Environmental Samples
The sample used in the present degradation study was collected from natural sources (soil) in and around Durgapur Steel Plant (DSP), West Bangal (WB), India. The site is in the location of Durgapur of latitude 51°50'43.8'' north and longitude 8°16'35.8'' west in the state of WB, India. Soil samples consisted of blackish fine to medium sub-angular gravel in the upper surface including fine sand and a high content of iron flecks. Samples were collected in a clean, sterile plastic container, transferred to the laboratory and stored at room temperature until further use for analysis.
Chemicals and Reagents
All chemicals and reagents were of analytical grade and were used as received without any further purification from local manufacturer, India. Milli-Q water (Elix 3, conductivity 0.12 mho) was used in the preparation of aqueous solution of reagents. However autoclaved double distilled water was used for microbial culture purposes.
Screening, characterization and sequence accession of morpholine degrading isolate
For the initial isolation and cultivation of bacteria, 10-10 fold serial diluted samples were spread onto nutrient agar (NA) plates. NA plates were prepared according to manufacturer’s instruction. Specific colonies obtained were sub-cultured further to find the pure bacterial colony. The selected pure bacterial isolate was identified based on morphological and molecular characterization. Morphological characterization was achieved by visual observation of colony in term of appearance, shape, color, arrangement, and motility by standard staining procedure and check growth in selected media (Hichrome M1353). Further, the pure colony was then identified by 16s rRNA analysis. In order to verify the phylogenetic affiliation of the selected isolate, a single colony was collected for DNA isolation (InstaGeneTM Matrix Genomic DNA isolation kit (Bio-Rad Catalog # 732-6030) as per the kit instruction and procedure) and subjected to Polymeric Chain Reaction (PCR) analysis using primers targeting the 16s rRNA gene [27F (AGAGTTTGATCMTGGCTCAG) and 1492R (TACGGYTACCTTGTTACGACTT)]. PCR (20 µL) contained 8 µL of Taq DNA Polymerase Master Mix, 1 μL each of 10 μM stock 27F/1492R primers, 9 µL DDW and 1 µL DNA template. PCR (MJ Research Peltier Thermal Cycler; Bio-Rad PTC 200) reaction was performed using following conditions as per Rahamat Unissa et al [12]. DNA was denatured at 94°C for 5 min, followed by 35 cycles of amplification, each of 94°C for 45 sec (denaturation), 55°C for 60 sec (annealing) and 72°C for 60 sec, (extension) followed by 10 min 72°C (final extension). The PCR product was sequenced (by Yaazh Xenomics, Chennai, Tamilnadu, India) and the 16s rRNA sequence was blast using NCBI blast search tool. The phylogeny analysis of the sequence with the closely related sequence of blast results was performed by multiple sequence alignment. The program MUSCLE 3.7 was used for multiple alignments of sequences [13]. The resulting aligned sequences were cured using the program Gblocks 0.91b. This Gblocks eliminates poorly aligned positions and divergent regions (removes alignment noise) [14]. Finally, the program PhyML 3.0 aLRT was used for phylogeny analysis and HKY85 as substitution model.
The nucleotide sequence of the isolated bacterium was deposited in GenBank, NCBI and was assigned the accession number having 2 letters and 6 numerals. [15].
Cultivation and acclimatization of isolate: Natural selection against morpholine
Bacterial inoculums were prepared by transferring the selected pure colonies aseptically to 10 ml enrichment media called Knapp buffer or mineral salt solution (MSS) having KH2PO4 = 100 mg, K2HPO4 = 100 mg, (NH4)2SO4 = 100 mg, MgSO4.7H2O = 4 mg and FeCl3 = 0.2 mg supplemented with 0.1% morpholine as described by author itself earlier [16]. Culture tubes were incubated at 37oC and 150 rpm for 1-2 weeks and absorbance at 600 nm was taken at different time points as a measure of growth. Based on the growth, when an optical density (OD) of 0.5 was reached (data not shown), a 1:100 dilution of culture was made with Knapp buffer and diluted culture was further spread on MSS Agar (amended with 2% Agar + 0.10% morpholine) to confirm the adaptation of isolate against morpholine stress. Further, the growing culture was centrifuged at 6500 rpm for 10 min and pellet was re-suspended in the Knapp buffer with gradual increase concentration of morpholine (at an interval of 0.01%) up to 0.20%. These were called as seeded acclimatized bacterial inoculums. For each increased acclimatization study, the tested bacteria were grown in MSS broth supplemented with increased concentration of morpholine and respective MSS Agar plate with same concentration of morpholine to confirm the acclimatization against increased morpholine concentration.
The acclimatized inoculums was later grown in presence of intermediate degradative product of morpholine to inspect the fact that this particular isolate either follows pathway 1 or 2. This can be further validated by performing in-vitro chemical and analytical assay of intermediate product of morpholine degradation in culture filtrate. Lastly, estimation of ammonia/ammonical nitrogen in cultural filtrate has been carried out to draw a conclusion for the complete degradation of morpholine by this isolate following the concern pathway.
