Kachia Military Shooting Range in situ Fungi

*Corresponding author email: [email protected] , [email protected] Group

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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

Journal of Advanced Research in Biotechnology Open AccessResearch Article

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: [email protected] , [email protected]

AbstractKachia 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 and analysed using EPA SW-846 Method 8095 contains TNT, RDX, HMX and PETN. White rot fungi analysed with amplified using 16srRNA gene of each isolates were processed for sequencing and characterization and contains Lignolytic fungi Aspergillus Niger, Rhizopus spp, Penicillium spp, Trametes versicolor and Phanarochate chrysoporium. Explosives ANOVA test shows significant difference for all locations in both dry and wet seasons (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 niger had the best ability to degrade explosives while Penicillium spp had the least ability in 1% explosive mineral salt broth supplemented with 1% v/v explosive (P>0.05) from the zero hour to the 5th day and beyond with significant difference in the overall growth pattern. Heavy metal reduction in increasing order were Aspergillus Niger > Phanorochate chrysoporium > Trametes versicolor > Rhizopus spp > Penicillium spp. Xenobiotic biodegradation occurred under co-metabolism with carcinogen potentials to biodiversity.

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

HighlightsTNT, RDX, HMX and PETN

Biotransformation

Biodegradation

Fungi 16srRNA Gene

Mineralization

Enzymes Biochemistry

Toxicity to ecosystem

IntroductionThe most widely used explosives in the world are

probably hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), 2,4,6-trinitrotoluene (TNT); octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX) and Pentaerythritol tetra nitrate [PETN;

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


Copyright: © 2019 Otaiku A. A and Alhaji I. A

C(CH2ONO2)]4its use in items such as exploding bridge wire (EBW) detonators and exploding bridge foil initiators (EFI) by Foltz, 2009; Ronen and Bernstein, 2014 (Figure 1). These compounds are characterized by relatively high thermal stability, high density and high detonation velocity, all of which promote their extensive use [40,237]. Some of their physical properties are summarized in Table 1. Explosive compounds are undesirable in the environment due to their toxicity. TNT, RDX and HMX have been defined as toxic to humans and animals, causes anaemia, abnormal liver function, spleen enlargement, nervous system and immune system (ATSDR 1996a, ATSDR 1996b) [12, 13].

RDX is composed of a triazinic ring to which three nitro functional groups are perpendicularly attached. In RDX, similar to TNT, the nitro groups are the main targets of the first degradation steps by sequential reduction or de nitration as key steps. Biodegradation clearly shows the potential for reducing the concentrations of explosives in the environment via microbial activity TNT [53, 134,195,234]; RDX [89,133] on the biodegradation of both RDX and TNT; and RDX and HMX by. Because of high electron deficiency on the nitro groups of TNT, its microbial degradation is often initiated by reductive rather than oxidative reactions, even under aerobic conditions in Figure 2. * Available for Fungi Species Study.

Nevertheless, further transformation of the mono and diamino derivatives toward the formation of the most reduced product-triaminotoluene (TAT) proceeds only under strictly anaerobic conditions with redox potential values below -200 mV [89].Thus, under oxic conditions, diamino derivatives tend to accumulate, while the presence of TAT is indicative of strictly reduced conditions. Under aerobic conditions, the formation of nitroso derivatives is normally not observed. This may be expected from thermodynamic considerations, where the calculated E0 shows a decrease from trinitro aromatics to nitramine, suggesting thermodynamic control of the reduction of RDX under aerobic conditions [215]. Only a few exceptions indicate formation of mono nitroso derivatives under aerobic conditions as reported for incubation of the white-rot fungus P. chrysosporium with RDX which, showed formation of MNX. This was followed by ring cleavage and the subsequent formation of methylene dinitramine (MEDINA), Figure3 [89].

TNT can be biodegraded via various pathways, which mainly involve transformation of the nitro functional group, while the aromatic ring remains intact [89]. The stability of the aromatic ring results from the strong electron-withdrawing properties of the nitro substituents which promote high electron deficiency and electrophilic characteristics on the p-electron system [43]. In the presence of oxygen, however, it has been shown that both the nitroso and the mono hydroxyl amino metabolites can follow an alternative abiotic transformation pathway to form tetra nitro azoxytoluene dimmers, also referred 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

shown that these derivatives themselves may further degrade in microcosm experiments with the fungal strain Phanerochaete chrysosporium [90], or in soil slurries with a mixed culture [92], as well as in other experimental systems and microbial transformations of nitrate esters PETN [232].

HMX is less amenable to biodegradation in the environment than RDX due to its lower water solubility and its relative chemical stability [89]. It is structurally similar to RDX, and thus follows analogous degradation pathways: both sequential reduction of the nitro group to nitroso derivatives and anaerobic de nitration followed by ring cleavage have been documented as HMX-degradation pathways limited microbial strains were found capable of degrading this compound. Similar to the sequential reduction of RDX to its corresponding nitroso derivatives following subsequent two-electron transfer steps [133], nitro groups of the HMX molecule can be reduced to nitroso derivatives. Nevertheless, while for RDX, all three nitroso derivatives are observed in high abundance, in HMX, only less reduced nitroso derivatives are normally detected. For example, the detection of only a mono nitroso derivative was documented by [59] with the fungal strain P.chrysoporium. The formation of the first two nitroso derivatives: mono- and dinitroso. HMX was observed by [91] following incubation of HMX with anaerobic sludge. Studied the transformation pathway of HMX with the metallo-flavo enzyme xanthine oxidase [25]. Based on the detected products, they proposed that HMX undergoes a single denitration step.

Looking at the reductive transformation of the nitro group from a purely energetic compounds, one would expect the decrease in reduction rate from TNT to RDX, and finally to HMX [215]. On the other hand, in complex biotic systems such as the sub-surface, factors other than energetic yield will dictate the rate at which explosives degrade. Nutrient availability is one important factor that plays a significant role in the rate of explosives biodegradation. Compared to other common pollutants, explosives, particularly RDX and HMX, are characterized by a higher nitrogen/carbon (N/C) ratio. Although, they may theoretically serve as sources of both carbon and nitrogen, only nitrogen is used by some organisms, and hence, another carbon source must be added [4].

