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Research Article Journal of Advanced Research in Biotechnology Open Access Kachia Military Shooting Range in situ Fungi Species Biodegradation of Explosives, Kaduna, Nigeria Alhaji Isyaku1, Ayodele A. Otaiku2*

1Department of Geography, Microbiology/Environmental Management, Faculty of Science, Nigerian Defence Academy Kaduna, 2Doctoral student, Nigerian Defence Academy (NDA), Faculty of Arts & Social Science

Received: September19, 2019; Accepted: September 20, 2019; Published: December 12, 2019

*Corresponding author: Ayodele A. Otaiku, Nigerian Defence Academy (NDA), Kaduna, Nigeria. P.No: +2348033721219; Email-id: otaiku.ajayiayo- [email protected] , [email protected]

Abstract Kachia 24.95 sq km military training (small arms and artillery weapons) shooting ranges, longitudes 90 55’ N and 70 58’ E at 732m elevation above sea level, was established 1965 Kaduna, Nigeria. Soil pollutants explosives Xenobiotic four locations was studied where soils samples collected

each isolates were processed for sequencing and characterization and contains Lignolytic fungi Aspergillus Niger, Rhizopus spp, Penicillium spp, and analysed using EPA SW-846 Method 8095 contains TNT, RDX, HMX and PETN. White rot fungi analysed with amplified using 16srRNA gene of (P<0.05) with higher values in dry season with RDX (68.19 mg/kg) highest for both seasons at locations 3 and 4 with artillery weapons. Aspergillus nigerTrametes had versicolorthe best ability and Phanarochate to degrade explosives chrysoporium. while Penicillium Explosives ANOVAspp had test the shows least ability significant in 1% difference explosive for mineral all locations salt broth in both supplemented dry and wet with seasons 1%

in increasing order were Aspergillus Niger > Phanorochate chrysoporium > Trametes versicolor > Rhizopus spp > Penicillium spp. Xenobiotic biodegradationv/v explosive (P>0.05) occurred from under the co-metabolismzero hour to the with 5th daycarcinogen and beyond potentials with significant to biodiversity. difference in the overall growth pattern. Heavy metal reduction

Keywords: Xenobiotic; Explosives; In situ Bioremediation; Explosives by turbidometry; Military shooting ranges; Nitrate reductase; Heavy metals; White rot fungi; Co-metabolism; 16srRNA gene.

Highlights Enzymes Biochemistry TNT, RDX, HMX and PETN Toxicity to ecosystem Biotransformation Introduction Biodegradation The most widely used explosives in the world are probably hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), Fungi 16srRNA Gene 2,4,6-trinitrotoluene (TNT); octahydro-1,3,5,7-tetranitro-1,3,5,7- Mineralization tetrazocine (HMX) and Pentaerythritol tetra nitrate [PETN;

Symbiosis Group *Corresponding author email: [email protected] , [email protected] Copyright: Kachia Military Shooting Range in situ Fungi Species Biodegradation of © 2019 Otaiku A. A and Alhaji I. A Explosives, Kaduna, Nigeria

C(CH2ONO2)]4its use in items such as exploding bridge wire shown that these derivatives themselves may further degrade (EBW) detonators and exploding bridge foil initiators (EFI) in microcosm experiments with the fungal strain Phanerochaete by Foltz, 2009; Ronen and Bernstein, 2014 (Figure 1). These chrysosporium [90], or in soil slurries with a mixed culture compounds are characterized by relatively high thermal stability, [92], as well as in other experimental systems and microbial high density and high detonation velocity, all of which promote transformations of nitrate esters PETN [232]. their extensive use [40,237]. Some of their physical properties HMX is less amenable to biodegradation in the environment are summarized in Table 1. Explosive compounds are undesirable than RDX due to its lower water solubility and its relative in the environment due to their toxicity. TNT, RDX and HMX have chemical stability [89]. It is structurally similar to RDX, and thus follows analogous degradation pathways: both sequential abnormal liver function, spleen enlargement, nervous system and reduction of the nitro group to nitroso derivatives and anaerobic immunebeen defined system as (ATSDR toxic to 1996a, humans ATSDR and animals,1996b) [12, causes 13]. anaemia, de nitration followed by ring cleavage have been documented as RDX is composed of a triazinic ring to which three nitro HMX-degradation pathways limited microbial strains were found functional groups are perpendicularly attached. In RDX, capable of degrading this compound. Similar to the sequential similar to TNT, the nitro groups are the main targets of the reduction of RDX to its corresponding nitroso derivatives following subsequent two-electron transfer steps [133], nitro as key steps. Biodegradation clearly shows the potential for groups of the HMX molecule can be reduced to nitroso derivatives. reducingfirst degradation the concentrations steps by sequential of explosives reduction in the or environment de nitration Nevertheless, while for RDX, all three nitroso derivatives are via microbial activity TNT [53, 134,195,234]; RDX [89,133] on observed in high abundance, in HMX, only less reduced nitroso the biodegradation of both RDX and TNT; and RDX and HMX by. derivatives are normally detected. For example, the detection of only a mono nitroso derivative was documented by [59] with microbial degradation is often initiated by reductive rather than oxidativeBecause of reactions, high electron even deficiency under aerobic on the conditions nitro groups in Figure of TNT, 2. its * nitroso derivatives: mono- and dinitroso. HMX was observed by Available for Fungi Species Study. [91]the fungal following strain incubation P.chrysoporium. of HMX withThe formationanaerobic ofsludge. the first Studied two Nevertheless, further transformation of the mono and diamino enzyme xanthine oxidase [25]. Based on the detected products, derivatives toward the formation of the most reduced product- theythe transformationproposed that HMX pathway undergoes of HMX a single with denitration the metallo-flavo step. triaminotoluene (TAT) proceeds only under strictly anaerobic conditions with redox potential values below -200 mV [89].Thus, Looking at the reductive transformation of the nitro group from under oxic conditions, diamino derivatives tend to accumulate, a purely energetic compounds, one would expect the decrease in while the presence of TAT is indicative of strictly reduced conditions. Under aerobic conditions, the formation of nitroso other hand, in complex biotic systems such as the sub-surface, derivatives is normally not observed. This may be expected from factorsreduction other rate than from energetic TNT to RDX, yield and will finally dictate to HMX the rate[215]. at Onwhich the thermodynamic considerations, where the calculated E0 shows explosives degrade. Nutrient availability is one important factor a decrease from trinitro aromatics to nitramine, suggesting thermodynamic control of the reduction of RDX under aerobic Compared to other common pollutants, explosives, particularly conditions [215]. Only a few exceptions indicate formation of RDXthat plays and aHMX, significant are characterized role in the rate by of explosivesa higher nitrogen/carbon biodegradation. mono nitroso derivatives under aerobic conditions as reported (N/C) ratio. Although, they may theoretically serve as sources for incubation of the white-rot fungus P. chrysosporium with of both carbon and nitrogen, only nitrogen is used by some RDX which, showed formation of MNX. This was followed by ring organisms, and hence, another carbon source must be added [4]. cleavage and the subsequent formation of methylene dinitramine The aim of this research is to identify bioremediation (MEDINA), Figure3 [89]. technology that can be adapted for the clean-up of munitions and TNT can be biodegraded via various pathways, which explosive compounds contaminated soils sites in Kachia, Kaduna mainly involve transformation of the nitro functional group, state, Nigeria. The objectives study are: To examine and identify while the aromatic ring remains intact [89]. The stability of the aromatic ring results from the strong electron-withdrawing sites; determine the physiochemical properties of soil in Kachia properties of the nitro substituents which promote high electron the munitions/explosive compounds in the weapons firing system [43]. In the presence of oxygen, however, it has been speciesmilitary to firing utilize range munitions and environs; compounds characterize ; screen and the isolate soil type genes at showndeficiency that and both electrophilic the nitroso characteristics and the mono on hydroxyl the p-electron amino responsiblefiring range andfor bio environs; remediating determined possibly the munitions ability of isolatedcontaminated fungi metabolites can follow an alternative abiotic transformation soil.; compare the rate of possibly munitions contaminated soil pathway to form tetra nitro azoxytoluene dimmers, also referred between dry and wet seasons. as azoxy tetra nitro toluene as shown in Figure 2[82,134]. These azoxy products were shown to cause a higher rate of mutations than TNT [65] and were thought to suppress the degradation of RDX and HMX which may coexist with TNT in the environment [92]. The accumulation of azoxy derivatives in the environment is hence clearly undesirable, but laboratory experiments have

