Bacterial Aspects of the Origin of Petroleum ROBERT W. STONE The Pennsyloania State College, State College, Pa.
CLAUDE E. ZOBELL The Scripps Institution of Oceanography, La
Jolla, Calif.
T h e role that microorganisms may play in the formation of petroleum has only begun to be explored experimentally. From small scale laboratory operations i t is possible to conclude that bacteria may enter the process at several stages in petroleum genesis. I t is certain that microorganisms are responsible for the initial processing
of the great mass of plant and animal matter which forms the raw material for petroleum. It is also evident that microorganisms are present and may survive for long periods in the vicinity of oil deposits. But the key questioncan bacteria produce significant amounts of hydrocarbons other than methane-still remains unanswered.
MONG the many theories proposed to account for the origin of petroleum (4), it is generally agreed that oil n a s formed from organic matter. The consensus of geologists (6) seems to be that petroleum was formed in a marine environment under conditions of moderate temperatures and pressures which mag be duplicated in countless sedimentary beds scattered over the earth’s surface. If we accept the geologists’ conclusion that organic matter is the mother substance of petroleum we add as a corollary to this premise t h a t microorganisms must have contributed to the process of formation. The soil and marine microbiologist expects to find evidence of extensive and complex microbial activity wherever he finds organic matter deposited, whether it be in fresh or salt water, cold or warm temperature, aerobic or anaerobic atmosphere, and in moderation, high pressure or low, From the standpoint of the microbiologist as well as the chemist and geologist the critical question appears t o be, “HOT far along the road to crude oil can microorganisms conduct the r a v organic matter?”
and Schwartz (11) have reported finding bacteria in reservoir fluids. Although some samples of both subsurface fluids and ancient sediments have been taken in ahich no bacteria nere detected, the frequency with which organisms are found adapted to high hydrostatic and osmotic pressure indicates that there may exist bacteria indigenous t o the older sediments. The fact that many of these organisms are able to grow anaerobically, utilizing the sulfate ion as a hydrogen acceptor, adds further evidence of specialization to local environment. Recently a sulfate-reducing bacteria was isolated from depths of 9000 feet which was most active a t a pressure of 5000 pounds per squarc inch (23). Therefore it is apparent that some organisms are present in older sediments TYith the capacity to effect changes in their chemical structure. The physiological action of these organisms over long periods of time is still an open question.
PRESENCE OF MICROORGANISMS IN MARINE SEDIMENTS
Recent marine sediments support great nunibers of inicroorganisms representing a wide range of physiological types. With the exception of the specialized plant and animal pathogens, most of the bacterial genera described from terrestrial isolations have some counterpart in marine sedinirnts (18) The species found in large numbers, however, are generally limited t o those that can gron anaerobically as well as aerobically and that can survive a highly competitive environment. For these reasons yeasts, molds, and higher fungi, although detected sporadically, are greatly outnumbered by the leas specialized bacterial genera such as Pseudomonas, Vibrio,Spirillwn, Achromobacter, and Flavobacterium The number of organisms varies greatly according to the depth and age of the sediment. Recent sediments may contain up to half a billion microorganisms per gram of mud. As the depth of sedimentary samples increases, the number of bacteria decreases, usually dropping abruptly after the first fen- centimeters. Hovever living bacteria have been found in carefully collected cores taken near shore a t depths exceeding 150 feet Several investigators. including Bastin and Greer (5)and Muller 2564
ACTION OF BACTERIA ON MARINE ORGANIC MATTER