The aim of this research is to identify bioremediation technology that can be adapted for the clean-up of munitions and explosive compounds contaminated soils sites in Kachia, Kaduna state, Nigeria. The objectives study are: To examine and identify the munitions/explosive compounds in the weapons firing sites; determine the physiochemical properties of soil in Kachia military firing range and environs; characterize the soil type at firing range and environs; determined the ability of isolated fungi species to utilize munitions compounds ; screen and isolate genes responsible for bio remediating possibly munitions contaminated soil.; compare the rate of possibly munitions contaminated soil between dry and wet seasons.




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

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

Composting in Explosive Remediation

Composting was the first biological process to be tested, approved and selected for use in remediating military sites [43].Composting has been identified as a feasible technology for the remediation of monition compound contaminated soils by the US Army Toxic and Hazardous Materials Agency and US Army Armament Research and Development Command [61]. Several types of composting systems exist, but static pile and window composting are the commonly used in explosive remediation [112]. A problem associated with composting is the high pile temperature sometimes resulting in volatilization of pollutants and also long incubation times. In situ bioremediation is generally utilized in areas where contamination is deep and excavation would be excessive and bioremediation involves treating the contaminated material at the site. The process involves the passing of water enriched with nutrients, oxygen and/or with microorganisms through the contaminated area and composting soils contaminated with organo nitro explosives, including PETN as well as extracellular enzymes Phanerochaete chrysosporium [45, 46].

Fungi Biodegradation

RDX degradation by Penicillium species and other soil fungi was strongly inhibited by ammonium but not by nitrate [222].Nitrate is a widely utilized nitrogen source but needs to be converted to ammonium after uptake. Nitrate reduction is

performed by the sequential action of two intracellular enzymes, nitrate reductase which converts nitrate (NO3) to nitrite (NO2), and nitrite reductase which reduces nitrite to ammonium (NH4).In Aspergillusnidulans and Neurospora crassa, which are the most thoroughly studied filamentous fungi in this respect, both enzymes are under the control of catabolite repression by ammonium, the actual regulator being intracellular glutamine. Further, nitrate acts as a signal which activates the transcription of enzymes, i.e. their genes are expressed only in the absence of ammonium and the simultaneous presence of nitrate regulatory mechanism has been proposed for A nidulans and reported that an unknown enzyme(s) was involved in the reduction of RDX by living fungi are, in fact, nitrate reductases, given that RDX degradation is repressed by ammonium and given that a purified commercially available nitrate reductase from Aspergillus niger has recently been shown to be capable of degrading RDX in vitro [41,103,129,130,213].

Activity of this pure nitrate reductase was sufficient to release the intermediate products hexahydro-1-nitroso-3,5-dinitro-1,3,5-triazine and methyl-enedinitramine which further decomposed to nitrous oxide, formaldehyde and ammonium and RDX degrader Penicillium sp. AK96151[24, 223]. Species of Penicillium and Aspergillus are phylogenetically closely related, and P.chrysogenum possesses an equivalent regulatory mechanism of nitrate assimilation to that outlined above for A.




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.

These results support the view first expressed by that the reduction of one nitro group by one specific enzyme is sufficient to bring about degradation of RDX. Unfortunately, since RDX degradation is such a slow process even with our highly efficient RDX degrader (Figure 2), it was impossible directly to test the ability of our protoplasts to degrade RDX [133]. The fungal nitrate reductase enzyme is therefore rather versatile, being capable of reducing ionic NO as well as chlorate and nitro groups of organic molecules. 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 degraded. Using radio labelled RDX, have shown that this substance is indeed present in an unmodified form in the mycelial fraction, although the uptake mechanism is unclear at present [223]. RDX degradation by nitrate reductase can be inhibited by as little as 150µM NH4+, and this was quite similar to the NH4+ concentration measured in the groundwater at the site which we studied (approx. 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.




Figure3: Postulated degradation pathways of HMX compounds in brackets are postulated intermediate

Source: Ronen and Bernstein, 2014Materials and Methods

Study Site

The study was conducted in the permanent military shooting/training range located at 5km east of Kachia town in Kaduna state, north central Nigeria. The range was established in 1965 and it covers an area of about 24.95sq km that lies between longitudes 90 55’ N and 70 58’ E, with a elevation of 732m above sea level and the topography is undulating and the vegetation is Guinea Savannah (Figure 4).The area where the munitions/explosive are fired (the impact area) is a valley consisting of about four large rocks, where the fired munitions/explosives land and explode during military training. Five military exercises involving the deployment of explosives are carried out annually by the Nigerian Defence Academy (NDA) Kaduna, Nigerian Air force (NAF) Kaduna, Nigerian Army School of Infantry (NASI) Jaji, Armed Forces Command and Staff College (AFCSC) Jaji and Nigerian Army School of Artillery (NASA) Kachia.

Sampling Points

Four sampling points selected for the study are locations 1, 2, 3 and 4, Table 2. Locations 1 and 2 are the twin smallest rocks closest to the road. While location 3 and 4 are much larger rocks heavily impacted by munitions/explosives between locations 1and 2 is a flat ground where rain run offs flow through to the stream in this site (map with Global positioning system (GPS) co-ordinates).Locations 1 and 2 approximately 200m from the table top that is where the small arms are fired such as Kalashinokov, FN, Greenade, GPMG, SMG and Pistols. The soil in locations 1 and 2 is made of 50% silt and a flat ground with shrubs and drainage that flow through to the farm lands near the sites. Location 3 is approximately 9000m away from the table top lies between 90 53’ 44.71” North and 70 53’ 17.87” East. The impact area of location 3 and 4 are mainly largely rocks containing high concentration of explosive due to the extensive use of bombardment by the Artillery weapons,155 mm nortwizer, and other heavy weapons while location 4 is ahead of location 3 is about 10,000m from the Plateau top where heavy weapons are fired too. The distance between locations 3 and 4 was covered with various shrubs and two major streams.

Sampling Technique and Soil Treatments

Soil Sampling

Sampling was done during both dry and wet season. Four locations located within NASA shooting/training range Kachia were earmarked as sampling sites for this study using soil iron auger.10 gram of soil sample (0-30 cm in depth) with diameter of 9cm were collected from 3 different points within a location 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 polythene bags until for analyses. Samples for microbial analyses were kept in a cool box refrigerated with ice pack to retain the original microbial activities.

