Accepted Manuscript

Title: A novel P450-initiated biphasic process for sustainable

biodegradation of benzo[a]pyrene in soil under
nutrient-sufficient conditions by the white rot fungus
Phanerochaete chrysosporium

Author: Sukanta S. Bhattacharya Khajamohiddin Syed Jodi

Shann Jagjit S. Yadav

PII: S0304-3894(13)00534-7
DOI: http://dx.doi.org/doi:10.1016/j.jhazmat.2013.07.055
Reference: HAZMAT 15280

To appear in: Journal of Hazardous Materials

Received date: 16-4-2013

Revised date: 12-7-2013
Accepted date: 25-7-2013

Please cite this article as: S.S. Bhattacharya, K. Syed, J. Shann, J.S. Yadav,
A novel P450-initiated biphasic process for sustainable biodegradation of
benzo[a]pyrene in soil under nutrient-sufficient conditions by the white rot
fungus Phanerochaete chrysosporium, Journal of Hazardous Materials (2013),
http://dx.doi.org/10.1016/j.jhazmat.2013.07.055

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 A novel P450-initiated biphasic process for sustainable biodegradation of benzo[a]pyrene

in soil under nutrient-sufficient conditions by the white rot fungus

Phanerochaete chrysosporium

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Sukanta S. Bhattacharya1, Khajamohiddin Syed1, Jodi Shann2, and Jagjit S. Yadav1*

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1
Department of Environmental Health, 2Department of Biological Sciences, University of

Cincinnati, Cincinnati, Ohio, USA

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*Corresponding Author: Environmental Genetics and Molecular Toxicology Division,

Department of Environmental Health, University of Cincinnati Medical Center, Cincinnati, OH

45267-0056, USA. E-mail: yadavjs@ucmail.uc.edu; Telephone: +1-513-558-4806; Fax: +1-513-

558-0925

Page 1 of 34
Abstract

High molecular weight polycyclic aromatic hydrocarbons (HMW-PAHs) such as benzo[a]pyrene

(BaP) are resistant to biodegradation in soil. Conventionally, white rot fungus Phanerochaete

chrysosporium has been investigated for HMW-PAH degradation in soil primarily using

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nutrient-deficient (ligninolytic) conditions, albeit with limited and non-sustainable

biodegradation outcomes. In this study, we report development of an alternative novel biphasic

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process initiated under nutrient-sufficient (non-ligninolytic) culture conditions, by employing an

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advanced experimental design strategy. During the initial nutrient-sufficient non-ligninolytic

phase (16 days), the process showed upregulation (3.6- and 22.3- fold, respectively) of two key

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PAH-oxidizing P450 monooxygenases pc2 (CYP63A2) and pah4 (CYP5136A3) and formation

of typical P450-hydroxylated metabolite. This along with abrogation (84.9%) of BaP degradation
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activity in response to a P450-specific inhibitor implied key role of these monooxygenases. The
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subsequent phase triggered on continued incubation (to 25 days) switched the process from non-
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ligninolytic to ligninolytic resulting in a significantly higher net degradation (91.6% as against

67.4% in the control nutrient-limited set) of BaP with concomitant de novo ligninolytic enzyme
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expression making it a biphasic process yielding improved sustainable bioremediation of PAH-

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contaminated soil. To our knowledge this is the first report on development of such biphasic
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process for bioremediation application of a white rot fungus.

Keywords: P450; Response surface methodology; Genetic algorithm; PAH biodegradation;

Transcript analysis in soil; Non-ligninolytic

Page 2 of 34
1. Introduction

Soil contaminated with persistent high molecular weight polycyclic aromatic hydrocarbons

(HMW-PAHs), including benzo[a]pyrene (BaP), poses human health hazard and ecological risks

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owing to the toxicity, mutagenicity and/or carcinogenicity of these chemicals [1-4] Among the

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few microorganisms that can degrade PAHs [5-8] white rot fungi including P. chrysosporium,

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have shown an extraordinary ability to degrade even higher (≥ 4 ring) PAHs including BaP [9-

12]. In conventional studies using white rot fungi, PAH biodegradation has been attempted by

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inducing ligninolytic conditions in nutrient-limited media. However, practical application of this

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nutrient starvation strategy to soil has yielded variable and less-than-efficient removal of PAHs

[13-15], probably because of inconsistent colonization and non-sustainable nature of the process
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due to short-lived fungal viability and enzyme activity as a result of exhaustion of nutrients. To

circumvent this sustainability problem and to achieve higher biodegradation, we employed a

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different strategy for developing a soil bioremediation process for PAHs, using BaP as the test
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PAH.
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Whole genome sequencing of P. chrysosporium has revealed a large repertoire (an estimated 149
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P450s) of Cytochrome P450 monooxygenases [9,16,17], seven of which have been shown to

have the capacity to oxidize a range of PAH compounds including low molecular weight
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(LMW)- and HMW- PAHs in our recent genome-to-function studies [18-20]. An upregulated

expression of majority of the P450 monooxygenases including these specific PAH-oxidizing

P450s under nutrient-sufficient culture conditions [21-23] and their induction by varying ring

size PAHs has been demonstrated in liquid cultures [20]. In light of the above facts, we

hypothesized that a greater and more sustained biodegradation of PAHs could be achieved if the

biodegradation process was initiated under P450-expressing nutrient-sufficient conditions and

Page 3 of 34
allowed to transition into nutrient-limited (ligninolytic) process to trigger the expression of

ligninolytic enzymes.

