Adhesion of Actinomyces viscosus to Porphyromonas (Bacteroides) gingivalis-coated hexadecane droplets.

Interbacterial adhesion (coadhesion) is considered a major determinant of dental plaque ecology. In this report, we studied several aspects of the adhesion of Porphyromonas (Bacteroides) gingivalis to hexadecane in order to use the liquid hydrocarbon as a convenient substratum for coadhesion assays. Washed suspensions of hydrophobic P. gingivalis 2561 cells were vortexed with hexadecane to yield highly stable cell-coated droplets. Kinetics of coadhesion between Actinomyces viscosus cells and P. gingivalis-coated hexadecane droplets (PCHD) was subsequently studied. Aliquots of PCHD were added to A. viscosus suspensions, and the mixtures were gently rotated. Avid adhesion of A. viscosus cells to the immobilized P. gingivalis layer could be readily measured by the decrease in turbidity in the aqueous phase, following phase separation. Despite the ability of A. viscosus cells to adsorb to hexadecane following vigorous mixing, gentle mixing did not appreciably promote adhesion to bare hexadecane. Moreover, extensive microscopic examinations revealed that A. viscosus cells adhered exclusively to the bound P. gingivalis cells rather than to exposed areas of hexadecane. Coadhesion of A. viscosus to the PCHD appeared to follow first-order kinetics, attaining 80% levels within 30 min. Electron micrographs revealed A. viscosus cells adhering to the P. gingivalis cell layer adsorbed at the hexadecane-water interface. Interestingly, P. gingivalis cells did not appear to penetrate the hexadecane. A viscosus mutants lacking type 1 or type 2 fimbriae or both were still able to bind to the PCHD. No obvious correlation was observed between relative hydrophobicity of A. viscosus strains and their binding to PCHD. However, defatted bovine serum albumin, an inhibitor of hydrophobic interactions, was the most potent inhibitor among those tested. The data suggest that this approach provides a simple, quantitative technique for studying kinetics of bacterial coadhesion which is amenable to both light and electron microscopic observation.     

Structure of the cell surface of the yeast Candida tropicalis and its relation to hydrocarbon transport

The surface structure of the hypdrocarbon-utilizing yeast Candida tropicalis was investigated by scanning and transmission electron microscopy (SEM and TEM respectively). The sample preparation technique was based on a rapid cryofixation without any addition of cryoprotectants. In subsequently freeze-dried samples the surface structure was analysed by scanning electron microscopy. Thin sections were prepared from freeze substituted samples. Both techniques revealed hair-like structures at the surface of hydrocarbon-grown cells. The hairy surface structure of the cells was less expressed in glucose-grown cells and it was absent completely after proteolytic digestion of the cells. When cells were incubated with hexadecane prior to cyryofixation a contrast-rich region occured in the hair fringe of thin sections as revealed by TEM. Since these structures were characteristic for hexadecane-grown cells and could not be detected in glucose-grown or proteasetreated cells it was concluded that they originate from hexadecane adhering to the cell surface and are functionally related to hexadecane transport. The structure of the surface and its relation to hydrocarbon transport are discussed in view of earlier results on the chemical composition of the surface layer of the cell wall.Abbreviations SEM Scanning electron microscopy - TEM transmission electron microscopy     

Adhesion of red blood cells to charged interfaces between immiscible liquids. A new method.

We have devised a method of making a flat oil/water interface which remains flat on inversion. Cell adhesion to the interface can be observed microscopically. Glutaraldehyde-fixed human red blood cells adhere to the interface between physiological saline and hexadecane containing surface-active behenic acid at pH values below about 7-5. At high pH values, cells are prevented from adhering due to dissociation of the carboxyl groups of behenic acid oriented in the interface. The negative red cells are driven away electrostatically. Adherent and non-adherent cells remain on the aqueous side of the interface and do not appreciably deform it when adherent. Cells are electrostatically attracted to a similar interface containing positively charged octadecyltrimethylammonium ions. Cells also adhere to an interface containing octadecanol, which carries no charge. Underlying both electrostatic repulsion and attraction between red cells and oil/water interfaces is an attractive force which may be of electrodynamic (van der Waals) origin.     

