Introduction

Numerous applications of military and supplementary civil areas, such as engineering, construction, extracting, mining, and rocket propellants, explosives, i.e., organic energetic compounds (nitro-amines and nitro-aromatic), are being used (Chatterjee et al. 2017; Nagar et al. 2018, 2021). 2,4,6-trinitrotoluene (TNT), is a reported universally utilized secondary explosive (Ayoub et al. 2010; Kao et al. 2016). As per Alothman et al. 2020, TNT, being a teratogenic, cytotoxic nitro-aromatic compound, may affect cellular mutations in humans, animals, plants, and microorganisms (Juhasz and Naidu 2007; Fahrenfeld et al. 2013; Kao et al. 2016). Throughout the two World Wars, because of the immense use of TNT, the ecosystem of soil and water has turned out to be utterly polluted (Lewis et al. 2004). The concentration of 10,000 mg per kg and 100 mg per liter of TNT was spotted in nitro-aromatic contaminated soil and water respectively. The lethal and mutagenic effect of TNT is being exhibited on eukaryotes and prokaryotes (Khan et al. 2013; Kao et al. 2016). Subsequently, TNT’s detrimental impacts on soil and groundwater are critical issue concerning environmental protection which is required to be controlled or minimalize as per the demand of the hour.

Plentiful reports have been recorded with numerous remediating methods for TNT polluted spots (Ayoub et al. 2010; Gümüscü and Tekinay 2013; Kao et al. 2016). Conventional treatment involves physical and chemical approaches, like incineration, adsorption, and advanced oxidation procedures, which are expensive and result in lethal by-products (Rodgers and Bunce 2001; Gümüscü and Tekinay 2013; Kao et al. 2016). On the other hand, biological-based remediation approaches are promising both ways; i.e., economically and organically (Chien et al. 2014; Kao et al. 2016). Bioremediation explores the potential characteristics of microorganisms to profitably bio-transform or biodegrades contaminants (Fahrenfeld et al. 2013; Kao et al. 2016). Bioremediation can be studied in situ and is frequently a cheap, less expertise practice which is up to the satisfaction of the general public (Kao et al. 2016). Microbial biotransformation can be approached for TNT biodegradation (Chien et al. 2014; Kao et al. 2016). Hence, TNT bioremediation through bacteriological degradation is a very optimistic practice.

Quite a lot of reviews concise widespread exploration of TNT biodegradation by fungi and bacteria (Van Aken and Agathos 2001; Heiss and Knackmuss 2002; Zhao et al. 2004; Serrano-González et al. 2018. In TNT, the existence of three electron-withdrawing nitro groups generates steric constraints accompanying elevation in electron shortage to the aromatic ring. Thus, the molecule resists oxidative degradation by microbes, nitro groups of TNT are used as a source of nitrogen by microbes (Claus et al. 2007). Anaerobic and aerobic degradation of TNT through a diversity of microorganisms have been testified, along with some anticipated metallic pathways (Esteve-Nunez et al. 2001; Kao et al. 2016). These comprised rigid anaerobic bacteria (such as Clostridium and Desulfovibrio), aerobic bacteria (such as Achromobacter, Bacillus, Citrobacter, and Pseudomonas), and fungi (Phanerochaete) (Duque et al. 1993; Montpas et al. 1997; Kalafut et al. 1998; Oh and Kim 1998; Esteve-Nunez et al. 2001; Lee et al. 2002; Kao et al. 2016).

The bacterium under study is a novel species and has not been used for TNT treatment. However, in the recent past, the authors have treated nitramine (octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine also known as HMX) using the same species but with the nomenclature Planomicrobium flavidum (Nagar et al. 2018) as submitted with accession number LT63187.2 (https://www.ncbi.nlm.nih.gov/nuccore/lt631687.2). However, in Jan 2019, the sequence has been updated and the nomenclature changed to Indiicoccus explosivorum gen. nov., sp. (https://www.ncbi.nlm.nih.gov/nuccore/LT631687.3; Pal et al. 2019). The degradation ability of I. explosivorum against nitro-aromatic-based explosives under aerobic conditions has not yet been explored. In the present study, for the first time native soil bacteria, i.e., Indiicoccus explosivorum (strain S5-TSA-19) was analyzed to get characterized for its ability to bio-transform TNT through bioremediation technique at a laboratory scale under aerobic conditions.

