To cite this article: Authors name, Article title, (2013), DOI: 10.1007/s11356-013-2040-z 1
Kinetics of phenol biodegradation at high
2
concentration by a metabolically versatile
3
isolated yeast Candida tropicalis PHB5
4
Bikram Basaka, Biswanath Bhuniab, Subhasish Duttaa,
5
Samayita Chakrabortya, Apurba Deya*
6 7
a
8
Gandhi Avenue, Durgapur-713209, India
9
b
10
Department of Biotechnology, National Institute of Technology Durgapur, Mahatma
Department of Bio Engineering, National Institute of Technology Agartala, Tripura-
799055, India
11 12
*Corresponding author
13
Tel No: +91-343-2754027
14
Fax No: +91-343-2547375,
15
Email:
[email protected],
[email protected]
16 17 18
Acknowledgement: This is the accepted version of the manuscript and
19
publishing this article to this public repository we acknowledge Springerlink.
20
This article is protected by copyright and all rights are held exclusively by
21
Springer-Verlag Berlin Heidelberg. "The final publication is available at
22
link.springer.com”. http://dx.doi.org/10.1007/s11356-013-2040-z
23
1
To cite this article: Authors name, Article title, (2013), DOI: 10.1007/s11356-013-2040-z 1
Abstract
2
A highly tolerant phenol degrading yeast strain PHB5 was isolated from wastewater effluent of a coke
3
oven plant and identified as Candida tropicalis based on phylogenetic analysis. Biodegradation
4
experiments with C. tropicalis PHB5 showed that the strain was able to utilize 99.4% of 2400 mg l-1
5
phenol as sole source of carbon and energy within 48 h. Strain PHB5 was also observed to grow on 18
6
various aromatic hydrocarbons. Haldane model was used to fit the exponential growth data and the
7
following kinetic parameters were obtained: µmax=0.3407 h-1, KS=15.81 mg l-1, Ki=169.0 mg l-1
8
(R2=0.9886). The true specific growth rate, calculated from µmax, was 0.2113. A volumetric phenol
9
degradation rate (Vmax) was calculated by fitting the phenol consumption data with Gompertz model
10
and specific degradation rate (q) was calculated from Vmax. The q values were fitted with Haldane
11
model, yielding following parameters: qmax= 0.2766 g g-1 h-1, KS´=2.819 mg l-1, Ki´=2093 (R2=0.8176).
12
The yield factor (YX/S) varied between 0.185 to 0.96 g g-1 for different initial phenol concentrations.
13
Phenol degradation by the strain proceeded through a pathway involving production of intermediates
14
such as catechol and cis,cis-muconic acid which were identified by enzymatic assays and HPLC
15
analysis.
16 17 18
Keywords: Candida tropicalis; Phenol biodegradation; Growth kinetics; Haldane
19
model; Degradation; ortho- pathway
20
2
To cite this article: Authors name, Article title, (2013), DOI: 10.1007/s11356-013-2040-z 1 2
Nomenclature
3
k
fitting parameter for Gompertz equation (h-1)
4
Kd
endogenous coefficient (h-1)
5
KS, Ki
half saturation coefficient and inhibition coefficient applied to growth rate (mg l-1)
6
KS´, Ki´
half saturation coefficient and inhibition coefficient applied to specific degradation rate (mg l-1)
7 8
M
cell maintenance coefficient
9
q
specific degradation rate (g g-1 h-1)
10
qmax
maximum specific degradation rate (g g-1 h-1)
11
Si, S
initial phenol concentration (mg l-1), phenol concentration (mg l-1)
12
S´
consumed phenol (mg l-1)
13
Sm
phenol concentration at which µ=µmax
14
S m´
phenol concentration at which q=qmax
15
tm
time of maximum volumetric phenol degradation rate
16
Vmax
maximum volumetric phenol degradation rate (mg l-1 h-1)
17
X 0, X
initial biomass concentration (mg l-1), biomass concentration (mg l-1)
18
X´
biomass concentration at t
19
YX/S
growth yield coefficient (g g-1)
20
YP/S
product yield coefficient (mg g-1)
21 22
Greek symbols
23
α, β
fitting parameters of the Gompertz equation (mg l-1)
24
µ
specific growth rate (h-1)
25
µmax
maximum specific growth rate in Haldane’s model (h-1)
26
µ*max
true maximum specific growth rate (h-1)
27
3
To cite this article: Authors name, Article title, (2013), DOI: 10.1007/s11356-013-2040-z 1
Introduction
2
Phenol is one of the major organic pollutants in wastewater from various phenol laden
3
industries such as coking, coal refining, petrochemicals, plastic, pharmaceutical industries etc. (Liu et
4
al. 2009; Kumar et al. 2013). Phenol containing effluents from these industries are potentially toxic,
5
and if discharged untreated it can pose critical health hazard to plants as well as other organisms (Yan
6
et al. 2005). Therefore, it is of utmost practical significance to reduce the phenol level in industrial
7
effluents to a tolerant level before its discharge into the environment (Gianfreda et al. 2006).
8
The effectiveness, economical and environmental convenience of biological treatment over
9
the physical and chemical treatment of phenol containing wastewater has been widely studied in the
10
last two decades (Christen et al. 2012; Banerjee and Ghoshal 2010a). However, because of the
11
inhibitory effects of phenol on microorganisms, biological treatment of phenol containing wastewater
12
has been facing up the challenge (Liu et al. 2011; Jiang et al. 2007a). There are reports that describe
13
the degradation of phenol at concentration from 500 mg l-1 to 2000 mg l-1 by different microorganisms
14
(Essam et al. 2010; Christen et al. 2012; Arutchelvan et al. 2006; Wang et al. 2010; Geng et al. 2006).
15
However, concentration of phenol can reach up to 6.8 g l-1 in some industrial wastewater, while
16
according to European-Union recommendation the upper limits of phenol concentration in potable
17
water and wastewater effluent are 0.5 µg l-1 and 0.5 mg l-1, respectively (Christen et al. 2012; Busca et
18
al. 2008). Hence, to remove higher level of phenol from industrial effluents it is especially important to
19
isolate and screen for appropriate microorganism that can tolerate as well as effectively degrade phenol
20
at relatively high concentration.
21
Phenol degrading microorganisms have been studied to show substrate inhibition at high
22
phenol concentration and dynamics of microbial growth on this substrate has been described by
23
different substrate-inhibition models. The growth kinetic parameters such as maximum specific cell
24
growth rate (µmax), substrate-affinity constant (KS) and substrate-inhibition constant (Ki) specify the
25
efficiency of biodegradation process and varies over a wide range depending upon the microorganism
26
and culture conditions (Banerjee and Ghoshal 2010a). Getting better insights about the kinetic behavior
27
of a microorganism growing on high concentration of phenol as sole source of carbon and energy is a
28
prerequisite as each microorganism has its unique growth dynamics which can foretell degradative
29
capability of that particular microorganism. As described in literature, phenol degradation by
30
microorganisms can follow either ortho- or meta- pathway. Ortho- pathway involves production of
31
intermediates such as catechol, cis,cis-muconic acid, while meta- pathway involves ring cleavage of
32
catechol to form 2-hydroxymuconic semialdehyde (2-HMSA) (Banerjee and Ghoshal 2010b).