Monolayer adsorption of a "bald" mutant of the highly adhesive and hydrophobic bacterium Acinetobacter sp. strain Tol 5 to a hydrocarbon surface

The affinity of microbial cells for hydrophobic interfaces is important because it directly affects the efficiency of various bioprocesses, including green biotechnologies. The toluene-degrading bacterium Acinetobacter sp. strain Tol 5 has filamentous appendages and a hydrophobic cell surface, shows high adhesiveness to solid surfaces, and self-agglutinates. A "bald" mutant of this bacterium, strain T1, lacks the filamentous appendages and has decreased adhesiveness but retains a hydrophobic cell surface. We investigated the interaction between T1 cells and an organic solvent dispersed in an aqueous matrix. During a microbial-adhesion-to-hydrocarbon (MATH) test, which is frequently used to measure cell surface hydrophobicity, T1 cells adhered to hexadecane droplet surfaces in a monolayer, whereas wild-type cells aggregated on the droplet surfaces. The adsorbed T1 cells on the hexadecane surfaces hindered the coalescence of the droplets formed by vortexing, stabilizing the emulsion phase. Following the replacement of the aqueous phase with fresh pure water after the MATH test, a proportion of the T1 cells that had adsorbed to the hydrocarbon surface detached during further vortexing, suggesting a reversible adsorption of T1 cells. The final ratio of the adhering cells to the total cells in the detachment test coincided with that in the MATH test. The adhesion of T1 cells to the hydrocarbon surface conformed to the Langmuir adsorption isotherm, which describes reversible monolayer adsorption. Reversible monolayer adsorption should be useful for green technologies employing two-liquid-phase partitioning systems and for bioremediation because it allows effective reaction and transport of hydrophobic substrates at oil-water interfaces.     

The enhancement by surfactants of hexadecane degradation by Pseudomonas aeruginosa varies with substrate availability.

The rhamnolipid biosurfactant produced by Pseudomonas aeruginosa influences various processes related to hydrocarbon degradation. However, degradation can only be enhanced by the surfactant when it stimulates a process that is rate limiting under the applied conditions. Therefore we determined how rhamnolipid influences hexadecane degradation by P. aeruginosa UG2 under conditions differing in hexadecane availability. The rate of hexadecane degradation in shake flask cultures was lower for hexadecane entrapped in a matrix with 6 nm pores (silica 60) or in quartz sand than for hexadecane immobilized in matrices with pore sizes larger than 300 nm or for hexadecane present as a separate liquid phase. This indicates that the availability of hexadecane decreased with decreasing pore size under these conditions. The rate-limiting step for hexadecane entrapped in silica 60 was the mass transfer of substrate from the matrix to the bulk liquid phase, whereas for hexadecane present as a second liquid phase it was the uptake of the substrate by the cells. Hexadecane degradation in batch incubations was accelerated by the addition of rhamnolipid or other surfactants in all experiments except in those where hexadecane was entrapped in silica 60, indicating that the surfactants stimulated uptake of hexadecane by the cells. Since rhamnolipid stimulated the degradation rate in batch experiments to a greater extent than any of the other 14 surfactants tested, hexadecane uptake was apparently more enhanced by rhamnolipid than by the other surfactants. Although rhamnolipid did not stimulate the release of hexadecane from silica 60 under conditions of intense agitation, it significantly enhanced this rate during column experiments in the absence of strain UG2. The results demonstrate that rhamnolipid enhances degradation by stimulating release of entrapped substrate in column studies under conditions of low agitation and by stimulating uptake of substrate by the cells, especially when degradation is not limited by release of substrate from the matrices.     

