Studies on the Utilization of Hydrocarbons by Microorganisms

The production of microbial cell substances from hydrocarbons has become the object of commercial attention. We have tested hydrocarbon-utilizing bacterial strains for cell production, and found out one strain of high cell yield, which is very similar to Pseudomonas aeruginosa. The effect of medium composition on cell yield and the utilization of individual hydrocarbons by this strain were investigated. Sixty percent of the added kerosene was converted into cell materials in the following medium of optimum composition: kerosene 2.5%, urea 0.13%, dipotassium phosphate 0.25%, magnesium sulfate 7 aq. 0.1% and tap water. Aliphatic series lower than C₁₀, aromatic and naphthenic hydrocarbons were not or very slightly assimilated. However, aliphatic members higher than C₁₂ were utilized with increasing case. Especially, n-docosane and octadecene-1 were utilized very effectively, and 70% of them were converted into cell substances.     

Studies on the Utilization of Hydrocarbons by Microorganisms

Taxonomical studies on ten strains of hydrocarbon-utilizing bacteria reported in previous paper, which produced various kinds of amino acid, were carried out. They were Achromo-bacter cycloclastes, Achromobacter delmarvae, Bacillus species, Corynebacterium species, Micrococcus species. Many of them were not identical with the species which are described in Bergey’s Manual of 7th Edition.     

Studies on the Utilization of Hydrocarbons by Microorganisms

Fumaric acid productivity of Candida hydrocarbofumarica in various culture conditions was investigated. Namely, the effects of pH, heavy metal ions, hydrocarbon concentration, aeration and surface active agents were studied.

The addition of CaCO₃ and the aeration were effective for fumaric acid production.

The rate of conversion of n-paraffin to fumaric acid gradually decreased as the concentration of n-paraffins (6%) was increased.

A very high yield, 84% was obtained with a culture medium containing 6% of n-paraffins for 7 days culture.     

Studies on the Utilization of Hydrocarbons by Microorganisms

Many strains of the hydrocabon-utilizing yeasts were isolated from various kinds of natural sources by accumulation culture.

Among those yeasts, two strains, S315YI and S131YI, which were identified with Candida tropilcais, assimilated hydrocarbons abundantly. As for type cultures, it was found that many strains of them could utilize hydrocarbons too, especially the strains which belonged to Genus Candida. However, as regards to the ability in utilizing hydrocarbons, no yeast from type culture collections utilized hydrocarbons better than the yeasts newly isolated from nature. Addition of the natural nutrients such as corn steep liquor to the cultural broth of Strain S315Y1 showed no effect on the production of yeast cells.

The yeast Strain S315Y1 assimilated the higher boiling points fraction of n-paraffins in comparison with the hydrocarbon-utilizing bacterium, Pseudomonas aeruginosa S7B1, which had been reported by the authors. Ribonucleic acid contents of dried cells of the yeast Strain S315Y1 and S131Y1 were 5.3 and 4.4% respectively by Schmidt-Thannhauser-Schneider method.     

Studies on the Utilization of Hydrocarbons by Microorganisms

During the course of an investigation of the microbial assimilation of aromatic hydrocarbons, several strains were found to produce a large amount of cumic acid from p-cymene.

Five strains, S449B1, B2, B3, B4 and B6, were isolated from soil with the aromatic hydrocarbon substrates. They all assimilated both p-cymene and cumene. The strain S449B3 grew also on p-xylene, and S449B6 on p-xylene, toluene, and ethylbenzene.

They were all shown to be capable of producing an ultraviolet-absorbing substance from p-cymene. This substance was isolated in crystalline form and identified as cumic acid by infrared absorption spectrum and other observations.

The superior strain, S449B6, produced the acid as much as 1000 mg/1 in shaking culture at 30°C after 24 hours. The yields were increased up to approximately 1700 mg/1 after further investigations. Addition of calcium carbonate and considerable agitation were favorable conditions for the acid production.

The taxonomical studies of these strains were carried out, and they were all identified as closely resembling Pseudomonas desmolytica.     

Studies on the Utilization of Hydrocarbons by Microorganisms

It is well known that a good many microorganisms can utilize hydrocarbons as a sole carbon source. We have as yt little information, however, as to what kinds of intermediate or end products are produced by the microbial dissimilation of hydrocarbons. Above all, the formation of amino acids from hydrocarbons has not been reported. We have isolated many strains of microorganisms from soil samples by selective culture techniques in a medium containing kerosene and mineral salts, in order to examine if the products of economical value such as amino acids, organic acids, fatty acids, steroids, nucleic acids and their related compounds, can be produced by those microorganisms. Shaking cultures of those microorganisms were carried out in the medium containing 3.5% kerosene, 3.5% liquid paraffin and mineral salts (K₂HPO₄ 0.25%; MgSO₄·7H₂O 0.1%; NH₄MO₈ 0.5% or urea 0.3%); we found that 55 strains out of 127 strains tested produced amino acids in their broths, after 4 days cultivation at 26.5°C. The kinds and maximum yields of amino acids were as follows: alanine 50mg/l, aspartic acid 23mg/l, glutamic acid 485 mg/1, glycine 8.5mg/l, isoleucine 15 mg/1, lysine 23.9 mg/1, and valine 38 mg/1.     

