microbiology in the petroleum industry · in problems of bacterial corrosion, microbial...

MICROBIOLOGY IN THE PETROLEUM INDUSTRY

JOHN B. DAVIS AND DAVID M. UPDEGRAFFMagnolia Petroleum Company, Field Research Laboratories, Dallas, Texas

CONTENTSI. Introduction....................................................................... 215

II. Petroleum genesis ........ 216A. Modification of organic marine sedimentary material ... 216

1. Oxidative processes................2162. Formation of hydrocarbons in marine sediments ...................... 217

B. Factors which affect bacterial activity in sedimentary rock ... 2181. Depletion of nutrients ................................... 2182. Thermodynamic considerations ............................... 2193. Temperature and pressure ................................. 219

C. Evidence regarding biogenesis of petroleum ... 2201. Constitution of crude oil as opposed to known bacterial hydrocarbon products....... 2202. Observations concerning bacteria in reservoir rock ..................... 2213. Comparison of petroleum genesis with coal formation .................... 221

III. Petroleum exploration.................................................................... 221A. Geomicrobiological prospecting for petroleum ... 221

1. Soil microorganisms as indirect indices of petroliferous emanations ............. 2212. Bacterial products as indices of petroliferous emanations .................. 223

B. Microbial activity as related to geochemical prospecting for petroleum ... 224IV. Production of petroleum.................................................................. 225

A. Bacterial corrosion of iron and steel ................................................... 2251. Bacteria concerned...................................................................2252. Mechanism of anaerobic bacterial corrosion ......................................... 2273. Importance of bacterial corrosion in drilling and production of oil ............. 2274. Remedies for bacterial corrosion .............................. 228

B. Microbial decomposition of organic drilling fluid additives ... 2291. Fermentation of starch and other natural carbohydrates .................. 2292. Decomposition of sodium carboxymethylcellulose ...................... 229

C. Microbiological plugging of injection wells ... 2301. Mechanisms ........................................................................ 2302. Organisms....................................................................... 2303. Remedies....................................................................... 230

D. Oil release from petroleum bearing rocks by bacterial action ... 231V. Refining and manufacturing of petroleum products .... 233

A. Deterioration of petroleum products.................................................... 233B. Bacterial desulfurization and denitrogenization of crude oil and petroleum products .... 233C. Petroleum as a substrate for the industrial manufacture of chemicals................... 234

VI. References ........................................................................ 234

I. INTMODUCTION stituents into useful products receives little

The microbiologist within the past decade h attention. On the other hand, although the rolejoined the maay other technologists serving the of microorganisms in petroleum genesis (thepetroleum industry. Hlis endeavors are not as process by which petroleum is formed in nature)highly specialized as might be presumed, and the has been a long range study of interest to ge-purpose of this review is to indicate the scope of ologists for more than twenty years, this subjectpetroleum microbiology. In time, certain aspects has yet to pass beyond the realm of speculation.within this scope will likely be pursued with Exploration for petroleum deposits wasmuch greater intensity of effort. Today, the pioneered by the rank wildcatter who wasmicrobial conversion of certain petroleum con- followed and surpassed by the geologist. The

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JOHN B. DAVIS AND DAVID M. UPDEGRAFF

geophysicist followed the geologist and addedfruitful physical techniques. On the heels of thegeophysicist came the geochemist, who, in turn,is followed by the geomicrobiologist. While thegeochemist searches for chemical evidences ofpetroleum in the surface soils, the geomicro-biologist investigates the effects of microbialactivity upon these chemicals and, in addition,looks for specific microorganisms which feedupon hydrocarbons emanating from petroleumreservoirs.

Petroleum production, by which is meantdrilling for petroleum and recovering the productas economically as possible, was, in the earlydays, a crude and wasteful process. Later im-provements in technology made by petroleum andmechanical engineers resulted in large increases inefficiency and in great increases in the yield of oilfrom a given reservoir. Still later, the need forscientific understanding of the physical prin-ciples of petroleum production led to the em-ployment of research engineers, physicists,chemists, and mathematicians, resulting infurther improvements in its technology. Themicrobiologist has now joined these othertechnologists and finds a fruitful field for researchin problems of bacterial corrosion, microbialplugging of oil reservoir formations, fermentationof drilling fluid additives, and even in attempts toincrease oil recovery by bacterial action withinpetroleum reservoirs.

Petroleum products are routinely stored intanks over water and are subject to microbialattack and modification at the oil-water inter-face, which may lead to deterioration of theproduct. Microorganisms which attack par-affinic hydrocarbons, in particular, are many andvaried. Although the mechanism of hydrocarbonoxidation is virtually an unexplored field, themethodology for such investigations is littledifferent from that used in other intermediarymetabolism studies.At least one university laboratory is engaged

in such studies under a grant from a petroleumcompany, and it is hoped that other academicmicrobiologists will be attracted to this field inthe future. An opportunity is here for fruitfulfundamental research, which could provide abasis for applications in the refining and manu-facturing of petroleum products. Although thepetroleum companies do a certain amount offundamental research, this is the type of in-

formation which must, at present, come prin-cipally from the academic laboratories, while inthe petroleum industry microbiologists pursueinformation of a more applied nature. As timepass, more microbiologists should swell thethin ranks of those employed in the petroleumindustry, and thus permit more fundamentalwork to be done, with results of mutual benefit toscience and the petroleum industry.Because of developments of possible competi-

tive advantage in this little-known field, in-dividual petroleum companies have restrictedthe publication of their research findings untilthey can be adequately protected by patents.Since patents require from two to five years toissue, many developments in petroleum micro-biology are undoubtedly being retained in theconfidential files of oil companies. The eventualpublication of this material should immediatelymake certain aspects of this review obsolete.

