1

2 Title:

4 Bacterial degradation of mixed-PAHs and expression of

5 PAH-catabolic genes
6

8 Authors:

9 Sakshi*, Santosh Kumar Singh, Anil Kumar Haritash

10

11 Affiliation:
12 Environmental Microbiology and Bioremediation Laboratory,
13 Department of Environmental Engineering, Delhi Technological University,
14 Shahbad Daulatpur, Delhi (110042), India
15

16

*
17 Corresponding author: saakshi1412@gmail.com

18 Mob: +91-9582725955
19

20

21
22 Acknowledgement
23 The authors acknowledge the help of Superworth Biodiscoveries, Delhi, India, for analysis of
24 gene expression.

1
25 Bacterial degradation of mixed-PAHs and expression of PAH-catabolic genes
26 Sakshi*, Santosh Kumar Singh, Anil Kumar Haritash
*
27 Corresponding author: saakshi1412@gmail.com
28
29
30 Abstract
31 Polyaromatic hydrocarbons (PAHs) are hazardous organic compounds with established
32 toxicity, carcinogenicity, and mutagenicity, ubiquitous distribution, and persistence in
33 different environmental matrices. In the present study, degradation of the mixture of PAHs
34 (phenanthrene, anthracene, fluorene, and pyrene) by Kocuria flava and Rhodococcus
35 pyridinivorans was investigated. The individual strains and consortium of both degraded
36 55.6%, 59.5%, and 59.1% of 10 mg L-1 of mixed PAHs, respectively, within 15 days. The
37 participation of catabolic enzymes (catechol 2,3-dioxygenase (C23O), dehydrogenase (DH),
38 and peroxidase (POD)) was confirmed during catalytic oxidation through meta-cleavage of
39 mixed PAHs in this study. The catabolic gene expression of naphthalene dioxygenase (NAH)
40 and catechol 2,3-dioxygenase (C23O) during degradation was confirmed using RT-qPCR in
41 the present study. This is the first study that shows significant gene expression of the
42 catabolic genes during degradation of mixed PAHs by selected bacterial strains. The C23O
43 gene showed a 6.02 log fold higher expression in Kocuria flava in comparison to
44 Rhodococcus pyridinivorans whereas NAH gene exhibited a 7.9 log fold higher expression in
45 Rhodococcus pyridinivorans in comparison to Kocuria flava. Hence it is likely to conclude
46 that combination of Kocuria flava and Rhodococcus pyridinivorans can effectively remove
47 hazardous mixture of PAHs from the contaminated environmental matrix.
48
49 Keywords: Bioremediation; Catabolic genes; catechol 2,3-dioxygenase; Naphthalene
50 dioxygenase; PAHs
51
52 Introduction
53 Environmental pollution with polycyclic aromatic hydrocarbons (PAHs) is a critical
54 global problem. PAHs are organic pollutants spread widely by various natural activities such
55 as volcanic eruption, forest fire, oil seeps; and anthropogenic activities like combustion of oil,
56 diesel, coal and oil products, burning of fossil fuel, agricultural waste etc.. PAHs are one of
57 the most hazardous, ubiquitous, carcinogenic, mutagenic, toxic and bioconcentrating
58 pollutants having environmental and human health concern (Lu et al. 2011). PAH compounds
59 are composed of two or more benzene rings fused together and categorized in two groups on
60 the basis of number of benzene rings i.e. low molecular weight polycyclic aromatic
61 hydrocarbons (LMW PAHs) consist two to three benzene rings and high molecular weight
62 polycyclic aromatic hydrocarbons (HMW PAHs) consist more than three benzene rings. The
63 degradation of PAH compounds is not easy under natural conditions due to chemical stability
64 and the persistence increases with increase in molecular weight (Bamforth and Singleton
65 2005). Low water solubility of PAHs results in deposition on soil/particulate matter which
66 acts as the ultimate sink for these compounds. Natural attenuation processes influencing the
67 fate of PAHs include volatilization, dispersion, solar dissociation, sorption, and
68 biodegradation (Rogers et al. 2002; Haritash and Kaushik 2009). Based on the toxicity, 16
69 PAH compounds have been listed as priority contaminants by the United States
70 Environmental Protection Agency (USEPA) (ATSDR 1990; Liu et al. 2001). Considering the
71 toxic effects of PAHs, researchers have investigated removal of PAHs from the contaminated

