HUNGARIAN JOURNAL OF INDUSTRY AND CHEMISTRY Vol. 50 pp. 33–43 (2022) hjic.mk.uni-pannon.hu DOI: 10.33927/hjic-2022-07 UNRAVELING THE NOVEL BACTERIAL ASSISTED BIODEGRADATION PATHWAY OF MORPHOLINE RUPAK KUMAR*1 , SUMAN KAPUR2 , AND SRINIVASA RAO VULICHI2,3 1Central Drugs Standard Control Organization, New Delhi, India 2Birla Institute of Technology and Science – Pilani, Hyderabad Campus, Hyderabad, India 3SVU College of Pharmaceutical Sciences, Sri Venkateswara University, Tirupati, India Most xenobiotics are biodegradable, persistent or recalcitrant in nature. Morpholine, a typical xenobiotic, was initially regarded as recalcitrant, however, later proved to be biodegradable by bacterial species like Mycobacterium and Pseu- domonas in particular. However, establishing the metabolic pathways involved for the successful biodegradation of mor- pholine is challenging because of its extreme level of water solubility that affects various analytical procedures. In addition, to date, no suitable analytical methods have been reported to directly estimate morpholine and its degradable products or intermediates. Nevertheless, methods, especially optical density, gas chromatography and mass spectrophotometric analysis, could indirectly estimate the degradation product(s) of morpholine formed as a result of its biotransformation. In the present study, the degradation pathway of morpholine was ascertained by selected bacterial isolates by measuring their capacity to degrade morpholine. Based on this analysis of culture filtrates, it was determined that the novel isolate is the genus Halobacillus blutaparonensis which follows the diglycolic acid route from the metabolic degradation pathway of morpholine to induce one of two branches of the morpholine biodegradation pathway. In the presence of concentration of morpholine, out of two branches of morpholine degradation one branch is induced, while the other branch is inhibited. Whatever the branches with regard to the degradation pathway of morpholine exhibited by bacteria are, ammonia is the final end product of degradation which might be biochemically utilized by the isolate. Keywords: morpholine, xenobiotic, recalcitrant, glycolic acid route, ammonia 1. Introduction Environmental pollution has become a global problem. Due to the indiscriminate and frequent release of xeno- biotics as a result of different anthropogenic activities, each and every day our environment becomes increas- ingly devastated by the pollutants. Morpholine (1-Oxa- 4-azacyclohexane) is one such heterocyclic xenobiotic organic chemical with different versatile applications in various processes in the rubber, paper, iron, textile, per- sonal care, pharmaceutical and agricultural industries amongst others. As a consequence of its vast operational usage, a significant amount of this chemical is released into the environment through the differential process of discharging at both micro- and macro-concentrations. Therefore, it is necessary to mention that anthropogenic environmental pollutants, even at low concentrations, of- ten produce deleterious effects on organisms, which are Recieved: 1 March 2022; Revised: 20 April 2022; Accepted: 26 April 2022 *Correspondence: rupakraman@gmail.com difficult to predict because measurable effects are ex- pressed only after prolonged exposure. In the environment, the majority of exposure to mor- pholine originates from water and leads to the forma- tion of the carcinogen N-nitrosomorpholine (NMOR) by the process of natural nitrosation [1] (Fig. 1). Further- more, it is pertinent to mention that this process of ni- trosation may occur in biological systems when directly consumed, ingested, inhaled and applied to the skin. In addition, NMOR is known as a mediator of various de- bilitating cancers associated with organs like the diges- tive tract, respiratory tract, kidneys and liver, which is eventually biomagnified through different trophic levels of biota by its application or the intake of polluted wa- ter leading to this carcinogen entering the food chain. In Figure 1: Formation of NMOR https://doi.org/10.33927/hjic-2022-07 