33
Depending upon the metabolic pathway employed by the organism to degrade the substrate and
4
To cite this article: Authors name, Article title, (2013), DOI: 10.1007/s11356-013-2040-z 1
consequently the intermediates generated, the situation and composition of wastewater also vary.
2
Therefore, it is important to know about the pathway and intermediates of phenol degradation.
3
In this study, we have described phenol degradation by a highly tolerant and metabolically
4
versatile yeast strain Candida tropicalis PHB5. This strain was able to grow on phenol and metabolize
5
this substrate at a maximum concentration of 2400 mg l-1. Different mathematical models were used to
6
determine the growth kinetics parameters (µmax, KS, Ki) and specific phenol degradation rate (q) for this
7
organism grown in batch cultures. Since phenol containing wastewater usually contains other related
8
organic toxicants, such as chlorophenols, m-cresol, and other aromatic hydrocarbons, degradative
9
versatility of this strain was evaluated by detecting growth and degradation in the presence of different
10
other toxicants as sole carbon source. To best of our knowledge, the literature lacks kinetic data for
11
phenol biodegradation at concentration as high as 2400 mg l-1 by a strain which is capable of degrading
12
18 various toxic substances. We have also determined the kinetics of phenol degradation pathway and
13
we have identified some of the metabolites produced during the degradation process.
14
Materials and Methods
15
Culture medium and growth condition
16
An inorganic medium, supplemented with trace element solution, was used for isolation and
17
biodegradation study and its composition was as follows (g l-1): NH4NO3 0.5; MgSO4.7H2O 0.2;
18
K2HPO4 0.5; KH2PO4 0.5; CaCl2.2H2O 0.02. The trace element solution was added to the inorganic
19
medium at 10 ml l-1 and contained (in g l-1) FeSO4,7H2O 0.3; MnSO4, H2O 0.05; CoCl2,6H2O 0.1;
20
Na2MoO4,2H2O 0.034; ZnSO4 0.04 and CuSO4,5H2O 0.05 (Basak et al. 2013b). Phenol at required
21
concentration served as sole source of carbon and energy. Chemicals used were of analytical and
22
HPLC grade and were purchased from Sigma Aldrich (USA), Himedia (India) and Merck (India).
23
Water used for the HPLC analysis was prepared by Ultrapure Water System (Arium® 611UF,
24
Sartorius, Germany). The initial pH of the medium was maintained at 6 and the working volume of
25
medium was 50 ml in 250ml Erlenmeyer flask in all experiments. The microorganism was maintained
26
by routine bimonthly transfer under aseptic conditions to an inorganic medium provided with phenol
27
(at concentration 2400 mg l-1) as sole source of carbon and energy and stored at 40 C after incubation at
28
300 C for 48 h.
29
5
To cite this article: Authors name, Article title, (2013), DOI: 10.1007/s11356-013-2040-z 1 2
Isolation and screening of phenol degrading strain
3
5 ml of sample (wastewater effluent of a coke oven plant in steel industry, Durgapur, India)
4
was added to 250 ml Erlenmeyer flask containing 50 ml of inorganic medium supplemented with 500
5
mg l-1 phenol and incubated at 300 C in New Brunswick Innova® 42 incubator shaker at 120 rpm for
6
120 h. After incubation, 5ml of culture was added to 50 ml of fresh medium with same concentration
7
of phenol. The optical density of the culture broth at 600 nm (OD600) was periodically monitored and
8
once microbial growth was established (OD600 ≈ 0.9 to 1.0) it was considered to be used as inoculum
9
for next inoculation. From then on, the culture was transferred successively to fresh inorganic media
10
using the same growth conditions at each transfer, except that the phenol concentration increased
11
stepwise, varying from 500 mg l-1 to 2000 mg l-1 at 500 mg l-1 interval and then from 2000 mg l-1 to
12
2400 mg l-1 at 100 mg l-1 interval. The transfer was repeated three times at each concentration of
13
phenol. After acclimatization for about 2 months the enriched culture obtained was duly plated on solid
14
inorganic medium supplemented with 2400 mg l-1 phenol. Colonies appeared were purified and each
15
separated colony was further screened for best phenol degradation capability and strain PHB5 was
16
selected for further study.
17
Identification and characterization of strain
18
The pure culture of isolated strain PHB5 was sent to Merck Millipore, India for identification
19
based on sequences of 18S rRNA gene (partial) and internal transcribed spacer (ITS) 1 and 2. The 18S
20
rRNA gene is highly conserved and was used for the phylogenetic analysis of higher taxonomic levels,
21
whereas the highly variable ITS region was used to differentiate the strain at lower taxonomic levels.
22
The genomic DNA was extracted and the 18S rRNA gene and the ITS regions were amplified by PCR
23
using the following primers: forward 5’ GGA AGT AAA AGT CGT AAC AAG G 3’ and reverse: 5’
24
GGT CCG TGT TTC AAG ACG G 3’. The obtained sequence was submitted to NCBI GenBank
25
database
26
(http://www.ncbi.nlm.nih.gov/). A maximum likelihood phylogenetic tree was generated and
27
evolutionary distance bootstrap values were determined by Jukes-cantor model of neighbor-joining
28
method in MEGA 4.
and
a
similarity
search
was
carried
out
using
online
BLAST
program
29
Morphological examination was done using Scanning Electron Microscopy (SEM) according
30
to Yu et al. (Yu et al. 2007). Biochemical tests were performed using HicandidaTM Identification Kit
31
(Himedia, India) according to the manufacturer’s instruction. The Biochemical tests included detection
32
of urease enzyme, and sugar assimilation test. The positive tests were confirmed by color changes in
6
To cite this article: Authors name, Article title, (2013), DOI: 10.1007/s11356-013-2040-z 1
the identification kit. The presence of oxidase was determined using Himedia DD018 Oxidase discs
2
(Himedia, India). Catalase activity was tested according to Zilouei et. al. (Zilouei et al. 2006).
3
Characterization of metabolic versatility
4
The metabolic versatility of the strain was evaluated by inoculating the strain into inorganic
5
medium supplemented with different organic compounds as sole source of carbon and energy. All the
6
compounds (Table 1) were sterilized by membrane filtration technique. Syringe filtration unit was used
7
with 25 mm diameter membrane filter for the filtration. The compounds which were polar and water
8
soluble (such as 4-chlorophenol, 3-chlorophenol, 2,4-dichlorophenol, 2,4,6-trichlorophenol, 4-
9
nitrophenol, m-cresol, o-cresol, catechol, resorcinol) were dissolved in distilled water to prepare stock
10
solution of desired concentration and then sterilized by filtration using 25mm diameter, hydrophilic
11
membrane filter (Durapore 22µ, Catalogue # GVWP02500, Millipore). The compounds which were
12
non-polar and solid (Naphthalene, anthracene, phenanthrene, pyrene) were dissolved in acetone or
13
methanol to prepare stock solution of desired concentration and then sterilized by filtration using
14
25mm diameter, hydrophobic membrane filter (Fluoropore 22µ, Catalogue # FGLP02500, Millipore).
