Aerobic degradation 2,4-dinitrotoluene: effect of raw organic wastes and nitrogen amendment

2,4-Dinitrotoluene (2,4-DNT), a major by-product of the synthesis of 2,4,6- trinitrotoluene, is widely used as a waterproofing, plasticizing and gelatinizing agent in propellants and explosives. Due to its toxicity, the compound is treated as a priority pollutant. Therefore, its removal from contaminated systems is a major focus of research and attention. Contaminated sites in Ibadan, Nigeria were screened for the presence of 2,4,-DNT degrading organisms. The technique of continual enrichment on NACs yielded bacterial isolates able to utilize 2,4-DNT as growth substrate. Based on phenotypic characteristics and 16S rRNA gene sequencing one of the isolates selected for further study was identified as Proteus sp. strain OSES2. Growth of the strain on 2,4-DNT resulted in exponential increase in biomass and complete substrate utilization within 72 h accompanied with NO 3 - elimination. Degradation competence enhanced in the presence of Corn steep liquor, molasses and Tween 80 compared to incubation without amendment. Conversely, amendment with nitrogen sources yielded no significant improvement in degradation. Use of this organism organic wastes as candidates in bioremediation strategy should be exploited. This would provide a cheaper organic source supplement for cleanup purposes with the ultimate aim of reducing the cost of bioremediation while reducing wastes intended for landfill.

Background 2,4-Dinitrotoluene, one of the several congeners of the dinitrotoluenes (DNTs) family is a major isomer generated from both the industrial synthesis of 2,4,6 Trinitrotoluene (TNT) and also a principal metabolite in the course of its microbial degradation [1,2]. They are not naturally present in the environment, but they are released into ground and surface water, and soils as a result of the widespread usage in ammunition manufacturing facilities and some chemical products [3]. These compounds are used by the military as an explosive intermediate, a component of gun powder and as a modifier of smokeless powders in moderate and high explosives. They are also used in the manufacturing of dyes, herbicides, polyurethane foam [4] and plasticizers. However, several national environmental regulators such as the U.S Environmental Protection Agency (USEPA) have described 2,4-DNT as a priority pollutant due to its carcinogenic and toxicological properties, widespread occurrence and recalcitrance to microbial attack which can potentially cause harm to public health [1,5,6,7].
Among several methods available for decommissioning of environments laden with nitroaromatic compounds (NACs), the use of microbial metabolic potential offers the most environmental compatibility and cost effectiveness. It is therefore not surprising that microbial degradation of NACs has been a subject of research owing to rapid adaptability of microorganisms for degradation of these xenobiotics. This is because, the presence of these pollutants such as 2,4-DNT in the habitat creates a strong selection force due to exposure of these soil microorganism to varying levels of the xenobiotics, which invariably results in the development of soil microbiota capable of utilizing 2,4-DNT, as a singular source of carbon, nitrogen and energy [8][9][10]. Interestingly, several organisms have been reported to metabolize 2,4-DNT and such organisms which have been reported to possess unique metabolic properties have been isolated from a polluted environmental matrix. For example, Pseudomonas sp., a bacterium strain isolated from Waconda Bay close to an Army ammunition factory, was reported as the first organism to utilize 2,4-DNT as the sole carbon and energy source with a concomitant evolution of nitrite [11]. Since this initial report, an abundance of 2,4-DNT degrading bacteria including; Burkholderia sp. strain DNT [12], Burkholderia cepacia strain R34 [13], Pseudomonas sp. strain VM908 [14], Bacillus sp. [15], Arthrobacter strain K1 [16] and Rhodococcus pyridinovorans NT2 [17] have been documented. The elucidated 2, 4-DNT degradative pathway utilized by these organisms showed that the substrate was degraded through a dioxygenation reaction in which 2,4-DNT is oxidized to 4-methyl-5-nitrocatechol (MNC) with nitrite removal. The MNC was then sequentially reduced to 2-hydroxy-5-methylquinone in a reaction mediated by a monooxgenase, which is eventually converted to 2,4,5-trihydroxytoluene which is subsequently transformed by meta cleavage [10,18].
Despite the isolation of organisms with inherent abilities to degrade NACs, the degradative competence of such strains can be improved or optimized in the presence of easily amenable cheap carbon sources that may sometimes constitute environmental nuisance. Remarkably, several investigators have reported the improved metabolism of contaminants when amended with additional carbon and nitrogen sources. For instance, Tharakan and Janice [19] documented enhancement of TNT degradation when fortified with 3000 mg L -1 of glucose and citric; according to the authors, only 9% of the 30 ppm TNT substrate was degraded by the bacterial consortium. However, the same concentration of the substrate was completely consumed in 24 h upon citric and glucose fortification. In another report, the degradation capability of Pseudomonas sp. strain HK-6 on some NACs (TNT, royal demolition explosive [RDX], atrazine and simazine) showed significant improvement when amended with different carbon sources [20] while fortification with nitrogen sources revealed no enhancement. Amendment with peptone and yeast also significantly improved the biodegradation of 2,4,6-trinitrophenol (TNP) and biomass formation using Rhodococcus pyridinivorans strain NT2 as a source of inoculum compared with non-fortification of the culture fluid [21]. The same trend was also observed in biodegradation of TNT by an immobilized Bacillus sp. strain YRE1 when amended with Tween 80 [22]. Irrespective of the available reports in the literature, it is obvious that amendment with carbon and nitrogen sources most likely results in meaningful improvement in metabolic capabilities of microorganisms to varying degrees with few exceptions.
Although there is to some extent an existing body of knowledge on microbial metabolism of 2,4-DNT, there is dearth of information on the availability of such phenotypes in tropical Africa particularly in Nigeria where there is unabated and indiscriminate release of the pollutant into the environment. Floyd et al [23] whilst reviewing the captured prokaryotic diversity in American Type Culture Collection (ATCC) indicated that geographical locations and environmental types of indigenous organism exhibited a significant difference between both geographical locations and environment that were represented in the culture collection. Accordingly, North America accounted for 24.1% of all the entries followed by Europe (14.7%) and Asia (11.5%). Quite unfortunately, only 2.8% was attributed to Africa despite the huge landmass and unregulated inflow of environmental contaminants. Therefore, it is the intent of the present study to showcase for the first time, the occurrence of microbial strains with 2,4-DNT degradation potential from a Nigerian polluted soil and to investigate the improvement of such metabolic capabilities by amendment with agro-allied waste such as molasses, corn steep liquor (CSL) and other nitrogen sources.