Soil Sample Pre-treatment

Sampling points were treated in the laboratory before digestion 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 soil sample was then ground in a porcelain mortar with pestle and sieved through 250 µg mesh size to obtain a homogenous sample. The soil sample was stored in sterilized polythene bags, label and kept for next stage of pre-treatment. This procedure was repeated for all the collected soil samples. The ground soil samples were used for analyzing heavy metal and explosives content for soil samples [99].

Digestion of Soil Sample for heavy metals and explosives analysis

1.0g of pre-treated soil sample was accurately weighed and placed in a 100 cm3 beaker. Then, 4.0cm3 of 70% of HCL04 and 2cm3 of concentrated HN03were added to the sample and the resultant mixture was put in the oven at 100 0C overnight. A white ash was formed which was dissolved in 2cm3 of 1m HCL. The digest was filtered into 50cm3 standard volumetric flask. The beaker was then rinsed with small portions of double distilled water and quantitatively transferred into the flask. The resultant




solution was made up to the mark with double distilled water, transferred into a clear dried plastic sample bottle, labelled and kept for heavy metal and explosives analysis. This procedure was repeated for all the samples collected at this site. A blank was prepared using the same procedure excluding the soil sample [166].

Heavy Metals Analysis Principle

The analysis was done using Atomic Absorption Spectrophotometer (AAS) at the laboratory of Geological Survey Kakuri-Schimadzu AAG50 model. The digested Soil Samples were analyzed for heavy metals. Heavy metals analyzed include Arsenic, Boron, cadmium, chromium, cobalt, copper, iron, lead, magnesium manganese, Nickel and zinc using AAS, according to APHA (1998). The technique makes use of absorption spectrometry to assess the concentration of analyzed in a sample by measuring the absorbance against a known concentration. It relies on the Bear Lambert Law.

ls Analysis Principle

Explosive Analysis

The analysis was carried out at National Research, Institute for Chemical Technology (NARICT) Zaria, Nigeria. The vials containing the AcN (Acetonitrite) soil extracts were placed into GC auto sampler trays that were continuously refrigerated by circulating zero OC glycol/water through the trays, the extracts were analyzed by gas chromatography using a micro-lectron capture detector (GC-µECD).

Figure 4: Map of Kachia within Kaduna state map, Nigeria Showing study area (Nigerian Army Shooting Range).

Source: Geography Department NDA, Kaduna, Nigeria

Results were obtained on HP-6890 GC equipped with a micro cell Ni63 detector at 2800C according to the general procedure outlined, in EPA SW-846 method 8095 [51]. Direct injection µL of soil extract was made into a purged packed inlet port, at 2500C, that was equipped with a deactivated Restek Uniiner.

Primary analysis was conducted on a 6 -m-x 0.32 mm ID-fused - silica column, with a 1.5-um film thickness of 5% (Phenyc) -methyl -siloxane (RTX -5 from Restek). The GC oven was temperature programmed as follows; 1000C for two minutes, 100C minutes ramp to 2600 C, two minutes hold. The carrier gas was helium at 10 ml/minute (Linear Velocity approximately 90 cm/second). The ECD make-up gas was nitrogen flowing at 40 ml/minute. If a peak was observed in the retention window for a specific signature compound, the extract was re-analyzed on a confirmation column, 6-mx 0.53-mm ID having a 0.1-mm film thickness of 50% cyanopropylmethyl-50% phenyl methyl- polysiloxane(RTX -225 from Restek ).If analyze Concentrations were taken from the determination on the primary column, unless they appear to be coalition with another compound, in, such cases reported concentration were taken from the determination of the confirmation column.

Table 2: Global positioning system (GPS) recorded in degrees, minutes and seconds (DMS) of

S.No Location Northing’s Easting’s Elevation

1 Kachia 90 52’24.42” 70 57’ 18.61” 715m

2Loc 1 & 2

90 53’ 50.23” 70 53’ 54.53” 818m

3 loc 3 & 4 9053’ 44.71” 7053’ 17.87” 773m

4 Control 9053’47.63” 7053’34.77” 644m

Screening for Explosive/Munitions Degrading Organisms

Preparation of Media

Nutrient Agar

Nutrient Agar (Antec/USA) by dissolving 28g of the Agar in 1 litre of distilled water in a conical flask. The conical flaks with the media was autodaved at 121OC at 15 Pressure Per Square much (PSI) for 15 minutes. Cooled to 400C before pouring or dispensing if a sterile petri dishes

Mac Conkey Agar

Mac Conkey Agar was prepared by dissolving 49.53g of dehydrated medium in 1000ml distilled water in a conical flask. It was heated to dissolve the medium completely. The medium was sterilized by autoclaving at 15 1bs pressure (1210C) for 15 minutes. Cooled to 45-500C, mixed well before pouring into sterile petri plates.

Potato Dextrote Agar (PDA)

Thirty nine (39) g of dehydrated medium suspended in 1000ml distilled water was heated to dissolve the medium completely. It was then sterilized by autoclaving at 15 1bs pressure (1210C) for 16 minutes. Cooled at 10% of tartaric acid was added and mixed well to achieve pH 3.5 before dispensing into petri plates. The media were spread with the specimen soon after solidification of the media. Plates were incubated at 25-300C in an inverted




position (agar slide up) with increased humidity. Cultures were examined weekly for fungal growth and were held for 4-6 weeks.

Serial Dilution

The sterile dilution blanks were marked in the following manner. 100 ml dilution blank was 102 and 9 ml tubes sequentially 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 vigorously shaken for at least one minute with the cap securely tightened. All the 10-2 dilution was allowed to sit for short Period. The 1ml\ from this dilution was aseptically transferred to the 10-3 dilution was again transferred to the 10-4 Dilution. The procedure was done to 10-5 and 10-6. A flask of nutrient agar from the 450 C water Bath was especially pounded into each petri plates for that set. 15ml was poured enough to cover the bottom of the plate and mixed with the 1 ml inoculum in the plate. Each set was gently swirled on the bench so that the inoculum gets thoroughly mixed with the ager.

All the plates were allowed to stand without moving so that the agar solidified and set completely. The plates were inverted and stacked into pipette canisters and placed in the incubator or at room temperature until after 48 hours the same procedures were applied for Mac Conkey agar and PDA.