Since the presence of bacteria in marine muds can be accepted as a matter of course and there appears to be adequate evidence of a t least some bacteria in the older sediments, it is of consequence to examine the mrtabolic activities of these organisms. The general nature of the bacterial processing of raw organic matter is in the direction of breaking down the cellulosic and protein structure to give simpler compounds such as carbon dioxide, hydrogen sulfide, hydrogen, ammonia, and the lower fatty acids, alcohols, and amines ( 1 9 ) . These low molecular weight organic compounds are subject to further breakdovn and resynthesis by microorganisms. The extent and variety of changes have been too inadequately investigated, but it is apparent from a cursory examination of the known products of metabolism that bacteria tend to leave organic compounds bearing carboxyl, hydroxyl, amine, and sulfhydryl groups rather than to accomplish the complete removal of the oxygen, nitrogen, or sulfur atoms required to produce hydrocarbons. The one notable exception is the production of methane. Methaneproducing bacteria are common in all types of mud samples, fresh water or marine, aiid have the ability to reduce carbon dioxide in the presence of hydrogen according to the reaction (8)
COS
+ 4H2 +CH, + 2H20
Methane production is demonstrable iindrr almost any type of
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PETROLEUM-ORIGIN anaerobic environment subject to the action of bacteria. If methane is any help in explaining petroleum genesis, the microbiologist can guarantee its supply in widespread locations and in quantity. I n addition to the simpler organic and inorganic products of bacterial metabolism there remains in marine sediments a heterogeneous and complex mass of organic matter subject t o slow microbial action. Waksman (17 ) applies the term “marine humus” t o the organic matrix of ocean sediments, and the key to the first steps of petroleum genesis may well lie in the chemical and physical changes taking place within this heterogeneous mixture. It is obvious and perhaps logical to suggest t h a t the complex organic plant and animal remains which form marine humus are slowly modified by bacterial action with the assistance of increasing pressure and possibly radioactivity to eliminate most of the oxygen and nitrogen a t o m , thus forming petroleum. The difficulty is t h a t a t present we do not have sufficient experimental evidence either to support or to deny this view. The complex nature of the chemical changes involved and a time factor somewhere in the range of IO8 times the usual laboratory interval with bacteria present formidable difficulties for the experimentalist. Extrapolation of biological data is hazardous even within reasonable limits and t o project our small amount of data over millions of years becomes mere guesswork. For these reasons the conclusions of the microbiologist concerning the ultimate fate of marine humus are highly speculative. PETROLEUM CONSTITUENTS PRODUCED BY BACTERIA
7 1 d
There have been a number of reports, especially in the last two decades, which make it apparent that bacteria do produce small amounts of higher hydrocarbons The hydrocarbons are for the most part unidentified mixtures, and the amount is of an entirely different order of magnitude than in the case of methane. Since methane is formed in quantity by bacterial action, it is of interest t o inquire whether microorganisms can produce any of the next higher homolog, ethane. Traces of ethane have been reported from time to time based on the analyses of swamp gases which may have been produced by bacterial action, and recently there has been evidence of traces of ethane in soil gas and fermenting plant material. In an experiment with fermenting algae, the senior author found ethane to be present in the evolved gas in a n amount equivalent to about 0.5 p.p.m. It is not certain that ethane in such gases is necessarily formed by bacteria and it would be unlikely to consider amounts of this quantity as a source of ethane in petroleum. The amount formed, however, is comparable in magnitude to some of the quantities of higher hydrocarbons found as a part of bacterial (or plant and animal) cell substances. There has been increasing evidence that bacteria as well as plants are capable of forming small amounts of hydrocarbon as part of their cellular substance or in connection with cell metabolism. During the analysis of large quantities of lipides from Mycobacterium tuberculosis, Anderson (1) found a small amount of nonsaponifiable matter which gave no color reaction for sterols. Although no further work was reported on this particular fraction, it is likely that a t least part of it was hydrocarbon. The nonpathogenic mycobacteria are fairly common in marine mud deposits and are high in lipoidal matter. The work of Haas et al. ( 7 ) in isolating certain carotenoid pigments from a species of Mycobacterium grown on mineral oil broth is of interest as many of these pigments are unsaturated hydrocarbons although not necessarily associated with petroleum. Jankowski and ZoBell (8) have reported small amounts of ether-soluble, nonsaponifiable material from cultures of Desuljvibrio cultivated on a sea water medium with fatty acids as sole carbon source. Carbon and hydrogen analysis showed t h a t the material corresponded closely to hydrocarbon.
