Oxidation of hydrocarbons by microorganisms isolated from soil1

JEROME J. PERRY AND H. WILLIAM SCHELD Department of Microbiology, North Carolina State University, Raleigh, North Carolir~a

Received October 31, 1967

Fertile soil contains significant numbers of microorganisms that can utilize hydrocarbons as a sole source of carbon and energy. Soils taken from oil fields contain an increased percentage of the total microbial population that can grow on petroleun~ hydrocarbons. Microorganisms isolated on a variety of non-hydrocarbon substrates were tested for the ability to grow a t the expense of n-tridecane. Those isolated on substrates that require an oxygenase for assimilation grew on the n-alkane in much greater numbers than organisms isolated on substrates that do not. One organism isolated on o-phthalic acid simultaneously induced to propane and o-phthalate after growth on glucose. The possible mechanism(s) involved are discussed.

Canadian Journal of Microbiology, 14, 403 (1968)

Most of the microbes used in investi- gations on hydrocarbon degradation and assimilation have been obtained by direct enrichment techniques (5). Such methods of isolation yield organisms with the desired property but tell little about the total micro- bial population iilvolved in the oxidation of these compounds under natural conditions. The capacity for hydrocarbon utilization in various pseudomonads (1, 16), actinomycetes (19), mycobacteria (12, 15), and multifarious stock culture microorganisms (5) has long been appreciated. The ubiquity of hydro- carbon-utiljzing organisms in culture collec- tions and in nature suggests that a predilec- tion for hydrocarbon substrates is widespread in microbial populatjons and not limited to a few unique organisms. The ability of micro- organisms selectively isolated on a broad spectrum of non-hydrocarbon substrates to oxidize hydrocarbons has not, as yet, been investigated (5).

We made this study to learn more about the relative numbers of organisms in soil that can use hydrocarbons as a sole source of carbon and energy. Organisms of numerous genera from diverse soils were isolated on an array of non-hydrocarbon substrates and tested for the ability to grow at the expense of hydrocarbon substrates. The inductive nature of the enzymes involved in the utiliza- tion of hydrocarbon and non-hydrocarbon substrates by one such isolate was investi- gated in some detail.

lPaper number 2248 of the Journal Series of the North Carolina State University Agricultural Ex- periment Station, Raleigh, North Carolina.

Materials and Methods Quantification of the microorganisnx in soil

samples that grow on selected substrates was obtained by diluting the soils in physiological saline and immediately pipetting 0.1-ml aliquots of the appro- priate dilutions onto the surface of a solid medium. The drop was spread over the agar surface with a sterile bent glass rod.

The plating media were nutrient agar (Difco) or L-salts (11) agar supplemented with the desired sub- strate. All non-hydrocarbon substrates were neutral- ized (where necessary) and added to the L-salts basal medium at a concentration of 0.2%. The inoculated plates were exposed to liquid hydrocarbons by plac- ing them in an air-tight container to which the appropriate substrate had been added (10 ml at the bottom of a 7-liter vessel). The vessel was sealed and 10% of the air removed to increase substrate vapor- ization. This method of substrate addition resulted in good growth with all n-alkanes and n-alkenes tested through CI8.

Organisms that utilized various non-hydrocarbon substrates for growth were isolated by selecting colonies from plates after 7 days growth and re- streaking on the same substrate until a pure culture was obtained. These isolates were tested for hydro- carbon utilization by inoculating a small amount of the agar-grown culture into 10 ml of L-salts medium in a 50-ml flask. Two drops of hydrocarbon were added and the flasks incubated on a rotary shaker for 5 days at 27 C. Since most hydrocarbon-utilizing organisms grow in irregular clumps on liquid media (particularly on n-alkanes), the measure of growth was turbidity.

