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African Journal of Environmental Economics and Management ISSN 2375-0707 Vol. 3 (5), pp. 244-248, October, 
2015. Available online at www.internationalscholarsjournals.org © International Scholars Journals 

 

Author(s) retain the copyright of this article. 

 

Full Length Research Paper 
 

A study of metabolism of para-nitrophenol in 
Arthrobacter sp. SPG 

 
Akash Mukul Jyothi   

 
Department of Environmental Sciences, School of Life Sciences, University of Hyderabad, P.O Central University, 

Hyderabad-500 046. Email : akash_jyothi@gmail.com 
 

Accepted 22 April, 2014 
 

Arthrobacter sp. SPG utilized p-nitrophenol (pNP) as the sole carbon, nitrogen, and energy source. The 
stoichiometric amounts of nitrite ions were released from pNP. The pathway of pNP degradation was studied for 
strain SPG. On the basis of thin layer chromatography, high performance liquid chromatography and gas 
chromatography-mass spectrometry, hydroquinone (HQ) was identified as major intermediate product. HQ 
dioxygenase activity was found in crude extract of pNP induced cells of strain SPG that suggested the cleavage 
of HQ into gamma-hydroxymuconic semialdehyde. This study clearly showed that strain SPG degraded pNP via 
HQ pathway. Arthrobacter sp. SPG degrades various nitroaromatic compounds including pNP, 2-chloro-4-
nitrophenol, 3-methyl-4-nitrophenol, 2-nitrocatechol and 2-nitrobenzoate. This strain would be a suitable 
candidate for bioremediation of nitroaromatic compounds contaminated sites. 
 
Keywords: Arthrobacter sp. SPG, Biodegradation, Bioremediation, hydroquinone, metabolism, p-nitrophenol, 
nitroaromatic compounds, sole carbon and energy source 
 
 
INTRODUCTION 
 
p-nitrophenol (pNP) is an environmental pollutant that is 
used for the manufacturing of dyes, explosives, 
pesticides, herbicides and drugs (Spain, 1995). It is also 
a hydrolytic product of the pesticide parathion (Munnecke 
and Hsieh, 1974). It may cause a blood disorder that 
reduces the ability of the blood to carry oxygen to tissues 
and organs due to methemoglobinemia (ATSDR, 1992). It 
is a potent uncouple of oxidative and photosynthetic 
phosphorylation. Further, it is also considered as 
mutagen (Cooper et al., 1997). Due to its high toxicity, 
United State Environmental Protection Agency (USEPA) 
has listed it as a priority pollutant.  

Bacteria that use pNP as sole carbon and energy 
source have been isolated and characterized (Mitra and 
Vaidyanathan, 1984; Hanne et al., 1993; Spain, 1995; 
Wan et al., 2007). Aerobic degradation of pNP was 
occurred either formation of hydroquinone (Spain and 
Gibson, 1991) or formation of nitrocatechol (Kadiyala and 
Spain, 1998). Previously, it has been supposed that 
Gram-positive bacteria degrade pNP via nitrocatechol 

 
 
 

 
pathway whereas Gram-negative utilize pNP via 
hydroquinone pathway. Literature studies showed that 
both Gram-positive and Gram negative bacteria can 
degrade pNP either via hydroquinone pathway or via 
nitrocatechol pathway (Jain et al., 1994; Hanne et al., 
1993; Spain, 1995; Pakala et al., 2006; Chauhan et al., 
2007). In this communication, we reported the 
degradation of pNP via hydroquinone pathway by  
Arthrobacter sp. SPG 
 
 
MATERIALS AND METHODS 
 
Isolation Of A pNP Degrading Bacterium 
 
Strain SPG was isolated from the soil collected from a 
pesticide contaminated site, India by enrichment method 
using 4NP as a substrate. Strain SPG utilized pNP as 
sole carbon and energy source. Strain SPG was 
screened to find out its degradation capacity for various  



Jyothi          244 
 
 

 
nitroaromatic compounds. For screening, strain SPG was 
streaked on minimal agar plates containing 0.3 mM test 
compound as sole carbon and energy source. Minimal 
agar plates were prepared by dissolving the 
followingcompounds in 100 mL of double distilled water: 

