Bioremediation of RDX and HMX contaminated soil employing a biochar-based bioformulation

Compounds like Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) and Octahydro-1, 3, 5, 7-tetranitro-1, 3, 5, 7-tetrazocine (HMX) are categorised as a secondary explosive. These secondary explosives are widely employed for defence and civil purposes worldwide. The release of explosive compounds in the environment during their production, storage and usage results in severe soil and water contamination. Pollution caused by explosives is a major concern as it is recalcitrant in nature and has toxic effects on human beings, animals and plants. There is a need to find an eco-friendly and sustainable approach to deal with explosive contaminated soil. In the present study, bioformulation was prepared with explosive degrading bacteria to treat explosive contaminated soil. Bioformulation consisted of coconut husk-derived biochar as a carrier material and Arthrobacter subterraneus as an active ingredient. The survivability of bacteria and performance of bioformulation with different concentrations of explosive compounds were analysed. Results showed that Arthrobacter subterraneus could immobilise with biochar and can survive up to 6 months. The prepared bioformulation was able to degrade up to 85.98% RDX and 80.4% HMX in contaminated soil in a time duration of 30 days. A significant increase in nitrite concentration, a major byproduct of RDX and HMX biodegradation, was found in soil treated with bioformulation. Thus, bioformulation can be applied to remediate explosive-contaminated sites as an eco-friendly technique. • Coconut husk-derived biochar and explosive degrading bacteria were used to develop a bioformulation. • Explosive degrading bacteria survived up to 6 months at 4°C temperature in biochar bioformulation. • Bacteria in bioformulation thrived with different concentrations of RDX and HMX. • Bioformulation was able to reduce 80.4% HMX and 85.98% RDX from contaminated soil in 30 days. • Eco-friendly and sustainable approach was proposed to remediate explosive-contaminated soil.