15
The non-polar liquid aromatic compounds were directly filtered through the aforementioned
16
hydrophobic membrane filter. The organic compounds (Table 1) were supplied under sterile condition
17
at concentration of 50-500 mg l-1 in 250 ml Erlenmeyer flasks containing inorganic medium. Each
18
flask was then inoculated with 5 ml of cells (OD600≈0.2) that were grown previously on inorganic
19
medium supplemented with phenol (sole source of carbon and energy), thereby making the final
20
medium volume 50 ml. Prior to inoculation, the cells previously grown were washed twice with
21
distilled water to remove traces of phenol and then resuspended in distilled water to make cell
22
suspension of OD600≈0.2. Flasks that were inoculated, but not supplied with any organic substrate,
23
were taken as negative control. Growth was considered positive if the optical density of the cultures at
24
600 nm was above 0.2. The residual concentrations of these compounds were measured by HPLC and
25
spectrophotometric analysis after incubation of 48 h.
26
Kinetics of cell growth and phenol biodegradation
27
We have presumed that aeration provided oxygen levels at sufficient concentration and does
28
not limit growth. Hence, the influence of oxygen was not considered and it was assumed that the
29
growth and phenol degradation rate of PHB5 strain was only inhibited by substrate concentration at
30
given initial pH, temperature and aeration rate.
31
Kinetics of cell growth in a batch reactor may be described as:
7
To cite this article: Authors name, Article title, (2013), DOI: 10.1007/s11356-013-2040-z 1
dX = µ X − kdX dt
2
(1) (Wang et al. 2010).
Kd can be assumed to be negligible during exponential growth. Hence, Eq. (1) can be written
3
as:
4
dX = µX dt
(2)
5
µ=
1 dX X dt
(3)
6
The Haldane’s kinetic model [Eq. (4)] has been frequently used to describe growth rates of
7
microorganisms on inhibitory substrates such as phenol (Monteiro et al. 2000; Yan et al. 2005; Wang
8
et al. 2010; Kumar et al. 2005).
9
µ=
µ maxS
(4)
KS + S + ( S 2 Ki )
10
Different other substrate inhibition models were also used to determine various kinetic
11
parameters viz. Aiba model, Edward’s model, Yano model etc. The equations of these models used are
12
as follows:
13
Aiba model
14
15
Edward’s model
Yano Model
16 17 18
19
µ max S
µ=
KS + S
µ=
µ=
exp (− S
Ki
)
(5)
µ maxS S + KS + ( S
2
Ki
)(1 + S
(6)
KS
)
µ maxS S + KS + ( S
2
Ki
(7)
)(1 + S ) K
The kinetic parameters of growth of the strain were calculated from experimental data from Eq. (8). The substrate utilization kinetics is given by:
dS 1 dX 1 dP =− − − MX X dt Y S dt Y P S dt
(8)
20
A carbon substrate is used to form cell material and metabolic products as well as used for
21
maintenance of the cell. However, in the present scenario, the substrate used for product formation and
22
cell maintenance is assumed to be negligible. Similar assumption was also made by Kumar et al.
23
(Kumar et al. 2005). Therefore, Eq. (8) can be reduced to:
8
To cite this article: Authors name, Article title, (2013), DOI: 10.1007/s11356-013-2040-z 1
dS 1 dX =− dt Y X S dt
2
now, YX/S is the ratio of cell mass growth and substrate concentration used for cell growth. YX/S can be
3
expressed as:
4
Y
5
YX/S was calculated from experimental data using the Eq. (11).
6
Y
X
X
S
S
=−
=
(9)
dX dS
(10)
X − X0 S0 − S
(11)
7
In most of the works reported, there is confusion between µmax which is one of the fitting
8
parameters and derived from kinetic models for growth, mistakenly considered as maximum specific
9
growth rate, and the true specific growth rate (µ*max) (Christen et al. 2012; Shareefdeen et al. 1993).
10
When dµ/dS=0, µmax occurs at:
11
Sm = KSKi
12
Replacing Sm in Eq. (4), we obtain:
13
µ * max =
14
(12)
µ max
(13) (Christen et al. 2012)
1 + 2 K SK i
To calculate the specific degradation rate (q), the phenol consumption data were fitted into the
15
integrated Gompertz equation (Acuna et al. 1999; Christen et al. 2012).
16
S´ = α exp [-β exp (-ktm)]
17
The maximum volumetric degradation rate is calculated as follows:
18
Vmax= 0.368 α k
19
For each Si, the corresponding time (tm) for Vmax is calculated as:
20
tm =
21
For a given Si, the corresponding X´ for tm is directly determined from the growth curve where growth
22
data was fitted to Gompertz model (Fig. 1a). The specific degradation rate (q) is then calculated as:
23
q=
(14)
(15)
lnβ k
(16)
Vmax X'
(17)
24
Haldane’s model was used to calculate q, KS´, Ki´, Sm´, and qmax and true q*max was then
25
calculated according to Eqs. (12) and (13). GraphPad Prism 5 software, based on Windows 7, was used
26
to run all the regression analysis.
9
To cite this article: Authors name, Article title, (2013), DOI: 10.1007/s11356-013-2040-z 1
Inocula of 5 ml were taken from cultures of late exponential growth and transferred into 250
2
ml Erlenmeyer flask containing 45 ml of sterilized inorganic media supplemented with 500-2400 mg l-
3
1
4
120 rpm. Samples were withdrawn at designated intervals in aseptic condition to determine cell growth
5
and residual phenol concentration. All the experiments were performed in triplicate.
6
Analytical procedures
phenol. All flasks were then incubated at 300 C in New Brunswick Innova® 42 incubator shaker at
7
Cell concentration was measured as cell dry weight method and expressed in g l-1. 4 ml of the
8
culture samples were taken in 15 ml centrifuge tube and centrifuged at 15,000×g for 10 min at 40 C.
9
The cell pellets harvested were washed with distilled water and dried at 1050 C to a constant weight for
10
48 h in a hot air oven and was used for growth study. The supernatant was filtered through 0.22µm
11
membrane filter (Millipore, India) and the filtrate was analyzed for determination of residual phenol
12
concentration by HPLC (WatersTM 600, USA) equipped with UV/Visible detector and a C18 hypersil
13
column (4.6 mm x 250 mm; particle size 5 µm) with a mobile phase of acetonitrile (70%): water (30%)
14
at a flow rate of 1 ml/min. An aliquot of 20 µl of the filtrate was injected and analyzed using the
15
UV/Visible detector (WatersTM 2489) at wavelength of 270 nm (λmax for phenol≈270 nm).