mailto:rupakraman@gmail.com 34 KUMAR, KAPUR, AND VULICHI this regard, it would be best to provide a solution for its efficient discharge or effective removal by different physical and chemical processes. Recently, photocataly- sis using catalysts irradiated by ultraviolet or visible light has been applied for the mineralization of toxic organic dyes in water and carbon dioxide [2, 3]. However, a cost- effective, environmentally-friendly biological tool pow- ered by microbes has been widely used as an ancient core concept for the purpose of conserving the natural environ- ment and resources to curb the negative impacts on biotic components. Therefore, a sustainable solution driven by microbes must be explored to elucidate the degradation pathway and measure how potent microbes are for the purposes of decontaminating a wide range of pollutants and their mitigation. In general, most pollutants are organic and may be biodegradable (transformed by biological mechanisms which might lead to mineralization), persistent (fail to undergo bioremediation in the environment or under a specific set of experimental conditions) or recalcitrant (inherently resistant to biodegradation) in nature. Bio- genic or naturally occurring compounds are biodegrad- able while man-made (anthropogenic) compounds may be biodegradable, persistent or recalcitrant. In terms of xenobiotics that are man-made, the microbial commu- nities present in the environment may not have evolved suitable mechanisms for their degradation. Many possi- ble mechanisms exist which differ from one xenobiotic to another. One common mechanism is the binding of en- zymes analogous to their natural substrates which con- tain xenobiotic functional groups, assuming these do not greatly alter or change the active site which catalyzes a reaction with the xenobiotic. The success of this enzy- matic reaction (as a biodegradation mechanism) also de- pends on other factors such as the ability of the xeno- biotic as an inducer or inhibitor and the nature of the product/intermediate formed. Specific to morpholine, the metabolic degradation pathway has been very difficult to establish because of the aforementioned technical limita- tion. 1.1 Sustainable remediation of morpholine and its degradation pathway Although morpholine was previously thought to be re- calcitrant, several microbes have proven to metaboli- cally degrade it. The majority of studies showed that the species Mycobacterium and Pseudomonas are the two po- tential bacterial isolates that utilize morpholine as their sole source of carbon and nitrogen, thereby undergo- ing degradation [4–7]. A few studies have been carried out to understand the biodegradation of morpholine and its regulation [8–10]. Later a hypothetical pathway was proposed for the complete mineralization of morpholine that could proceed via 2-(2-aminoethoxy)acetate to pro- duce its diglycolate salt and/or ethanolamine [5, 11, 12]. These two different routes of degradation are called the ethanolamine/monoethanolamine pathway (Pathway 1) (a) (b) Figure 2: (a) Hypothetical pathway of morpholine degra- dation where X = 2-(2-aminoethoxy)acetaldehyde, Y = 2(2-aminoethoxy)acetate and a, b, c indicate the position of carbon atoms in the ring. (b) Postulation of the morpho- line degradation pathway after 1H-NMR and ion spectro- scopic analyses where 1 = 2-(2-aminoethoxy)acetate, 2 = diglycolic acid and 3 = glycolic acid. and diglycolic acid/glycolate pathway (Pathway 2), re- spectively (Fig. 2a). The illustrated degradation pathway might start with the cleavage of the C-N bond, leading to the formation of an intermediary amino acid which is fol- lowed by deamination and oxidation of this amino acid to form a diacid [11, 12]. The degradation of morpholine via the ethanolamine or glycolate pathways has been described in the presence of Mycobacterium chelonae and M. aurum MO1 [8, 9] (Fig. 2a). The degradation of morpholine is likely to be- gin with the breakage of a bond between a heteroatom and an adjacent carbon atom by the enzyme morpho- line monooxygenase, which is responsible for the ring cleavage. Morpholine monooxygenase