Nitroaromatics-contaminated sites are known reservoir for many microorganisms with specific metabolic capabilities. It is therefore, not surprising that such sites are actively explored for isolation of competent degraders since prior exposure may enhance the catabolic potentials of such bacteria strains . Using explosive contaminated soil samples from a quarry site in Ibadan, Nigeria, an enteric bacterium identified as Proteus sp. strain OSES2 was successfully isolated amongst other species following the technique described. Extensive study on the degradation of NACs and particularly, 2,4-DNT over the past two decades has culminated in the identification of a diverse collection of bacterial species, e.g., Alcaligenes denitrificans JS867 and JS871, A. xylosoxidans, and Burkholderia cepacia JS872 ; Pseudomonas fluorescens sp. ; P. mandelii HC88  that can metabolize the compounds and utilize them as a sole carbon and energy source. The metabolic capability of an enteric organism to utilize 2, 4-DNT as a principal carbon and energy source could be regarded as a novel trend as there has been no previous documentation of such metabolic abilities by enteric bacterial strains. Enteric organisms are predominantly intestinal in origin and are not known for degradation of relatively recalcitrant xenobiotic organic compounds, although there are few exceptions. For instance, analysis of the Escherichia coli whole genome has shown that the bacterium harbor unknown genes that are suspected to be involved in the aerobic degradation and transformation of aromatic compounds . Accordingly, E. coli cells was demonstrated to reduce two of the TNT nitro groups under aerobic and anaerobic conditions, with the formation of 2,4-diamino–6-nitrotoluene . Likewise a strain of E. coli has been documented to cause the dechlorination of the 1,1,1-trichlor–2,2-bis(p-chlorophenyl)ethane (DDT) to 1,1,-dichloro–2,2-bis(p-chlorophenyl)ethane (DDD)  just as the E. coli mediated conversion of γ-hexachlorocyclohexane (lindane) to γ-pentachlorocyclohexene under aerobic condition was documented for the first time . Adebusoye et al.  reported the isolation of an Enterobacter sp. SA2 from a PCB-contaminated soil with an attributed capability to metabolize a spectrum of PCB and chlorobenzene congeners. Recently, Proteus vulgaris strain CPY1 was unambiguously demonstrated to possess competent pyrene metabolic functions . In contrast to strain OSES2, CPY1 was isolated from an animal waste, a clearly unusual place for sourcing of xenobiotic degraders. Since OSES2 was isolated from an explosive contaminated soil due to human activities, the presence of this recalcitrant compounds may have resulted in adaptability of the organism over time and development of its capabilities for utilization of 2,4-DNT. This suggests that some contaminated soils in sub-Saharan African environment may contain exotic bacterial strains whose metabolic capabilities and potentials are previously unknown and are yet to be discovered.
Strain OSES2 exhibited an unusual ability to utilize 2,4-DNT as a sole source of carbon, energy and nitrogen since the culture fluid contained no additional fortification with nitrogen or carbon sources. The percentage utilization of this substrate consumed by strain OSES2 was within the range previously reported for some 2,4-DNT degraders, and perhaps, superior to other phenotypes . Mariela et al.  reported that 2,4-DNT was completely utilized in 90 h by Pseudomonas fluorescens when supplied as the only carbon, energy and nitrogen source. More recently, Rhodococcus pyridinovorans NT2 recovered from a pesticide polluted soil was shown to degrade both 2,4- and 2,6-DNT within 48 h of incubation . It is noteworthy that, while the authors reported 70% degradation of 2,4-DNT in extended cultivation period of 10 days, strain OSES2 utilized over 90% of the same compound in 72 h. By implication, the catabolic repertoire in OSES2 may be unique and superior to that of K1 reinforcing its relevance and desirability as candidates for cleanup of contaminated systems. Aerobic metabolism of NACs is often accompanied by the release NO3-, NO2- and sometimes NH4+ [33, 34]. However, a thorough evaluation of the data obtained in this study showed a non-stoichiometric recovery of these metabolites. The reason for this observation is not farfetched. Since strain OSES2 depended on the DNT substrate both as sources of nitrogen and carbon, the difference in nitrogen mass balance could have been incorporated into the cellular architecture. In this case, the recovery of NO3- and NO2- in the culture fluid and the observable increase in biomass, undoubtedly indicate the metabolism of 2,4-DNT as previously documented by other workers [8, 17]. A plausible explanation for the extreme low level of NO2-could be due to its rapid conversion to NO3- .
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 [37–39]. 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 . 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 (43–45].
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 KNO3 awesomely supported proliferation of cells which, unfortunately, was not translated to 2,4-DNT consumption. Similar trend was previously documented by Cho et al.  and Shen et al.  while investigating conditions necessary for optimization of TNT degradation but not 2,4-DNT. By implication, amendment by exogenous nitrogen sources in systems inoculated with OSES2 may not be necessary since the organism could also utilize the substrate as a nitrogen source. Under this condition, the presence of additional nitrogen sources may present some metabolic bottlenecks to the organism thus impairing DNT degradation.
Considering the hydrophobic nature of 2,4-DNT, the addition of Tween 80 in combination with KNO3 increased the rate of 2,4-DNT degradation, biomass production and decreased the degradation time by 12 h in comparison with when the surfactant was used alone. The effectiveness of Tween 80 in enhancing pollutant degradation by microorganisms was reported by Boopathy and Manning . 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.