2
 72 environmental matrix using various physical, chemical, and biological methods (degradation
73 by microbial species (bacteria, fungi, and algae) and plants). As, the physical and chemical
74 processes used are technologically challenging and cost-prohibitive, the biological methods
75 have gained attention as it is environment-friendly, cost-efficient, most acceptable and
76 sustainable way for degradation/removal of hydrocarbons under diverse environmental
77 conditions (Chen et al. 2015; Cameselle et al. 2019; Sakshi et al. 2019; Ławniczak et al.
78 2020). The microbial degradation of contaminants is catalysed by catabolic enzymes encoded
79 by catabolic genes and microbial degradation of hydrocarbons, toxic organic compounds and
80 PAHs depends on adaptation of microbes through molecular level changes resulting in the
81 expression of catabolic genes induced by long-term exposure to contaminants (Keum et al.
82 2008; Naether et al. 2013; Sakshi and Haritash 2020).
83 It has been observed that catabolic gene expression was positively correlated with
84 biodegradation of hydrocarbons (Gennaro et al. 2009; Afzal et al. 2011) which indicated that
85 enhanced expression of catabolic genes is required for contaminant degradation. The bacterial
86 species isolated from PAH-polluted sites are adapted to contaminated environment and have
87 the capability to degrade/transform a range of PAHs (Cerniglia 1993; Reddy and Adams
88 2015). Most of such studies investigated PAH-degradation using a single PAH compound,
89 however, the contaminated site contains a mixture of several PAH compounds. It difficult to
90 achieve the desired level of degradation of mixture of PAHs since the toxicity and
91 recalcitrance are substantially enhanced with increasing structural complexity of PAHs,
92 besides the inter-PAH chemical interference. Therefore, the degradation of mixed PAHs is
93 significant to remove the toxicity from the environmental matrix.
94 Primarily, microbial degradation of PAHs depends on catalytic action of catabolic
95 enzymes such as mono-oxygenases, dioxygenases, peroxidases and lipases (Lyu et al., 2014;
96 Pandey et al., 2012). The catabolic enzymes like oxygenases, dehydrogenases (DH), and
97 peroxidases (POD) are involved in PAH-transformation/degradation under aerobic conditions
98 (Sakshi et al. 2021a; Sakshi et al. 2021b), and catabolic genes such as catechol 2,3-
99 dioxygenase (C23O), naphthalene dioxygenase (NAH), and polycyclic aromatic hydrocarbon-
100 ring hydroxylating dioxygenase (PAH-RHD), etc. are responsible for producing PAH-
101 catabolising enzymes (Sakshi et al. 2020). Genetic investigation of microbial species studied
102 during the degradation process reported the presence of PAH-catabolic genes which are
103 responsible for degradation (Van Hamme et al. 2003; Wang et al. 2007), but most of the
104 studies lack the analysis of expression of PAH-catabolic genes during degradation (Hesham
105 et al. 2014; Sangkharak et al. 2020). Since the catabolic genes are involved in the PAH-
106 degradation pathway, identification and expression of such genes during degradation of
107 multiple PAHs may provide a better perceptive of the degradation pathway. In this study, the
108 catabolic gene expression of NAH, C23O, and PAH-RHD in selected bacterial strains isolated
109 from petroleum-contaminated soil during degradation of a mixture of PAHs containing three-
110 ring PAHs and four-ring PAHs (phenanthrene, anthracene, fluorene, and pyrene) was
111 examined. Further, catabolic enzyme activity i.e. C23O, DH, and POD was also investigated
112 during degradation analysis.
113
114 Materials and Methods
115 Chemicals, culture medium, and bacterial species
116 Three-ring PAHs (Phenanthrene-PHEN, anthracene-ANTH, fluorene-FLUO) and
117 four-ring PAH (pyrene-PYRN) with ≥98% purity, and acetonitrile (HPLC grade) were
118 purchased from Sigma-Aldrich, India. The analytical grade (AR) chemicals used to prepare
119 mineral salt medium (MSM: KNO3, KH2PO4, K2HPO4.3H2O, (NH4)2SO4, NaCl,
120 MgSO4.7H2O, CuSO4.7H2O, and FeSO4.7 H2O), other chemicals, and silica (100-200 mesh

