Extracellular polysaccharides of Rhodococcus rhodochrous S-2 stimulate the degradation of aromatic components in crude oil by indigenous marine bacteria

Abstract

Rhodococcus rhodochrous S-2 produces extracellular polysaccharides (S-2 EPS) containing D-glucose, D-galactose, D-mannose, D-glucuronic acid, and lipids, which is important to the tolerance of this strain to an aromatic fraction of (AF) Arabian light crude oil (N. Iwabuchi, N. Sunairi, H. Anzai, M. Nakajima, and S. Harayama, Appl. Environ. Microbiol. 66:5073-5077, 2000). In the present study, we examined the effects of S-2 EPS on the growth of indigenous marine bacteria on AF. Indigenous bacteria did not grow significantly in seawater containing AF even when nitrogen, phosphorus, and iron nutrients were supplemented. The addition of S-2 EPS to seawater containing nutrients and AF resulted in the emulsification of AF, promotion of the growth of indigenous bacteria, and enhancement of the degradation of AF by the bacteria. PCR-denaturing gradient gel electrophoresis analyses show that addition of S-2 EPS to the seawater containing nutrients and AF changed the composition of the bacterial populations in the seawater and that bacteria closely related to the genus Cycloclasticus became the major population. These results suggest that Cycloclasticus was responsible for the degradation of hydrocarbons in AF. The effects of 15 synthetic surfactants on the degradation of AF by indigenous marine bacteria were also examined, but enhancement of the degradation of AF was not significant. S-2 EPS was hence the most effective of the surfactants tested in promoting the biodegradation of AF and may thus be an attractive agent to use in the bioremediation of oil-contaminated marine environments.

FIG. 1.

Degradation of AF in the presence and absence of S-2 EPS. □, weight of oil extracted from cultures of NSW containing no EPS; ▪, weight of oil extracted from cultures of NSW containing S-2 EPS; ○, weight of oil extracted from filter-sterilized NSW medium; •, weight of oil extracted from filter-sterilized NSW medium containing S-2 EPS. Each value is the average of at least two independent experiments, and the standard deviation is indicated if it is larger than 10% of the mean value.

FIG. 2.

Effects of S-2 EPS on degradation of AF components. (A) Dibenzothiophenes (DBTs): squares, C₂-DBT (C_n indicates the carbon number in the side chain; C₀ indicates no alkyl substitution); diamonds, C₃-DBT; circles, C₄-DBT. (B) Fluorenes: squares, C₀-fluorene; circles, C₂-fluorene. (C) Phenanthrenes: squares, C₃-phenanthrene; diamonds, C₄-phenanthrene; circles, C₅-phenanthrene; triangles, C₆-phenanthrene; inverted triangles, C₇-phenanthrene. (D) Naphthalenes: squares, C₃-naphthalene; circles, C₄-naphthalene. Open symbols show the amounts in the absence of S-2 EPS; solid symbols show the amounts in the presence of S-2 EPS. Each value is the average of at least two independent experiments. The standard deviation is not indicated for clarity. It was less than 25% of the average value for all the data at day 15, one exception being that of C₇-phenanthrene, which was 60% of the average value.

FIG. 3.

Effects of S-2 EPS on the growth of marine bacteria on AF. (A) DAPI counts. (B) CFU on 1/5 MA. Symbols: ▴, in NSW; □, in NSW containing S-2 EPS; ○, in NSW containing AF; •, in NSW containing AF and S-2 EPS. The standard deviation is also indicated.

FIG. 4.

Effects of S-2 EPS on the bacterial community structure established in the NSW cultures. (A) DGGE profiles of PCR-amplified partial 16S rDNA fragments. (B) Drawing of the DGGE gel (panel A). The bands used for the sequence analyses are shown. SW, seawater sample at day 0. +EPS, NSW was supplemented with S-2 EPS; +AF, NSW was supplemented with AF; +(AF and EPS), NSW was supplemented with AF and S-2 EPS. Cultures were withdrawn on days 5, 10, and 15 for DNA extraction.

FIG. 5.

Bacterial community structure in the presence of a synthetic surfactant. The NSW either containing AF or not was supplemented with a surfactant and cultivated at 30°C. DNA was isolated from each culture after incubation for 15 days. Partial 16S rDNA fragments were PCR amplified from the DNA and separated by DGGE. The bands used for the sequence analyses are shown, and the sequences are listed in Table 2. Lane 1, CHAPS; lane 2, CHAPSO; lane 3, BIGCHAP; lane 4, deoxy-BIGCHAP; lane 5, n-octyl-β-d-glucoside; lane 6, n-heptyl-β-d-thioglucoside; lane 7, n-octyl-β-d-thioglucoside; lane 8, n-dodecyl-β-d-maltoside; lane 9, MEGA-8; lane 10, MEGA-9; lane 11, sucrose monocaprate; lane 12, sucrose monolaurate; lane 13, sodium cholate; lane 14, digitonin; lane 15, sodium dodecyl sulfate. In each lane, A shows the band pattern of cultures grown on the NSW supplemented with a surfactant, while B shows the band pattern of cultures grown on the NSW supplemented with a surfactant and AF (10 mg/ml). See Table 1 for the chemical names of the surfactants.