Abstract: A bacterial strain, DN1, degrading p-nitrophenol (PNP) was isolated from garden soil by selective enrichment in M63 medium. Repeated subculturing in Nutrient Agar (NA) plates, NA slants and Basal Salts Medium (BSM) containing PNP (BSM+ PNP) led to isolation of pure colonies. The organism is Gram negative, aerobic, catalase positive, oxidase positive and rod shaped with mostly single arrangement. It shows bluish green pigmentation on various specialized media such as Pseudomonas P medium, Pseudomonas F medium, Modified F medium, Pseudomonas Isolation Agar (PIA) and HiFluoro Pseudomonas Agar. DN1 gave positive results with motility, citrate utilization, urease, Nitrate Reduction (NR) and gelatin liquefaction tests but negative results with Methyl Red (MR),Voges Proskauer (VP) and indole tests. It was casein hydrolysis and lipase positive but starch hydrolysis negative. Acid production from carbohydrates tested (glucose and lactose) was negative. It can grow at 42°C but not at 4°C and tolerates <5% NaCl concentration. Optimum pH for PNP degradation was found to be 7.0. Among several media tested such as M9, M63 and BSM, BSM was found to be the optimum medium for biodegradation of PNP. DN1 could degrade upto 100 mg L-1 PNP using the xenobiotic as sole carbon or carbon and nitrogen sources. On the basis of gross morphological, micromorphological, physiological and biochemical tests DN1 was definitively identified as Pseudomonas aeruginosa strain DN1. To our knowledge this is the first report of a Pseudomonas aeruginosa strain able to degrade p-nitrophenol (PNP).
INTRODUCTION
Nitroaromatics are environmentally significant xenobiotics widely used as or in the production of dyes, explosives, pesticides, herbicides, polymers, plasticizers and solvents (Kulkarni and Chaudhari, 2007; Ye et al., 2004; Spain, 1995; Marvin-Sikkema and de Bont, 1994; Spain and Gibson, 1991; Bruhn et al., 1987). They pose serious health and environmental risks as majority of them are highly toxic to human beings, animals, plants and microorganisms. Several nitroaromatic compounds are powerful carcinogens (Kulkarni and Chaudhari, 2007) and several of them are listed as priority pollutants (US EPA, 2007). Nitroaromatics are important industrial chemicals, with estimated annual production of 108 tons.
p-Nitrophenol (PNP) is a nitroaromatic compound widely used as raw material in the manufacture of pesticides, pharmaceuticals and dyes etc. It is also a breakdown product of the degradation of parathion and methyl parathion, organophosphate pesticides widely used as agricultural insecticides in developing countries including India. The US EPA (2007) has listed PNP along with several other nitroaromatics as priority pollutants. Thus biodegradation studies of PNP are of prime importance (Ningthoujam, 2005).
Microbial degradation of PNP has been reported for several bacteria including strains of Arthrobacter and Nocardia (Hanne et al., 1993), Arthrobacter sp. (Jain et al., 1994), Arthrobacter protophormae (Chauhan et al., 2000), Bacillus sphaericus (Kadiyala and Spain, 1998), Brevibacterium linens (Ningthoujam, 2005), Moraxella (Spain et al., 1979), Nocardiodes nitrophenolicus (Yoon et al., 1999), Pseudomonas sp. (Munnecke and Hsieh, 1974), Pseudomonas cepacia (Prakash et al., 1996), Pseudomonas putida (Loser et al., 1998; Kulkarni and Chaudhari, 2006), Rhodobacter capsulatus (Roldan et al., 1998), Sphingomonas sp. (Zablotowicz et al., 1999). Towards the aim of isolating robust and novel PNP degrading strains we have set up enrichment cultures from several pristine and contaminated sites. We have recently isolated a new p-nitrophenol degrading Pseudomonas strain DN1 from garden soil by selective enrichment in M63 medium. The present study deals with the isolation and characterization of this PNP degrading strain.
MATERIALS AND METHODS
Enrichment
The bacterial strain was recently isolated from a local garden soil. Soil
sample was collected from the Biochemistry Department Campus, Manipur University,
Canchipur, India. Aerobic shake flask cultures were set up in M63 medium containing
(L-1), 5.8 g Na2HPO4, 3.0 g KH2PO4,
0.5 g NaCl, 1.0 g NH4Cl, with 20 mg L-1 PNP. It was adjusted
to pH 7.0 and inoculated with filtered soil suspension (10% v/v) derived from
10 g garden soil mixed with 100 mL distilled water and shaken (170 rpm) for
1 h. The enrichment culture was incubated at ambient temperature.