3
121 size) were procured from Thermo Fisher Scientific, India. The MSM was prepared as
122 reported in earlier studies (Sakshi et al. 2021a, Sakshi et al. 2021b), and the bacterial strains
123 Kocuria flava DTU-1Y (MTCC 13093) (Kf) and Rhodococcus pyridinivorans DTU-7P
124 (MTCC 13094) (Rp) having the capability to use PAH compounds as a source of carbon for
125 their growth with accession number (16S rRNA): MN841976 and MN841977, respectively,
126 (Sakshi et al. 2020) were used for degradation of multiple PAHs in a mixture in the present
127 study.
128
129 Bacterial degradation of PAHs
130 Assessment of degradation of mixed PAHs (PHEN, ANTH, FLUO, and PYRN) was
131 conducted in MSM broth under sterilized and controlled conditions in the laboratory during
132 15 days of the incubation period at regular time intervals of 3 days. Bacterial strains (Kf and
133 Rp) were used to prepare the inoculum for degradation of mixed PAHs in MSM. Each
134 selected bacterial strain was grown individually in MSM broth till exponential growth
135 (absorbance of 0.6 at OD600) was reached. The MSM broth (100 ml) supplemented with a
136 mixture of four PAHs (PHEN, ANTH, FLUO, and PYRN dissolved in acetone, each 2.5 mg
137 L-1) to attain a concentration of 10 mg L -1 of mixed PAHs and aseptically inoculated with
138 MSM broth (1% (v/v)) containing bacterial cell suspension individually and in a consortium
139 of both strains each having OD600 of 0.6) for degradation study. An abiotic control (blank)
140 was also prepared in a similar way without any inoculum. Each flask containing bacterial
141 monoculture, consortium, and blank was incubated (30°C with 120 rpm) in a shaking
142 incubator for 15 days. The degradation study was performed in triplicates. The residual
143 concentration of mixed PAHs from each flask was obtained using high-performance liquid
144 chromatography (HPLC). To assess residual PAHs during degradation process, 5 ml of
145 sample from each flask was taken. The PAH from each sample was extracted thrice
146 thoroughly with dichloromethane (DCM). The extracts were concentrated by evaporating
147 DCM under dark conditions. Conclusively the extracted residual mixed PAHs were
148 quantified by HPLC (Shimadzu, LC-20AD) (Haritash and Kaushik 2016; Sakshi et al.
149 2021b).
150 The kinetics of degradation of mixed PAHs was assessed using first-order kinetics
151 (Kachieng'a and Momba 2017) and the bacterial biomass growth was also examined during
152 degradation study by measuring the absorbance (OD600) (Labtronics, model LT-290) of
153 medium.
154
155 Determination of catabolic Enzyme activity
156 The catabolic enzyme activities for catechol 1,2-dioxygenase (C12O), catechol 2,3-
157 dioxygenase (C23O)), dehydrogenase (DH), and peroxidase (POD) during degradation were
158 analysed over a UV–Visible spectrophotometer (double beam, Lab India make UV 3092
159 model). A volume of 5 ml bacterial cell suspension was taken during degradation process for
160 enzyme extraction as reported earlier (Sakshi et al. 2021a, Sakshi et al. 2021b). Briefly, to
161 examine C12O and C23O activities, catechol was used as a substrate and the enzymatic
162 reaction of C12O and C23O were analyzed by monitoring the increase in absorbance at 260
163 nm and 375 nm due to products cis-cis muconic acid and 2-hydroxymuconic semialdehyde
164 formation, respectively. For POD enzyme activity 5 mM guaiacol and 0.6 mM hydrogen
165 peroxide were used as substrate and the POD enzymatic reaction was analyzed by monitoring
166 increased absorbance at 470 nm due to the product (tetraguaiacol) formation. The DH
167 enzyme activity analysis was done using 2,3,5-triphenyl tetrazoilum chloride (TTC), which
168 was reduced to red-colored triphenyl formazan (TPF) and TPF concentration was measured at
169 OD484 (Sakshi et al. 2021a; Sakshi et al. 2021b).