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The preparation of large quantities of bacterial cells grown on a sea water medium containing peptone and a small amount of glucose was carried out by Stone and co-workers ( l e ) . About 400 grams of moist cells of a culture of Serratia marinorubrum, isolated a t Scripps Institution from marine sediments, yielded 6.5 grams of extract when treated with an ethanol-ether mixture. On saponification, 0.6 gram of a reddish oil was obtained which resulted in 0.2 gram of a colorless mixture of hydrocarbons after treatment with succinic anhydride and alumina. On a dry weight basis the following approximate analysis was found: total fatty extract, 6.5%; nonsaponifiable oil, 0.75%; hydrocarbon, 0.25%. A total of 10 kilograms of another marine isolate, Vabrio ponticus, was collected over several months. Chemical extraction yielded 78 grams of extract, giving 3.0 grams of nonsaponifiable oil. Further purification resulted in 0.6 gram of bacterial hydrocarbon. On a dry weight basis the approximate percentages for this organism were: total extract, 3.4%; nonsaponifiable oil, 0.15%; and hydrocarbon, 0.03%. An elementary analysis of the hydrocarbon showed 85.96% carbon and 1$.99% hydrogen. I n addition to the small amount of hydrocarbon found in the bacterial cell, there are generally small amounts t h a t may be extracted from fermentation liquors. Such experiments have been carried out a t both Scripps Institution and The Pennsylvania State College. I n a series of experiments using mixed cultures isolated from marine muds, a n attempt was made to show the formation of small amounts of volatile hydrocarbon from the breakdown of certain lower molecular weight organic acids. The fermentations were carried out with mixed cultures of marine bacteria that had been developed by several successive transfers on a sea-water medium enriched with a small amount of yeast extract and the particular compound under study. The experiments were conducted under nitrogen to provide anaerobic conditions, and the apparatus was constructed so t h a t nitrogen b a s slowly passed through a train to produce a gas-sweeping action on the fermentation liquor. The effluent gas was passed through appropriate absorbents for hydrogen sulfide, carbpn dibxide, and water and then through a liquid air trap t o condense any volatile hydrocarbon such as ethane. No detectable amounts of volatile hydrocarbons were found in experiments with lactic, propionic, and glutamic acids, leucine, tyrosine or phenylalanine. However, ether extraction of the fermentation liquors with subsequent treatment on silica gel columns to remove alcohols and nitrogen- and sulfur-containing compounds did show small amounts of hydrocarbon present. For example, the extraction of 3 liters of fermented broth made up with sea water and 0.4% Z-glutamic acid produced about 28 mg. of extract of which 9.4 mg. survived the silica gel treatment and could be labeled as “probably hydrocarbon.” A similar experiment with fermented sea water broth containing 0.4% I-leucine resulted in 4.5 mg. of hydrocarbon residue. Similar experiments have been carried out at Scripps Institution, and it has always been possible to find small amounts of nonsaponifiable oily fractions-a portion of which is presumably hydrocarbon. These small concentrations of hydrocarbon from fermentation liquor appear to be present in the same range of magnitude as those fractions found by extraction of the bacterial cells, and may be related. It is evident t h a t in all serious attempts to analyze the lipoidal fraction of the bacterial cell or fermentation liquor, either a small amount of hydrocarbon has been found or a t least a definite amount of nonsaponifiable extract t h a t presumably contains some hydrocarbon. It is suggested, therefore, that we may expect all bacteria t o form small amounts of hydrocarbon, the type and amount of compounds varying with the organism. We have as yet little information as t o the nature or purpose of this cellhydrocarbon or the mechanism by which it is formed. The lack of success in finding volatile hydrocarbons to complement the heavy nonvolatile oils and waxes from ether extract is disturbing
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to any one wishing to establish a case for the bacterial production of petroleum; but it is possible that the experimental methods used in researches have not been sensitive enough to detect the minute quantities present. In studies on the action of marine bacteria on amino acids, the chief products have been acetic acid and fatty acids corresponding to one carbon atom less than the particular amino acid under study. An interesting exception to this general behavior is found in the case of organisms attacking tyrosine. Although many cultures form chiefly p-hydroxyphenylacetic acid from this compound, some are able to form phenol and a few, especially in mixed cultures, form p-cresol (16). Machamer and Stone (IO) isolated two cultures that were able to form p-cresol from tyrosine in addition to others forming phenol It was reasoned that if a n analogous series of reactions could be carried out with phenylalanine, benzene or toluene should be produced.