To develop a process based on this biphasic strategy, understanding of the interaction of various

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physicochemical and nutritional parameters, their modeling and optimization for biodegradation

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was critical for scale up and deployment of the process in field conditions. For this, we

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employed response surface methodology (RSM) to investigate the interaction between

parameters over a wide range of conditions encompassing both nutrient-limited (ligninolytic) and

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nutrient-sufficient (non-ligninolytic) conditions. RSM has been frequently used for such

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optimizations in biological systems [24]. However owing to its limitation of prediction beyond

user-defined boundary parameters and often restricting to local optima, genetic algorithm (GA),
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an advanced computational tool capable of arriving to global optima [25] has been used. GA

uses the biological principles of mutation, selection, and recombination (using the
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physicochemical factors as genes embedded in a chromosome) to find the best viable solution to
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the problem. As a part of evaluating the process optimization, the two oxidative enzyme types
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(P450 monooxygenases and peroxidases) known to be involved in the PAH degradation process
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(albeit under differing nutrient conditions) were also measured to get an insight into the

relationship between their expression and the associated BaP biodegradation changes during the
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process. The optimized biphasic process yielded sustainable and high biodegradation of BaP in

soil. To the best of our knowledge, this is the first attempt at exploiting the nutrient-sufficient

soil conditions and the P450 monooxygenases expressed therein for advanced in-silico based

optimization of a sustainable biphasic PAH biodegradation process for white rot fungus.

2. Materials and Methods

Page 4 of 34
2.1 Fungal strain, chemicals, and treatment matrices

Phanerochaete chrysosporium strain BKM-F-1767 (ATCC 24725) was used. The fungus was

subcultured and maintained on malt extract (ME) agar. BaP, analytical grade was obtained from

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Sigma chemicals, Inc., St. Louis, Mo. All organic solvents were HPLC grade and obtained from

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Spectrum Chemical Mfg. Corp., NJ. A sandy loam type soil obtained from a field site was used.

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This soil type, containing low total organic carbon (TOC) of 0.93 and a cation exchange capacity

(CEC) of 6.3, was used in the present study to minimize the effect of variables such as organic

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carbon and other contributing ionic factors. The soil composition was 56% sand, 16% clay and

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28% silt and the pH was 8.3. Initial pH of soil matrix in the treatment process was adjusted per

the experimental conditions using 0.1 M acetate buffer of an appropriate pH. Wood sawdust was
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used as a source of lignocellulose substrate for soil supplementation to support colonization by

the fungus. The sawdust material was procured from the carpentry of the University of
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Cincinnati located in the Department of Biological Sciences; it was a mixture derived from hard
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and soft woods, mainly from pine, and had the following composition: 41.81 % cellulose, 23.57
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% lignin, 400:1 C:N ratio.

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2.2. Fungal inoculum preparation

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P. chrysosporium inoculum was grown on sawdust to minimize the lag period in colonizing the

main substrate (soil amended with sawdust) in the process optimization experiments. Dried and

sieved (15 mesh size) sawdust (5 g) was supplemented with basal III salts solution of the defined

low-nitrogen medium [26] buffered with 0.1M sodium acetate buffer pH 5, 2% glucose, and 20

mM ammonium tartrate to achieve a liquid:solid ratio of 2.5:1. The resulting substrate matrix

Page 5 of 34
was inoculated with 9 agar plugs (0.5 mm diameter) cut from a 5-day old malt extract (ME) agar

culture plate of P. chrysosporium and incubated at 30oC for 4 days.

2.3. Design of experiments

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The design of experiments (DOE) was based on four key physiological factors (variables),

namely carbon (C) (in the form of dextrose), nitrogen (N) (in the form of ammonium tartarate),

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pH, and sawdust (as a source of lignocellulose). These factors varied over a wide range, with

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respective boundary parameters as follows: C (2% w/v and 6% w/v), N (12.5 mM and 37.5 mM),

initial pH (3.5 and 5.5) and sawdust (2.5% w/w to 7.5% w/w). RSM was employed for modeling

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the process and studying the interaction between the parameters for the biodegradation of BaP.

The regression equation generated was subsequently used as objective function for process
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optimization in GA (Fig. 1).
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2.3.1 Modeling and parameter interaction analysis using RSM

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Central Composite design (CCD) was selected, owing to its better prediction capability in the
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entire design space, for the optimization of biodegradation of BaP under different nutritional
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conditions. This design was applied using Minitab® program with four variables at five levels.

The four variables used were approximated by the quadratic model equation as follows:
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Y=k0+kaA+kbB+kcC+kdD+kaaA2+kbbB2+kccC2+kddD2+kabAB+kacAC+kadAD+kbcBC+kbdBD+ kcdCD

(Equation 1)

where Y is the predicted response; k0 is a constant; ka, kb, kc, kd are the linear coefficients; kab, kac,

kad, kbc, kbd, kcd are the cross-coefficients; kaa, kbb, kcc, kdd are the quadratic coefficients. A total of

31 experiments were carried out to estimate the 15 coefficients for the BaP biodegradation as

Page 6 of 34
well as BaP metabolite (3OH-BaP) formation (Table 1). Data were analyzed using Minitab® to

find out the interaction between the variables and the response. The quality of the fit of this

model was expressed by the coefficient of determination (R2) using the same program.