Graphical Abstract 1 Introduction
High energetic munition compounds that release a huge amount of gases and energy on an explosion are called explosives.Nitramines like Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) and Octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX) are classified as secondary explosives.Generally, these high energetic compounds are used for defence activities due to their high detonation power, density and thermal stability (Zapata and García-Ruiz 2020).RDX and HMX are anthropogenic compounds not found naturally in the environment.These compounds enter our environment and contaminate air, water, and soil during their manufacturing, transportation, disposal, and use during military activities like training and war (Chatterjee et al. 2017).The agglomeration of these harmful and toxic explosives in the ecosystem has become a matter of concern as the toxicity of RDX and HMX on plants, invertebrates, and animals has been described by many researchers (Robidoux et al. 2003(Robidoux et al. , 2000)).United States Department of Health and Human Services has identified RDX as a potential carcinogen (U.S.Department of Health and Human Services, Public Health Service 2012).Also, HMX can affect the central nervous system, liver, kidney, brain and heart in animals (Kanekar et al. 2009).US Environmental Protection Agency (USEPA) has listed a health advisory of lifetime exposure limit of 0.002 mg/L for RDX and 0.4 mg/L for HMX in potable water (USEPA 2018).RDX and HMX are sparingly soluble in water and have less soil sorption coefficients.Therefore, most soils do not significantly retain these compounds, resulting in their seepage from the soil surface to the groundwater table (U.S.Department of Health and Human Services, Public Health Service 2012; EPA 2005; Singh and Singh 2012).The concentration of RDX and HMX in soil varies hugely at different explosive-contaminated sites.The RDX and HMX contamination can range from 800 to 1900 mg/kg and 600 to 900 mg/kg respectively.In some places, contamination up to 74000 mg/kg RDX and 5700 mg/kg HMX has been found.Conversely, concentration of 10 mg/kg RDX and HMX has been found at some training bases of air and land force.At antitank firing sites, concentration of HMX varies from 2.1 to 1640 mg/kg (Panz and Miksch 2012;Hawari 2000).EPA has established a residential soil screening level (SSL) of 390 mg/kg, industrial SSL of 5700 (mg/kg) and risk-based SSL of 0.13 mg/kg for HMX, and a residential SSL of 8.3 mg/kg, industrial SSL of 38 mg/ kg and risk-based SSL of 0.00037 mg/kg for RDX (NCBI 2021a, b).The explosive-contaminated soil needs to be treated to maintain safe SSL.
Conventional physico-chemical technologies employed for the remediation of explosive-polluted soil are not very cost-effective or efficient and can result in toxic by-products (Singh and Singh 2012).Application of bioremediation techniques can also have challenges like being a time intensive process, requiring a longer time frame compared to conventional physico-chemical remediation methods.The effectiveness of bioremediation can be influenced by site-specific factors, such as soil composition, pH, moisture content, and the presence of inhibitory substances.Variations in these factors can impact the performance of the bioremediation process, requiring careful site assessment and optimization (Kumar et al. 2018).With regular assessment of the bioremediation process, environmental conditions, and the effectiveness of contaminant degradation, bioremediation can be an eco-friendly, cost-effective and sustainable method to clean up explosive-polluted soil.Microbial remediation of xenobiotic compounds has emerged as an effective bioremediation method to clean up explosive-polluted soil (Chatterjee et al. 2017).Some microorganisms can metabolise these xenobiotics, often mineralising or converting them into lesser toxic by-products (Sangwan et al. 2018).However, the efficiency of microbes highly depends on various factors like nutrient availability, the bioavailability of the target compound, suitable temperature and pH.The xenobiotic degradation ability of microbes can be further enhanced by the use of biocarriers, as biocarriers can improve the chances of survival of selected microbes.A variety of carriers like biochar, eggshell, diatoms, cocopeat etc. have been used by researchers to enhance the chances of survival of desired microbes and degradation of targeted contaminants.For example, Liu et al. (2017) used a combination of biochar and immobilized bacteria to treat cypermethrin-contaminated soil.Similarly, Kahla et al. (2021) employed benthic diatom-associated bacteria for remediation of fluoranthene and benzo(a)pyrene.Biochar has been proven to enhance the quality of soil, and its benefits in the agricultural as well as the environmental field were reported by various studies (Yavari et al. 2015).Biochar is a carbon-rich and porous substance prepared by burning biomass under oxygen-deprived conditions.The porous structure of biochar has a high internal surface area which helps to retain water and nutrients in the soil and create an appropriate niche for microbial growth (Ab Aziz et al. 2015).Tan et al. (2023) have also highlighted the co-benefits of biochar in soil remediation and agronomics.The sorptive properties of biochar also increase the availability of contaminants to the microbes.Therefore, biochar has been regarded as a better carrier material for degradation microbes, as it also increases the bioavailability of pollutants (Liu et al. 2017).In a study conducted by Ren et al. (2021), it was found that biochar derived from peanut shell could reduce stress caused by cadmium pollution in tobacco plants.Many researchers have analysed the utilisation of biochar for cell immobilisation to remediate contaminants like pesticides (Liu et al. 2017) and polycyclic aromatic hydrocarbons (Chen et al. 2012).However, the usage of biochar for bacterial cell immobilisation in explosive remediation has not yet been reported.
Earlier research conducted to remediate explosive contamination using bioformulation focused on the elimination of explosives only by increasing the survival of desired microbes, but neglected the long-term impact on soil and environment.The present study aims to develop a sustainable bioremediation method for explosivecontaminated soil, in which a bioformulation consisting of native explosive degrading bacteria immobilised on a coconut husk-derived biochar was evaluated.An explosive-degrading bacteria Arthrobacter subterraneus, isolated from an explosive-contaminated site was used to prepare bioformulation.The explosive degradation potential of this isolate in the water was demonstrated in our previous work (Sharma et al. 2021).The present study is the first report on employing Arthrobacter subterraneus immobilised on coconut husk biochar to remove RDX and HMX in soil.Coconut husk biochar was chosen as a carrier material because of its well-known long-term benefits to increase the productivity of the soil.RDX and HMX degradation in soil by bioformulation during a time period of 30 days was reported.The survivability of bacteria in contaminated soil was analysed by colony forming units (CFU), and major degradation by-products, i.e.Nitrite concentration in soil, were also reported during the degradation of RDX and HMX.The viability of explosive degrading bacteria in biochar bioformulation was also observed for up to six months.

