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In Mexico, waste motor oil (WMO), a product of the petrochemical industry, is a mixture of hydrocarbons (HYCO): linear aliphatic, branched and polycyclic aromatics [1]. WMO is generated during the lubrication cycle of automobile engines, which when not properly disposed of by garages that change the oil [2], throw it into the drainage system and prevent its treatment for reuse [3], causing environmental pollution, in part because some components of WMO are toxic to all life forms [4]. By impacting surface water and aquifers, WMO violates the Ley General de Equilibrio Ecológico y Protección Ambiental (LGEEPA), which classifies it as a hazardous waste; specifically in domestic water according to regulations: NOM-001-SEMARNAT-1996, NOM-002- ECOL-1997 and NOM-003-ECOL-1997. The spillage of WMO into the drainage system is an environmental problem because the treatment plants do not solve it, and it is important because of the growing scarcity of water for and industrial use and garden irrigation, since by exceeding concentrations of: 25 ppm for discharges of wastewater and national assets, 75 ppm for wastewater to urban sewage systems, and 15 ppm for treated wastewater for public reuse, they contaminate it and it is impossible to reuse it. An alternative solution to reverse the environmental damage is biostimulation (BIS) of water polluted by WMO. The first factor required in the BIS of water polluted by WMO is a detergent that emulsifies the HYCO, followed by BIS with a mineral solution (M)S, to enrich it with essential minerals of: N (nitrogen), P (phosphorus), K (potassium), so that native microorganisms mineralize the WMO [5]. In general, BIS from water impacted by HYCO with MS is useful for eliminating petroleum derivatives, but it is relatively little known [6] and limited, in aquatic environments, simultaneously with BIS with O₂ (oxygen) for maximum oxidation of WMO [5], since WMO mineralization is better and greater in aerobic conditions [7]. In nature, specifically in water, the mineralization of aromatic HYCO is complicated and slow [8,9]. Therefore, the objective of this work was the integral BIS of domestic water to eliminate WMO at a value lower than those established by NOM-001, NOM-002 and NOM-003.

Integral biostimulation of domestic water impacted by WMO

For this test, 500 ml Bartha flasks were used, with 100 ml of WMO (obtained from an oil change workshop in Morelia, Mich, Mexico) diluted 1:100, equivalent to 10,000 ppm biostimulated with 0.01% Tween 20 and a mineral solution (MS) (g/L): K₂HPO₄: 5; KH₂PO₄: 4; MgSO₄: 3; NH₄NO₃: 10; CaCO₃: 1; KCl: 2; ZnSO₄: 0.5; CuSO₄: 0.5; FeSO₄: 0.2, and EDTA 8. The flasks were shaken at 100 rpm and incubated at 30 ± 2ºC/3 weeks, the experiment was performed in triplicate. As relative control was used: a flask with domestic water impacted by diluted WMO without biostimulant; as absolute control a flask with distilled water, biostimulated with Tween 20 and the MS; as sterile control a flask with sterile water (121°C/15min), with Tween 20, impacted by WMO biostimulated with the MS. As treatment 1 a flask with domestic water impacted by WMO, biostimulated with Tween 20 and MS; as treatment 2 a flask with domestic water impacted by WMO, biostimulated with Tween 20, MS and H₂O₂, as shown in Table 1. To demonstrate the mineralization of WMO in the biostimulated water, 10 ml of 0.1N KOH was added to each of the arms of the flask to capture the CO₂, every 24 h the 0.1N KOH was taken from each flask, the production of CO₂ being quantified by titration with 0.1N HCl [10].

Table 1: Experimental design for the biostimulation of domestic water impacted by waste motor oil, with Tween 20, enriched with a mineral solution and H₂O₂ in agitation.

Quantification of WMO aliphatic hydrocarbons in biostimulated domestic water

In wastewater impacted by WMO biostimulated with Tween 20, MS, with H₂O₂ was observed by GC-MS analysis, as well as by the Soxhlet method. A Hewlett-Packard (Waldbroon, Germany) 6890 series gas chromatograph coupled with a 5792 A series mass spectrophotometer was used, with a 30 m long HP-5MS capillary column with an internal diameter of 0.25 mm and a film thickness of 0.25 mm, the injection was split mode, the carrier gas was Helium, with a flow rate of 37 cm/ sec^-1, the oven temperature was 40º C / 8min, with an increase to 180º C / 6º C min-1 and the injector temperature was 250ºC [11].

Figure 1 shows the production of CO₂ during BIS of domestic water with Tween 20, MS and H₂O₂ in agitation, where the mineralization of ARA was observed, measured indirectly by the increase in CO₂ concentration, with a maximum of 3.5 mmoles/mL at 24 h. The above result supports that, in water, the limiting factor of WMO oxidation was the nutritional imbalance, which, in balance, allows the various aerobic heterotrophic populations to eliminate in a relatively short time, most of the aliphatic WMO, which according to the analysis by Soxhlet was 10 ppm, a value lower than the maximum accepted by NOM-001, NOM-002 and NOM-003.

Figure 1: CO2 Production in domestic water contaminated by waste motor oil biostimulated with Tween 20, mineral solution, H₂O₂ at 100 rpm.

This was confirmed with the ARA impacted water experiment in which, without BIS with MS, where heterotrophic water consortia were unable to mineralize the WMO HYCO, consequently, CO₂ production was minimal. While it is demonstrated that the disappearance of WMO is due to the activity of aerobic heterotrophic microorganisms, since the sterilization of domestic water eliminated the microorganisms and thus totally suppressed the production of CO₂, despite the BIS with MS; according to statistical analysis, the values of CO₂ in the water impacted with WMO were statistically different, since the biostimulation of water with MS showed that the nutritional factor is limiting or essential in the bioremediation of water with WMO. In contrast to the test where the domestic water impacted by WMO, only biostimulated with Tween 20, without the MS, proved that the detergent, was responsible only for the solubility of WMO, where without the BIS with the MS, there was no increase in the production of CO₂, in this case only evidence of the mineralization of WMO. A critical aspect in the analysis of these results is the lack of information in the literature regarding water contaminated by WMO. Therefore, the data shown in this research are among the few that support the bioremediation of domestic water as a feasible strategy for its reuse. One related research is on bioremediation impacted by WMO that reported a 92.5% disposal rate [12].