Growth and Chemical tests of different hypothetical degradation intermediate compounds
The growth of isolate on various substrates (intermediate degradation products) was investigated by adding the corresponding compounds (0.15%) to MSS. The pH of the medium was adjusted to 7 and growth was carried out at 370C and 150 rpm for 48 hours in triplicate test tubes. At different time points absorbance was measured in term of OD (optical density) to establish that these degradation products might be formed and if so, it would support the growth of the isolated bacteria in their presence.
Chemical tests of degradation products mainly monoethanolamine (primary amine) and morpholine (secondary amine) were carried out by standard Simon- 1 (Rimini test) and Simon-2 (modified Rimini test) respectively to distinguish primary and secondary amine [17] in culture filtrate. The assay is based on the fact that amine undergoes nucleophilic addition reaction with nitroprusside in presence of acetaldehyde or ketone to give a characteristic color of blue and violet in case of primary and secondary amine respectively.
Gas Chromatography (GC) and Mass spectroscopy (MS) studies of degradation intermediate/s
A GC system (Shimadzu GC-2010) equipped with standard oven, injection ports, flame ionization detector (FID) and column RTX-35 (30 mm x 0.32 mm x 1µm film thickness) with nitrogen as carrier gas by direct injection at split condition has been used for analysis of monoethanoline (MEA). The analytical parameters for analysis of monoethanolamine was summarized in Table 1 as per method (modified with change in column and its parameter) reported by Gerster FM et al [18]
Standard solution of MEA of 0.125 to 0.5% (prepared in methanol) was injected along with processed culture supernatant (1:10 with methanol) as per method developed below. GC of test samples was run against media blank and positive control to estimate the presence of monoethanolamine in the culture filtrate (discuss elsewhere in the manuscript) by analyzing the area under curve (AUC), given by the machine.
Table 1 GC parameters for the estimation of monoethanolamine
Parameters
|
Specificity
|
Column and its configuration
|
RTX-35 30m x 0.32mm x 1µm
|
Oven/Column temperature
|
Initial 60 0C
Hold: 1 min
Ramping rate: 30◦C/min
Final tem: 240◦C hold for 3 minutes
Linear velocity: 37.6 cm/sec (for nitrogen)
|
Injector port
|
Temperature: 200◦C
Split Ratio: 30:1
Injection Volume: 1µl
|
Carrier gases
(Mobile Phase)
|
Column Gas Flow: 2 ml/min
Purge Flow: 1 ml/min
Hydrogen: 40 ml/min
Zero air: 400 ml/min
Nitrogen: 15 ml/min
|
Stationary phase
|
60% Dimethyl polysiloxane and 35% Diphenyl polysiloxane
|
Detector
|
Flame ionization detector @ 300◦C
|
Analysis time
|
10.0 min
|
Software
|
GC solution
|
Workstation
|
Window 8
|
MS system of an integrated LCMS (Liquid Chromatography Mass Spectroscopy) instrument (Shimadzu LCMS-2020) equipped with inlet interface, ion source, mass analyzer and detector has been used for analysis of degradation products of morpholine based on their m/z peak. The analytical parameters for estimation of morpholine degradation product are summarized in Table 2.
Table 2 MS operating parameters for intermediate/s
Parameter
|
Specificity
|
Ionization
|
electron spray ionization ESI
Needle voltage 4.5 kv
|
Interface temperature
|
350ºC
|
Heat block temperature
|
200ºC
|
Sheath/Drying gas flow rate
|
15 l/min
|
The nebulizer gas flow rate
|
1.5 l/min
|
Acquisition time
|
2 min
|
Acquisition mode
|
Positive/Negative
|
Scan m/z 50-200
|
Scan speed 52 unit/sec
|
Sampling acquisition time 1.56 Hz (640 msec)
|
Detector
|
Electron multiplier
|
Software
|
Lab solutions
|
Workstation
|
Window 7
|
Sample for injection was prepared with solvent (with methanol/acetonitrile; 1:10) and without solvent system as per combourieu et al [4]. Culture sample (5 ml) was centrifuged at 10,000 rpm for 10 min and supernatant was filtered through 0.22 µm pore size nylon filter (Axiva Sichem Biotech, India) to avoid any bacterial cells. 1 ml neat filtrate (1:10 diluted) was directly used to inject into MS system. Similarly, MSS culture media was also prepared accordingly.
Estimation of ammonia
The presence of ammonia in culture supernatant was estimated by standard Nessler’s method [19]. This method involves the coupling of ammonium to Nessler’s reagent to produce a yellow color under strong alkaline condition. A resulting yellow color was formed in proportion to ammonium (NH4+-N) concentration and was measured at 425 nm using an Elisa reader (ELX50/8MS BioTek India) against reagent blank and expressed as mg/L (ppm). A standard solution of 10 ppm of NH4+-N were prepared by dissolving 4.773 mg ammonium chloride in 125 ml of double distilled water to make it further dilution to a concentration of 1-5 ppm NH4+-N solution. A calibration curve was plotted and ammonia has been quantified in accordance with standard curve.