A Bacillus Sp. Strain Bgsc1 That Efficiently Degrades p-Nitrophenol

ISBN: 978-93-7143-285-6 174 Chapter - 10 A Bacillus Sp. Strain Bgsc1 That Efficiently Degrades p-Nitrophenol Dr. Ningthoujam Shovarani Deptt. of Environmental Sciences, Y.K. College, Wangjing-795148, Thoubal District, Manipur Corresponding Email: [email protected] ABSTRACT: A bacterium with rhizoid pattern of growth degrading pnitrophenol (PNP) was isolated from local garden soil. This strain was identified as a member of the genus Bacillus based on morphological, biochemical, physiological and 16S rDNA sequence analyses. The strain BGSC1 is a gram-positive rod arranged in fragmented chain forms. It is positive in catalase, oxidase, casein hydrolysis, starch hydrolysis, and tyrosine clearing tests. The strain produces acid from glucose and sucrose and shows positive results in nitrate reduction, gelatin liquefaction, Tween 80 hydrolysis, and glucose O/F tests. 16S rDNA sequence analysis established strain BGSC1 as most closely related to Bacillus anthracis and designated as Bacillus sp. strain BGSC1. Neutral pH (7.0) was found to be the optimal pH for degradation of PNP. PNP conc. was measured at 400nm and nitrite was released almost stoichiometrically along with depletion of PNP during the biodegradation process. To our knowledge BGSC1 strain may be a potent agent for biodegradation of nitroaromatic xenobiotics as well as a good A Bacillus Sp. Strain Bgsc1 That Efficiently Degrades 175 promising addition to the repertoire of PNP degrading microbial isolates. Keywords: Bacillus anthracis, P-nitrophenol, Biodegradation, Nitroaromatic compound, Xenobiotics 1. Introduction Nitroaromatic compounds exist in nature due to their extensive use in the production of dyes, plastics and explosives and also as intermediate products from partially transformed insecticides, organophosphate pesticides and herbicides (Qureshi et al., 2001). They exhibit high toxicity and/or mutagenicity to plants, animals and microbes and pose health and environmental risks, either directly or through some of their catabolic metabolites (Rehman et al., 2007). Several nitroaromatic compounds are powerful carcinogens (Kulkarni and Chaudhari, 2007) and several of them are listed as priority pollutants (US EPA, 2007). p-Nitrophenol (PNP) is a nitroaromatic compound and US EPA (2007) has listed PNP along with several other nitroaromatics as priority pollutants. It is also a breakdown product of parathion and methyl parathion, organophosphate pesticides widely used in developing countries including India. Thus, biodegradation studies of p-nitrophenol (PNP) are of prime importance (Debananda and Ningthoujam, 2008). Recent research had reported several bacteria capable of degrading PNP including the strains of Arthrobacter and Nocardia (Hanne et al., 1993), Bacillus sphaericus (Kadiyala and Spain, 1998), Brevibacterium linens (Ningthoujam, 2005), Dr. Ningthoujam Shovarani 176 Moraxella (Spain et al., 1979), Nocardiodes nitrophenolicus (Yoon et al., 1999), Rhodobacter capsulatus (Roldan et al., 1998), Sphingomonas sp. (Zablatowicz et al., 1999), Pseudomonas aeruginosa strain DN1 (Ningthoujam and Shovarani, 2008). We have recently isolated a new p-nitrophenol degrading Bacillus sp. strain BGSC1 by selective enrichment in M63 medium. The present study deals with the isolation, characterization and biodegradation study of this PNP degrading strain. 