Chemicals
RDX and HMX with a purity of more than 99% were supplied by an explosive manufacturing facility in north India.Acetonitrile of HPLC gradient grade having purity > 99% was used as a solvent and procured by Sigma Aldrich.All chemicals utilized in this research were of analytical grade and bought from standard manufacturers.

Microorganism, culture media and growth conditions
Arthrobacter subterraneus (MTCC No.12883, Isolate name-S5-TSB-17) was isolated from the explosive-contaminated soil near a manufacturing facility in India and was received from the Institute of Microbial Technology (IMTECH), Chandigarh, India, in lyophilised form.A consortium of Paenibacillus aestuarii and Arthrobacter subterraneus was able to remove 80.4% RDX in 12 days, in RDX-contaminated water with an initial concentration of 40 mg/L (Sharma et al. 2021).In this research, Arthrobacter subterraneus was used to degrade explosives in contaminated soil due to its better survivability in soil.The isolate was revived on culture plates with trypticase soy agar (TSA) as a culture medium.TSA was prepared by adding 2.5 g di-potassium phosphate, 5 g sodium chloride, 15 g agar, 2.5 g dextrose, 3 g soya peptone and 17 g casein peptone per liter of demineralised water.The isolate was, later on, cultured with trypticase soy broth (TSB) as a growth medium.The broth cultures were incubated in an orbital shaker incubator at 120 rpm and a temperature of 33 ± 2 °C in aseptic conditions (Sharma et al. 2023).Afterwards, the culture was supported in the minimal salt medium (MSM), which had nutrients in a limited quantity.At first, isolate was made adaptive to the minimal salt medium.MSM was composed of 0.061 g MgSO 4 •7H 2 O, 0.053 g NH 4 Cl,0.278 mg FeSO 4 •7H 2 O,  11.09 mg CaCl 2 •2H 2 O, 0.015 mg MnCl 2 •4H 2 O, 0.024 mg  H 3 BO 3 , 0.0028 mg ZnSO 4 •7H 2 O,1.74 g K 2 HPO 4 ,  1.36 g KH 2 PO 4 , 0.0024 mg CuSO 4 •5H 2 O, 0.0071 mg CoCl 2 •6H 2 O, 0.59 g succinic acid, 0.900 g glucose, and 0.92 g glycerol in one litre of demineralised water.Then isolate was enriched with increasing RDX and HMX concentration as the only source of nitrogen in MSM.The enriched isolate was subcultured and maintained for further studies.

Preparation of biochar bioformulation
Waste coconut husk was obtained from the local market.It was sundried to remove unnecessary moisture and was cleaned to remove any unwanted material or debris like soil, small pebbles and plastic pieces.Cleaned coconut husk was subjected to an average temperature of 500 °C in the absence of oxygen for 3 h to convert it into biochar.The biochar was sieved through a 500-micron sieve.The powdered coconut husk biochar was added with 2% starch as an additive.The biochar and starch mixture was sterilised two times at 121 °C temperature for 20 min in an autoclave.The sterile carrier mixture was inoculated with explosive degrading isolate Arthrobacter subterraneus as an active ingredient and mixed thoroughly.Prepared bioformulation was left in a laminar airflow cabinet overnight to air dry and later on packed in sterile autoclave packets leaving about 25% airspace to give proper aeration to the inoculants (El-Hadidy 2019).The initial count of cells in biochar-based bioformulation was made to obtain 10 14 cells/g at the time of storage.