II. PETROLEUM GENESIS

We shall first present a critical analysis ofpresent views regarding the role of bacteria in theactual formation of petroleum. No attempt hasbeen made here to compile an exhaustive reviewincluding a multitude of observations or state-ments, many of which would appear to beirrelevant based on present knowledge. Prac-tically all geologists agree that petroleum has anorganic marine sedimentary origin, but the modeof its formation is not known. Bacterial activityhas undoubtedly been involved in petroleumgenesis, but the extent to which bacteria havecontributed to the formation of petroleum isdebatable. Attempts to demonstrate hydro-carbon formation by bacteria under highlyartificial conditions have yielded only smallamounts of paraffinic hydrocarbons other thanmethane and practically none of the othermyriad compounds present in petroleum. Theconservative viewpoint is that bacterial action islimited to producing reduced organic mattermore closely resembling petroleum than theoriginal material and that the final stages ofpetroleum genesis are physicochemical.

A. Modification of Organic MarineSedimentary Material

1. Oxidative processes. It is axiomatic thatbacteria will oxidize sedimentary organic matterfor the purpose of gaining energy as long as

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physicochemical conditions permit. The mostefficient means of gaining energy from organiccompounds is for the bacteria to oxidize them inthe presence of oxygen, the carbon compoundsbecoming completely oxidized to carbon dioxideand water, thus yielding the maximum ofenergy. Such oxidation can take place only at thesurface of marine sediments since below thefirst few centimeters most sediments rich inorganic matter are depleted of oxygen. Thebacteria which are active in the oxidation ofsedimentary organic material in the presence offree oxygen are common forms found in soil andfresh water, usually facultative anaerobes such asPeeudmonas, Achromobacter, Flavobacterium, andSpirillum (75).

In the absence of free oxygen strictly anaerobicbacteria are active as well as the facultativeanaerobes. Certain anaerobic bacteria such as theDeoulfovibrio have been given much attentionregarding their role in petroleum genesis, es-pecially by ZoBell (104). These bacteria oxidizeorganic compounds in sediments and con-comitantly reduce oxidized forms of sulfur, usingthem as hydrogen acceptors. This process takesplace in the absence of oxygen resulting inoxidized compounds, energy for the Detdfovibrioand hydrogen sulfide. Because hydrogen sulfidereacts with metals to give a black sulfide pre-cipitate, the blackening of organic sediments isusually an indication of the activities of De-sulfovibrio. Other anaerobic bacteria may beactive in sediments, but little attention has beengiven them. Anaerobes other than Desufovibriooxidize organic compounds in the absence ofoxygen by using other organic compounds ashydrogen acceptors rather than sulfur com-pounds.The hydrolysis products of protein and

carbohydrate materials are the most rapidlymetabolized compounds, yielding C02, NH3,H2S, CHI and fatty acids depending upon thebacteria and the conditions involved (75).Other materials such as chitin and lignin aremore slowly decomposed by bacterial action andform the basis for the accumulation of marinehumus (92). Marine humus, like soil humus, ischemically ill-defined and may be describedsimply as a colloidal residual of undecomposedorganic matter which because of its resistance tooxidation very slowly succumbs to bacterialdecomposition processes.

Marine sediments are somewhat analogous tosoil in the sense that the bacterial flora and con-sequently bacterial activity are regulated by thetype of organic material available and theconditions existing at a given time. The bacteriafunction in both soil and marine sediments as abiochemical means of regenerating the elementsconcerned with the carbon, nitrogen, sulfur, andphosphorus cycles of nature, thereby prohibitingthe accumulation of dead organic matter on thesoil surface as well as on the ocean floor.

2. Formtion of hydrocarbnm8 in marine sedi-mente. The formation of petroleum hydrocarbonsin recent marine sediments by bacteria has notbeen demonstrated although it is known that thesediments do contain methane producingbacteria (83), and certain bacteria found insediments contain minute amounts of hydro-carbon as a part of their cell substance (75, 100).Trask and Wu (85) were unable to detect liquidhydrocarbons in sediments twenty years ago butreported small amounts of solid hydrocarbons.Smith (69) recently has detected small amountsof hydrocarbons in marine sediments usingchromatographic methods. Smith extractedsediments of the Gulf of Mexico with fat solventsand obtained about 0.031 per cent extractableswhich contained from 16 to 25 per cent paraffinhydrocarbons besides other hydrocarbons. Traskand Wu extracted sediments of the Florida Bayand obtained 0.062 per cent extractables whichcontained 8.9 per cent "paraffinaceous" material,and another of their sediment samples yielded0.087 per cent extractable material containing 27per cent paraffin. Trask and Wu apparently werelooking for liquid petroleum in the sediments anddid not attach much significance to their findings.Smith, on the other hand, with the modernmethods of chromatography has been able tostudy the characteristics of the sediment extractsand has found them actually to resemble pe-troleum, although, admittedly, not identicalwith it. The role of bacteria in the formation ofthese hydrocarbons is not known, but it is knownthat bacterial cells contain very small amounts ofhydrocarbons. In Stone's laboratory (75) 400grams of one bacterial cell mass yielded 0.25per cent hydrocarbons, but analysis of 10kilograms of another mass of bacteria revealedonly 0.03 per cent hydrocarbons. ZoBell in1951 (99) reported an "oily" material producedby the anaerobe De&dfovibrio, but so little of the

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material was available that an accurate identi-fication of it was imposible. In 1952, Dr. Hansonof the Mellon Institute examined a small amountof unsaponifiable material (67 milligras)submitted to him by ZoBell, which was describedas having been produced by Deulfoibrio as itgrew autotrophically in a synthetic mediumconsisting of carbonate, sulfate and other mineralalts in a hydrogen gas atmosphere. Dr. Hansonremarked: "Although it was necessary to foregosome of the usual techniques employed in han-dling materials of this type because of the smallamount available, some information on thechemical constitution of this oily extract wasobtained. Chromatography made possible theseparation of the total mateil into five distinctfractions. Although the first of these fractionscould not be analyzed further, it seems likely thatit was composed entirely of hydrocarbon material.The second fraction, as shown by infrared ab-sorption and the elementary analysis, is largelyhydrocarbon of paraffinic character, and if anynon-hydrocarbon components are present, theymust make up a very small part of the cut. Thethird chromatographic fraction was the first tocontain any amount of non-hydrocarbon con-stituents and these were largely oxygen-contain-ing substances. Unfortunately, the remainder ofthe fractions could not be studied furtherbecause of the small amounts, but they areundoubtedly composed of non-hydrocarbonmaterials. If any nitrogen or sulfur componentswere present in the original sample, they musthave been concentrated in the last fractions"(100). Thus, the material was apparently, inpart, the hydrocarbon fraction of the bacterialcells, similar to that of the bacteria examined byStone (75).This hydrocarbon material is synthesized by