2. Materials and Methods 2.1 Media and cultural conditions M63 medium had the following composition (per litre): 5.8g Na2HPO4, 3.0g KH2PO4, 0.5g NaCl, 1.0g NH4Cl (Gunasekaran,2000) and was used for enrichment studies. Basal Salts Medium (BSM) had the composition (per litre): 0.25g FeCl3.6H2O, 22.5g MgSO4.7H2O, 27.5g CaCl2, 40.0g (NH4)2SO4 and phosphate buffer (pH 7.0) and was used for degradation studies in liquid as well as solid media. The sole carbon source used in cultivation media was p-nitrophenol (20mg/L). The culture temperature was kept at ambient conditions with shaking at 170 rpm and pH 7.0. 2.2 Enrichment and Isolation A local garden soil sample was used as an inoculum for enrichment of PNP-degrading microorganisms. Aerobic shake flask cultures were set up in M63 medium (pH, 7.0) and inoculated with filtered soil suspension (10% v/v) derived from 10g garden soil mixed with 100ml distilled water and shaken for 1 hour at ambient temperature. After 2 to 3 months of A Bacillus Sp. Strain Bgsc1 That Efficiently Degrades 177 incubation, 0.1mL aliquot from the enrichment flask was taken and spread on sterile Nutrient Agar (NA) plates and incubated at 300C (24-48 h). Visible colonies were picked up and then subcultured on NA plates and slants. We obtained four different isolates growing as bluish green, white (with rhizoid pattern), tan color and whitish colonies. On further investigation, two strains were found to be promising PNP degraders i.e. bluish green strain now reporte d as Pseudomonas aeruginosa DN1 (Ningthoujam and Shovarani, 2008) and white strain with rhizoid pattern of growth (BGSC1). Degradation was monitored by visible turbidity and/or disappearance of characteristic yellow color of PNP. PNP depletion was also monitored by following the absorbance of alkalinized culture supernatants at 400 nm. The biodegrading strains were then sub-cultured for several generations in BSM containing various concentrations of PNP. It was maintained on NA slants and BSM + PNP slants. 2.3 Characterization of BGSC1 2.3.1: Phenotypic characterization Phenotypic characteristics include all those features of a strain such as morphological, biochemical and physiological properties. Phenotypic characterization of the strain BGSC1 was done according to standard protocols (Halebian et al., 1981; Gunasekaran, 2000; Cappuccino and Sherman, 2004; Atlas, 1997, Atlas, 2010.Gross morphology was observed by visual inspection. Growth was observed at different pH, temperatures and salt concentrations on NA plates as well as on various growth media such as peptone water agar, tap water agar, 0.1% Dr. Ningthoujam Shovarani 178 yeast agar, acetamide agar etc. All the other tests, when not specifically mentioned, are as per Bergey’s Manual of Determinative Bacteriology and other standard procedures. Liquid cultures of BGSC1 were analyzed with the Bio Merieux API 50CH test strips as per manufacturer’s guidelines for carbohydrate utilization. 2.3.2 Genotypic characterization 2.3.2.1. 16S rDNA sequencing The 16S rRNA gene sequence of the strain BGSC1 was submitted to EzTaxon server (Chun et al., 2007) and aligned with the 16S rRNA gene sequences of other Bacillus species using Clustal X2 version 2.1 (Larkin et al., 2007). Phylogenetic analysis was done using the software package MEGA version 4.0 (Tamura et al., 2007). Distances (using distance options according to Kimura’s two-parameter model; Kimura, 1983) were calculated and clustering was performed with the neighbour joining method (Saitou and Nei, 1987). To determine the support of each clade, bootstrap analysis was performed with 1000 resamplings (Felsenstein, 1985). The 16S rDNA sequence of BGSC1 was performed with the help of Institute of Microbial Technology (IMTECH) Chandigarh, India. 