Polymerase Chain Reaction (PCR) for Amplification of Catabolic genes

Principle

PCR consist of an exponential amplification of DNA fragment and the principle is based on the mechanism of DNA replication in vivo, double stranded DNA is denatured to single stranded DNA, each single strand DNA is anneal by the forward and reverse primers of known sequence and elongated using tag DNA polymerase to produce copies of DNA template.

Procedure

Isolated explosive fungi DNA were amplified using catechol 2,3dioxygenase gene primers. The genomic DNA, the primers and PCR master mix were added into a PCR tube. The tubes were spun to collect the droplets. The tubes were then inserted into the PCR machine while maintaining the regulation for initial denaturation (45 seconds at 940C), annealing (1minute at 550C) extension (1minute at 720C) and final extension (10 minutes at 720C). The amplified catabolic genes were resolved in 1.5% agarose gel electrophoresis stained with ethidium bromide and viewed under ultra-violet (UV light).

Bioremediation of Heavy Metals in Soil

The soil samples solution was sterilized using an autoclave at 1200C for 15 minutes. 100ml of the sterilized soil

Solution was poured into conical flasks. The first flask was not inoculated with fungi (control), the second, third, fourth,

Fifth, were each inoculated with Aspergillus spp, Penicillium

spp, Rhizopus spp; Trametes versicolor, and Phanerochate chrysoporium, respectively. The experiment was left to stand for 24 hours after which the concentration of cadmium, chromium, nickel, zinc, cobalt, iron, lead, cupper and arsenic were measured using Atomic Absorption Spectrophotometer (AAS).

Bioremediation of Explosive in Soil

Nutrient broth and soil solution were added in a ratio 4:1.8 set of 250 ml flask were Set for the work. The broth and soil solution were sterilized in an autoclave at 1210C for 15 minutes. 120ml of nutrient broth and 30 ml of sterilized solid solution were added into each of the fifteen flasks. The first flask was left inoculated, but the second flask was inoculated with the isolated fungi from explosive/munitions which are Aspergillus Niger, Rhizopus spp, Penicillium spp, Trametes versicolor and Phanarochate chrysoporium. The experiment set-up was left to stand for 14 days and the micro remediation was measured by taking the optical density (O.D) reading at 595minutes from zero hour-14 days at regular intervals of 2 days against the control [217].

Isolation of Genomic DNA from Fungi

The genomic DNA of each fungus with observed remediation capabilities was isolated. 1ml of each fungal culture was

Pelleted by centrifuging at 12,000 rpm for 2 minutes, the pellet was treated with Iysis solution and proteinases K and incubated at 600C for 30minutes. Deoxyribose nucleic acid (DNA) was 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 purity and quantity of (DNA) of each sample was examined using UV absorption spectrum and\agarose gel (1%) electrophoresis as described by [106, 204].

Polymerase Chain Reaction Amplification of 16srRNA Gene

The PCR reaction mixture containing 10 X PCR buffer, 25mm, magnesium chloride, 25mm dNTP’s, 10pm/ul Primer concentrations and template DNA were used for the amplification of the 16srRNA gene for each isolates. PCR conditions were optimized using lab net thermal cycler. The PCR Program began with an initial 5-minutes denaturation step at 940C: 35 cycles of 940C for 45seconds, annealing (1 minute at 550C), and 10 minutes’ extension step at 720C. All reaction mix were preserved at 40C until it was time for analysis as reported by [114].The amplified 16srRNA gene of each isolates was further characterized using gel electrophoresis, Plate 1.

Sequence Determination of 16srRNA Gene

The amplified 16srRNA gene of each isolate was processed for sequencing and characterization. The sequencing Kit (Applied Bio systems) with the product was analyzed with ABI prism DNA sequence (ABI). The gene sequence of each isolates obtained in this study were compared with known16srRNA gene sequences in the Gene Bank database as described by [106].




Determination of Explosive/Munition Biodegradability of 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 Density (O.D) readings at 595nm from 0 hours -40 days at regular intervals of 5 days against Basal salt broth medium as blank as demonstrated by [106], Figure 13.

Statistical Analysis

Data collected from the study were analyzed using general descriptive statistics, one way Analysis of variance (ANOVA) at 95% probability level of Significance. If significant differences were found, Duncan’s Multiple Range test was used to compare the different experimental groups. Computer Software Statistical Package for Social Scientists (SPSS) and Microsoft-Excel were used for the statistical analysis.

Results and Discussion Physio-Chemical Properties of the Military Firing Ranges

The temperatures of 22 - 34oC during the dry season and 27.16oC during the wet season were recorded respectively. The values obtained fell within the acceptable Federal Ministry of Environment (FMENV), Nigeria limit of <40. Similarly, the pH of 6-8 has been suggested to be appropriate for microbial 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 report of Jenkins et al., (2001) on characterization of explosives contamination at military firing ranges with soils characterization of silt, clay and loamy.

The increased temperature obtained during the dry season can be attributed to the high concentration of explosives due to the frequent exercises by the personnel of Armed forces of Nigeria. Compared with the wet season that the temperature is less as a result of rain washing away heavy metals and explosives drown the streams. The high value of electric conductivity EC recorded during the wet season is in agreement with the finding of , respectively on their physico- chemical of the soils samples within the FMENV and WHO permitted limits of 500/µ5/cm and within the value of 2000mg/l which was contrary to the finding of, whose values were within 2400-5900 mg/l. and is in agreement with the high values of TDS would be due to the deposit of organic and inorganic matters necessitated by the

military activities [29,50,153,154,216].

Figure 5: Percentage of Soil texture in various locations, Kachia, Nigeria. L10m Location (1) – zero metre, L1 200m Location 1 200 metre, Loc 1, 400 Location1 400 metre, Loc 20m – locations (2).0 metre, Loc 2- Location (2)

