Utilization of phenol by hydrocarbon assimilating yeasts

77 Ascomycetous, basidiomycetous as well as imperfect yeast strains of 46 different species and 20 genera were tested for growth with the substrates n-octane, n-hexadecane, and phenol. Of 59 yeast strains with ascomycetous cell wall structure 33 grew on hydrocarbons and 32 on phenol. No yeast strain out of 26 which are unable to use n-alkanes as a source of carbon and energy grew on phenol. In comparison with the latter 32 out of 33 n-hexadecane assimilating yeasts were also capable of using phenol. All n-octane utilizing yeasts of this group also assimilate phenol as a carbon source for growth.The correlation of the hydrocarbon assimilation with the phenol assimilation seems to be not so strong in the basidiomycetous yeasts. 7 out of 18 strains from this group grew on n-hexadecane and 13 on phenol.Furthermore, it could be shown that the use of hydrocarbons and phenol (as well as methanol) is strongly correlated with the coenzyme Q structure of the respective yeast strain.The results are discussed with respect to the particular chemical properties of the substrates used and the fact that coenzyme Q structure is considered to be an important marker of evolutionary relationships among yeasts.     

Ascopore formation in yeasts during active growth on hydrocarbons

Summary Induction of ascospore formation in hydrocarbon utilizing ascosporogenous yeasts was observed during the growth of the yeasts on gas oil, diesel oil, white kerosene and n-alkanes. Studies of relationships between cell morphology and cell growth showed that ascospores were formed during the active growth phase on gas oil but not on glucose. Contact of yeast cells with hydrocarbons may be the possible reason for sporogenesis on hydrocarbons.     

VARIABILITY OF CELL WALL STRUCTURE AND HYDROCARBON TYPE IN DIFFERENT STRAINS OF BOTRYOCOCCUS BRAUNII1

Different samples of Botryococcus braunii Kütz., freshly collected from nature or laboratory-grown from culture collection strains, were studied by electron microscopy and their hydrocarbon content analyzed. Although the general internal structure of the cells was rather constant, the organization of the outer walls forming the hydrocarbon-rich matrix of the colonies differed greatly from one sample to another. In the majority of cultivated strains, the colonies were rather small, the different successive external walls remained distinct and all strains contained dienic or trienic hydrocarbons. In contrast, most of the collected samples possessed large colonies with a rather compact matrix formed by the hydrocarbon-rich part of the successive closely appressed external wall layers. These samples contained polyunsaturated hydrocarbons, i.e. botryococcenes. Well defined cell caps which sheared off the cells were observed only in those strains with a compact matrix. The Austin strain and some collected samples, however, were intermediate with rather small colonies, dense matrix, definite cell caps and dienic hydrocarbons. Thus, the hydrocarbon composition did not correlate directly with the variations in wall structure; however, the occurence of dienic and botryococcene-like hydrocarbons together in one strain was never observed, although analyzed at various stages of growth. Thus, the existence of distinct strains of Botryococcus braunii, some synthesizing dienes, others botryococcenes, appears highly probable.     

Microbial assimilation of hydrocarbons

1. The fine-structure analysis of the hydrocarbon oxidizing microorganism, Acinetobacter sp., demonstrated a cytoplasmic modification resulting from growth on paraffinic and olefinic hydrocarbons.
2. Intracytoplasmic hydrocarbon inclusions were documented by electron microscopy with chemical identifications obtained by gas chromatography and X-ray diffraction.
3. These results demonstrate the ability of a micro-organism to accumulate hydrocarbon substrates intracellularly which, in turn, indicates transport across the cell membrane.

     

Microbial assimilation of hydrocarbons. I. The fine-structure of a hydrocarbon oxidizing Acinetobacter sp.

1. The fine-structure analysis of the hydrocarbon oxidizing microorganism, Acinetobacter sp., demonstrated a cytoplasmic modification resulting from growth on paraffinic and olefinic hydrocarbons. 2. Intracytoplasmic hydrocarbon inclusions were documented by electron microscopy with chemical identifications obtained by gas chromatography and X-ray diffraction. 3. These results demonstrate the ability of a microorganism to accumulate hydrocarbon substrates intracellularly which, in turn, indicates the transport across the cell membrane.     