WMO: Waste motor oil, Dil. 1:100, biostimulated with MS: mineral solution, Tween 20 0.01% Temperature: 30 ± 2ºC. Shaking: 100 rpm. Different letters are statistically significant, equal letters with no statistical difference (Tukey: P<0.05%).

Figure 2 shows that water BIS with MS, and with H₂O₂, which by increasing the concentration of available O₂ for a longer time and reducing rapid volatilization (Kunucku, 2017), induced increased WMO mineralization and simultaneously increased CO₂ production, to a value of 5. 88 mMoles/ml at 24 h; compared to domestic water with WMO biostimulated with MS; but without H₂O₂, there aerobic heterotrophic microorganisms without sufficient O₂ generated less CO₂, with 3.5 mMoles/mL; (Figure 3), indicating that, being aerobic mineralization, H₂O₂ is a limiting factor for the efficient removal of WMO.

Figure 2: CO2 production during biostimulation of domestic water contaminated by waste motor oil with Tween 20, mineral solution, and intermittent H₂O₂ agitation at 100 rpm.

Figure 3: CO2 production during biostimulation of domestic water contaminated by waste motor oil with Tween 20, mineral solution and regular application of H₂O₂.

WMO: waste motor oil, MS: mineral solution ^*Dil. 1:100 (10,000 ppm) Temperature: 30 ± 2ºC. Shaking: 100 rpm. Different letters are statistically significant, equal letters with no statistical difference (Tukey: P<0.05%).

WMO: Waste Motor Oil, MS: Mineral Solution ^*Dil. 1:100 (10,000 ppm) Temperature: 30 ± 2ºC. Shaking: 100 rpm. Different letters are statistically significant, equal letters with no statistical difference (Tukey: P<0.05%).

Figure 4 shows the chromatographic profile of domestic water impacted by WMO biostimulated with Tween 20, MS, H₂O₂ and 100 rpm agitation, where it was observed that the dominant aliphatic HYCO of WMO were of 11-20 carbon chains, as well as the domestic water used as absolute control, in which, the concentration of the aliphatic HYCO of WMO was similar (data not shown).

Figure 4: Chromatogram of domestic water contaminated with unsterilized waste motor oil before biostimulation with Tween 20, MS and H₂O₂.

Figure 5 shows the BIS of domestic water impacted by the WMO with Tween 20, MS and H₂O₂ where 100% of aliphatic 11,12 and 20 carbon chain HYCO were removed, contrary to what is reported in the literature which indicates that the lower carbon chain HYCO are the first to oxidize, since short chain alkanes mineralize faster than long chain ones [13]. This supports that in domestic water impacted by WMO, the wide diversity of aerobic heterotrophic microorganisms eliminated 96% of the WMO aliphatics constituted by 13-19 carbons, according to the chromatographic analysis after the biostimulation of domestic water with Tween 20, MS, H₂O₂, were responsible for the mineralization of the different WMO aliphatics.

Figure 5: Chromatogram of domestic water contaminated by waste motor oil after biostimulation with Tween 20, MS and H₂O₂ in agitation at 100 rpm at 30°C.

According to the Soxhlet analysis, the final concentration of the HYCO of the WMO was close to 10 ppm, below the maximum permissible limits of NOM-001, NOM-002 and NOM-003, which demonstrated the recovery of this water to be reused for garden and industrial irrigation. A critical aspect for the discussion of these results was the lack of specific information in the literature associated with the bioremediation of domestic water contaminated by WMO. In general, to solve a problem of this type, bioaugmentation with previously selected collection microorganisms is used because they mineralize the WMO aliphatics, exclusively in the culture medium, relatively few in an environment close to what happens in the wastewater [14].

Figure 6 shows the chromatogram of the biostimulation of domestic water contaminated by WMO with Tween 20, MS and H₂O₂, in 100 rpm agitation at 30°C on the mineralization of aliphatics with carbon chains between (C11-C20). This indicates that the aerobic heterotrophic microbial consortiums existing in domestic water, eliminated them in 96% in the 21 days of the experiment, while it was detected the elimination in 100% of the short chain aliphatic HYCO: undecane, dodecane, contrary to what was detected for the aliphatic WMO of C13-C19 in a residual concentration of 4% at the end of the experiment [15-19].

Figure 6: Percentage of remaining aliphatic from household water biostimulation impacted by waste motor oil with Tween 20, mineral solution, H₂O₂.

These results indicate something similar to what was reported by Hassanshahian et al., in 2014, which proved that seawater bioaugmentation impacted by 1000 ppm of oil with Alcanivorax borkumensis 95% mineralization. In the same way as Gopinath et al. demonstrated that bioaugmentation of water contaminated by HYCO with Bacillus sp and achieved a reduction to 92.5%.

WMO: waste motor oil, MS: mineral solution

The results of this research support that it is possible to apply the strategy of recovery of black water impacted by WMO is the integral BIS through actions of restitution of the chemical-physical environment to induce the microorganisms to oxidize the WMO that eliminate their main aliphatic and aromatic HYCO that allow the industrial and/or reactive reuse.

We would like to thank Project 2.7 (2020) of CIC-UMSNH, BIONUTRA, SA de CV Maravatio, Mich, Mexico for the support.