Table 3: Explosives sites analyses of wet and dry seasons, Kachia, Nigeria

N/S Explosives Wet Season µg Kg-1Dry Season µg

kg-1

1 TNT 0.12 – 26.6 0.11 – 10.29

2 HMX 1.43 – 68.19 0.39 – 35.67

3 RDX 0.38- 15.33 0.49 – 68.19

4 PETN 1.17 – 7.12 0.39 - 4.38

The results of seasonal variation of total explosive contents in various locations of NDA shooting range Kachia, Table 3 with impacts of distance on the concentration of the various explosives indicated high order of artillery bombardment in locations 3 and 4 compared to locations 1 and 2 which is the closest distance to the Plateau top where small arms are fired does not explode explosives such as TNT, RDX, HMX and PETN. It was revealed that RDX had the highest concentration of 68.19 µgkg-1 in dry season compared to wet season that was not very significant. ANOVA test for total explosive contents shows a significant difference for all locations in both dry and wet seasons (P<0.05). In zero metre of the objective TNT RDX and HMX were consistently found at the highest values of 49.39mgkg-1, 68.19mgkg-1 and 35.67mgkg-1, respectively (location 4). An impact area and beyond within the hand grenade range, a 105mm howitzer firing point and the heavy artillery and mortar range that had been analyzed by GC-ECD.TNT concentrations ranged from 0.11 to 49.39 mgkg-1 in dry season and 0.12 to 26.6 mgkg-1 in wet season. Among all the concentration of explosives both in dry and wet season RDX recorded the highest concentration of 68.19 mgkg-1.Which is suggestive to us that the Kachia shooting range is polluted with explosives most especially the RDX and agreed by EPA according to that RDX was formerly used as a rat poison, has also been listed as a priority pollutant and as a possible carcinogen [124] (Tables 4 and 5 respectively).




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

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-laden soils and waters [79]. The filamentous white-rot fungus Phanerochaete chrysosporium has been shown to hydrolyze the nitrate ester nitro-glycerine (glyceryl trinitrate) [46]. As long 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 Metals

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 chrysoporium. This result agrees with the finding of which demonstrated that Lignolytic fungi such as Penicillium spp [60]. Aspergillus spp, Rhizopus and Phanerochate chrysoporium 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. And 20% of an initial HMX concentration in 58 days when added

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

Figure 8: Fe (Iron) was the highest soil impacted with mean standard vale of 458.30 Fe>Mn>Cr>As>Zn>Pb>Cd>Co>Cu

Figure 9: Fe (Iron) was the highest soil impacted with mean standard vale of 374.57





Figure 11: Fe (Iron) was the highest soil impacted with m e a n standard vale of 169.71


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].

However, different fungi harbour chemical and structural properties which are relatively different like Aspergillus niger cell lack of chitosan in the cell wall exhibit better metals biosorption efficiency as compared other fungi isolates in Figures 6 and 15 respectively, similar to the reports of [10, 62,223,240]. Heavy metal ions are observed to influence the metabolism of the fungal activities and can subsequently affect the fermentation processes and fungi as potential biosorbents and had led 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 important role in the metal-binding process [94]. The fungus 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 shooting range Kachia Kaduna. Five (5) fungal genera were 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 from the study site in Kachia military firing range shows Lane of 100bp – DNA market (Ladder) lane F1-Aspergillus niger, lane F2-Rhizopus spp, lane F3-Penicillium spp, lane F4-Trametes versicolor and lane F5 -Phanorochate chrysoporium.

Plate 1: Unexploded Rocket launcher

to soil slurries of ammunition contaminated soil to yield the same N product [59]. Similarly, Aspergillus spp, Rhizopus, Penicillium and Trametes Vesicolor isolated in the study sites in Plate 1. Collaborated the finding of having biodegradation potentials on heavy metals Cd, Zn, Co, Pb, Cu, and Ni.., [71]. Table 5




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 15) as affirmed by similar work of Rhizopus spp had maximum growth peak at optical density 8.000 - 8.500 on the 25th, 35th and 40th respectively, Penicillium spp had the lowest growth peak at optical density2.500 on the 30th day while the maximum was 6.010 on the 10th day [223]. The maximum growth peak of Trametes versicolor was at 7.800 on the 20th day and the lowest growth peak 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 had the least ability. The analysis of variance result shows that there is a significant difference in the overall growth pattern of fungi in explosive mineral salt (Basal Salt Medium)broth supplemented with 1% v/v explosive (P>0.05). The overall result also indicates that the growth rate increased significantly from the zero hour to the 5th day and beyond. Means on the same column having different superscripts are significantly different (P<0.05) according to Duncan multiple range test. Stewart and Cullen (1999) also reported that lignin peroxidases of P. chrysosporium are encoded by a minimum of 10 closely related genes, designated from A to J. These are lipA, lipB, lipC, lipD, lipE, lipF, lipG, lipH, lipI and lipJ. (Table 6).

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

Metal Effects on Human Effects on Plants Microrganisms

Impacts Fungi Isolates Bioremedation References

CadmiumBone disease, coughing,

emphysemaChlorosis, decrease in

plant nutrientDamage nucleic

acid Asp. sp> Rhz sp> Pen sp.>

T.Vesicolor >

Nagajyoti et al, 2010; Fashola, et al.,

2018

headache,

hypertension, etcs

Inhibits metabolism

> P.chrysoporiumChibuike et al., 2014

; Sebogodi et al., 2011

Ayangbenro and Babalola ,2017

ArsenicBrain damage,

cardiovascular andPhysiological disorders

Deactivation of enzymes

P.chrysoporium > T.Vesicolor >Abdul-Wahab, and

Marikar, 2012 ;

conjunctivitis,

dermatitis, skin cancerLoss of fertility,

Inhibition of growth, Pen sp.> Asp. sp > Rhz sp

Finnegan and Chen, 2012

Destory metabolic

processes

CopperAbdominal pain,

anemia, diarrhea,Chlorosis, oxidative

stress,Disrupt cellular

function T.Vesicolor >

P.chrysoporium>Asp. sp > Dixit et al., 2015; Salem et al., 2000

kidney damage,

metabolic disorders,retard growth

inhibit enzyme activities

Rhz sp> Pen sp.Salem et al., 2000 ;