Isolation of PNP Degrading Species
After 2 to 3 months of incubation, 0.1 mL inoculum from the enrichment flask
was taken and spread on sterile NA plates and incubated at 30°C (24-48 h).
If the growth of the colony was slow, it was kept for further incubation. Visible
colonies were then picked and subcultured on NA plates and slants. We obtained
four different isolates exhibiting whitish, transparent colourless, white (branching
filaments) and bluish green colonies.
On further investigation, the bluish green strain (DN1) was found to be a promising PNP degrader. Degradation was monitored by visible turbidity and/or disappearance of characteristic yellow colour of PNP. PNP depletion was also monitored by following the absorbance of alkalinized culture supernatants at 405 nm (data not shown). The biodegrading strain was then subcultured for several generations in BSM (L- 1), 0.25 g FeCl3. 6H2O, 22.5 g MgSO4.7H2O, 27.5 g CaCl2, 40.0 g (NH4)2SO4 and Phosphate buffer (pH 7.0) containing various concentrations of PNP. It was maintained on NA slants with or without PNP and on BSM slants with or without PNP.
Morphological, Biochemical and Physiological Tests
Gross morphology was observed by visual inspection. Micromorphology was
observed by light microscopy (PRIOR, UK). Motility was determined using hanging
drop method (Gunasekaran, 2000). Catalase activity was determined by bubble
production in H2O2 solution. Oxidase activity was determined
by oxidase discs (HiMedia, India) as well as by oxidation of 0.2% 2, 6- Dichlorophenolindolphenol
in 0.1% ascorbic acid. Hydrolysis of casein and starch was determined as per
standard procedures (Gunasekaran, 2000). Citrate utilization, Indole, MR, VP,
NR, Gelatin Liquefaction tests, Urea Broth test for production of urease, Peptonization
of Ulrich milk etc. were also determined (Gunasekaran, 2000; Cappuccino and
Sherman, 2004). Growth with or without the production of pigmentation was determined
with various growth media such as LB (Luria Bertani), Pseudomonas F medium (proteose
peptone; 20 g L-1, tryptone; 10 g L-1, K2HPO4;
1.5 g L-1, MgSO4.7H2O; 0.73 g L-1,
glycerol; 10.0 g L-1, agar; 15.0 g L-1, pH 7.0), Pseudomonas
P medium (Atlas, 1997), Modified F medium (peptone; 20 g L-1, K2HPO4;
1.5 g L-1, MgSO4.7H2O; 1.5 g L-1,
agar; 15.0 g L-1, pH 7.2), Pseudomonas Isolation Agar (PIA) [HiMedia
(http://www.himedialabs.com),
India], HiFluoro Pseudomonas Agar (HiMedia, India). Growth on succinate medium
with or without FeCl3 and acetamide agar medium were also tested
to see if this strain can utilize succinate and acetamide as carbon sources
with production of greenish blue pigmentation. All other tests, when not specifically
mentioned, are as per Bergey’s Manual of Determinative Bacteriology and
other standard procedures.
Optimization of Medium and Substrate Tolerance Limits
Various culture media (M63, M9 and BSM) were tested to find out the most
suitable medium for PNP degradation. For study of substrate tolerance limits,
various acclimation studies at different concentrations of PNP were determined
with or without added nutritional supplements e.g., Yeast Extract (YE). Effects
of various inoculum sizes for further optimization of degradation were also
studied.
Nitrite Assay
Nitrite (NO¯2) liberation during PNP degradation was assayed
colorimetrically according to standard protocols (Montgomery and Dymock, 1961;
Ningthoujam, 1998).