is an important enzyme in the degradation of morpholine as it cat- alyzes the biotransformation of morpholine to form 2- (2-aminoethoxy)acetic acid and contains a catalytic sub- unit of cytochrome P450 [1, 10]. Morpholine could serve as a substrate for flavin-containing monooxygenases or cytochromes P450 which is associated with oxygen con- sumption [13]. Further inhibitory effects of metyrapone on the degradation of the Mycobacterium strain RP1 have been attributed to the involvement of cytochromes P450 Hungarian Journal of Industry and Chemistry BACTERIAL ASSISTED BIODEGRADATION PATHWAY OF MORPHOLINE 35 in the biodegradation of morpholine [5]. Depending on the concentration of morpholine in the culture medium, one pathway could be expressed while the other might be inhibited [11]. Recently, a new approach was applied in which the culture filtrate was analyzed by 1H-NMR spec- troscopy and ion spectroscopy to identify the metabolic intermediates of morpholine degradation by M. aurum MO1 [11, 12] (Fig. 2). Although many different species of Mycobacterium have been shown to degrade morpho- line via this shared group of degradation reactions, little information is known about the enzymes involved (Fig. 2b). Furthermore, the byproducts of the microbial pro- cesses can be indicative of a successful bioremediation process. Consequently, since only hypothetical pathways have been proposed, limited interpretations of various ex- perimental designs can be made to establish the degrada- tion pathway that follows the route of degradation path- way that follows the route of Pathway 1 and /or Pathway 2 via the shared formation of 2-(2- aminoethoxy)acetate. 2. Materials and methods 2.1 Environmental samples The sample used in the present degradation study was collected from natural sources (soil) in and around Dur- gapur Steel Plant, West Bengal, India. The site is located in Durgapur at a latitude of 51◦50’43.8” north and a lon- gitude of 8◦16’35.8” west in the state of West Bengal, 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 col- lected in a clean, sterile plastic container before being transferred to the laboratory and stored at room tempera- ture until used for further analysis. 2.2 Chemicals and reagents All chemicals and reagents were of analytical grade and used as received without any further purification. Even though Milli-Q water (Elix Essential 3 Water Purification System with a conductance of 0.12 Siemens) was used to prepare an aqueous solution of reagents, autoclaved dou- ble distilled water was used because of the microbial cul- tures. 2.3 Screening, characterization and se- quence accession of the morpholine- degrading isolate For the initial isolation and cultivation of bacteria, ten- fold serial diluted samples were spread onto nutrient agar plates, which were prepared according to the manufac- turer’s instructions. The specific colonies obtained were subcultured further to isolate the pure bacterial strain. The selected pure bacterial isolate was identified based on morphological, biochemical and molecular character- ization. Morphological characterization was achieved by visually observing colonies in terms of their appearance, shape, color, arrangement, optical nature, margin, texture and elevation. However, the biochemical tests were per- formed as per standard methods [14]. Furthermore, the pure colony was then identified by 16S rRNA gene se- quence analysis. In order to verify the phylogenetic affiliation of the selected isolate, a single colony was collected for the purpose of DNA isolation (InstaGeneTM Matrix Genomic DNA isolation kit (Bio-Rad Catalog # 732- 6030) as per the kit instructions and procedures) and subjected to Polymeric Chain Reaction (PCR) analy- sis using primers targeting two 16S rRNA genes [27F (5’-AGAGTTTGATCMTGGCTCAG-3’) and 1492R (5’- TACGGYTACCTTGTTACGACTT-3’-). A PCR reaction (20 µL) was performed containing 8 µL of Taq DNA Polymerase Master Mix, 1 µL of both 10 µM stock 27F/1492R primers, 9 µL of double distilled water and 1 µL of a DNA template. The PCR (MJ Research PTC- 200 Peltier Thermal Cycler; Bio-Rad PTC-200) reaction