Odum,1971

ChromiumBronchopneumonia, chronic bronchitis,

Chlorosis, delayed, senescence

inhibition of oxygen uptake

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

Barakat, 2011; Mohanty et al., 2012

reproductive toxicity,

lung cancer,stunted growth, oxidative stress

Pen sp.> Rhz sp Cervantes, 2001

LeadAnorexia, chronic

nephropathy, insomnia, reduce enzyme

activities Denatures nucleic

acid andP.chrysoporium > T.Vesicolor

>Rhz sp> Nagajyoti et al.,

2010 ; Mupa, 2013




Bioremediation Potentials of Fungi Isolates to Heavy Metals

Figure 6 shows the fungal isolates showed very good activity in the reduction of heavy metals when observed. Lead concentration was reduced by Aspergillus niger to 20%, Rhizopus to 31.5%, Penicillium spp to 25%, Trametes versicolor to 54.5%, Phanorochate chrysoporium to 57% .Cadmium was reduced by A. niger to 16.5% Rhizopus spp to 17.5%, Penicillium spp to 15.3%, T.versicolor to 24.8%, Phanorochate chrysoporium to 20.5% .Zinc was reduced by A. niger to 32.49%, Rhizopus spp, to 38.8%, Penicillium spp to 34.2%, T.versicolor to 32.8%, P. Chrysoporium to 37.85% .Cobalt was reduced A. niger to 100%, Rhizopus spp to 19.7%,Penicillum spp 21.8%, T. versicolor to 100%, P. chrysoporium to 100%..Copper was reduced by to 22.1%, Rhizopus spp to 6.5%, Penicillium spp 23.19%, T. versicolor to 34.5%, P. Chrysoporium to 32.8%.Managanase was reduced by A. niger to 60.4%, Rhizopus spp to 7%,

Penicillium spp to 16%, T. versicolor to 61.6%, P.chrysoporium to 63.3%.Nickel was reduced by A. niger to 17.1%, Rhizopus spp to 8%, Penicillium spp to 18.6%, T. versicolor to 26.8%, P. chrysoporium to 28.90%.Chromium was reduced by A. niger

to 14.8%, Rhizopus spp to 5%, Penicillium spp to 10.9%, T. versicolor to 43.2%, P. chrysoporium to 41.7% Arsenic was reduced by A. niger to 13.3%, Rhizopus spp to 14%, Penicillium spp to 19.4%, T. versicolor to 19.8%, P. chrysoporium to 19.8% . The best fungal isolates for heavy metal reduction in increasing order were Aspergillus niger > Phanorochate chrysoporium > Trametes versicolor > Rhizopus spp > Penicillium spp while, in the overall, ANOVA test for total explosive contents shows a significant difference for all locations in both dry and wet seasons (P<0.05) .

Enzymes Biochemistry: Explosives and Heavy metals Bioremediation

The white rot fungus contains all three enzymes lignin peroxidase (LiP), manganese-dependent peroxidase (MnP) and lactase (Lac) called lignin modifying enzymes (LMEs) and non-selective [31,120,182]. LiP is unable to oxidize lignin to CO2 in a cell-free system.It requires certain radicals, Veratryl alcohol (VA) is a secondary metabolite produced by P. chrysosporium under nitrogen-limited conditions in parallel with the onset of LiP production. LiP genes have been characterized in P.

damage to neurons, risk

Alzheimer ’s disease,Affects photosynthesis

and growth, inhibits enzymes

activitiesPen sp.> Asp. sp

Wuana and Okieimen, 2011

NickelCardiovascular

diseases, chest pain, dermatitis,

reduced nutrient uptake

Disrupt cell membrane

T.Vesicolor >P.chrysoporium > Chibuike and Obiora, 2014

kidney diseases, lung

and nasal cancer, Decrease chlorophyll

content

inhibits enzyme activities Pen sp.> Asp. sp .> Rhz sp Malik, 2004

Zinc Ataxia, depression,

gastrointestinal irritation,

Reduced chlorophyll content

inhibits growthP.chrysoporium >T.Vesicolor>

Rhz spGumpu et al., 2015

icterus, impotence,

kidney and liver failure,impacts on plant

growthDeath, decrease in

biomass, Asp. sp >Pen sp.

Cobalt T.Vesicolor>P.chrysoporium >

Asp. sp

>Pen sp.> Rhz sp

Manganese oxidation of dopamine,

resulting in free radicals,

Plant growth affected and

fitness of the organisms

P.chrysoporium >T.Vesicolor>Pen sp.>

Roth, 2006; Takeda, 2003; Mena et al.,

1967

cytotoxicity nervous

system (CNS) pathology

poor quality yielddamage to

mitochondrial DNA> Rhz sp Asp. sp

Rollin, 2011; Wedler, 1994; Barbeau ,1984




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 Species

Explosives Conditions References

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

Electrophilic attack by microbial oxygenases Rylott and Bruce, 2009 ;

P. chrysosporium spp TNTMineralization to C02 by mycelium and 90%

removal of TNTFernando et al.,1990 ; Rho et al.,

2001;Sublette et al.,1992

Mineralization to C02 by sporesSpiker et al.,1992 ; Donnelly et

al.,1997;

Mineralization to C02 by myceliumMichels and Gottschalk, 1994 ; Huang

and Zhou,1999;

Mineralization correlated with appearance of

peroxidase activitySublette et al.,1992; Stahl & Aust,

1993a, b

Mineralization to C02 in soil-corn cob cultures

and stable Fernando and Aust, 1991 ; Yinon and

Zitrin,1993

Compost microbes TNTBiotransformation by Thermophile

microorganismsKaplan and Kaplan,1982;Isbister et

al.,1984

Transformed ring-[ 14C]-labeled

TNT,humification reactions Griest et al., 1991; Binks, et al. 1995 ;

Williams et al.,1992

P.chrysoporium spp RDX Biotransformation under nitrate-reducingFreedman and Sutherland, 1998 ; Alic

et al., 1997

Reduction by nitrogen-limiting conditionsFernando and Aust ,1991; Fernando

and Aust, 1990

Reduction by sulfate-reducing conditionBoopathy et al.,1998a, 1998b ;

McCormick et al.,1981

Methylenedinitramine formation under nerobic

conditionHawari et al., 2000 ; Kitts et al. 1994;

Aerobic denitration of RDX and highly mobile in

soilFournier et al, 2002; Clausen et al.,

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

hydrazines Hawari et al., 2000b ;McCormick et

al.,1976

White rot fungus RDXCoagulation in contaminated water by nitrate

reductase Sullivan et al., 1979 ; Bhushan et

al.,2002

TNTTreated TNT in wastewater and achieved 99%.

degradationHuang and Zhou,1999 ; Bennett ,

1994

TNT First step was degradation to OHADNT and ADNT, Aken et al., 1999 ; Stahl and Aust,

1993a




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).

