3.1 Isolation of bacteria from rhizospheric soil
The soil sample generated mixed colonies on nutrient agar (NA) plate on initial screening. There were four morphologically distinguished colony that appeared on the NA plate. The control showed no growth on the culture plate. The colonies were succoured as pure cultures and reserved for further screening in glycerol stocks at -80℃.
3.2 Screening of isolates potential for dimethoate degradation
3.2.1 Growth potential in pesticide supplemented media
The pure cultures were screened for dimethoate degradation potential. The initially screened isolates when grown in MSM with pesticide as sole carbon source, yielded one isolate which was found to grow well in pesticide supplemented media. Rest of the isolates (obtained initially in nutrient agar media) showed no growth in this stage of screening and hence were opted out.
3.2.2 Pesticide tolerance and dimethoate degradation
The selected isolate was tested for pesticide toxicity tolerance for a range of 25, 50,100,150,300,500 and 1000ppm. A good growth was observed up to 100ppm concentration of dimethoate and later cases, the cell density has been decreased. Though quite less, yet the isolate growth was observed even in pesticide concentration as high as 1000ppm. Hence, it can be assured that the isolate has good tolerance to pesticide toxicity and can be considered as a potential pesticide-degrading bacteria. Negative control (without inoculum) showed no turbidity, whereas positive control (inoculum with added glucose, no pesticide) showed more turbidity(growth) comparing to the other tubes where pesticide was the sole carbon source. The growth pattern has been demonstrated in Table I and figure I(a) from the observation in UV-spectroscopic reading. The degradation analysis was perceived upto 5 days (FigI (b)) and dimethoate degradation % was calculated as per the formula mentioned in section 2.6. It was found that the strain is capable of degrading 97.6% of dimethoate in 5 days.
Pesticide concentration (ppm)
|
(+) Control
|
(-) Control
|
25
|
50
|
100
|
150
|
300
|
500
|
1000
|
+++
|
-
|
+++
|
+++
|
+++
|
++
|
++
|
+
|
+
|
Table I: Cell density of isolate in different pesticide concentrations
+++very good growth; ++less growth; +negligible growth; - No growth
For further advancements, degradation efficiency of isolate was also performed by FTIR-analysis. Any
difference in spectrum observed between parent compound and isolate-treated sample, might denote a degradation by bond breaking or conformational changes or alterations occurring due to isolate activity on pesticide dimethoate. The spectrum has been shown in figure II(a) and (b). Figure (a) shows the spectrum for parent compound dimethoate. Peaks were noticed at 3330 cm-1, 2930 cm-1, 2850 cm-1, 1721 cm-1, 1470 cm-1, 1050 cm-1, 687 cm-1 and 585 cm-1. Whereas, in the treated sample (figure b), peaks were observed at 3290 cm-1, 2915 cm-1, 2850 cm-1, 1627 cm-1 and 1050 cm-1. Peaks 1470 cm-1, 687 cm-1 and 585 cm-1 had completely disappeared in tested sample which otherwise was seen in spectrum of parent compound. The peak at 1050 cm-1 have been shortened indicating some chemical alterations in phosphate group. There was a shift in peaks ranging between 3000-3500 cm-1, 2500-3000 cm-1, 1500-2000 cm-1 and 500-1000 cm-1. The probable functional group pertaining to the peaks obtained for parent compound were interpreted by the frequencies listed by Nandiyanto et al. [17] and the details have been listed in table II.
Peaks obtained
|
Peak range
|
Probable functional group
|
3330
|
3360-3310
|
N-H stretch
|
2930
|
2935-2915
|
Methylene C-H stretch
|
2850
|
2850-2810
|
Methoxy C-H stretch
|
1470
|
1480-1375
|
CH-Bending
|
1050
|
1050-990
|
Aliphatic phosphate stretch
|
1721
|
1725-1705
|
Ketone
|
687
|
710-685
|
Thiols
|
585
|
705-570
|
Disulphide(C-S) stretch
|
Table II: Details of peak obtained in FTIR-spectrum
3.2 Characterization and identification of the isolate
3.2.1 Morphological and biochemical characterization
The selected strain was observed for morphological characters and Gram staining was performed. The strain WS3 forms white colonies and was found to be rod shaped, positive for Gram stain (Table III). Biochemical characterizations were performed as standard protocols and the observations have been tabulated in table IV.