C H z C H ( ” K ~ ) C O H0QH3 I
or
HO Tyrosine
p-Cresol
0
()C“
CH,CH(NH&OLH
u
Phenylalanine
Toluene
0
Phenol
or
Benzene
In view of this possibility extensive studies were made on phenylalanine, both by the use of enrichment cultures developed from marine mud with phenylalanine as the carbon source and with similar cultures which attacked tyrosine inoculated in a phenylalanine medium. The neutral extracts from large quantities of fermentation liquor were subjected t o analysis using the ultraviolet spectrum to detect the presence of an aromatic ring, but no trace of toluene or benzene could be found. Thus a n organism which was’able to effect the decarboxylation and reduction of tyrosine to give rise to an end product, p-cresol, that was close to a hydrocarbon, was not able to carry out the analogous reaction with phenylalanine even to a minute degree. Another example can be cited in the process of methane formation from acetic acid. Barker ( 9 ) has shown that methane may be formed by Methanobacterium omelianski, for the most part, a t least, by the reduction of carbon dioxide. Such a mechanism would entail no close analogous counterpart in the formation of homologs such as propane. Recently, Buswell and Sol10 (4a), using a mixed culture of organisms from anaerobic digestion, have shown that most of the methane formed from Cl*-labeled acetate was derived from the methyl group, and concluded that the mechanism may be decarboxylation. Stadtman and Barker ( I S ) using strains of both Methanosarcina and Methanococcus have likewise concluded that methane is derived from the methyl group of acetate. They believe that these compounds are more readily converted to methane than is carbon dioxide, but if an organism cannot utilize acetate or methanol it may resort to the reduction of carbon dioxide. If it is possible that the precursor of methane obtained from acetate is produced by some type of decarboxylation, this reaction would offer a n attractive mechanism for obtaining aliphatic hydrocarbons. This reaction can be represented by the general formula, RCOOH
+RH
+ COz
When K is H--, the equation becomes the decomposition of formic acid to COz and Hz, a reaction carried out by a large number of bacteria. When R is CH3-, the equation depicts the decarboxylation of acetic acid as described above. If R is 2566
CH3CH2CHz-, the reaction indicates the decarboxylation of butyric acid to give propane. Such a reaction would provide a plausible mechanism for producing hydrocarbons of higher molecular weight than methane. However, all attempts to demonstrate this reaction experimentally with bacteria have failed to produce any of the methane homologs. BACTERIAL MODIFICATION OF MARINE SEDIMENTS
One of the large gaps in our knowledge of bacterial metabolism is the effect of microorganisms on the complex organic matrix known as marine humus. It is entirely possible that biochemical changes which cannot be demonstrated to any extent JTith small molecular m i g h t compounds are much more likrly to be accomplished with complex colloidal material, especially over long periods of time. The difficulties of demonstrating these changes chemically are quite apparent. I n regard to the possible slow modification of complex marine organic matter, the action of bacteria on hydrogen is of particular interest. It is known that hydrogen is one of the common end products of the anaerobic decomposition of organic matter by many facultative and anaerobic bacteria; therefore a constant supply is available. Molecular hydrogen is used by bacteria for several types of reactions (21) and is probably even more active under conditions of the increased hydrostatic pressure existing in marine deposits. Many of the anaerobic and facultative types of bacteria, in which marine sediments abound, are known to produce hydrogenase, the enzyme which activates molecular hydrogen. The possiole effect of bacterial hydrogenase in contact with the complex organic matter of marine humus over long periods of time may have important implications in petroleum genesis. The tremendous time factor plus the colloidal and complex nature of the substrates involved make the hydrogen effect a most difficult one to evaluate experimentally. Researchers a t both Scripps Institution and Pennsylvania State College have studied the effect of hydrogen under varying gaseous and hydrostatic pressures, and