Citation: Otaiku A. A and Alhaji I . A (2019) .Kachia Military Shooting Range In situ Fungi Species Biodegradation of Explosives, Page 2 of 27 Kaduna, Nigeria. J Adv Res Biotech 4(2):1-26. Copyright: Kachia Military Shooting Range in situ Fungi Species Biodegradation of © 2019 Otaiku A. A and Alhaji I. A Explosives, Kaduna, Nigeria

Figure1: Molecular structures of TNT, RDX, HMX and PETN

Table 1: Physical and chemical properties of TNT, RDX and HMX

Composting in Explosive Remediation performed by the sequential action of two intracellular enzymes, nitrate reductase which converts nitrate (NO3) to nitrite (NO2), approved and selected for use in remediating military sites [43]. Composting was the first biological process to be tested, and nitrite reductase which reduces nitrite to ammonium (NH4). In Aspergillusnidulans and Neurospora crassa, which are the remediation of monition compound contaminated soils by the USComposting Army Toxic has and been Hazardous identified Materialsas a feasible Agency technology and US for Army the both enzymes are under the control of catabolite repression by Armament Research and Development Command [61]. Several ammonium,most thoroughly the actual studied regulator filamentous being intracellular fungi in this glutamine. respect, types of composting systems exist, but static pile and window Further, nitrate acts as a signal which activates the transcription composting are the commonly used in explosive remediation of enzymes, i.e. their genes are expressed only in the absence of [112]. A problem associated with composting is the high pile ammonium and the simultaneous presence of nitrate regulatory temperature sometimes resulting in volatilization of pollutants mechanism has been proposed for A nidulans and reported that and also long incubation times. In situ bioremediation is generally an unknown enzyme(s) was involved in the reduction of RDX utilized in areas where contamination is deep and excavation by living fungi are, in fact, nitrate reductases, given that RDX would be excessive and bioremediation involves treating the contaminated material at the site. The process involves the commercially available nitrate reductase from Aspergillus niger passing of water enriched with nutrients, oxygen and/or with hasdegradation recently isbeen repressed shown byto beammonium capable of and degrading given that RDX a purified in vitro microorganisms through the contaminated area and composting [41,103,129,130,213]. soils contaminated with organo nitro explosives, including PETN as well as extracellular enzymes Phanerochaete chrysosporium [45, 46]. release the intermediate products hexahydro-1-nitroso-3,5- dinitro-1,3,5-triazine Activity of this and pure methyl-enedinitramine nitrate reductase was which sufficient further to Fungi Biodegradation decomposed to nitrous oxide, formaldehyde and ammonium RDX degradation by Penicillium species and other soil fungi and RDX degrader Penicillium sp. AK96151[24, 223]. Species was strongly inhibited by ammonium but not by nitrate [222]. of Penicillium and Aspergillus are phylogenetically closely Nitrate is a widely utilized nitrogen source but needs to be related, and P.chrysogenum possesses an equivalent regulatory converted to ammonium after uptake. Nitrate reduction is mechanism of nitrate assimilation to that outlined above for A.

Citation: Otaiku A. A and Alhaji I . A (2019) .Kachia Military Shooting Range In situ Fungi Species Biodegradation of Explosives, Page 3 of 27 Kaduna, Nigeria. J Adv Res Biotech 4(2):1-26. Copyright: Kachia Military Shooting Range in situ Fungi Species Biodegradation of © 2019 Otaiku A. A and Alhaji I. A Explosives, Kaduna, Nigeria nidulans [27, 81]. Xanthine reductase and sulphite reductase are the only ones which are known to occur in fungi and are repressed by ammonium. However, on mechanistic grounds neither can be postulated to play a role in RDX degradation, and both nitrate reduction by protoplasts and RDX degradation by mycelial cultures are best explained on the basis of our knowledge of the regulatory mechanism of nitrate reductase activity. Whereas, a pure fungal nitrate reductase has been shown to degrade RDX into formaldehyde, N2O and NH4+1 augmented with µmol mg [24, 43 and 67], and the enzyme is also involved in RDX degradation by living fungi.

(FigureThese results 2), it was support impossible the view directly first expressed to test the byability that ofthe our reduction protoplasts of one to degradenitro group RDX by [133]. one specificThe fungal enzyme nitrate is reductasesufficient toenzyme bring isabout therefore degradation rather ofversatile, RDX. Unfortunately, being capable since of reducing RDX degradation ionic NO isas such well a as slow chlorate process and even nitro with groups our highlyof organic efficient molecules. RDX degrader Averill (1995) has pointed out that all these substrates are structurally related to each other [27]. The only previous report of a fungal nitro reductase attacking the nitro groups of explosives is that of on TNT reduction by Phanerochaete. However, since these authors assayed neither the ability of their enzyme to reduce NO nor any inhibitory effect of NH4+ on or the requirement of molybdenum for nitro group reduction, their enzyme might well have been a nitrate reductase[168]. Fungal nitrate reductases reported so far appear to be intracellular enzymes, being bound to membranes in contact with the cytoplasm such as the plasma membrane and tonoplast or membranes of mitochondria, or they are located in the soluble cytoplasm [116,171and181]. Xenobiotic such as RDX or TNT therefore, have to be taken up across the plasma membrane before they can be bio transformed or although the uptake mechanism is unclear at present [223]. RDX degradation by nitrate reductase can be inhibited by as little as 150µMdegraded. NH4+, Using and radio this labelledwas quite RDX, similar have to shown the NH4+ that concentration this substance measured is indeed in present the groundwater in an unmodified at the site form which in the we mycelial studied (approx.fraction, 100 µM; Kuhn, 2001). In general, mitosporic genera such as Penicillium and Trichoderma as well as Zygomycota such as Absidia and

Figure 2: Reduction pathway of TNT, and further transformation of the reduced derivatives under aerobic and anaerobic conditions.

Citation: Otaiku A. A and Alhaji I . A (2019) .Kachia Military Shooting Range In situ Fungi Species Biodegradation of Explosives, Page 4 of 27 Kaduna, Nigeria. J Adv Res Biotech 4(2):1-26. Copyright: Kachia Military Shooting Range in situ Fungi Species Biodegradation of © 2019 Otaiku A. A and Alhaji I. A Explosives, Kaduna, Nigeria

