Noriyuki Iwabuchi

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.

Relationships between Colony Morphotypes and Oil Tolerance in Rhodococcus rhodochrous

ABSTRACT A mucoidal strain of Rhodococcus rhodochrous was resistant to 10% (vol/vol) n-hexadecane, while its rough derivatives were sensitive. When the extracellular polysaccharide (EPS) produced by the mucoidal strain was added to cultures of the rough strains, the rough strains gained resistance ton-hexadecane. Thus, EPS confer tolerance ton-hexadecane in members of the genusRhodococcus.

Cell-surface hydrophobicity and scum formation of Rhodococcus rhodochrous strains with different colonial morphologies.

Rhodococcus rhodochrous has been reported to be one of the micro‐organisms responsible for the formation of scum which is thick and viscous biological foam in activated sludge plants. The hydrophobicity of mycolic acids present on the cell surface and the long‐branched shape of the hyphae have been thought to contribute to the scum formation. Cell surface hydrophobicity and scum formation of four R. rhodochrous strains with different colony morphologies were determined, and the results showed that the two rough strains had strong cell surface hydrophobicity and produced scum, whereas the weakly hydrophobic smooth strain and the hydrophilic mucoidal strain did not. All four strains displayed long, branched hyphae, and their electrophoretic mobilities were similar, between pH 4 and 9. These data suggest that changes in the cell surface hydrophobicity of the R. rhodochrous result in changes in the culture characteristics and the formation of scum.

Relationships among colony morphotypes, cell-surface properties and bacterial adhesion to substrata in Rhodococcus

To elucidate the function of extracellular polysaccharide (EPS) produced by bacteria with hydrophobic surfaces we investigated relationships among colony morphotypes, cell-surface properties and bacterial adhesion to substrata in Rhodococcus. Rough strains adhered well to various materials and formed cell aggregates, while their mucoidal derivatives did so poorly. Cell surfaces of the rough strains were more hydrophobic than those of the corresponding mucoidal strains. Production of the EPS by the rough strains was far less than that by the corresponding mucoidal strains. Adhesion of the rough strains to quartz, glass or hydrocarbon was inhibited by the addition of EPS. Thus, rhodococcal EPS plays an important role in determination of the cell-surface hydrophobicity by acting as a hydrophilin, and its addition to the rough strains resulted in inhibition of their adhesion to the substrata.

Inhibitory effects of odor substances, geosmin and 2-methylisoborneol, on early development of sea urchins

Geosmin (1α,10β-dimethyl-9α-decanol) and 2-methylisoborneol ((1-R-exo)-1,2,7,7-tetramethyl-bicyclo-(2,2,1)-heptan-2-ol) (MIB) are volatile terpene derivatives, and have received a great deal of attention because they can cause musty/muddy off-flavor in water and food resources. By the Ames test, these metabolites showed no mutagenicity but antimicrobial activity toward tester strains. While these compounds are produced by various organisms living in aquatic environments, there are few reports of their effects on aquatic organisms. The effects of geosmin and MIB on sea urchin development were examined. The estimated IC50 (50% inhibitory concentration) values for the formation of the fertilization membrane were 16.67 mg geosmin l−1 and 68.77 mg MIB l−1; those for the cell cleavage were 16.58 mg geosmin l−1 and 66.86 mg MIB l−1, suggesting that the toxicity of geosmin and MIB toward sea urchins are comparable to their toxicity toward Salmonella tester strains in the Ames test. These values are far greater than concentrations of these substances observed in aquatic environments with severe muddy off-flavor problems.

Bacillus trypoxylicola sp. nov., xylanase-producing alkaliphilic bacteria isolated from the guts of Japanese horned beetle larvae (Trypoxylus dichotomus septentrionalis)

Three xylanase-producing alkaliphilic strains, SU1(T), 36AC4 and 36AC6, were isolated from the guts of larvae of the Japanese horned beetle (Trypoxylus dichotomus septentrionalis). The isolates stained Gram-positive and were aerobic, spore-forming, non-motile and rod-shaped and grew optimally at 30 degrees C and pH 9. They contained MK-7 as the major isoprenoid quinone and iso-C(15 : 0), anteiso-C(15 : 0), anteiso-C(17 : 0) and iso-C(17 : 0) as the major fatty acids. The DNA G+C contents of the strains were 37.4-37.7 mol%. On the basis of 16S rRNA gene sequence similarity, these strains were shown to belong to the genus Bacillus. Although their 16S rRNA gene sequence similarity to the type strains of the alkaliphilic species Bacillus pseudalcaliphilus and B. alcalophilus was 97 %, the novel isolates formed a distinct group in the phylogenetic trees and DNA-DNA relatedness values to the type strains of these species were less than 30 %. Results of physiological and biochemical tests, including salt preference, enabled these strains to be differentiated phenotypically from described Bacillus species. Therefore, strains SU1(T), 36AC4 and 36AC6 represent a novel species for which the name Bacillus trypoxylicola sp. nov. is proposed; the type strain is SU1(T) (=NBRC 102646(T) =KCTC 13244(T)).