Pot experiment for explosive-contaminated soil remediation with bioformulation
Garden soil was dug out from the top layer (5-20 cm) and screened through a 2 mm sieve.The experimental setup is depicted in Table 1, and the experiments were carried out in the lab.The soil was contaminated using RDX and HMX dissolved in acetonitrile separately to make the final concentration of 50 mg/kg HMX, 100 mg/ kg HMX, 50 mg/kg RDX and 100 mg/kg RDX.Later on, acetonitrile from the soil was allowed to evaporate overnight.1 kg of garden soil contaminated with RDX and HMX was filled in each plastic pot of 15.5 cm diameter and 14.5 cm high.Contaminated soil was mixed thoroughly with 2% (w/w) prepared bioformulation, while no bioformulation was added in controls.The controls with biochar only and bacteria only mixed with contaminated soil are provided in the Supplementary file.All the pots were sprinkled with water regularly to maintain 40% moisture of water holding capacity by adding distilled water (Liu et al. 2017).Soil samples were taken from each pot on the 0 th , 5 th , 10 th , 20 th and 30 th days.Each soil sample was analysed in duplicate.

Analytical methods
The viable number of cells in bioformulation was analysed by adding 1 g of bioformulation in 50 mM phosphate buffer having pH 7 in a sterile 50 mL polypropylene tube.The suspension was shaken vigorously for 10 min to separate bacterial cells attached to the biochar, followed by serial dilution and spreading on TSA plate (Chuaphasuk and Prapagdee 2019).Similarly, soil treated with bioformulation was also enumerated for a viable cell count of explosive degrading isolate Arthrobacter subterraneus at regular intervals for up to 30 days.The colony forming units (CFU) in the bioformulation and treated soil were determined by the standard plate count technique using a colony counter.The surface of the biochar before and after the immobilisation of bacterial cells was monitored under a Scanning Electron microscope (SEM).The method used for preparing samples for SEM to analyse immobilised bacteria on the surface of biochar has been previously described by Prapagdee and Wankumpha (2017).Briefly, the samples were fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer for 1 h, followed by washing with phosphate buffer and then deionised water.The sample was later on dehydrated by serial application of ethanol at concentrations varying from 30 to 99.99%.The samples were dried with Hexamethyldisilazane (HMDS) as an alternative to critical point drying.The dried sample was mounted on a stub with graphite tape and coated with gold.The concentration reduction of RDX and HMX was evaluated employing the High-Performance Liquid Chromatograph (HPLC) from Perkin Elmer Inc., USA.C18 column was used as a stationary phase with 50:50 v/v mixture of acetonitrile: triple distilled water with 1 mL/min flow rate as mobile phase.Detection of RDX and HMX was performed at 254 nm.Samples for HPLC analysis were prepared by following the USEPA 8330 B method (EPA 2006).Briefly, 2 gm of soil sample was added in 10 ml of acetonitrile and vortex shaken for 1 min.The suspensions of soil and acetonitrile were sonicated in a cooled (18 °C) ultrasonic bath for 18 h.Samples were kept to settle for 30 min and then passed from a 0.45 μm polytetrafluorethylene (PTFE) filter.The quantification was done by preparing a standard curve with known RDX and HMX concentrations.
Soil nitrite was extracted by adding 0.05 g calcium sulphate in 5 g of soil with 10 ml distilled water (Norman and Stucki 1981).The soil suspension was shaken on a mechanical shaker for 10 min, and then it was centrifuged at 10,000 rpm for 10 min.Nitrite released was detected by UV VIS spectrophotometer at 540 nm, as illustrated by Nyanhongo et al. (2006).

Statistical analysis
Single-factor analysis of variance (ANOVA) was employed to conduct statistical analysis.P-value less than 0.05 was regarded as significant.The effectiveness of bioformulation in degrading RDX and HMX present as contaminant in soil were evaluated in term of confidence interval.The contaminant degradation data are depicted as mean values ± standard deviation (SD).The data processing and analysis were conducted using Microsoft Excel.