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170 Analysis of catabolic gene expression
171 The selected bacterial strains having the capability for PAH-degradation were studied
172 for catabolic gene expression for C23O, PAH-RHD, and NAH involved in the degradation of
173 PAHs. For gene expression study, during mixed PAH-degradation, 25 ml of bacterial cell
174 suspension of each strain was taken aseptically from each flask on the 15 th day of the
175 incubation period. Total RNA from bacterial strains was isolated using Trizol method of
176 RNA isolation (Rio et al. 2010) followed by DNase treatment to remove contaminations of
177 DNA molecules. The purified RNA was stored at -20°C till further analysis at Superworth
178 Biodiscoveries, Delhi, India, for gene expression study by quantitative reverse
179 transcription polymerase chain reaction (RT-qPCR). For catabolic gene expression
180 examination by RT-qPCR, gene-specific primers giving an amplicon of 150-300 bp and
181 anneals at 60°C temperature (Table 1) were designed using Integrated DNA Technologies
182 (IDT) real-time assay design (https://www.idtdna.com/scitools/Applications/RealTimePCR/).
183 The quality of purified RNA samples was analyzed on a 1.2 % denaturing agarose gel run in
184 MOPS (3-(N-morpholino) propane sulfonic acid) buffer and quantified using NanoDrop 8000
185 Spectrophotometer (Thermo Scientific). A total of 500 ng of total RNA was used in the
186 cDNA preparation reaction using the iScript kit (BioRad) following the manufacturer’s
187 protocol. The total obtained cDNA was used and diluted up to10 ng/µl and 50 ng was used in
188 total reaction mixture of 20 µl (Table 2) containing Power SYBR® Green PCR Master Mix
189 (Life Technologies).
190 The RT-qPCR was performed on a real-time PCR system i.e. StepOnePlus™ Real-
191 time PCR system (Thermo Fisher Scientific, Applied Biosystems™). The total 20 µl reaction
192 mixture for RT-qPCR contained 10 µl 2X Sybr green buffer, 5 µl cDNA diluted to 10 ng/µl,
193 1 µl primer 10 µM Fwd, 1 µl primer 10 µM Rev, and 3µl nuclease-free water. For each
194 reaction, three technical replicates were set in a 96 well format. Simultaneously, a no
195 template control (NTC) for each set of primer was kept to put a check for contaminants or
196 non-specific amplicons or primer dimmers, if any. The thermal cycling program was set as 10
197 min at 95 °C for enzyme activation, 10 sec at 95 °C for cyclic denaturation, and 30 sec at 60
198 °C for annealing/extension, for 40-45 cycles. A dissociation curve/melting curve analysis was
199 carried out from 55°C-95°C in 0.1 °C increments, each lasting for 5 sec, to ensure the
200 presence of a specific product. CT values for each gene were recorded to examine gene
201 expression. The RNA concentration in different samples was normalized using housekeeping
202 gene 16S rRNA transcript abundance for both the microbial strains.
203
204 Results
205 Degradation of mixed PAHs
206 Degradation experiments showed that these bacterial strains can efficiently degrade a
207 mixture of PAHs containing LMW PAHs i.e. PHEN, ANTH, FLUO as well as HMW PAH
208 i.e. PYRN, ranging from 56%-59% after 15 days. As far as available data, this is the first
209 report to examine the capability of Kf and Rp to use PAH-mixture as the carbon source for
210 their growth. Both strains were found to have great potential in utilizing mixed PAH
211 compounds as the sole carbon source as revealed by their growth on a mixture of PAHs in
212 MSM (Fig. 1). Biodegradation of mixed PAHs was maximum during the first nine days of
213 incubation by monoculture and consortium of Kf and Rp It was observed that the degradation
214 ability of Kf was slightly lower than that of consortium and Rp. After 15 days, the strains Kf,
215 Rp, and consortium degraded 55.6%, 59.5%, and 59.1% of 10 mg L-1 of mixed PAHs,
216 respectively. The strain Rp had maximum degradation efficiency for mixed PAHs, however,
217 the degradation efficiencies of Rp and consortium were almost similar. Bacterial growth
218 during degradation had a significant role to understand the degradation abilities of bacterial