Figure3: Postulated degradation pathways of HMX compounds in brackets are postulated intermediate Source: Ronen and Bernstein, 2014 Materials and Methods Sampling Technique and Soil Treatments Study Site Soil Sampling The study was conducted in the permanent military shooting/ Sampling was done during both dry and wet season. Four training range located at 5km east of Kachia town in Kaduna locations located within NASA shooting/training range Kachia state, north central Nigeria. The range was established in 1965 and it covers an area of about 24.95sq km that lies between were earmarked as sampling sites for this study using soil iron longitudes 90 55’ N and 70 58’ E, with a elevation of 732m above auger.10 gram of soil sample (0-30 cm in depth) with diameter sea level and the topography is undulating and the vegetation of 9cm were collected from 3 different points within a location is Guinea Savannah (Figure 4).The area where the munitions/ and harmonized to form a composite sample at various locations of the sites. All samples collected were sieved using a 63 (106m) mesh size laboratory sieve and then stored in black labelled landexplosive and explode are fired during (the military impact training. area) is Five a valley military consisting exercises of polythene bags until for analyses. Samples for microbial analyses involvingabout four the large deployment rocks, where of explosives the fired are munitions/explosives carried out annually were kept in a cool box refrigerated with ice pack to retain the by the Nigerian Defence Academy (NDA) Kaduna, Nigerian Air original microbial activities. force (NAF) Kaduna, Nigerian Army School of Infantry (NASI) Jaji, Armed Forces Command and Staff College (AFCSC) Jaji and Soil Sample Pre-treatment Nigerian Army School of Artillery (NASA) Kachia. Sampling points were treated in the laboratory before digestion Sampling Points as follows. 10 grams of the soil sample was weighed into a clean dried beaker and put into an oven at about 100 0C for 1 hour. The Four sampling points selected for the study are locations 1, soil sample was then ground in a porcelain mortar with pestle 2, 3 and 4, Table 2. Locations 1 and 2 are the twin smallest rocks and sieved through 250 µg mesh size to obtain a homogenous closest to the road. While location 3 and 4 are much larger rocks sample. The soil sample was stored in sterilized polythene bags, heavily impacted by munitions/explosives between locations label and kept for next stage of pre-treatment. This procedure stream in this site (map with Global positioning system (GPS) co- was repeated for all the collected soil samples. The ground soil ordinates).Locations1and 2 is a flat ground 1 and where 2 approximately rain run offs 200mflow through from the to table the samples were used for analyzing heavy metal and explosives content for soil samples [99]. FN, Greenade, GPMG, SMG and Pistols. The soil in locations 1 and top that is where the small arms are fired such as Kalashinokov, Digestion of Soil Sample for heavy metals and explosives analysis approximately2 is made of 50% 9000m silt and away a flat from ground the table with top shrubs lies between and drainage 90 53’ 1.0g of pre-treated soil sample was accurately weighed and 44.71”that flow North through and 70to 53’the 17.87”farm lands East. near The impactthe sites. area Location of location 3 is placed in a 100 cm3 beaker. Then, 4.0cm3 of 70% of HCL04 and 3 and 4 are mainly largely rocks containing high concentration 2cm3 of concentrated HN03were added to the sample and the of explosive due to the extensive use of bombardment by the resultant mixture was put in the oven at 100 0C overnight. A Artillery weapons,155 mm nortwizer, and other heavy weapons white ash was formed which was dissolved in 2cm3 of 1m HCL. while location 4 is ahead of location 3 is about 10,000m from beaker was then rinsed with small portions of double distilled between locations 3 and 4 was covered with various shrubs and The digest was filtered into 50cm3 standard volumetric flask. The twothe Plateau major streams. top where heavy weapons are fired too. The distance water and quantitatively transferred into the flask. The resultant Citation: Otaiku A. A and Alhaji I . A (2019) .Kachia Military Shooting Range In situ Fungi Species Biodegradation of Explosives, Page 5 of 27 Kaduna, Nigeria. J Adv Res Biotech 4(2):1-26. Copyright: Kachia Military Shooting Range in situ Fungi Species Biodegradation of © 2019 Otaiku A. A and Alhaji I. A Explosives, Kaduna, Nigeria solution was made up to the mark with double distilled water, Primary analysis was conducted on a 6 -m-x 0.32 mm ID-fused transferred into a clear dried plastic sample bottle, labelled and kept for heavy metal and explosives analysis. This procedure was -methyl -siloxane (RTX -5 from Restek). The GC oven was - silica column, with a 1.5-um film thickness of 5% (Phenyc) repeated for all the samples collected at this site. A blank was temperature programmed as follows; 1000C for two minutes, prepared using the same procedure excluding the soil sample 100C minutes ramp to 2600 C, two minutes hold. The carrier [166]. gas was helium at 10 ml/minute (Linear Velocity approximately

Heavy Metals Analysis Principle 40 ml/minute. If a peak was observed in the retention window The analysis was done using Atomic Absorption 90 cm/second). The ECD make-up gas was nitrogen flowing at Spectrophotometer (AAS) at the laboratory of Geological Survey Kakuri-Schimadzu AAG50 model. The digested Soil Samples for a specific signature compound, the extract was re-analyzed were analyzed for heavy metals. Heavy metals analyzed include polysiloxane(RTXon a confirmation -225 column, from 6-mx Restek 0.53-mm ).If analyze ID having Concentrations a 0.1-mm Arsenic, Boron, cadmium, chromium, cobalt, copper, iron, lead, werefilm thicknesstaken from of the 50% determination cyanopropylmethyl-50% on the primary phenyl column, methyl- unless magnesium manganese, Nickel and zinc using AAS, according they appear to be coalition with another compound, in, such cases to APHA (1998). The technique makes use of absorption reported concentration were taken from the determination of the spectrometry to assess the concentration of analyzed in a sample by measuring the absorbance against a known concentration. It confirmation column. relies on the Bear Lambert Law. Table 2: Global positioning system (GPS) recorded in degrees, minutes and seconds (DMS) of ls Analysis Principle S.No Location Northing’s Easting’s Elevation Explosive Analysis 1 Kachia 90 52’24.42” 70 57’ 18.61” 715m The analysis was carried out at National Research, Institute Loc 1 for Chemical Technology (NARICT) Zaria, Nigeria. The vials 2 90 53’ 50.23” 70 53’ 54.53” 818m & 2 containing the AcN (Acetonitrite) soil extracts were placed into GC auto sampler trays that were continuously refrigerated by 3 loc 3 & 4 9053’ 44.71” 7053’ 17.87” 773m circulating zero OC glycol/water through the trays, the extracts 4 Control 9053’47.63” 7053’34.77” 644m were analyzed by gas chromatography using a micro-lectron capture detector (GC-µECD). Screening for Explosive/Munitions Degrading Organisms Preparation of Media Nutrient Agar Nutrient Agar (Antec/USA) by dissolving 28g of the Agar in 1

media was autodaved at 121OC at 15 Pressure Per Square much (PSI)litre of for distilled 15 minutes. water Cooled in a conical to 400C flask. before The pouring conical flaksor dispensing with the if a sterile petri dishes

Mac Conkey Agar Mac Conkey Agar was prepared by dissolving 49.53g of

It was heated to dissolve the medium completely. The medium wasdehydrated sterilized medium by autoclaving in 1000ml at distilled 15 1bs water pressure in a conical(1210C) flask. for 15 minutes. Cooled to 45-500C, mixed well before pouring into sterile petri plates. Figure 4: Map of Kachia within Kaduna state map, Nigeria Showing study area (Nigerian Army Shooting Range). Potato Dextrote Agar (PDA) Source: Geography Department NDA, Kaduna, Nigeria Thirty nine (39) g of dehydrated medium suspended in 1000ml distilled water was heated to dissolve the medium completely. It Results were obtained on HP-6890 GC equipped with a micro was then sterilized by autoclaving at 15 1bs pressure (1210C) for cell Ni63 detector at 2800C according to the general procedure 16 minutes. Cooled at 10% of tartaric acid was added and mixed outlined, in EPA SW-846 method 8095 [51]. Direct injection well to achieve pH 3.5 before dispensing into petri plates. The µL of soil extract was made into a purged packed inlet port, at 2500C, that was equipped with a deactivated Restek Uniiner. of the media. Plates were incubated at 25-300C in an inverted media were spread with the specimen soon after solidification

Citation: Otaiku A. A and Alhaji I . A (2019) .Kachia Military Shooting Range In situ Fungi Species Biodegradation of Explosives, Page 6 of 27 Kaduna, Nigeria. J Adv Res Biotech 4(2):1-26. Copyright: Kachia Military Shooting Range in situ Fungi Species Biodegradation of © 2019 Otaiku A. A and Alhaji I. A Explosives, Kaduna, Nigeria position (agar slide up) with increased humidity. Cultures were spp, Rhizopus spp; Trametes versicolor, and Phanerochate examined weekly for fungal growth and were held for 4-6 weeks. chrysoporium, respectively. The experiment was left to stand for 24 hours after which the concentration of cadmium, chromium, Serial Dilution nickel, zinc, cobalt, iron, lead, cupper and arsenic were measured The sterile dilution blanks were marked in the following using Atomic Absorption Spectrophotometer (AAS). manner. 100 ml dilution blank was 102 and 9 ml tubes sequentially Bioremediation of Explosive in Soil Were 103, 104, 10-6 one gram of soil sample was weighed from each sample located and added to the 12-2 dilution blank and Nutrient broth and soil solution were added in a ratio 4:1.8 set vigorously shaken for at least one minute with the cap securely tightened. All the 10-2 dilution was allowed to sit for short were sterilized in an autoclave at 1210C for 15 minutes. 120ml of of 250 ml flask were Set for the work. The broth and soil solution Period. The 1ml\ from this dilution was aseptically transferred to nutrient broth and 30 ml of sterilized solid solution were added the 10-3 dilution was again transferred to the 10-4 Dilution. The