Russian Article pp. 213–225

The kinetics of assimilation of hydrocarbons having a different structure by Candida yeasts was studied. The rates of oxidation of various carbon atoms in the molecules of isoalkanes, alkylaromatic hydrocarbons and n-alkanes were evaluated in the range of C₁₁ to C₂₈. Metabolic inhomogeneity of carbon atoms in the molecules of isolakanes and alkylaromatic hydrocarbons was observed. A competition in the assimilation of the called hydrocarbons and n-alkanes and also a competition in the assimilation of n-alkanes of a different molecular weight, i.e. metabolic inhomogeneity of carbon atoms of n-dodecane and n-tetracosane was found out for yeasts.     

Studies on Utilization of Hydrocarbons by Yeasts

The production of microbial cell substances from hydrocarbons has been attracting attention of people for many years. Production of bacterial cell from hydrocarbons is disadvantageous because of the difficulty in separating cell from the broth.

We have tested hydrocarbon-utilizing yeasts isolated from garden soil for cell production. The effect of medium composition on yeast growth and the utilization of individual hydrocarbon by yeast, strain Y-3, were investigated.

As a nitrogen source, urea was more effective than ammonium nitrate. When a very smal! amount of corn steep liquor was added, yeast growth was very improved. Aliphatic series of hydrocarbon lower than C₉ were not or very slightly assimilated by this yeast.

Generally speaking, series of even-number hydrocarbons were more effective than those of odd-number hydrocarbons.

We found that the yeast Y-3 strain reported in the previous paper¹⁾ has a diterminal oxidation system of hydrocarbon.

This yeast capable of growing in mineral-salts solution with hydrocarbons as sole source of carbon produced a series of dioic acid from n-undecane. These acids are 1,11-undecane dioic acid, 1,9-nonane dioic acid (azelaic acid), 1,7-heptane dioic acid (pimelic acid) and 1,5-pentane dioic acid (glutaric acid). 1,10-Decane dioic acid (sebacic acid) was also isolated from n-decane cultures.

Azelaic acid was partially transformed into pimelic acid and glutaric acid by treating it with resting cells of this yeast.

1,11-Undecane dioic was also transformed into azelaic acid pimelic acid, and glutaric acid by the same treatment as described above.     

Sites of accumulation and composition of hydrocarbons in Botryococcus braunii

Raman spectrometry and electron microscopy show that, in the hydrocarbon-rich alga Botryococcus braunii, hydrocarbons accumulate in two distinct sites; internally in cytoplasmic inclusions and externally in successive outer walls and derived globules. No other classes of lipid are present in noticeable amounts in the cytoplasmic inclusions and in the external globules. The same hydrocarbons are observed in the internal and external pools but with different relative abundances, the shorter hydrocarbons being more abundant in the internal pool. The bulk of B. braunii hydrocarbons (ca 95%) is located in the external pool. Such an extracellular location allows this species to exhibit both an unusually high hydrocarbon content (15% of dry wt) and a normal level (0.75%) within the cells. The hydrocarbon pattern and location of B. braunii were compared with that of other organisms; a close relation appears between higher plant epidermal cells and this green alga. The trilaminar outer walls of B. braunii, at whose contact external hydrocarbon globules accumulate, contain a sporopollenin-like compound.     

Colony Organization in the Green Alga Botryococcus braunii (Race B) Is Specified by a Complex Extracellular Matrix