Isolation and characterization of 2,4-DNT degrading bacteria
Thirty-four (34) distinct bacterial colonies were picked from the MSM agar plates after a preliminary enrichment on nitroaromatic mixtures. After screening of individual isolates for growth on 2, 4-DNT, five (5) isolates with unique metabolic ability to utilize this substrate as the singular carbon and energy source were obtained. Among them, strain OSES2, a rod-shaped Gram-negative bacterium with swarming motility on nutrient agar plate which was the most efficient degrader was subsequently selected for further study.
A positive genotypic identification was conducted by sequencing of the 16S rDNA fragment. Using the BLAST algorithm, the sequence showed 96.86% identity with the type strain P. mirabilis PWN3A, as illustrated in Fig. 1. The 16S rRNA gene of Archaeoglobus fulgidus a clearly unrelated organism used as an out-group. Strain OSES2 clustered more cohesively with members of the genus Proteus, than with genera Enterobacter and Achromobacter. The organism was subsequently putatively identified as Proteus sp. strain OSES2.

Degradation of 2,4-DNT by Strain OSES2
Strain OSES2 grew readily on 2,4-DNT under aerobic batch conditions. Growth of the isolate was exponential; it commenced immediately without a display of lag phase (Fig. 2).
The cell population density increased by eight orders of magnitude within 48 h, yielding a mean generation time of 25.14 h. Metabolism of the substrate is evidenced by gradual disappearance concomitant with increase in population densities and release of NO 3 - (Fig.   3). Lack of meaningful change in 2,4-DNT concentration in control flasks showed that the reduction observed in experimental flasks were essentially due to microbial action and not physiochemical factors. Since no carbon and nitrogen sources other than 2,4-DNT were introduced into the culture medium, the obtained data suggests that the organism could utilize the substrate both as the only source of carbon and nitrogen source. A critical examination of the results showed that nearly 65% of the 2,4-DNT substrate was utilized by the organism, at which time points biomass production was five folds even though the amount of NO 3 recovered was less than 1 mg L -1 (Fig 3). By implication, only 28% of the substrate was utilized between 24 and 72 h of cultivation yielding a degradation rate of 0.032 mg L -1 h -1 as against 0.035 mg L -1 h -1 . Overall, nearly 93% of the substrate was utilized in 72 h of incubation yielding a degradation rate of 0.035 mg L -1 h -1 assuming a constant rate of utilization of the substrate per time ( Table 1). The amount of NO 2 recovered throughout the incubation period was significantly low, whereas that of NO 3 picked at 2.9 mg L -1 and declined slowly to 2.5 mg L -1 at the very end of the incubation. It is noteworthy, that incubation beyond 48 h produced no significant cell increase, in fact, the population density took a downward trend with a characteristic color change of the medium from pale yellow to colorless. This observation notwithstanding, utilization of substrate did not cease, although the rate of consumption slowed considerably.