Materials and methods

Experimental preparations

TNT (> 99% purity) was from a reliable manufacturing unit in India and was obtained only for the study purpose. During the current study, all analytical and gradient grade chemicals together with the standard of 2-ADNT were from Sigma-Aldrich. Extracted soil samples from an explosive manufacturing site located in India were sent to the Institute of Microbial Technology (IMTECH), Chandigarh for the isolation and characterization process through 16 s rRNA sequencing (Krishnamurthi et al. 2009; Pal et al. 2019).

Initially, tryptone soya agar (TSA) plates were used to maintain the primary cultures of Indiicoccus explosivorum. Throughout the aqueous phase study, minimal salt medium (MSM) for microbial proliferation and associated experimentations was favored (Cook and Huetter 1981; Thompson et al. 2005). Essential media enhancement was done through 40 mM potassium phosphate buffer (pH 7.2), MgSO4.7H2O (61.61 mg), NH4Cl (50 mg), and trace elements. Trace element mixture supplementation was done with a composition (per liter) as reported earlier by Lamba et al. 2021, with an aim to study the proliferation and degrading potential of I. explosivorum, TNT (120 mg/L) solution was prepared in MSM medium.

Characteristics and tolerance assessment of the microbial strain

Strain S5-TSA-19 (Microbial type culture collection number 12608) with GenBank accession number LT631687 was received in lyophilized form and was characterized through 16S ribosomal RNA sequence practice. The optical density (OD) of the bacterial biomass was observed at 600 nm of absorbance with a UV–Vis spectrophotometer from Perkin Elmer, Model Lambda 650S. For obtaining culture, bacteria were initially nurtured in the minimal salt medium till the third generation. For the degradation study, the cell suspension in the log phase through an OD 0.8 (≈ 108 cells/mL) was used.

The isolated strain was assessed regarding its tolerance level against TNT by growing it in minimal salt media comprising different concentrations of TNT (60, 80, 100, and 120 ppm) and also with an additional nitrogen source in 2 sets along with pH and temperature variations.

Aqueous phase experiment preparation

Under sterile conditions, the isolate I. explosivorum (in its log phase) was inoculated in MSM medium in 250-mL Erlenmeyer flasks already spiked with 120 mg/L of TNT along with 3 replications per study. 5% (v/v) of the bacterium was inoculated in TNT containing MSM. Two sets of controls, viz., MSM containing TNT but excluding bacterial strain and MSM with isolate excluding TNT were also prepared. Flasks were nurtured for 30-day duration (at 30 °C and 120 rpm) in an orbital incubator shaker. Throughout the incubation period, regularly (i.e., on each alternative day) aliquots (2 mL) were drawn off from individual experimental arrangements and were centrifuged (10,000 rpm; 10 min). Further, the supernatant solution was filtered with a 0.45-μm Teflon filter before quantification of TNT and intermediates concentrations (USEPA 2007) with a photodiode array detector of model e2695, Perkin Elmer Inc., high-performance liquid chromatography (HPLC). C18 reversed-phase column (4.6 mm × 150 mm) was employed with acetonitrile and water (at a ratio of 1:1, v/v) as mobile phase and a flow rate of 1 mL/min.

Nitrite, nitrate, ammonia, TNT, and converted compounds analysis

Further, the conversion, disappearance, and tolerance against TNT were evaluated and quantified. To get brief evidence about the disappearance and conversion of TNT along with the formation of secondary metabolites, liquid chromatography–mass spectrophotometric (LC–MS) analysis was carried out. LC–MS analyses were accomplished via Waters, Micro mass Q-TOF microsystem (Waters Alliance 2795) comprising Waters X Bridge Column C18 with negative ion (ES-). 20 µL injection volume through a flow rate of 0.8 mL/min was utilized to analyze individual TNT peaks and its degradation by-products. For solvent purposes, acetonitrile, formic acid (0.1%), and methanol were utilized. Apart from spectrophotometric analyses, strain’s tolerance toward explosive was also viewed using scanning electron microscopy (SEM) detailed earlier by Prasad et al., 2016. Au–Pd layering was done through Sputter Coater (Quorum SC7620 Mini) and detected via ZEISS EV˚18 scanning electron microscope.

Nitrate and nitrite, being important metabolites of TNT degradation, were analyzed regularly (every alternate day). The amount of nitrite ion was measured at OD 540 nm and nitrate ion was analyzed at OD 220 and 275 nm via UV–VIS Spectrophotometer (Mercimek et al. 2013; Lamba et al. 2021).