into each of the fifteen flasks. The first flask was left inoculated, from the 450 C water Bath was especially pounded into each petri explosive/munitions which are Aspergillus Niger, Rhizopus but the second flask was inoculated with the isolated fungi from platesprocedure for that was set. done 15ml to was10-5 poured and 10-6. enough A flask to cover of nutrient the bottom agar spp, Penicillium spp, Trametes versicolor and Phanarochate of the plate and mixed with the 1 ml inoculum in the plate. Each chrysoporium. The experiment set-up was left to stand for 14 set was gently swirled on the bench so that the inoculum gets days and the micro remediation was measured by taking the thoroughly mixed with the ager. optical density (O.D) reading at 595minutes from zero hour-14 days at regular intervals of 2 days against the control [217]. All the plates were allowed to stand without moving so that the Isolation of Genomic DNA from Fungi stacked into pipette canisters and placed in the incubator or at agar solidified and set completely. The plates were inverted and The genomic DNA of each fungus with observed remediation room temperature until after 48 hours the same procedures were capabilities was isolated. 1ml of each fungal culture was applied for Mac Conkey agar and PDA. Pelleted by centrifuging at 12,000 rpm for 2 minutes, the pellet Polymerase Chain Reaction (PCR) for Amplification of was treated with Iysis solution and proteinases K and incubated Catabolic genes at 600C for 30minutes. Deoxyribose nucleic acid (DNA) was Principle extracted from each fungus precipitated with Isopropanol by centrifuging at 10,000 rpm for 10 min, washed with 1 ml of a 70% (v/v) ethanol solution and dissolved in 0.1 ml of a T.E buffer. The and the principle is based on the mechanism of DNA replication PCR consist of an exponential amplification of DNA fragment purity and quantity of (DNA) of each sample was examined using in vivo, double stranded DNA is denatured to single stranded UV absorption spectrum and\agarose gel (1%) electrophoresis DNA, each single strand DNA is anneal by the forward and as described by [106, 204]. reverse primers of known sequence and elongated using tag DNA polymerase to produce copies of DNA template. Polymerase Chain Reaction Amplification of 16srRNA Gene

Procedure The PCR reaction mixture containing 10 X PCR buffer, 25mm, magnesium chloride, 25mm dNTP’s, 10pm/ul Primer

2,3dioxygenase gene primers. The genomic DNA, the primers and Isolated explosive fungi DNA were amplified using catechol of the 16srRNA gene for each isolates. PCR conditions were PCR master mix were added into a PCR tube. The tubes were spun optimizedconcentrations using and lab template net thermal DNA were cycler. used forThe the PCR amplification Program to collect the droplets. The tubes were then inserted into the PCR began with an initial 5-minutes denaturation step at 940C: 35 machine while maintaining the regulation for initial denaturation cycles of 940C for 45seconds, annealing (1 minute at 550C), (45 seconds at 940C), annealing (1minute at 550C) extension and 10 minutes’ extension step at 720C. All reaction mix were preserved at 40C until it was time for analysis as reported by (1minute at 720C) and final extension (10 minutes at 720C). gel electrophoresis stained with ethidium bromide and viewed characterized using gel electrophoresis, Plate 1. underThe amplified ultra-violet catabolic (UV light). genes were resolved in 1.5% agarose [114].The amplified 16srRNA gene of each isolates was further Sequence Determination of 16srRNA Gene Bioremediation of Heavy Metals in Soil

The soil samples solution was sterilized using an autoclave at for sequencing and characterization. The sequencing Kit (Applied 1200C for 15 minutes. 100ml of the sterilized soil Bio systems)The amplified with the16srRNA product gene was of analyzed each isolate with ABIwas prism processed DNA sequence (ABI). The gene sequence of each isolates obtained in inoculated with fungi (control), the second, third, fourth, this study were compared with known16srRNA gene sequences Solution was poured into conical flasks. The first flask was not in the Gene Bank database as described by [106]. Fifth, were each inoculated with Aspergillus spp, Penicillium

Citation: Otaiku A. A and Alhaji I . A (2019) .Kachia Military Shooting Range In situ Fungi Species Biodegradation of Explosives, Page 7 of 27 Kaduna, Nigeria. J Adv Res Biotech 4(2):1-26. Copyright: Kachia Military Shooting Range in situ Fungi Species Biodegradation of © 2019 Otaiku A. A and Alhaji I. A Explosives, Kaduna, Nigeria

Determination of Explosive/Munition Biodegradability of military activities [29,50,153,154,216]. Fungal Isolates Turbidometry was used to determine the growth pattern of the fungal isolates, utilizing TNT in the mineral Salt medium as carbon source. The biodegrading activities of each fungal isolate were determined by using Basal salt broth to which was added 1% of the explosive of samples. The inoculated fungus in each tube containing the medium composition was incubated for 40 days at room temperature (28-300C). The test tubes were shaken constantly throughout the duration of the experiment to facilitate phase contact. The ability to biodegrade the explosive products (based on the growth rate of the organisms in the Basal salt medium was measured every 5 days using the turbidity of the Basal salt measured by the photoelectric calorimeter. The fungal growth rate of each fungus was measured by taking the optical Figure 5: Percentage of Soil texture in various locations, Kachia, Density (O.D) readings at 595nm from 0 hours -40 days at regular Nigeria. L10m Location (1) – zero metre, L1 200m Location 1 intervals of 5 days against Basal salt broth medium as blank as 200 metre, Loc 1, 400 Location1 400 metre, Loc 20m – locations demonstrated by [106], Figure 13. (2).0 metre, Loc 2- Location (2)

Statistical Analysis Table 3: Explosives sites analyses of wet and dry seasons, Kachia, Nigeria Data collected from the study were analyzed using general Dry Season µg descriptive statistics, one way Analysis of variance (ANOVA) at N/S Explosives Wet Season µg Kg-1 kg-1 were found, Duncan’s Multiple Range test was used to compare 1 TNT 0.12 – 26.6 0.11 – 10.29 95% probability level of Significance. If significant differences the different experimental groups. Computer Software Statistical 2 HMX 1.43 – 68.19 0.39 – 35.67 Package for Social Scientists (SPSS) and Microsoft-Excel were 3 RDX 0.38- 15.33 0.49 – 68.19 used for the statistical analysis. 4 PETN 1.17 – 7.12 0.39 - 4.38

Results and Discussion The results of seasonal variation of total explosive contents in Physio-Chemical Properties of the Military Firing Ranges various locations of NDA shooting range Kachia, Table 3 with impacts of distance on the concentration of the various explosives The temperatures of 22 - 34oC during the dry season and indicated high order of artillery bombardment in locations 3 and 27.16oC during the wet season were recorded respectively. 4 compared to locations 1 and 2 which is the closest distance The values obtained fell within the acceptable Federal Ministry of Environment (FMENV), Nigeria limit of <40. Similarly, the explosives such as TNT, RDX, HMX and PETN. It was revealed that pH of 6-8 has been suggested to be appropriate for microbial RDXto the had Plateau the highest top where concentration small arms of 68.19are fired µgkg-1 does in not dry explode season bioremediation (US-EPA, 2006). In the current investigation, the soil pH range for all the soil samples falls well within the range suggested by US-EPA (2006) and range of 27-35oC similar to the locationscompared in to both wet seasondry and that wet was seasons not very (P<0.05). significant. In zero ANOVA metre test of report of Jenkins et al., (2001) on characterization of explosives thefor totalobjective explosive TNT RDXcontents and showsHMX were a significant consistently difference found forat the all highest values of 49.39mgkg-1, 68.19mgkg-1 and 35.67mgkg- of silt, clay and loamy. 1, respectively (location 4). An impact area and beyond within contamination at military firing ranges with soils characterization The increased temperature obtained during the dry season can be attributed to the high concentration of explosives due to the the heavy artillery and mortar range that had been analyzed by the hand grenade range, a 105mm howitzer firing point and frequent exercises by the personnel of Armed forces of Nigeria. GC-ECD.TNT concentrations ranged from 0.11 to 49.39 mgkg-1 Compared with the wet season that the temperature is less in dry season and 0.12 to 26.6 mgkg-1 in wet season. Among all as a result of rain washing away heavy metals and explosives the concentration of explosives both in dry and wet season RDX drown the streams. The high value of electric conductivity EC recorded the highest concentration of 68.19 mgkg-1.Which is suggestive to us that the Kachia shooting range is polluted with of , respectively on their physico- chemical of the soils samples explosives most especially the RDX and agreed by EPA according withinrecorded the during FMENV the wetand seasonWHO permittedis in agreement limits with of 500/µ5/cm the finding to that RDX was formerly used as a rat poison, has also been listed and within the value of 2000mg/l which was contrary to the as a priority pollutant and as a possible carcinogen [124] (Tables 4 and 5 respectively). in agreement with the high values of TDS would be due to the depositfinding of,of whoseorganic valuesand inorganic were within matters 2400-5900 necessitated mg/l. by and the is

Citation: Otaiku A. A and Alhaji I . A (2019) .Kachia Military Shooting Range In situ Fungi Species Biodegradation of Explosives, Page 8 of 27 Kaduna, Nigeria. J Adv Res Biotech 4(2):1-26. Copyright: Kachia Military Shooting Range in situ Fungi Species Biodegradation of © 2019 Otaiku A. A and Alhaji I. A Explosives, Kaduna, Nigeria

Figure 6: Bioremediation potential of fungal Isolates to heavy

Mean Seasonal Variation of Heavy Metal Contamination The results of the seasonal variation of heavy metals contamination of soil samples from various locations of shooting range Kachia is presented in Figure 7. The overall highest heavy metal contamination was (Fe) and (Pb) recorded at Zero metre in dry season (814.13 mgkg-1) and (25.28 mgkg-1) respectively. The lowest were observed in (Fe) and (Cd, Zn, Co, As) recorded at 400m during wet season (67.93mgkg-1) in Figure 11.