16
Determination of intermediates of phenol degradation pathway
17
Enzymatic assay and HPLC analysis were carried out to determine the possible phenol
18
metabolic pathway of C. tropicalis PHB5. To detect intermediates of the pathway by enzymatic assay,
19
the cultures were taken periodically and centrifuged at 15,000×g and 40 C. The cell pellet was washed
20
with 50 mM phosphate buffer (KH2PO4:K2HPO4, pH 7.0) and resuspended in the same buffer. Then
21
the cells were disrupted using a sonicator (Sartorius LABSONIC® M, Germany) to prepare the crude
22
extract. The crude extract was centrifuged at 15,000×g and 40 C to remove the cell debris. The reaction
23
mixture of catechol was prepared in same phosphate buffer according to Banerjee et al. (Banerjee and
24
Ghoshal 2010b). The cell extract was added to it and formation of the reaction products of catechol 2,
25
3-dioxygenase and catechol 1, 2-dioxygenase (HMSA and cis, cis-muconic acid) were detected
26
spectrophotometrically (Rayleigh 2601, China) at 375 nm and 260 nm respectively (Neumann et al.
27
2004). In the cell free supernatants the intermediate products of degradation pathway were also
28
analyzed and quantified by comparing with standards using HPLC as described in our recent work
29
(Basak et al. 2013a).
30
Results and discussion 10
To cite this article: Authors name, Article title, (2013), DOI: 10.1007/s11356-013-2040-z 1
Isolation and characterization of phenol degrading strain
2
A yeast strain PHB5 was successfully isolated from effluent of a coking wastewater treatment
3
plant. The 18S rRNA and ITS region sequences of PHB5 strain were found to be 1103 bp and were
4
submitted to NCBI Genbank database under accession number JN542555. A maximum likelihood
5
phylogenetic tree was generated (see supplementary data). The strain PHB5 was found to be
6
phylogenetically closely related to Candida tropicalis strain KB-41 (GenBank accession number:
7
FJ947158) and Candida tropicalis strain XJ-5 (GenBank accession number: JQ686913), showing
8
>99% sequence identity. Therefore, the isolated yeast was designated as Candida tropicalis PHB5.
9
Scanning electron micrographs revealed strain PHB5 was around 4.5 µm in length and was
10
present singly or in cluster (see supplementary data). The cells were ovate or elliptical in shape and the
11
colonies appeared creamy-white and non-glistening with rough edge. The complete details of
12
biochemical and physiological characteristics are given in Table 1. C. tropicalis and other species of
13
Candida are well known for its ability to degrade phenol and chlorophenols at high concentration (Yan
14
et al. 2005; Basak et al. 2013a; Jiang et al. 2007b; Jiang et al. 2007c; Tsai et al. 2005). Although the
15
ability of C. tropicalis to tolerate and mineralize different other aromatic compounds like benzene,
16
toluene, ethylbenzene, xylene (BTEX), Polycyclic Aromatic Hydrocarbons (PAHs), and substituted
17
aromatics (chlorophenols, nitrophenols etc.) have been reported separately (Jiang et al. 2010; Ahmed
18
and Song 2011; Das and Chandran 2011; Krastanov et al. 2013), to best of our knowledge strain PHB5
19
is the first isolate to be reported as capable of metabolizing phenol as well as 18 different aromatic
20
compounds (including phenol) (Table 1). This metabolic versatility makes C. tropicalis PHB5 an
21
excellent candidate for the bio-treatment of industrial wastewater contaminated with different types of
22
pollutants.
23
Kinetic studies of growth and phenol biodegradation
24
Growth and biodegradation studies were carried out under parameters that were optimized in
25
our previous work (Basak et al. 2013b). Time courses of growth and phenol consumption were plotted
26
in Fig. 1a. Gompertz sigmoidal function was used to fit the growth and phenol consumption data of the
27
strain (Zwietering et al. 1990). Under optimized condition, C. tropicalis PHB5 was able to grow on
28
2400 mg l-1 phenol and could metabolize 99.4% of this substrate within 48 h. Fig. 1a shows that there
29
was lag phase of about 18 h, indicating substrate inhibition on the cells. However, phenol was depleted
30
quickly as cell growth increased at the log phase. This result is in conjunction with the previous reports
31
of Yan et al. where degradation of 2000 mg l-1 phenol within 66 h with a lag phase of about 24 h (Yan
11
To cite this article: Authors name, Article title, (2013), DOI: 10.1007/s11356-013-2040-z 1
et al. 2005) and 2600 mg l-1 phenol within 70 h with lag phase of 16 h (Jiang et al. 2007b) have been
2
reported.
3
To study the effect of Si on µ, µ for each Si of phenol was calculated using Eq. (3) while X was
4
obtained at different interval of incubation period. The Haldane’s model fitted well with the
5
experimental values of µ obtained for each Si (Fig. 1b), since a correlation coefficient (R2) of 0.9886
6
was found. The values of µmax, KS and Ki were found to be 0.3407 h-1, 15.81 mg l-1 and 169 mg l-1,
7
respectively. Sm of 51.69 mg l-1 and µ*max of 0.2113 h-1 were calculated with Eqs. (12) and (13)
8
respectively. The values of Sm and µ*max found match with those observed in Fig 1b. Since, Sm is the
9
concentration of phenol where µ*max occurs, it can be considered as the value below which growth is
10
limited by substrate concentration and above which growth is increasingly inhibited by higher
11
substrate concentration. Several substrate inhibition kinetic models were used to fit the experimental
12
data and compared in this work (Eq. 5, 6, 7) (Table 2). The (R2) values obtained with different models
13
suggest that all model fitted the experimental data well (Table 2). However, we selected Haldane
14
model to compare the kinetic data of the studied strain with that of different organisms found in
15
literature. When we calculated the value of µmax for strain PHB5 and compared with values reported in
16
literature, we observed that the value of µmax was fairly high among those obtained for strains capable
17
of degrading phenol above concentration of 2000 mg l-1 (Table 3). When we compared Sm and µmax
18
value found with the studied strain with that of reported in literature, it was found that these values
19
were in quite lower and upper middle range respectively. However, this shortcoming is circumvented
20
by the strain’s ability to tolerate and degrade phenol at high concentration as well as its ability to
21
degrade different types of toxicants.
22
In most of the articles in literature, there is a discrepancy between the graphical
23
determinations of µmax and the calculated value of µmax (Christen et al. 2012; Khleifat 2006; Yan et al.
24
2005). Therefore, we followed the difference between µmax and µ*max reported by Shareefdeen et al.