Using UV–VIS Spectrophotometer at 490 nm of absorbance, the concentration of ammonium ion was evaluated at regular intervals of time. Quantification of ammonium ion was done using Nessler’s reagent. The supernatant (0.5 mL) was incubated at 37 ̊C for 3 h, succeeding with Nessler’s reagent (0.1 mL) addition (Mackie and MacCartney 1989; Lamba et al. 2021).

Statistical analysis

Through OriginPro 8.5 software, one-way analysis of variance (ANOVA) was taken out to significantly examine the differences between sample data’s mean obtained from MSM comprising I. explosivorum with and without the explosive. At 0.05, a significant level descriptive statistical normality test of the sample data was evaluated. Shapiro–Wilk data specified that samples were drawn from a normally distributed population because the significant value was greater than 0.05. Further, at a 0.05 significance level, the means of the data were compared employing Tukey’s post hoc test and homogeneity of variance was derived with Levene’s test.

Results and discussion

A key emphasis of the current study was to explore the efficiency of a native bacterial isolate in remediating nitro-aromatic explosive i.e., TNT aerobically. The strain S5-TSA-19 is a soil bacterium named Indiicoccus explosivorum isolated from the sample collected from an explosive manufacturing site containing up to a concentration of 900–1000 mg/Kg TNT. I. explosivorum can sustain in temperature between 4 and 37 ˚C.

Tolerance level of P. flavidum

The tolerance or sustaining capacity of the isolate with the explosive was evaluated in MSM at various concentrations of TNT (60, 80, 100, 120 mg/L), temperature (20 to 35 ˚C), and pH (5 to 9). Satisfactory results were detected up to higher concentrations of TNT i.e., 120 mg/L at 30 ˚C (± 5 ˚C), and neutral pH i.e., 7. Survival of the isolate at the higher concentration was indicative of its potential for further study.

Isolate’s morphology

SEM images of S5-TSA-19 isolate (SEM images have been detailed in Online Resource 1) with and without the nitro-aromatic explosive depicted that (a) strain is coccoid-shaped which is comparable to the morphology of Gram-positive Indiicoccus explosivorum (Pal et al. 2019), (b) no variation was observed in the isolate’s shape neither in presence or absence of TNT, confirming that I. explosivorum thrives flexibly well in the minimal medium comprising TNT and (c) 120 mg/L TNT was non-lethal to the strain.

Co-metabolism factor

Cells of I. explosivorum exhibited alike growth patterns in MSM with and without TNT (Fig. 1) and proliferate very well in both circumstances. This depicts the unaffected behavior of the isolate S5-TSA-19 against the nitro-aromatic explosive and co-metabolic pathway for degrading TNT as mentioned elsewhere (Dalton et al.1982; Nagar et al. 2018, 2020; Lamba et al. 2021). It attained extreme proliferation in the 12 days as is confirmed by the rise in absorbance at 600 nm from 0.086 initially to 1.073 on the 12th day (Fig. 1). Levene’s test for homogeneity of variance of growth of S5-TSA-19 depicted that there is an insignificant difference in the variances in the isolate’s population with and without TNT at 0.05 level since the p value is 0.7857 which means p < 0.05. Similarly, no significant difference among the pair of means was detected in Tukey’s test for paired mean comparison at a 0.05 level.

Fig. 1
figure 1

Growth comparison of Indiicoccus explosivorum with and without TNT during incubation in MSM. Error bars specify the standard deviation at the specified time

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Degradation kinetics

To evaluate the degradation rate of TNT with bacterial strain S5-TSA-19, the kinetics model, with first order was exercised against residual TNT concentrations (in treatment sets). Equation (1) depicted the “First-order rate equation”.

$$\mathit{ln}A=-kt+\mathit{ln}{A}_{0}$$
(1)

where Ao is the nitro-aromatic explosive concentration initially, A is TNT concentration at specified incubation duration, ‘t’ and ‘k’ represent rate constant for degradation. t0.5 is represented as follows:

$$t_{{0.5}} = 0.693k - 1$$
(2)

Through the rate of reaction, the value of k = 0.027 per day and t0.5 = 25.67 h.