0.4 g Na2HPO4, 0.2 g KH2PO4, 0.08 g (NH4)2SO4, 0.08 g 

MgSO4.7H2O, 0.1 mL trace element solution and 1.8 g 

agar. The composition of trace element solution was 
exactly same as described previously (Arora and Jain, 
2011a). The media was autoclaved at 15 lbs for 20 min. 
After autoclave, the desired concentration (0.3 mM) of the 
filter sterilized test compound was added to the media 
and media was allowed to cool at room temperature and 
poured into petri plates. 2-Nitrophenol, 2-chloro-4-
nitrophenol, 3-methyl-4-nitrophenol, 3-nitrophenol, 2-
amino-4-nitrophenol, 4-nitrocatechol and 2-nitrobenzoate 
were used as test compounds. Decolourization and/or 
growth of strain SPG on minimal agar plates was/were 
considered as positive results. 

 
 

 
UV radiation and sprayed with Folin-Ciocalteu’s reagent. 
HPLC analysis was carried out using a Waters 600 model 
high performance liquid chromatography (HPLC) 
equipped with a photodiode array detector system. The 

compounds were separated on a C18 reverse-phase 
silica column using 1% glacial acetic acid in methanol 
and 1% glacial acetic acid in HPLC grade water at a ratio 
of 80:20 as the mobile phase. Flow rate was 1.0 mL/min; 
injection volume was 15 µL, and the compounds were 
detected at 280 nm and 300 nm.  

GC-MS analysis was carried out using a GC-MS-
QP5000 instrument (Shimadzu, Tokyo, Japan) equipped 
with quadrupole mass filter and DB-1 capillary column 
with ionization of 70 eV, scan interval 1.5 s and mass 
range of 50-550 Da. The column temperature was initially 
increased from 90°C to 180°C at the rate of 5°C/min and 
then from 180°C to 280°C at the rate of 10°C/min. The 
carrier gas (nitrogen) flow rate was 10 mL/min. 

 
 
Identification Of Bacteria 
 
Strain SPG was identified by the 16S rRNA gene 
sequencing using the universal primers as described 
previously (Arora et al., 2011). 
 
 
Growth Of Strain SPG On PNP 
 
Strain SPG was grown on 1L Erlenmeyer flask containing 
250 mL minimal media and 0.3 mM pNP as sole carbon 
and energy source. The flask was incubated at 30°C 
under shaking condition at 200 rpm. Samples were 
collected at regular intervals and growth of 
microorganism was measured by taking optical density at 
600 nm. For estimation of nitrite release, samples were 
centrifuged at 8000 rpm and supernatant was used for 
nitrite detection by classical method (Spain and Gibson, 
1991). 
 

 
Identification Of Metabolites 

 
Samples collected at different intervals were centrifuged 
and supernatants were extracted with ethyl acetate. 
Extracted samples were analyzed by thin layer 
chromatography (TLC), high performance liquid 
chromatography (HPLC) and gas chromatography-mass 
spectrometry (GC-MS).  
TLC was performed using pre-coated silica gel 60 F254 
plates (20 X 20 cm, 0.25 mm; Merck, Germany) with  
solvent (toluene: ethyl acetate: glacial acetic acid, 
60:30:5). The compound(s) was/were visualized under  

 
CRUDE EXTRACT PREPARATION 
 
Strain SPG was grown on 200 mL minimal media, 10 mM 
sodium succinate and 0.3 mM pNP. Cells of strain SPG 
were centrifuged just prior to decolourization and washed 
twice with phosphate buffer (20 mM, 7.4 pH) and 
resuspended in the same buffer. The cells were 
sonicated in a sonicator by twenty 30s burst with 
intermittent 30s cooling on ice. The cell extracts were 
centrifuged (12000 rpm) at 4°C for 15 minutes to remove 
cell debris and the supernatant was used for enzyme 
assay. Protein contents were estimated according 
Bradford method (Bradford, 1976). 
 