Cell immobilization on biochar
SEM analysis illustrated the external morphology of coconut husk-derived biochar before (Fig. 1a) and after immobilisation of explosive degrading bacteria Arthrobacter subterraneus (Fig. 1b).The number of viable cells of Arthrobacter subterraneus adhered to coconut husk biochar was 15 log CFU/g of biochar.The survival of bacterial cells for up to six months shows that carrier material was able to maintain the survival of cells stored under 4 °C temperature.The viability of bacteria over the period of six months is depicted in Fig. 2. The CFU of the prepared bioformulation was found to be sufficient, with a loss of 4.39 log units in the sixth month.El-Hadidy (2019) also reported the viability of bacterial strain immobilised on biochar for six months.Physicochemical and elemental constituents of coconut husk-derived biochar at an average temperature of 500 °C are shown in Table 2.
Prepared biochar shows high water holding capacity of 103.75%, which also helped in maintaining soil moisture.The elemental composition of biochar was analysed by a CHNS analyser.Besides carbon and nitrogen, some amount of elements like Sodium, Chlorine, Sulphur and Potassium are also found in coconut husk-derived biochar (Suman and Gautam 2017).Therefore, the coconut husk biochar used as carrier material provided the required nutrients and suitable habitat for the survival of the isolate.Hence, biochar can be considered a suitable carrier material for the cell immobilisation of desired microbe (Chuaphasuk and Prapagdee 2019).

Survival ability of bacteria in contaminated soil
The ability of Arthrobacter subterraneus to thrive in RDX and HMX-contaminated soil was studied to further apply biochar-based bioformulation for RDX and HMX remediation from the soil.Figure 3 demonstrates the survivability of bacteria in contaminated soil after mixing bioformulation in soil for 30 days.It was observed that the number of Arthrobacter subterraneus viable cells increased from 6.44 to 7.83 Log CFU/g in 50 mg/kg HMX contaminated soil, while 100 mg/ kg HMX contaminated soil showed an increase from 6.76 to 7.04 Log CFU/g in time duration of 10 days.A decline in the number of viable cells was observed after 10 days in both cases.Similarly, viable cells of explosive degrading isolate increased from 6.69 to 8.24 Log CFU/g in 50 mg/kg RDX-contaminated soil for 10 days, after which there was a decline in their number.In the case of 100 mg/kg RDX contaminated soil, there was an increase of viable cells from 6.28 to 8.15 Log CFU/g in a time duration of 20 days, after which the number of viable cells showed a decline.The CFU of bacteria increased from 6.13 to 7.06 Log CFU/g in the case of soil contaminated with a mixture of RDX and HMX.A similar biochar-immobilised bacteria growth trend in cadmium-contaminated soil was observed by Chuaphasuk and Prapagdee (2019).The decline in number of viable cells in RDX and HMX spiked pots can be due to increased nitrite concentration in the soil as increased nitrite can have an inhibitory effect on the growth of bacteria.Nitrite concentration was highest during 10 th day sampling in most of the cases.Moreover, metabolites formed during RDX and HMX degradation can also adversely affect the bacterial population in the pots.The maximum number of viable cells was comparatively observed more in a lower concentration of RDX and HMX.The growth of Arthrobacter subterraneus in RDX spiked soil was higher than that of HMX spiked soil.This can be due to lower chemical stability of RDX than HMX and better susceptibility to degradation by the bacteria.The higher stability of HMX can be attributed to its molecular structure, which contains eight nitro groups that provide steric hindrance and enhance its resistance to decomposition.The tightly packed structure of HMX molecules contributes to their greater stability to withstand higher temperatures, mechanical stress, and other environmental conditions without significant decomposition (Song et al. 2021).The results showed that biochar assisted Arthrobacter subterraneus cells in RDX and HMX-contaminated soil to withstand toxicity stress from RDX and HMX.This can be elucidated by cell immobilisation enhancing microbial cells' resistance to toxic or stressful environments (Jézéquel et al. 2005;Dzionek et al. 2016).Moreover, biochar can assimilate and hold nutrients in the soil and create suitable soil moisture and temperature to enhance microbial survival (Jiang et al. 2017;Chen et al. 2018).