5
219 strains. It was observed that the biomass of studied bacterial strains was steadily increased
220 during the initial days of incubation and maximum biomass growth of Kf and Rp was
221 achieved during six to nine days which matched with the PAH degradation profile.
222 Stabilization of microbial biomass/growth from 12th day onward resulted in gradual
223 stabilization of degradation as well. This may be attributed to limited/scarce availability of
224 growth nutrients (by the 15th day) in the MSM used for degradation studies. HPLC analysis
225 of initial and final concentration confirmed significant degradation of mixed PAHs within 15
226 days (Fig. 1). The maximum degradation was attained during the logarithmic growth phase of
227 bacterial strains resulting in more than 43% mixed PAH-degradation within first 9 days.
228 The linear graphs of the natural log of mixed PAHs concentration against the time of
229 incubation (Fig. 3) were plotted for Kf, Rp, and consortium with R2 values of 0.98, 0.93, and
230 0.95, respectively. For mixed PAH-biodegradation the rate constant k were 0.0572 day -1,
231 0.0618 day-1, and 0.0595 day-1, respectively for Kf, Rp, and consortium, and half-life for
232 biodegradation were 12.1, 11.2, and 11.6 days, respectively.
233
234 Enzyme activity
235 As a part of this study, the function of catalytic enzymes during the degradation of
236 mixed PAHs was examined. Catabolic enzyme activity for C12O, C23O, DH, and POD
237 during degradation by monocultures of Kf and Rp, and consortium were analyzed at 0, 3, 6, 9,
238 12, and 15 days of incubation period when samples were investigated for PAH-degradation.
239 In the current investigation, it was observed that none of the bacterial strains exhibited C12O
240 activity during degradation. Both bacterial strains were found to express C23O, DH, and
241 POD activity during the degradation. The range of enzyme activity for C23O, DH, and POD
242 was noted to gradually enhance during the early degradation phase and drop off to some
243 extent at the 12th and 15th day of incubation (Fig. 4; Table 3). Maximum enzyme activity for
244 C23O, DH, and POD was observed during the 9th day of incubation for Kf and Rp. For Kf
245 maximum catabolic enzyme activity of C23O, DH and POD was 10.8±1.97*10^-4
246 µmol/ml/min, 8.93±0.81*10^-4 µmol/ml/min, and 2.22±0.10*10^-4 µmol/ml/min
247 respectively, for Rp maximum C23O, DH and POD activity was 11.93±1.70*10^-4
248 µmol/ml/min, 12.69±1.41*10^-4 µmol/ml/min, and 1.94±0.10*10^-4 µmol/ml/min,
249 respectively. For consortium enzyme activity for C23O, DH and POD were maximum on the
250 9th day (10.8±1.97*10^-4 µmol/ml/min, 10.34±2.15*10^-4 µmol/ml/min, and
251 1.94±0.19*10^-4 µmol/ml/min, respectively). The activity of POD was observed to be higher
252 than the activity of C23O and DH enzymes for both bacterial strains.
253
254 Catabolic gene expression during PAH-degradation
255 In the present study, three genes i.e. NAH, C23O, and PAH-RHD, along with the 16S
256 rRNA gene as a housekeeping gene, were analyzed to investigate the expression level of
257 three selected genes in two selected bacterial strains during PAH-degradation. The isolated
258 RNA from both bacterial strains was examined over 1.2% MOPS containing denaturing
259 Agarose gel. Two distinct bands, 23S, and 16S rRNA molecules were observed in each of the
260 RNA sample of the strains ensuring the good quality of RNA for cDNA synthesis (Fig. 5A).
261 The purity of isolated RNA was confirmed with DNase treatment to remove the residual
262 DNA, if any. Further, the presence of DNA contamination was checked using 16SrRNA
263 gene-specific primers. No amplicon was observed in the samples (Fig. 5B) indicating DNA
264 free preparation of RNA samples. The RNA samples from each strain were converted to
265 cDNA, followed by analysis for the presence of the 16S rRNA transcript. In both the
266 samples, a specific transcript of 245 bp size (Fig. 5C) was observed indicating that cDNA
267 could be used for expression analysis of the selected genes. The RT-qPCR assay was