Within the first 7 days of the incubation period (30 days), 120 ppm TNT was observed to be 100% degraded by isolate S5-TSA-19. In the control samples containing 120 mg/L TNT in MSM but no bacterial strain, no explosive degradation was noticed (Fig. 3b).

Fig. 2
figure 2

Pattern of nitrite release, nitrate transformation and ammonium formation during the incubation period of I. explosivorum (data represented in triplicate). Error bars specify the standard deviation at the specified time

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Correlation between nitrite, nitrate, ammonium, and TNT degradation

The end product of TNT degradation is composed of ammonium ions; thus, detection and analysis of the concentration of nitrite, nitrate, and ammonium ions and further correlating them along are important in TNT degradation. According to Mercimek et al. 2015, TNT’s demineralization in the culture with the advances in bacterial growth corresponds to the liberation of nitrite ions. In the present experiment too, an assemblage of nitrite ions was detected with the rise of S5-TSA-19 strain in the media which corresponds to TNT degradation escalation till complete explosive degradation (Fig. 2). Likewise, a consistent pattern was observed in the case of ammonium ion concentration, i.e., after incubation originally, concentration unswervingly amplified through the first 3 days, which subsequently becomes persistent (Fig. 2). Earlier reports support the observation made for nitrite, nitrate, and ammonium ions production along with the pattern of nitro-aromatic explosive degradation (Lotufo and Lydy 2005; Mercimek et al. 2015; Lamba et al. 2021).

Fig. 3
figure 3

a HPLC chromatographs of supernatant of culture media all through the degradation period along with the standard of metabolite (2ADNT) b TNT degradation in treated medium with the absence and presence of I. explosivorum. Error bars specify the standard deviation at the specified time

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A reductive pathway is responsible for TNT degradation where electron-withdrawing nitro groups (03) of TNT expedite the nucleophilic attack of a hydride ion. This leads to the development of a changeable Meisenheimer complex among TNT and the hydride ion (Esteve-Nunez et al. 2001; Conder et al. 2004). Further, the formation of DNT is confirmed with the release of nitrite ions. Within 7 days of TNT degradation through I. explosivorum (Fig. 3a), the formation of 2ADNT was depicted in the HPLC chromatogram. Furthermore, the LC–MS study confirms the TNT degradation pathway along with the formation of transitional compounds (Fig. 4). As per Lachance et al. 2004, nitro group conversion to an amino group during TNT degradation leads to the formation of 2-amino-4,6-dinitrotoluene (2-ADNT) and the same was evident in the current study (Fig. 4). A steady rise in nitrite concentration is indicative of the emergence of DNT as the initial intermediate detected at the preliminary phase of the catabolism of TNT (Lamba et al. 2021) but in the concluding phase, no DNT was noticed that put forward the increased metabolism and bacterium accelerating potential in degrading the compound ultimately producing toluene. Earlier, diverse researches recorded the transformation of DNT to toluene continuing to the TCA cycle via toluene cis-dihydrodiol formation (Esteve-Nunez et al. 2001; Serrano-González et al. 2018) and here in this study also LC–MS peaks confirmed the same (Fig. 4).

Fig. 4
figure 4

LC–MS spectra of TNT and its transformation products during the growth of isolate I. explosivorum in MSM supplemented with TNT

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Nitrate ions concentration showed an escalation at the early phase of the study followed by a gradual decrease which might be because of the primary transformation of NO2− ions to NO3−. But due to the instability of NO3− ions, they further get converted to NH4+ ions.

The disappearance of TNT concurrently through different pathways

TNT mineralization by pure bacterial system as compared to mixed or undefined bacterial cultures under aerobic or anaerobic conditions is missing in the literature (Kalderis et al. 2011; Serrano-González et al. 2018). The degradation rate of TNT in soil bacteria through nitrate is the best in contrast with other electron acceptors, such as sulfate and carbon (Boopathy et al.1993). TNT is transformed through mutually occurring aerobic and anaerobic circumstances reducing to amino-derivatives via non-specific NAD(P)H-dependent nitro-reductase (Kalderis et al. 2011). Although most of the bacteria follow a lone initial metabolic pathway for TNT transformation, some of them follow two different pathways at the same time (Vorbeck et al. 1994; HaÏdour and Ramos 1996).