Partition of hexadecane to the cell surface of Candida tropicalis: Mechanism for the transport of water-insoluble substrates

The partition of hexadecane to the cell surface of Candida tropicalis was measured by incubating heat-inactivated cells with hexadecane-1-¹⁴C on a gyratory shaker. The free hexadecane was separated by centrifuging the cells through a 15% sucrose solution, and the partitioned hexadecane was quantified by scintillation spectrometry of the samples from the resulting cell sediment. Heat-inactivated cells did not take up hexadecane as determined by a membrane filtration technique involving organic solvent washing. The partitioning was a time-dependent process. The velocity increased by increasing the shake rate of te shaker. At 360 rpm and with baffled flasks, saturation of the cell surface with hexadecane was obtained after a 20 min incubation period. The amount of hexadecane partitioned depended on the initial hexadecane-to-cell concentration ratio. At a ratio of 5 μmol/mg cell protein the highest amount of hexadecane partitioned was measured at 2100 μmol/mg cell protein. At ratios higher than 6 μmol/mg cell protein the cells were no longer sedimentable by centrifugation. The partition of hexadecane to the cell surface was affected by removing the surface layer of the cell wall by Pronase treatment and by using detergents in the partition assay. Pronase treatment lowered the amount of hexadecane partitioned as a consequence of the removal of the lipophilic layer of the cell surface. Detergents influence the partition coefficient and also lowered the amount of hexadecane partitioning to the cell surface. At a low shaking intensity (280 rpm, unbaffled flasks), after Pronase treatment, and in the presence of detergents he uptake of hexadecane by the cells was limited by the partitioning.     

水-有机溶剂两相体系中甲基单孢菌Z201细胞催化丙烯环氧化的初步研究

Laabe于1987年提出了生物催化剂工程(Biocatalyst engineering)和介质工程(Medium engineering)的概念^[1]。有机相生物催化中溶剂的选择也是介质工程的内容之一。纯酶在有机相中的催化作用已有大量报道^[2],但对完整细胞研究甚步。本文以甲基单胞菌(Methylomonas Z201)完整细胞为生物催化剂．丙烯环氧化为指标反应．研究有机溶剂对活性的影响并对催化活性——溶剂疏水性进行了相关性分析。研究了水一十六烷两相体系中十六烷含量和搅拌速度对丙烯环氧化速度的影响和细胞的操作稳定性。     

水-有机溶剂两相体系中甲基单胞菌Z201催化丙烯环氧化的初步研究

Lanne于1987年提出了生物催化剂工程（Biocatalyst engimeering）和介质工程（Medium enineering）的概念^[1].有机相生物催化中溶剂的选择也是介质工程的内容之一。纯酶在有机相中的催化作用已有大量报道^[2],但对完整细胞研究甚少。本文以甲基单胞菌（Methylomonos）Z201完整细胞为生物催化剂,丙烯环氧化为指标反应,研究有机溶剂对活性的影响并对催化活性-溶剂疏水性进行了相关性分析。研究了水-十六烷两相体系中十六烷含量和搅拦速度对丙烯环氧化速度的影响和细胞的操作稳定性。     

Concentration of larvicidal Bacillus spores at the water surface by adherence to oil droplets

Adherence of two larvicidal spores, Bacillus sphaericus 2297 and Bacillus thuringiensis israelensis, to hexadecane and corn oil was determined. Adherence was greatly promoted in the presence of 2 M ammonium sulphate, reaching levels of up to 89%. The data suggest that larvicidal spores may be concentrated at their operational niche (the water surface) by flotation, adhering to the surface of oil droplets. The data suggest that adherence of the spores to oil droplets may enable their concentration at the water surface, where they can effectively kill mosquito larvae.     

Comparison of contact angles and adhesion to hexadecane of urogenital, dairy, and poultry lactobacilli: effect of serial culture passages.