bacteria as part of the baeterial cell and, as such,very probably exists in sediments a bacteriallyproduced constituent of the hydrocarbon foundthere. Furthermore, bacterial flora under thereduced conditions of recent marine sedimentswould have a tendency to attack the moreoxidized constituents of the sediments, thuspreserving the more reduced organic materialsuch as the lipid fracoion including the hydro-carbons. Smith (69) recently has shown that thepercentage of less polar (reduced) compoundsincreases with the depth of sediments; therefore,with time. Bacteria, because of their growth

requirement for nitrogen, would be expected toattack preferentially the nitrogenous compounds;the sediments, therefore, become progressivelyless rich in nitrogenous compounds with time anddepth of burial (33). Trask in his extensive work(86) showed that ancient sediments contain acarbon/nitrogen ratio of about 14 whereas thisratio for recent sediments is 8.5. These obser-vations may be considered as circumstantialevidence for bacterial activity, but the formationof petroleum by bacteria under adequatelysimulated or actual geological conditions has yetto be observed.

Treibs (87), who has studied organically richrecent deposits such as are found in the BlackSea, is of the opinion that oil is generated fromthe nonlipid organic constituents in the sedimentsas well as from the lipid constituents. Treibscalculated petroleum generally to be 85.7 percent carbon and 14.3 per cent hydrogen. Theatom ratios are thus 7.15 to 14.3, and the em-pirical formula can be considered (CH2)1 forall practical purposes. Organic matter was cal-culated by Treibs to be 55 per cent carbon, 7 percent hydrogen, 5 per cent oxygen and 3 per centnitrogen (based upon a logical mixture of carbo-hydrate, protein and fat of which living thingsare composed). The atom ratios of carbon, hydro-gen and oxygen then are 4.6-7-2.2. If oneassumes that carbon dioxide is the most logicaldecomposition product of this organic mixture,the organic material thus becomes depleted ofoxygen, and the ratio of carbon to hydrogenbecomes 3.5 to 7 or (CH2)1, the same as theempirical formula for petroleum. It may be con-cluded with regard to bacterial action that itdefnitely can and does remove carbon dioxidefrom dead organic matter under anaerobic con-ditions and thereby contributes to its ultimatereduction making it more like petroleum.

B. Factors Which Affect Bactrial Actiiy inSedimentary Rock

1. Depletion of nutrients. The first limitingfactor of bacterial activity in organic sedimentarymaterial is a lack of free oxygen. The oxygendemand of the sediments is apparently greatenough to deplete free oxygen at an early stage insedimentation (26). Lack of free oxygen resultsin the accumulation of sedimentary orgaicmatter which otherwise would be oxidized (ormineralized) ultimately to carbon dioxide,

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mineraLs and water (93). Bacterial decompositionunder anerobic conditions proceeds at a rela-tively slow rate, the hydrogen from the decom-posable (oxidizable) organic compounds beingtransferred through the bacterial enzyme systemto hydrogen acceptors such as oxidized organiccompounds or to forms of oxidized sulfur. Thus,general anaerobic bacterial activity ultimatelyleads to an accumulation of more reduced or-ganic material and hydrogen sulfide. As pointedout earlier, the activities of the sulfate reducingbacteria (De8ulfovibrio spp.) have received aa great deal of attention (104) whereas otheranaerobic bacteria which may be active in marinesediments have received little. Desulfovibrio,because of its peculiar metabolism, primarilyreduces oxidized forms of sulfur rather thanorganic matter.' If sulfate becomes limiting inthe environs, activity of De8ulfovibrio spp.ostensibly ceases. Connate waters associated withpetroleum reservoirs are notably low in sulfatealthough there are many exceptions (29).

Nitrogen in available form must be present inorder for bacterial activity to proceed. As thebacteria incorporate nitrogen into their celLs, it islargely converted into protein. Upon death ofthe cell and its subsequent decomposition theprotein nitrogen is converted into ammonia andis therefore susceptible to dissipation. In this waythe sediments could become depleted of avail-able nitrogen, and the consequence would be adecrease in bacterial activity.

Actually very little is known about the bac-terial activity that ensues in recent marinesediments, and practically nothing is known ofsuch activity in source beds productive ofpetroleum as we know it. The various stages ofpetroleum formation have yet to be clearly de-fined, and the bacterial flora, bactei activity,or the nutritive factors influencing such activityhave not been determined.

2. Thermodynamic conierations. It can bedemonstrated in the laboratory that anaerobicbacteria convert fatty acids into methane al-though the production of significant amounts ofhigher paraffin homologs has not been ac-complished (17, 83). This indicates that a bac-teriological reduction of already relatively

I There are small amounts of reduced organicmatter in the Desulfovbrio bacterial cells in theform of lipids and hydrocarbons, as already men-tioned.

