Unraveling the Bacterial Assisted Degradation Pathway of Morpholine- a Xenobiotic Micropollutant: A Sustainability for Zero Pollution Environment

Biodegradation is the process by which chemicals both natural and xenobiotics are metabolized by microorganisms. Most naturally occurring chemical compounds are biodegradable while xenobiotic may be biodegradable, persistent or recalcitrant. Xenobiotic chemicals, because they are manmade and have developed recently, are present in the environment for comparatively shorter periods of time from its geological presence. This in turn means that the microbial communities present in these environments may not have evolved specic mechanisms for their degradation. Morpholine, a known xenobiotics micropollutant initially believes to be recalcitrant but later prove to be biodegradable by specic set of bacterium species most likely Mycobacterium and Pseudomonas sp in particular. However, the metabolic pathways involved in the successful biodegradation of morpholine stand challenging to establish because of its extreme water solubility and the lack of any chromophore group in morpholine which does not allow easy extraction process. Consequently, no tool for direct estimation of intermediates or metabolites of morpholine has been well reported and only indirect strategies have been developed like presence of microbial growth on intermediates, chemical/analytical assay for intermediate and ammonia measurements to elucidate the degradation pathway for zero pollution environment. In this present study degradation pathway has been ascertained by some selected bacterial isolate for their capacity to degrade morpholine. Based on the said analysis of culture ltrate, it has been revealed that the isolate namely Halobacillus utilizes glycolic route of the metabolic degradation pathway of morpholine and supports the fact that in presence of morpholine, one of two branches of morpholine biodegradation pathway namely ethanolamine and glycolate was was induced while the other branch was inhibited. Whatever the degradation pathway of morpholine exhibited by bacteria, ammonia is the end product of degradation which would be biochemically utilized by isolate.


Introduction
Environmental pollution in any form causes foremost peril to the ecosystem and many health hazards.
Industrialization, urbanization, exploration of natural resources and technological advances are some of the major contributors to release various anthropogenic chemicals like xenobiotics, plasticizers, pesticides, hydrocarbons, detergents, pharmaceuticals etc to air, water and soil. In general, these chemicals are pollutants and organic in nature and may be behave as biodegradable (transformed by biological mechanisms which might lead to mineralization), persistent (fail to undergo bioremediation in an environment or under speci c set of experimental conditions) or recalcitrant (inherently resistant to biodegradation) (Fig. 1). However, most naturally occurring (or biogenic) compounds are biodegradable.
Morpholine (1-oxa-4-azacyclohexane) is one such heterocyclic xenobiotic compound with physical feature of oiliness, volatility and hygroscopic whereas chemically characteristic amine smell and is completely miscible with water as well as with many organic solvents. It has extremely versatile application in different industrial purposes like rubber vulcanization, corrosion control (plating industry), the synthesis of many drugs and personal care products, crop protection agents, glazing agent for coated fruits and vegetables, solvent for lyocell process (textile industry), optical brightener (paper industry). Consequently to its wide range of applications, signi cant amount of morpholine and its salts is released via different industrial process into the environment where its secondary amine functionality leads to nitrosation to form N-nitrosomorpholine (NMOR), a well-characterized carcinogen [1,2] (Fig. 2).
The large-scale industrial usage and its potentially carcinogenic effects thus have environmental interest for its biodegradation to allow industrial e uents to be disposed of without unacceptable damage to the natural environment by utilizing bene cial and effective microbes for a sustainable remediation.
Morpholine was earlier thought to be recalcitrant but later several microbes have been explored to be metabolically able to degrade it. Most of the reported studies showed that Mycobacterium and Pseudomonas sp. are the two potential bacterial isolates that utilize morpholine for their sole source of carbon, nitrogen and energy and hence underwent degradation [3,4,5].
Explore the degradation pathway for sustainable remediation of morpholine Nature friendly microbial system has been revolved for the remediation of many pollutants, waste, spill and e uents etc. through the route of biodegradation. Many possible mechanisms exist which differs with different xenobiotics. Some enzymes can bind analogous to their natural substrates which contain xenobiotic functional groups, if these do not greatly alter the change of the active site. So, it is necessary for the enzyme to catalyze a reaction with the xenobiotic as substrate. The success of this reaction metabolism (as a biodegradation mechanism) depends on other factor also, such as the ability of the xenobiotic to act as an inducer and the nature of product/intermediate formed. In speci c to morpholine, the metabolic degradation pathway has been very di cult to establish, because of the technical limitation discussed above.
A few studies were carried out to understand the morpholine biodegradation pathway. In these studies, authors have established that for the complete mineralization of morpholine, a hypothetical pathway had been proposed that could proceed via 2-(2 aminoethoxy) acetate to enter either ethanolamine (called pathway 1) or glycolate (called pathway 2) route of degradation [6,7]. Depending on the available morpholine concentration in the cultural medium, one pathway could be used while the other may be inhibited. They further illustrated that degradative pathway might be starts with the rst step of the cleavage of the C-N bond; this leads formation of an intermediary amino acid, which is further followed by deamination and oxidation of this amino acid to a diacid.
In brief, degradation of morpholine is likely to begin by the breakage of a bond between the hetero atom and an adjacent carbon atom by the enzyme morpholine monooxygenase; responsible for the ring cleavage. Morpholine monooxygenase is an important enzyme in morpholine degradation as this enzyme catalyzes the biotransformation of morpholine to 2-(2-aminoethoxy) acetic acid and contains a cytochrome P450 catalytic subunit [1,6]. Knapp et al [8] showed that morpholine could serve as substrates for avin-containing monooxygenase or cytochrome P450 which is associated with oxygen consumption. Poupin et al [9] further, attributed the inhibitory effects of metyrapone on the degradation ability of Mycobacterium strain RP1 to prove the involvement of cytochrome P-450 in the bio-degradation of morpholine.
Many different species of Mycobacterium have been shown to degrade morpholine via this shared group of degradation reactions with little information of other enzymes involved in the reactions. Swain et al [10] found proposed hypothetical pathway in Mycobacterium chelonae (Fig. 3). Mazure and Truffaut 1994 described the degradation of morpholine via the ethanolamine and glycolate routes in M. aurum MO1.
Recently, a new approach, in which 1 H-NMR spectroscopy and ion spectroscopy have been performed directly with cultural ltrate to identify some metabolic intermediates of morpholine degradation by M. aurum MO1 [4,11] (Fig. 4). Furthermore, the byproducts of microbial processes can provide indication for successful bioremediation process. Since, only hypothetical pathways have been proposed, limited interpretations of various experimental designs have been made (in material method section) to establish the degradation pathway by an isolate to check either it follows an ethanolamine and/or glycolate route for the complete removal of morpholine at lab scale.

