Growth of a Dehalococcoides-like microorganism on vinyl chloride and cis-dichloroethene as electron acceptors as determined by competitive PCR

Abstract

A competitive PCR (cPCR) assay targeting 16S ribosomal DNA was developed to enumerate growth of a Dehalococcoides-like microorganism, bacterium VS, from a mixed culture catalyzing the reductive dehalogenation of cis-1,2-dichloroethene (cDCE) and vinyl chloride (VC), with hydrogen being used as an electron donor. The growth of bacterium VS was found to be coupled to the dehalogenation of VC and cDCE, suggesting unique metabolic capabilities. The average growth yield was (5.2 +/- 1.5) x 10(8) copies of the 16S rRNA gene/ micromol of Cl(-) (number of samples, 10), with VC being used as the electron acceptor and hydrogen as the electron donor. The maximum VC utilization rate (q) was determined to be 7.8 x 10(-10) micromol of Cl(-) (copy(-1) day(-1)), indicating a maximum growth rate of 0.4 day(-1). These average growth yield and q values agree well with values found previously for dechlorinating cultures. Decay coefficients were determined with growth (0.05 day(-1)) and no-growth (0.09 day(-1)) conditions. An important limitation of this cPCR assay was its inability to discriminate between active and inactive cells. This is an essential consideration for kinetic studies.

FIG. 1.

Example of the cPCR assay. (A) A constant amount of extracted DNA was coamplified with the serially diluted competitor sequences (2.9 × 10⁵, 1.4 × 10⁵, 0.7 × 10⁵, and 0.4 × 10⁵ copies/μl) and subjected to gel electrophoresis. (B) The ratios of the intensities of the target to the competitor (diamonds) are plotted against the concentrations of the competitor on a log scale. The target copy number equals the competitor number when the log ratio equals zero.

FIG. 2.

Quantification of 16S rDNA copies. Numbers of determined copies were counted via cPCR and compared to actual copy numbers (previously quantified with a fluorometer—see Materials and Methods). The lighter line represents a 1:1 ratio of actual numbers of copies to determined numbers of copies.

FIG. 3.

VC (filled squares) removal and ethene (open squares) formation (A) with the resulting increase in copy number (filled circles) (B). Additional VC (10.5 μmol) was added on day 15. Points are measured values, and lines are those predicted by a nonlinear least-squares fit to the model (equations 1 and 2) to determine decay rate and q̂. An increase in the number of copies is correlated with the formation of ethene, resulting in a yield coefficient of 5.1 × 10⁸ copies/μmol of Cl⁻ (r² = 0.93) (C). The duplicate culture behaved similarly, with a yield coefficient of 5.9 × 10⁸ copies/μmol of Cl⁻ (r² = 0.83) (data not shown).

FIG. 4.

Application of model (lines) to cultures containing a range of initial inocula. The points are the measured values with 0.5 ml (triangles) (A), 1 ml (diamonds) (B), 3 ml (squares) (C), and 9 ml (circles) (D) of initial inoculum (8 × 10⁶ copies/ml). Additional VC (5.6 μmol) was added on day 40 to the 0.5-ml (A), 1-ml (B), and 9-ml (D) samples.

FIG. 5.

Ethene formation following VC addition to duplicate cultures lacking an electron acceptor for 0, 8, 14, and 21 days. Points (diamonds) are measured values, and lines are those predicted by a nonlinear least-squares fit to the model (equations 1 and 2) to determine decay rate.