Degradation of RDX and HMX by bioformulation
The novel biochar-based bioformulation was evaluated for its efficiency in remediating RDX and HMX-contaminated soil.Samples were taken at regular intervals of time for HPLC analysis to determine the decrease in the concentration of RDX and HMX. Figure 4 shows a reduction in the concentration of HMX in soil with bioformulation and control conditions.The pot containing an initial concentration of 50 mg/kg HMX was reduced to 9.98 mg/kg in 30 days after the application of bioformulation, while in the control condition, HMX was reduced to just 38.06 mg/kg.Similarly, the pot having an initial concentration of 100 mg/kg HMX was reduced to 46.3 mg/  kg HMX in 30 days after the application of bioformulation while in the control condition, HMX was reduced to just 78.4 mg/kg.Therefore, bioformulation was able to remove 80.4% and 53.7% HMX in the pot containing an initial concentration of 50 mg/kg HMX and 100 mg/ kg HMX, respectively.Pots which did not contain bioformulation showed a decrease of only 23.88% and 21.6% for 50 mg/kg and 100 mg/kg initial concentration of HMX, respectively.Single factor ANOVA was applied to find significance of HMX degradation in the control pot and bioformulation-containing pot.In case of pots containing 50 mg/kg HMX, a p-value of 0.038 was observed which is less than 0.05.Hence, bioformulation was effective in degrading 50 mg/kg HMX with 95% confidence interval.
Similarly, in the case of 100 mg/kg HMX p-value was 0.062 which is less than 0.1.Therefore, bioformulation was effective in degrading 100 mg/kg with 90% confidence interval.
Figure 5 shows the reduction in the concentration of RDX in contaminated soil with bioformulation and control conditions.The pot containing an initial concentration of 50 mg/kg RDX was reduced to 7.01 mg/ kg in a time duration of 30 days after the application of bioformulation, while in the control condition, RDX was reduced to just 35.3 mg/kg.Similarly, the pot having an initial concentration of 100 mg/kg RDX was reduced to 34.84 mg/kg RDX in 30 days after the application of bioformulation, while in the control condition, RDX was reduced to just 74.87 mg/kg.Therefore, bioformulation was able to remove 85.98% in the pot containing an initial concentration of 50 mg/kg RDX and 65.14% RDX in the pot containing an initial concentration of 100 mg/kg RDX.Pots which does not have bioformulation showed a decrease of only 29.4% and 25.13% for 50 mg/kg and 100 mg/kg initial concentration of RDX, respectively.On application of ANOVA to compare the degradation of 50 mg/kg RDX, a p-value of 0.0439 was obtained which is less than 0.05.The p-value, in this case, shows that bioformulation was effective in degrading the contaminant with a 95% confidence interval.Similarly, in pots containing 100 mg/kg RDX, a p-value of 0.048 was obtained which again showed the bioformulation was effective in degrading RDX with 95% confidence interval.The decrease in the concentration of RDX and HMX in soil contaminated with a mixture of RDX and HMX is shown in Fig. 6.The concentration of 50 mg/Kg RDX was reduced to 13.73 mg/Kg and the concentration of 50 mg/ Kg HMX was reduced to 18.15 mg/Kg.Bioformulation was able to reduce 72.54%RDX and 63.7% HMX in soil contaminated with a combination of RDX and HMX.In the case of soil contaminated with RDX and HMX mix, its degradation percentage was lower than the soil contaminated with RDX and HMX separately.On comparing control and treatment sets, a p-value of 0.030 and 0.021 (< 0.05) for RDX and HMX was obtained respectively.Therefore, in bi-pollutant pots, the bioformulation was effective to degrade RDX and HMX with 95% confidence interval.The degradation percentage of a bi-pollutant system can be lower, compared to a single-pollutant system due to several reasons like competitive biodegradation, bacterial growth inhibition or increased toxicity and prevention of any enzymatic activity by one of the pollutants.Moreover, in soil contaminated with both RDX and HMX, the degradation percentage can also be affected by the metabolic priority of microbes and complex degradation pathways (Sagi-Ben Moshe et al. 2009).
The percentage removal of RDX and HMX after inoculation of bacteria only into the soil was just 57.54% and 43.68%, respectively, after 30 days.While the percentage removal of RDX and HMX after the application of biochar only was 37.10% and 28.42%, respectively (Supplementary Fig. 1).The results showed that the application of bioformulation in RDX and HMX-contaminated soil significantly increased the rate of RDX and HMX biodegradation.The combined action of biochar and bacteria in bioformulation resulted in better degradation as the availability of RDX and HMX to the immobilised bacteria increased due to the absorption of RDX and HMX on biochar.In a study conducted by Roh et al. 2015, it was found that alginates biochar Beads could absorb up to 28.09 mg/g RDX.Moreover, the high initial concentration of RDX and HMX affected the efficiency of bioformulation due to the higher toxicity of explosives at increased concentrations.On comparing Figs. 4 and 5, it can be inferred that bioformulation was slightly more efficient in degrading RDX than HMX.This can be due to the high chemical stability of HMX, which makes it less susceptible to biodegradation than RDX (Sagi-Ben Moshe et al. 2009;Hawari et al. 2000).