The aim of this study was to examine the hydrophobicities of 23 urogenital, dairy, poultry, and American Type Culture Collection isolates of lactobacilli and to determine the effect on hydrophobicity of serially passaging the strains in liquid medium. To this end, strains were grown after isolation and identification and then serially passaged up to 20 times. Hydrophobicity was assessed through contact angle measurements on lawns of cells by using water, formamide, methylene iodide, 1-bromonaphthalene, and hexadecane as wetting agents and through measurement of their partitioning in a hexadecane-water system. The hydrophobicities of these strains varied widely, with Lactobacillus casei strains being predominantly hydrophilic and L. acidophilus strains being mostly hydrophobic. For some isolates, serial passaging was accompanied by a clear loss of hydrophobic surface properties, whereas for other strains, cultures became heterogeneous in that some cells had already lost their hydrophobic surface properties while others were still hydrophobic. Adhesion of this collection of lactobacilli to hexadecane droplets in microbial adhesion to hexadecane (MATH) tests was driven by their aversion to water rather than by their affinity for hexadecane, as concluded from the fact that hexadecane contact angles were zero for all strains. Furthermore, adhesion of the lactobacilli to hexadecane in MATH tests occurred only when the water contact angle on the cells was above 60 degrees.     

Comparison of contact angles and adhesion to hexadecane of urogenital, dairy, and poultry lactobacilli: effect of serial culture passages.

The aim of this study was to examine the hydrophobicities of 23 urogenital, dairy, poultry, and American Type Culture Collection isolates of lactobacilli and to determine the effect on hydrophobicity of serially passaging the strains in liquid medium. To this end, strains were grown after isolation and identification and then serially passaged up to 20 times. Hydrophobicity was assessed through contact angle measurements on lawns of cells by using water, formamide, methylene iodide, 1-bromonaphthalene, and hexadecane as wetting agents and through measurement of their partitioning in a hexadecane-water system. The hydrophobicities of these strains varied widely, with Lactobacillus casei strains being predominantly hydrophilic and L. acidophilus strains being mostly hydrophobic. For some isolates, serial passaging was accompanied by a clear loss of hydrophobic surface properties, whereas for other strains, cultures became heterogeneous in that some cells had already lost their hydrophobic surface properties while others were still hydrophobic. Adhesion of this collection of lactobacilli to hexadecane droplets in microbial adhesion to hexadecane (MATH) tests was driven by their aversion to water rather than by their affinity for hexadecane, as concluded from the fact that hexadecane contact angles were zero for all strains. Furthermore, adhesion of the lactobacilli to hexadecane in MATH tests occurred only when the water contact angle on the cells was above 60 degrees.     

Plasma membrane from Candida tropicalis grown on glucose or hexadecane. II. Biochemical properties and substrate-induced alterations.

Isolated plasma membranes from the yeast Candida tropicalis grown on two different carbon sources (glucose or hexadecane), had similar contents of protein (60% of total dry weight), lipid (21-24%) and carbohydrates (16-21%). Sodium dodecyl sulphate gel electrophoresis of the membrane proteins revealed 17 and 19 protein bands, respectively, for glucose and hexadecane grown cells. There were marked differences in RF values and relative peak heights between the two gels. Sterols and free fatty acids were the major components of the plasma membrane lipids. Phospholipid content was less than 2% of total plasma membrane lipids. Membrane microviscosity, as determined by fluorescence polarization, was very high (16.6 P). Fatty acid determination of membrane lipids by gas chromatography showed a significant increase of C16 fatty acids in plasma membranes of cells grown on hexadecane. Reduced-oxidized difference spectra demonstrated the presence of a b-type cytochrome in both Saccharomyces cerevisiae and C. tropicalis plasma membranes. Its concentration in C. tropicalis plasma membranes was three-fold greater in cells grown on hexadecane than in glucose grown cells.     

