model

qmax (day1)

Ks (mg/l)

KI (mg/l)

Haldane

0.012

300.99

53.24

0.99

0.02

Yano

0.018

220.6

200.6

40.54

0.48

0.04

K (mg/l)

R2

SD

Webb

0.107

350.93

100.9

400.06

0.11

0.09

Aiba
Edward

0.054
0.058

208.13
77.94

30.4
100.9

0.33
0.19

0.05
0.08

Appl Biochem Biotechnol

Infrared spectrum shows that the percent transmittance in this region decreases (Supp. Fig. 4a)
while peak area increases (Supp. Fig. 4b) with phenol incubation attaining prominence on the
third day. However, there is no such prominent transmittance (Supp. Fig. 4a) or area (Supp.
Fig 4b) variation within this wave number range in control cells. The results suggest increased
intracellular phenol accumulation with incubation time. Since the microalgae completely
removed 125 mg/l phenol from the culture medium by the second day (Fig. 2a), its high
intracellular accumulation is quite evident. An increased peak area indicates increase in
concentration of functional groups whose stretching/bending is responsible for the peak. Thus,
peak area differences have been successfully used to monitor change in concentration of
different biomolecules as monosaccharides in Enterobacter cloacae [46], amide I and II,
cellulosic compounds, nonstructural carbohydrates in different barley varieties [47], as well
as erythromycin quantification in pharmaceutical formulations [48]. Similarly, comparatively
higher percent transmittance as well as low peak area due to low intracellular phenol uptake on
day 1 is explained by lower phenol removal rate till day 1. The first metabolic intermediate of
phenol degradation pathway is catechol. The metabolic intermediates formed by breakdown of
catechol contains carboxylic acid group (COOH) [49]. The infrared spectra region 1754
1710 cm1 is associated to carbonyl group vibration in COOH group [44]. Transmittance
(Supp. Fig. 4a) in this region is found to decrease, and peak area (Supp. Fig. 4c) increases with
phenol incubation, which is not evident in control cells. This shows that the accumulated
intracellular phenol is metabolized into intermediate products downstream of catechol. Wharfe
et al. [49] reported similar findings of an increased infrared absorbance due to carbonyl group
vibration of intermediate products of phenol metabolism in a microbial consortium. The
infrared region 14401380 cm1 is attributed to CH bending of aliphatic groups. Decreased
percent transmittance (Supp. Fig. 4a) and an increased peak area (Supp. Fig 4d) in this region
suggest an increased accumulation of aliphatics in phenol-incubated cells. Intracellular accumulation of aliphatic intermediates of phenol metabolism may be associated to the decreased
transmittance and increased peak area. Thus, infrared analysis suggests intracellular uptake of
phenol by C. pyrenoidosa, and then phenol is broken down into intermediate products. This
breakdown of intracellular uptaken phenol into its intermediate metabolites confirms the
process of phenol removal to be biodegradation.

Elucidation of Phenol Degradation Pathway

The phenol metabolic pathway was characterized by identifying the different intermediates
produced during phenol degradation using HPLC, UVvisible spectrophotometry, and LCMS. Supplementary Fig. 5a and b suggests appearance of catechol peak (11.9 min) with
progressive decrease in phenol peak (10.69 min), which confirms catechol accumulation in
media (extra cellular). The present results also accord with the observation reported by ElSheekh et al. [11] for different algal species as V. aureus, N. linkia, and O. rubescens. It proves
that phenol is degraded through an enzymatic pathway in C. pyrenoidosa. Intracellular phenol
hydroxylase activity was determined by HPLC (Fig. 5a). Control incubations carried out by
Fig. 5 a Hydroxylation of phenol to catechol by phenol hydroxylase activity. b Ortho-cleavage of catechol to
cis,cis-muconic acid by catechol-1,2-dioxygenase activity. c Meta cleavage of catechol to 2-hydroxymuconic
semialdehyde (2-HMS) by catechol-2,3-dioxygenase activity. d LC-MS analysis of catechol dioxygenase assay
mixture at 0 min (before incubation). e LC-MS analysis of catechol dioxygenase assay mixture at 20 min (after
incubation). f Proposed pathway of phenol degradation in Chlorella pyrenoidosa (NCIM 2738)