Nitrite released during RDX and HMX degradation
Nitrite is one of the major byproducts formed during the biodegradation of RDX and HMX (Coleman et al. 2002).Figures 7, 8 and 9 depict the nitrite formed during the Fig. 6 Decrease in concentration of RDX and HMX mix in contaminated soil treated with bioformulation and control condition degradation of RDX and HMX.Nitrite formation in the case of samples containing bioformulation was comparatively more than in the control samples.Maximum nitrite formation of 8.4 mg/kg was seen in samples of the pot containing an initial concentration of 100 mg/kg HMX along with bioformulation on the 10 th day followed by nitrite release of 7.93 mg/kg in the pot containing contaminated soil with an initial concentration of 50 mg/ kg HMX treated with bioformulation (Fig. 7).In case of RDX contaminated soil, maximum nitrite formation of 9.19 mg/kg was seen in the pot containing an initial concentration of 100 mg/kg RDX along with bioformulation on 10th day, followed by nitrite release of 7.93 mg/kg in the pot containing an initial concentration of 50 mg/ kg RDX treated with bioformulation (Fig. 8).Similarly, in case of soil contaminated with both RDX and HMX, highest nitrite release of 8.52 mg/Kg was seen with soil treated with bioformulation on 20 th day.The increased nitrite concentration can slow down RDX and HMX degradation rates because of competitive inhibition as nitrite can compete with RDX and HMX for microbial degradation pathways or enzyme systems.While in the control sample, maximum nitrite release was just 4.03 mg/Kg on the 10 th day (Fig. 9).Nitrite concentration continued to increase up to 10 days after which it started declining in almost all cases.The reduction in nitrite concentration could be due to the uptake or conversion of nitrite by soil microbes.It can be concluded that there was comparatively less nitrite formation in controls which depicts very low RDX and HMX degradation in control samples.Nitrite formation was dependent on the available concentration of RDX and HMX to the isolates along with their ability of RDX and HMX degradation (Cho et al. 2016).It can also be inferred that there was a continuous increase in the concentration of nitrite during the reduction in the amount of RDX and HMX.In most of the studies, nitrite is the first metabolite released which is produced by denitration before the ring cleavage.Other secondary metabolites like methylenedinitramine (MEDINA), bishydroxymethylnitramine, N-methyl-N,N′-dinitromethanediamine, 5-Nitro-1,3,5-triazinane-1,3-diamine, 1,3,5-trinitroso-1,3,5-triazinane (TNX) and nitro-3,5-dinitroso-1,3,5-triazinane (DNX) have also been reported during biodegradation of RDX and HMX (Sharma et al. 2021).