Direct reduction of two-electron pathway

With the increase of time in the incubation period, a noticeable variation in the color of the TNT-enriched medium was detected. Explosive-enriched medium changed its color from white to reddish brown (darkened gradually till the end). The conversion of the culture’s color from red to brown is the validation for dihydride Meisenheimer complex formation (Pak et al. 2000; Nyanhongo et al. 2009; Gün Gök et al. 2019; Lamba et al. 2021). The product of direct reduction of two electrons of the aromatic ring via hydride addition reaction forms dihydride Meisenheimer complex, hydride Meisenheimer complex, and protonated tautomers (Serrano-González et al. 2018). This confirms the release of nitro groups during TNT degradation from the nitro-aromatic structural ring of the explosive. The unstable nature of the dihydride Meisenheimer complex contributes to the release of intermediate products like nitrite ions and DNT during TNT degradation (Serrano-González et al. 2018). The consecutive reduction in TNT concentration was in synergy with Meisenheimer complex formation and isolate’s activity which steadily led to the darkening of MSM color comprising TNT (Lamba et al. 2021).

Consecutive reduction of two-electron pathway

This metabolic pathway of TNT degradation initiates via successive reduction of two electrons of the nitro groups of the aromatic ring. Removal of electrons in the nitro groups produces reduced intermediates like di-amino-nitrotoluene (DANT), 2-amino-4,6-dinitrotoluene (2-ADNT), 4-amino-2,6-dinitrotoluene (4-A-2,6-DNT), isomers, and azo and azoxy dimers (Serrano-González et al. 2018).

Collective aerobic and anaerobic degradation of TNT

Aerobic degradation followed two initial biochemical routes. During the initial reduction process, TNT transforms to 2-hydroxylamino-4,6-dinitrotoluene or 2-hydroxylamino-2,6-dinitrotoluene. This is intervened by nitro-reductase/ nitrobenzene nitro-reductase/ dihydropteridinereductase, N-ethylmaleimidereductase, and nitrobenzene nitro-reductase (Serrano-González et al. 2018) and this led to the formation of 4-amino-2,6-dinitrotoluene or 2-amino-4,6-dinitrotoluene along with tetranitro-azoxybenzene compound. The aerobic route leads to the production of 2-amino-4-nitrosotoluene and 2,6-diamino-4-nitrotoluene as it progresses, further continuing with anaerobic degradation producing 4-amino-2,6-dinitrotoluene and 2-ADNT to conclude the process. Through NAD(P)H, nitro-reductase (non-specific) conversion of both 4-amino-2,6-dinitrotoluene and 2-ADNT to DANT takes place with anaerobic bacteria. With the involvement of hydrogenase or carbon monoxide dehydrogenase, DANT converts to 2,4-diamino-6-hydroxyl-aminotoluene (DAHAT), along with TAT formation by dissimilatory sulfite reductase. On the other hand, DANT get directly converted to TAT as also reported earlier in presence of C. sordelli, C. bifermentans, and C. sporogenes (Serrano-González et al. 2018). TAT is sequentially reduced to toluene and then to toluene cis-dihydrodiol. Toluene cis-dihydrodiol is a transitional compound within the pathway of toluene degradation. Further, toluene cis-dihydrodiol gets transformed into 4-hydroxybenzaldehyde as confirmed through the LC–MS chromatogram. Finally, 4-hydroxybenzaldehyde followed the pathway resulting in the trichloroacetate cycle, i.e., the TCA cycle (Serrano-González et al. 2018). Figure 5 depicts the proposed pathway.

Fig. 5
figure 5

Projected Pathway for TNT degradation

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Conclusion

The present study depicts the first reporting of both aerobic and anaerobic degradation of TNT in the presence of a novel soil isolate I. explosivorum, strain S5-TSA-19 in an aerobic environment. Throughout the study, satisfactory multiplication of the bacterium was observed in the growth medium containing TNT (120 mg/L). TNT, as known to be a xenobiotic compound, showed complete degradation after I. explosivorum action. Irrespective of the fact that the study was planned for 30 days, the complete disappearance of TNT (120 mg/L) was observed on the 7th day of incubation. Through the elucidation of the degradation pathway, DNT and 2-ADNT were observed to be the two key metabolites of this bioconversion process. SEM results confirmed the adaptable nature of the isolates, as the cell morphology did not change in presence of the toxic compound TNT. Admirable outcomes of the study prove the outstanding efficiency of I. explosivorum for TNT degradation. Therefore, the bacterium can be used on a large field scale along with suitable organic and inorganic amendments.