Induction experiments were carried out in respirom- eter vessels using 0 2 uptake as the measure of in- duced enzyme synthesis. Substrate was added to respirometer vessels at 20 pmoles per vessel in the case of sodium o-phthalate; propane was added by flushing 100 ml of a 50150 v/v air-propane mixture through the respirometer vessels. A control vessel without cells was included to measure pressure changes from added propane other than those caused by substrate assimilation. Chloramphenicol was added to appropriate control vessels at a concentration of

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404 CANADIAN JOURNAL OF MICROBIOLOGY. VOL. 14, 1968

45 I.rg/ml to prevent the synthesis of inducible en- of organisms ca~ab le of hvdrocarbon utiliza- u

zymes. tion were in evidence. Simultaneous induction to propane and o-phthalate

oxidation was studied by incubating glucose-grown The microorganisms use cells in a ]-liter flask containing 250 ml of L-salts. hydrates, ketones, organic acids, amino 0.2% sodium o-phthalate, and 0.01% yeast extract acids, alcohols, and unsaturated cyclic com- (cell-concentration 1 mg/ml dry weight). The flask pounds as the sole source of carbon and was shaken for 3 hours at 30 C. The cells were energy. The organisms were obtained in pure harvested by centrifugation in the cold, then re- sus~ended in L-salts medium at a concentration of and tested for the grow 8 i g / m l dry weight. These cells were added to res- On wtridecane. n-Tridecane was chosen as pirometer vessels. After a 10-minute equilibration substrate because studies illdicate that it is period, substrates were added from the sidearm o r the most frequently used hydrocarbon (5). propane was flushed through the vessels. Oxygen uptake was followed at 30 C. Representative data on the proportion of

Conventional manometric techniaues were those isolates from each substrate that grew 011 the described in Umbreit et al. (17). n-alkane are resented in Table 11. More

The organism used in the induction studies was than 100 microorganisms were isolated on isolated from soil on o-phthalic acid (sodium salt). each of the substrates given in the table and It has tentatively been assigned to the genus Artl~ro- bacter since it produces Gram-variable rods and each was tested for growth On n- branched cells in young culture changing to coccoid tridecane. Various other isolation substrates - - cells as the culture ages. Large coccoid cells (cystites) occur in older cultures.

Results The microbial population of selected

fertile soil that grew on n-decane, n-hexadec- ane, n-pentane, and n-hexadecene-1 was compared with the numbers developing on nutrient agar and glucose-L-salts agar. Results are presented in Table I. The number of organisms growing on ilutrient agar and glucose agar was substantially the same. Organisms using 11-decane, 11-hexadecane, and n-hexadecene-1 are more prevalent in soil sainple 1 than organisms that grew on n-pentane. This observation was confirmed with other soil samples. Soils from the vicinity of active oil wells and from oil dumps were examined and a significantly greater number

TABLE I Bacterial population per gram of fertile garden

soil capable of growth on hydrocarbons and other substrates

Soil sample

Substrate* 1 2

Nutrient agar 1 090 000 1 180 000 Glucose 860 000 1110000 n-Hexadecane 25 000 24 000 n-Pentane 8 500 26 000 n-Decane 18 000 8 1 000 n-Hexadecene-l 28 000 9 000

*The basal medium, for all except nutrient agar, was composed of Lsalts with substrate added as described under Methods. Plates were examined after 10 days growth at 30 C.

were used, i.e., lactate, n-aIcohols, n-fatty acids, and 2-methyl pentane, but fewer organisms were isolated on these substrates. The number of microorganisins isolated on carbohydrates, alcohols, or organic acids that could use n-tridecane as a sole source of carbon and energy was less than 20y0 and usually about lOyo of the total. In every case microorganisms isolated on branched chain or cyclic saturated hydrocarbons grew on the n-alkane. Approximately 80% of the organisms isolated on I-~ui~decanone, cyclo- pentanone, and cyclohexailone grew on n- tridecane but a consistently low number did

TABLE I1 Ability of microorganisms

isolated on various substrates to utilize n-tridecane as the sole

source of carbon and energy

% of isolates capable of

Isolation growth on substrate n-tridecane*

Glucose 9 Glycerol 0 Tryptophan 16 Tyrosine 49 'esorcinol 3 8 Ca tech01 5 3 o-Phthalate 46 Acetone 44 2-Pentanone 80

'Tested by inoculating agar-grown cells into ID ml of L-salts + 2 drops substrate in a 5&ml Erlenmeyer flask. Growth was determined after 5 days shaking on a rotary shaker at 30 C.