2.4. Biodegradation studies of p-nitrophenol 2.4.1. Identification of maximum absorbance for PNP For the identification of maximum absorbance for PNP, 50 mg/L was used as the standard. It was then scanned through UV-VIS spectrophotometer at various wavelengths and the maximum absorbance for PNP was determined. A Bacillus Sp. Strain Bgsc1 That Efficiently Degrades 179 2.4.2. Optimization of medium Optimization of medium is necessary for effective degradation of xenobiotics. Various minimal media were examined for PNP degradation at pH 7.0 such as BSM (Ningthoujam, 1998), Minimal Salts Medium (MSM) (Liu et al., 2007) and Minimal Medium (MM) (Li et al., 2008). 5% inoculum from the cell suspension of PNP induced cells (A600 = 1) were inoculated in the different media and the degradation times were measured. Among the media tested the medium in which degradation occurred in minimum time was selected as the optimal medium for further degradation studies. 2.4.3. Effect of pH on degradation Optimization of different environmental and cultural parameters is necessary along with medium optimization for efficient biodegradation. pH, temperature, salt concentration etc. are some of the parameters influencing biodegradation. Various pH values (pH 5.5-12) were checked for their effects on degradation. Optimum pH for degradation of PNP (50 mg/L) was then determined. 2.4.4. Effect of different inoculum sizes on degradation Inoculum density is also one of the cultural conditions that affect biodegradation. Sufficient amount of inoculum is necessary for successful degradation. Effect of different inoculum sizes on degradation of PNP was studied to identify the optimum inoculum size for effective PNP degradation. Dr. Ningthoujam Shovarani 180 2.4.5. PNP depletion assay To determine the time course of PNP biodegradation, samples were collected at various time intervals. The samples were then centrifuged at 8,000 rpm for 15 minutes and the supernatants were collected. The pH of the supernatants was brought to 10.0 using 1N NaOH. The supernatants were checked for PNP depletion by measuring the absorbance at 400 nm. 2.4.6. Nitrite liberation assay Nitrite liberation during PNP degradation was measured spectrophotometrically according to standard protocol (Montgomery and Dymock, 1961: Ningthoujam 1998). It was determined through the formation of a reddish purple dye produced by coupling sulphanilic acid and αnapthylamine and used by comparing values with those of standard curve prepared using sodium nitrite. Higher concentration of NO2can be determined by diluting the samples. 3. Results 3.1. Isolation of BGSC1 From enrichment culture, four different isolates were obtained exhibiting bluish green, dirty white (with rhizoid pattern of growth), pitch colour and whitish colonies. Three isolates were found to be promising PNP degraders. Of these, BGSC1 is the PNP degrading isolate which formed rhizoid pattern of growth (Fig A). A Bacillus Sp. Strain Bgsc1 That Efficiently Degrades 181 Fig A: BGSC1 growing on peptone water agar 3.2. Phenotypic characterization of BGSC1 3.2.1. Morphological characterization BGSC1 colony was whitish with dry surface. The colonies were flat and rhizoid (having margins with root-like filaments). The organism was gram positive, exhibiting rods with fragmented chain arrangement. The organism was non-motile with ellipsoidal endospores located centrally. The fragmented chain arrangement was further confirmed by microscopic observations using methylene blue, crystal violet and negative staining procedures (Table 1a). It showed luxuriant filamentous and fragmenting mycelia on poor nutrient media such as 0.1% yeast agar, peptone water agar and tap water agar and luxuriant filamentous growth on Muller Hinton agar, Nutrient agar, Sabouraud agar and Streptomyces agar (Table 1b). Table 1a: Morphological characteristics of BGSC1 Sl.No. Tests Results 1. Colony Morphology i) Elevation