25
(Shareefdeen et al. 1993) to find out the true maximum specific growth rate (µ*max) (Eq. (13)). µ*max
26
values of strain PHB5 and other organisms are presented in Table 3. We found that the values reported
27
for µmax were overestimated with respect to µ*max. In some cases, it has been found to be overestimated
28
by 10-fold (Table 3) (Geng et al. 2006). We have also found an overestimation of Sm when we
29
calculated Sm (Table 3) according to Eq. (12) using KS and Ki given by Khleifat (Khleifat 2006) and
30
determined it graphically from the plot presented in that article (Khleifat 2006).
31
The KS value obtained in this study was comparatively larger than that of previously reported
32
for C. tropicalis (Yan et al. 2005; Jiang et al. 2007b), indicating that strain PHB5 was half saturated by
33
phenol at relatively higher concentrations. Although the Ki value for this strain is relatively mediocre,
34
this combination of KS and Ki suggests that C. tropicalis PHB5 is able to grow on phenol containing
12
To cite this article: Authors name, Article title, (2013), DOI: 10.1007/s11356-013-2040-z 1
wastewaters within a wide range of concentrations in comparison to other microorganisms grown on
2
phenol.
3
In order to calculate the specific degradation rate (q) for each Si, the phenol consumption data
4
for each Si was fitted into Gompertz model (R2> 0.98). Eq. (15) and (16) were applied to determine
5
parameters (Vmax, tm) used for calculating q (Eq. (17)) by using different coefficients obtained (α, β, k
6
Eq. (14)) when Gompertz model was used for modelizing the phenol consumption data for each Si
7
(Table 4). Then, for each Si, X’ (Table 4) was obtained from the corresponding tm in Fig 1a. Adav et al.
8
(Adav et al. 2007) reported that Vmax was independent of Si probably because they used aerobic
9
granules for phenol degradation where microbial population remained constant throughout the process.
10
However, in our study we found Vmax to be a function of Si, since biomass also increased with
11
increasing substrate concentration up to the maximum level tolerated and phenol degradation is
12
accompanied by growth of the organism. This observation is also in accordance with other reports
13
(Christen et al. 2012).
14
The values of q, obtained for each Si, were fitted into Haldane model (R2 = 0.8176) and the
15
pattern of q vs. Si showed substrate-inhibition characteristic (Fig. 1b). As for µ, q was also restrained
16
by high concentrations of phenol and thus the relationship between q and Si was also satisfactorily
17
described by different substrate inhibition models (Table 2), as reported in other works (Yan et al.
18
2005; Bai et al. 2007; Jia et al. 2006). A qmax value of 0.2766 g g-1 h-1 was found using Haldane model
19
and true specific degradation rate (q*max) of 0.257 g g-1 h-1 was calculated according to Eq. (13).
20
Fitting parameters (KS, Ki, KS´ and Ki´) for different models determined are presented in Table 2. Sm´
21
for q was also calculated using Eq. (12) and its value was found to be 76.81 mg l-1. Sm and Sm´ values as
22
well as Fig 1b show that µmax and qmax occurred at low initial phenol concentrations (Si) and upon
23
further increase of Si, µmax and qmax values significantly decreased. As for µmax in case of Haldane
24
model, although qmax is also quite low, inhibition coefficient (Ki´) value for phenol degradation rate
25
(Table 2) is relatively higher which implies that degradation rate can be less inhibited at higher
26
concentrations. Thus, despite the display of a mediocre µmax and qmax values, Ki´ foretells the ability of
27
C. tropicalis PHB5 in terms of phenol degradation in long range of concentrations.
28
The yield coefficient (YX/S) was estimated by plotting the biomass yield against phenol
29
consumed (S´), as previously estimated by Eq. (11). An YX/S varying between 0.185 g g-1 and 0.96 g g-1
30
was found for different phenol concentration up to 2000 mg l-1 (Fig. 3). It is evident from Fig. 3 that
31
YX/S value was maximal at low Si and minimal at highest Si used in the range, with a considerable
32
decrease noticed beyond Si of 700 mg l-1. This type of phenomenon of decreasing YX/S with increasing
33
phenol concentration (Si) in the inhibitory range was also reported (Christen et al. 2012; Li et al. 2010)
34
and can be attributed to the fact that beyond inhibitory substrate concentration energy required by the
13
To cite this article: Authors name, Article title, (2013), DOI: 10.1007/s11356-013-2040-z 1
organisms to overcome the inhibitory effects of phenol is maximum. The amount of substrate
2
consumed and converted to energy for cell maintenance increases as µ decreases, while the amount of
3
substrate assimilated into biomass decreases as µ decreases with increasing Si (Wang et al. 2010). The
4
maximum value of YX/S (0.96) was found at very low phenol concentration (20 mg l-1) (Fig. 3) and this
5
relatively high value of YX/S may be attributed to the higher metabolic efficiency of C. tropicalis PHB5
6
(Li et al. 2010; Adav et al. 2007). This finding is consistent with Adav et al. (Adav et al. 2007) and
7
Wang and Loh (Wang and Loh 1999) who have also reported high YX/S values of 0.823 g g-1 and 0.94 g
8
g-1, respectively.
9
Intermediates of phenol degradation pathway
10
To determine the possible phenol degradation pathway involved in C. tropicalis PHB5
11
enzymatic assays were carried out for detection of products of catechol 2, 3-dioxygenase and catechol
12
1, 2-dioxygenase. The absence of catechol 2, 3-dioxygenase activity was confirmed, since absorbance
13
at 375 nm due to the formation of 2-HMSA was absent. This finding suggested that C. tropicalis PHB5
14
did not metabolize phenol through meta-cleavage pathway (Banerjee and Ghoshal 2010b) However,
15
the presence of absorbance at 260 nm indicated the possible formation of cis,cis-muconic acid due to
16
the activity of catechol 1, 2-dioxygenase and ensuing ortho-cleavage pathway. During phenol
17
degradation by C. tropicalis PHB5, HPLC analysis of culture supernatant taken from batch system
18
containing various concentration of phenol revealed the presence of catechol and cis,cis-muconic acid
19
as intermediates. These two major metabolites produced from phenol were identified on the basis of
20
their HPLC retention time (see supplementary data) when compared with that of the standards as
21
described in our previous work (Basak et al. 2013a). HPLC elution profile of these two intermediates
22
of phenol degradative pathway recorded for sample taken after 30 h incubation of the medium. In
23
addition, the formation of another product was also observed in HPLC chromatogram (see
24
supplementary data). Although, this product (peak 01 in HPLC chromatogram) was not identified, it
25
could be assumed to be any other intermediates of the ortho-cleavage pathway such as muconolactone
26
or 3-oxoadipic acid. Fig. 3 represents the kinetics of products accumulation and decomposition during
27
growth of C. tropicalis PHB5 on phenol. It can be seen in Fig. 3, catechol was formed at the earlier
28
stage of reaction, followed by formation of cis,cis-muconic acid peaked at relatively later stage of the
29
biodegradation as phenol concentration decreased. Similar trends were also found in our previous
30
study (Basak et al. 2013a) with 4-chlorophenol and by Chung et al. (Chung et al. 2003) working with
31
phenol-Pseudomonas putida system.