reduced compounds is possible under anaerobicconditions. Furthermore, Stadtman and Barker(72) and Buswell and Mueller (17) have eluci-dated two mechansms for bacterially formedmethane dependent upon the bacteria involved.One mechanism involves a reduction of carbondioxide, the other a reduction of the methylgroup of methanol or acetic acid. Thus, it isconceivable that still other, longer alkyl radicalscan be reduced to corresponding paraffinichydrocarbons by anaerobic bacteria. Whilemost attempts to demonstrate this have failed-(17, 83), recently Davis and Squires (23) foundother gaseous hydrocarbons, including ethane,in the order of a few parts per million in methanefermentations.As organic matter becomes more reduced in

the sediments, presumably because of hydrogentransfer resulting from anaerobic oxidations, itbecomes progressively more difficult to oxidizebecause it is less susceptible to activation from athermodynamic standpoint. The anaerobic con-version of compounds such as tyrosine to yieldphenol or cresol, the alleged production of evenbenzene (33), and the already mentioned methaneformation from fatty acids indicate a bacteri-ological means of carrying organic matter to astate as reduced as petroleum; but these obser-vations are not indicative of anaerobic bacterialactivity in general or of such activity in sedi-mentary rock. There is a tendency for highlyreduced organic matter to resist bacterialdecomposition or modification under anaerobicconditions. Experimental work designed tosubject sedimentary material in various stagesof petroleogenesis to anaerobic bacterial actionshould serve to elucidate the affect of suchaction. Various ways of accelerating bacterialactivity may be used, such as adjustment of themineral concentration, temperature, pH, moistureand bacterial flora. Under optimal conditions foranaerobic bacterial activity a reasonable estimateof their potential function at various stages ofpetroleogenesis may be made, provided the dataare extrapolated as realistically as possible togeological conditions. The foregoing is no easytask, but approaches in the past have maderealistic extrapolation of data impossible due to adistinct separation of the bacterial system beingstudied from the sedimentary system beingconsidered.

3. Temperature and pressure. In 1946, Cox (21)

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proposed a "geological fence" secured to "posts",namely, organic matter, marine environment,temperature, pressure and time, within which theherd of facts pertaining to petroleum formationshould be brought. Observations relative tobacterial activity should logically be considered inthe light of known temperature and pressureranges existent in sedimentary rock. Definiteranges of temperature and pressure exist beyondwhich bacteria are no longer physically stable norbiochemically active.Cox points out that petroleum is probably

formed in sedimentary sections not exceeding5,000 ft in thicknes. The minimum temperatureexpected would be about 65 C and the maximumwould be slightly higher than 100 C. Maximumpressure due to an overburden of 5,000 ft wouldbe about 5,000 lb/sq in, hydrostatic head wouldbe 2,000 psi. Certain bacteria can metabolize attemperatures of 55 to 75 C, and some sporeform-ing bacteria can resist temperature up to 100 C(55). Furthermore, certain bacteria which do noteven form spores can apparently withstand amechanical pressure of 75,000 psi. However,definite changes in bacterial activity can beobserved under the influence of 3,000 psi. ZoBelland Johnson (106) give data to show that certainbacteria including sporeformers are killed atpressures of 7,500 and 9,000 psi in 48 hours.

Isolated observations of bacterial resistance torelatively high temperature and pressure areinsufficient evidence of potential bacterialactivity related to petroleum formation undergeological conditions. The term "barophilic"has been coined by ZoBell and Johnson (106) todescribe certain bacterial strains (some ofmarine origin) that grow at a pressure of 9,000psi. Careful scrutiny of their data reveals that nomarked differences exist in the pressure tolerancesof some terrestrial bacteria as compared with themarine bacteria. The interesting feature of theirexperiments was the concomitant increase inpressure tolerance with temperature over theranges of 1-600 atmospheres and 20-40 C.While bacterial activity may not be completely

prevented by geological conditions of temperatureand pressure as we know them, we have noknowledge as yet concerning such activity underthese conditions. What knowledge is availablepertains to very recent sediments which have nogreat amount of overburden, and even thisknowledge is extremely limited regarding the

specific bacterial flora and activities involved.

C. Eviden Regarding Biogenesis of Petroleum1. Constitution of crude oil as opposed to known

bacterial hydrocarbon products. Van Nes and VanWesten (91) point out that it is logical to assumecrude oil to contain cyclic compounds similar inbasic structure to those which occur in livingorganisms. Terpenes, sesquiterpenes, and poly-terpenes which appear to be polymerized iso-prene units occur abundantly in nature (es-pecially in plants), and these type compounds areamply represented in petroleum. Furthermore,the sulfur, nitrogen and oxygen containing com-pounds of petroleum very likely are similar tocompounds found in living nature although littlepertinent information regarding this is available.Bacteria could hardly be responsible for thebiosynthesis of the myriad compounds in crudeoil, e.g., the hydrocarbon components whichmake up about 95 per cent of petroleums con-sisting of varying amounts of paraffinic, naph-thenic and aromatic groups. While the con-stitution of the hydrocarbon fraction of bacterialcells is not known in detail (75, 100), it is certainlynot analogous with crude oil. Methane is theonly hydrocarbon known to be produced ex-tracellularly in any quantity by bacteria. Itappears, therefore, that their function in petrole-ogenesis is confined to some modification of theprecursor organic material rather than actualconversion of this material into crude oil.Another possible assumption, which seems

farfetched, is that bacteria utilize all proto-petroleum, converting it into their own cell sub-stance (containing small amounts of hydro-carbon), the nonhydrocarbon fraction of whichis reconverted again by other bacteria into cellsubstance containing small amounts of hydro-carbon, and so ad infinitum. The result,ostensibly, is an eventual accumulation ofhydrocarbons, a disappearance in proto-pe-troleum and a small residual bacterial flora. Itwould follow however, that the hydrocarbonfraction of bacterial cells very closely resemblespetroleum, while actually it appears to bealmost exclusively paraffinic (75, 100).

It is difficult to visualize the process of eventsjust described for many reasons, among thembeing the observation that crude oil containsmany compounds, including chlorophyll por-phyrin (87), which could not be formed by

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bacterial synthesis after sedimentation. Bacterialaction must at least be limited to the formationin sediments of those compounds which areconceivably formed by bacteria, regardless of thetime which bacteria are active in the sediments.

2. Observation concerning bacteria in reservoirrock. In 1952, Schwartz and Mueller (66) re-ported anaerobic bacteria in oil bearing sands inWestern Germany where "oil is recovered bymining". While they failed to find aerobicmicrobial forms such as mold fungi, actino-mycetes or strictly aerobic bacteria, they thinkthat the anaerobes could have invaded the oilfields "after opening of the mines". The authorsreferred to the observations made by certainUSSR and USA scientists regarding bacteria incrude oil and associated brines. They maintainedthat a discrepancy exists between the presence ofso many kinds or species of bacteria in reservoirfluids taken from oil wells and the presence ofonly a small number of strictly anaerobic formsin marine source beds. Schwartz and Muellerthink this may be caused by a secondary in-vasion of the oil reservoirs during drilling oper-ations. Drilling muds sometimes contain manymillions of bacteria per milliliter.