6
268 performed using three technical replicates of each sample for which cDNA synthesis was
269 performed separately and independently. The amplification plot and melting curve plots were
270 examined for catabolic genes (C23O, PAH-RHD, and NAH) (Fig. 6). This study shows
271 significant gene expression of the catabolic genes during mixed PAH-degradation by selected
272 bacterial strains. It was observed two genes i.e. C23O and NAH were yielding a specific and
273 single melting curve (Fig. 6A, 6C), whereas, PAH-RHD yielded multiple peaks (Fig. 6B).
274 PAH-RHD gene in both Rp and Kf did not exhibit any gene expression (their melting curves
275 exhibited a mixture of peaks). C23O and NAH gene expression exhibited in Rp and Kf strains.
276
277 Discussion
278 Based on this study, catabolic enzymes producing bacterial strains Kf and Rp were
279 found to efficiently degrade the mixture of PAHs. Both strains were found to produce C23O,
280 DH, and POD enzymes during degradation of a mixture of PAHs signifying the crucial role
281 of these strains in the metabolism of PAHs. It was analysed that the biodegradation efficiency
282 of the studied bacterial strains for a PAH compound was not affected by the existence of
283 other PAH compounds and bacterial strains have the ability to utilise different PAHs
284 simultaneously when present in a mixture. Some other studies on degradation of mixed PAHs
285 by bacterial strains examined simultaneous utilization of different PAHs present as a mixture
286 (Kelley and Cerniglia 1995; Juhasz et al. 1996). The range of three-ring PAHs (ANTH,
287 PHEN, and FLUO) and four-ring PAH (PYRN) degradation in the mixture was 53%-61% in
288 15 days of the incubation period. The strain Kf degraded 54.1%, 60.7%, 53.3% of 2.5 mg L-1
289 of three-ring PAHs (PHEN, ANTH, and FLUO, respectively), Rp degraded 60.0%, 61.1%,
290 61.2%, whereas, consortium of both strains degraded 59.8%, 60.8%, 60.3% of 2.5 mg L -1 of
291 respective PAHs. Kf, Rp and consortium degraded 53.2%, 55.9%, 55.7% of 2.5 mg L-1 of
292 PYRN, respectively (Fig. 2). The consortium of both strains exhibited similar degradation
293 efficiency with Rp and slightly higher degradation efficiency as compared to Kf indicating
294 that there was no inhibitory or synergistic consequence of the strains over one another. The
295 degradation efficiency of studied bacterial strains for three-rings PAHs and four-ring PAH
296 investigated in this study is observed to be comparable to previous studies (Sakshi et al.
297 2021a; Sakshi et al. 2021b) where both the strains were found to have the ability to utilize
298 PHEN, ANTH, FLUO, and PYRN as the source of carbon when present as a single PAH
299 compound or as a mixture of PAHs. The degradation efficiency of both strains (monoculture
300 and consortium) was more than 53% for three-ring and four-ring PAHs (10 mg L -1) when a
301 single PAH compound was studied using the same microbial strains. The degradation
302 efficiency of 55.1%, 59.0%, and 63.5% for PHEN, ANTH, and FLUO (10 mg L -1)
303 respectively, was observed with Kf, whereas, Rp could degrade 62.0%, 65.0%, and 66.8% of
304 PHEN, ANTH, and FLUO (10 mg L-1). The 61.3%, 64.7%, and 66.6% degradation of 10 mg
305 L-1 of PHEN, ANTH, and FLUO was observed with consortium of both strains within 15
306 days of incubation (Sakshi et al., 2021a). The Kf, Rp, and consortium were observed to
307 degrade 53.8%, 56.2%, and 56.5% of 10 mg L-1 of PYRN, respectively (Sakshi et al. 2021b).
308 In the present study, Kf, Rp, and consortium degraded 55.6%, 59.5%, and 59.1% of 10 mg L -1
309 of mixed PAHs, respectively, in liquid MSM within 15 days of incubation. The linear plots of
310 the natural log of mixed PAHs concentration against the time of incubation (Fig. 3) indicate
311 first-order reaction kinetics similar to a study by Dutta et al., 2017, where first-order reaction
312 kinetics was studied during degradation of mixed PAHs. The degradation kinetics indicates
313 that the bacterial strains used in the degradation of mixed PAHs have considerable potential
314 towards the eradication of hazardous mixture of PAHs from polluted environment.
315 The catabolic enzyme activity investigated in the present study for C23O, POD, and
316 DH enzyme was found similar to catabolic enzyme activity investigated for bacterial strains

