Chemosphere 308 (2022) 136269

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Chemosphere
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Valorization of phenol contaminated wastewater for lipid production by

Rhodosporidium toruloides 9564T
Sangeeta Singh, Tanmay Bharadwaj, Devendra Verma, Kasturi Dutta *
Department of Biotechnology and Medical Engineering, National Institute of Technology, Rourkela, Odisha, 769008, India

H I G H L I G H T S

• R. toruloides 9564T is a potential yeast for phenol degradation upto 0.75 g/L.
• R. toruloides 9564T follows an ortho cleavage pathway for phenol degradation.
• Haldane’s model shows μmax and qmax to be 0.0717 h− 1 and 0.01523 h− 1 respectively.
• > 70% cell and seed viability observed for treated samples (0.25–0.50 g/L).

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

Handling editor: Veeriah (Jega) Jegatheesan Phenol is one of the most common hazardous organic compound presents in several industrial effluents which
directly affects the aquatic environment. The present study envisaged the phenol biodegradation and simulta­
Keywords: neous lipid production along with its underlying mechanism by oleaginous yeast Rhodosporidium toruloides
Cytotoxicity 9564T. Experiments were designed using simulated wastewater by varying phenol concentration in the range of
Lipid production
0.25–1.5 g/L and inoculum size of 1, 5, and 10% with and without glucose. The oleaginous yeast was found to
Ortho cleavage pathway
completely degrade up to 0.75 g/L phenol with lipid accumulation of 26.3%. Phenol at > 0.5 g/L severely
Phenol biodegradation
Phytotoxicity inhibited the growth of R. toruloides 9564T at 1% and 5% inoculum size. Phenol toxicity up to 0.75 g/L can be
Rhodosporidium toruloides overcome by increasing inoculum size to 10%. The maximum specific growth rate (μmax) and phenol degradation
rate (qmax) were found to be 0.0717 h− 1 and 0.01523 h− 1, respectively. The enzymatic pathway study suggested
that R. toruloides 9564T follows an ortho cleavage pathway for phenol degradation and lipid accumulation.
Phytotoxicty and cytotoxicity tests for treated and untreated samples clearly demonstrated a decline in toxicity of
the treated wastewater. R. toruloides brought about an important paradigm shift toward a circular economy in
which industrial wastewater is considered a valuable resource for bioenergy production.

1. Introduction 2005), phenolic compounds are the key elements of effluents released
from petrochemicals, coal processing, pulp and paper, insecticides, and
Over the last decades, waste management has become a serious pharmaceuticals sectors, pesticides, steel plants, plastics, polymeric
concern due to the fast growth in population and hurried urbanization. resins, and textile industries. . Every year these industries release from 1
Industrial operations produce a range of effluents that contain various mg/L to 7000 mg/L phenol in their wastewater effluents (Ahmad,
chemicals that may contaminate air, water, and soil. It’s also been 2015a; Panigrahy et al., 2022a).
claimed that around 75–95% of the water released as effluent from According to the United Nations Environmental Protection Agency
various industries contains organic, inorganic, and colorant contami­ (EPA), a safe limit for phenol is 1 μg/L while the EU directive specifies an
nants (Sachan et al., 2019). These contaminants have undesirable admissible concentration of 0.5 μg/L of phenol in drinking water
human and environmental health effects due to their toxicity, carcino­ (Krastanov et al., 2013). Phenol has a complex and persistent chemical
genicity, and mutagenicity. According to (Pazarlioǧ lu and Telefoncu, structure. Phenol and its derivatives may change the taste and odor of

Abbreviations: EPA, Environmental Protection Agency; MSM, Minimal salt medium; MTT, 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide; 4-
NP, 4-Nitrophenol; TAG, Triacylglycerol; TCA, Tricarboxylic acid cycle.
* Corresponding author.
E-mail addresses: [email protected], [email protected] (K. Dutta).

https://doi.org/10.1016/j.chemosphere.2022.136269
Received 21 April 2022; Received in revised form 28 July 2022; Accepted 27 August 2022
Available online 31 August 2022
0045-6535/© 2022 Elsevier Ltd. All rights reserved.
S. Singh et al. Chemosphere 308 (2022) 136269