Appl Biochem Biotechnol

Phenol
Phenol hydroxylase

Catechol

Catechol-1,2-dioxygenase

cis,cis-muconate

Catechol-2,3-dioxygenase

2-hydroxymuconic semialdehyde
2-hydroxymuconate semialdehyde
hydrolase

Cis-2-hydroxypenta-2,4-dienoate
-ketoadipate

Citrate cycle

Acetaldehyde

Pyruvate

Appl Biochem Biotechnol

heat-killed enzyme extract showed no phenol hydroxylation activity (no catechol and no
phenol utilization). Similar observations have also been reported in the literature for algae
[8], fungi [50, 15] and bacteria [12, 51]. Catechol can be ortho-cleaved (if it follows orthopathway) or meta-cleaved (if it follows meta-pathway) by catechol-1,2-dioxygenase and
catechol-2,3-dioxygenase, respectively. Catechol-1,2-dioxygenase activity was characterized
by identifying its ortho-cleavage product namely cis,cis-muconic acid using HPLC as shown
in Fig. 5b. The reaction product was identified to be cis,cis-muconic based on identical
retention time of 4.2 min with that of standard cis,cis-muconic acid. Control incubations
carried out by heat-killed enzyme extract showed no catechol-1,2-dioxygenase activity. Catechol-2,3-dioxygenase activity was also determined by identifying 2-hydroxymuconic semialdehyde as the meta-cleavage product of catechol, using UV-visible spectrophotometry as
shown in Fig. 5c. Control incubations by heat-killed enzyme extract showed no accumulation
of meta-cleavage product 2-hydroxymuconic semialdehyde, indicating no catechol-2,3dioxygenase activity. Both catechol-1,2-dioxygenase and catechol-2,3-dioxygenase activity
suggests that phenol metabolism involves both ortho- as well as meta-pathway. The catabolic
efficiencies of phenol hydroxylase, catechol-1,2-dioxygenase, and catechol-2,3-dioxygenase
were estimated on basis of specific enzyme activities, which are tabulated in Table 5. Comparatively higher activity of catechol-1,2-dioxygenase against that of catechol-2,3-dioxygenase
suggests efficiency of ortho- over meta-pathway in C. pyrenoidosa. Similar results about
simultaneous activity of meta as well as ortho-pathway were reported in P. fluorescens PU1
[12]. They reported higher meta-activity over ortho-activity. Cai et al. [52] also reported
similar findings of coexistence of both ortho- and meta-pathway in Fusarium species. Semple
and Cain [8] reported involvement of meta pathway in golden brown chrysophyte alga
O. danica, whereas most eukaryotes generally utilize ortho-pathway [53]. Evidence of ortho-activity in other eukaryotes as Trichosporon cutaneum [15], Penicillium sp. [54], Fusarium sp., Aspergillus sp., Penicillium sp. and Graphium sp. [55], and Candida sp. [17]
correlates with the present finding.
The breakdown products of cis,cis-muconic acid (ortho-pathway) and 2-hydroxymuconic
semialdehyde (meta-pathway) were identified by LC-MS analysis. In order to identify the
target metabolites, we are analyzing the catechol dioxygenase assay mixture both at the start
and end of the reaction, and the chromatograms are shown in Fig. 5d and e, respectively.
Figure 5d shows a highly abundant (abundance=95,828) m/z signal of 131. The m/z signal of
131 is consistent with the molecular ion mass of [M+Na2H] adduct of catechol. Two more
m/z signals of low abundance at 177 (abundance=2989) and 195 (abundance=3060) are also
noted. The m/z signal at 177 corresponds the molecular ion mass of [M+K2H] adduct of
cis,cis-muconic acid (ortho-cleavage product of catechol). Similarly, the m/z signal at 195 is
consistent with the molecular ion mass of [M+Cl] adduct of -ketoadipate, a metabolite of
the ortho-pathway. The occurrence of adduct ions are common occurrence in LC-MS analysis.
Biological samples generally have high endogenous concentration of various salts, while other
Table 5 Enzyme activities in C. pyrenoidosa (NCIM 2738) cell lysate grown in phenol-containing media
Enzyme assayed

Activity (U)

Specific activity (U/mg)

