Quantitative detection of pentachlorophenol-degrading Sphingomonas sp. UG30 in soil by a most-probable-number/polymerase chain reaction protocol

Abstract

Traditionally, biodegradation of pentachlorophenol (PCP) in soil is studied by disappearance of the parent compound and its intermediates. Because of the lack of methods to enumerate PCP-degrading bacteria in environmental samples, little is known about the dynamics of these organisms in soil. In this study, the efficiency of a modified most-probable-number/polymerase chain reaction (MPN/PCR) protocol was compared to the traditional MPN/[ 14C]PCP mineralization assay to quantify the density of PCP-degrading Sphingomonas sp. UG30 cells inoculated in an agricultural soil. A 753-bp tetrachlorohydroquinone reductive dehalogenase gene ( pcpC) fragment of UG30 was targeted for the MPN/PCR amplification. The MPN/PCR protocol had a detection limit of 3 CFU/g dry soil. A good correlation was established between the MPN/PCR estimations and initial inoculum densities ranging from 30 to 2×10 9 CFU/g of soil. However, the MPN/[ 14C]PCP mineralization protocol underestimated the inoculum density by 70 to 740-fold. Survival of UG30 in soil was monitored by the MPN/PCR assay. Cell density of the UG30 inoculum decreased from 1.8×10 8 to 1.9×10 5 cells/g of soil in the first 20 days of incubation and stabilized at 1.9×10 4 cells/g of soil after 50 days. When the soil was autoclaved prior to inoculation, UG30 cell density remained at 6.7×10 7 cells/g of soil after 50 days of incubation.