water and render it very hazardous to aquatic and terrestrial life (Pan­ yeast) uses different carbon sources and produces biomass around 130
igrahy et al., 2022b; Shazryenna et al., 2015). Phenol on prolonged g/L. Additionally, it can accumulate up to 70% of the lipids in the
exposure can cause severe skin damage, muscle pains, gastrointestinal exponential and stationary phase of growth (Park et al., 2018). The
disorders, headache, renal failure, peripheral nerve damage, inhibition bioproducts produced by R. toruloides are also of interest, mainly lipids
of photosynthesis in diatoms and mutagenesis in several living organ­ (mainly palmitic, stearic, oleic, and linoleic acids), enzymes, and ca­
isms and might even to lead to cancer when present in water above the rotenoids (Osorio-González et al., 2019). So far, limited studies have
concentrations of 100 μg/L (Ahmad, 2015b). Phenol has also been found been done using oleaginous yeast R. toruloides for aromatic compounds
to be lethal to certain trout fishes and humans at a concentration of 5–25 degradation.
mg/L (Kumar et al., 2005; Sachan et al., 2019). The quantity of phenol Therefore, the present work is aimed (1) to evaluate the potential of
can alter the natural microbiota of the surrounding. Phenol and phenolic oleaginous yeast R. toruloides 9564T for simultaneous phenol degrada­
compounds have proven to cause renal failure, peripheral nerve dam­ tion and lipid production, (2) to study the effects of inoculum size and
age, inhibition of photosynthesis in diatoms and mutagenesis in several phenol concentration on the yeast growth, lipid production and phenol
living organisms. The deadly quantity of phenol in the blood is about degradation, (3) to identify the phenol degradation pathway of
1500 mg/L (Sunil J. Kulkarni, 2013). Due to its harmful effects EPA R. toruloides 9564T, and (4) to assess the phytotoxicity and cytotoxicity
considered it as one of the most common toxic contaminants which of the simulated phenol containing wastewater treated by R. toruloides
acquired 11th position out of 126 compounds (Weiner, 2000). 9564T.
Nowadays, the most common method used for wastewater treatment
and reducing organic chemicals from wastewater effluent is biological 2. Materials and methods
treatment that utilizes the potential of microorganisms (bacteria, fun­
gus, yeasts, and algae) (Patel et al., 2017a). Several research studies The reagents, medium, and solvents consumed in the experiments
(Mostafa et al., 2019) have been published on the microbial decompo­ were procured from Himedia and were of analytical grade.
sition of wastewater in aerobic environments using bacteria, fungi,
algae, and yeasts. Bacteria, in general, are unable to digest the complex 2.1. Microorganism growth conditions and seed culture preparation
polyphenols that cause wastewater to be black.
The difficulty of creating a homogenous culture due to the produc­ Oleaginous yeast R. toruloides 9564T was procured from MTCC (Mi­
tion of fungal pellets and other aggregations and the necessity of long- crobial Type Culture Collection) Chandigarh, India. The YPD agar plate
lasting fermentation cycles of at least 2 weeks limit the use of filamen­ (consisting of yeast extract 3 g/L, peptone 5 g/L, glucose 10 g/L, malt
tous fungi on a wide scale (Ayed et al., 2019). Yeasts have emerged as extract 3 g/L, and 2% agar) was prepared for pure or single colonies at
potential microorganisms for removing phenol from wastewater, over­ 25 ◦ C in an orbital shaker at 160 rpm for 48 h, and the pH was main­
coming the above constraints. The lipid accumulated by Saccharomyces tained at 6.2. Erlenmeyer flask (250 mL) containing YPD medium (50
cerevisiae (non-oleaginous yeast) and Candida utilis is lesser than 10% of mL) was inoculated with a loopful of culture from the agar plate. Later,
their biomass. Oleaginous yeast may synthesize triacylglycerol (TAG) as the flask was incubated in an orbital shaker at 25 ◦ C, 160 rpm for 48 h.
lipid droplets, which can account for up to 70% of their biomass (Qin This activated seed culture was used as inoculum to study growth profile
et al., 2017). They may quickly adapt to the harsh circumstances of (spectrophotometer) and lipid productions (Bligh and Dyer extraction
wastewaters by utilizing hazardous chemicals as an energy source, and it method).
has a high degradability to certain organic poisons (Abdel-Shafy and
Mansour, 2016). Yeasts have been found to destroy a wide range of 2.2. Biomass and lipid production by R. toruloides 9564T in simulated
macromolecular compounds, including phenol (Wang et al., 2018) and wastewater (MSM media)
glyceride (Hashem et al., 2018).
However, oleaginous yeasts are less explored for their application in The growth and lipid production profile of R. toruloides 9564T was
aromatic compound degradation than bacteria and algae. In addition to performed in duplicates using Erlenmeyer flasks containing minimal salt
the above-stated advantages, yeasts are also resistant to phages and medium (MSM) (100 mL). The composition of MSM media used is as
showed higher tolerance to high osmolarity, pH, and solvents (Yaguchi follows (g/L): Na2HPO4, 6.6; CaCl2, 0.12; MgSO4, 0.11; KH2PO4, 3.7;
et al., 2020). NaCl, 5.2; and NH4Cl, 2.6. The pH of the media was adjusted to 6.2
Around 100 of the approximately 1600 yeast species are classified as before autoclaving (15 psi at 121 ◦ C and for 15 min). The sterile medium
oleaginous. They accumulate above 20% of their dry cell biomass as was inoculated with fresh seed culture of 5% inoculum size and kept for
neutral lipids, primarily triacylglycerides (TAGs) (Garay et al., 2016). incubation in an orbital shaker at 160 rpm 25 ◦ C for 144 h. The samples
These yeasts are being investigated more and more for the generation of were collected every 12 h intervals to study the growth profile and lipid
biofuels and oleochemicals on a long-term basis. production.
A variety of oleaginous yeast species such as Saccharomyces rosinii,
Trichosporon spps, Rhodosporidium spps., Cryptococcus spps., Candida 2.3. Phenol degradation, lipid production, and biomass production by
spps., and Rhodotorula spps (Gargouri et al., 2015; Margesin et al., 2003; R. toruloides 9564T
Patel et al., 2017b). were used to degrade aromatic compounds like
phenols. Interestingly, the data reveals that phenol metabolism differs Batch shake flasks experiments were performed to study the growth,
significantly between ascomycetes and basidiomycetes (Yaguchi et al., phenol degradation, and lipid production by R toruloides 9564T. A total
2020). of 39 sets of flasks (250 mL) containing 100 mL MSM medium were used
The degradation of phenol by oleaginous yeast follows the meta and in this study using either glucose (control), phenol (0.25–1.5 g/L) or
ortho cleavage pathways. The end product of these two pathways is the glucose (50 g/L) and phenol (0.25–1.5 g/L) as carbon source(s). The
formation of triacylglycerol (TAG) by utilizing the acetyl-CoA and py­ media compositions of different flasks are described in Table 1.
ruvate produced during the breakdown of phenol. These two precursors The negative control (flasks A, H & O) with inoculum sizes 1%, 5%,
help in fatty acid synthesis for TAG formation. Therefore, finding and 10% contained MSM with 50 g/L glucose, 5 g/L ammonium sulfate,
oleaginous yeasts that can metabolize aromatic compounds might boost 1.7 g/L complete supplement mixture, and 0.79 g/L yeast nitrogen base
yields and produce a high quantity of lipid by protecting the environ­ without ammonium sulfate. The flasks (B-G, I–N, and P–U) contained the
ment from hazardous chemicals such as phenol. R. toruloides species MSM along with the varying phenol concentration (0.25, 0.5, 0.75, 1,
naturally have physiological advantages in terms of substrate utiliza­ and 1.5 g/L) at three different inoculum sizes (1%, 5%, and 10%). Flasks
tion, lipid accumulation, and inhibitor resistance. R. toruloides (red (B*- G*, I*- N* and P*- U*) contained MSM along with glucose (50 g/L)