Ekzertzev (25) in 1951 described observationsmade of the bacterial flora in oil reservoirs nearVtoroi Baku in Russia. The depth of the samplesranged from 1,000 to 6,000 ft. He reported finding12 to 117 million bacterial cells per gram of drysample in oil bearing rock, but no bacteria fromhorizons devoid of oil. Ekzertzev mentionedtechnical difficulties in making the microscopicbacterial counts and gave no descriptions of thebacteria observed. It is conceivable that bac-terial cells would be difficultly differentiated fromoil globules in the oil bearing rock sample prepa-ration. Microscopic examination by bacteri-ologists of oil reservoir rock from other regionswould be of interest.

3. Comparison of petroleum genesis with coalformation. Plant materials consisting primarily oflignin and cellulose, which have accumulatedunder conditions adverse to microbial decompo-sition, appear to be the source of coal (36).One outstanding feature of these accumulationsis the preponderance of organic matter relativeto inorganic matter. The most accepted mecha-nism for coal formation is through the peat statewhere microbial action, though slow, operatesover long periods of time modifying the organic

matter and converting it into "humus". Theconversion of peat into lignite, then bituminouscoal, and finally anthracitic coal is conceded to bedue to physicochemical changes brought on bycompaction and heat during geologic time. Coalformation certainly is largely an in situ process,and the observed fossil imprints of leaves andother organized plant structures, e'ven in theadvanced bituminous statie of coal, point to itsorigin. It is assumed that while bacterial actionhas had some part in the modification of coal inthe peat state, such action could not be respon-sible for the later changes in physicochemicalcomposition which result in lignite, bituminousand anthracitic coal.

Petroleum formation, on the other hand, is notso well outlined. Without regard to a discussionof the differences in source material leading toeither coal or petroleum, suffice it to say thatpetroleum may or may not be formed in situand modification of it may actually take placeduring migration. The organic source materialof petroleum has very probably undergone somemodification by bacteria, just as has coal in thepeat state, but a most important distinctionexists in the respective environments of thesource materials during petroleum and coalgenesis. As mentioned above, coal originates fromorganic accumulations which contain relativelylittle inorganic matter, e.g., as in swamp con-ditions; therefore little inorganic surface is incontact with the organic matter. Petroleum in itsvarious stages of formation is presumed to havebeen constantly in intimate contact with a largeinorganic surface as a result of its marine sedi-mentary origin (15). The catalytic action ofsurface might influence a conversion into pe-troleum of the trapped organic matter whichescapes bacterial decomposition. Brooks (15)discusses the possible role of active surfaceminerals in petroleum formation at the moderatetemperatures prevailing in oil producing reser-voirs.

m. PETROLIEUM EXPLORATION

A. Geomicrobiological Prospecting for Petroleum1. Soil microorganims as indirect indices of

petroliferous emanations. Sohngen, one of thefirst bacteriologists to become interested inhydrocarbon oxidizing microorganisms, in 1906described an enrichment method of isolatingmethane oxidizing bacteria from soil (70).

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Methods for determining the presence of hydro-carbon oxdizing bacteria in soil have since beenpatented (34, 78) and assigned to petroleumcompanies. The premise is that detection ofhydrocarbon oxidizers will serve as an index ofhydrocarbons in the soil. Gaseous hydrocarbonsare believed to emanate from subsurface pe-troleum reservoirs into the soil.In 1943, Hassler obtained the first U. S.

patent (34), and in 1954, Strawinski obtained thelatest U. S. patent (78) describing methods ofprospecting for oil based upon measuring gasuptake by hydrocarbon oxidizing bacteria insystems containing soil, gaseous hydrocarbonsand oxygen. Russian workers, particularly thegeologist Mogilevskii, had proposed in 1940 theutilization of data obtained in bacteriologicalstudies of the subsoil for the purpose of detectingand contouring gas emanating areas (50).Bacterial surveys of oil and gas fields were madeby Mogilevskii and co-workers during the years1937-1939 in conjunction with gas surveys. TheRussian microbiologist, V. S. Butkevich, head ofthe Microbiology Department of the TimiryazevAgricultural Academy, participated in thiswork in which a total of more than 3,000 soilsamples was studied. Gas surveys previouslycarried out by the Russians had established thatonly negligible concentrations of gaseous hydro-carbons could be found in the soil, even overknown gas deposits, and they questioned whetherthese gases could serve as a medium for bacteria.Furthermore, as pointed out by Mogilevskii, thebacterial surveys, like the gas surveys, werecomplicated by the presence of mete in thesurface soi layers, the result of organic matterdecomposition rather than seepage from crude oiland gas reservoirs.Some of the physiological properties of the

methane oxidizing bacteria (found in the subsoillayers at a depth of two to three meters) werestudied under the direction of ProfessorButkevich. The bacteria were capable of de-veloping in an atmosphere containing methaneand oxygen in the presence of moisture andmineral salts. Hence, it was concluded that a lowconcentration of methane, in a steady supply, isthe determining factor making it possible formethane oxidizing bacteria to grow in the sub-soil.At the suggestion of Butkevich, Mogilevskii

had the soil samples analyzed for both methane

oxidizing bacteria and cellulose decomposing bac-teria (ostensibly methane forming bacteria).Particularly significant were those samples whichcontained methane oxidizers, in the absence ofcellulose decomposers. The ceilulose decomposerswere detected by observation of paper decompo-sition in a mineral salts medium together withthe soil samples during a prescribed incubationperiod. The determination of methane oxidizingbacteria was likewise qualitative. Samples of soilwere added to test tubes with a mineral saltsmedium and the tubes placed under a bell jar.A water seal was used through which methanewas introduced in admixture with oxygen. In-cubation at 34-35 C lasted for 12-14 days, andmethane oxidizing bacteria, when present, char-acteristically formed a pellicle on the surface ofthe mineral medium.