both groups have found that hydrogen affects fermentation and growth. I n mixed cultures of marine bacteria under an atmosphere of hydrogen, i t is common to note an increase of hydrogen sulfide production. Machamer (9) also noted a change of type of products with bacterial fermentations conducted under hydrogen pressure. I n general, a pressure of 10 atmospheres of hydrogen increased by 50 to 100% the amount of neutral ether extract formed over the nitrogen control, and pressures in the range of 50 atmospheres caused some inhibition. At the end of the fermentation the mixtures were swept with nitrogen gas conducted through a liquid air trap, but there was no evidence of any tendency to form detectable amounts of volatile hydrocarbons under these conditions. The fact that in cases where tyrosine, phenylalanine, leucine, and valine were fermented under hydrogen pressure and found to give small but definite increase in extractable lipids is indicative that the presence of hydrogen under hydrostatic pressure may be important in evaluating the complex reactions in marine sediments. BACTERIAL MODIFICATION OF PETROLEUM HYDROCARBONS
Virtually all kinds and classes of petroleum hydrocarbons have been shown to be susceptible to bacterial oxidation undep favorable conditions ( 2 0 ) . I n addition, various petroleum fractions and crude oils have been found to be attacked by bacteria. Although in marine sediments conditions are perhaps too anaerobic for most of the organisms that are commonly associated with hydrocarbon oxidation to function with material success, it has been shown that the sulfate-reducing organisms which use the sulfate ion as a hydrogen acceptor may still carry on some hydrocarbon oxidation in a n anaerobic environment. It is also possible t h a t in the rapid initial breakdown to which
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PETROLEUM-ORIGIN marine organic matter is subjected, a portion of the cellular hydrocarbon may be destroyed in the complex variety of oxidation-reduction reactions involved. Scattered experiments on the amount of hydrocarbon or lipides remaining after marine algae have been subject t o decomposition by bacteria (6, 1 2 ) indicate that these fractions may increase; however there is not enough experimental data to make any conclusive statement on the fate of small droplets of oil in colloidal sediment over a long period of time. Tausson and Shapiro (16) found that bacterial activity affected the index of refraction, iodine number, saponification number, density, and physical appearance of petroleum. Similar effects have been noted by the authors. One point concerning bacterial modification of petroleum should be emphasized -namely, that no bacterium can attack any hydrocarbon or oil droplet unless it has a water interface available for growth. Therefore, once the individual droplets of oil are able t o coalesce and collect in sufficient quantity t o present a limited surface, the danger of destruction or modification from bacteria becomes of decreasing importance. Bacteria may be responsible in part for the formation of water emulsions of oil by the formation of fatty and naphthenic acids from hydrocarbons a t the oil-water interface. I n an aerobic environment under favorable conditions, bacteria are capable of bringing about considerable destruction of petroleum hydrocarbons. This may help to account for the absence of oil under many environments and its disappearance from sediment samples stored in the presence of free oxygen. I n anaerobic environments, characteristic of petroliferous sediments, crude oil may be slowly and perhaps selectively attacked a t the oil-water interface. Certain heavy metals, low redox potentials, high concentrations of hydrogen sulfide and toxic products of bacteria themselves would tend t o limit bacterial activity. I n a closed system such as a petroleum deposit, the extent of bacterial action would be limited by such factorg, the lack of sufficient oil-water interface and competition for essential nutrients. Consequently the few surviving organisms would reach a state of equilibrium in which the rate of reproduction would be drastically curtailed and regulated t o some degree by the rate of diffusion of toxic products. The over-all effect of microorganisms on any well stabilized petroleum deposit during such a period would be relatively slight.