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S. Singh et al. Chemosphere 308 (2022) 136269

Table 1 cess as well as to study the cell growth and phenol degradation kinetics
Experimental design to study the effect of substrate concentration and inoculum using the following inhibitory kinetic equations
size.
μmax S
Sl. No Marking Glucose (g/l) Inoculum Size (%) Phenol (g/l) μ= 2 (3)
S + Ks + (SKi )
1 A (control) 50 1 0

2 B 0 1 0.25 qmax S
q= (4)
3 C 0 1 0.5 2
S + Ks + (SKi )
4 D 0 1 0.75
5 E 0 1 1
6 F 0 1 1.25 where S is the phenol concentration (g/L), μmax is the maximum specific
7 G 0 1 1.5 growth rate (h− 1), qmax is the maximum specific phenol degradation rate
8 Ba 50 1 0.25 (h− 1), Ks is the half-saturation coefficient (mg/L), and Ki inhibition co­
9 Ca 50 1 0.5 efficient (mg/L) (Wang et al., 2016; Zhao et al., 2021).
10 Da 50 1 0.75
11 Ea 50 1 1
12 Fa 50 1 1.25
13 Ga 50 1 1.5 2.5. Analytical methods
14 H (control) 50 5 0
15 I 0 5 0.25 2.5.1. Estimation of residual phenol
16 J 0 5 0.5 The collected sample was centrifuged at 6000 rpm for 10 min, and
17 K 0 5 0.75
18 L 0 5 1
the collected supernatant was used for the estimation of residual phenol.
19 M 0 5 1.25 The residual phenol amount in the collected samples was estimated
20 N 0 5 1.5 using the 4-aminoantipyrine (4-AAP) colorimetric technique. The
21 Ia 50 5 0.25 amount of phenol in the supernatant was determined by the amount of
22 Ja 50 5 0.5
red antipyrine dye produced.
23 Ka 50 5 0.75
24 La 50 5 1 To the specified volume of supernatant, 0.2 mL of ammonium
25 Ma 50 5 1.25 chloride was added, and pH 10 was adjusted using ammonia solution.
26 Na 50 5 1.5 Then 0.2 mL of 4-AAP reagent and 0.2 mL potassium ferricyanide re­
27 O (control) 50 10 0 agent were added one after another and the final volume made up to 10
28 P 0 10 0.25
mL. Samples were kept for 15-min incubation at room temperature. A
29 Q 0 10 0.5
30 R 0 10 0.75 blank was prepared by mixing all the reagents in distilled water.
31 S 0 10 1 Absorbance was measured in UV–vis spectrophotometer at 510 nm. The
32 T 0 10 1.25 phenol concentration was calculated using the standard curve of the
33 U 0 10 1.5
phenol. All the experiments were performed in duplicate and the final
34 Pa 50 10 0.25
35 Qa 50 10 0.5 results were presented in averaged (Patel et al., 2017b).
36 Ra 50 10 0.75
37 Sa 50 10 1 2.5.2. Determination of dry cell biomass (g/L), total lipid (g/L), and lipid
38 Ta 50 10 1.25 content (%)
39 Ua 50 10 1.5
The dry cell biomass was obtained by harvesting culture (50 mL)
a
Phenol with glucose. using centrifugation at 10,000 rpm for 15 min. The pellet was washed
using distilled water and dried in a hot air oven at 60 ◦ C. Modified Bligh
and phenol, to know the effect of dual carbon source on the biodegra­ and Dyer’s method was used for total lipid estimation (Behera et al.,
dation of phenol by R. toruloides 9564T. The media pH was adjusted to 2019).
6.2 for all flasks. All flasks were steam sterilized in an autoclave for 15
min at 15 psi and 121 ◦ C. Phenol was added after autoclaving by ster­
ilizing with the help of a syringe filter (0.22 μm). 2.6. Enzymatic assay for the estimation of the phenol degradation
The experiments were performed using the inoculum size of 1%, 5%, pathway
and 10% having cell density (OD~ 0.538 @600 nm). The incubation of
culture flasks (A to U*) was done using a rotary orbital shaker at 25 ◦ C, The most prevalent mechanism for phenol biodegradation involves
160 rpm for 144 h. The growth and phenol degradation profiles were either ortho or meta-cleavage pathway, using catechol 1, 2-dioxygenase
studied using a 2 mL incubated sample every 12 h. All the experiments and catechol 2, 3-dioxygenase enzymes (Cao et al., 2008). The current
were performed in duplicate, and the final results were presented on study utilized these two for the enzymatic experiment. Cell-free extracts
average. produced were used in the enzymatic experiment to investigate the
biodegradation route in oleaginous yeast. After harvesting by centrifu­
gation, the cells were sonicated for 5 min at 20 kHz, followed by
2.4. Cell growth and phenol degradation kinetics centrifugation for 5 min at 10,000 rpm. The cell-free supernatant was
maintained at 4 ◦ C and used to determine the enzymatic activities
In the exponential phase, the following equations described by (Deeba et al., 2018a; Shetty and Shetty, 2016).
(Banerjee and Ghoshal, 2010; Li et al., 2010; Wang et al., 2016) were
used to calculate microbe growth (Eq. (1)) and phenol degradation (Eq. 2.6.1. Enzymatic activity of catechol 1, 2-dioxygenase
(2)) kinetics. Phenol degradation through the ortho cleavage pathway was studied
X = X0 ​ eμt (1) using 2 mL (50 mM) Tris HCl buffer of pH 8, 0.1 mL cell-free supernatant
in 0.7 mL distilled water, 0.1 mL (100 mM) 2- mercaptoethanol taken in
S = S0 ​ e− qt
(2) a 4 mL cuvette. After proper mixing of the constituents, 0.1 mL (1 mM)
catechol was added. The absorbance of the solution was measured at
where X is the biomass (g/L), μ is the specific growth rate (h− 1), S is the 260 nm for 5 min. An increase in absorbance or straight line formation
phenol concentration (g/L), q is the specific degradation kinetics (h− 1). with time was used to depict the formation of cis-cis muconic acid
The Haldane model was used to describe the phenol inhibition pro­ (Shetty and Shetty, 2016).

