Isolation and molecular identification of landfill bacteria capable of growing on di-(2-ethylhexyl) phthalate and deteriorating PVC materials

Abstract

Waste materials containing Di-(2-ethylhexyl) phthalate (DEHP), a suspected endocrine disruptor and reasonably anticipated human carcinogen, are typically disposed of in landfills. Despite this, very few studies had been conducted to isolate and identify DEHP-degrading bacteria in landfill leachate. Therefore, this study was conducted to isolate and characterize bacteria in landfill leachate growing on DEHP as the sole carbon source and deteriorating PVC materials. Four strains LHM1, LHM2, LHM3 and LHM4, not previously reported as DEHP-degraders, were identified via 16S rRNA gene sequence. Gram-positive strains LHM1 and LHM2 had a greater than 97% similarity with Chryseomicrobium imtechense MW 10(T) and Lysinibacillus fusiformis NBRC 15717(T), respectively. Gram-negative strains LHM3 and LHM4 were related to Acinetobacter calcoaceticus DSM 30006(T) (90.7% similarity) and Stenotrophomonas pavanii ICB 89(T) (96.0% similarity), respectively. Phylogenetic analysis also corroborated these similarities of strains LHM1 and LHM2 to the corresponding bacteria species. Strains LHM2 and LHM4 grew faster than strains LHM1 and LHM3 in the enrichment where DEHP was the sole carbon source. When augmented to the reactors with PVC shower curtains containing DEHP, strains LHM1 and LHM2 developed greater optical densities in the solution phase and thicker biofilm on the surfaces of the shower curtains.

Fig. 1

Molecular structure of di-(2-ethylhexyl) phthalate (DEHP), a suspected endocrine disruptor and reasonably anticipated human carcinogen.

Fig. 2

Growth trend of total heterotrophic bacteria (THB) in fed batch reactors (FBRs) with DEHP inoculation (treatment FBR) and without it (control FBR). Data are shown the average values with standard deviations (n=3).

Fig. 3

Growth of Gram-positive strains LHM1 and LHM2 and Gram-negative strains LHM3 and LHM4 during the enrichment in a mineral salt medium with DEHP as the sole carbon source.

Fig. 4

Agarose gel electrophoresis of the extracted DNA (top) and polymerase chain reaction (PCR) amplification product (bottom). S1: strain LHM1, S2 and S3: strain LHM2, S4: strain LHM3, and S5: strain LHM4.

Fig. 5

Neighbor-joining distance tree using the 16S rRNA gene sequences of strains LHM1 and LHM2 and closely related species. Bootstrap values higher than 35% are shown. Accession numbers are shown in the parenthesis. Pseudomonas aeruginosa LMG 1242T (Z76651) was used as the outgroup. The scale bar indicates 0.02 substitutions per nucleotide position.

Fig. 6

Growths of strain LHM1 and LHM2 on PVC shower curtain (type 3). Optical densities at 610 nm are measured in the solution phase during the 34-day experiment and the results are shown in (a). Images of scanning electron microscope (x3,000) taken at the end of the 34-day experiment are shown: (b) virgin shower curtain 3, (c) shower curtain 3 from the control reactor, (d) shower curtain 3 from the reactor bioaugmented with strain LHM1, and (e) shower curtain 3 from the reactor bioaugmented with strain LHM2.