Phenol hydroxylase

833

11.41

Catechol-1,2-dioxygenase

514

7.04

Catechol-2,3-dioxygenase

13

0.18

Appl Biochem Biotechnol

salts may be added during sample preparation. This justifies the high probability of formation
of adduct ions during LC-MS of biological samples [56]. One probable source of potassium
leading to formation of potassium ion adduct in our samples is possibly the utilization of
potassium phosphate buffer during cell free extract preparation. The chloride ion adduct
formed is one of the commonly formed metal adduct ion during negative ion electrospray
analysis [56]. Figure 5e shows that the abundance of the m/z signal 131 decreases (abundance=3236) after 20-min incubation of the reaction mixture, which confirms utilization of
catechol. The increase in abundance of m/z signal 177 (abundance=263,655) shows increase
in accumulation of ortho-cleavage product of catechol, i.e., cis,cis-muconic acid. This indicates active ortho-pathway for phenol metabolism. Similarly, the increase in abundance of m/z
signal at 195 (abundance=373,154) shows increase in accumulation of ortho-pathway intermediate, -ketoadipate. Figure 5e shows an additional abundance at m/z 338 (abundance=
35,223) with a molecular ion mass identical to [3MH] adduct of cis-2-hydroxypent-2,4dienoate, a metabolite from the meta-cleavage pathway. Identification of adduct ion of cis-2hydroxypent-2,4-dienoate suggests the presence of meta-pathway along with ortho-pathway.
The literature also supports present observation as Tsai et al. [17] identified a LC-MS signal at
m/z 163 corresponding to molar mass of sodium adduct of cis,cis-muconic acid in the enzyme
activity reaction mixtures, which is due to active ortho-pathway in Candida albicans TL3.
On the basis of the enzyme activity and metabolite analysis study, the pathway proposed for
phenol degradation in C. pyrenoidosa has been shown in Fig. 5f. In the pathway, phenol
hydroxylase is involved in initial attack on phenol hydroxylating phenol to catechol. The
resulting catechol is ortho-cleaved by catechol-2,3-dioxygenase as well as meta-cleaved by
catechol-1,2-dioxygenase. Catechol-1,2-dioxygenase ortho-cleaves catechol into its reaction
product cis,cis-muconic acid. Determination of 3-oxoadipate, a metabolite downstream of
cis,cis-muconic acid in the ortho-pathway indicates breakdown of cis,cis-muconic acid.
Catechol also undergoes meta-cleavage into 2-HMS by catechol-2,3-dioxygenase activity. 2Hydroxymuconate semialdehyde hydrolase causes hydrolysis of 2-HMS to cis-2Hydroxypenta-2,4-dienoate in the meta-pathway. Thus, both ortho- as well as meta-pathway
is involved in phenol degradation in C. pyrenoidosa. However, ortho-pathway is significantly
active over meta-pathway. The proposed pathway is found to possess similarities with algal
[8], fungal [17, 52, 55], as well as bacterial [12, 51] catabolic mechanisms.

Biomolecular Characterization of the Biomass for Potential Applications

This section analyses the usefulness of the phenol-degrading algal biomass as potential
feedstock for applications as biodiesel and animal feed. Microalgal biomass with high lipid
and protein content could serve as biodiesel feedstock [57] and protein supplement in animal
diet, respectively [58]. Liu et al. [59] characterized lipid content in algal strains in a bid to
identify high lipid producing strains. They reported C. pyrenoidosa to be one of the best oil
producers whose total lipid content varies between 18.67 % to as high as 52.08 % of dry
biomass. Chlorella sp. have been reported to have high protein content between 51 and 58 %
of dry biomass, and so it is one of the species selected for large scale production [60]. The
biochemical characteristic of the algal biomass was analyzed using FTIR. FTIR analysis
shown in Supp. Fig. 4a depicts a snapshot of the changes in biomolecular level in response
to phenol stress. The infrared region 28752850 cm1 corresponds to symmetric stretching of
CH2 and CH3 of lipids [61]. With phenol incubation, there is prominent decrease in transmittance (Supp. Fig. 4a) and increase in peak area (Supp. Fig. 6a) in this region indicating higher