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S. Singh et al. Chemosphere 308 (2022) 136269

2.6.2. Enzymatic activity of catechol 2, 3-dioxygenase

The degradation pathway involving the meta-cleavage pathway was
studied using 2 mL Tris-HCl buffer (pH 7.5) and distilled water (0.6 mL)
along with cell-free extract (0.2 mL). Later, the mixture was added with
100 mM catechol resulting in the formation of 2-hydroxymuconic semi-
aldehyde (absorbance at 375 nm) (Shetty and Shetty, 2016).

2.7. Toxicological studies

Organic and inorganic substances released into industrial waste­

water effluents have a variety of environmental consequences. Phenol,
an organic chemical found in wastewater at concentrations of more than
100 μg/L, inhibits the growth of plants and animals (Ahmad, 2015a). As
a result, phytotoxicity and cytotoxicity assays were used to assess the
toxicity of phenol in both untreated and treated samples (Ali et al.,
2021).

2.7.1. Cytotoxicity test

The cytotoxicity of phenol was tested on L929 (mice fibroblast) and
LN229 (glioblastoma) cell line using 3-(4,5-dimethyl thiazol-2-yl)-2,5- Fig. 1. The growth curve of oleaginous yeast R. toruloides in MSM media
diphenyl-2H-tetrazolium bromide (MTT) cytotoxicity assay in minimal (inoculum size, 5%) along with the dry cell biomass, total lipid, and
lipid content.
salt media (MSM) before and after degradation of phenol by oleaginous
yeast R. toruloides 9564T. The degraded samples were centrifuged for 10
min at 6000 rpm. The samples were then sterilized using a 0.22 μm R. toruloides-1588, the maximum biomass formed using hardwood hy­
syringe filter and kept in UV for sterilization. The treated and untreated drolysates was 5.2 g/L and softwood hydrolysates were 6 g/L. However,
phenol samples (100 μL) were used for the test (Ali et al., 2019; Kenawy a significant increase in biomass of 9 g/L was observed using synthetic
et al., 2019). For this assay 0.5 mg/mL concentration of MTT was used. media (MSM) where R. toruloides-1588 remained in its log phase for 72 h
Eq. (5) was used to calculate the viability percentage of cells. (Saini et al., 2020). Another group reported maximum biomass and lipid
content of 18 g/L and 74.5%, respectively in Mandi waste media and 9.6
Cell Viability =
At − Ab
× 100 (5) g/L and 60% in minimal media (Singh et al., 2018) using R toruloides.
Ac − Ab Qian et al. (2021) used volatile fatty acids (VFA) derived from waste
organics in their study of oleaginous yeast Apiotrichumporosum
where, At = Absorbance of the test sample, Ac = Absorbance of the
DSM27194. They found that the maximum biomass and lipid contents
control sample, and Ab = Absorbance of the blank. The statistical
were 26.5 g/L and 36.2%, respectively for the yeast (Qian et al., 2021).
analysis was performed in GraphPad prism 8 by using a one-way and
two-way ANOVA test where the main factor is the phenol concentration.
3.2. Phenol degradation and growth curve by R. toruloides 9564T in
Data indicated ±mean standard error difference at * for p ≥ 0.05, ** for
phenol containing MSM media
p ≤ 0.05, *** for p ≤ 0.001 and **** for p ≤ 0.0001.