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2.3.2 In silico optimization of treatment conditions using GA

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GA, a stochastic computational optimization tool where the input variables (C, N, pH, and

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sawdust) were treated like the genes “embedded” in a chromosome, was used. The fitness

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function (Equation 1) obtained from the RSM was used as input in GA to create the initial

population of the solutions (% degradation outputs), evaluation of these candidate solutions in

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light of feasibility, creation of the genetic variants (of the parameters) in the offspring using

genetic operators (crossover and mutation), and evaluation of the altered offspring (parameter
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combination variants).
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“Mutation, recombination, and selection” were carried out for this “chromosome” as in the
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biological systems to find out the best fit solution (maximum biodegradation) subject to a
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constraint (biodegradation < 100%). GA was initiated with a randomly generated initial
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population size of 100 individuals (initial genetic variants of the parameter set). Random

mutation was carried out with tournament selection (based on fitness). Here each parent was
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selected by randomly choosing individuals, the number of which was specified by the

tournament size that is 4, and then choosing the best individual out of the set to be a parent. A

uniform mutation function was used with scattered crossover which created a random binary

vector. These randomizations in the GA were more likely to overcome the constraints of local

optima thereby attaining the global optima for the biodegradation of BaP.

2.4. Experimental conditions

Page 7 of 34
All biodegradation experiments were set up using a preselected basic set of conditions (60%

moisture level, 5% inoculum level w/w, and 31oC incubation temperature). Levels of added

nutrients (C, N), sawdust, and initial pH were varied over an entire range in the soil-sawdust

matrix using the Tien and Kirk’s defined medium base (without C, N, trace elements solution,

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veratryl alcohol, and Tween-80) prepared as described previously [27,28]. The numerical values

for C, N, and pH parameter levels were those achieved in the supplementation medium whereas

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the sawdust level values were those relative to the soil amount used. These test parameters (C, N,

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sawdust, initial pH) were varied based on 31 different parameter sets generated by the DOE as

described above.

2.4.1. Soil treatment conditions

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The soil was prepared into a uniform material by grinding and sieving (20 mesh size) before use.

For all experiments, the soil was spiked with BaP at a final concentration of 100 mg/Kg, using a
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working stock (1 mg/ml) in acetonitrile. Degradation studies were carried out in 150-ml
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Erlenmeyer flasks, each flask containing 10g soil supplemented with media and subjected to
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aeration on alternate days to maintain the aerobic environment. The culture conditions (levels of
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C, N, pH, sawdust) were varied using the statistical design of experiments (Central Composite

Design). The modeling was based on 16 days incubation period from which the optimal
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degradation conditions were selected (Table 1). The optimized treatment sets (nutrient-limited

set and nutrient-sufficient set) were then further incubated for 25 days to understand the biphasic

nature of the process.

2.4.2. P450 inhibitor studies

Page 8 of 34
To ascertain the role of P450(s) in the degradation of BaP in nutrient-sufficient conditions, the

known P450 inhibitor piperonyl butoxide, was added to the optimal treatment process by

incorporation (0.5 mM) in the aqueous base medium used for soil supplementation.

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2.4.3. Extraction conditions

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Simultaneous extraction of BaP and its P450 metabolite 3-hydroxybenzo[a]pyrene (3-OH BaP)

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and ergosterol (used as an index of fungal growth) was carried out using a microwave-assisted

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extraction method by Montgomery et al. [29] with some modifications. Briefly, 0.5 g soil was

treated with 0.5 ml 2M NaOH followed by the addition of 2 ml methanol in a 15 ml extraction

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vial. The contents were heated in a microwave oven (highest power setting) using three pulses of

30 sec each with intermittent cooling for 30 sec each. In the resulting mixture, phenol red was
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added as an indicator and the contents were neutralized with 1M HCl. Methanol (2 ml) was

added to the mixture followed by extraction (3 x 2ml) with pentane. The extract was dried under
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a stream of nitrogen gas and resolubilized in a small volume of methanol (500 µl).
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2.4.4. Analyses
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The treated and untreated soil-sawdust samples were analyzed for total biodegradation, enzyme

activities, and fungal biomass yield.

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2.4.4.1. HPLC analysis:

Organic extracts of the soil-sawdust samples were simultaneously analyzed for BaP, 3-OH-BaP,

and ergosterol using a Varian Prostar 210/215 HPLC unit (Varian Analytical Instruments,

Walnut Creek, CA) equipped with a C18 column (250mm x 4.6mm, 5µm) run with a solvent

mobile phase containing methanol and acetonitrile (50:50). Sample elution was carried out in the

Page 9 of 34
gradient mode at a flow rate of 1.5 ml/min using a gradient of methanol (50% to 100% methanol)

in 8 min. Detection was based on a UV-Vis detector set at 282 nm. BaP and 3-OH-BaP standards

(Sigma) were used for identification and quantification of the parent compound and its P450

metabolite 3-OH-BaP as described in our recent studies [18,20]. Dried mycelial mass obtained

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from liquid cultures of P. chrysosporium was used to prepare a standard curve [mycelial dry

weight versus ergosterol content] for quantification of fungal biomass in the samples (expressed

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as µg mycelial biomass/g matrix substrate).