Role of Interfacial Tensions in the Translocation of Rhodococcus erythropolis during Growth in a Two Phase Culture

Rhodococcus erythropolis PR4 is an alkane-degrading bacterium, which grows well in media containing high concentrations of alkanes. These properties give the organism potential in the bioremediation of various environments contaminated by alkanes. In this study, we report the translocation of R. erythropolis PR4 from an aqueous phase to an alkane phase during growth in a two phase culture medium. When the alkane chain length was between C10 and C12, PR4 was located at the aqueous-alkane interface, but when the alkane chain length was above C14, PR4 translocated into the alkane phase. Complete translocation into alkane phase was accompanied by normal growth, whereas interfacial localization hampered growth, indicating that localization among other possible factors, play an important role in the growth of R. erythropolis PR4 in two phase cultures. The PR4 cell surface was physico-chemically characterized in terms of its cell surface charge and surface free energy. Contact angles were measured on bacterial lawns, followed by thermodynamic analyses of Gibbs free energies for localization of PR4 in the aqueous or alkane phase or at the interface. Although entry into the alkane phase of PR4 grown in the presence of both C12 and C19 was thermodynamically favorable, translocation from the inside of the alkane phase to the interface was only favorable for PR4 grown in the presence of C12. In line with these thermodynamic analyses, two phase partitioning showed that PR4 grown in the presence of C12 and C19 were more hydrophobic than PR4 grown in the presence of lower alkanes, while C12 grown bacteria were less lipophilic than C19 grown bacteria. In conclusion, the localization of R. erythropolis PR4 in a two phase culture medium is thermodynamically driven to facilitate its optimal growth.

Enhanced Translocation and Growth of Rhodococcus erythropolis PR4 in the Alkane Phase of Aqueous-Alkane Two Phase Cultures Were Mediated by GroEL2 Overexpression

We previously reported that R. erythropolis PR4 translocated from the aqueous to the alkane phase, and then grew in two phase cultures to which long-chain alkanes had been added. This was considered to be beneficial for bioremediation. In the present study, we investigated the proteins involved in the translocation of R. erythropolis PR4. The results of our proteogenomic analysis suggested that GroEL2 was upregulated more in cells that translocated inside of the pristane (C19) phase than in those located at the aqueous-alkane interface attached to the n-dodecane (C12) surface. PR4 (pK4-EL2-1) and PR4 (pK4-ΔEL2-1) strains were constructed to confirm the effects of the upregulation of GroEL2 in translocated cells. The expression of GroEL2 in PR4 (pK4-EL2-1) was 15.5-fold higher than that in PR4 (pK4-ΔEL2-1) in two phase cultures containing C12. The growth and cell surface lipophilicity of PR4 were enhanced by the introduction of pK4-EL2-1. These results suggested that the plasmid overexpression of groEL2 in PR4 (pK4-EL2-1) led to changes in cell localization, enhanced growth, and increased cell surface lipophilicity. Thus, we concluded that the overexpression of GroEL2 may play an important role in increasing the organic solvent tolerance of R. erythropolis PR4 in aqueous-alkane two phase cultures.

Rhodococcus rhodochrous ATCC12674 Becomes Alkane-Tolerant upon GroEL2 Overexpression and Survives in the n-Octane Phase in Two Phase Culture

We recently reported that the overexpression of GroEL2 played an important role in increasing the alkane tolerance of Rhodococcus erythropolis PR4. In the present study, we examined the effects of the introduction of groEL2 on the alkane tolerance of other Rhodococcus strains. The introduction of groEL2 into Rhodococcus strains led to increased alkane tolerance. The translocation of R. rhodochrous ATCC12674 cells to and survival in the n-octane (C8) phase in two phase culture were significantly enhanced by the introduction of groEL2 derived from strain PR4, suggesting that engineering cells to overexpress GroEL2 represents an effective strategy for enhancing organic solvent tolerance in Rhodococcus.

Selective Stimulation of Aromatic Compound Degradation by the Indigenous Marine Bacterium Cycloclasticus for Bioremediation of Oil Spills in the Marine Environment

S-2 EPS, an extracellular polysaccharide produced by Rhodococcus rhodochrous S-2, is a functional biopolymer that increases the organism’s organic solvent tolerance. We previously demonstrated that addition of S-2 EPS and minerals to oil-contaminated seawater enhanced the degradation of an aromatic fraction (AF) of Arabian light crude oil by Cycloclasticus, a polycyclic aromatic hydrocarbon (PAH)-degrading indigenous marine bacterium, and stimulated its growth in AF-contaminated seawater. Moreover, Cycloclasticus growth and PAH degradation were selectively activated by the association of S-2 EPS with Cycloclasticus cells. In this chapter, the effects of S-2 EPS on the growth and PAH degradation activity of Cycloclasticus in hydrocarbon-degrading marine bacterial consortia are summarized. S-2 EPS regulates the interaction between living bacteria and oils, and because it selectively stimulates degradation of aromatic compounds by the indigenous marine bacterium Cycloclasticus, S-2 EPS is useful for bioremediation of oil spills in the marine environment.