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

1993b

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

Low water solubilitIity in anaerobic conditionsMchllan et al., 1988b ;Kaplan ,1993;McCormick et al.,1984

PETNMono-nitrated pentaerythritol to un-nitrated

pentaerythrito productsWilliams and Bruce ,2000;

Mineralization PETNBiodegraded by sequential denitration to

pentaerythritolBinks et al.,1996 ; Barr and Aust,

1994 ; de Boer et al ., 1987

P.chrysoporium spp PETNNitroglycerin, or glycerol trinitrate, degraded co-

metabolismWendt, et al., 1978 ; Barr and Aust,

1994 ;Stahl et al., 2001

by sequential removal of nitro groups Angermaier et al., 1981 ; Angermaier

and Simon, 1983

White rot fungus Explosives Dissolution rates: TNT > HMX > RDX>PETNTownsend and Myers, 1996 ;Brannon

and Pennington , 2002

Degraders glycerol trinitrate and isosorbide

dinitrate : nitrate estersWhite & Snape, 1993 ; Have and

Teunissen ,2001

plate 2: Location 2 traces of heavy metals and explosives.




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

LiP, nitrogen-deficient conditions favour the production of MnP (Manganese peroxidase hydrogen peroxide-generating systems [3, 26, 83, 117and 118]. MnP is also glycosylated heme-containing extracellular peroxidase. Its catalytic cycle is similar to those of LiP and horseradish peroxidase (HRP), but it uses absolute Mn(II) as a substrate that is widespread in lignocellulose and soil.A catalytic cycle is initiated by binding H2O2 or an organic peroxide to the native ferric enzyme, forming an iron–peroxide complex, as depicted in Figure 16 [93]. Peroxide oxygen–oxygen bond cleaves producing MnP compound I (Fe4+ oxo-porphyrin-radical complex). Subsequent reduction takes place through MnP compound II (Fe4+-oxo-porphyrin complex), and Mn(II) oxidizes to Mn(III), and this continues with the generation of native enzyme and release of the water molecule. MnP compound I is similar to LiP and HRP and can be reduced by electron donors such as ferrocyanide, phenolics, and Mn(II), whereas MnP compound II is very slowly reduced by other substrates and requires Mn(II) for completion of the catalytic cycle [220]. Transcription of different MnP isozymes exhibits variable dependencies in the presence of Mn (II) [66]. MnP gene sequences and cDNA have been cloned and sequenced, including Trametes versicolor [103,104]; MnP genes and reporter genes can be constructed that confirm these putative regulating elements [69]. Responses from a MnP promotor and applications in determining conditions for optimal MnP 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

as compared to nitrogen-sufficient (non-ligninolytic) conditions. They also reported that RDX was directly amenable to degradation by MnP, but not LiP [155].Pre-grown cultures have previously been suggested to apply to white rot fungi co-metabolism [22]. Besides these functions, fungal lactases may play a major role in the de polymerization of lignin MnP from P. chrysosporium has been studied extensively by a variety of biochemical and biophysical methods [72, 73, 86,159, 228 and 239] and the crystal structure and the heme environment of MnP is similar to that of other plant and fungal peroxidases by [197]. MnP is unique among enzymes using manganese as a redox cofactor. Rather than permanently sequestering Mn II in an interior binding site, MnP selectively binds Mn II on the surface of the protein, oxidizes it, and then releases the Mn III product in complex with organic acids such as oxalate and malonate. However, the crystal structure of MnP- Mn II in the absence of chelators suggests that the later alternative is more likely [18, 119,120,197, and 228].

However, molecular modelling and kinetic studies indicate that Mn II binding and oxidation are sensitive to subtle changes in ligand geometry [118, 122, 196, 238, and 239]. Indeed, the flexibility of the Mn-binding site in MnP allows a wide variety of metal ions to bind. However, it appears that only Cd II exhibits a dissociation constant similar to that of MnII and other divalent cations, such as CO II [84,239].




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

Figure 17: Overall structure of MnP-MnII refined at 1.45 Å resolutions. Source: Sundaramoorthy et al., 2005

Figure 18: Overall structure of MnP-MnII refined at 1.45 Å resolutions. Source: Sundaramoorthy et al., 2005




The results presented here provide a more accurate model of native MnP at very high resolution (Figure 17). The 2.0 Å resolution crystal structure of MnP, determined at room temperature, shows that the substrate, Mn II, binds to one heme propionate and the side chains of three amino acids, Glu35, Glu39, and Asp179, as well as two solvent ligands [200] (Figure 17). This site was confirmed by kinetic and biophysical studies of wild-type MnP and of proteins containing point mutations in the putative binding site. Figure17. Overall structure of MnP-MnII refined at 1.45 Å resolutions. A second glycosylation site at Ser336 with O-linked R-mannose is shown. The FO - Fc omit map around Ser336 is contoured at 3.0σ showing density for R-mannose. This is a new feature in the 1.45 Å cryo maps that was not present in the 2.0 Å room-temperature structure. Figure18. Comparison of the C-termini of MnP-Mn II, MnP-Cd II, and MnP-Sm II structures. The Fo-Fc omit maps were calculated omitting all atoms near the C-terminus within a sphere with a radius of 5 Å, including Ala357 and Asp84. The density is contoured at 3σ for MnP-Mn II (A) and MnP-Sm III (C) and 3σ and 20σ for MnP-Cd II (B). The large peak in the MnP-Cd II map is modelled as Cd II, and its four potential ligands include the terminal carboxylate of the polypeptide, Ala357, and the side chain carboxylate of Asp84.

Table 6: Peroxidase-catalyzed Degradation of Lignin

Fungus/ Peroxidase

Lignin Model Compound

Medium/Reaction Conditions

Degradation / Mineralization Rate

(%)

Metabolic Products

Incubation (hours/ Weeks)