3.2.1. Growth profile of R. toruloides 9564T in phenol containing MSM

2.7.2. Phytotoxicity test
media
The phytotoxicity test was carried out using the seed germination
Experiments were carried out using different inoculum sizes (1, 5,
method of Phaseolus mungo and Cicer arietinum. A total of 10 lentil seeds
and 10%) with constant cell density (0.538 OD) and phenol concen­
of Phaseolus mungo and 5 lentil seeds of Cicer arietinum were employed,
trations ranging from 0.25 to 1.5 g/L, with a 0.25 g/L interval to
and the seeds were irrigated with 15 mL of both treated and untreated
determine the tolerance of R. toruloides 9564T to phenol. The growth
samples. Tap water and distilled water were used as controls in this
profile of R. toruloides 9564T is shown in Fig. 2, at different phenol
experiment, and the samples were stored at room temperature at 25 ◦ C.
concentrations with or without glucose. Cell growth was substantially
The proportion of seed germination, shoot production, and root for­
suppressed when the medium phenol concentration was higher than 0.5
mation was observed after 7 days (Ali et al., 2020; Pandey et al., 2022).
g/L at inoculum size of 1% and 5%. In Fig. 2a and b, the maximum cell
The % seed germination was calculated using the following formula (Eq.
growth was found between 24 and 72 h, and there was less cell growth in
(6)):
the media after 0.5 g/L phenol concentration. When the flasks with
No. of seeds sprouted inoculum size 1% and 5% were inoculated with 5% glucose, the log
Germination % = × 100 (6)
total no. of seedsused phase started from 48 h, and maximum growth was achieved at 120 h
(Fig. 2d and e). Increase in inoculum size from 5 to 10%, the log phase of
3. Results and discussions oleaginous yeast lasted between 12 and 72 h in MSM media with
different phenol concentrations. When MSM media was supplemented
3.1. Biomass and lipid production by R. toruloides 9564T in simulated with 5% glucose, the log phase was extended up to 132 h (Fig. 2c and f).
wastewater (MSM media) However, maximum biomass production of 9 g/L was observed at 60 h
when phenol concentration was 0.5 g/L, and the media was supple­
The growth curve of oleaginous yeast R. toruloides 9564Twas studied mented with 5% glucose. Whereas when media was not supplemented
in MSM media. Fig. 1 shows the growth profile, dry cell biomass, and with glucose the maximum biomass was found to be only 1.088 g/L.
lipid profile of R toruloides 9564T. It can be observed that the log phase Data interprets that this oleaginous yeast utilizes phenol as a carbon
started after 12 h of growth and lasted up to 132 h. After every 12 h, the source, resulting in its fast degradation compared to when the media was
dry cell biomass was also increased, and the maximum biomass of supplemented with glucose in addition to phenol. Since the microbes
14.775 g/L was obtained at 144 h. Additionally, maximum total lipid utilize glucose as their carbon source. The results suggested that dual
and lipid content were found to be 3.075 g/L and 38.70%, respectively carbon source leads to increase in the biomass production, total lipid,
at 60 h. As studied by Saini et al. (2020) on biomass growth of lipid content, along with an extended log phase. Moreover, yeasts were

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S. Singh et al. Chemosphere 308 (2022) 136269

Fig. 2. The growth profile of R toruloides in MSM media when grown on various concentrations of phenol (0.25–1.5 g/L) without glucose in Fig. 2 (a, b, and c with
inoculum sizes 1%, 5%, and 10%) and with glucose-containing media in Fig. 2 (d, e, and f with inoculum sizes 1%, 5%, and 10%).

able to grow in higher phenol concentration in presence of glucose in the (Deeba et al., 2018a).
media. In previous studies, Patel et al. (2017a) reported that when
oleaginous yeast R. kratochvilovae HIMPA1 was grown in MSM media 3.2.2. Phenol degradation by R. toruloides 9564T
with and without glucose the yeast was able to grow in both the medium The yeast R. toruloides 9564T completely degraded phenol at con­
with phenol concentration up to 1 g/L (Patel et al., 2017b). Deeba et al., centration ranges between 0.25 and 0.5 g/L with 1% inoculum size
(2018a) studied aromatic hydrocarbon degradation using oleaginous within 48–60 h (without glucose in the medium). However, when the
yeast Cryptococcus psychrotolerans IITRFD and reported that among four media was supplemented with glucose the degradation of phenol was
different aromatic compounds (naphthalene, anthracene, pyrene, and achieved at 60–120 h (Fig. 3a and d). As glucose is an easily metabo­
phenol), this yeast can completely degrade 1 g/L phenol from the media lizable carbon source and microbes prefer glucose over other substrates.

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S. Singh et al. Chemosphere 308 (2022) 136269

Fig. 3. Residual phenol estimation in MSM media without glucose in (Fig. 3a, b, and c with inoculum sizes 1%, 5%, and 10%, respectively) and with glucose-
containing MSM media (Fig. 3d, e, and f with inoculum size 1%, 5%, and 10%, respectively).