Toxicity of Explosives to life forms in Kachia, Kaduna, Nigeria Figure 7: Fe (Iron) was the highest soil impacted with mean standard vale of 500.69 Fe>Pb>Cu>Mn>Zn>Cr>As>Cd>Co The ability of fungi to produce extracellular enzymes and factors that can degrade complex organic compounds has also sparked research on their use in decontamination of explosives-

Phanerochaete chrysosporium has been shown to hydrolyze the nitrateladen soils ester and nitro-glycerine waters [79]. (glyceryl The filamentous trinitrate) white-rot [46]. As long fungus as the nitro-glycerine extracellular concentration was under the lethal dose, metabolite formation and regioselectivity depend nature of the strains used, Table 4.

Fungi Isolates Biodegradation of Explosives and Heavy Figure 8: Fe (Iron) was the highest soil impacted with mean Metals standard vale of 458.30 Fe>Mn>Cr>As>Zn>Pb>Cd>Co>Cu Fungi Isolated Characterization The results of this study indicate that many of the fungal species isolated from toxic metals and explosives were Aspergillus spp, Penicillium spp, Rhizopus, Trametes Vesicolor and Phanorochate demonstrated that Lignolytic fungi such as Penicillium spp [60]. Aspergilluschrysoporium. spp, This Rhizopus result and agrees Phanerochate with the findingchrysoporium of which a white dot fungus was capable of oxidizing cyclic nitramines and mineralizing RDX and HMX. This is in conformity with Sheremata and [89]. Where the fungus mineralizing 52.9% of an initial RDX concentration in 60 days in liquid culture to mainly Co2 and N20. Figure 9: Fe (Iron) was the highest soil impacted with mean And 20% of an initial HMX concentration in 58 days when added standard vale of 374.57

Citation: Otaiku A. A and Alhaji I . A (2019) .Kachia Military Shooting Range In situ Fungi Species Biodegradation of Explosives, Page 9 of 27 Kaduna, Nigeria. J Adv Res Biotech 4(2):1-26. Copyright: Kachia Military Shooting Range in situ Fungi Species Biodegradation of © 2019 Otaiku A. A and Alhaji I. A Explosives, Kaduna, Nigeria to soil slurries of ammunition contaminated soil to yield the same However, different fungi harbour chemical and structural N product [59]. Similarly, Aspergillus spp, Rhizopus, Penicillium properties which are relatively different like Aspergillus and Trametes Vesicolor isolated in the study sites in Plate 1. niger cell lack of chitosan in the cell wall exhibit better metals heavy metals Cd, Zn, Co, Pb, Cu, and Ni.., [71]. Table 5 6 and 15 respectively, similar to the reports of [10, 62,223,240]. Collaborated the finding of having biodegradation potentials on biosorption efficiency as compared other fungi isolates in Figures fungal activities and can subsequently affect the fermentation processesHeavy metal and ions fungi are observed as potential to influence biosorbents the metabolism and had of theled Kapoor and Viraraghavan (1997a). fungi are easy to grow and produce large amount of biomass, and it can also be manipulated morphologically and genetically to suit the interest of study [80, 240]. For example, Aspergillus strains have been used in the production of gallic acid, kojic acid, ferrichrome, itaconic acid, citric acid, glucose and enzymes such as amylase and lipases. Fungal cells can be altered genetically and morphologically to produce better raw biosorbent materials [226]. Fungal biomass provides high percentage of cell wall content which play an Figure 10: Fe (Iron) was the highest soil impacted with mean important role in the metal-binding process [94]. The fungus standard vale of 155.45 Penicillium adsorbent material in removing heavy metal ions Aspergillus niger is a popular microorganism used in the biotechnological applications [143, 198]. Rhizopus nigricans was studied Kogej and Pavko, 2001 to show metal biosorption capacity Thus, showing fungal cell wall play a crucial role in the heavy metal biosorption capacity. This work focused on indigenous fungi microbes found in the study site (Kachia) with a view to ascertaining their bioremediation capabilities and recommending ways by which such indigenous microorganisms can be stimulated to enhance biodegradation of such toxic metals and munitions contaminants. Characteristic of fungi isolated from twelve locations in NASA Figure 11: Fe (Iron) was the highest soil impacted with m e a n shooting range Kachia Kaduna. Five (5) fungal genera were standard vale of 169.71 isolated in all the twelve sampled NASA shooting range (Plate 3). The growth appearances of those fungal isolates observed on Potato Dextrote Agar (PDA) were dark, pigment, grey or, yellowish green to dark green, yellowish-brown dark grey centre with light periphery, mycelia and spores. Microscopic examination of acid fuschin stain of the fungal isolate revealed probable features of Rhizopus spp, Aspergillus niger, Penicillium spp Trametes versicolor, and Phanorochate chrysoporium blow. Plate 3Agarose gel electrophoresis of the 16srRNA gene for the fungal isolates

of 100bp – DNA market (Ladder) lane F1-Aspergillus niger, lane F2-Rhizopusfrom the study spp, site lane in Kachia F3-Penicillium military firingspp, rangelane F4-Trametes shows Lane versicolor and lane F5 -Phanorochate chrysoporium. Figure 12: Fe (Iron) was the highest soil impacted with mean standard vale of 160.86 Fungi Implicated in Biotransformation and Mineralization The fungal cells are mostly multicellular and protected by a rigid cell wall (Figure 13), and play an important site in sequestering the metal ions and made of various polysaccharides, which are made of 90% of the cell wall, include lipids, proteins, pigments, polyphosphates , inorganic ions and N-acetylglucosamine residues[223, 224]. Plate 1: Unexploded Rocket launcher

Citation: Otaiku A. A and Alhaji I . A (2019) .Kachia Military Shooting Range In situ Fungi Species Biodegradation of Explosives, Page 10 of 27 Kaduna, Nigeria. J Adv Res Biotech 4(2):1-26. Copyright: Kachia Military Shooting Range in situ Fungi Species Biodegradation of © 2019 Otaiku A. A and Alhaji I. A Explosives, Kaduna, Nigeria

Fungi Species (Explosives by Turbidometry): The Growth Potential The Figure 14 shows the optical density reading of biodegrading activity of each fungal isolate on 1% explosive mineral salt medium (MSM) broth (Basal salt medium). It was revealed that Aspergillus niger had a maximum growth peak at 9.004 on the 30th day (Figure

40th respectively, Penicillium spp had the lowest growth peak at optical density2.500 on the 30th day while the maximum was 6.010 on15) the as affirmed10th day by[223]. similar The workmaximum of Rhizopus growth spppeak had of Trametesmaximum versicolor growth peak was at at optical 7.800 ondensity the 20th 8.000 day - 8.500and the on lowest the 25th, growth 35th peak and was at 1.500 on the 10th day. Phanorochate chrysoporium had the maximum growth peak at 6.000 on the 20th day while the lowest for P. chrysoporium was at 1.000 on the 10th day. Aspergillus niger had the best ability to degrade explosive while Penicillium spp in explosive mineral salt (Basal Salt Medium)broth supplemented with 1% v/v explosive (P>0.05). The overall result also indicates had the least ability. The analysis of variance result shows that there is a significant difference in the overall growth pattern of fungi ligninthat the peroxidases growth rate of increased P. chrysosporium significantly are encodedfrom the byzero a minimum hour to the of 5th 10 dayclosely and related beyond. genes, Means designated on the same from column A to J. having These differentare lipA, lipB,superscripts lipC, lipD, are lipE, significantly lipF, lipG, differentlipH, lipI (P<0.05)and lipJ. (Table according 6). to Duncan multiple range test. Stewart and Cullen (1999) also reported that