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2.4.4.2. Enzyme Assays

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An aqueous extract of treated or untreated soil-sawdust matrix was prepared by suspending 1 g

of the matrix substrate in 3 ml distilled water by vortexing and centrifugation (10,000 rpm for 10
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min.). The resulting extract was subjected to enzyme activity assays for lignin peroxidase (LiP),

manganese-dependent peroxidase (MnP). and cellobiose dehydrogenase (CBD) using standard

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spectrophotometric methods as described by Tien and Kirk [26], Paszczynski et al. [30] and
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Baminger et al. [31] respectively. One unit (expressed as International Unit) of enzyme activity
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was defined as 1 µM of substrate oxidized per min. The final activity was calculated with respect
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to the dry weight of the matrix substrate.

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2.4.4.3 Estimation of carbon

Total carbon in the matrices (soil, sawdust, soil+sawdust mixture) was estimated by the

Anthrone method as described elsewhere [32] .

2.4.4.4 Estimation of total nitrogen

Page 10 of 34
Total nitrogen content in the matrices was estimated using the Kjeldahl method [33] with slight

modifications. Briefly, 1 g of sample was digested in concentrated H2SO4 supplemented with 2

Kjeldahl buffer tablets followed by mixing with 25 ml of 35% (w/w) of NaOH and heating in the

Kjeldahl flask for ~15 min till a clear solution was obtained. The evolved ammonia was distilled

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over and trapped in 0.05 M boric Acid. The total nitrogen content was determined in terms of the

amount of ammonia produced by titrating the excess boric acid against 0.1 M NaOH.

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2.4.4.5. Quantitative RT-PCR analysis for expression of P450 monooxygenases

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Transcriptional quantification for the P450 monooxygenases pc2 and pah4, recently identified

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for their role in PAH oxidation in P.chrysosporium [18,20], was carried out in soil-sawdust

samples using real-time qRT-PCR. Total fungal RNA was extracted from soil-sawdust matrix
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using a modified version of the method for soil nucleic acid isolation by Griffiths et al.[34]

coupled with further purification using RNeasy kit per manufacturer’s instructions (Qiagen,
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Inc.,Valencia, CA). Purified RNA prep showing a A260/A280 value over 2 was considered
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acceptable. The purified total RNA (25 ng) was subjected to qRT-PCR quantification of the
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target P450 transcripts using gene-specific primers (Table 2) and the Brilliant SYBR green
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QRT-PCR master mix kit (Stratagene, La Jolla, CA) per manufacturer’s specifications using

7900 HT ABI Prism unit (Applied Biosystems, Forest city, CA). A fold-change in P450
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transcript level for pc2 or pah4 was determined by using the formula 2ΔCt in comparison to the

housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (gpd) an internal control, which

is known to have a constitutive expression in the fungus. The qRT-PCR reaction conditions

involved the reverse transcription step carried out at 50°C for 30 min followed by the PCR

reaction performed using denaturation at 95oC for 10 min and target amplification for 40 cycles.

Each amplification cycle involved denaturation at 95°C for 30 s, annealing at 60°C for 30 s, and

Page 11 of 34
extension at 72°C for 1 min. [22]. A melting curve analysis was also carried out to rule out the

possibility of a crosslinking between the primers and a non-specific amplification.

3. Results and Discussion

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Soil treatment process for biodegradation of BaP was first optimized for both nutrient-limited

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and nutrient-sufficient conditions in a 16-day treatment using RSM coupled with GA. The latter

set of conditions eventually led to development of a 25-day biphasic process with improved and

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sustained BaP biodegradation activity. These optimizations encompassed the entire non-

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ligninolytic to ligninolytic range of conditions. The biphasic process was based on a metabolic

switch from non-ligninolytic to ligninolytic state as interpreted in terms of the associated enzyme
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activities and P450 metabolite formation and disappearance.

3.1. Understanding the biodegradation parameters and their interactions using RSM-
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assisted model:
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The RSM-assisted experimental design (Table 1), meant to investigate the effect of varying the
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process parameters on BaP biodegradation, led to the following regression equation (expressed

in terms of uncoded variables) as a part of the modeling process:

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Biodegradation = 95.631 - 4.83.x1 - 1.94.x2 -4.95.x3 + 0.17.x4 -0.45.x1.x1 +0.01.x2.x2 - 1.51.x3.x3

- 0.67.x4.x4 - 0.12.x1.x2 + 2.38.x1.x3 + 0.34.x1.x4 + 0.31.x2.x3 + 0.16.x2.x4 - 0.05.x3.x4

(Equation 2)

where x1 is the added carbon level, x2 is the added nitrogen level, x3 is the initial pH and, x4 is the

sawdust level. The RSM model showed significance based on ANOVA (Table A.1) and its

Page 12 of 34
experimental validation showed the R2 and R2adj to be 84.2% and 70.3%, respectively; these

values are considered acceptable for the biological experiments involving soil owing to the

problem of uniform mass transfer and extraction of PAHs for such complex matrix.

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In order to gain further insights on the effect of interaction of the parameters on BaP

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biodegradation (%), the individual response surface plots were critically analyzed. From the C

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and N response surface plot (Fig. 2a), an elevated degradation was observed in the high N

condition over a moderate range of C (2%-5% w/v). A similar increase in the degradation was

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observed in the low nitrogen condition at elevated carbon levels. However, at very low levels of

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both the nutrients (C and N), a decrease in BaP degradation was observed which may be

attributed to the poor colonization/growth of the fungus. Also at higher levels of C (> 7 % w/v),
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when N was increased beyond 40 mM, a decrease in the BaP degradation was observed which

may be attributed to the physiological capacity of the fungus to perform optimally in a certain
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range of nutrient conditions. This response plot therefore represents one of the most important
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interactions as this is indicative of the two systems of degradation (ligninolytic and non-
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ligninolytic) coming into play. Low N conditions typically induced the ligninolytic enzyme
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system [26] which is known to catalyze oxidation of PAHs [33,11], in this case BaP. On the

other hand, an increased degradation in high C and high N condition during the initial phase of
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incubation, which is essentially a non-ligninolytic condition, highlights the presence of an

alternative oxidizing enzyme system responsible for the degradation of the PAH compound.