When inoculum size increased from 1% to 5% the degradation time was inoculated with 10% inoculum size. The yeast could able to degrade
reduced to 24–36 h in media without glucose, while degradation time 48% phenol at a concentration of 1 g/L after 60 h with 10% inoculum
for glucose containing media was 72–96 h. At 5% inoculum size size (Fig. 3c). Supplementation of 5% glucose to the media the extended
degradation of phenol at 0.75 and 1 g/L was found to be 13% and 6.7% the degradation time from 60 h to 84 h for 1 g/L phenol concentration.,
(Fig. 3b and e). Complete phenol degradation at a concentration of 0.75 with increase in phenol degradation rate from 48% to 62.5% (Fig. 3f).
g/L was achieved after 48 h when the glucose-free medium was Further, increase in the concentration of phenol to 1.25 and 1.5 g/L the

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S. Singh et al. Chemosphere 308 (2022) 136269

cells could not able to grow and degrade the phenol even in the presence (Yaguchi et al., 2020). Deeba et al. (2018b), in their study on phenol
of glucose in the media. This could be due to phenol’s harmful effect at degradation by Cryptococcus psychrotolerans IITRFD have found that the
these doses on cell viability. Similar study was also done by Deeba et al. 13.75 g/L biomass, 6.40 g/L total lipid and 46.54% lipid content (Deeba
(2018a) using oleaginous yeast Cryptococcus psychrotolerans IITRF for et al., 2018b).
aromatic compounds (naphthalene, anthracene and pyrene) degrada­
tion. They reported that above 0.5 g/L concentration of aromatic com­ 3.2.4. Kinetics of cell growth and phenol degradation by R. toruloides
pounds were having lethal effect on cell viability (Deeba et al., 2018b). 9564T
Hence it is concluded that the increase in initial inoculum size will The kinetics of growth and phenol degradation by R. toruloides 9564T
improve the phenol degradation rate and time. The overall degradation at the exponential stage were studied using the Haldane model at
of phenol depends upon the inoculum size, incubation period, the different initial phenol concentrations (0.1–0.75 g/L). The experimental
phenol concentration, and glucose utilization. data were fitted to the model equations (3) and (4), and kinetic pa­
Cryptococcus psychrotolerans IITRFD was able to completely degrade rameters like Ks, μmax, Ki, and Ks were estimated (Supplementary Fig. 1a
1 g/L phenol concentration after 120 h from the media (Deeba et al., and b). The regression coefficient of both the kinetics was found to be
2018a). Study on phenol degradation by R. kratochvilovae HIMPA1, at 0.999 and 0.978 which confirms the good fit of the experimental data
three different cell densities (0.063, 0.126 and 0.189 @ OD 600 nm) has with the model. Growth kinetic parameters obtained from model were
shown that increases cell densities improve the phenol degradation rate 0.071731 h− 1 μmax, 405.93 mg/L, KS, 190.84 mg/L Ki and for degra­
from 62.2% to 100% also lowers the degradation time from 96 h to 36 h dation kinetics parameter were 0.01523 h− 1qmax, 102.21 mg/L KS’,
at varying phenol concentration from 0.5 to 1 g/L (Patel et al., 2017a). 473.68 mg/L Ki’. Similarly, Haldane kinetics was also performed on
The study also showed that supplementation of glucose extend the Candida tropicalis PHB5 (Basak et al., 2014) to determine the growth
phenol degradation time. Sun et al. (2020) recently studied phenol kinetics parameters. The parameters obtained were 0.3407 h− 1μmax,
degradation in saline wastewater using oleaginous yeast T. cutaneum 15.81 mg/L KS, 169.0 mg/L Ki and the regression coefficient (R2) was
MP11 with inoculum size 10% (v/v). They observed that T. cutaneum 0.9886, and the degradation kinetics parameters were 0.2766 g/g h− 1
MP11 could completely degrade 800 mg/L phenol at 48 h of incubation qmax, 2.819 mg/L KS’, 2,093Ki’ and the regression coefficient (R2) was
(Sun et al., 2021). 0.8176. In another investigation, a halotolerant Candida sp. was
immobilized in alginate and nano-SiO2 for effective phenol degradation.
3.2.3. Lipid accumulation and biomass production by R. toruloides 9564T This study also confirms the degradation kinetic with the following re­
in phenol containing MSM media sults: 15.92 h− 1qmax, 42,603 mg/L Ks, 1.10 mg/L Ki and R2 was 0.993
Fig. 4 represents the dry biomass (g/L), total lipid (g/L), and lipid respectively (Jiang et al., 2018).
content (%; wt/wt) of the R. truloides 9564T culture at various phenol Panigrahy et al. (2020) used Pseudomonas citronellolis NS1 to study
concentrations (0.25, 0.5, and 0.75 g/L) with 10% inoculum size. R phenol biodegradation kinetics and found that, 0.246 h− 1μmax, 14.85
toruloides 9564T, when grown in minimal salt media containing phenol mg/L Ks, 890 mg/L Ki and R2 0.955 (Panigrahy et al., 2020). Zhao et al.
concentration of 0.25 g/L can produce dry biomass of 1.08 g/L, total (2021) studied phenol biodegradation using Rhodococcus ruber C1 and
lipid of 0.267 g/L, and lipid content of 24.77%. While, when supple­ reported that the 1.527 h− 1 μmax, 69.74 mg/L Ks, 4895 mg/L Ki and R2
mented with glucose, there was an increase in dry biomass, total lipid was 0.9385 and 3.674 h− 1 qmax, 76.89 mg/L Ks, 3652 mg/L Ki and R2
and lipid content of 9 g/L, 3.175 g/L, and 35.27%. Further increase in was 0.9436, respectively (Zhao et al., 2021).
phenol concentration to 0.75 g/L dry biomass and total lipid decreased
to 1.035 g/L and 0.24 g/L, respectively. However, lipid content
improved from 24.77% to 26.3%. In contrast, when glucose was added 3.3. Metabolic pathway of phenol degradation by R. toruloides 9564T
along with 0.75 g/L phenol, dry biomass and total lipid increased by
6.76 g/L and 1.58 g/L but a decrease in lipid content of 23.37%. In R. toruloides 9564T, both ortho- and meta-cleavage routes were
Oleaginous yeast R. kratochvilovae HIMPA1 was able to degrade 1 g/ investigated for phenol degradation. The product generation in the
L phenol and produce dry biomass, total lipid and lipid content of 3.81 ortho cleavage pathway was studied using catechol 1, 2-dioxygenase
g/L, 1.09 g/L, and 29.47%, respectively (Patel et al., 2017a). In another enzyme, which shows absorbance at 260 nm, whereas 2-HMSA was
study, oleaginous yeast C. oleaginosus was used to degrade 1.0 g/L studied using catechol 2, 3-dioxygenase, which shows absorbance at
phenol, which resulted in the maximum biomass, total lipid and lipid 375 nm for metacleavage pathway. Phenol degradation by oleaginous
accumulations of 0.72 ± 0.02 g/L, 0.21 ± 0.03 g/L, 29.2 ± 5.4% yeast may occur in either of these two routes. For the identification of
the catechol ring fission pathway, C1,2-D and C2,3-D enzyme assays