In spite of its simplicity, use of the methodresulted in detecting anomalies of methaneoxidizing bacteria in the subsoil which wereasociated with gas and oil producing areas.Some of these bacterial surveys preceded drillingoperations. Mogilevskii (50) concluded themethod had promise, but that a development ofa quantitative interpretation was desired. Astudy of bacterial indicators for higher hydro-carbons was suggested as well as a determinationof the optimum depths for soil g.

Later Russian workers followed the lead ofMogilevskii. In 1947 Bokova et al. (11) andSubbota (79) described experiments and fieldsurveys involving methane oxidizing bacteria aswell as other gaseous hydrocarbon oxidizingbacteria. Subbota continued to compare thecellulose decomposing bacterial flora with themethane oxidizing bacterial flora as had Mogilev-skii. Bokova and co-workers isolated not onlymethane oxidizing bacteria from the soil butalso ethane and propane oxidizing bacteria.These workers were particularly interested inspecificity relative to the particular hydrocarbonswhich could be utilized by the different bacteria.They reported that all methane oxidizing bac-teria isolated failed to utilize ethane or propane.These they classified as Methanomonas methanicadespite former reports, e.g., of Tausz and Donath(82), that this organism was capable of uti-lizing these hydrocarbons. Bokova and co-workers also reported the isolation of an ethaneoxidizing bacterium which could not utilizemethane, and a propane oxidizing bacterium

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which could not utilize ethane or methane.Subbota (79) pointed out that the bacterial

method of oil prospecting proposed by geologistMogilevskii recently came into use in oil ex-ploration in Russia, in conjunction with gassurveys. It was also used independently by aspecialized office of the Central Department ofEastern Oil Exploration and All-Union ScientificResearch Institute of Hydraulic Geology andGeological Engineering. Bokova and his associ-ates (11) reported the discovery of a gas field inStavropol Kavkaz and an oil pool in Ikhta, theresults of drilling to check bacteriological pros-pecting data.German workers, Schwartz and Mueller, have

likewise reported that bacteriological prospectingfor petroleum has promise and claim some successusing a quantitative dilution method. In a re-view (66) they refer only briefly to their ownunpublished observations, and no details aregiven.Another approach toward exploitation of

bacteria in petroleum prospecting has beenproposed by Sanderson (64), namely, the plantingof hydrocarbon oxidizing bacteria in the soil andobserving their growth in response to emanatinghydrocarbons. He maintained that it was pre-ferable to bury pure cultures at a depth of four orfive feet between sterile layers of permeablematerial (e.g., asbestos) and keep them out ofcontact with the soil. Technical difficulties of sucha procedure, particularly in view of the slow rateof growth of the bacteria in the presence of theminute amounts of emanating hydrocarbons,would be anticipated.' Varying. water level in thesoil because of unpredictable seasonal rainswould likely inundate planted bacterial culturesin many areas where such a method is employed.Practical success in detecting hydrocarbon gasemnation by the Sanderson method has notbeen reported in the scientific literature. For thatmatter, success in geomicrobiological prospectingfor petroleum on a commercial basis has not beenreported in scientific journals apparently, exceptby the Russians already referred to.

Several factors influence results of soil analysisfor hydrocarbon oxidizing bacteria. Methane oxi-dizing bacteria, particularly, have been observedand their function described in ecological rela

2 Ethane has been found by Rosaire (60) to bein the order of a few parts per billion in soils overoil and gas fields.

tionships totally unrelated to petroliferousemanations (1). Anomalies in the abundance ofmethane oxidizing bacteria in the soil must there-fore be scrutinized carefully before they are givensignificance as an index of petroleum-gas ema-nation. The adaptive ability of bacteria to utilizeorganic compounds, including hydrocarbons,must likewise be considered. Therefore, thedetection in the soil of bacteria which can oxidizethe various hydrocarbons in natural gas is notnecessarily an index of natural gas emanation.Seasonal fluctuations in the soil bacterial flora,including the hydrocarbon oxidizing flora, mustlikewise be considered, as pointed out by Sub-bota (79).

2. Bactrial products as indices of petroliferousemanations. In 1942, Blau (9) described a methodfor detecting a "color change" in the soil as anindex of bacterial action upon hydrocarbon gasesemanating from subterranean petroleum de-posits. The best reagent used for this purpose wasreported to be sodium peroxide although avariety of reagents were employed. According toBlau, the "color change" resulted with soilsamples containing hydrocarbon consumingbacteria which converted hydrocarbons intopolymerized and oxidized compounds of highmolecular weight that appeared to be carboxylicacids. He intimated that bacterial cells themselvescould account for the color reaction, describedas "deep red to light yellow", depending uponthe reagent employed.

In 1943, he pointed out further that these"bodies of high molecular weight" apparentlyfluoresce under the influence of ultraviolet light(10). Slavina (68) more recently studied thefluorescence of certain soil bacteria includnghydrocarbon oxidizers. Bacterium aliphaticumliquef which utilized pentane, hexanes,and heptane fluoresced brilliant green, whileMethanomonas methanica reportedly active onmethane, ethane, and propane did not fluorescein ultraviolet light. Evidence of practical successutilizing the above methods as prospectingparameters of petroliferous emanation ap-parently has not been published.A manifestation of surface soils, described as

"paraffin dirt", has long been associated withcertain oil and gas producing areas by petroleumgeologists. One assumption prevailed that adeposition of high concentrations of paraffingemanating from petroleum deposits resulted in