Botryococcus braunii is a colonial green alga whose cells associate via a complex extracellular matrix (ECM) and produce prodigious amounts of liquid hydrocarbons that can be readily converted into conventional combustion engine fuels. We used quick-freeze deep-etch electron microscopy and biochemical/histochemical analysis to elucidate many new features of B. braunii cell/colony organization and composition. Intracellular lipid bodies associate with the chloroplast and endoplasmic reticulum (ER) but show no evidence of being secreted. The ER displays striking fenestrations and forms a continuous subcortical system in direct contact with the cell membrane. The ECM has three distinct components. (i) Each cell is surrounded by a fibrous β-1, 4- and/or β-1, 3-glucan-containing cell wall. (ii) The intracolonial ECM space is filled with a cross-linked hydrocarbon network permeated with liquid hydrocarbons. (iii) Colonies are enclosed in a retaining wall festooned with a fibrillar sheath dominated by arabinose-galactose polysaccharides, which sequesters ECM liquid hydrocarbons. Each cell apex associates with the retaining wall and contributes to its synthesis. Retaining-wall domains also form “drapes” between cells, with some folding in on themselves and penetrating the hydrocarbon interior of a mother colony, partitioning it into daughter colonies. We propose that retaining-wall components are synthesized in the apical Golgi apparatus, delivered to apical ER fenestrations, and assembled on the surfaces of apical cell walls, where a proteinaceous granular layer apparently participates in fibril morphogenesis. We further propose that hydrocarbons are produced by the nonapical ER, directly delivered to the contiguous cell membrane, and pass across the nonapical cell wall into the hydrocarbon-based ECM.     

Microbial degradation of model petroleum at low temperatures

Two areas of Chesapeake Bay, Colgate Creek in Baltimore Harbor and Eastern Bay, are presently under study, with routine sampling of water and sediment for petroleum-degrading microorganisms (bacteria, yeasts, and fungi) by direct plating and enrichment culture. Selected physical and chemical parameters are recorded for each sampling site, and water and sediment samples are extracted for hydrocarbons. Numbers of petroleum-degrading microorganisms enumerated by direct plating were found to correlate with the concentration of benzene-extractable material and were higher for the Colgate Creek than for the Eastern Bay site. Petroleum-degrading microorganisms were isolated from water and sediment samples at environmental temperatures of 0°, 5°, and 10°C. A salts medium supplemented with nitrate and phosphate was used to provide optimum conditions for petroleum degradation, whereas Chesapeake Bay water was used to simulate natural environmental conditions. Use of a model petroleum permitted quantitative measurement of utilization of individual hydrocarbons ranging in complexity from simple alkanes to polynuclear aromatic hydrocarbons. Higher growth yields and maximum hydrocarbon degradation was observed for microorganisms in the salts medium at 0°, 5°, and 10°C, although significant quantities of hydrocarbons were utilized in some samples grown in a medium for which Chesapeake Bay water was the diluent. Bacterial hydrocarbon degradation accounted for most of the model petroleum utilization at 0° and 5°C. However, oscillations of bacterial populations, with significant growth of yeasts, was observed at 10°C. Photomicroscopy and scanning electron microscopy revealed aggregates of bacteria, yeasts, and fungi associated with oil globules. From preliminary identification and classification of the hydrocarbon-utilizing bacteria, members of the generaVibrio, Aeromonas, Pseudomonas, andAcinetobacter were present in the enrichment cultures. From results of this study, it is concluded that utilization of model petroleum at low temperatures is a function of the types and numbers of microorganisms present in an original inoculum taken from the natural environment.     

Emulsifier production and microscopical study of emulsions and biofilms formed by the hydrocarbon-utilizing bacteria Acinetobacter calcoaceticus MM5

A bacterial strain was isolated from a sample of contaminated heating oil and identified as a strain of Acinetobacter calcoaceticus, named MM5. The bacterial isolate was able to grow on petroleum derivatives and brought about an emulsification of those compounds. A bioemulsifier was extracted from the culture medium of MM5 strain and partially characterized. This compound was able to emulsify petroleum fuels and both aliphatic and aromatic pure hydrocarbons and was stable over a wide range of temperatures. Studies developed by light, scanning electron and transmission electron microscopy showed that, during the growth on petroleum derivatives, the microorganisms were orientated on the surface of drops enclosed in a skin or membranous polymer produced by the bacteria. These droplets may represent the hydrocarbon/water emulsion of the liquid culture. The growth of A. calcoaceticus MM5 on media containing both hydrocarbon and water-soluble substrates as carbon sources also results in the formation of a film, consisting of amorphous and membranous layers. The bacteria were connected to the biofilm and showed intercellular contacts through cell-surface appendages, forming a complex network. The importance of the biofilms for bacterial adhesion to oil droplets and for its nourishment is discussed.     