was conducted using specified conditions from the liter- ature [15]. DNA was denatured at 94◦C for 5 mins, fol- lowed by 35 cycles of amplification, each consisting of the following components: 94◦C for 45 secs (denatura- tion), 55◦C for 60 secs (annealing), 72◦C for 60 secs, (extension) followed by 72◦C for 10 mins (final exten- sion). The PCR product was sequenced by Yaazh Xenomics, Chennai, Tamil Nadu, India. The 16S rRNA gene was sequenced using the National Center for Biotechnol- ogy Information’s Basic Local Alignment Search Tool (BLAST). The phylogenetic analysis of the sequence us- ing the closely related sequence of BLAST results was performed by multiple sequence alignment. The program MUSCLE 3.7 was used for multiple sequence align- ments [16]. The resulting aligned sequences were fil- tered using the program Gblocks 0.91b, which eliminates poorly aligned positions and divergent regions, that is, re- moves alignment noise [17]. Finally, the program PhyML 3.0 aLRT was used for phylogenetic analysis and HKY85 as a substitution model. The nucleotide sequence of the isolated bacterium was included in NCBI’s GenBank and assigned an acces- sion number consisting of 2 letters and 6 numbers [18]. 2.4 Cultivation and acclimatization of the iso- late: Microbial adaptation against mor- pholine Bacterial inocula were prepared by aseptically transfer- ring the selected identified pure colonies to 10 mL of an enriched media called Knapp Buffer. Alternatively, a mineral salt solution (MSS) comprised of 100 mg of KH2PO4, 100 mg of K2HPO4, 4 mg of MgSO4.7H2O and 0.2 mg of FeCl3 was used as previously described by the author supplemented with 0.1% v/v morpholine as previously described by the author [19]. Cultures were incubated at 37oC as well as 150 rpm for 1−2 weeks and 50 pp. 33–43 (2022) 36 KUMAR, KAPUR, AND VULICHI Table 1: GC parameters for the estimation of the monoethanolamine concentration Parameters Specificity Column and its configuration Rtx-35 30 mm × 0.32 mm × 1 µm Oven/Column temperature Initial temp.: 60 ◦C Hold: 1 min Ramp rate: 30 ◦C/min Final temp.: 240 ◦C maintained for 3 mins Linear velocity: 37.6 cm/sec (for nitrogen) Injection port Temp.: 200 ◦C Split Ratio: 30:1 Injection Volume: 1 µL Carrier gases (mobile phase) Column gas flow rate: 2 mL/min Purge gas flow rate: 1 mL/min Hydrogen gas flow rate: 40 mL/min Zero air flow rate: 400 mL/min Nitrogen gas flow rate: 15 mL/min Stationary phase 60% Dimethylpolysiloxane and 35% Diphenyl polysiloxane Detector Flame ionization detector at 300 ◦C Analysis time 10 mins Software GC Solution Workstation Windows 8 their absorbance at 600 nm was taken regularly as a mea- sure of growth. Based on their growth, when an optical density of 0.5 was reached (data not shown), the culture was diluted to 1 : 100 before being further spread onto MSS-agar plates (treated with 2% agar + 0.1% morpho- line) to confirm the acclimatization of the isolate against morpholine stress. Furthermore, the growing culture was centrifuged at 6500 rpm for 10 mins and the pellet was re- suspended in the MSS medium while gradually increas- ing the concentration of morpholine to 0.2% which was referred to as a seeded acclimatized bacterial inoculum. For each increased acclimatization study, the tested bac- teria were grown in an MSS broth supplemented with an increased concentration of morpholine and a respective MSS-agar plate with the same concentration of morpho- line to confirm the said acclimatization. The acclimatized inoculum was later grown in the presence of an intermediate degradation product of mor- pholine to explore whether this particular isolate follows Pathway 1 or 2. This was further validated by performing in-vitro chemical and analytical assay(s) with the avail- ability of intermediate product of morpholine degradation in the culture filtrate. Lastly, estimation of the ammonia- cal nitrogen (measure of the amount of ammonia) in the culture filtrate revealed the complete degradation of mor- pholine by this isolate following the concerned pathway. 