Studies by us and others have highlighted the importance of cytochrome P450 enzyme system in

the oxidation of PAHs under nutrient-sufficient culture conditions [20,34,35]. Our gene-specific

qRT-PCR analysis indeed confirmed the induction of two PAH degradation-relevant P450

monooxygenases in the nutrient-sufficient conditions (discussed in later sections). Enhanced

Page 13 of 34
initial growth of the fungi in high C and low N may be responsible for the increased depletion of

nutrients which in turn causes induction of the ligninolytic conditions in extended incubation.

The presence of ligninolytic and non-ligninolytic systems in the degradation of BaP was further

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indicated in the response plot between C and pH (Fig. 2b). Low C (< 2% w/v) and low pH (< 3)

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showed enhanced BaP degradation, indicating the role of a typical ligninolytic system known to

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be triggered under carbon-limited conditions in acidic medium such as the Tien and Kirk’s

defined low N (LN) medium. In contrast, another scenario supporting enhanced BaP degradation

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was observed at elevated levels of C (> 4.5% w/v) and relatively higher pH (> 5.5). This is a

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condition that may be indicative of the expression and role of PAH-oxidizing P450 systems in

the degradation, such as that observed in nutrient-sufficient media [36,20].

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The response surface plot between C and sawdust (Fig. 2c) revealed an optimum BaP

degradation with added C at ~5% (w/v) and sawdust at ~5% (w/w). Critical analysis of the plot
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showed that, at higher levels of sawdust, an increase in the added C (glucose) leads to enhanced
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BaP degradation. This observation may be explained by the fact that increase in the added C
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level increases the biomass of the fungus thereby enabling a greater access of the fungus to the
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matrix-adsorbed BaP, as observed earlier [36,37] where sawdust was used as a source of

supplemental carbon and observed here in the optimal nutrient-sufficient degradation sets
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compared to the nutrient-limited sets (Table 3). This is despite the fact that excess sawdust may

result in a lower bioavailability of the BaP (possibly due to increased sequestration) to the fungus

[38] which is also evident from the plot between pH and sawdust (Fig. 2d).

Analysis of the response surface plot between N and pH (Fig. 2e) depicted two distinct sets of

conditions supporting enhanced BaP degradation viz. low N-low pH set and high N-high pH set.

Page 14 of 34
This is similar to the plot between sawdust and N (Fig. 2f) which also depicts two optimal sets

one at high N and the other at low N. These two sets of conditions are known to represent

typical ligninolytic conditions triggering exo-peroxidases [26] and typical non-ligninolytic

conditions upregulating the P450 monooxygenase systems [22] respectively. Hence, the

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observed degradation under these two sets of physiological conditions may be attributed to the

two respective enzyme systems, as discussed later in this section with the enzyme data.

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3.2. Understanding the parameter interactions using RSM-assisted model for the formation

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of P450-generated BaP metabolite:

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The degradation experiments based on DOE (described above) also allowed us to understand the

parameter interactions for the nature of biodegradation when interpreted in terms of formation of
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P450-generated metabolite 3OH-BaP; this can be represented by the following regression

equation:
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te

3OH-BaP= -9061 + 21 x a1 - 1.94 x a2 -4.95 x a3 + 0.17.x4 -0.45.x1.x1 +0.01.x2.x2 - 1.51.x3.x3 -

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0.67.x4.x4 - 0.12.x1.x2 + 2.38.x1.x3 + 0.34.x1.x4 + 0.31.x2.x3 + 0.16.x2.x4 - 0.05.x3.x4

ce

(Equation 3)

Where x1 is the added carbon level, x2 is the added nitrogen level, x3 is the initial pH and, x4 is
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the sawdust level. Greater amounts of 3OH-BaP formation for the parameter interactions that are

representative of nutrient-sufficient conditions (Fig. 3) implied a higher expression of P450

activity and its role in BaP degradation under these non-ligninolytic conditions. For instance, the

response surface plot on the effect of C and N on the production of 3OH-BaP (Fig. 3a ) reveals

that high levels of nitrogen (both at low and high levels of carbon) lead to greater amounts of

3OH-BaP. This indicates the nitrogen concentration is a critical factor for the accumulation of

Page 15 of 34
3OH-BaP (or expression of P450 monooxygenases). The response surface plot between C and

pH (Fig. 3b) reveals that a carbon concentration of 5-6% w/v and a pH over 4.5 favor an increase

in the 3OH-BaP concentration; these represent non-ligninolytic conditions. An increase in 3OH-

BaP formation at a lower carbon concentration (1.5 - 2.5%) in conjunction with a lower pH may

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apparently seem contrary to the trend but actually these conditions in conjunction with the hold

value of 25mM nitrogen, also represent non-ligninolytic conditions. Evaluation of the surface

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plot between sawdust and C (Fig. 3c) revealed that an increase in the sawdust level leads to an

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increase in the concentration of 3OH-BaP, with maximum amount being present at high C and

high sawdust concentrations. This may be attributed to the nutrient-rich conditions and facilitated

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aeration. Surface plot of N and pH showed that high nitrogen - low pH (Fig. 3d) and low

nitrogen- high pH combinations with a hold value of high C favored a higher accumulation of
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3OH-BaP. Sawdust and N plot showed that higher sawdust levels favored the production of
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3OH-BaP with the maximum accumulation being at higher levels of N and sawdust (Fig. 3e).
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The response surface plot between sawdust level and pH (Fig. 3f) with hold values of high C and

high N revealed that an increase in the sawdust level and pH resulted in an increase in 3OH-BaP
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(Fig. 3f). Taken together, higher 3OH-BaP formation in various parameter combinations could in
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general be attributed to nutrient-sufficient conditions favoring better growth and/or metabolic

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status conducive for expression of P450 monooxygenase enzymes.

