ABSTRACT
Environmental pollution is principally caused by human activities that usually result in the release of man–made pollutants such as biological, chemical and radioactive in such states as solid, liquid and gaseous substances into the biosphere via, land, water and air. Globally, the increasing rate of environmental contamination by ammunition/explosives resulting from their increasing deployments in the rising spites of military conflicts and training has certainly become a matter of great concern for every nation. The aim of this research was to molecularly characterize some isolated bacteria from an apparent munitions contaminated sites in Kachia Military Firing Range, Kaduna State. DNA from each isolated bacteria was extracted and 16s rRNA Gene amplified from each isolated bacteria DNA using thermo cycler. The amplified genes were run on Agarose gel plate and visualized. Amplified gene bands were sequenced and Basic Local Alignment Search Tool (BLAST). 16s rRNA gene sequences result aligned with BLAST search of NCBI databases that revealed the presence of Lysinibacillus pakistanensis, Eschericia coli, Achromobacter spanius, Achromobacter animicus, Escherichia fergusonii and Shigella sp. The results identified bacteria that were isolated from munitions contaminated sites and that could also be useful for their bioremediation potential against munitions contaminant.
Key words: Environment, pollution, contamination, bacteria, bioremediation.
Globally, the increasing rate of environmental contamination by ammunition/explosives resulting from their increasing deployments in the rising spites of military conflicts and training has certainly become a matter of great concern for every nation. Therefore, there is an urgent need to make provisions for efficient and effective mediation measures to save the affected nation(s) from the danger of environmental contamination by ammunition/explosive toxic substances. Environmental regulations have however not been established for trinitrotoluene (TNT), royal demolition explosive (RDX) and high melting explosive (HMX) in many countries including Nigeria (Rya et al., 2007). United States Environmental Protection Agency (EPA) classified TNT as a possible human carcinogenic (class C). Explosives are being used by different military formations in test firing, during training of military personnel’s which take place in all the military firing ranges across the country in Nigeria and the Nigerian Army School of Artillery (NASA) Kachia, Kaduna State which is the area of study in this research. The formations that carried out training and firing ranges in NASA includes the Nigerian Defence Academy (NDA), Nigerian Air Force Base (NAF), Nigerian Army School of Infantry (NASI) Armed Forces Staff and Command College, Jaji and Defence Industrial Corporation (DICON). So far, no current practice is taking place by the military of Armed Forces of Nigeria for the remediation of the contaminated soil apart from the normal cleaning and burning of the munitions contaminated sites.
The primary human exposure pathways for explosive contaminated soils are dust inhalation, soils ingestion and dermal absorption (Craig et al., 1995). Inhalation of air and consumption of farm products may increase the liveliness of shooters transporting heavy metals and explosives on their hands, hair, skin, clothing, shoes and shooting equipment from the range into their vehicles and homes. Also, wind interaction and rain run offs flow through to the stream in the site of firing range leading to the contamination of air, ground water, soil, plants, animals and other ecological parameters within the sites and its vicinity. In order for a contaminated site to be treated properly, the magnitude, impacts and risks the pollutants impose on the ecological parameters and human health must be assessed. Clean up of the soil and groundwater in such sites is necessary to prevent the spread of contaminants and subsequent injury to the ecosystems. Contaminated soil that contains heavy metals or volatile organic compounds (VOCs) have in the past been remediated utilizing the techniques of land farming, soil washing, or soil flushing (described under soil reclamation section) (Fall, 2003).
The chemical, mechanical and thermal treatments (incineration) of lands and water bodies to destroy hazardous wastes have proved to be economically and environmentally unsustainable, hence the shift of focus to biological methods which are cost-effective, environ-mentally sustainable as well as socially acceptable (Rittmann et al., 1994).
Microorganisms have developed many different strategies for removal and degrading of these xenobiotic compounds; also, oxidative or reductive pathways for the degradation of nitroaromatics have been widely studied (Spain, 1995). Microorganisms with distinctive features of catabolic potential and/or their products such as enzymes and bio surfactant direct use is a novel approach to enhance and boost their remediation efficacy (Le et al., 2017). Microorganisms used for remediation of polluted environment can either be bacteria, fungi, algae or combination of different species. Bacteria rely on various enzymes such as mono and dioxygenases, nitroreductases, haloreductases and esterase to degrade chemicals. These enzymes often need to be induced by the pollutant for degradation to occur (Axtell, 1998). In general, those enzymes metabolize contaminants and are located within the cell. The aim of the research was to molecularly characterize bacteria isolated from munitions contaminated sites in Kachia Military Firing Range, Kaduna.