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 ne to medium sub-angular gravel in the upper surface including ne sand and a high content of iron ecks. 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 puri cation 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. Speci c colonies obtained were sub-cultured further to nd the pure bacterial colony. The selected pure bacterial isolate was identi ed 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 identi ed by 16s rRNA analysis. In order to verify the phylogenetic a liation 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 ampli cation, 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 ( nal 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 KH 2 PO 4 = 100 mg, K 2 HPO 4 = 100 mg, (NH 4 ) 2 SO 4 = 100 mg, MgSO 4 .7H 2 O = 4 mg and FeCl 3 = 0.2 mg supplemented with 0.1% morpholine as described by author itself earlier [16]. Culture tubes were incubated at 37 o C 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 con rm 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 con rm 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 ltrate. Lastly, estimation of ammonia/ammonical nitrogen in cultural ltrate 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 37 0 C 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 (modi ed Rimini test)  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. 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 ltered through 0.22 µm pore size nylon lter (Axiva Sichem Biotech, India) to avoid any bacterial cells. 1 ml neat ltrate (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 quanti ed in accordance with standard curve.

Morphological and Molecular Identi cation
The morphological feature of the isolate was found to be white colour, rod shaped, dull opaque appearance, smooth texture and convex elevation. Standard gram staining nds it a gram negative with high motility and also shows growth on selective media of Hichrome M1353.
The primary sequence of the 16s rRNA from present bacterial isolate was determined. The program PhyML 3.0 aLRT for phylogeny analysis and HKY85 as substitution model on 16s rRNA gene sequences showed the phylogenetic position of this isolate to be a closely related species from the genus Halobacillus blutaparonensis with the sequence representative of E. coli (Fig.5).
Nucleotide sequence accession was assigned by Gene Bank, NCBI, and an accession number namely KC345029 was found to this bacterial isolate of genus Halobacillus blutaparonensis.

Growth on intermediates
The isolate showed growth in presence of morpholine as well as in intermediate aminoethoxy ethanol (reduced product of aminoethoxy acetate) by taking it as carbon source. However, no growth was recorded in presence of ethanolamine in culture media as shown in Fig. 6. The count of bacterial cells was adjusted to 1×10 8 cells/mL (1 unit absorbance = 5×10 8 cells) by varying incubation period up to 48 hours.