Collectively, the above observations on biodegradation and metabolite formation highlighted the

role of interaction of various parameters in the biodegradation process and were strongly

indicative of a dual degradative enzyme system operating in this process over the tested ranges

of parameters. In order to obtain an enhanced efficiency of such process, optimization was

carried out using GA, as it can easily overcome the user-defined boundary parameters, is less

Page 16 of 34
likely to be limited by local optima and thus has a greater chance to arrive to the global optima

than the conventional RSM programs.

3.2 Process optimization and validation using RSM-GA combination strategy

t
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The regression equation (equation 2) obtained in the second order RSM-based model above was

utilized as the objective function for optimization using GA. After running the GA for a set of

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parameters, two optimal points were obtained involving very different sets of parameters. One

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optimal point gave a predicted response of 61.88% degradation (the experimental value was

60.3%) at C 2.0% (w/v), N 2.5 mM, pH 3.5, sawdust 2% (w/w). Temperature (31oC) and initial

an
moisture content (60% of the water holding capacity of soil) were kept constant. This is

essentially a ligninolytic condition scenario. Using the same RSM regression equation, a very
M
different set of conditions was observed in the other GA-predicted response. At the higher values

of the parameters viz. C 5% (w/v), N 55 mM, pH 6.5, sawdust 7.83% (w/w), a 69.04%
d

degradation was predicted (the experimental value was 66%). This set of parameters coincides
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with the non-ligninolytic condition, where alternative enzyme system(s) such as P450
p

monooxygenases may be responsible for the degradation activity. These assumptions were
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confirmed by the enzyme assays for the known ligninolytic enzymes (LiP and MnP) and

transcript analysis for the specific PAH-oxidizing P450s pc2 (CYP62A2) and pah4
Ac

(CYP5136A3) as shown in Table 3. In this context, the two subject P450 enzymes were

identified in our recent studies as the most promising P450 monooxygenases for role in PAH

oxidations in this fungus [18,19,20] and pc2 was found to be critical in oxidation of higher

molecular weight PAHs including BaP [18].

Page 17 of 34
On day 16, samples from the optimal set of nutrient-limited conditions showed high ligninolytic

enzyme activities (LiPs, MnPs). On the other hand, the optimal set of nutrient-sufficient

conditions showed no LiP activity and very low MnP activity. In contrast to peroxidases, P450

analysis relative to the housekeeping control gene revealed a much higher expression of both pc2

t
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(3.6-fold) and pah4 (22.3-fold) in nutrient-sufficient conditions as against the low or constitutive

level of expression under nutrient-limited ligninolytic conditions. Further validation of the role

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of P450 monooxygenases under nutrient-sufficient conditions was carried out using piperonyl

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butoxide, a known P450 inhibitor [23]. Abrogation (84.9%) of degradation activity was observed

in presence of the inhibitor as compared to the control treatments without the inhibitor. This

an
along with a higher concentration of 3OH-BaP (3036.84 µg Kg-1), the known P450 metabolite of

BaP [18], in the nutrient-sufficient conditions confirmed the significant role of P450
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monooxygenases under these conditions. CBD expression was higher and more sustained under
d

nutrient-sufficient conditions, consistent with that in liquid cultures [39], although its role in
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PAH biodegradation is not clear.

p

Importantly, further incubation of the optimal sets of both nutrient-sufficient and nutrient-limited
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sets until day 25 resulted in a much higher degradation in nutrient-sufficient set (91.6%) but only

slightly higher degradation in nutrient-limited set (67%). The presence of 3OH-BaP was not
Ac

detected in either of the sets in the 25-day process, implying its disappearance due to amenability

to further degradation in the system. Enzyme analysis revealed no LiP and CBD activities and

low residual MnP activity (10.52 IU/gds) for the nutrient-limited set which may be attributed to

the loss of fungal viability due to lack of nutrients. Enzyme analysis for nutrient-sufficient set

revealed much higher activity for LiPs (302.39 IU/gds), MnPs (48.96 IU/gds) and CBD

(131.6IU/gds) and a 2.4-fold expression of pah4. Also, this set showed sustained fungal growth

Page 18 of 34
as depicted in Table 3. These observations collectively indicate that the 25-day nutrient-

sufficient process could support an enhanced initial degradation of PAHs due to expression of

PAH-oxidizing P450s in the first 16 days and could provide a sustained and more extensive

degradation in the extended incubation (25 days) by switching to ligninolytic conditions

t
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(wherein ligninolytic enzymes are expressed). The low carbon and nitrogen levels in the nutrient-

limited conditions may be the major limiting factors for a sustainable growth of the fungus in

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extended incubation as compared to the nutrient sufficient set of conditions that result in more