Study site
The study was conducted in the permanent military shooting/ training range located at 5 km east of Kachia town in Kaduna State. The range was established in 1965 and it covers an area of about 24.95 km2 that lies between longitudes 9° 55’ N and 7° 58’ E, with an elevation of 732 m above sea level (Figure 1). The topography is undulating and the vegetation is Guinea Savannah. The area where the munitions/explosives are fired (the impact area) is a valley consisting of about four large rocks as objectives where the fired munitions/explosives land and explode.
Sampling points
Four sampling points selected for the study are locations 1, 2, 3 and 4. Location 1 and 2 are approximately 200 m from the table top, that is, where the small arms are fired such as Kalashinokov, FN, Grenade, GPMG, SMG and Pistols. The soil in Location 1 and 2 is made of 50% silt and a flat ground with shrubs and drainage that flows through to the farm lands near the sites. Location 3 is approximately 9000 m away from the table top and lies between 9° 53’ 44.71” Northings and 7° 53’ 17.87” Easting’s while Location 4 is ahead of Location 3 and is about 10,000 m from the table top where heavy weapons are aimed at. 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.
Soil sampling
This was done at particular sampling areas using soil iron auger. Four locations within NASA shooting/training range Kachia were earmarked as sampling sites for this study. Samples for bacteria analysis were kept in a cool box refrigerated with ice pack to retain the original microbial activities.
Screening for explosive/munitions degrading bacteria
Nutrient agar media preparation
Nutrient Agar (Antec/USA) was prepared by dissolving 28 g of the agar in 1 L of distilled water in a conical flask. The conical flaks with the media was autoclaved at 121°C at 15 Pressure Per Square inch (PSI) for 15 min, cooled to 40°C before dispensing into sterile petri dishes.
Isolation of possible explosives degrading bacteria
Thirty gram samples of soil from selected positive screening tests were placed into flasks and amended with 1% cometabolite (Sodium acetate) and 5 ml of distilled water. Flasks were plugged and incubated at 28°C. After one week of incubation, samples of the acetate primed sterile water were plated onto basal salts medium supplemented with 15 g/L yeast extract that cannot solidify medium Nutrient Agar and the cometabolite at a concentration of 20 g/L and 60 g/ml nystatin to suppress fungal growth. Plates were incubated at 28°C for two weeks. Representative colonies were picked from each plate and transferred to liquid culture media containing 100 mg/L TNT and incubated for 10 days at 28°C on a gyrorotary shaker at 150 rpm. Bacteria Isolates were identified by molecular characterization.
Isolation of genomic DNA from bacteria
Bacteria with proven bioremediation capacity were selected and their DNA isolated. DNA of bacteria strains isolated was extracted from 1 ml of bacteria culture; the culture was pelleted by centrifuging at 12,000 rpm for 2 min. Further, the pellet was treated with lyses solution and proteinase K and incubated at 60°C for 30 min. Nucleic acid was 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 TE buffer (Tris-EDTA buffer). The purity and quantity of DNA were examined by recording its agarosegel electrophoresis (Jyothi et al., 2012; Thenmozhil et al., 2013).
Polymerase chain reaction amplification of 16S rRNA gene
The PCR reaction mixture containing 10 X PCR buffer, 25 mM, Magnesium chloride, 25 mM dNTP’s, 10 pm/ul Primer concentrations and template DNA were used for the amplification of the 16s rRNA gene for each isolates. PCR conditions were optimized using 96-well PCR thermocycler. The PCR Program began with an initial 5-min denaturation step at 94°C: 35 cycles of 94°C for 45 s, annealing (1 min at 55°C), and 10 min extension step at 72°C. All reaction mixtures were preserved at 4°C until it was time for analysis as reported by Kloos et al. (2006).
The amplified 16S rRNA gene of each isolates was further characterized using gel electrophoresis.
Sequence determination of 16s rRNA gene
The amplified 16s rRNA gene of each isolate was processed for sequencing and characterization. The sequencing Kit (Applied Biosystems) with the product was analyzed with ABI prism DNA sequence (ABI). The gene sequence of each isolate obtained in this study was compared with known 16s rRNA gene sequences in the Gene Bank database as described by Jyothi et al. (2012).