RESULTS AND DISCUSSION
Morphological, Biochemical and Physiological Tests
DN1 forms round, smooth, bluish green colonies with entire margins and convex
elevations. The organism is gram negative, exhibiting rods with mostly single
arrangements. The gram negative property was further confirmed by growth on
MacConkey’s agar and Eosin Methylene Blue (EMB) agar and also positive
result in 3% KOH (String) test. It was motile, non-spore former and can grow
at 42°C but not 4°C. Various biochemical and physiological tests (Table
1) showed that DN1 is catalase, urease, casein hydrolysis, NR, citrate utilization,
gelatin liquefaction, oxidase and lipase positive and starch hydrolysis, lactose
fermentation, MR, VP and indole negative. Based on these tests, PNP degrading
DN1 isolate is definitively identified as Pseudomonas aeruginosa which
was further confirmed by culturing it on media specific for Pseudomonas
species (Table 2) such as Pseudomonas P medium, Pseudomonas
F medium, Modified F medium, Pseudomonas Isolation Agar (PIA) medium, HiFluoro
Pseudomonas Agar. The organism has been designated as Pseudomonas aeruginosa
strain DN1. It can use succinate, acetamide and phenol as carbon sources as
shown by growth tests with succinate medium, acetamide agar medium and BSM containing
phenol.
| Table 1: | Biochemical and physiological tests of the PNP degrading isolate Pseudomonas aeruginosa DN1 |
| MR: Methyl Red, VP: Voges Proskauer, NR: Nitrate Reduction | |
| Table 2: | Growth on various media |
| Table 3: | Degradation time (h) of 20-100 mg L-1, PNP with and without 0.02% Yeast Extract (YE) |
| Table 4: | Effect of inoculum size on degradation of PNP (20 mg L-1) |
| Table 5: | Nitrite released at different time intervals during complete degradation of 50 mg L-1 |
Optimization and Substrate Tolerance Limit
Among various culture media tested such as M63 (Gunasekaran, 2000), M9 (Ford
et al., 1994) and BSM (Ningthoujam, 1998); BSM was found to be the most
satisfactory medium for degradation studies. Initial acclimation studies indicated
that degradation time for 20-100 mg L-1 PNP took about five days
to one week. On further acclimation, degradation occurred in 24 to 72 h when
PNP was used as sole carbon and energy source (Table 3). That
prior exposure to the xenobiotic (PNP) leads to accelerated biodegradation has
been shown earlier (Shinozaki et al., 2002; Labana et al., 2005).
To further optimize PNP degradation, studies were undertaken to establish the
effects of various inoculum sizes (Table 4) on the degradation
rate.
Nitrite Liberation
Nitrite was released almost stoichiometrically during degradation of 50
mg L-1 of PNP by DN1 (90% of nitrite were released at 62 h, Table
5 and Fig. 1). Aerobic degradation of PNP is accompanied
with oxygenolytic removal of nitro groups in the first or subsequent steps with
hydroquinone (HQ) or nitrocatechol (NC) as intermediates of degradation (Spain
and Gibson, 1991; Jain et al., 1994; Kadiyala and Spain, 1998; Chauhan
et al., 2000). Present study also confirms oxygenolytic removal of the
nitro group during PNP degradation. However, detailed characterization of the
degradative pathway awaits further experimentation.
| Fig. 1: | Standard graph for |
Present studies showed that DN1 can degrade PNP upto 100 mg L-1 though there was increasing lag periods corresponding with increasing substrate concentrations. It can use PNP, succinate, phenol and acetamide as C sources but not m-nitrophenol, o-nitrophenol and other higher nitrophenols. It can also use PNP as sole nitrogen source. DN1 thus seems to be an interesting strain of Pseudomonas aeruginosa degrading PNP. Though Pseudomonas aeruginosa strain has been reported earlier as a PNP degrader in the context of a biodegrading consortium (Daughton and Hsieh, 1977), this is the first report-to our best knowledge of a pure culture of Pseudomonas aeruginosa that can biodegrade PNP.
DN1 seems to be a promising addition to the repertoire of PNP degrading microbial isolates and may be a potential agent for biodegradation of nitroaromatic xenobiotics and for possible production of value-added compounds of biotechnological significance (e.g., catechols or siderophores). Studies are now being undertaken to optimize PNP biodegradation and analyze substrate range, optimum pH and metabolic pathways for PNP degradation. Detailed biochemical and genetic characterizations of DN1 will form the target of our further studies, towards the aim of exploiting it for bioremediation or production of biotechnological products (DN1 produces a blue-green siderophore with potential antimicrobial activity, data not shown here).
Further studies on biodegradation of PNP (kinetics, substrate range and xenobiotic tolerance limits etc.) or PNP as part of mixtures with other co-contaminant xenobiotics are necessary as PNP may exist in the environment in various concentration ranges as well as mixtures with other nitroaromatic or non-nitroaromatic xenobiotics.