 
Enzyme Assay 
 
Hydroquinone dioxygenase activity was determined by 
measuring decreased the absorbance of substrate at 289 
nm and increased the absorbance of product at 320 nm. 
The reaction mixure contanined ( in a final volume of 
1mL) 20 mM phosphate buffer, 0.1 mM hydroquinone, 0.1 
mM ferrus sulphate and 0.5-1.0 mg crude extract of the 
proteins. 
 
 
RESULTS 
 
Isolation And Identification Of Strain SPG 
 
A pNP degrading bacterium, strain SPG was isolated from a 
pesticide contaminated site, India and utilized following 
compounds as sole carbon and energy source:  
pNP, 2-chloro-4-nitrophenol (2C4NP), 2-nitrobenzoate, 3-

methyl-4-nitrophenol and nitrocatechol strain SPG was 



245         Afr. J. Environ. Econ. Manage. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

Figure 1. (a) Growth of the strain SPG on pNP and depletion of the pNP by strain SPG. (b) Stoichiometric release of 
nitrite ions when strain SPG grown on minimal salt media and 0.3mM pNP and non-stoichiometric release of nitrite ions 
when strain SPG grown on nitrogen free minimal media and 0.3mM pNP. 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
Figure 2.TLC analysis of samples of pNP degradation 
by Arthrobacter sp. SPG. TLC showing the presence of 
hydroquinone in blue colour spots after spraying with 
Folin-Ciocalteu’s reagent. 
 
 
 
identified as Arthrobacter sp. on the basis of the 16S 
rRNA gene sequencing. The 16S rRNA gene sequence 
of strain SPG has been deposited in the 
 
 
Growth Of Strain SPG On pNP: 
 
When strain SPG was grown on minimal media 
containing 0.3 mM pNP as sole carbon and energy 
source, the yellow color of pNP (0.3 mM final 
concentration) changed to colourless indicating its 
utilization by the microorganism. Microbial growth was 
measured by the increased in optical density (OD) at 600 

 

 
nm and depletion of pNP was measured by taking the OD 
at 420 nm (Figure. 1a). The microorganism released 
nitrite ions in the medium, suggesting the involvement of 
an initial oxidative step in the degradation of pNP. The 
stoichiometric amounts of nitrite ions was observed when 
strain SPG was grown on minimal media containing pNP 
as sole carbon and energy source. When strain SPG was 
grown on nitrogen free minimal media (without GenBank 
database under the accession number 
HM027882.ammonium sulphate) containing 0.3 mM pNP, 
non-stoichiometric release of nitrite ions was observed 
(Figure. 1b). This is due to the rapid utilization of nitrite 
ions as a nitrogen source in the absence of ammonium 
sulphate. This data suggested that strain SPG utilize pNP 
as sole carbon, nitrogen and energy source. 
 
 
 
Metabolic Pathway For pNP Degradation: 

 
TLC analysis indicated the presence of HQ (Rf 0.65) and 
pNP (Rf 0.75) in samples drawn from 8 and 12 h 
intervals. However, the samples drawn after 16 hours 
growth did not show the presence of pNP or another 
intermediate indicating the whole degradation of pNP. 
Furthermore, when TLC plates were sprayed with Folin-
Ciocalteu’s reagent an immediate blue coloration was 
apparent in case of suspected HQ spot since 
dihydroxylated benzene gives an immediate blue 
coloration with this reagent (Figure 2). HPLC analysis 
also showed the presence of HQ along with pNP in the 
samples drawn from 8 and 12 h period (Figure. 3). 

 



Jyothi          246 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

Figure 3. HPLC analysis of samples showing degradation of pNP by 
Arthrobacter sp. SPG with appearance of intermediate hydroquninone. 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
Figure 4. Mass fragement of metabolite hydroquinone. 

 
 

 
However, no pNP or HQ peaks were observed in 16 h 
drawn samples, again suggesting the whole degradation 
of pNP. GC-MS analysis was also carried out to confirm 
the presence of HQ in the degradation pathway of pNP. 
GC-MS(Figure 4). showed that mass fragment of the 
metabolite was observed at 110 m/z that was exactly 
matching that of the standard HQ (Figure. 3). Inhibition 
studies were also carried out using 2,2'-dipyridyl since the 

 
 

 
presence of this compound in the growth mediumchelates 
ferrous ions required for the ring-cleavage of aromatic 
compounds. Strain SPG was grown on minimal media 
containing pNP, sodium succinate and 2,2'-dipyridyl and 
samples were taken at regular interval and extracted with 
ethyl acetate and extracted samples were analyzed by 
TLC and HPLC. Results of this experiment showed that 
there was accumulation of HQ even after 48 h of growth,

.  



