Journal of Methods Microbiological Journal of Microbiological Methods 31 (1997) 59–66 Quantitative detection of pentachlorophenol-degrading Sphingomonas sp. UG30 in soil by a most-probable-number / polymerase chain reaction protocol * * Kam Tin Leung, Andrew Watt, Hung Lee , Jack T. Trevors Department of Environmental Biology, University of Guelph, Guelph, Ontario N1G 2 W1, Canada Received 1 August 1997; accepted 9 September 1997 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 / 14 polymerase chain reaction (MPN / PCR) protocol was compared to the traditional MPN / [ C]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 9 between the MPN / PCR estimations and initial inoculum densities ranging from 30 to 2310 CFU / g of soil. However, the 14 MPN/[ C]PCP mineralization protocol underestimated the inoculum density by 70 to 740-fold. Survival of UG30 in soil 8 5 was monitored by the MPN / PCR assay. Cell density of the UG30 inoculum decreased from 1.8310 to 1.9310 cells/g of 4 soil in the first 20 days of incubation and stabilized at 1.9310 cells / g of soil after 50 days. When the soil was autoclaved 7 prior to inoculation, UG30 cell density remained at 6.7310 cells / g of soil after 50 days of incubation.  1997 Elsevier Science B.V. Keywords: Enumeration; MPN; PCR; Pentachlorophenol; Soil; Sphingomonas sp. 1. Introduction sp. [8] are capable of degrading and mineralizing PCP. Recently, evidence shows that Arthrobacter sp. Pentachlorophenol (PCP) is a wide-spectrum ATCC 33790, Flavobacterium sp. ATCC 39723 and biocide used world-wide as a wood preservative. Pseudomonas spp. SR3, and UG30 should be re- Despite its toxicity, some microorganisms, such as classified under the genus Sphingomonas based on Arthrobacter sp. ATCC 33790 [1], Flavobacterium their biochemical characteristics, cellular fatty acid sp. ATCC 39723 [2], Pseudomonas spp. SR3, UG25 profiles, the presence of sphingolipids and / or 16S and UG30 [3–5], Sphingomonas sp. RA2 [6], rDNA sequence information [5,9,10]. Mycobacterium sp. PCP-1 [7], and Phanerochaete Although PCP-degrading microbial inoculum can be used to degrade PCP in soil and groundwater, the * success of this approach depends on survival of the Corresponding author. Tel.: 11 519 8244120; fax: 11 519 8370442; e-mail: hlee@uoguelph.ca or jtrevors@uoguelph.ca inoculum in the environment. Several methods have 0167-7012 / 97 / $17.00  1997 Elsevier Science B.V. All rights reserved. PII S0167-7012(97)00082-1