Chemical assay for intermediate/s
Based on Simon test 1 and 2, the presence of MEA and morpholine in the culture supernatant is shown below in Table 3.  (Table 4). However, absentia of MEA peak in diluted culture ltrate, supporting the fact that this bacterium prefers the glycolic route of degradation of morpholine which was further proven by MS analysis.
MS was run directly with cultural ltrate (with and without solvent). Each sample was performed separately in positive and negative mode (Table 5 and Fig.7).  Further, it is also noticed that when methanol is used as a solvent system, a peak of m/z = 75 has been recognized indicates the presence of glycolic acid (having formula C 2 H 4 O 3 and molecular weight = 76) as  (Fig. 7) which supports the earlier fact that this particular isolate prefer the path of glycolic route (pathway 2) of degradation similar to mycobacterium strain as reported by Combourieu et al [4].
MS analysis also supports the GC ndings of no availability of ethanolamine in culture ltrate. Based on this, it is envisaged that ethanolamine might be inhibitory to bacteria. Hence, this bacterial isolate prefers the glycolic route of metabolic pathway with supporting the fact that in presence of morpholine, one of two branches of morpholine biodegradation pathway was induced while the other branch was inhibited.
The illustrated degradative pathway might start with the cleavage of the C-N bond; this leads formation of an intermediary amino acid, which is further followed by deamination and oxidation of this amino acid to a diacid as shown in Fig. 4.
Ammonia release: As end product of morpholine degradation Morpholine can be degraded by bacteria, with the liberation of ammonia which can biochemically utilized as a nitrogen source. Whatever the degradation pathway of morpholine, ammonia is the end product.
Based on Nessler quanti cation, ammoniacal nitrogen produced by isolate is calculated (as shown in the Table 6 and Fig. 8) by the regression equation (y= 0.145x-0.028 with r 2 =0.984) and found to be 5.2 ppm.
Initial morpholine concentration in culture supernatant (before degradation) was reported to be 2000 ppm.
The molar conversion ratio of morpholine to ammonia was found to be 1:0.014. Also, it was shown that the nal pH of the media throughout the experiment did not change supporting the fact that low concentration of ammonia was released as end product of morpholine degradation.

Discussion
Based on the result summarized, it has been reported that the isolate prefers to undergo glycolic route of degradation instead of ethanolamine route as it inhibits the bacterial growths. The illustrated degradative pathway might start with the rst step of the cleavage of the C-N bond; this leads to the formation of an intermediary amino acid, which is further followed by deamination and oxidation of this amino acid to form diacid (Fig. 9) which is metabolically consumed by isolate.
More ever, the presence of other intermediate compounds also supports this nding with a conclusion that the diglycolic route of biodegradation might be a common degradation mechanism shown by other groups of bacteria too. This was well reported by other authors [4,9,11] to prove our nding. Whatever the degradation pathway was exhibited by bacteria, the end product was ammonia which would biochemically use by bacteria. Our studies con rm the presence of ammonia as an end product and the molar ratio of morpholine to ammonia was found to be 1:0.014. Due to the low concentration of ammonia produced, the pH of the culture media did not change throughout the experiment. However, higher molar ratio of morpholine to ammonia results in the inhibitory effect on the growth of bacteria because it increases the pH of media to shift it toward alkalinity.  [21].

Conclusion
Environmental pollution has become a global problem. Every day, everybody, every sector, every organization/industry is responsible for environmental pollution due to indiscriminate and frequent release of xenobiotics by different anthropogenic activities. Morpholine is one such xenobiotic chemical.
The heterocyclic morpholine has versatile application in different industrial setup processing and so it is released into the environment through the differential process of discharge at a micro concentration to macro concentration. It is necessary to highlight that anthropogenic environmental pollutants even at low concentrations often produce deleterious effects on organisms, which are di cult to be predicted because measurable effects are expressed only after prolonged exposure.
In the environment, the majority of morpholine exposure comes through water and leads to form human carcinogen NMOR by the process of natural nitrosation. Also, it is mention to note that this nitrosation process is also carried out in the biological system when it is directly ingested, inhaled, or applied to the skin acting as a mutagen or carcinogen. In addition, NMOR is known to act as a mediator for various debilitating cancers associated with organs like the digestive tract, respiratory tract, kidney, and liver when it biomagni es through different tropic levels of biota by consumption or in taking of pollutant water and so eventually perturbing into the food-chain.
In this concern, it is better to provide a solution for e cient discharge or effective remediation by costeffective, nature-friendly biological tools powered by microbes to conserve the natural environment and resources, and to curb the negative impacts on biota. This sustainable solution is an ancient core concept (ACC) that continues to be the only solution for healthy and productive life into the distant future. The workhorses of this effective removal are microbes and so it must be explored and elucidate its degradation pathway to know how potent the microbes are for the cleaner, greener, and safer solution to decontaminating a wide range of pollutants and its mitigation.

Declarations
Con ict of interest The authors undertake that there is no con ict of interest.
Consent for Publication The authors give consent for the manuscript to be published.
Research involving the Human subject/Animal studies No such studies has been conducted in this manuscript.
Ethics committee approval The scope of article does not involve animal/ human subject studies. Hence, no institutional ethics committee approval has been required.
Funding Source I would like to acknowledge my former institute BITS Pilani, Hyderabad Campus, Hyderabad, India for providing the all necessary lab infrastructure and nancial support. Molecular phylogeny of 16sRNA sequenced and sequences from identi ed bacteria in the database. The sequence of E.coli served as the out group for rooting the tree.

Figure 6
Growth curve of isolate in presence of intermediates of morpholine degradation The complete illustration of the degradation pathway of morpholine