16S rRNA gene amplification from bacteria isolates
16s rRNA gene band size of 100bp was observed for each bacteria on the agarose gel isolates from each munition contaminated site (Figure 1). The positive results were obtained as shown in the following DNA samples BB1, BB2, BB3, BB6, BB9, BB10 and BB11 while the others have negative results. Meanwhile, the identity of the isolates were further confirmed by 16S rRNA sequencing and BLAST bacterial samples.
16S rRNA gene sequences result
The bacterial 16S rRNA genes were sequenced and the sequenced results were BLAST (Basic Local Alignment Search Tool) using NCBI databases. The sequences aligned showed 100% result similarity with Achromobacter animicus and Esherichia fergusonii, 99% similarity with Lysinibacillus pakistanensis, 98% similarity with Esherichia coli, while Achromobacter spanius had 95% similarity. In this result, different species of bacteria strains involved in munition/explosive degradation were highlighted (Table 1).
Different metal types are used in munition bodies for steel casing and copper driving band that can lead to accelerated galvanic corrosion, although authigenic mineral precipitation and biological overgrowth may slow corrosion rates (Jurczak and Fabisiak, 2017). Even at low concentration, toxic and mutagenic effects of some explosives to various organisms ranging from microorganisms and humans have been described (Ayoub et al., 2010). Microorganisms have developed many different strategies to remove and degrade these xenobiotic compounds and oxidative or reductive pathways for the degradation of nitro aromatics.
Out of the 14 isolates, 16S rRNA gene was amplified from seven bacteria isolates (Plate 1) and sequenced (Figure 2). The gene sequence from each of the isolates were BLAST (Basic Local Alignment Search Tool) using NCBI databases and identified as L. pakistanensis, E. coli, A. spanius, A. animicus, E. fergusonii and Shigella sp (Table 1). The result showed similarity of 100, 99, 98 and 95% with the uploaded gene in the NCBI database gene bank. The differences in genetic makeup might be due to the mutation of their genes caused by the adaptation of the organisms to the environmental change. This work conformed to that of Rajbanshi (2008) who isolated E. coli, Klebsiella sp., Enterobacter sp., Flavobacterium sp., Bacillus sp., Arcobacter sp. and Psendomonas sp. as heavy metal tolerant bacteria in treatment plant in Nepali.
Among the seven bacteria isolates, three of the genera, viz; Achromobacter sp., Escherichia sp. and Lysinibacillus sp. were predominant when compared to the other bacteria isolate from the munition contaminated soil samples. This finding is in agreement with Philip et al., (2000) and Gunasekaran et al. (2003), whose report revealed that Bacillus sp. showed maximum tolerance to Zinc, Copper, Cadium, Nickel, Lead, and Arsenic. The result is also similar with the study that was carried out by Kitts et al. (1994) and Kafilzadeh et al. (2011) respectively where bacteria strains were isolated indigenously from ammunition contaminated site and identified as follows: Bacillus, Citrobacter, Escherichia, Klesbsiella, Enterobacter, Achromobacter and Shigella
The dominant groups of bacteria isolated from munition contaminated soil samples were gram negative bacteria and this result corroborates with the report of Kaplan and Kitts (2004). In general, the gram negative bacteria have been reported as the most effective group of hydrocarbon degrading bacteria. Lipopolysaccharide produced in the bacterial membranes of gram negative bacteria supports the formation and stabilization in aqueous system and contributes by increasing the attack surface on the pollutant for subsequent assimilation (Van Hamme et al., 2003).
Bacteria are known to mineralize explosive by primary reduction of nitro group that are amenable to reduction of electrons. It is the combination of these oxidants together with the carbon skeleton that gives an explosive the capability for explosive autocatalytic oxidation. The isolated bacteria can be used to bioremediate munition contaminants in varying degrees and may be used as consortia for bioremediation of soils contaminated with heavy metals and explosive products.
Bioremediation is a rapidly establishing technology for contaminated soil and ground water treatment. For some compounds, it may be the best technology for treatment, particularly in sites where it is difficult to access the contamination such as in deeper aquifers. It was observed from this study that munitions-explosives degrading bacteria are ever-present in the study site and they can be isolated as indigenous bacteria. It is observed from this study that bacterial strain isolated from contaminated soil can be good heavy metal and explosives degraders. Therefore, isolated bacteria can be capable of bioremediation of munition contaminants in varying degrees and should be used as consortia for bioremediation of soils contaminated with heavy metals and explosive products.
The authors have not declared any conflict of interests.
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