Table 4: Toxicity of heavy metals to life forms in Kachia, Kaduna, Nigeria

Microrganisms Metal Effects on Human Effects on Plants Fungi Isolates Bioremedation References Impacts Nagajyoti et al, Bone disease, coughing, Chlorosis, decrease in Damage nucleic Asp. sp> Rhz sp> Pen sp.> Cadmium 2010; Fashola, et al., emphysema plant nutrient acid T.Vesicolor > 2018 Chibuike et al., 2014 headache, Inhibits > P.chrysoporium ; Sebogodi et al., hypertension, etcs metabolism 2011 Ayangbenro and

Babalola ,2017 Brain damage, Deactivation of Abdul-Wahab, and Arsenic Physiological disorders P.chrysoporium > T.Vesicolor > cardiovascular and enzymes Marikar, 2012 ;

conjunctivitis, Loss of fertility, Finnegan and Chen, Pen sp.> Asp. sp > Rhz sp dermatitis, skin cancer Inhibition of growth, 2012

Destory metabolic

processes

Abdominal pain, Chlorosis, oxidative Disrupt cellular T.Vesicolor > Dixit et al., 2015; Copper anemia, diarrhea, stress, function P.chrysoporium>Asp. sp > Salem et al., 2000

kidney damage, inhibit enzyme Salem et al., 2000 ; retard growth Rhz sp> Pen sp. metabolic disorders, activities Odum,1971

Bronchopneumonia, Chlorosis, delayed, inhibition of P.chrysoporium> T.Vesicolor > Barakat, 2011; Chromium chronic bronchitis, senescence oxygen uptake Asp. sp > Mohanty et al., 2012

reproductive toxicity, stunted growth, Pen sp.> Rhz sp Cervantes, 2001 lung cancer, oxidative stress

Anorexia, chronic reduce enzyme Denatures nucleic P.chrysoporium > T.Vesicolor Nagajyoti et al., Lead nephropathy, insomnia, activities acid and >Rhz sp> 2010 ; Mupa, 2013

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damage to neurons, risk Affects photosynthesis inhibits enzymes Wuana and Pen sp.> Asp. sp Alzheimer ’s disease, and growth, activities Okieimen, 2011

Cardiovascular reduced nutrient Disrupt cell Chibuike and Nickel diseases, chest pain, T.Vesicolor >P.chrysoporium > uptake membrane Obiora, 2014 dermatitis,

inhibits enzyme kidney diseases, lung Decrease chlorophyll activities Pen sp.> Asp. sp .> Rhz sp Malik, 2004 and nasal cancer, content

Ataxia, depression, Reduced chlorophyll P.chrysoporium >T.Vesicolor> Zinc gastrointestinal inhibits growth Gumpu et al., 2015 content Rhz sp irritation,

icterus, impotence, impacts on plant Death, decrease in Asp. sp >Pen sp. kidney and liver failure, growth biomass,

T.Vesicolor>P.chrysoporium > Cobalt Asp. sp

>Pen sp.> Rhz sp

oxidation of dopamine, Roth, 2006; Takeda, Plant growth affected P.chrysoporium Manganese resulting in free 2003; Mena et al., and organisms >T.Vesicolor>Pen sp.> radicals, fitness of the 1967 cytotoxicity nervous Rollin, 2011; damage to system (CNS) poor quality yield > Rhz sp Asp. sp Wedler, 1994; mitochondrial DNA pathology Barbeau ,1984

Bioremediation Potentials of Fungi Isolates to Heavy Metals to 14.8%, Rhizopus spp to 5%, Penicillium spp to 10.9%, T. versicolor to 43.2%, P. chrysoporium to 41.7% Arsenic was Figure 6 shows the fungal isolates showed very good reduced by A. niger to 13.3%, Rhizopus spp to 14%, Penicillium activity in the reduction of heavy metals when observed. Lead spp to 19.4%, T. versicolor to 19.8%, P. chrysoporium to 19.8% . concentration was reduced by Aspergillus niger to 20%, Rhizopus The best fungal isolates for heavy metal reduction in increasing to 31.5%, Penicillium spp to 25%, Trametes versicolor to 54.5%, order were Aspergillus niger > Phanorochate chrysoporium > Phanorochate chrysoporium to 57% .Cadmium was reduced by A. Trametes versicolor > Rhizopus spp > Penicillium spp while, niger to 16.5% Rhizopus spp to 17.5%, Penicillium spp to 15.3%, in the overall, ANOVA test for total explosive contents shows a T.versicolor to 24.8%, Phanorochate chrysoporium to 20.5% .Zinc was reduced by A. niger to 32.49%, Rhizopus spp, to 38.8%, (P<0.05) . Penicillium spp to 34.2%, T.versicolor to 32.8%, P. Chrysoporium significant difference for all locations in both dry and wet seasons to 37.85% .Cobalt was reduced A. niger to 100%, Rhizopus Enzymes Biochemistry: Explosives and Heavy metals spp to 19.7%,Penicillum spp 21.8%, T. versicolor to 100%, Bioremediation P. chrysoporium to 100%..Copper was reduced by to 22.1%, The white rot fungus contains all three enzymes lignin Rhizopus spp to 6.5%, Penicillium spp 23.19%, T. versicolor to peroxidase (LiP), manganese-dependent peroxidase (MnP) and 34.5%, P. Chrysoporium to 32.8%.Managanase was reduced by A. lactase (Lac) called lignin modifying enzymes (LMEs) and non- niger to 60.4%, Rhizopus spp to 7%, selective [31,120,182]. LiP is unable to oxidize lignin to CO2 in Penicillium spp to 16%, T. versicolor to 61.6%, P.chrysoporium a cell-free system.It requires certain radicals, Veratryl alcohol to 63.3%.Nickel was reduced by A. niger to 17.1%, Rhizopus (VA) is a secondary metabolite produced by P. chrysosporium spp to 8%, Penicillium spp to 18.6%, T. versicolor to 26.8%, P. under nitrogen-limited conditions in parallel with the onset chrysoporium to 28.90%.Chromium was reduced by A. niger of LiP production. LiP genes have been characterized in P.

Citation: Otaiku A. A and Alhaji I . A (2019) .Kachia Military Shooting Range In situ Fungi Species Biodegradation of Explosives, Page 12 of 27 Kaduna, Nigeria. J Adv Res Biotech 4(2):1-26. Copyright: Kachia Military Shooting Range in situ Fungi Species Biodegradation of © 2019 Otaiku A. A and Alhaji I. A Explosives, Kaduna, Nigeria chrysosporium [11,47]. The number of introns in genes encoding LiP are eight to nine for P. chrysosporium and have to six for T. versicolor [97]. Mature protein lengths of LiPs of P. chrysosporium and T. versicolor 342 to 344,338 to 346, amino acids, respectively. T. versicolor better stability of enzyme production of all the fungi isolates as reported by found that lactase production by the white rot fungus T. versicolor was enhanced by several Xenobiotic and their transformation products [139]. The lignin peroxidase of Phanerochaete (Table 6) and demonstrated that in a cell-free culture medium with broth, the lignin peroxidase activity could reach 2800 U/L. by and immobilization enhancing enzyme production techniques to biodegradation of TNT was reported by and potentials for ex situ bio-augmentation products because degradation of TNT by more than 99% in TNT wastewater [96, 147and 166].

Table5: Fungi isolate Implicated in Biotransformation and Mineralization. Kachia, Kaduna, Nigeria

Fungi Isolates Explosives Conditions References Species

Rhizopus spp TNT TNT disappearance in malt extract broth Klausmeier et al., 1974 ;

Electrophilic attack by microbial oxygenases Rylott and Bruce, 2009 ;

Mineralization to C02 by mycelium and 90% Fernando et al.,1990 ; Rho et al., P. chrysosporium spp TNT removal of TNT 2001;Sublette et al.,1992 Spiker et al.,1992 ; Donnelly et Mineralization to C02 by spores al.,1997; Michels and Gottschalk, 1994 ; Huang Mineralization to C02 by mycelium and Zhou,1999; Mineralization correlated with appearance of Sublette et al.,1992; Stahl & Aust,

peroxidase activity 1993a, b Mineralization to C02 in soil-corn cob cultures Fernando and Aust, 1991 ; Yinon and

and stable Zitrin,1993 Biotransformation by Thermophile Kaplan and Kaplan,1982;Isbister et Compost microbes TNT microorganisms al.,1984 Transformed ring-[ 14C]-labeled Griest et al., 1991; Binks, et al. 1995 ;