Flat Dr. Ningthoujam Shovarani 182 ii) Margin` Rhizoid pattern iii) Surface Dry iv) Colour Dirty white 2. Gram’s Reaction +ve i) Shape Rod ii) Arrangement Fragmented chain 3. Spore Production Ellipsoidal endospore 4. Motility -ve 5. String test (3% KOH) -ve 6. Capsule Stain -ve 7. Motility -ve 8. Methylene blue stain, crystal violet stain, negative stain Rods in fragmented chains Table 1b: Growth characteristics of BGSC1 on various media Sl. no. Growth media Growth result 1. Peptone water agar Growth with branching fibres (rhizoid form) within 24hr. 2. Tap water agar Thin growth with light branching fibres (rhizoid form) 3. Yeast(0.1%) agar Thin growth with branching fibres (rhizoid form) within 24 hr. 4. Nutrient agar Growth with branching fibres (rhizoid form) within 24 hr. A Bacillus Sp. Strain Bgsc1 That Efficiently Degrades 189 Dr. Ningthoujam Shovarani 190 Fig. B: Phylogenetic tree based on 16S rRNA gene sequences showing the position of strain BGSC1 among the type species of the genus Bacillus. Bootstrap values (in percentage) are shown at the node of each branch 3.4 Biodegradation studies of p-nitrophenol 3.4.1. Optimization of medium Among various culture media tested such as MSM, MM and BSM. BSM was found to be the optimal medium for degradation of PNP by BGSC1 (Table 2a). Among the various concentrations of BSM media tested, normal strength BSM was observed to be optimal for degradation of PNP and it was used as the standard medium for further degradation studies (Table 2b). Table 2a: Degradation of PNP (50 mg/L) in various media at pH 7.0. Sl. no. Medium Degradation time (h) Final OD (A600) nm 1 BSM 39. 8 ± 0.115 0.083 ± 0.007 2 MSM 42.3± 0.347 0.066 ± 0.024 3 MM 42.3 ± 0.824 0.085 ± 0.006 A Bacillus Sp. Strain Bgsc1 That Efficiently Degrades 191 (Note: Initial OD at 600nm = 0.0640.070) Table 2b. Degradation of PNP (20 mg/L) in various concentrations of BSM at pH 7.0. (Note: Initial OD at 600nm = 0.065-0.070.) 3.4.2. Effect of pH on degradation Effect of pH on degradation of PNP (20 mg/L) is shown in Table 3. Degradation of PNP occurred between pH 6 - 10 but no degradation was observed at pH 5.5 and pH 11.0. Optimal pH for degradation of PNP by BGSC1was found to be pH 7.0. Table 3. Effect of pH on degradation of PNP (20 mg/L). pH values Degradation time (h) Final OD (A600) nm Rate of PNP degradation mg L-1 h-1 5.5 No degradation ND ND 6.0 37.4 ± 0.108 0.065 ± 0.010 0.53 7.0 19.3 ± 0.108 0.086 ± 0.002 1.04 8.0 20.3 ± 0.216 0.087 ± 0.004 0.99 Sl. no. Medium Degradation time (h) Final OD (A600) nm 1 BSM 24.1 ± 0.414 0.080 ± 0.010 2 5X BSM 24.5 ± 0.374 0.085 ±0.008 3 10x BSM 31.0 ± 0.148 0.072 ±0.015 Dr. Ningthoujam Shovarani 192 9.0 21.1 ± 0.674 0.088 ± 0.007 0.95 10.0 24.7 ± 0.738 0.071 ± 0.008 0.81 11.0 No degradation ND ND (Note: Initial OD at 600 nm = 0.072-0.077; ND= Not Determined.) 3.4.3. Effect of different inoculum sizes on degradation 5% inoculum was found to be optimal for degradation of PNP (20 mg/L). Detailed findings on the effects of inoculum sizes on PNP biodegradation are shown in Table 4 and Fig C. Table 4. Effect of different inoculum sizes on degradation of PNP (20 mg/L) Inoculum sizes (%) Degradation time (h) Initial OD (A600) nm Final OD (A600) nm Rate of PNP degradation mgL-1 h-1 1 32.6± 0.071 0.014±0.005 0.014 ± 0.001 0.613 2 27.4 ± 0.492 0.036± 0.010 0.023 ± 0.006 0.729 4 21.6 ± 0.070 0.058±0.007 0.043 ± 0.012 0.925 A Bacillus Sp. Strain Bgsc1 That Efficiently Degrades 193 5 19.8 ± 0.247 0.065±0.004 0.060 ± 0.001 1.010 6 19.9 ± 0.105 0.068±0.002 0.070 ± 0.005 1.005 7 19.9 ± 0.050 0.075±0.005 0.075 ± 0.003 1.005 Fig. C: Effect of different inoculum sizes on degradation of PNP (20 mg/L). Dr. Ningthoujam Shovarani 194 