Fig. 4. The graph shows dry cell biomass production (g/L), lipid yield (g/L), and lipid content (%; wt/wt) of R toruloides 9564T in MSM media (a) MSM medium with
different concentrations of phenol (b) MSM medium with different concentration of phenol along with glucose (50 g/L).

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S. Singh et al. Chemosphere 308 (2022) 136269

were performed. From Fig. 5 it can be observed that this oleaginous After treatment with oleaginous yeast, the samples showed the cell
yeast follows the ortho cleavage pathway for phenol degradation as viability of L929 was found to be 97%, 95%and 76% and for LN299, cell
absorbance at 260 nm follows a straight line with respect to time. In the viability were 84.47%, 80.38%, and 71.46% at phenol concentrations of
ortho cleavage pathway, the enzyme hydroxylase converts phenol to 0.25 g/L, 0.5 g/L, and 0.75 g/L respectively (Fig. 6). Bacterial con­
catechol (Tuah et al., 2009). Catechol is broken down into cis,cis-mu­ sortium was used by Sarkar et al. (2022) to degrade 4-Nitophenol (4-NP)
conic acid, which is the main intermediate product of ortho cleavage and reported that 700 mg/L 4-NP was degraded from the media. They
pathway. Cis, cis-muconoci acid converted into muconolactone (ML) by used the treated and untreated samples for the cytotoxicity assay using
the cis,cis-muconate lactonizing enzyme (MLE). With the help of en­ HEK293 cell line. Data interprets that, in degraded metabolites con­
zymes like isomerase, hydrolase and transferase the end intermediate centration (100 μg/mL) cell viability was nearly 83.5% (Sarkar et al.,
formed was 3-oxo adipyl-CoA (OA-CoA), which is then converted to 2022).
metabolites of anabolic pathways such as fatty acid biosynthesis and the
TCA cycle (acetylCoA and succinyl-CoA) by 3-oxo adipate CoA thiolase 3.4.2. Phytotoxicity
(TH) and these two end products follow the TCA cycle and Fatty acid Phytotoxicity at different phenol concentrations (0.25–0.75 g/L) was
biosynthesis pathway for the formation of TAG (Wells and Ragauskas, evaluated using dicot seeds of P. mungo and Cicer arietinum. The un­
2012). The metabolic pathway for phenol breakdown is strain-specific treated phenol sample (at a concentration of 0.25–0.75 g/L) inhibited
and dependent on the concentrations of phenol. Previous research has P. mungo seed and Cicer arietinum seed germination completely,
shown that both ortho and meta pathways might be active for the same compared to the control (distilled water and tap water). However, after
microbe depending on the substrate. treatment with this oleaginous yeast, out of 10 lentil seeds, 9 seeds
R. kratochvilovae HIMPA1 degrades 1 g/L phenol by following meta formed radicles, but germination was slow and radicle formation was
cleavage pathway (Patel et al., 2017b). Nocardia Otitidiscaviarum TSH1 minimal in the case of P. mungo and in case of Cicer arietinum total 5
was found to degrade naphthalene also by meta cleavage pathway seeds were taken for germination out of which 4 seeds were formed
(Zeinali et al., 2008). radicles (90% germination) in 0.25–0.5 g/L, and 3 seeds formed radicles
According to Deeba et al., (2018b), oleaginous yeast Cryptococcus (80% germination) in 0.75 g/L phenol treated sample, when compared
psychrotolerans IITRFD degrades aromatic compounds like phenol, to the control (Supplementary Fig 2). These data indicate that the phenol
naphthalene by following ortho cleavage pathway whereas, anthracene is hazardous to P. mungo and Cicer arietinum a common agriculturally
and pyrene by meta cleavage pathway (Deeba et al., 2018a). significant crop. However, the biodegraded products were found to be
less toxic because the seeds were assimilating the degraded samples. In
previous research, Naraginti and Yong (2019) did phytotoxicity study on
3.4. Toxicological studies the untreated p-bromophenol (p-BP) and treated p-bromophenol (p-BP)
using dicot seeds (Vigna radiata (V. radiata)) and observed that before
It is well understood that inappropriate treatment of industrial ef­ treatment the germination were 40% but after treatment with photo­
fluents containing aromatic compounds such as phenol can endanger the catalyst the germination rate was increased to 100% (Naraginti and
environment and cause health problems. As a result, determining the Yong, 2019). An isolated bacterial consortium was used to degrade
toxicity of phenolic wastewater effluents and their biodegraded com­ 4-nitrophenol (4-NP) after degradation, the toxicity of the 4-NP and
pounds is crucial. In this study, different phenol concentrations in syn­ their degraded metabolites was done using three different seeds Vigna
thetic media were prepared and their toxicities were assessed before and radiata, Vigna mungo, and Cicer arietinum, and observed that before
after treatment by oleaginous yeast R. toruloides 9564T using various treatment the seed germination were 0% but after treatment with bac­
bioassays such as phytotoxicity and cytotoxicity. terial consortium the germination rate was increased to 100, 40 and 80%
(Sarkar et al., 2022). In another study Jayapal et al. (2018) used plant
3.4.1. Cytotoxicity microbes for the degradation of azo dye, their aromatic amine. The
The untreated and treated samples were evaluated for cell cytotox­ treated samples were used for phytotoxicity test using Vigna radiata. The
icity (MTT bioassay). The cell used for the experiment was L929 (mice results showed that up to 96% germination occurred in treated sample
fibroblast) and LN229 (glioblastoma). The percentage of cell viability of (Jayapal et al., 2018).
L929 for the untreated phenolic sample was found to be 69%, 63%, and
60% and for LN299 was found to be 64.90%, 54.11% and 39.21% at 4. Conclusion
phenol concentrations of 0.25 g/L, 0.5 g/L, and 0.75 g/L respectively.
In the present study, the oleaginous yeast R. toruloides 9564T was
successfully able to degrade phenol up to 0.75 g/L completely and
accumulated 26.3% lipids per dry cell weight at 60 h incubation period
with 10% inoculum size. Maximum specific growth rate (μmax) and
phenol degradation rate (qmax) were found to be 0.0717 h− 1 and
0.01523 h− 1, with regression coefficient of 0.999 and 0.978, respec­
tively using Haldane model. The degraded product of phenol was found
to be less toxic, as depicted in phytotoxicity and cytotoxicity studies.
Therefore, the present research confirmed that R. toruloides 9564T is a
promising oleaginous yeast for treating phenol-contaminated waste­
water with simultaneous capacity for lipid production, which can be a
potential feedstock for biodiesel production.