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PERRY AND SCHELD: USE OF HYDROCARBONS BY MICROORGANISMS

500- PROPANE

400 - N

m u 300 - W

0 120 240 360 MIN MIN

FIG. 1. Induction of glucose-grown Arthrobacter sp. cells to the oxidation of propane and o-phthalate. Each vessel contained in a total of 2.4 ml; 8 mg dry weight cells, 20 pmoles substrate (0-phthalate) or a 50150 gaseous atmosphere of propane, 0.2 ml 20% K O H in center well, and 0.01% yeast extract. Open circles represent the vessel containing substrate and the closed circles the endogenous.

not grow on propane or tlie n-alkane cor- responding in carbon chain length to the isolation substrate.

When the substrate used for isolation was one that requires a molecular oxygenase for initial enzymatic attack, i.e. catechol, res- orcinol, or o-phthalate, a greater proportion of the isolates grew readily on iz-tridecane. A direct relationship between the chemical structure of the isolation substrate and the percentage of isolates that utilized iz-tri- decane is evident from the data presented in Table 11.

This observation was investigated further with one isolate, an Arthrobucter, isolated on o-phthalic acid. The organism grew readily on glucose and after growth on this sub- strate, induced to the oxidation of propane and o-phthalic acid as shown in Fig. 1. Control vessels with chloramphenicol (45 pg/ml) added did not induce to the oxidation of propane or o-phthalate. An experiment was conducted to determine whether cells grown on glucose and induced to o-phthalate utilization were simultaneously induced to propane oxidation. Results are presented in Fig. 2. The glucose-grown, o-phthalate- induced cells oxidized propane without lag and were unaffected by the presence of chloramphenicol.

MIN

FIG. 2. The oxidation of propane by glucose-grown Arthrobncter sp. cells after induction to o-phthalate oxidation. Open circles represent substrate and closed circles endogenous.

Discussion

The rapidly expanding literature on the oxidation and assimilation of hydrocarbon substrates by soil microorganisms attests to the widespread occurrence and ease of isolat- ing these organisms from nature (13, 18). Plate counts confirm the presence of a signifi- cant number of hydrocarbon-oxidizing organ- isms in fertile soil. Comparative counts on

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406 CANADIAN JOURNAL OF MICROBIOLOGY. VOL. 14, 1968

nutrient agar and hydrocarbons indicate grow on n-tridecane or the normal saturated that 1 to 3% as many as develop on nutrient alkane of the equivalent chain length. agar will grow on n-alkanes (Table I). Other Approximately 80y0 of the 2-undecanone and experiments have shown that the organisms 2-pentanone isolates did grow on the satur- isolated on hydrocarbon substrates are also ated hydrocarbon. capable of growth on nutrient agar. The A striking feature of the isolation experi- number that developed on the longer chain ments was the high incidence of n-alkane normal CI0-Cl8 alkanes was greater than oxidation by microorganisms isolated on those that grew on the intermediate length diverse aromatic substrates. lilcorporation n-pentane. This can be explained by the lipid of tyrosine, catechol, resorcinol, or o-phthal- solvent characteristics of the C5-C8 n-alkanes ate as the sole source of carbon and energy (10). The presence of unsaturation did not in the isolation medium always resulted in a adversely affect the number of organisms that grew provided the substrate was of long chain length.