Reference

Phanerochaete [14C]Methylated Crude culture liquid,

De polymerization

1 hTien and

Kirk,1983chrysosporium /LiP (crude)

spruce lignin

tartrate, H2O2 , Tween,

pH 3.0, 37°C Glycolate, VA, 30 – 40%

P. chrysosporium /LiP

14C [DHP] or [α-13C] DHP

Co-solvent, H2O2 De polymerization

Low-molecular-

weight products

16 hHammel et

al.,1993

P. chrysosporium /LiP, MnP

Hardwood lignin Aqueous medium, 37°C 5% Degradation

12 hThompsonet al.,

1998

P. chrysosporium /MnP

Four [14C]DHPsMalonate, 100% O2, Mn (II), glucose, oxidase/glucose, pH 4.5, 37°C

Partially degraded

0.5–7 hWariishi et

al.,1991

P. chrysosporium /MnP

Chlorolignin Lactate, Mn(II), H2O2 De polymerization

Lackner et al.,1991

P. chrysosporium / Recombinant

MnP

Non phenolic lignin model dimer

MnP-Mn(II) -linoleic acid

20% as 14CO2

Water-soluble

products48 h

Kapich et al.,1999b

The MnP-CdII structure also exhibits a possible additional metal-binding site near the C-terminus (Figure 18). The carboxy-terminal oxygen, the side chain of Asp84, and two solvent molecules provide ligands to a second cadmium ion, displaying tetrahedral geometry. Ligands in the two metal sites, Glu39 and Asp84, are linked through hydrogen bonds mediated by Asp85 and water. This network of hydrogen bonds is possible only with the open conformation of Glu39. Although Mn II is not observed to bind at this site in the crystal structures, a second, lower-affinity Mn-binding site has been postulated on the basis of pH binding analysis of the protein in solution Mauk et al., 1998. It is possible that MnII does bind to this site under physiological conditions, but not under the conditions for crystal formation (pH 6.5 in cacodylate buffer). The site is exposed to the solvent and could accommodate a MnII ion with a bound chelator such as oxalate or malonate. Alternatively, Cd II may exhibit a higher affinity for this site than Mn II because of its larger mass-to-charge ratio and its ability to adopt a wider range of ligation geometries [44]. Atomic absorption analysis indicated only 1 equiv. of Cd II binding per mole of protein in solution, possibly due to partial occupation of both sites by this metal at pH 4.5 [239].




Previously, reported was the extensive characterization of the effects of Cd II on manganese peroxidase as a reversible, competitive inhibitor by [239]. Steady-state kinetic analysis showed that Cd II is uncompetitive for H2O2 and MnP-Cd II forms compound I under transient-state conditions. Cadmium cannot be oxidized by MnP intermediates based on our analyses (Figure 6) and acts as a reversible, competitive inhibitor for Mn II oxidation with a Ki of 10 µM and confirmed by similar works in Figure 18 [200, 239]. Similar changes in the MnP heme spectrum upon binding of Cd II and Mn II and very close apparent dissociation constants (Kd ≈ 8 µM for CdII and 10 µM for Mn II) indicate that these metals bind at the same site. The lack of a Glu39-Arg42 interaction in the MnP-Cd II structure increases the net negative charge, which might be responsible for tighter binding of Cd II than of Mn II as reported by Sundaramoorthy et al., 2005.Ethanol and arsenite are known to induce the synthesis of heat shock proteins in fungi [111,146]. However, in P. chrysosporium, H2O2compound increases MnP transcript levels only in nitrogen-limited cultures [185].The copper-thio¬ ether bond and non-coordination residue strongly influence the redox potential of the enzyme. Lactases from different sources display a wide range of redox potentials. The T1 site of lactase of T. versicolor shows a high redox potential of 780-800mV which supports the results of Table 5T. Versicolor biodegrade copper metal in the study area. Figure 19 showing all fungal lactases show a similar architecture consisting of three sequentially arranged domains of a-barrel type structure. The active site is well conserved with four copper sites T1 is located in domain 3 with copper lying in a shallow depression and trinuclear copper cluster is at the interface between domainaI and 3 with each domain providing ligand residues at the coordination of copper ions. The T1 copper is coordinated with His-Nand Cys-S as conserved equatorial ligands [160].

Due to their powerful degrading capabilities towards various recalcitrant chemicals, white-rot fungi and their lignin degrading enzymes have long been studied for biotechnological applications such as bio bleaching, bio decolourisation and bioremediation [33, 48, 209 and 244]

Fungal Decomposition of Nitro-compounds

White-rot fungi and are primary microbes decomposing extremely recalcitrant lignin [145,245]. Their effectiveness in its 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 and expression of peroxidase genes, Table 6 [3,107,152, 161and

163]. Restriction fragment length polymorphism (RFLP) and allele specific marker demonstrated that linkage of eight genes lipA, lipB, lipC, lipE, lipG, lipH, lipI and lipJ, reside within 3% recombination, which corresponds to 96 kb region of scaffold 19 [142].The promoter region of MnP has heat shock elements and putative metal response elements (MREs) [75, 103].

ConclusionAmong all the concentration of explosives both in dry and

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 impacts (Tables 4,5 and 6 respectively). Lignolytic fungi such as Penicillium spp. Aspergillus spp, Rhizopus and Phanerochate chrysoporium a white dot fungus was capable of oxidizing cyclic nitramines and mineralizing RDX, HMX, TNT and PETN where Aspergillus niger had the best ability to degrade explosive while Penicillium spp had the least ability using the analysis of variance result shows that there is a significant difference in the overall growth pattern of fungi in Explosive Mineral Salt (Basal Salt Medium) broth supplemented with 1% v/v explosive (P>0.05).

The best fungal isolates for heavy metal reduction in increasing order were Aspergillus niger > Phanorochate chrysoporium > Trametes versicolor > Rhizopus spp > Penicillium spp while, in the overall, ANOVA test for total explosive contents Shows a Significant difference for all locations in both dry and wet Seasons (P<0.05) and bioremediation will occur via biotransformation where the fungi enzymes biochemistry is very significance to biodegradation pathway correlated with the appearance of peroxidase activity [22].

There is clearly a need to better define the electron acceptor conditions that promote RDX biotransformation, so that treatment systems can be designed with a greater assurance of high treatment efficiency as reported by [63].There is a great possibility to develop P. chrysosporium strains for munitions wastes management because white rot fungi for biodegradation of TNT has been patented [182]. Also, several LiP genes have also been characterized from other species of fungi, including four Trametes versicolor clones and P. Chrysosporium LiPs are encoded by a family of at least10 closely related genes, designated lipA through lipJ [74, 207,208]. LiP genes show a high degree of sequence conservation for 2,4,6- trinitrotoluene (TNT) and other nitro explosives, and reduction improves in more reduced environments[186].

Figure17: Source: Trametes versicolor http://www.wisconsinmushrooms.com/) as grown in nature.




Figure19: Metabolic reduction of 2, 4, 6-trinitrotoluene by Phanerochaete chrysosporium.

Source: Baker et al., 2015

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