Author contribution

SS: Conducted experiments, collected data, analysed samples;

Writing – draft, review, and editing. TB: Conducted experiments,
collected data; Writing – draft. DV: Supervision, Writing – draft, review,
Fig. 5. Analysis of ortho and meta cleavage pathway for phenol degradation in and editing, KD: Supervision, Writing – draft, review, and editing,
oleaginous yeast by enzymatic assay. Conceptualization.

8
S. Singh et al. Chemosphere 308 (2022) 136269

Fig. 6. a) Cell viability test of L929 and b) cell viability test of LN299.

Declaration of competing interest as potential biodiesel feedstock. J. Environ. Manag. 252, 109686 https://doi.org/
10.1016/j.jenvman.2019.109686.
Cao, B., Geng, A., Loh, K.C., 2008. Induction of ortho- and meta-cleavage pathways in
The authors declare that they have no known competing financial Pseudomonas in biodegradation of high benzoate concentration: MS identification of
interests or personal relationships that could have appeared to influence catabolic enzymes. Appl. Microbiol. Biotechnol. 81, 99–107. https://doi.org/
the work reported in this paper. 10.1007/s00253-008-1728-3.
Deeba, F., Pruthi, V., Negi, Y.S., 2018a. Aromatic hydrocarbon biodegradation activates
neutral lipid biosynthesis in oleaginous yeast. Bioresour. Technol. 255, 273–280.
Data availability https://doi.org/10.1016/j.biortech.2018.01.096.
Deeba, F., Pruthi, V., Negi, Y.S., 2018b. Aromatic hydrocarbon biodegradation activates
neutral lipid biosynthesis in oleaginous yeast. Bioresour. Technol. 255, 273–280.
Data will be made available on request. https://doi.org/10.1016/j.biortech.2018.01.096.
Garay, L.A., Sitepu, I.R., Cajka, T., Chandra, I., Shi, S., Lin, T., German, J.B., Fiehn, O.,
Acknowledgments Boundy-Mills, K.L., 2016. Eighteen new oleaginous yeast species. J. Ind. Microbiol.
Biotechnol. 43, 887–900. https://doi.org/10.1007/s10295-016-1765-3.
Gargouri, B., Mhiri, N., Karray, F., Aloui, F., Sayadi, S., 2015. Isolation and
The authors would like to thank NIT Rourkela for institute funding characterization of hydrocarbon-degrading yeast strains from petroleum
and Institute’s Central Instrumental Facility (CIF) access. contaminated industrial wastewater. BioMed Res. Int. https://doi.org/10.1155/
2015/929424, 2015.
Hashem, M., Alamri, S.A., Al-Zomyh, S.S.A.A., Alrumman, S.A., 2018. Biodegradation
Appendix A. Supplementary data and detoxification of aliphatic and aromatic hydrocarbons by new yeast strains.
Ecotoxicol. Environ. Saf. 151, 28–34. https://doi.org/10.1016/j.
ecoenv.2017.12.064.
Supplementary data to this article can be found online at https://doi.
Jayapal, M., Jagadeesan, H., Shanmugam, M., Danisha J, P., Murugesan, S., 2018.
org/10.1016/j.chemosphere.2022.136269. Sequential anaerobic-aerobic treatment using plant microbe integrated system for
degradation of azo dyes and their aromatic amines by-products. J. Hazard Mater.
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