7
317 Kf and Rp for the presence of C23O, DH, and POD activity which involved the degradation
318 of individual three-ring PAHs (PHEN, ANTH, and FLUO) and four-ring PAH (PYRN)
319 (Sakshi et al. 2021a; Sakshi et al. 2021b). Catalytic enzymes such as dioxygenases and
320 peroxidase have an important role in the oxidation of PAHs (Pinyakong et al. 2000; Sakshi et
321 al. 2020), and dihydroxy aromatic intermediates (formed during aromatic-ring oxidation) get
322 rearomatized by DH to possible formation of diols making the ring susceptible to opening by
323 the subsequent activity of dioxygenases (Margesin et al. 2000). Ring-cleaving dioxygenases
324 play a significant role in the process of degradation of aromatic compounds by O2
325 incorporation which results in aromatic ring oxidation (Goyal and Zylstra 1996). As the
326 activity of C23O enzyme was examined during the biodegradation process, thus meta-
327 cleavage through benzene ring-opening by C23O was most common for the lower pathway of
328 PAH degradation among these isolates (Meyer et al. 1999). In the present study, the activity
329 of the three catabolic enzymes were examined simultaneously during the degradation of
330 mixed PAHs (PHEN, ANTH, FLUO, PYRN) by Kf and Rp. The results of the present study
331 signify that bacterial strains Kf and Rp have the capability to remove/degrade a mixture of
332 PAHs in the natural environment where these pollutants are present in a mixture and have a
333 great potential for use in bioremediation.
334 The RT-qPCR has also been used in previous studies on degradation of aromatic
335 pollutants to examine the catabolic gene expression, and it is induced in the presence of
336 substrates. The RT-qPCR technique was used to study the expression of catabolic genes
337 (phdF, phdI, pcaG, and pcaH) responsible for pyrene degradation in M. gilvum PYR-GCK
338 strain during degradation of pyrene (Badejo et al. 2013). The catabolic gene
339 (nidAB and phdFIJ) expression in four different strains of Mycobacterium sp. was analysed
340 during phenanthrene and pyrene degradations as single and mixed-PAH (Hennessee and Li
341 2016). Increased expression level of a catabolic gene (baaA) related to PAH-degradation was
342 examined in Rhodococcus sp. P14 when grown with ANTH, PYRN, PHEN, or benz[a]-
343 anthracene (Peng et al. 2018). The CT values (Table 4) of the catabolic genes were also
344 analyzed in Rp and Kf in the present study. Both of the strains showed a similar level of
345 expression of the housekeeping gene i.e. 16SrRNA, which is relevant for the housekeeping
346 gene (internal control) to normalize the catabolic gene expression. After calculating the log
347 (base 2) expression values using 2-ddCT method (Livak and Schmittgen 2001), it was observed
348 that C23O gene showed a 6.02 log fold higher expression in Kf in comparison to Rp and NAH
349 gene exhibited a 7.9 log fold higher expression in Rp in comparison to Kf (Fig. 7). The study
350 concluded that the C23O gene expressed higher activity in Kf, and NAH gene expressed
351 higher activity in Rp. Significant catabolic gene expression for C23O and NAH during
352 degradation of mixed-PAHs concluded that Kf and Rp are efficient HMW-PAHs degraders
353 and can be utilised for the development of an efficient bioremediation method for cleaning of
354 PAH-contaminated environmental matrix.
355 The outcomes of gene expression analysis indicated that the presence and expression
356 of catabolic genes for PAH-degradation in PAH-degrading bacterial strains may be suitable
357 for the biodegradation process and can make available opportunities for analysis of bacterial
358 abilities to degrade PAHs. The study also concludes that microorganisms exposed/adapted to
359 higher concentration of PAHs may develop expression of PAH-catabolising genes. The
360 genetic studies help in the elucidation of degradation pathways involving catabolic genes
361 which encode catabolic enzymes responsible for PAH-degradation.
362
363 Conclusion
364 Based on this study, catabolic enzymes producing bacterial strains Kf and Rp were
365 found to efficiently degrade the mixture of PAHS. Both strains were found to produce C23O,