Soils taken from oil well sites where sur- face petroleum hydrocarbons are present or from areas where oil spillage occurs contain a significantly greater proportion of hydro- carbon utilizers as compared with garden soil and also have a much higher population that will grow on nutrient agar. Komagata and co-workers (8, 9) obtained similar results from extensive studies on the microflora of oil fields in Japan.

The capacity for n-alkane oxidation in microorganisms isolated on various non- hydrocarbon substrates was examined. There is a wide variation in the capacity for n- tridecane utilization in these isolates depend- ent on the nature of the substrate used for primary isolation. Such isolation substrates as carbohydrates, fatty acids, various n- alcohols, and aliphatic amino acids yielded organisms that were capable of hydrocarbon utilization in the same order of magnitude as reported by Foster (5) for stock culture organisms. Use of branched chain alkanes always resulted in the isolation of organisms capable of n-alkane utilization as reported by Fredericks (6). Ketones gave varied results although earlier work (12) demonstrated that propane-oxidizing organisms must induce to utilization of this alkane after growth on any non-hydrocarbon substrates tested except acetone or isopropanol. This suggests that the methyl group of acetone induces the oxygenase necessary for hydrocarbon oxida-

high percentage of organisms capable of growth on n-tridecane. A colnmon feature of growth on these substrates is the requirement for an oxygenase to degrade and assimilate the coinpound (14). This observation was expanded to ascertain whether the assimila- tion of these compounds was inducible. An Artlzrobacter sp. that was isolated on o- phthalic acid, grew on glucose and induced to both propane and o-phthalic acid oxida- tion (Fig. 1). When glucose cells were in- duced to o-phthalic acid oxidation they oxidized propane without an induction period a n d the oxidation of propane was un- affected by chloramphenicol. The reverse experiment (propane induced to phthalate) was impractical because of the high accumu- lation of assimilation products of propane, resulting in an endogenous rate that obscured any possible phthalate oxidation. Similar results were reported by Gronlund and Campbell (7) in Pseudolnonas aeruginosa.

The sin~ultaneous induction of enzymes involved in propane oxidation during in- duction to o-phthalate oxidation is not readily explained on the basis of a single oxyge;lase-of such broad specificity. It has previously been demonstrated that oxygen- ases can be induced by the product as well as by the substrate of enzyme activity (3). Different catechol oxygenases can be induced in the same organism by different aromatic substrates (2) and an oxygenase for propane might be induced by a product of phthalate oxidation.

The high incidence of the hydrocarbon- tion. However, not all organisms isolated on oxidizing property in many stock culture acetone grow on propane or n-tridecane. A organisms (5) might be explained by frequent similar result was obtained with 2-undeca- exposure in their natural habitats to metabolic none and 2-pentanone isolates that did not intermediates that induce hydrocarbon oxy-

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PERRY AND SCHELD: USE OF HYDROCARBONS BY MICROORGANISMS 407

genases. The loss of hydrocarbon-oxidizing 7. GRONLUND, A. F. and CAMPBELL, J. J. R. 1966. Influence of exogenous substrates on the en- capacity in cells that were "'Iated On ethane do~enous res~iration of Pserrrlon~onns aer.lrpinosn.

after continued erowth on acetate (4) mieht J. gacteriol. 91: 1577-1581. -

u \ , u

also be explailled by the absence of such 8. I~ZUKA, H. and KOMAGATA, K. 1965. Micro- biological studies on petroleum and natural gas.

intermediates under these growth conditions 1". ~ i ~ ~ ~ f l ~ ~ ~ of Higashiyama oil mining and some selective advantage for mutants field. J. Gen. Appl. Microbial. 11: 15-23. that lack this unused capability. 9. IIZUKA, H. and KOMAGATA, K. 1965. Micro-

biological studies on uetroleum and natural gas.

Acknowledgments The assistance of Brenda F. Wishon during

this study is gratefully acknowledged. We are grateful for- suggestions and discussions with the late Dr. J. W. Foster of the Univer- sity of Texas during the early part of this work.

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