Williams et al.,1992 Freedman and Sutherland, 1998 ; Alic P.chrysoporium spp RDX BiotransformationTNT,humification under reactionsnitrate-reducing et al., 1997 Fernando and Aust ,1991; Fernando Reduction by nitrogen-limiting conditions and Aust, 1990 Boopathy et al.,1998a, 1998b ; Reduction by sulfate-reducing condition McCormick et al.,1981 Methylenedinitramine formation under nerobic Hawari et al., 2000 ; Kitts et al. 1994; condition Aerobic denitration of RDX and highly mobile in Fournier et al, 2002; Clausen et al.,

soil 2004 ; Alavi et al.,2010; Reduction under methanogenic conditions Boopathy et al., 1998b ; Kaplan, 1996

Reductions lead to destabilization, ring cleavage, McCormick et al.,1976; Clausen,.2005

Final products may include methanol and Hawari et al., 2000b ;McCormick et

hydrazines al.,1976 Coagulation in contaminated water by nitrate Sullivan et al., 1979 ; Bhushan et White rot fungus RDX reductase al.,2002 Treated TNT in wastewater and achieved 99%. Huang and Zhou,1999 ; Bennett , TNT degradation 1994 Aken et al., 1999 ; Stahl and Aust, TNT First step was degradation to OHADNT and ADNT, 1993a

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Axtell et al. 2000 ; Stahl and Aust, Second step was to DANT including HMX and RDX. 1993b

Aspergillus niger spp HMX Sequential denitration chemical substitution Urbanski, 1963 ; Kaplan, 1996

Mchllan et al., 1988b ;Kaplan Low water solubilitIity in anaerobic conditions ,1993;McCormick et al.,1984 Mono-nitrated pentaerythritol to un-nitrated PETN Williams and Bruce ,2000; pentaerythrito products Biodegraded by sequential denitration to Binks et al.,1996 ; Barr and Aust, Mineralization PETN pentaerythritol 1994 ; de Boer et al ., 1987 Nitroglycerin, or glycerol trinitrate, degraded co- Wendt, et al., 1978 ; Barr and Aust, P.chrysoporium spp PETN metabolism 1994 ;Stahl et al., 2001 Angermaier et al., 1981 ; Angermaier by sequential removal of nitro groups and Simon, 1983 Townsend and Myers, 1996 ;Brannon White rot fungus Explosives Dissolution rates: TNT > HMX > RDX>PETN and Pennington , 2002 Degraders glycerol trinitrate and isosorbide White & Snape, 1993 ; Have and

dinitrate : nitrate esters Teunissen ,2001

plate 2: Location 2 traces of heavy metals and explosives. Figure 13: The rigid fungal cell wall composed of complex layers of various polysaccharides and proteins. Picture taken from Skowronski etal, (2001)

Figure 14: The growth potential of explosive utilizing fungi species (explosives by turbidometry).

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Figure 15: The best fungal isolates for heavy metal reduction in increasing order were Aspergillus Niger > Phanorochate chrysoporium > Trametes versicolor > Rhizopus spp > Penicillium spp.

(Manganese peroxidase hydrogen peroxide-generating systems They also reported that RDX was directly amenable to degradation LiP, nitrogen-deficient conditions favour the production of MnP [3, 26, 83, 117and 118]. MnP is also glycosylated heme-containing byas MnP,compared but not to LiP nitrogen-sufficient [155].Pre-grown (non-ligninolytic)cultures have previously conditions. been extracellular peroxidase. Its catalytic cycle is similar to those suggested to apply to white rot fungi co-metabolism [22]. Besides of LiP and horseradish peroxidase (HRP), but it uses absolute these functions, fungal lactases may play a major role in the de Mn(II) as a substrate that is widespread in lignocellulose and polymerization of lignin MnP from P. chrysosporium has been soil.A catalytic cycle is initiated by binding H2O2 or an organic studied extensively by a variety of biochemical and biophysical peroxide to the native ferric enzyme, forming an iron–peroxide methods [72, 73, 86,159, 228 and 239] and the crystal structure complex, as depicted in Figure 16 [93]. Peroxide oxygen– and the heme environment of MnP is similar to that of other plant oxygen bond cleaves producing MnP compound I (Fe4+ oxo- and fungal peroxidases by [197]. MnP is unique among enzymes porphyrin-radical complex). Subsequent reduction takes place using manganese as a redox cofactor. Rather than permanently through MnP compound II (Fe4+-oxo-porphyrin complex), sequestering Mn II in an interior binding site, MnP selectively and Mn(II) oxidizes to Mn(III), and this continues with the binds Mn II on the surface of the protein, oxidizes it, and then generation of native enzyme and release of the water molecule. releases the Mn III product in complex with organic acids such as MnP compound I is similar to LiP and HRP and can be reduced oxalate and malonate. However, the crystal structure of MnP- Mn by electron donors such as ferrocyanide, phenolics, and II in the absence of chelators suggests that the later alternative is Mn(II), whereas MnP compound II is very slowly reduced by more likely [18, 119,120,197, and 228]. other substrates and requires Mn(II) for completion of the However, molecular modelling and kinetic studies indicate that catalytic cycle [220]. Transcription of different MnP isozymes Mn II binding and oxidation are sensitive to subtle changes exhibits variable dependencies in the presence of Mn (II) [66]. in ligand geometry [118, 122, 196, 238, and 239]. Indeed, the MnP gene sequences and cDNA have been cloned and sequenced, including Trametes versicolor [103,104]; MnP genes and metal ions to bind. However, it appears that only Cd II exhibits a dissociationflexibility of theconstant Mn-binding similar site to thatin MnP of MnIIallows and a wide other variety divalent of regulating elements [69]. Responses from a MnP promotor and cations, such as CO II [84,239]. applicationsreporter genes in candetermining be constructed conditions that confirmfor optimal these MnP putative gene expression from certain loci have been demonstrated. Putative regulatory elements in the promoter region of a P. chrysosporium gene-encoding MnP contain both metal response (MRE) and heat-shock response (HSE) elements [3]. P. chrysosporium which produces several MnP iso enzymes [68, 123]. Stahl et al., 2001 reported that the amount of RDX degraded by growing Phanerochaete chrysosporium cultures was approximately 10 times higher under nitrogen-limited conditions

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Figure 16: The catalytic cycle of manganese peroxidase (MnP) Source: Hofrichter (2002)

Figure 17: Source: Sundaramoorthy et al., 2005

Overall structure of MnP-MnII refined at 1.45 Å resolutions.

Figure 18: Source: Sundaramoorthy et al., 2005

Overall structure of MnP-MnII refined at 1.45 Å resolutions. Citation: Otaiku A. A and Alhaji I . A (2019) .Kachia Military Shooting Range In situ Fungi Species Biodegradation of Explosives, Page 16 of 27 Kaduna, Nigeria. J Adv Res Biotech 4(2):1-26. Copyright: Kachia Military Shooting Range in situ Fungi Species Biodegradation of © 2019 Otaiku A. A and Alhaji I. A Explosives, Kaduna, Nigeria

The results presented here provide a more accurate model The MnP-CdII structure also exhibits a possible additional metal- of native MnP at very high resolution (Figure 17). The 2.0 binding site near the C-terminus (Figure 18). The carboxy- terminal oxygen, the side chain of Asp84, and two solvent temperature, shows that the substrate, Mn II, binds to one heme molecules provide ligands to a second cadmium ion, displaying propionateÅ resolution and crystal the side structure chains ofof MnP,three determinedamino acids, at Glu35, room tetrahedral geometry. Ligands in the two metal sites, Glu39 and Glu39, and Asp179, as well as two solvent ligands [200] (Figure Asp84, are linked through hydrogen bonds mediated by Asp85 and water. This network of hydrogen bonds is possible only with of wild-type MnP and of proteins containing point mutations in the open conformation of Glu39. Although Mn II is not observed to the17). putativeThis site bindingwas confirmed site. Figure17. by kinetic Overall and biophysicalstructure of studies MnP- Mn-binding site has been postulated on the basis of pH binding bind at this site in the crystal structures, a second, lower-affinity at Ser336 with O-linked R-mannose is shown. The FO - Fc omit analysis of the protein in solution Mauk et al., 1998. It is possible MnII refined at 1.45 Å resolutions. A second glycosylation site that MnII does bind to this site under physiological conditions, but not under the conditions for crystal formation (pH 6.5 in map around Ser336 is contoured at 3.0σ showing density for cacodylate buffer). The site is exposed to the solvent and could ComparisonR-mannose. Thisof the is aC-termini new feature of MnP-Mn in the 1.45 II, ÅMnP-Cd cryo maps II, and that MnP- was accommodate a MnII ion with a bound chelator such as oxalate or Smnot IIpresent structures. in the The 2.0 Fo-Fc Å room-temperature omit maps were structure.calculated Figure18. omitting all atoms near the C-terminus within a sphere with a radius of site than Mn II because of its larger mass-to-charge ratio and its abilitymalonate. to adopt Alternatively, a wider rangeCd II may of ligation exhibit geometries a higher affinity [44]. forAtomic this absorption analysis indicated only 1 equiv. of Cd II binding per II5 Å,(B). including The large Ala357 peak inand the Asp84. MnP-Cd The II density map is ismodelled contoured as Cdat 3σII, mole of protein in solution, possibly due to partial occupation of andfor MnP-Mn its four potentialII (A) and ligands MnP-Sm include III (C) andthe terminal3σ and 20σ carboxylate for MnP-Cd of both sites by this metal at pH 4.5 [239]. the polypeptide, Ala357, and the side chain carboxylate of Asp84.