Role of emulsification in the process of hydrocarbon absorption by Pseudomonas aeruginosa cells

Lipophilic biopolymers from the cell walls of saprophytic mycobacteria were shown to stimulate the process of hydrocarbon assimilation by Pseudomonas aeruginosa cells. This should be attributed to the fact that bacterial peptidoglycolipids emulsify a hydrocarbon facilitating the contact between it and the cells. It has been found experimentally that P. aeruginosa cells growing in the medium with n-alkanes release a factor into the medium. The factor appears to contain peptide chains and is responsible for hydrocarbon emulsification.     

The biosynthesis of long-chain hydrocarbons in the green alga Botryococcus braunii

The green colonial alga Botryococcus braunii has unusually high levels of hydrocarbons. Two distinct sites of hydrocarbon accumulation are present in the species: an internal pool present in cytoplasmic inclusions and an external pool in the trilaminar outer walls and associated globules. It is generally assumed that the hydrocarbons are produced within the cells and then excreted into the external pool to maintain the intracellular content at a normal value. Various feeding experiments showed, however, that the radioactivity of the external pool is much higher than the internal one. On the other hand, there was no decrease in the labelling of internal hydrocarbons in chase experiments. Therefore, an excretory process apparently does not take place in B. braunii. The outer wall, therefore, is the main site of hydrocarbon accumulation and also the place where the bulk of B. braunii hydrocarbons are produced. The outer wall also is involved in the matrix of colony formation and the above findings account for the sharp decrease of hydrocarbon production which is associated with the loss of colonial habit. The cultures were also shown to be unable, under usual growth conditions, to catabolize their own hydrocarbons. Such a feature, along with the extracellular location of the main site of production, may account for the abnormally high content of hydrocarbons typical of B. braunii.     

The significance of hydrocarbon assimilation in yeast identification

A large number of yeasts were screened for the ability to assimilate hydrocarbons. Not only representatives of the genusCandida, but also species from other perfect and imperfect genera are able to usen-alkanes as sole carbon and energy source. The significance of this feature in yeast systematics is discussed. In general, all strains of a species share either the ability to assimilate hydrocarbons or the failure to do so. Exceptions are found in species regarded as heterogeneous, likeCandida sake, Candida diddensii andCandida zeylanoides. In cases where the usual criteria used in identification seem to be inadequate, the simple hydrocarbon assimilation test may be useful. Also in subgrouping the generaCandida andTorulopsis the test may be of value, because some perfect genera likeHansenula, Kluyveromyces andSaccharomyces lack hydrocarbon-assimilating representatives.     

Structural and immunological studies on the protoplast membrane of the yeast Candida utilis

Cell membranes of the yeast Candida utilis isolated by lysis of protoplasts have been shown to be lipoprotein in nature. Electron microscopy shows that Mg⁺⁺ is responsible for maintaining the integrity of the membrane. A close serological relationship was found between membranes and cell walls isolated from the yeast. This relationship was exhibited not only by membranes obtained by strepzyme treatment but also by those obtained from the action of helicase enzyme. No such relationship existed between membranes and whole cells. Related data have been obtained by treatment of yeasts with different digestive enzymes. All of the results suggest that the protoplast membrane possesses traces of structural cell wall material. This material is detectable by serological tests, but not by electron microscopy.     

Selenite removal and reduction by growing <Emphasis Type="Italic">Aspergillus</Emphasis> sp. J2

In this study, the removal and reduction of selenite [Se(IV)] by growing Aspergillus sp. J2 were investigated. The lag phase, growth rate and biomass of J2 was not significantly influenced by the presence of 100 mg/L Se(IV). A rapid Se(IV) removal process took place from the 3rd to the 4th day during the growth of J2. Scanning electron microscopy, transmission electron microscopy, X-ray photoelectron spectroscopy and X-ray diffraction analyses showed that the Se(IV) did not cause any visible effects on cell morphology and the reduced amorphous elemental selenium [Se(0)] nanoparticles were mainly on the surface of the mycelial cell walls. The macromolecules containing amine groups also interact with Se(IV) and could play an important role in Se(IV) removal by J2.     