    
 

 OH O OH  
 

     

    COOH 
 

    CHO 
 

 NO2 O OH OH 
 

 4-Nitrophenol 1,4-Benzaquinone Hydroqionone γ−Hydroxymuconic semialdehyde 
 

 
Figure 5. Proposed pathway of pNP degradation by Arthrobacter sp. SPG 

 

 
suggesting the inhibition of the ring cleavage of HQ (data 
not shown). To identify the ring cleavage product, 
enzyme assay for HQ dioxygenase activity was carried 
out. The spectrophotometric analysis of HQ dioxygenase 
assay showed that the peak of the HQ at289 nm 
disappeared gradually and a transient peak of 4-
hydroxymuconic semialdehyde around 320 nm appeared. 
 
 
DISCUSSION 
 
Arthrobacter sp. SPG is a versatile bacterium that able to 
degrade various nitroaromatic compound including pNP, 
2C4NP, 3-methyl-4-nitrophenol, nitrocatechol and 2-
nitrobenzoate. This strain would be a suitable candidate 
for bioremediation of a nitroaromatic contamitaned site.  

Several members of the genus Arthrobacter are known 
to degrade phenolic compounds. Arthrobacter 
chlorophonolicus A6 was characterized to degrade 
mixture of phenol, pNP and 4-cholrophenol (Westerberg 
et al., 2000). Arthrobacter ureafaciens CPR706 has been 
reported to degrade 4-chlorophenol via HQ pathway (Bae 
et al., 1996). The degradation of phenol by immobilized 
cells of Arthrobacter citrus has also been achieved 
(Karigar et al., 2006). Arthrobacter sp. SJCon utilized 2-
chloro-4-nitrophenol as the sole carbon and energy 
source (Arora and Jain, 2011b). Furthermore,  
Arthrobacter protophormiae RKJ 100 (Chauhan et al, 
2007), Arthrobacter sp. JS443 (Jain et al., 1994), and 
Arthrobacter aurescens TW17 (Hanne et al., 1993) have 
been reported to degrade pNP. This study also report 
degradation of pNP by Arthrobacter sp. SPG.  

The members of genus Arthrobacter degrade pNP either 
by HQ pathway (Jain et al., 1994) or nitrocatechol pathway 
(Chauhan et al., 2007). In nitrocatechol pathway, pNP was 
converted to nitrocatechol by monooxygenation at ortho 
position. Nitrocatechol was further converted to 1,2,4-
benzentriol (BT) by monooxygenation at para position with 
release of nitrite ions (Chauhan et al., 2007). BT then 
cleaved into maleylacetate by ring cleavage activity of BT 
dioxygenase (Chauhan et al., 2007). In HQ pathway, pNP  

 

 
was first converted to 1,4-benzoquinone (BQ) by a 
monooxygenase and BQ was further reduced to 
hydroquinone (HQ) that was further cleaved to gamma-
hydroxymuconic semaldehyde by HQ dioxygenase (Jain et 
al., 1994). This study clearly showed that Arthrobacter sp. 
SPG degraded pNP via HQ pathway (Figure. 5). 
 

 
CONCLUSION 
 
A pNP degrading bacterium Arthrobacter sp. SPG was 
isolated from soil collected from a pesticide contaminated 
site. Strain SPG utilized pNP as sole carbon, nitrogen 
and energy source and degraded it via formation of HQ. 
Strain SPG was also able to degrade 2-chloro-4-
nitrophenol (2C4NP), 2-nitrobenzoate, 3-methyl-4-
nitrophenol and nitrocatechol. This strain may be used for 
bioremediation of nitroaromatic compounds contaminated 
sites. 
 

 
ACKNOWLEDGEMENTS 
 
Author is thankful to the University Grants Commission, 
New Delhi for financial supports. 
 

 
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