Table 6: Peroxidase-catalyzed Degradation of Lignin

Degradation / Incubation Fungus/ Lignin Model Medium/Reaction Metabolic Mineralization Rate (hours/ Reference Peroxidase Compound Conditions Products (%) Weeks)

Phanerochaete [14C]Methylated Crude culture liquid,

tartrate, H O , Tween, Tien and 2 2 De polymerization 1 h chrysosporium / Kirk,1983 spruce lignin LiP (crude) pH 3.0, 37°C Glycolate, VA, 30 – 40%

Low- P. chrysosporium molecular- Hammel et Co-solvent, H2O De polymerization 16 h /LiP DHP 2 weight al.,1993 14C [DHP] or [α-13C] products P. chrysosporium Thompsonet al., Hardwood lignin Aqueous medium, 37°C 5% Degradation 12 h /LiP, MnP 1998

Malonate, 100% O , Mn P. chrysosporium 2 Wariishi et Four [14C]DHPs (II), glucose, oxidase/ Partially degraded 0.5–7 h /MnP al.,1991 glucose, pH 4.5, 37°C

P. chrysosporium Lackner et Chlorolignin Lactate, Mn(II), H O De polymerization /MnP 2 2 al.,1991

P. chrysosporium Water- Non phenolic lignin MnP-Mn(II) -linoleic Kapich et / Recombinant 20% as 14CO soluble 48 h model dimer acid 2 al.,1999b MnP products

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Previously, reported was the extensive characterization of 163]. Restriction fragment length polymorphism (RFLP) and the effects of Cd II on manganese peroxidase as a reversible, competitive inhibitor by [239]. Steady-state kinetic analysis lipA, lipB, lipC, lipE, lipG, lipH, lipI and lipJ, reside within 3% allele specific marker demonstrated that linkage of eight genes showed that Cd II is uncompetitive for H2O2 and MnP-Cd II forms recombination, which corresponds to 96 kb region of scaffold 19 compound I under transient-state conditions. Cadmium cannot be [142].The promoter region of MnP has heat shock elements and oxidized by MnP intermediates based on our analyses (Figure 6) putative metal response elements (MREs) [75, 103]. and acts as a reversible, competitive inhibitor for Mn II oxidation Conclusion

[200, 239]. Similar changes in the MnP heme spectrum upon Among all the concentration of explosives both in dry and bindingwith a Ki of of Cd 10 II µM and and Mn confirmed II and very by close similar apparent works indissociation Figure 18 wet season RDX recorded the highest concentration of 68.19 mgkg-1 in Kachia shooting range is polluted with explosives and the toxicity will affects the ecosystems with carcinogen these metals bind at the same site. The lack of a Glu39-Arg42 impacts (Tables 4,5 and 6 respectively). Lignolytic fungi such interactionconstants (Kd in the≈ 8 MnP-CdµM for CdII II structure and 10 µM increases for Mn theII) indicatenet negative that as Penicillium spp. Aspergillus spp, Rhizopus and Phanerochate charge, which might be responsible for tighter binding of Cd II chrysoporium a white dot fungus was capable of oxidizing cyclic than of Mn II as reported by Sundaramoorthy et al., 2005.Ethanol nitramines and mineralizing RDX, HMX, TNT and PETN where and arsenite are known to induce the synthesis of heat shock Aspergillus niger had the best ability to degrade explosive while proteins in fungi [111,146]. However, in P. chrysosporium, Penicillium spp had the least ability using the analysis of variance H2O2compound increases MnP transcript levels only in nitrogen- limited cultures [185].The copper-thio¬ ether bond and non- growth pattern of fungi in Explosive Mineral Salt (Basal Salt Medium)result shows broth that supplemented there is a significant with 1% v/vdifference explosive in (P>0.05).the overall enzyme. Lactases from different sources display a wide range of coordination residue strongly influence the redox potential of the The best fungal isolates for heavy metal reduction in increasing redox potentials. The T1 site of lactase of T. versicolor shows a order were Aspergillus niger > Phanorochate chrysoporium > high redox potential of 780-800mV which supports the results of Trametes versicolor > Rhizopus spp > Penicillium spp while, Table 5T. Versicolor biodegrade copper metal in the study area. in the overall, ANOVA test for total explosive contents Shows a Figure 19 showing all fungal lactases show a similar architecture consisting of three sequentially arranged domains of a-barrel (P<0.05) and bioremediation will occur via biotransformation type structure. The active site is well conserved with four copper Significant difference for all locations in both dry and wet Seasons sites T1 is located in domain 3 with copper lying in a shallow biodegradation pathway correlated with the appearance of depression and trinuclear copper cluster is at the interface peroxidasewhere the fungiactivity enzymes [22]. biochemistry is very significance to between domainaI and 3 with each domain providing ligand residues at the coordination of copper ions. The T1 copper is conditions that promote RDX biotransformation, so that coordinated with His-Nand Cys-S as conserved equatorial ligands treatmentThere issystems clearly cana need be designedto better definewith a thegreater electron assurance acceptor of [160]. Due to their powerful degrading capabilities towards various possibility to develop P. chrysosporium strains for munitions wasteshigh treatment management efficiency because as reportedwhite rot byfungi [63].There for biodegradation is a great recalcitrant chemicals, white-rot fungi and their lignin degrading of TNT has been patented [182]. Also, several LiP genes have enzymes have long been studied for biotechnological applications also been characterized from other species of fungi, including such as bio bleaching, bio decolourisation and bioremediation four Trametes versicolor clones and P. Chrysosporium LiPs are [33, 48, 209 and 244] encoded by a family of at least10 closely related genes, designated lipA through lipJ [74, 207,208]. LiP genes show a high degree Fungal Decomposition of Nitro-compounds of sequence conservation for 2,4,6- trinitrotoluene (TNT) and White-rot fungi and are primary microbes decomposing other nitro explosives, and reduction improves in more reduced extremely recalcitrant lignin [145,245]. Their effectiveness in its environments[186]. degradation is based of cooperation of three enzymes: Manganese peroxidase (MnP), lignin peroxidase (LiP) and lactase. The most intensively studied P. chrysosporium and T.versicolor are the most intensively studied fungi from the isolates fungi the study area. The fungi are capable of complete degradation of nitro compounds, primarily due to reduction of nitro groups and further decomposition via oxido reductases [140]. A fragment of such pathway is presented in Figures 19 and 20. All genomic sequences of P. Chrysosporium strain T. versicolor MPGI are available for expanding our knowledge about the regulation Figure17: Source: Trametes versicolor http://www. and expression of peroxidase genes, Table 6 [3,107,152, 161and wisconsinmushrooms.com/) as grown in nature.

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Figure19: Metabolic reduction of 2, 4, 6-trinitrotoluene by Phanerochaete chrysosporium. Source: Baker et al., 2015

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Citation: Otaiku A. A and Alhaji I . A (2019) .Kachia Military Shooting Range In situ Fungi Species Biodegradation of Explosives, Page 26 of 27 Kaduna, Nigeria. J Adv Res Biotech 4(2):1-26. Copyright: Kachia Military Shooting Range in situ Fungi Species Biodegradation of © 2019 Otaiku A. A and Alhaji I. A Explosives, Kaduna, Nigeria

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Citation: Otaiku A. A and Alhaji I . A (2019) .Kachia Military Shooting Range In situ Fungi Species Biodegradation of Explosives, Page 27 of 27 Kaduna, Nigeria. J Adv Res Biotech 4(2):1-26.