Catalase Activities of Hydrocarbon-utilizing Candida Yeasts

The catalase activities of the Candida cells grown on hydrocarbons were generally much higher than those of the cells grown on Iauryl alcohol, glucose or ethanol. Km values for hydrogen peroxide of the enzymes from the glucose- and the hydrocarbon-grown cells of Candida tropicalis were the same level. The enzyme activities of the yeasts were higher at the exponential growth phase, especially of the hydrocarbon-grown cells, than at the stationary phase. Profuse appearance of microbodies having homogeneous matrix surrounded by a single-layer membrane has also been observed electronmicroscopically in the hydrocarbon- grown cells of several Candida yeasts. Cytochemical studies using 3,3′-diaminobenzidine (DAB) revealed that the catalase activity was located in microbodies. These facts suggest that the catalase activities would be related to the hydrocarbon metabolism in the yeasts.     

Physiological adaptations involved in alkane assimilation at a low temperature by Rhodococcus sp. strain Q15.

We examined physiological adaptations which allow the psychrotroph Rhodococcus sp. strain Q15 to assimilate alkanes at a low temperature (alkanes are contaminants which are generally insoluble and/or solid at low temperatures). During growth at 5 degrees C on hexadecane or diesel fuel, strain Q15 produced a cell surface-associated biosurfactant(s) and, compared to glucose-acetate-grown cells, exhibited increased cell surface hydrophobicity. A transmission electron microscopy examination of strain Q15 grown at 5 degrees C revealed the presence of intracellular electron-transparent inclusions and flocs of cells connected by an extracellular polymeric substance (EPS) when cells were grown on a hydrocarbon and morphological differences between the EPS of glucose-acetate-grown and diesel fuel-grown cells. A lectin binding analysis performed by using confocal scanning laser microscopy (CSLM) showed that the EPS contained a complex mixture of glycoconjugates, depending on both the growth temperature and the carbon source. Two glycoconjugates [beta-D-Gal-(1-3)-D-GlcNAc and alpha-L-fucose] were detected only on the surfaces of cells grown on diesel fuel at 5 degrees C. Using scanning electron microscopy, we observed strain Q15 cells on the surfaces of octacosane crystals, and using CSLM, we observed strain Q15 cells covering the surfaces of diesel fuel microdroplets; these findings indicate that this organism assimilates both solid and liquid alkane substrates at a low temperature by adhering to the alkane phase. Membrane fatty acid analysis demonstrated that strain Q15 adapted to growth at a low temperature by decreasing the degree of saturation of membrane lipid fatty acids, but it did so to a lesser extent when it was grown on hydrocarbons at 5 degrees C; these findings suggest that strain Q15 modulates membrane fluidity in response to the counteracting influences of low temperature and hydrocarbon toxicity.     

A convenient microtitre tray procedure for yeast identification

Media for yeast identification tests were incorporated into the wells of a microtitre tray. The tests included fermentation and assimilation of carbohydrates, assimilation of nitrogen compounds, growth in vitamin-free medium, resistance to cycloheximide, and observations for cell morphology and sporulation. Results of tests conducted in the trays showed very good agreement with those obtained by conventional methods. Eighteen reference yeasts were correctly identified from tests conducted in the trays. The trays of media could be stored, and provided a convenient system for yeast identification.     

Localization of mannoprotein in Cryptococcus neoformans.

Cell wall mannoprotein of nonpathogenic yeasts is surface exposed, since the cells are agglutinated by concanavalin A and antimannoprotein antibodies. However, nonencapsulated cells of Cryptococcus neoformans were agglutinated neither by concanavalin A nor by antimannoprotein antibodies. Immunogold electron microscopy located most mannoprotein in the inner cell wall. Chemical analysis of purified cell walls showed the lack of mannose, xylose, and galactose residues. These data indicate that cryptococcal mannoprotein recovered from the cultural supernatant is a nonstructural element of the cell wall.