8
366 DH, and POD enzymes during degradation of a mixture of PAHs signifying the crucial role
367 of these strains in the metabolism of PAHs. The microbial transformation of mixed PAHs is
368 observed through meta-cleavage of benzene ring by C23O and subsequent oxidation by POD
369 enzyme produced by both bacterial strains. Significant catabolic gene expression for catechol
370 2,3-dioxygenase (C23O) and naphthalene dioxygenase (NAH) during degradation of mixed-
371 PAHs concluded that Kf and Rp are efficient HMW-PAHs degraders and can be utilised for
372 the development of an efficient bioremediation method for cleaning of PAH-contaminated
373 environmental matrix. The study also concludes that microorganisms exposed/adapted to
374 higher concentration of PAHs may develop expression of PAH-catabolising genes. The
375 genetic studies help in the elucidation of degradation pathways involving catabolic genes
376 which encode catabolic enzymes responsible for PAH-degradation.
377
378 References
379 Afzal M, Yousaf S, Reichenauer TG, Kuffner M, Sessitsch A (2011) Soil type affects plant
380 colonization, activity and catabolic gene expression of inoculated bacterial strains during
381 phytoremediation of diesel. J Hazard Mater 186(2-3): 1568-75. doi:
382 10.1016/j.jhazmat.2010.12.040. Epub 2010 Dec 15. PMID: 21216097
383 Agency for Toxic Substances and Disease Registry (ATSDR) (1990) Toxicological profile
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509
510 Statements & Declarations
511 Funding
512 The authors declare that no funds, grants or other support was received during the preparation
513 of the manuscript.
514
515 Competing interest
516 The authors declare that they have no known competing financial interests or personal
517 relationships that could have appeared to influence the work reported in this paper.
518
519 Author Contributions
520 The study was conceptualised and executed by Sakshi and Anil Kumar Haritash; and drafted
521 by all the authors together.
522
523 Ethical approval
524 This article does not contain any studies with human participants or animals performed by
525 any of the authors.
526
527 Data Availability Statement
528 All data generated or analysed during this study are included in this article.
529
530
531 Figure Captions
532 Fig. 1 Dynamics of microbial growth (MG) against PAH-degradation, and HPLC
533 chromatogram of initial and residual concentration of PAHs
534 Fig. 2 Degradation of phenanthrene, anthracene, fluorene, and pyrene individually and
535 mixed PAHs after 15 days at initial concentration of 10 mg/L
536 Fig. 3 The graphs for mixed PAH-degradation indicating first-order reaction kinetics
537 Fig. 4 Catabolic enzyme activities ((*10-4) µmoles/ml/min) with average residual mixed
538 PAHs (PHEN, ANTH, FLUO, and PYRN) during biodegradation
539 Fig. 5 Agarose gels showing quality of RNA isolated (5A); Check for DNA
540 contamination in RNA preparation (5B) and a control RT-PCR for 16S rRNA showing
541 amplification of specific size amplicons (5C)
542 Fig. 6 Melting curves and amplification plots showing the specific and non-specific
543 amplifications of C23O, PAH-RHD and NAH in R. pyridinivorans and K. flava samples
544 Fig. 7 Relative quantification of C23O and NAH catabolic gene expression in Kocuria
545 flava and Rhodococcus pyridinivorans following induction by a mixture of PAHs (PHEN,
546 ANTH, FLUO, and PYRN) on the 15th day of the incubation period

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