Effects of carbon and nitrogen sources on 2,4-DNT degradation
The effect of different carbon sources (molasses, CSL, glucose, glycerol, maleic acid, acetate, sucrose and aspartate) on biodegradation of 2,4-DNT was evaluated over a period of 48 h to determine the one that best enhanced degradation. The data obtained are summarized in Fig. 4. Addition of these co-substrates to the culture media resulted in a significant improvement in degradation competence of the organism, although to a varying degree. Amendment with all the carbon sources yielded over 80% degradation of 2,4-DNT except for aspartate. Interestingly, molasses and CSL produced the highest enhancement compared to other substrates utilized.
A more detail growth analysis of the isolates in MSM fortified with CSL and molasses is shown in Fig 5. Higher growth and degradation rates were recorded in the presence of these amendments in comparison with when 2,4-DNT was supplied alone. Growth and catabolic patterns of strain OSES2 in flask amended with CSL paralleled observations in flask containing molasses. In both cases, maximum cell densities of over fifteen orders of magnitude were observed within 36 h of incubation, which was three times higher than flasks without fortification. A detailed assessment of the data obtained indicated that between 60% -70% of the initial NAC supplied was used by the organism within the first 12 h of cultivation cumulating in approximately 0.074-0.085 mg L -1 h -1 as against 0.072 to 0.087 mg L -1 h -1 overall degradation rate (Table 1). 2,4-DNT utilization was relatively faster in CSL compared with molasses, nevertheless, complete degradation was accomplished in 36 h in both cases. When subjected to statistical analysis, both molasses and CSL amendments yielded lack of significant difference at P <0.05 confidence level.
Conversely, a significant difference was obtained when both incubations were compared with flask without amendment. By implication, both molasses and CSL significantly improved 2,4-DNT degradation competence of strain OSES2.
In contrast to the enhanced degradation observed with carbon sources, amendment with nitrogen sources stimulated bacterial growth ( Fig. 6), but there was relatively no effect on DNT degradation with reference to Fig. 2. Although, complete degradation of the substrate was not achieved until 72 h of incubation, the data was comparable with Fig 2 which lacked nitrogen sources. This may not be unconnected with the fact that the organism exhibited an ability to utilize 2,4-DNT both as the sole source of carbon, energy and nitrogen. Fortification with exogenous nitrogen sources appears to be insignificant.
Interestingly, statistical analysis showed that the activity of strain OSES2 with or without alternative nitrogen sources yielded lack of significant differences.
Since amendment with nitrogen sources did not significantly improve degradation of 2,4-  [17].
Microbial enzymatic activity is reliant on the organismal physiological nature as well as the physico-chemical characteristics the environment process [35,36]. Nutritional consumption by microorganism is an important criterion for microbial activity which in turn can influence their enzymatic activity. Amendment with carbon source such as starch, molasses, pyruvate, sucrose, lactate, glucose, ethanol, and citric acid, does not only increase the population density of microorganisms, but also their effectiveness by producing enzymes, elimination of lag phases and ultimately shortening the degradation time [37][38][39]. Interestingly, strain OSES2 metabolic activity was greatly enhanced when amended with different carbon sources particularly, CSL and molasses resulting in complete DNT utilization in few hours as previously documented for other organisms [40][41]. Specifically, CSL was found to be the most effective carbon source. One possible reason for enhanced degradation of 2,4-DNT in CSL amended incubation was the increased biomass. This increased biomass resulting in higher growth rate may not be unconnected with the fact that CSL is rich in amino acids, minerals, co-factors and vitamins and other nutrients required by microorganisms. The composition of molasses on the other hand is very complex containing sugars (30% of glucose, 43% sucrose), organic nitrogen, vitamins, amino acids, proteins, vitamins and minerals [42]. The components of these carbon sources make them conducive for microbial growth and therefore, could have in turn stimulated enzymatic activity and biodegradation of the xenobiotic. When strain OSES2 was grown on 2,4-DNT alone, very little additional biomass could be produced because of the limited amount of carbon substrate available in the medium. Since CSL or other easily degradable substrate, was present, the organism grew on it while producing biomass for degradation of the DNT. Amendment with CSL, molasses, surfactant and various supplemental sources have also been investigated to enhance the biodegradation rate of TNT (43)(44)(45).
It is noteworthy that CSL in addition to molasses and acetate are good supplemental carbon sources for enhanced DNT degradation more so, owing to the fact that they are among the inexpensive sources of carbon and are readily available as waste emanating agro-allied industries [42,44]. Furthermore, since bioremediation is a sustainable waste management system that primarily details the usage of microorganisms or cheaper raw materials from waste as biostimulants to clean-up pollutants from a contaminated matrix; the use of CSL and molasses is likened to killing two birds with a stone. While reducing the overall cost of remediation on one hand, it is a means of waste management for industries generating them.
In contrast to carbon sources, amendment with nitrogen sources yielded no significant improvement in the metabolic capability of strain OSES2. It is noteworthy that fortification of the growth medium with yeast extract and KNO 3  reported by Boopathy and Manning [46]. According to the authors, the surfactant can be utilized by microorganisms as an additional carbon source owing to the presence of long chain fatty acids, which include, oleic acid, myristic acid, palmitic acid, arachidic acid, linoleic acid and stearic acid while at the same time improving mass transfer and bioavailability of the pollutant to the degrading organisms. This inference was also reinforced by several researchers [47,48]. Furthermore, it has now been established that both the growth and degradation rates are usually not dependent on a single substrate in a multi-substrate system.

