Isolation and characterization of Novosphingobium sp. strain MT1, a dominant polychlorophenol-degrading strain in a groundwater bioremediation system

Abstract

A high-rate fluidized-bed bioreactor has been treating polychlorophenol-contaminated groundwater in southern Finland at 5 to 8 degrees C for over 6 years. We examined the microbial diversity of the bioreactor using three 16S ribosomal DNA (rDNA)-based methods: denaturing gradient gel electrophoresis, length heterogeneity-PCR analysis, and restriction fragment length polymorphism analysis. The molecular study revealed that the process was dependent on a stable bacterial community with low species diversity. The dominant organism, Novosphingobium sp. strain MT1, was isolated and characterized. Novosphingobium sp. strain MT1 degraded the main contaminants of the groundwater, 2,4,6-trichlorophenol, 2,3,4,6-tetrachlorophenol, and pentachlorophenol, at 8 degrees C. The strain carried a homolog of the pcpB gene, coding for the pentachlorophenol-4-monooxygenase in Sphingobium chlorophenolicum. Spontaneous deletion of the pcpB gene homolog resulted in the loss of degradation ability. Phenotypic dimorphism (planktonic and sessile phenotypes), low growth rate (0.14 to 0.15 h(-1)), and low-copy-number 16S rDNA genes (single copy) were characteristic of strain MT1 and other MT1-like organisms isolated from the bioreactor.

FIG. 1.

Bioreactor function and time points of sample collection (arrows). Feed (▵) is the total concentration of 2,4,6-TCP, 2,3,4,6-TeCP, and PCP in the influent water. Chlorophenol removal (•) is shown as the removal percentage. Bioreactor sand samples (s) and influent groundwater samples (g) are marked. Dates are given below the graph as day.month.year. (Data reproduced with permission from Häme Regional Environment Centre, Lahti, Finland.)

FIG. 2.

Proportions of the main fragment size classes in LH-PCR analysis of the bioreactor samples with primer pair 27F-PRUN518. The bars represent relative band densities in each size class in samples 1s (solid), 2, 3, 4s (solid), 6, 7, 9, 10, 11, 12, 13, 14, 15, and 16s (solid) from left to right.

FIG. 3.

16S RFLP patterns of MspI digestions from bioreactor samples 1s to 14 compared to the patterns obtained from strain MT1 and the MT1 Chl⁻ mutant. The molecular size marker bands (lane M) are indicated on the right.

FIG. 4.

DGGE separation patterns of amplified DNA fragments coding for eubacterial 16S rDNA. Lane M, reference fragments (the G + C contents of these fragments from top to bottom were 51.7, 53.7, 54.2, 55.9, and 57.7%); lane GW, influent groundwater sample 5 g; lane BW, bioreactor water sample 3; lane BS, carrier sand sample 4s. Band D from lane GW and bands A, B, and C from lane BW were reamplified and sequenced (Table 1).

FIG. 5.

Degradation of the mixture of 2,4,6-TCP, 2,3,4,6-TeCP, and PCP by Novosphingobium sp. strain MT1 at 8°C. Chlorophenols (CP) were spiked four times (indicated with arrows). The error bars indicate standard deviations.

FIG. 6.

Southern blot analysis of genomic DNAs hybridized with a pcpB gene-specific probe. Each lane was loaded with 1 μg of DNA digested with EcoRI: lane 1, S. chlorophenolicum ATCC 39723; lane 2, strain MT1; lane 3, Chl⁻ mutant of strain MT1; lane 4, strain MT101; lane 5, strain MT103; lane 6, strain MT104.