2.5 Growth on different hypothetical degrada- tion intermediate compounds The growth of the isolate on various substrates (degrada- tion intermediate compounds) was investigated by adding the corresponding compounds (0.15%) to the MSS. The pH of the media was adjusted to 7 and growth carried out at 37◦C as well as 150 rpm for 48 hours. At regu- lar time intervals, the absorbance was measured in terms of optical density to establish whether these degradation products might have been formed to facilitate the growth of the isolated bacteria. 2.6 Chemical tests of intermediate(s) in the degradation pathway Chemical tests on degradation products, mainly mo- noethanolamine (primary amine) and morpholine (sec- ondary amine), were carried out by the standard Simon test - 1 (Rimini test) and Simon test - 2 (modified Rim- ini test) on the culture filtrate to determine the presence of primary and secondary amines [20]. The amine under- goes a nucleophilic addition reaction with nitroprusside ions in the presence of acetaldehyde or a ketone to yield the characteristic color of primary amines (blue) or sec- ondary amines (violet). 2.7 Gas Chromatography (GC) studies of degradation intermediate(s) A GC system (Shimadzu GC-2010) equipped with a stan- dard oven for temperature ramping, split condition, in- jection ports, a flame ionization detector and a Rtx-35 amine column (30 mm × 0.32 mm × 1µm film thick- ness) in the presence of nitrogen as a carrier gas by the direct injection method was used for the analysis of mo- noethanolamine (MEA). The analytical parameters for the analysis of MEA are summarized in Table 1, as per the method (by modifying the column and its parameters) reported in the literature [21]. Hungarian Journal of Industry and Chemistry BACTERIAL ASSISTED BIODEGRADATION PATHWAY OF MORPHOLINE 37 Table 2: MS operating parameters for intermediate(s) Parameter Specificity Ionization electrospray ionization Needle voltage = 4.5 kV Interface tem- perature 350 ◦C Temperature of heating block 200 ◦C Sheath/Drying gas flow rate 15 L/min Nebulizer gas flow rate 1.5 L/min Acquisition time 2 mins Acquisition mode Positive/Negative Scan m/z 50 − 200 Scan speed = 52 units/sec Sampling acquisition time = 1.56 Hz (640 msec) Detector Electron multiplier Software Lab Solutions Workstation Windows 7 A standard solution of 0.125 to 0.5% v/v MEA (corre- sponding to ppm and prepared in methanol) was injected along with the processed culture supernatant (1:10, fil- trate volume of 1 and 9 volumes of methanol), as per the method described above. GC of the test samples was run against blank media using positive controls to quantify or estimate the presence of MEA in the culture filtrate by analyzing the Area Under the Curve (AUC) calculated by the machine. 2.8 Mass spectrometry studies of degrada- tion intermediate(s) The mass spectrometry (MS) system of an integrated Liquid Chromatography-Mass Spectrometry instrument (Shimadzu LCMS-2020) equipped with an inlet inter- face, ion source, mass analyzer and detector was used to analyze the degradation products of morpholine. The analytical parameters for ascertain the morpholine degra- dation products are summarized in Table 2. The sample for injection was prepared without using a solvent, as per the method reported in the literature [12]. The culture sample (5 mL) was centrifuged at 10,000 rpm for 10 mins before the supernatant was filtered through a nylon filter with a pore size of 0.22 µm (Axiva Sichem Biotech, India) to remove any bacterial cells. 1 mL of neat filtrate was injected directly into the MS instrument. 2.9 Estimation of the ammonia concentration The presence of ammonia in the culture supernatant was estimated by the standard Nessler’s method [22], which involves coupling of ammonium to the Nessler’s reagent Figure 3: Estimation of the ammoniacal nitrogen concen- tration by Nessler’s method to produce a yellow color under strongly alkaline con- ditions (Fig. 3). The resulting yellow color was formed in proportion to the ammonium (NH+4 ) concentration and was measured at a wavelength of 405 nm using an Elisa reader (ELx50/8MS BioTek India) against a reagent blank. The ammonia level in terms of ammoniacal ni- trogen was expressed in mg/L (ppm). A standard solu- tion of 10 ppm of NH+4 −N was prepared by dissolving 4.773 mg of ammonium chloride in 125 ml of double- distilled water and further diluted to make solutions of 1 − 5 ppm NH+4 −N. A calibration curve was plotted and is presented in the results section. 