Role of Thin Fimbriae in Adherence and Growth of Acinetobacter calcoaceticus RAG-1 on Hexadecane

Acinetobacter calcoaceticus RAG-1, a hydrocarbon-degrading bacterium which adheres avidly to hydrocarbons and other hydrophobic surfaces, possesses numerous thin fimbriae (ca. 3.5-nm diameter) on the cell surface. MR-481, a nonadherent mutant of RAG-1 which is unable to grow on hexadecane under conditions of limited emulsification and low initial cell density, lacks these fimbriae. Prolonged incubation of MR-481 in hexadecane medium enriched for partial adherence revertants. The reappearance of thin fimbriae was observed in all such revertant strains. RAG-1 cells and partial revertant strains were agglutinated in the presence of antibody, whereas MR-481 cells were not. Another mutant, AB15, which was previously isolated on the basis of its nonagglutinability in the presence of antibody, also lacked thin fimbriae and was conditionally nonadherent. Furthermore, strain AB15 was unable to grow on hexadecane medium. Adherence of RAG-1 cells to hexadecane was considerably reduced after shearing treatment. The material removed from the cell surface by shearing of RAG-1 and the partial revertant strains yielded a single antigenic band in RAG-1 and partial revertant strains, as observed by crossed immunoelectrophoresis. This band was absent in both fimbriae-less mutants, MR-481 and AB15. The data demonstrate that the thin fimbriae of RAG-1 (i) are a major factor in adherence to polystyrene and hydrocarbon, (ii) may be crucial in enabling growth of cells on hexadecane, and (iii) constitute the major cell surface agglutinogen.     

Effect of Cetylpyridinium Chloride on Microbial Adhesion to Hexadecane and Polystyrene

Microbial adhesion at the oil-water interface is a subject of both basic interest (e.g., as a technique for the measurement of hydrophobicity) and applied interest (e.g., for use in two-phase oil-water mouthwashes for the desorption of oral microorganisms). In general, surfactants inhibit microbial adhesion to oils and other hydrophobic surfaces. In the present study, we demonstrated that the cationic surfactant cetylpyridinium chloride (CPC) significantly enhanced microbial adhesion to hexadecane and various oils, as well as to the solid hydrophobic surface polystyrene. CPC increased adhesion to hexadecane of Escherichia coli, Candida albicans and Acinetobacter calcoaceticus MR-481 and of expectorated oral bacteria from near 0% to over 90%. The CPC concentration required for optimal enhancement of adhesion was a function of the initial cell density. This phenomenon was inhibited by high salt concentrations and, in the case of E. coli, by a low pH. CPC-pretreated cells were able to bind to hexadecane, but CPC-pretreated hexadecane was unable to bind untreated cells. Another cationic, surface-active antimicrobial agent, chlorhexidine gluconate, was similarly able to promote microbial adhesion to hexadecane. The results suggest that (i) CPC enhances microbial adhesion to hexadecane by binding via electrostatic interactions at the cell surface, thus diminishing surface charge and increasing cell surface hydrophobicity, and (ii) this phenomenon may have applications in oral formulations and in the use of hydrocarbon droplets as a support for cell immobilization.     

Fatty aldehyde dehydrogenases in Acinetobacter sp. strain HO1-N: role in hexadecanol metabolism