32
The results of enzymatic assay and HPLC revealed that C. tropicalis PHB5 could metabolize
33
phenol via ortho-cleavage pathway. Cis,cis-muconic acid is the gateway intermediate for this pathway
34
which is converted to muconolactone and subsequently leads to the formation of succinyl-CoA and
14
To cite this article: Authors name, Article title, (2013), DOI: 10.1007/s11356-013-2040-z 1
acetyl-CoA. This postulated ortho-cleavage pathway for phenol degradation by C. tropicalis PHB5
2
could be similar to one reported on other C. tropicalis strains by Bastos et al. (Bastos et al. 2000).
3
Further investigation on characterization of other intermediate metabolites may provide real insight.
4
Conclusion
5
The present study mainly focused on the kinetics of growth and biodegradation and
6
determination of phenol metabolic pathway employed by the isolated Candida tropicalis PHB5. Strain
7
PHB5 was capable of growing on various monocyclic and polycyclic aromatic hydrocarbons. In
8
particular, it was able to tolerate and consume phenol as sole source of carbon and energy up to
9
concentration of 2400 mg l-1 and its growth kinetics using phenol as a sole carbon source was well
10
characterized by Haldane model (µmax=0.3407 h-1, KS=15.81 mg l-1, Ki=169.0 mg l-1, R2=0.9886). Most
11
reports in the literatures wrongly interpreted the fitting parameter of Haldane model µmax as true
12
specific growth rate (µ*max). This confusion overestimates the µmax, while µ*max must be calculated as
13
described in section 2.5 of this paper. This is of practical significance if subsequent application of a
14
strain in bioremediation of a toxicant is intended. The relationship between specific degradation rate
15
(q) and initial phenol concentration (Si) was also described by Haldane model (qmax= 0.2766 g g-1 h-1,
16
KS´=2.819 mg l-1, Ki´=2093 mg l-1, R2=0.8176) and found to be subject to substrate inhibition.
17
Depending on degradation pathway of a toxicant and consequent production of intermediates,
18
the circumstances and composition of wastewater being treated can also vary. Therefore, the present
19
study revealed the production of a key intermediate of ortho-cleavage pathway, indicating involvement
20
of this pathway in phenol biodegradation.
21
Finally, the relatively high values of various kinetic parameters indicate the capability of C.
22
tropicalis PHB5 to degrade phenol at relatively high concentration and possibility of potential
23
application of the whole cell for bioremediation of phenol contaminated wastewater. The ability of this
24
strain to utilize various other aromatic compounds confirms its potential as versatile and efficient
25
microorganism for application when aromatic mixtures have to be treated.
26
References
27
Acuna ME, Perez F, Auria R, Revah S (1999) Microbiological and kinetic aspects of a biofilter for the
28
removal
29
doi:10.1002/(SICI)1097-0290(19990420)63:23.0.CO;2-G [pii]
of
toluene
from
waste
gases.
15
Biotechnol
Bioeng
63
(2):175-184.
To cite this article: Authors name, Article title, (2013), DOI: 10.1007/s11356-013-2040-z 1 2 3 4
Adav SS, Chen MY, Lee DJ, Ren NQ (2007) Degradation of phenol by aerobic granules and isolated yeast Candida tropicalis. Biotechnol Bioeng 96 (5):844-852. doi:10.1002/bit.21148 Ahmed Z, Song J (2011) Removal of gaseous toluene using immobilized Candida tropicalis in a fluidized bed bioreactor. 3 Biotech 1 (2):111-116. doi:10.1007/s13205-011-0015-7 15 [pii]
5
Arutchelvan V, Kanakasabai V, Elangovan R, Nagarajan S, Muralikrishnan V (2006) Kinetics of high
6
strength phenol degradation using Bacillus brevis. J Hazard Mater 129 (1-3):216-222.
7
doi:S0304-3894(05)00525-X [pii] 10.1016/j.jhazmat.2005.08.040
8 9 10
Bai J, Wen J-P, Li H-M, Jiang Y (2007) Kinetic modeling of growth and biodegradation of phenol and m-cresol using Alcaligenes faecalis. Process Biochem 42:510-517 Banerjee A, Ghoshal AK (2010a) Isolation and characterization of hyper phenol tolerant Bacillus sp.
11
from
12
doi:10.1016/j.jhazmat.2009.11.002 S0304-3894(09)01780-4 [pii]
oil
refinery
and
exploration
sites.
J
Hazard
Mater
176
(1-3):85-91.
13
Banerjee A, Ghoshal AK (2010b) Phenol degradation by Bacillus cereus: pathway and kinetic
14
modeling. Bioresour Technol 101 (14):5501-5507. doi:10.1016/j.biortech.2010.02.018S0960-
15
8524(10)00289-0 [pii]
16
Basak B, Bhunia B, Dutta S, Dey A (2013a) Enhanced biodegradation of 4-chlorophenol by Candida
17
tropicalis PHB5 via optimization of physicochemical parameters using Taguchi orthogonal
18
array approach. Int Biodeterior Biodegrad 78:17-23
19
Basak B, Bhunia B, Mukherjee S, Dey A (2013b) Optimization of physicochemical parameters for
20
phenol biodegradation by Candida tropicalis PHB5 using Taguchi Methodology. Desalin
21
Water Treat :DOI:10.1080/19443994.19442013.19770638
22
Bastos AER, Tornisielo VL, Nozawa SR, Trevors JT, Rossi A (2000) Phenol metabolism by two
23
microorganisms isolated from Amazonian forest soil samples. J Ind Microbiol Biotechnol
24
24:403-409
25
Busca G, Berardinelli S, Resini C, Arrighi L (2008) Technologies for the removal of phenol from fluid
26
streams: a short review of recent developments. J Hazard Mater 160 (2-3):265-288.
27
doi:10.1016/j.jhazmat.2008.03.045 S0304-3894(08)00417-2 [pii]
28
Christen P, Vega A, Casalot L, Simon G, Auria R (2012) Kinetics of aerobic phenol biodegradation by
29
the acidophilic and hyperthermophilic archaeon Sulfolobus solfataricus 98/2. Biochem Eng J
30
62:56-61
31
Chung T-P, Tseng H-Y, Juang R-S (2003) Mass transfer effect and intermediate detection for phenol
32
degradation in immobilized Pseudomonas putida systems. Process Biochemistry 38:1497–
33
1507
16
To cite this article: Authors name, Article title, (2013), DOI: 10.1007/s11356-013-2040-z 1 2
Das N, Chandran P (2011) Microbial degradation of petroleum hydrocarbon contaminants: an overview. Biotechnol Res Int 2011:941810. doi:10.4061/2011/941810
3
Essam T, Amin MA, El Tayeb O, Mattiasson B, Guieysse B (2010) Kinetics and metabolic versatility
4
of highly tolerant phenol degrading Alcaligenes strain TW1. J Hazard Mater 173 (1-3):783-
5
788. doi:10.1016/j.jhazmat.2009.09.006 S0304-3894(09)01452-6 [pii]
6
Geng A, Soh AE, Lim CJ, Loke LC (2006) Isolation and characterization of a phenol-degrading
7
bacterium from an industrial activated sludge. Appl Microbiol Biotechnol 71 (5):728-735.