Table III: Observation of colony morphology and microscopy
Colony morphology
|
Microscopic analysis of isolate
|
Shape
|
Margin
|
Elevation
|
Surface
|
Color
|
Opacity
|
Texture
|
Round
|
-
|
Raised
|
Smooth, mat
|
White
|
Opaque
|
Moist
|
Shape
|
Gram staining
|
Spore
|
Rod
|
Positive
|
-
|
Table IV: Biochemical characterizations of the isolated strain
Biochemical tests
|
Observation
|
Catalase
|
+
|
Urease
|
-
|
Indole
|
-
|
Methyl red
|
+
|
Voges-Proskauer
|
+
|
Oxidase
|
-
|
Citrate utilization
|
+
|
3.2.2 Scanning electron microscopy (SEM) imaging of isolates
SEM micrograph at 7k X and 14K X shows that the isolate is rod shaped in structure (Figure III(a) and (b)). The batch of isolate that was cultured in pesticide treated media was observed to be intact and without any alterations in cell surface morphology. This indicates that the strain bears the capacity to withstand the pesticide influenced environment unlike the cases reported of cell morphology deformities in presence of pesticide stress [28].
3.2.3 Molecular identification by 16S rRNA sequencing
The 16S rRNA sequence analysis was implemented for molecular detection of the culture WS3 and was identified as Bacillus paramycoides, accession number, OR230269, which showed similarity of 100%. The phylogenetic tree has been demonstrated in figure IV.
3.2.4 Effects of environmental conditions on growth of isolates
The isolate was tested against various pH, salt and temperature variations to optimize their ideal growth conditions by UV-spectrophotometer analysis (Table V). The isolate was found to be growing best with pH range 5-7 at 30℃. However, the strain was found to be not so tolerant to high salinity. The isolate had shown good growth at 2% salinity, but the growth was decreased as the salinity was increased. The graphical representation of growth in different salt and pH ranges have been shown in figure V(a) and (b).
Table V: Growth of isolate in response to different environmental conditions
|
Test samples with adjusted conditions
|
Temperature
|
RT(28-30℃)
|
RT(30℃)
|
100℃
|
|
|
|
-
|
+++
|
-
|
|
|
|
pH
|
*
|
3
|
5
|
7
|
9
|
-
|
++
|
+++
|
+++
|
++
|
Salinity (%)
|
*
|
2
|
4
|
6
|
8
|
10
|
-
|
+++
|
++
|
+
|
+
|
-
|
*not adjusted; +++very good growth; ++less growth; +negligible growth; - No growth
3.2.6 Identification of metabolites produced by isolate
The identification of the compounds present in the chloroform crude extract of isolate WS3 was done by GC–MS. The GC-MS spectrum showed the presence of several biologically significant compounds like Dibutyltin dibromide, 5-epi-Callystatin A, 2-hydroxyethaneperoxoic acid exhibiting antimicrobial, anti-cancer and many other pharmaceutical properties. The metabolites are identified on the basis of molecular formula, molecular weight, structure and retention time comparing the MS spectrum database. The chemical compounds obtained are listed in table VI and the chromatogram have been presented in figure VI.
Table VI: Chemical compounds obtained in crude metabolite extract by GC-MS analysis
RT
|
Compound name
|
Molecular weight(g/mol)
|
Molecular formula
|
21.03
|
Dibutyltin dibromide
|
392
|
C8H18Br2Sn
|
22.69
|
5-hydroxy-4-(3-methylbutanoyl)-2,6,6-tris(3-methylbut-2-enyl)cyclohex-4-ene-1,3-dione
|
414
|
C26H38O4
|
22.69
|
5-epi-Callystatin A
|
456
|
C29H44O4
|
22.69
|
Isononyl isooctyl phthalate
|
404
|
C25H40O4
|
22.70
|
2-hydroxyethaneperoxoic acid
|
347
|
C19H25O5N
|
22.71
|
5,8-dihydroxy-2-(4-methylpent-3-enyl)naphthalene-1,4-dione
|
272.29
|
C16H16O4
|
23.73
|
1',1'-Dimethylheptyl-delta-8-tetrahydrocannabinol-11-oic acid
|
400
|
C25H36O4
|
24.73
|
5,6-dihydroxy-1,1-dimethyl-7-(propan-2-yl)-1,2,3,4,4a,9,10,10a-octahydrophenanthrene-4a-carboxylic acid
|
332
|
C20H28O4
|
25.73
|
1,2-benzene dicarboxylic acid
|
334
|
C20H30O4
|
3.2.7 FTIR spectroscopy analysis to detect functional group of metabolites
The peaks obtained in FTIR spectrum (Figure VII) depicts the presence of functional groups associated to the chemical components obtained by GC-MS analysis of crude metabolite extract. The peaks seen in 2855cm-1, 2925cm-1, 2950 cm-1 denotes C-H stretch, 1655cm-1 indicating C=O groups, 1259cm-1 for OH group. There were also peak frequencies obtained for halogen groups like C-Cl (Chloro) at 796cm-1 and C-Br (Bromo) at 654cm-1. The peaks were compared according to standard FTIR peak frequency table.