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sustainable growth. Comparison of the data on fungal biomass, total C and total N (Table 3) in

16-day versus 25-day bioprocess supports this phenomenon. Taken together, the above data

an
demonstrate that the optimized 25-day nutrient-sufficient treatment set is a biphasic process

involving initial P450-initiated (non-ligninolytic) oxidation (phase I) followed by a ligninolytic

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degradation (phase II) and is an alternative, more sustained approach for bioremediation.
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4. Conclusions
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This study, based on an advanced experimental design using RSM coupled with GA, led to
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optimization of two separate sets of conditions for biodegradation of BaP in soil in a 16-day
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process, one for a nutrient-limited (ligninolytic) process and the other for a nutrient-sufficient

(non-ligninolytic) process. A notable overexpression of HMW-PAH specific genes pc2 and pah4
Ac

in the nutrient-sufficient process as compared to the nutrient-limited process confirmed the role

of P450 monooxygenases in the degradation of BaP under the nutrient-sufficient conditions,

which was also confirmed by analysis of the known P450 metabolite 3OH-BaP. More

importantly, the study led to the development of a 25-day biphasic process initiated under

nutrient-sufficient (non-ligninolytic) conditions and transitioned into nutrient-limited

(ligninolytic) conditions. This biphasic process sustained better fungal colonization/growth and

Page 19 of 34
led to a much greater PAH biodegradation efficiency due to a concerted activity of the non-

ligninolytic (P450) and ligninolytic (peroxidase) enzyme systems expressed in tandem during the

process. The developed biphasic process overcomes the bottlenecks in the conventional

strategies currently adopted for soil bioremediation by white rot fungi and is a significant step in

t
ip
the direction of designing field applications of these promising bioremediation agents. Further

studies on scale up of this process, extrapolation of the process to other soil conditions, as well as

cr
elucidation of the degradation pathways during the process are being pursued.

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Acknowledgement

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The work was supported by the National Institute of Environmental Health Sciences (NIEHS)

grants R01ES10210 and R01ES015543 to JSY.

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d

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te

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Figure captions

Fig. 1. Overall design of the optimization strategy for the developed Biphasic process for BaP

degradation in soil.

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Fig. 2. Response Surface plots depicting influence of parameter interactions on the

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biodegradation of BaP by P. chrysosporium in the soil treatment process. (a).Carbon and

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Nitrogen interaction; (b).Carbon and pH interaction; (c). Carbon and Sawdust interaction; (d).

pH and Sawdust interaction; (e). Nitrogen and pH interaction; (f). Nitrogen and Sawdust

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interaction. In addition to the variables considered in the individual plots, hold values were 4%

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(w/v) Carbon, 25 mM Nitrogen, 4.5 initial pH, and 5% (w/w) Sawdust.

Fig. 3. Response Surface plots depicting influence of parameter interactions on the concentration
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of the P450 monooxygenase metabolite 3OH-BaP generated by P. chrysosporium in the soil

treatment. (a).Carbon and Nitrogen interaction; (b).Carbon and pH interaction; (c). Carbon and
d

Sawdust interaction; (d). Nitrogen and pH interaction; (e). Nitrogen and Sawdust interaction; (f)
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Sawdust and pH interaction. In addition to the variables considered in the individual plots, hold
p

values were 4% (w/v) Carbon, 25 mM Nitrogen, 4.5 initial pH, and 5% (w/w) Sawdust.
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Page 27 of 34
 Selection of Parameters
(Carbon, Nitrogen, pH, Sawdust)

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Design of Experiments using
Central Composite Design

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Response Surface Plot Generation of Model for Optimization of BaP
analysis to assess the degradation of BaP using biodegradation using
effect of parameter Response Surface Methodology Genetic Algorithm
interactions on

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biodegradation
NUTRIENT-
SUFFICIENT PROCESS
Process
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Non-Ligninolytic phase
(P450-mediated) Validation

Biphasic Process Extended incubation

d
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Ligninolytic phase
(Peroxidase-mediated)
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Fig. 1
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Page 28 of 34
 t
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ed
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Fig.2

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1
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2 Fig. 3
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3
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Page 30 of 34
 3 Table 1
4 Optimization of biodegradation of BaP in soil by P. chrysosporium using various sets of
5 treatment conditions designed using the central composite design of experiments*.
6
Run C N Initial Sawdust X1 X2 Y1 Y2
Order (%w/v) (mM) pH (%w/w)
(Treatment

t
set)

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1 2 12.5 3.5 2.50 59.89 54.74 1701.56 1599.07
2 6 12.5 3.5 2.50 46.47 51.61 2021.59 1782.25

cr
3 2 37.5 3.5 2.50 44.06 43.42 2836.31 2909.25
4 6 37.5 3.5 2.50 31.81 27.84 2935.58 2859.95
5 2 12.5 5.5 2.50 36.77 34.83 3010.71 3215.83

us
6 6 12.5 5.5 2.50 47.07 50.76 3272.35 3002.45
7 2 37.5 5.5 2.50 37.14 39.17 2303.24 2286.72
8 6 37.5 5.5 2.50 41.25 42.65 1856.05 1840.85