8
doi:10.1007/s00253-005-0199-z
9 10 11 12 13 14
Gianfreda L, Iamarino G, Scelza R, Rao MA (2006) Oxidative catalysts for the transformation of phenolic pollutants: a brief review. Biocatal Biotransform 24:177-187 Jia X, Wen J, Jiang Y, Bai J, Cheng X, Zheng Y (2006) Modeling for batch phenol biodegradation with immobilized Alcaligenes faecalis. AIChE J 52:1294–1303 Jiang Y, Cai X, Wu D, Ren N (2010) Biodegradation of phenol and m-cresol by mutated Candida tropicalis. J Environ Sci (China) 22 (4):621-626
15
Jiang Y, Wen J, Bai J, Jia X, Hu Z (2007a) Biodegradation of phenol at high initial concentration by
16
Alcaligenes faecalis. J Hazard Mater 147 (1-2):672-676. doi:S0304-3894(07)00749-2 [pii]
17
10.1016/j.jhazmat.2007.05.031
18
Jiang Y, Wen J, Jia X, Caiyin Q, Hu Z (2007b) Mutation of Candida tropicalis by irradiation with a
19
He-Ne laser to increase its ability to degrade phenol. Appl Environ Microbiol 73 (1):226-231.
20
doi:AEM.00677-06 [pii] 10.1128/AEM.00677-06
21
Jiang Y, Wen J, Lan L, Hu Z (2007c) Biodegradation of phenol and 4-chlorophenol by the yeast
22
Candida tropicalis. Biodegradation 18 (6):719-729. doi:10.1007/s10532-007-9100-3
23
Khleifat KM (2006) Biodegradation of phenol by Ewingella americana: Effect of carbon starvation
24 25 26
and some growth conditions. Process Biochem 41:2010-2016 Krastanov A, Alexieva Z, Yemendzhiev H (2013) Microbial degradation of phenol and phenolic derivatives. Eng Life Sci 13:76-87
27
Kumar A, Bhunia B, Dasgupta D, Mandal T, Dey A, Datta S, Bhattacharya P (2013) Optimization of
28
culture condition for growth and phenol degradation by Alcaligenes faecalis JF339228 using
29
Taguchi Methodology. Desalin Water Treat 51:3153-3163
30 31
Kumar A, Kumar S, Kumar S (2005) Biodegradation kinetics of phenol and catechol using Pseudomonas putida MTCC 1194. Biochem Eng J 22:151-159
17
To cite this article: Authors name, Article title, (2013), DOI: 10.1007/s11356-013-2040-z 1
Li Y, Li J, Wang C, Wang P (2010) Growth kinetics and phenol biodegradation of psychrotrophic
2
Pseudomonas
3
doi:10.1016/j.biortech.2010.03.083 S0960-8524(10)00560-2 [pii]
4 5 6 7 8 9
putida
LY1.
Bioresour
Technol
101
(17):6740-6744.
Liu H, Yu QJ, Wang G, Ye F, Cong Y (2011) Biodegradation of phenol at high concentration by a novel yeast Trichosporon montevideense PHE1. Process Biochem 46:1678-1681 Liu YJ, Zhang AN, Wang XC (2009) Biodegradation of phenol by using free and immobilized cells of Acinetobacter sp. XA05 and Sphingomonas sp. FG03. Biochem Eng J 44:187-192 Monteiro AA, Boaventura RA, Rodrigues AE (2000) Phenol biodegradation by Pseudomonas putida DSM 548 in a batch reactor. Biochem Eng J 6 (1):45-49. doi:S1369703X00000723 [pii]
10
Neumann G, Teras R, Monson L, Kivisaar M, Schauer F, Heipieper HJ (2004) Simultaneous
11
degradation of atrazine and phenol by Pseudomonas sp. strain ADP: effects of toxicity and
12
adaptation. Appl Environ Microbiol 70 (4):1907-1912
13 14 15
Shareefdeen Z, Baltzis BC, Oh YS, Bartha R (1993) Biofiltration of methanol vapor. Biotechnol Bioeng 41 (5):512-524. doi:10.1002/bit.260410503 Tsai SC, Tsai LD, Li YK (2005) An isolated Candida albicans TL3 capable of degrading phenol at
16
large
17
doi:JST.JSTAGE/bbb/69.2358 [pii]
concentration.
Biosci
Biotechnol
Biochem
69
(12):2358-2367.
18
Wang L, Li Y, Yu P, Xie Z, Luo Y, Lin Y (2010) Biodegradation of phenol at high concentration by a
19
novel fungal strain Paecilomyces variotii JH6. J Hazard Mater 183 (1-3):366-371.
20
doi:10.1016/j.jhazmat.2010.07.033 S0304-3894(10)00918-0 [pii]
21 22 23 24
Wang S-J, Loh K-C (1999) Modeling the role of metabolic intermediates in kinetics of phenol biodegradation. Enzyme Microb Technol 25:177-184 Yan J, Jianping W, Hongmei L, Suliang Y, Zongding H (2005) The biodegradation of phenol at high initial concentration by the yeast Candida tropicalis. Biochem Eng J 24:243-247
25
Yu J, Zhang X, Tan T (2007) An novel immobilization method of Saccharomyces cerevisiae to
26
sorghum bagasse for ethanol production. J Biotechnol 129 (3):415-420. doi:S0168-
27
1656(07)00159-9 [pii]10.1016/j.jbiotec.2007.01.039
28
Zilouei H, Soares A, Murto M, Guieysse B, Mattiasson B (2006) Influence of temperature on process
29
efficiency and microbial community response during the biological removal of chlorophenols
30
in a packed-bed bioreactor. Appl Microbiol Biotechnol 72 (3):591-599. doi:10.1007/s00253-
31
005-0296-z
18
To cite this article: Authors name, Article title, (2013), DOI: 10.1007/s11356-013-2040-z 1 2
Zwietering MH, Jongenburger I, Rombouts FM, van 't Riet K (1990) Modeling of the bacterial growth curve. Appl Environ Microbiol 56 (6):1875-1881
3 4 5
Figure legends:
6
Fig. 1. (a) Time course of growth and phenol consumption by C. tropicalis PHB5 at initial phenol
7
concentration 2400 g l-1, (b) Relationships between specific growth rates (µ) and initial substrate
8
concentration (Si) and between specific degradation rates (q) and Si. Haldane model was fitted to the
9
experimental values of µ and q.
10 11
Fig. 2. Relationship between growth yield (YX/S) and initial phenol concentrations (Si).