3. Results and discussion 3.1 Morphological, biochemical and molecu- lar identification Morphologically, the isolate was found to be white in color with a dull opaque appearance, rod-shaped, have a smooth texture and grow as a convex elevation colony. Standard staining reported it to be a Gram-negative bac- terium with high motility which also showed signs of growth on a selective medium, namely HiCrome UTI Agar M1353. The primary sequence of the 16S rRNA from the present bacterial isolate was determined. The program PhyML 3.0 aLRT for phylogenetic analysis and HKY85 as a substitution model on the 16S rRNA gene sequences determined the phylogenetic position of said isolate to be a species closely related to the genus Halobacillus bluta- paronensis with a sequence representative of E. coli (Fig. 4). Nucleotide sequence accession was assigned by Gen- Bank, NCBI and an accession number of KC345029 was Figure 4: Molecular phylogeny of the 16S rRNA gene sequence and sequences from identified bacteria in the database. The sequence of E. coli served as the outgroup for rooting the tree. 50 pp. 33–43 (2022) 38 KUMAR, KAPUR, AND VULICHI Figure 5: Growth of the isolate in the presence of interme- diates of morpholine degradation Figure 6: GC (Rtx-35)- flame ionization detector chro- matogram of MEA assigned to this bacterial isolate of genus Halobacillus blutaparonensis. 3.2 Growth on intermediates The isolate grew in the presence of morpholine and the intermediate, namely aminoethoxy ethanol (reduced product of aminoethoxy acetate) by consuming it as a source of carbon and nitrogen. However, no growth was recorded in the presence of ethanolamine in the cul- ture media shown in Fig. 5. The count of bacterial cells was adjusted to 1×108 cells/mL (1 unit of absorbance = 5×108 cells) by varying the incubation periods up to 48 hours. 3.3 Chemical assay of intermediate(s) Based on Simon tests - 1 and 2 [20], the presence of MEA and morpholine in the culture filtrate is shown in Table 3. 3.4 GC studies of MEA in the culture super- natant GC of the culture supernatant was run at different con- centrations (ppm) of a standard MEA solution. Table 4 and Fig. 6 indicate a retention time of MEA equal to 2.2 mins which was absent in the diluted culture supernatant. GC analysis revealed that no MEA was present in the cul- ture supernatant suggesting that bacteria might prefer the diglycolic route (Pathway 2) of morpholine degradation which was later confirmed by MS analysis. 3.5 MS studies of the culture filtrate MS was run directly with a neat culture filtrate. Each sam- ple was analyzed separately in both the positive and neg- ative ion modes (Table 5 and Fig. 7). It was observed that the m/z peak of the neat cul- ture filtrate (Fig. 7) indicates the presence of 2-(2- aminoethoxy)acetate (C4H9NO3, molecular weight = 119.119 and m/z = 120 as [M+H]+) and an anion of diglycolic acid (C4H6O5, molecular weight = 134.09 and m/z = 133 as [M-H]–) which supports the fact that this particular isolate prefers the degradation path- way of diglycolic acid (Pathway 2), similar to a strain of mycobacterium reported earlier by conducting elec- trospray ionization mass spectrometry on the culture fil- trate [12]. Further MS analysis supports the GC findings that MEA is not present in the culture filtrate because it might have an inhibitory effect on the bacteria. Therefore, the said bacterial isolate prefers the diglycolic acid route of the metabolic pathway given the fact that in the presence of morpholine, one of the two branches of morpholine biodegradation was induced while the other was inhib- ited. The illustrated degradation pathway might start with the cleavage of C-N bond, leading to the formation of an intermediary amino acid followed by deamination and oxidation of this amino acid to form a diacid as is