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9 2 12.5 3.5 7.50 32.41 34.88 3679.30 3581.94
10 6 12.5 3.5 7.50 41.68 38.49 4848.40 4474.58
11 2 37.5 3.5 7.50 48.68 43.83 5237.12 5116.69
12 6 37.5 3.5 7.50 29.19 34.99 6094.54 5776.85
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13 2 12.5 5.5 7.50 11.64 14.45 2714.35 2399.65
14 6 12.5 5.5 7.50 32.61 37.12 3081.24 2895.73
15 2 37.5 5.5 7.50 40.33 39.05 1568.33 1695.11
16 6 37.5 5.5 7.50 45.28 49.27 2246.55 1958.71
d

17 0 25.0 4.5 5.00 31.22 35.85 2927.63 2799.51

18 8 25.0 4.5 5.00 50.27 42.93 2615.27 3246.29
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19 4 00.0 4.5 5.00 51.85 49.03 2576.38 3013.93

20 4 50.0 4.5 5.00 49.76 49.87 3321.74 3387.09
p

21 4 25.0 2.5 5.00 39.79 43.34 2937.80 3313.26

22 4 25.0 6.5 5.00 43.97 37.72 984.44 1111.88
ce

23 4 25.0 4.5 0.00 35.44 36.52 2393.50 2362.56

24 4 25.0 4.5 10.00 27.06 23.28 3929.45 4463.29
25 4 25.0 4.5 5.00 43.04 46.55 2688.56 2745.20
26 4 25.0 4.5 5.00 47.73 46.55 2813.21 2745.20
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27 4 25.0 4.5 5.00 49.54 46.55 2707.98 2745.20

28 4 25.0 4.5 5.00 41.20 46.55 2813.25 2745.20
29 4 25.0 4.5 5.00 48.21 46.55 2704.21 2745.20
30 4 25.0 4.5 5.00 48.03 46.55 2770.84 2745.20
31 4 25.0 4.5 5.00 48.11 46.55 2718.35 2745.20
7 *Experimental variables included dextrose as carbon source [C], ammonium tartarate as nitrogen source [N], pH, and sawdust. Boundary
8 parameters were C (2% w/v and 6% w/v), N (12.5 mM and 37.5 mM), pH (3.5 and 5.5) and sawdust (2.5% w/w to 7.5% w/w); X1=Experimental
9 degradation, X2= Predicted degradation, Y1= Experimental 3OH-BaP level, Y2=Predicted 3OH-BaP level
10
11
12

32
Page 31 of 34
13 Table 2
14 Primers used in the study
Target Forward primer (5’to 3’) Reverse primer (5’ to 3’) Amplicon
gene* (bp)

pah4 CAAGCAAGTGCAGCACATCC CCGCGTGCAACGATTTCG 354

CGACTTCCAGGACCTCATCTCG CACTCTCCCACCGTTCTCAC 346

t
pc2

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gpd GGCATTGTGCAGGGTCTCATG GAGTAGCCCCACTCGTTGTC 458

pc2, pah4: Cytochrome P450 genes; gpd: Glyceraldehyde-3-phosphate dehydrogenase gene

15

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16

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17

18

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19

20
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21

22
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23
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24
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25
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26

27
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28

29

30

31

32

33
34
35

33
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36 Table 3
37 Relative BaP biodegradation activity and associated fungal enzymatic activities and growth in
38 the optimized nutrient-limited and nutrient-sufficient processes for soil treatment with P.
39 chrysosporium.
40
Test parameter Nutrient-limited process** Nutrient-sufficient processΔ
day 16 day 25 day 16 day 25

t
(Biphasic process)

ip
BaP degradation (%) 60.27 67.39 66.14 91.60

cr
LiPs (IU gds-1 #) 106.43 - - 302.39

MnPs (IU gds-1) 40.83 10.52 12.13 48.96

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CBD (IU gds-1) 16.48 0 124.5 131.6

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P450 pc2 (fold- change) 0.44 - 3.58 -

P450 pah4 (fold-change) 2.39 - 22.3 2.4

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Fungal biomass (µg g-1)* 331.59 387.37 513.28 753.84

Total C € (mg gds-1) 13.94 6.39 45.37 30.43

d

Total NΩ (µg gds-1) 34.54 29.47 1320.62 1162.21

p te

#IU gds-1 means International units per gram dry substrate

41
*Fungal growth was estimated in terms of biomass yield, measured based on ergosterol analysis (µg biomass per gram dry substrate)
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42
43
The initial levels of added C (as glucose), added N (as ammonium tartarate), and pH indicated for the two processes are as follows; these levels
44
were those achieved in the liquid base medium for supplementation.
45
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**Nutrient-limited process: 2.0% w/v C, 2.5 mM N, 2.0% w/w sawdust, 3.5 pH

46
47 Δ Nutrient-sufficient process: 5.0 % w/v C, 55.0 mM N, 7.8% w/w sawdust, 6.5 pH
48
€ Final total carbon was measured in terms of total reducing sugar. The amounts represent their added form plus the native form present in the
49
matrices (soil and sawdust). Initial total carbon levels were as follows: nutrient deficient set 21.28 mg/gds; nutrient sufficient set 56.96 mg/gds.
50
51
Ω Total N measured using Kjeldahl method represents the added nitrogen plus the native nitrogen present in the matrices.
52
53
54
55
56
57

34
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57 Highlights
58 A novel biphasic process developed for soil bioremediation using white rot fungus
59 Bioprocess optimization by experimental design based on RSM coupled with GA
60 Report on biodegradation of a normally recalcitrant higher PAH (5-rings) in soil
61 First report on soil bioremediation by a ligninolytic fungus in a nutrient-rich process
62 First demonstration of expression of PAH-oxidizing P450s of white rot fungus in soil
63

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