Appl Biochem Biotechnol

cellular lipid accumulation compared to control biomass. Gracia et al.[57] reported increased
lipid production in Phaeodactylum tricornutum UTEX-640 when cultured mixotrophically
with carbon sources glycerol and fructose. Kong et al. [62] reported stimulation of lipid
biosynthesis in C. vulgaris when mixotrophically cultured in glucose and glycerol. These
findings accords with present finding of increased lipid accumulation in the presence of phenol
as additional carbon source. However, high neutral lipid accumulation in algal biomass is
necessary for its commercial applicability as biodiesel feedstock [32]. The profile for neutral
lipid biosynthesis in C. pyrenoidosa cells (Fig. 6a) shows 50 % increased neutral lipid
accumulation in phenol-degrading biomass as compared to control on fourth day of incubation. Hamed and Klock [63] reported similar results of enhanced neutral lipid accumulation in
Chlorella sorokiniana during mixotrophic culture on glycerol. Fluorescence microscopy of
phenol-degrading cells (Fig. 6b) showed enhanced yellow gold fluorescence of neutral lipid
bodies in cell cytoplasm compared to that from control cells (Fig. 6c). This further supports the
finding of enhanced neutral lipid accumulation in phenol-degrading biomass. Therefore, the
algal biomass (after phenol biodegradation) could serve as potential raw material for biodiesel
production. Although mixotrophic cultivation allows microalgae to accumulate higher proportion of lipids within less time, its commercial applicability is hindered by high substrate cost.
The cost of carbon source represents 50 % of the cost of the medium used in mixotrophic algal
cultivation. This makes the process of production of algal biomass feedstock for biodiesel
costly [64]. Thus, cheap alternative carbon sources for mixotrophic cultivation of algae could
help commercialize algal biodiesel by reducing the cost of production of algal biomass
feedstock. The present study shows that C. pyrenoidosa accumulates high proportion of lipids
while metabolizing phenol, which is a waste product of various industrial processes. Thus,
mixotrophic cultivation of C. pyrenoidosa using industrial waste phenol as a carbon source
could provide exciting possibility to decrease the production cost of algal biomass feedstock
for biodiesel.

Fig. 6 a Neutral lipid accumulation in phenol-degrading and control biomass of C. pyrenoidosa. b Fluorescence
microscopy image of Nile red stained phenol-degrading C. pyrenoidosa cells. c Fluorescence microscopy image
of Nile red stained control cells of C. pyrenoidosa

Appl Biochem Biotechnol

The infrared region 1200900 cm1 corresponds to symmetric stretching of COC of

polysaccharides [61]. The infrared region 16101685 cm1 is associated to C=O
stretching of proteins [61]. Phenol incubation causes increased intracellular protein
synthesis compared to control as evident from decreased transmittance (Supp. Fig. 4a)
as well as increased peak area (Supp. Fig. 6b) in this region (16101685 cm1) in
phenol-degrading biomass. Thus, the protein rich algal biomass (after phenol degradation) could be used as an animal feed supplement. Since the amino acid profiles of
microalgal protein is comparable to other food proteins, it could serve as a protein
supplement in animal feed [65]. Although microalgae have been widely used as protein
source to supplement animal diets, recent research trend in this area is supplementation
of animal diets with defatted (lipid extracted) microalgal biomass from biodiesel production processes [66]. Since phenol-degrading biomass of C. pyrenoidosa is lipid as
well as protein rich (increased accumulation of lipid and protein during the phenol
degradation process), the biomass after lipid extraction (for biodiesel production) could
serve as a protein supplement in animal diets.

Conclusion
This study showed photodependent phenol degradation capability of C. pyrenoidosa with
complete degradation till 200 mg/l phenol concentration under the optimal nutrient
conditions of Fogs medium. The maximum specific rate of degradation was achieved
at 125 mg/l phenol due to maximum specific growth rate at this concentration. However,
the strain could metabolize 38.32 % of 23.33 mg/l phenol along with removal of
aliphatics from petroleum refinery wastewater. Biokinetic parameters obtained by kinetic
modeling shows the differences in growth and phenol degradation dynamics of
C. pyrenoidosa in nutrient media and petroleum refinery wastewater. Low max values
obtained by kinetic modeling in refinery wastewater is probably related to lack of
optimal growth factors as well as other inhibitory constituents, which may be present
in refinery wastewater. Cometabolism of alkanes along with decreased max values may
be responsible for decreased phenol degradation rates (low qmax value) as well as
decreased phenol affinity (high Ks value)in refinery wastewater. SEM analysis indicates
that the cellular membrane morphology gets wrinkled on phenol exposure.
C. pyrenoidosa metabolizes phenol simultaneously by both ortho- as well as metapathway. The ortho-pathway is significantly predominant over the meta pathway. The
phenol-degrading biomass has 50 % higher neutral lipid accumulation compared to
control cells, suggesting exciting possibility to utilize the spent biomass as biodiesel
feedstock. The defatted biomass could additionally serve as animal feed owing to its
enhanced protein content. Thus, the mixotrophic growth of C. pyrenoidosa on industrial
waste phenol could prove to be an environmentally sustainable process as it will cause
remediation of the toxic waste phenol along with generation of biodiesel feedstock with
decreased production costs solving a major bottleneck in commercialization of algal
biodiesel.
Acknowledgments Bhaskar Das acknowledges Indian Institute of Technology, Guwahati, for providing research fellowship to pursue doctoral studies at the Centre for the Environment, Indian Institute of Technology,
Guwahati. The present work is not financially supported by any funding agency.

Appl Biochem Biotechnol

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