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the waxy appearing nature of the soil. However,Milner (49) gave a good description of thispeculiar material and pointed out its low hydro-carbon content more than twenty-five years ago.Recent studies by Davis (22) on a "paraffindirt" bed in Texas confirmed suspicions of otherobservers that microorganisms were responsiblefor a conversion of hydrocarbon gases intomicrobial cell material, thus accounting for thewaxy appearance of the soil in a localized area.Analyses of a representative "paraffin dirt"sample showed the dried soil to contain 17.6per cent organic carbon, 1.2 per cent organicnitrogen, 0.27 per cent lipid (organic mattersoluble in CCII), and 0.0038 per cent saturatedhydrocarbons. Microscopic examination of thesoil revealed an abundance of microorganismsincluding protozoa, filamentous fungi, yeasts,actinomycetes and bacteria. Among the bacteria,especially, were varieties capable of utilizingmethane and other gaseous hydrocarbons ascarbon sources. Mass spectrometer analysis of thesoil gas collected about six feet below the surfaceof the "paraffin dirt" bed showed the presenceof 1.4 per cent methane and 0.13 part per millionof ethane. Traces of other gaseous hydrocarbonswere indicated. It is believed that the organicmatter of "paraffin dirt" consists largely ofmicrobial cells, living and dead.Laboratory experiments consisting of passing

natural gas through two ordinary surface soilsfor a period of months resulted in a marked in-crease in organic content of the soils. The numberof microorganisms also increased markedly asthe gas flow continued. Both soils acquired awaxy, gummy appearance, and one of the soilsupon microscopic (wide field binocular) examina-tion was indistinguishable from specimens of"paraffin dirt" collected in the field. The othertreated soil, while similar, was not identical incharacter with the field samples, primarily, it isbelieved, because of an original difference in soiltexture.The fixation of organic matter in the form of

hydrocarbon oxidizing microbial cells as theyconsume the emanating hydrocarbons ostensiblyresults in a food source for other microorganisms.The latter thus feed indirectly upon hydrocarbonemanations. "Paraffin dirt" is a misnomer sincethe waxy appearance of the soil is not caused byparaffin, as is borne out by its low lipid andhydrocarbon content.

B. Microbial Acivity as Related to GeochemicalProspecting for Petroleum

Visible seepages of hydrocarbon gases andcrude oil at the surface of the earth have servedman as an index of subsurface accumulation ofpetroleum for many years. Practically all of suchseepages have been observed by this time, atleast in this country. Invisible seepages whichalso may serve as a means of finding oil must bedetected by technical means. Sokolov (71) andLaubmeyer (42) were among the first to investi-gate methods of soil gas surveying as a meansof geochemical prospecting for petroleum.Sokolov, in about 1930, began investigating gassurveying in Russia. Soil gas was assayed forgaseous hydrocarbons, including methane, usingan intricate hot filament (combustion) means ofmeasurement. Over known oil and gas depositsthe range of hydrocarbons found was from0.0001-0.2 per cent of the soil gas. It is significantthat among Sokolov's collaborators was Mogilev-skii who later, in 1937, proposed that bacterialsurveys be made as a means of prospecting forpetroleum. While Sokolov appreciated the factthat anaerobic bacteria in the soil producedmethane which could mask the micro appearancesof petroleum gases coming from subsurfacereservoirs, it was his associate, Mogilevskii, whomaintained that due to the preponderance ofmethane in natural gas, anomalies in methaneoxidizing bacteria were significant if observed atdepths ordinarily below organic matter decom-position in the soil (50).American investigators (37, 45, 61) became

interested in the observations of Sokolov and ofLaubmeyer and began their own geochemicalsurveys. Rosaire (61) in particular was an activeproponent of geochemical prospecting based uponsoil analyses. He was especially interested inhydrocarbon gases, such as ethane, propane,and butane, which may be considered "direct"indices of petroleum because of their practicallyunique origin. He showed further interest insecondary products arising from the oxidationand polymerization of these emanating gases.Rosaire points out that these secondary products(called "soil waxes") resemble hydrocarbons butchemically they are not true hydrocarbons. Theirmolecular composition, mode or rate of forma-tion has not been clarified. It is interesting thatRosaire, Horvitz (37), and McDermott (45)along with others (62), in discussing factors

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MICROBIOLOGY IN PETROLCUM INDUSTRY

affecting geochemical prospecting, did not con-sider microbial activity as a possible means ofeither modifying or destroying the index hydro-carbons.

Since it has long been agreed that methane insoil may have either a biological or a petroliferousorigin, geochemists in the U. S. have had atendency to shun measurements of methane asbeing nonsignificant. It should be pointed out,however, that there is no knowledge of theactual amounts of biomethane produced inordinary soils. Rosaire (61) likewise referred toethylene of biological origin (e.g., ripening fruits,plant tissues) as a factor to be considered ingeochemical prospecting. More recently ethyleneformation by filamentous fungi has been shownby Nickerson (51) and Williamson (94). Ethyleneand other olefins have been observed in naturalgases only rarely and in small amounts. Buswelland Mueller (17) in 1952 reiterated that ethaneand higher hydrocarbons had not been observedin bacterial methane fermentations and that ifpresent must be in concentrations less than 20parts per million of the partially purifiedmethane.3 Thus, for all practical purposes onewould assume that ethane in the soil is principallyof petroleum origin and that ethylene has prin-cipally a biological origin. Interestingly enough,McDermott (45) reported both ethane andethylene in concentrations of 0.02 to 0.10 ppmby weight in the soil over oil fields.

Horvitz (37) in discussing "soil wax" indicatedthat it was observed in a thousand to ten thou-sandfold greater concentration in soil than thelighter constituents such as ethane, propane, andbutane. While a true knowledge of "soil wax"was admittedly lacking, he maintained it was"empirically significant material", implying thatit was a geochemical parameter of importance.Knowledge of the chemical characteristics ofthis organic material would be required beforeeither speculation or experiments could relate itto microbial activity in soil.