Conclusions
The unique finding of this study lies in the demonstration of 2,4-DNT metabolic phenotypes for the first time in Nigerian contaminated system and the fact that this function is found in quite an unusual bacterium. It is therefore, reasonable to hypothesize that diversity of microbial populations may harbor bacterial strains with novel metabolic capabilities in unexplored tropical soils in other regions and that the genes responsible for metabolism of these compounds may be more widely distributed across different microbial genera than earlier documented. Also, the data presented here readily suggest that CSL, molasses, acetate are potential stimulants for DNT degradation at least in strain OSES2.
Therefore, the utilization of this organism as candidate in addition with the amended carbon sources in bioremediation strategy should be exploited. This will help reduce the amount of waste intended for landfill, thus reducing landfill gas emissions. It will also provide a cheaper source of organic supplement for cleanup purposes with the ultimate aim of reducing the cost of bioremediation. Nevertheless, further research into the biochemical attributes and genotypic profile of this isolate is necessary to determine its possible activity in the natural attenuation or overall engineered bioremediation of NACs.

Microorganism and Culture Conditions
Top soils were sourced from a quarry site in Ibadan, Oyo State, Nigeria which have a history of contamination with explosive materials. The samples were kept in a clean plastic bag, conveyed to the laboratory immediately and stored in 4 o C prior to analysis.
Bacterial strain able to degrade 2,4-DNT were cultured on mineral salt medium (MSM) [49] with some modifications by continual enrichment technique. The medium contained the Nucleotide sequence were aligned using mega X software, phylogenetic tree was drawn using Neighbor-joining algorithm and the branch points were determined using boot strap method in Mega X software.

Nutrients and carbon sources amendment on 2,4-DNT degradation
Washed cells of strain OSES2 was seeded into a flask which contained modified MSM supplemented with 100 mg L -1 of 2,4-DNT. The Flasks were amended with 0.5% of different carbon sources (e.g., molasses, CSL, glucose, glycerol, maleic acid, acetate, sucrose and aspartate). Flask inoculated with heat inactivated cells served as control.
Incubation and sampling were handled as previously described. A parallel experiment in which inoculated flasks were amended with 1% (w/v) either of yeast extract, KNO 3 or 1.5% (v/v) of surfactant (Tween 80) were set up to monitor the roles of nitrogen sources on 2,4-DNT degradation.

Analytical methods
The amounts of NO   In the control flask, 2,4-DNT was not utilized and minimal abiotic loss occurred.
Data points are average of three replicate determinations, with error bars above and below the mean. Values were determined with reference to 2,4-DNT recovered from heat activated cells.