The role of fatty aldehyde dehydrogenases (FALDHs) in hexadecane and hexadecanol metabolism was studied in Acinetobacter sp. strain HO1-N. Two distinct FALDHs were demonstrated in Acinetobacter sp. strain HO1-N: a membrane-bound, NADP-dependent FALDH activity induced 5-, 15-, and 9-fold by growth on hexadecanol, dodecyl aldehyde, and hexadecane, respectively, and a constitutive, NAD-dependent, membrane-localized FALDH. The NADP-dependent FALDH exhibited apparent Km and Vmax values for decyl aldehyde of 5.0, 13.0, 18.0, and 18.3 microM and 537.0, 500.0, 25.0, and 38.0 nmol/min in hexadecane-, hexadecanol-, ethanol-, palmitate-grown cells, respectively. FALDH isozymes ald-a, ald-b, and ald-c were demonstrated by gel electrophoresis in extracts of hexadecane- and hexadecanol-grown cells. ald-a, ald-b, and ald-d were present in dodecyl aldehyde-grown cells, while palmitate-grown control cells contained ald-b and ald-d. Dodecyl aldehyde-negative mutants were isolated and grouped into two phenotypic classes based on growth: class 1 mutants were hexadecane and hexadecanol negative and class 2 mutants were hexadecane and hexadecanol positive. Specific activity of NADP-dependent FALDH in Ald21 (class 1 mutant) was 85% lower than that of wild-type FALDH, while the specific activity of Ald24 (class 2 mutant) was 55% greater than that of wild-type FALDH. Ald21R, a dodecyl aldehyde-positive revertant able to grow on hexadecane, hexadecanol, and dodecyl aldehyde, exhibited a 100% increase in the specific activity of the NADP-dependent FALDH. The oxidation of [3H]hexadecane byAld21 yielded the accumulation of 61% more fatty aldehyde than the wild type, while Ald24 accumulated 27% more fatty aldehyde, 95% more fatty alcohol, and 65% more wax ester than the wild type. This study provides genetic and physiological evidence for the role of fatty aldehyde as an essential metabolic intermediate and NADP-dependent FALDH as a key enzyme in the dissimilation of hexadecane, hexadecanol, and dodecyl aldehyde in Acinetobactor sp. strain HO1-N.     

Inhibition of glucose metabolism by n-hexadecane in Cladosporium (Amorphotheca) resinae.

When Cladosporium resinae is provided with n-hexadecane and glucose, n-hexadecane is used preferentially. Studies using [14C]glucose indicated that n-hexadecane did not inhibit glucose uptake but did retard oxidation of glucose to CO2 and assimilation of glucose carbon into trichloroacetic acid-insoluble material. Glucose could be recovered quantitatively from hydrocarbon-grown cells that had been transferred to glucose. Four enzymes that may be involved in glucose metabolism, hexokinase, glucose-6-phosphate dehydrogenase, glucose-phosphate isomerase, and succinate dehydrogenase, were not detected in cells grown on hexadecane but were present in cells grown on glucose. Addition of hexadecane to extracts of glucose-grown cells resulted in immediate loss of activity for each of the four enzymes, but two other enzymes did not directly involved in glucose metabolism, adenosine triphosphatase and alanine-ketoacid aminotransferase, were not inhibited by hexadecane in vitro. Cells grown on hexadecane and transferred to glucose metabolize intracellular hexadecane; after 1 day, activity of hexokinase, glucose-6-phosphate dehydrogenase, glucosephosphate isomerase, and succinate dehydrogenase could be detected and 22% of the intracellular hydrocarbon had been metabolized. Hexadecane-grown cells transferred to glucose plus cycloheximide showed the same level of activity of all the four enzymes as cells transferred to glucose alone. Thus, intracellular n-hexadecane or a metabolite of hexadecane can inthesis of those enzymes is not inhibited.     

Rhamnolipid stimulates uptake of hydrophobic compounds by Pseudomonas aeruginosa

The biodegradation of hexadecane by five biosurfactant-producing bacterial strains (Pseudomonas aeruginosa UG2, Acinetobacter calcoaceticus RAG1, Rhodococcus erythropolis DSM 43066, R. erythropolis ATCC 19558, and strain BCG112) was determined in the presence and absence of exogenously added biosurfactants. The degradation of hexadecane by P. aeruginosa was stimulated only by the rhamnolipid biosurfactant produced by the same organism. This rhamnolipid did not stimulate the biodegradation of hexadecane by the four other strains to the same extent, nor was degradation of hexadecane by these strains stimulated by addition of their own biosurfactants. This suggests that P. aeruginosa has a mode of hexadecane uptake different from those of the other organisms. Rhamnolipid also enhanced the rate of epoxidation of the aliphatic hydrocarbon alpha,omega-tetradecadiene by a cell suspension of P. aeruginosa. Furthermore, the uptake of the hydrophobic probe 1-naphthylphenylamine by cells of P. aeruginosa was enhanced by rhamnolipid, as indicated by stopped-flow fluorescence experiments. Rhamnolipid did not stimulate the uptake rate of this probe in de-energized cells. These results indicate that an energy-dependent system is present in P. aeruginosa strain UG2 that mediates fast uptake of hydrophobic compounds in the presence of rhamnolipid.     