shown in Fig. 2b. 3.6 Ammonia release: As the end product of morpholine degradation Morpholine can be degraded by bacteria which releases ammonia. Whichever degradation pathway of morpho- line is followed, ammonia is produced as an end product. The concentration of ammoniacal nitrogen produced by the isolate was calculated (Table 6 and Fig. 8) by the regression equation of a standard curve (y = 0.137x with r2 = 0.98) and found to be present at a concentration of 5.2 ppm based on Nessler’s quantification. The ini- tial morpholine concentration in the culture supernatant (before degradation) was reported to be 2000 ppm. The molar ratio with regard to the conversion of morpholine into ammonia was found to be 1 : 0.014. Furthermore, it was shown that the final pH of the media throughout the experiment did not change, supporting the fact that a low concentration of ammonia was released as an end product of morpholine degradation. Hungarian Journal of Industry and Chemistry BACTERIAL ASSISTED BIODEGRADATION PATHWAY OF MORPHOLINE 39 Table 3: Simon tests for the presence of the primary amine MEA and secondary amine morpholine in the culture supernatant Sample Test Feature Remark Result Morpholine Simon 1 Characteristic blue color of the secondary amine Morpholine positive MEA Simon 2 Characteristic violet color of the primary amine MEA positive Culture media Simon 1 No characteris- tic blue color Morpholine negative Simon 2 No characteris- tic violet color MEA negative Culture Supernatant (Filtrate) Simon 1 No characteris- tic blue color Morpholine negative Simon 2 No characteris- tic violet color MEA negative 50 pp. 33–43 (2022) 40 KUMAR, KAPUR, AND VULICHI Table 4: GC analysis of the diluted culture filtrate Vial Retention time (mins) AUC Interpretation (Compound) Methanol 1.331 378534920.9 Methanol 5000 ppm MEA 1.333 2.218 366649701.7 2748948.5 Methanol MEA 2500 ppm MEA 1.331 2.216 374551161.2 2397300.9 Methanol MEA 1250 ppm MEA 1.331 2.211 378803557.4 1149593.1 Methanol MEA Culture Supernatant (1:10) 1.334 2.331 310947764.4 92353.6 Methanol No/Negligible MEA Table 5: Expected intermediate according to the MS analysis of the culture filtrate. Sample m/z Positive mode m/z Negative mode Remark Neat Culture filtrate 120 [M+H] + 2,2 Aminoethoxy acetate 133 [M-H] – Anion of diglycolic acid Figure 7: Electrospray ionization - MS spectra recorded under positive and negative ionization of the neat culture filtrate. Hungarian Journal of Industry and Chemistry BACTERIAL ASSISTED BIODEGRADATION PATHWAY OF MORPHOLINE 41 Table 6: Estimation of ammoniacal nitrogen concentration by Nessler’s reagent Well 10 ppm Stock NH4-N+ (µL) Milli-Q water (µL) Culture media (µl) 50% Na-K Tartrate (µL) Nessler’s reagent (µL) Net absorbance at 405 nm 1 ppm 25 225 — 5 5 0.091 2 ppm 50 200 — 5 5 0.284 3 ppm 75 175 — 5 5 0.353 4 ppm 100 150 — 5 5 0.552 5 ppm 125 125 — 5 5 0.725 Culture supernatant 250 — 5 5 0.725 Figure 8: Standard curve of ammoniacal nitrogen concen- tration by Nessler’s reagent 4. Discussion Based on the results summarized, it has been reported that the isolate prefers to undergo the diglycolic acid route of degradation instead of the ethanolamine pathway, which might be an inhibitory effect on bacterial growth. The il- lustrated degradation pathway starts with cleavage of the C-N bond, leading to the formation of an intermediary amino acid which is followed by deamination and oxi- dation to form the diacid (Fig. 9). This diacid, namely diglycolate, later participates in intermediate metabolism and is converted indirectly into TCA by the Krebs cycle, which is beyond the scope of the present article. Moreover, the presence of degradation intermediate compounds in culture filtrate also favors this finding with the conclusion that the diglycolic acid route of biodegradation might be a common degradation mech- anism, which is also shown by other strains of bacteria, proceeding via 2-(2-aminoethoxy)acetate. The said inves- tigation to reveal the degradation pathway of morpholine is supported by similar findings published by other au- thors [5, 10–12]. Furthermore, whatever the degradation pathway