theory t h a t oil was formed from marine organic matter then we must also accept the fact that bacteria are responsible for the initial processing of this raw material. Bacteria produce large quantities of methane and in addition small amounts of higher oils and waxes as part of their cellular substance. The slow accumulation of these small packets of hydrocarbon from both bacteria and the decomposition of marine plant life offers a possible means of petroleum genesis. One obstacle to this theory is that no low molecular weight or volatile hydrocarbons except methane have been detected from bacterial cells or fermentation liquor. Another objection is the difficulty of evaluating the long-time destructive effect of microorganisms on such heterogeneous material. Many of the bacteria present in marine sediments are able to utilize hydrogen and since this gas with hydrogen sulfide is a common bacterial product it is to be expected that a slow reducing action on the complex organic matrix termed marine humus may be maintained over long periods of time. It is attractive to suggest, that such modification of marine humus would eventually give rise to petroleum. There is nothing in our knowledge of bacterial metabolism t o deny this hypothesis; however, at present we have almost no experimental evidence as t o what constitutes the ultimate bacterial end product from marine organic matter. Bacteria may also function in modifying or destroying petroleum when it is possible for them to gain access t o it, and by their action may aid in the accumulation and flow of oil. ACKNOWLEDGMENT
Much of the work reported here was carried out in connection with Project 43 sponsored by the American Petroleum Institute. The authors wish to acknowledge the generous support of the member companies and their unstinting cooperation in providing samples and analytical facilities. LITERATURE CITED (1) Anderson, R. J., Physiol. Revs., 12, 166-87 (1932). (2) Barker, H. A., Proc. Natl. Acad. Sci. U.S., 29,184-90 (1943) (3) Bastin, E. S., and Greer, F. E., Bull Am. Assoc. Petroleum Geol., 14,153-9 (1930). (4) Brooks, B. T., Ibid., 20,238-300 (1936). (4a) Buswell A. M., and Sollo. F. W., J . Am. Chem. Soc., 70, 1778-80 (1948). (5) Clarke, H. T., and Mazur, A., J . Biol. Chem., 141, 283-9 (1941). (6) Cox, B. B., Bull. Am. Assoc. Petroleum Geol., 30, 645-59 (1946). (7) Haas, H. F., Busnell, L. D., and Peterson, W. J., Science, 95, 631-2 (1942). (8) Jankowski, G. J., and ZoBeil, C. E., J . Bact., 47, 447 (1944). (9) Machamer, H. E., “Effect of Gaseous Hydrogen Pressure Upon Dissimilation of Glucose by Bacteria,” thesis, The Pennsylvania State College (1950). (10) Machamer, H. E., and Stone, R. W., J . Bact., 54, 39 (1947). (11) Miller, A., and Schwartz, W., Arch. Mikrobiol., 14, 291-308 (1949). (12) Stadnikow, G . , Brennstof-Chem., 10, 477-80 (1929). (13) Stadtman, T. C., and Barker, H. A., J . Bact., 61, 81-6 (1951) (14) Stone, R. W., Proc. Am. Petrol Inst., 25, (No. IV) 101-2 (1945). (15) Tausson, W. O., and Shapiro, S. L., Mikrobiologia 3, 79-87 (1934). (16) Updegraff, D. M., J . Bact., 57,555-64 (1948). (17) Waksman, S. A., Soil Sci., 36, 125-47 (1933). (18) ZoBeli, C. E., “Marine Microbiology,” pp. 124-8, Waltham, Mass., Chronioa Botanica Co., 1946. (19) Ibid., Chap. X. (20) ZoBell, C. E., Bact. Revs., 10, 1-49 (1946). (21) ZoBell, C. E., Bull. Am. Assoc. Petroleum Geol., 31, 1709-51 (1 947). (22) ZoBell, C. E., World Oil, 127, No. 1, 35241 (1947). (23) ZoBell, C. E., and Johnson, F. H., J. Bact., 57, 179-89 (1949).
LIBERATION AND MIGRATION OF OIL
Bacteria growing in marine sediments may contribute t o the liberation of oil from oil-bearing materials in various ways. Of obvious import is the bacterial decomposition of the organic complex in which oil may be trapped. A second mechanism is the dissolving of carbonate or sulfate particles on which oil is absorbed by the action of bacterial acids or reduction (22). Thirdly, bacterial gases may tend to decrease the viscosity of oil, particularly when under pressure. Bacterially produced carbon dioxide, methane, and hydrogen would, in addition, help toexpel entrapped oil from pore spaces in reservoir formations. Fourthly, the tendency of many bacteria to grow on solid surfaces may help t o effect a removal of adherent oil from the surface of sedimentary particles. If the environment were suitable for significant bacterial growth, the production of fatty acids and other emulsifying compounds would help to bring about water emulsions of oil that may promote its flow. SUMMARY AND CONCLUSIONS Great numbers of bacteria of various types and biochemical activity are found in recent marine sediments and t o a less degree in older deposits and a t increasing depths. If we accept the
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for review March 1, 1952. RECEIEVED
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ACCEPTED September 22, 1952.
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