Rhamnolipid (biosurfactant) effects on cell aggregation and biodegradation of residual hexadecane under saturated flow conditions.

The objective of this research was to evaluate the effect of low concentrations of a rhamnolipid biosurfactant on the in situ biodegradation of hydrocarbon entrapped in a porous matrix. Experiments were performed with sand-packed columns under saturated flow conditions with hexadecane as a model hydrocarbon. Application of biosurfactant concentrations greater than the CMC (the concentration at which the surfactant molecules spontaneously form micelles or vesicles [0.03 mM]) resulted primarily in the mobilization of hexadecane entrapped within the sand matrix. In contrast, application of biosurfactant concentrations less than the CMC enhanced the in situ mineralization of entrapped hexadecane; however, this effect was dependent on the choice of bacterial isolate. The two Pseudomonas isolates tested, R4 and ATCC 15524, were used because they exhibit different patterns of biodegradation of hexadecane, and they also differed in their physical response to rhamnolipid addition. ATCC 15524 cells formed extensive multicell aggregates in the presence of rhamnolipid while R4 cells were unaffected. This behavior did not affect the ability of the biosurfactant to enhance the biodegradation of hexadecane in well-mixed soil slurry systems but had a large affect on the extent of entrapped hexadecane biodegradation in the sand-packed-column system that was used in this study.     

Characterization of intracytoplasmic hydrocarbon inclusions from the hydrocarbon-oxidizing Acinetobacter species HO1-N.

The ultrastructure of Acinetobacter sp. strain HO1-N grown on hydrocarbon and nonhydrocarbon substrates was compared using thin sections and freeze-etching. Hydrocarbon-grown cells were characterized by the presence of intracytoplasmic membrane-bound hexadecane inclusions. This membrane did not exhibit a typical unit membrane structure but appeared as a monolayer. The freeze-etch technique revealed the internal structure of the hexadecane inclusions and provided evidence for the presence of a smooth-surfaced limiting membrane. Freeze-etching also revealed intracytoplasmic membranes in the hexadecane-grown cells. These ultrastructural modifications were not present in nonhydrocarbon-grown cells. The hexadecane inclusions were isolated from Acinetobacter. Negative-staining of the inclusions revealed electron-transparent vesicles approximating the size of the inclusions seen in whole cells. Freeze-etching of the purified inclusions revealed membrane-bound vesicles. The purified inclusions exhibited a relatively high value of lipid phosphorus to protein. The lipid composition and the electrophoretic banding pattern of the inclusions on sodium dodecyl sulfate-polyacrylamide gels were determined and compared with other membrane fractions (outer membrane and cytoplasmic membrane) previously isolated from this organism.     

Alcohol oxidation by Candida guilliermondii yeasts grown on hexadecanol

A number of enzyme systems involved in the first steps of hexadecane oxidation can be induced by hexadecanol, an intermediate product of hexadecane degradation. It has also been found that, in Candida guilliermondii cells and in their mitochondrial fraction, an oxidase system is induced when the cells are grown on hexadecanol. This system is similar to that in cells grown on hexadecane; it oxidises higher alcohols at a high rate and is not inhibited by the inhibitors of the man phosphorylating respiration chain. The membrane-bound alcohol dehydrogenase and aldehyde dehydrogenase activities resistant to pyrazole, an inhibitor of cytosol ethanol dehydrogenase, are induced together with the oxidase activity when the cells are grown on hexadecanol as well as on hexadecane. The oxidation of higher alcohols by whole cells is entirely inhibited by azide although their oxidation by mitochondria is resistant to the action of azide; apparently, azide inhibits the transport of alcohols into the cell.