12 13
Fig. 4. Time course of intermediate metabolites accumulation and decomposition during phenol
14
degradation by C. tropicalis PHB5.
15
19
To cite this article: Authors name, Article title, (2013), DOI: 10.1007/s11356-013-2040-z
1 2
Fig. 1. (a) Time course of growth and phenol consumption by C. tropicalis PHB5 at initial phenol
3
concentration 2400 g l-1, (b) Relationships between specific growth rates (µ) and initial substrate
4
concentration (Si) and between specific degradation rates (q) and Si. Haldane model was fitted to the
5
experimental values of µ and q.
6 20
To cite this article: Authors name, Article title, (2013), DOI: 10.1007/s11356-013-2040-z
1 2
Fig. 2. Relationship between growth yield (YX/S) and initial phenol concentrations (Si).
3
4 5
Fig. 4. Time course of intermediate metabolites accumulation and decomposition during phenol
6
degradation by C. tropicalis PHB5.
7
21
To cite this article: Authors name, Article title, (2013), DOI: 10.1007/s11356-013-2040-z 1
Table 1
2
Physiological characteristics and substrate screening test of Candida tropicalis PHB5. Characteristics
Enzyme production
Sugars assimilation
Result/Growth Urease
-
Oxidase
+
Catalase
+
Glucose
+
Melibiose
+
Lactose
-
Maltose
+
Sucrose
+
Galactose
+
Cellobiose
+
Inositol
+
Xylose
+
Dulcitol
+
Raffinose
-
Trehalose
+ +
99.81
-1
+
94.13
2,4-Dichlorophenol (50 mg l )
+
86.83
-1
+
72.10
+
81.04
+
99.44
+
83.63
+
84.18
Anthracene (250 mg l )
+
63.99
-1
+
79.43
+
89.31
+
99.54
+
99.44
+
99.06
Xylene (200 mg l )
+
99.43
-1
+
99.95
-
0.12
+
99.34
3-Chlorophenol (250 mg l ) -1
2,4,6-Trichlorophenol (50 mg l ) -1
4-Nitrophenol (50 mg l ) -1
m-Cresol (500 mg l ) -1
o-Cresol (500 mg l ) -1
Naphthalene (250 mg l ) -1
Phenanthrene (100 mg l ) -1
Pyrene (50 mg l ) -1
Benzene (200 mg l ) -1
Toluene (200 mg l ) -1
Ethylbenzene (200 mg l ) -1
Catechol (500 mg l ) -1
Resorcinol (50 mg l ) -1
n-Hexadecane (100 mg l )
3 4
a
NA
-1
4-Chlorophenol (500 mg l )
Substrate screening
Percentage of substrates degradeda
after incubation of 48 h.
22
KS + S
µ max S
µ=
Ki
2
Ki
)
Ki
)
K
)(1 + S )
µ maxS
)(1 + S
S + KS + ( S
2
µ maxS KS
exp (− S
KS + S + ( S 2 Ki )
µ max S
Edward model:
µ=
µ=
S + KS + ( S
Yano model:
µ=
Aiba model:
Haldane model:
Mathematical Model
0.2364
0.1953
0.2254
0.3407
µmax
4.890
0.5935
0.0015
15.81
23
644.7
5248
3.343
169.0
Ki
0.2013
0.1948
0.2162
0.2113
µ*max
0.9949
0.9883
0.9846
0.9886
R2 qmax
0.2671
0.2454
0.27
0.2766
2.165
0.6489
0.0003
2.819
KS’
3369
106
7.28 X
2.357
2093
Ki’
0.2542
0.2452
0.2636
0.257
q*max
specific degradation rate (q)
specific growth rate (µ)
KS
Parameters obtained for
Parameters obtained for
Kinetic parameters for phenol biodegradation by C. tropicalis PHB5 obtained by different models.
Table 2
To cite this article: Authors name, Article title, (2013), DOI: 10.1007/s11356-013-2040-z
0.8399
0.8483
0.8403
0.8176
R2
< 2000 1000 100 745 2000 1800 1750 1000 2400 2600 2400
Bacillus cereus MTCC 9817
Ewingella americana
Pseudomonas putida DSM 548
Sulfolobus solfataricus 98/2
Candida tropicalis
Paecilomyces variotii JH6
Bacillus brevis
Acinetobacter sp.
Candida albicans TL3
Candida tropicalis mutated strain CTM 2
Candida tropicalis PHB5
c
b
Calculated according to Eq. (13)
Calculated according to Eq. (12)
Maximum value.
1600
Alcaligenes faecalis
a
Sia (mg l-1)
Microorganisms
6.7 15.81
0.3407
160
1167
2.2-29.3
130.4
11.7
77.7
6.19
5.16
129.4
2.22
24
KS (mg l-1)
0.54
0.66
0.28
0.026-0.078
0.312
0.48
0.094
0.436
0.29
0.4396
0.15
µmax (h-1)
169
234
3760
58.5
868-2434
200
207.9
319.4
54.1
1033.7
637.8
245.37
Ki (mg l-1)
0.467
775.62
51.69
0.2113
0.403
0.028
261.28
39.59
Arutchelvan et al. (2006)
0.0230.063
43.69-267.05
This study
Jiang et al. (2007)
Tsai et al. (2005)
Liu et al. (2009)
Wang et al. (2010)
Yan et al. (2005)
Christen et al. (2012)
Monteiro et al. (2000)
Khleifat (2006)
Banerjee et al. (2010b)
Jiang et al. (2007a)
References
0.119315
0.325
0.047
0.26
0.254
0.2312
0.126
µ*maxc (h-1)
161.493
49.31
157.5
18.3
73.0
287.28
23.33
Smb (mg l-1)
Different parameters (µmax, KS, Ki) for Haldane equation and calculated parameters (Sm, µ*max) of various microorganisms grown on phenol.
Table 3
To cite this article: Authors name, Article title, (2013), DOI: 10.1007/s11356-013-2040-z
c
b
a
29.82 51.91 42.17 59.47 68.84 78.41 76.74 95.52 110.36 101.03
155
366
453
803
1197
1472
1610
2200
2349
2380
Read from Fig. 1a.
Calculated according to Eq. (16)
Calculated according to Eq. (15)
Vmaxa (mg l-1 h-1)
Si (mg l-1)
25
15.97
12.04
16.68
12.34
10.91
3.9
8.83
3.84
4.11
2.44
tmb (h)
by C. tropicalis PHB5 grown at different initial phenol concentration (Si).
428
390
430
391
380
0.9937
0.9952
0.9816
0.9866
0.9853
0.9896
0.9994
362 331
0.9962
0.9909
0.9987
R2
330
333.1
322
X´ c (mg l-1)
Kinetic parameters (Vmax, tm and X) used for determination of specific degradation rate (q), obtained with the Gompertz model for phenol consumption
Table 4
To cite this article: Authors name, Article title, (2013), DOI: 10.1007/s11356-013-2040-z