Practical implications of this study
The results of the study can have practical applications to develop bioformulations based on biochar which can facilitate the biodegradation of RDX and HMX in polluted environments.The study outcomes can be relevant in military or defence applications where RDX and HMX are frequently used as explosives.Biochar-based bioformulations offer a potential avenue for the sustainable management of RDX and HMX waste and can lead to the development of eco-friendly and cost-effective approaches for the treatment and disposal of waste containing RDX and HMX residues, thereby reducing the need for conventional and energy-intensive methods.The major challenge in current research is the transition from laboratory-scale studies to real-world field applications.The effectiveness and feasibility of the biochar-based bioformulation need to be assessed under varying environmental conditions, in different contaminated sites, and in the presence of other co-contaminants.For successful large-scale implementation, optimization of production processes, guarantee of consistent quality, and determination of the appropriate dosage and application techniques are important parameters to be considered.Understanding the long-term stability and persistence of biochar-based bioformulation is also crucial.It is necessary to evaluate the degradation efficiency over extended periods and assess any potential adverse effects or by-products that might accumulate in the environment.
In future work, field trials are required to evaluate the effectiveness of the bioformulation in actual contaminated sites which can provide valuable insights into the performance, applicability, and limitations of the biocharbased bioformulation under realistic conditions.Also, to ensure the environmental safety of the bioformulation, comprehensive ecotoxicological assessments need to be conducted.These assessments will evaluate the potential impacts on non-target organisms and ecosystems.
Besides this, conducting a cost-benefit analysis is important to evaluate the economic feasibility and sustainability of implementing biochar-based bioformulation on a larger scale is recommended.Assessing the potential benefits, including environmental remediation and cost savings compared to traditional methods, will aid in decision-making and wider adoption.By addressing the challenges, focusing on future work, and providing recommendations, the study can pave the way for practical implementation, facilitate the transition from lab to the field, and contribute to the broader application of biochar-based bioformulations for the biodegradation of RDX and HMX contaminants.

Conclusion
In this study, an eco-friendly bioformulation was developed to remediate RDX and HMX-contaminated soil.The waste coconut husk-derived biochar acted as a suitable carrier material for the immobilisation of an explosive-degrading bacterium, Arthrobacter subterraneus isolated from explosive-contaminated soil.Bioformulation was able to sustain viable cells of bacteria for up to six months.Moreover, biochar-based bioformulation helped Arthrobacter subterraneus to survive and proliferate in RDX and HMX-contaminated soil.The bioformulation efficiently degraded 85.98% RDX and 80.4% HMX in soil spiked with an initial concentration of 50 mg/kg RDX and HMX.The sorption properties of biochar in the bioformulation increased the availability of RDX and HMX for biodegradation by the bacteria.The concentration of the explosives and the presence of bi-pollutant affected the rate of degradation.It can also be concluded that RDX was more susceptible than HMX to degradation by Arthrobacter subterraneus.Singlefactor ANOVA showed significant results for the degradation of RDX and HMX by the prepared bioformulation.A higher amount of nitrite formation in soil treated with bioformulation also confirmed better biodegradation of RDX and HMX in the soil as nitrite is one of the major biodegradation by-products.Moreover, biochar amendments are well known to increase the productivity of the soil.Therefore, the bioformulation remediated soil can be used for planting trees and agricultural purposes.Thus, biocharbased bioformulation can be potentially used as a sustainable approach to remediate and manage explosive residues, waste disposal, and environmental stewardship within military facilities or training grounds to reclaim contaminated areas.A further intricate study is required to investigate the detailed mechanism, enzymes involved and secondary metabolites formed during RDX and HMX biodegradation and its applications in field conditions.

Fig. 1
Fig. 1 SEM image of biochar surface before (a) and after immobilization with explosive degrading isolate Arthrobacter subterraneus (b)

Fig. 4
Fig. 4 Decrease in concentration of HMX in contaminated soil treated with bioformulation and control condition

Fig. 7 Fig. 8
Fig. 7 Nitrite released during biodegradation of HMX in soil

Fig. 9
Fig. 9 Nitrite released during biodegradation of RDX and HMX mix in soil

Table 1
Experimental design for remediation of explosives using bioformulation

Table 2
Physicochemical and elemental compositions of coconut husk derived biochar at an average temperature of 500 °C Fig. 3 CFU of Arthrobacter subterraneus in bioformulation treated soil contaminated with HMX and RDX