Detection of Sphingobium sp. strain P2 in wastewater treatment system of petrol station by qPCR

Abstract

Conventional techniques are inefficient for car wash wastewater treatment because it cannot remove oil-in-water emulsion formed by admixture of lubricant oil with emulsifier and wash water. Therefore, bioremediation is another choice for treating this problem. In the present work, Sphingobium sp. strain P2 was used to degrade lubricant oil emulsion. This study aimed to develop a quantitative real-time PCR method to monitor survival of this strain during oil biodegradation. The PCR primer set specific for ferredoxin gene involved in biodegradation of aromatic compounds in strain P2 was designed. Lubricating oil degradability test by strain P2 was done in both flask-laboratory and reactor scales. These experiments were examined for amount of oil recovery by TLC-FID, COD concentration by COD reagents, and survival of strain P2 by real-time PCR. First, the survival of strain P2 during lubricating oil degradation was determined in air supply and without air supply condition. Strain P2 in air supply was able to grow in lubricant oil emulsion, showing high lubricant oil elimination (75.16% of the initial concentration of lubricant oil of 200 ppm) in 5 days. In contrast, those in the without air supply condition were tend to decrease and could only eliminate 43.38%. While, chitosan-immobilized strain P2 could degrade lubricating oil to 59.84% and 36.76% in air supply and without air supply conditions. However, chitosan-immobilized strain P2 still survived at 2.13×10⁶ to 2.15×10⁷ adhA3 gene copies number/0.2 g chitosan. These indicated that the immobilization could improve bacterial survival better than in the form of free cells. Moreover, chitosan-immobilized strain P2 was applied to treat real wastewater in 3 l and 350 l airlift continuous bioreactor systems (2.5 g/l) within 30 and 60 days, respectively. Hydraulic retention time was 2 hours. The result showed that both of reactors had high efficiency to remove oil (91.50 and 68.18% average oil removal, respectively). Furthermore, COD concentration was reduced to 26.76 and 32.96% COD removal, respectively. The presence of strain P2 was 1.92×10⁷-3.87×10⁸ adhA3 gene copies number/7.5 g chitosan and 2.63×10⁶-3.57×10⁷ adhA3 gene copies number/3 l in 3 l aitlift bioreactor, 9.08×10⁸-1.18×10¹º adhA3 gene copies number/875 g chitosan and 2.87×10⁷-1.75×10⁹ adhA3 gene copies number/350 l in 350 l reactor. The ratio of number adhA3 gene copies in chitosan was higher than in wastewater for both systems. In conclusion, chitosan-immobilized strain P2 could degrade lubricant oil emulsion, and it could be maintained in all conditions performed in this study. The monitoring method developed here provided more specific assessment survival of strain P2 and can be further used to develop a biological treatment system for car wash wastewater in petrol station.