Studies on the Utilization of Hydrocarbons by Microorganisms

In the course of study on the utilization of methyl-substituents of mono-cyclic aromatic hydrocarbons by Pseudomonas aeruginosa S668B2, some organic acids and phenolic compounds were found to be produced in culture broth.

Strain S668B2 was capable of producing ultraviolet absorbing and fluorescent substances from m-xylene. These substances were isolated in the form of crystal and identified as 3-methyl salicylic acid and m-toluic acid.

Strain S668B2 also produced ultraviolet absorbing and fluorescent substances from pseudocumene (1,2,4-trimethyl benzene). These substances were isolated in the crystalline form and identified as 3,4-dimethyl benzoic acid and 3,4-dimethyl phenol.

Strain S668B″ did not attack o-xylene. Under the similar conditions Pseudomonas desmolytica S449B3, which produced a large amount of cumic acid from p-cymene, did not oxidize o-xylene, but grew on p-xylene, m-xylene and 1,2,4-trimethyl benzene.

None out of 364 soil samples gave microorganisms which utilize o-xylene as a sole carbon source.     

Utilization of Hydrocarbons by Microorganisms

The accumulation of vitamin B₂ by Pichia guilliermondii Wickerham grown on hydrocarbon was investigated. Addition of the following materials stimulated vitamin B₂ production: metal ions such as ferrous, cobalt, manganese, and calcium ions; organic nutrients such as yeast extract and casamino acid; amino acids such as proline and arginine; vitamins such as B₁, nicotinic acid, inositol, and p-aminobenzoic acid. Optimal aeration rate for vitamin B2 production was obtained in a 500-ml shaking flask containing 75 ml of the medium.     

Utilization of Hydrocarbons by Microorganisms

As already reported, Corynebacterium hydrocarboclastus S10B1 was able to accumulate a good deal of l-glutamate in a thiamine-deficient medium at the sole expense of n-alkanes, but unable to form l-glutamate in a thiamine-sufficient medium though an abundant cell growth was observed.

α-Ketoglutaric acid and dl-alanine were found to be produced in the same thiamine-deficient medium in which l-glutamate was accumulated. Both products formed from n-tetradecane by this organism were isolated from culture broth, purified and identified. The optimum concentration of thiamine in the culture medium was 3 to 5 µg per liter for their production. The maximum yields of α-ketoglutaric acid and dl-alanine reached 16 g and 1.5 g per liter in the calcium carbonate-added medium, respectively. However, the addition of more than 30 μg per liter of thiamine extremely repressed their accumulation.     

Utilization of Hydrocarbons by Microorganisms

Corynebacterium hydrocarboclastus S10BI or S489BI can accumulate a good deal of Lglutamate in a thiamine-deficient medium at the expence of hydrocarbon, but can not form L-glutamate in a thiamine-sufficient medium in spite of rapid cell growth, as already reported. In order to establish the optimal culture condition for L-glutamate formation, the influence of the following factors was first studied: hydrocarbon concentration, pH control, nitrogen sources, temperature, aeration and supplement of metal ions or amino acids. Then L-glutamate production from a variety of hydrocarbons or petroleum fractions was examined. Sodium oleate was found to stimulate growth remarkably instead of thiamine. Ferrous ion was found to be obligatory for L-glutamate formation and was suggested to have taken part in the earliest step of n-alkane oxidation.     

Utilization of Hydrocarbons by Microorganisms

As already reported, strain S1OB1 was found to accumulate l-glutamic acid in a thiamine-deficient medium at the sole expense of hydrocarbon. In order to elucidate the biosynthetic pathway of l-glutamic acid, first of all, the incorporation of molecular oxygen into l-glutamic acid was examined. l-Glutamic acid accumulated under ¹⁸O-enriched atmosphere was separated, purified, identified and found to have been enriched with ¹⁸O. This results indicate the occurrence of oxygenase reaction involving addition of molecular oxygen. From a postulated biosynthetic pathway of l-glutamic acid, theoretical ¹⁸O content was calculated and compared with experimental one. ¹⁸O content of cells grown on n-alkane or glucose was also examined.     

Studies on the Utilization of Hydrocarbon by Microorganisms

An uracil-requiring mutant (KY7122) of Arthrobacter paraffineus KY4303 (ATCC15591) was found to accumulate orotic acid and orotidine on n-paraffine as a sole carbon source.

Both substances were definitely indentified as orotic acid and orotidine, from the results on column and paper chromatography, UV and IR absorption spectra, elementary analysis and analyses of hydrolysate.

Cultural conditions for orotic acid and orotidine fermentation were then investigated. As the carbon source n-paraffines from C₁₄ to C₁₆ were the most suitable for the fermentation, and sorbitol, fructose and mannitol were best utilized for the growth, and orotidine produced from them were twice as much as those from hydrocarbon. The addition of 200 mg of uracil and 2 g of C. S. L to 1 liter of medium was most optimal for orotic acid and orotidine fermentation.