ex- hibited by the bacterial isolate, the end product, that is, ammonia, will be biochemically produced and used. Our studies confirm the presence of ammonia as an end prod- uct in a molar conversion ratio of morpholine to ammonia of 1 : 0.014. Due to the low concentration of ammonia produced, the pH of the culture medium did not change throughout the experiment. However, a higher molar ra- tio of morpholine to ammonia brought about an inhibitory effect on the growth of bacteria by increasing the pH of the medium and making it more alkaline. The molar ra- tio of morpholine to ammonia was found to be different for different strains of bacteria as viz., namely 1 : 0.5 for Mycobacterium sp. HE5 [6], 1 : 0.89 for Mycobacterium sp. [7] and 1 : 0.82 for Mycobacterium sp. MO1 [9]. 5. Conclusions The large scale industrial applications of morpholine and its known carcinogenic effect thus have an environmental interest for its biodegradation and exploring the degrada- tive pathway so that unrevertable damage to the natural environment and biota can be minimized. Along with the Mycobacterium and Pseudomonas sp. another po- tential isolate namely Halobacillus blutaparonensis has been investigated for its ability to removal of morpho- line by adopting the diglycolate degradation pathway. Hence, sustainable remediation practice by utilizing ef- fective microbes should be applied to bring the environ- mental cleanup or facilitate the existing system of effluent treatment mechanism incorporation with biological ap- proaches to minimize the impact of xenobiotic pollutants in the anthropocentric epoch. Conflicts of interest The authors confirm no conflicts of interest with regard to the results derived from this study on the sustainable re- mediation of morpholine and its micro-scale degradation pathway. REFERENCES [1] Sielaff, B.; Andreesen, J. R.; Schräder, T. A.: Cy- tochrome P450 and a ferredoxin isolated from My- cobacterium sp. strain HE5 after growth on Mor- pholine. Appl. Microbiol. Biotechnol., 2001, 56(3-4), 458–464 DOI: 10.1007/s002530100634 [2] Dhiwahar, A. T.; Maruthamuthu, S.; Marnadu, R.; Sundararajan, M.; Manthrammel, M. A.; Shkir, M.; Sakthivel, P.; Reddy, V. R. M.: Improved photo- catalytic degradation of rhodamine B under visi- ble light and magnetic properties using microwave 50 pp. 33–43 (2022) https://doi.org/10.1007/s002530100634 42 KUMAR, KAPUR, AND VULICHI Figure 9: The complete illustration of a possible degradation pathway of morpholine. The isolate, namely Halobacillus bluta- paronensis, prefers Pathway 2 for the successful removal of morpholine. 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Analyst, 1986, 93(1107), 406–408 DOI: 10.1039/AN9689300406 50 pp. 33–43 (2022) https://doi.org/10.1111/j.1472-765X.1996.tb00202.x https://doi.org/10.1111/j.1472-765X.1996.tb00202.x https://www.wjpr.net/abstract_show/2951 https://doi.org/10.1093/nar/gkh340 https://doi.org/10.1093/nar/gkh340 https://doi.org/10.1080/10635150701472164 https://doi.org/10.1080/10635150701472164 https://www.ncbi.nlm.nih.gov/genbank https://www.unodc.org/pdf/scientific/SCITEC20-fin.pdf https://doi.org/10.1002/jssc.201200196 https://doi.org/10.1039/AN9689300406 https://doi.org/10.1039/AN9689300406 Introduction Sustainable remediation of morpholine and its degradation pathway Materials and methods Environmental samples Chemicals and reagents Screening, characterization and sequence accession of the morpholine-degrading isolate Cultivation and acclimatization of the isolate: Microbial adaptation against morpholine Growth on different hypothetical degradation intermediate compounds Chemical tests of intermediate(s) in the degradation pathway Gas Chromatography (GC) studies of degradation intermediate(s) Mass spectrometry studies of degradation intermediate(s) Estimation of the ammonia concentration Results and discussion Morphological, biochemical and molecular identification Growth on intermediates Chemical assay of intermediate(s) GC studies of MEA in the culture supernatant MS studies of the culture filtrate Ammonia release: As the end product of morpholine degradation Discussion Conclusions