3.4.4. PNP depletion assay The time course for degradation of PNP (20 mg/L) with and without nitrogen source are shown in Figs D(a) and D(b). The complete degradation of PNP (20 mg/L) was observed at 22 h in BSM with nitrogen source (Table 5 and Fig Da) and at 20 h in BSM without nitrogen source (Table 5 and Fig Db). Fig.D(a): Absorption spectra of PNP (20mg/L) in BSM medium without N-source at various time periods Fig. D(b): Absorption spectra of PNP (20 mg/L) in BSM medium with N-source at various time periods. A Bacillus Sp. Strain Bgsc1 That Efficiently Degrades 195 3.4.5. Nitrite Liberation Nitrite was released almost stoichiometrically during degradation of 20 mg/L of PNP. 98.4% of nitrite was released at the end of degradation (Table 6a and Fig Fa) in presence of nitrogen source and 91.1% in absence of nitrogen source (Table 6b and Fig Fb). The concentration of nitrite was calculated based on the standard curve as shown in Fig E. Table 6a: Nitrite liberation in BSM with n-source Time (h) A543 (nm) NO2-(µmoles/L) Percentage (%) 0 0.012 0.6 0.42 4 0.122 7.2 5.00 8 0.411 24.2 16.83 12 0.705 41.4 28.79 16 0.834 48.9 34.01 18 0.867 51.6 35.89 19 0.880 52 36.16 20 0.988 58.4 40.62 22 1.068 63 43.81 24 1.180 70 48.68 26 1.760 104 72.33 28 2.070 122 84.85 30 2.185 129 89.72 32 2.270 134 93.20 34 2.310 136.5 94.94 36 2.400 141.5 98.42 Dr. Ningthoujam Shovarani 196 Fig. F(a): PNP depletion and nitrite liberation in BSM with N-source Fig. F(b): PNP depletion and nitrite liberation in BSM without N-source Table 6b: Nitrite liberation in BSM without N-source Time (h) A543 (nm) NO2- (µ moles/L) Percentage (%) 0 .004 0.2 0.14 4 .247 14.6 10.15 8 .671 39.6 27.54 12 1.280 75 52.16 16 1.335 78 54.25 18 1.360 81 56.33 19 1.385 81.5 56.68 20 1.410 83.5 58.07 22 1.475 86.5 60.16 A Bacillus Sp. Strain Bgsc1 That Efficiently Degrades 197 24 1.550 91 63.29 26 1.565 92 63.99 28 1.605 95 66.07 30 1.695 100 69.55 32 2.044 119.7 83.25 34 2.060 122 84.85 36 2.220 131 91.11 Fig. E: Standard graph for NO2 – assay. 4. Discussion BGSC1 is an aerobic, gram positive, rod-shaped, spore forming and non-motile organism. The colonies show rhizoid growth on agar plates. Similar morphotypes of rhizoidal growth exhibited by some Bacillus spp. have been reported in the Dr. Ningthoujam Shovarani 198 literature (Flugge, 1886; Gause, 1939; Nakamura and Jackson, 1995; Nakamura, 1998; Aarthi and Ramana, 2011). Cells are arranged in fragmented chains. The strain showed luxuriant filamentous growth on poor nutrient media such as peptone water agar, yeast extract agar and tap water agar. The organism showed positive results in catalase, oxidase, casein and starch hydrolysis tests. Acid was produced from carbohydrates such as glucose and sucrose but not from lactose and maltose. BGSC1 did not hydrolyze lipid and could not utilize citrate. Aarthi and Ramana (2011) reported Bacillus mycoides and Bacillus cereus strains which showed biochemical and physiological characteristics similar to those of BGSC1. Nakamura (1998) also reported a strain of Bacillus pseudomycoides which showed characteristics similar to that of BGSC1. Overall, most of the biochemical characteristics of BGSC1 resembled those of Bacillus cereus group as reported by Claus and Berkeley (1986). The optimum temperature for growth of BGSC1 was 300C. Similarly, Ajithkumar et al. (2002) had also reported that the optimum temperature for growth of Bacillus funiculus was 300C but in contrast a strain of Bacillus pseudomycoides showed optimum temperature for growth at 280C (Nakamura, 1998). BGSC1 could grow at pH 5.0 – 9.0 with optimum at pH 7.0. Ajithkumar et al. (2001) and Saman et al. 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