Orotic acid and orotidine accumulations were enhanced by the addition of either l-tyrosine, l-leusine, l-threonine, gluconate or meat extract.     

Studies on the Utilization of Petrochemicals by Microorganisms

During the investigation of petrochemical-utilizing microorganisms, 60 strains of 1,2- propanediol-utilizing microorganisms were isolated from soil. Among these microorganisms, the strain PG-21-1 was found to produce lactic acid from 1, 2-propanediol and β-hydroxybutyric acid from 1, 3-butanediol. From the results of taxonomical studies, the strain PG-21-1 was identified to Arthrobacter oxydans.

The yield of lactic acid was increased up from 0,18 g/liter to 9.02 g/liter by improving the cultural conditions. The residual 1, 2-propanediol recovered from culture broth was found to be optically active.     

Studies on the Utilization of Petrochemicals by Microorganisms

During the investigation of petrochemical-utilizing microorganisms, two strains (EGB-17b, EGB-24b) were found to produce two kinds of exocellular polysaccharides from ethanediol.

From the results of taxonomical studies, the strains EGB-17b and EGB-24b were named to Arthrobacter simplex (Jensen) Lochhead var. viscosus n. var. Namely, the strain EGB-17b was very similar to the strain EGB-24b in taxonomy.

But main components of the polysaccharide produced by the strain EGB-17b were rhamnose, mannose and glucose, while main component of the polysaccharide produced by the strain EGB-24b was glucose.

The maximum yield of the polysaccharide produced by the strain EGB-17b was 10.6 g/liter, while the maximum yield of the polysaccharide produced by the strain EGB-24b was 6.3 g/liter after the investigation of cultural conditions.     

Studies on the Utilization of Hydrocarbons by Yeasts

The growth of M. japonica in hexadecane or decane medium was markedly improved by dialysis culture and there existed a “growth-inhibitory factor” in the dialyzable material. This material inhibited the growth in hydrocarbon medium but not in glucose containing medium. Free fatty acids excreted extracellularly by dialysis culture in hexadecane medium were mostly palmitic, myristic and lauric acids in the culturing chamber, but almost exclusively lauric acid was found in the dialyzable material. These acids, assimilated themselves by the organism, inhibited the growth in hexadecane medium but did so only weakly in glucose. It was concluded that lauric acid was at least partly an active principle of “growth-inhibitory factor” in the dialyzable material in hexadecane medium.     

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.     

Utilization of Drilling Fluid Base Oil Hydrocarbons by Microorganisms Isolated from Diesel-Polluted Soil

Hydrocarbon-degrading microorganisms identified as Pseudomonas luteola, Pseudomonas alcaligenes, Pseudomonas aeruginosa, and Actinomyces sp. were isolated from diesel oil-polluted soils using an enrichment culture technique. The isolates grew luxuriantly on hydrocarbons, including crude oil, diesel, kerosene, engine oil, cyclohexane, and dodecanol. Naphthalene and pyrene were poorly utilized, while there was no growth on benzene. The organisms utilized drilling fluid base oil as the sole source of carbon and energy, with rapid exponential growth at a rate ranging from 0.015 to 0.094 h^?1. The concomitant doubling time was between 7.4 and 45.5 h. Gas chromatographic analyses of the culture revealed reduction in the height of the n-alkane peaks, confirming biodegradation of the compounds. Among the isolates, P. alcaligenes had the highest (99.4%) percentage hydrocarbon degradation. Remarkable (99.2% and 98.7%) hydrocarbon removal was also noted for P. luteola and P. aeruginosa, while the lowest (92.3%) value was recorded in Actinomyces sp. These bacteria with high degradative capacity for hydrocarbons in oil-based drilling fluids would be useful in bioremediation of a tropical environment, polluted with spent drilling mud and drill cuttings.     

Effects of Corn Steep Liquor and Thiamine on l-Glutamic Acid Fermentation of Hydrocarbons: IV. Utilization of Hydrocarbons by Microorganisms

The effect of nutrients of natural source, such as corn steep liquor, peptone, and yeast extract, on the fermentative production of L-glutamic acid from hydrocarbons by a Corynebacterium was studied. Corn steep liquor and meat extract were found to be remarkably stimulatory to L-glutamic acid production; about 5 g per liter of L-glutamic acid were accumulated in a culture broth containing 3% n-paraffins, 0.01% corn steep liquor, and mineral salts. Among nutritional factors contained in corn steep liquor, biotin had very little effect on the accumulation of L-glutamic acid, but thiamine was highly stimulatory to L-glutamic acid production. The optimal concentration of thiamine for L-glutamic acid production was 3 to 5 μg per liter, and for cell growth, 50 μg per liter. L-Glutamic acid was accumulated in negligible quantity when the amount of thiamine in the culture broth was sufficient to support abundant growth of bacterial cells.