IV. PRODUCTION OF PETROLEUM

A. Bacterial Corrosion of Iron and SteelIron and steel, as well as other metals, corrode

in aqueous media principally because of electro-lytic action resulting from differences in potential

I Davis and Squires (23) detected ethane inmethane fermentations in concentrations rangingfrom 0.1 to 7 ppm.

between anodic and cathodic areas. ZoBell (105)pointed out many ways in which bacteria maycontribute to the corrosion of iron and steel.He emphasized the multiplicity of interrelatedchemical, mechanical, electrical, and biologicalmechanisms that combine to cause corrosion, andconcluded that the worst and most extensivework of bacteria is of a nonspecific nature suchas producing acidic microspheres, oxygen con-centration cells, surface charges, or hydrogensulfide. This is no doubt true of the marineenvironments with which the author was pri-marily concerned, and the petroleum industryhas to contend with this severely corrosiveenvironment in its offshore drilling structures,pipe lines, and tankers. Marine paints andcathodic protection are the principal methods ofcombatting marine corrosion. The complexnature of this environment usually makes itimpossible to evaluate the extent to whichbacteria contribute to corrosion. This mayaccount for the paucity of published informationabout the corrosion of iron and steel underaerobic conditions.The role of bacteria in the corrosion of iron

and steel under anaerobic conditions is betterunderstood. Although the oil and gas industriessustain an enormous annual loss through theanaerobic corrosion of iron and steel (31, 32, 74),it is only recently that the role of bacteria in thisprocess has been appreciated by the petroleumindustry (see figure 1). As early as 1934, however,von Wolzogen Kuhr and Van der Vlugt (39)presented an explanation of anaerobic bacterialcorrosion which is generally accepted today.

1. Bacteria concerned. Sulfate reducing bacteriacapable of utilizing molecular and cathodichydrogen are the principal agents of anaerobicbacterial corrosion. Since their discovery byBeijerinck in 1895, investigations have revealedthat these bacteria are abundant in soil, sedi-ments of fresh water and marine origin, sulfursprings, and mineral waters, including oil wellwaters. Starkey and Wight (74) and ZoBell andRittenberg (107) have reviewed this literature indetail. The sulfate reducing bacteria are obligateanaerobes. Shturm (67) has reported the aerobicgrowth of sulfate reducing bacteria, but Grossmanand Postgate (30) pointed out that Shturm'sresults may be explained by the fact that sulfatereducing bacteria will grow in culture mediaexposed to the air, provided that sufficientsulfide or other reducing agent is present. Our

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Fig. 1 Fig. 2Figure 1. Section of oil well tubing cut apart longitudinally to show p)itting andi l)erforat ion

characteristic of anaerobic corrosion by sulfate reducing bacteria.Figure 2. Apparatus for the study of oil release by sulfate reducing bacteria. The glass tube in the

center is packed with Ottawa sand, the vessel to the upper left contains crude oil, and that to theright aqueous nutrient medium. The tube at the right goes to It vacuum p)ump. The entire aPparatusmay be autoclaved, and the sand pack can then be saturate(l with meassured volumes of oil andwater under aseptic conditions.

owni experience with sulfate Ireducing bacteriafrom widely scattered habitats confirms the factthat aerobic sulfate reducing bacter ia are riareoI nonexistent (89).Breed et al. (12) list three accepted species of

sulfate rieducing bacteria: Desulfovibrio desul-furicans, D. rubentschickii, and D. aestuarii. D.desulfuricans and D. rubentschickii are character-ized as species preferring a low salinity medium,i.e., less than two per cent sodium chloride, whileD. aestuarii grows preferentially in sea water oIthree per cent salt media. D. rubentschickiidiffers from D. desulfuricans only in being ableto utilize certain organic acids (acetic, propioinic,and butyric) as energy sources which are notutilized by D. desulfuricans. Starkey (73) de-scribed Sporovibrio desulfuricans, a thermophilicsporeforming strain. ZoBell, cited in Breed et al.(12), concludedl that sporefoimatioin is the excep-

tion rather than the rule among sulfate reducingbacteiia, a statement with which we concur.M\1iller has shown that both fresh water andmarine strains prooduce the greatest amount ofhydrogen sulfide in media containing about oneper cent sodium chloride. All strains teste(lproduced more than 2,000 mg of hydrogen sulfideper liter of medium when supplie(l with essentialminerals, lactate as an energy source, and sulfateas a hydrogen acceptor. :Miller (47) reported thatsulfate rieducing bacteria require an unknow!ngrowth factor (or factors) found in yeast extractor other natural materials. None of the knownbacterial growtth factors or amino acids could besubstituted for the natural material. Baumaniiand Denk (5) and Postgate (56) reported es-sentially the same results.

In nature, sulfate reducing bacteria, normallyutilize sulfate as a hydrogen acceptor, thus

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oxidizing organic matter or molecular hydrogen(19) as an energy source. In addition to sulfate,sulfite, thiosulfate, tetrathionate, metabisulfite,and dithionite (57) and colloidal sulfur (18)may be used as hydrogen acceptors for growth.Contrary to earlier belief that some reduciblesulfur compound is essential for growth, Bau-mann and Denk (5) reported growth of purecultures of DesulfoMbrio utilizing nitrate as theonly hydrogen acceptor. Postgate (58) found 8strains of sulfate reducing bacteria of 12 testedwhich required no reducible sulfur or nitrogencompounds when grown with pyruvate. Ourown observations confirm this finding (89).

2. Mechanism of anaerobic bactrial corrosion.The mechanism of anaerobic bacterial corrosionproposed by von Wolzogen Kuhr and Van derVlugt (39) has not been amply confirmed (16,74). The following equations summarize theprocess:

1. 4Fe -- 4Fe+ + 8e (Anodic solution of iron)2. 8e + 8H+ -) 8H (Cathode)3. HsSO4 + 8H n H2S + 4H20

Depolarization by the oxidation ofcathodic hydrogen by sulfate reducingbacteria

4. Fe+ + H2S ± FeS + 2H+ Formation5. 3Fe+ +6 (OH)- ± 3Fe(OH)2 of corrosion

products6. 4Fe + H2SO4 +2H20 ! FeS + 3Fe (OH)2

Equation 3, implying the oxidation of cathodichydrogen by sulfate reducing bacteria, was con-firmed by Starkey and Wight (74) using enrich-ment cultures and by Butlin, Vernon, andWhiskin (20) employing pure cultures of sulfatereducing bacteria. In these experiments thebacteria grew in mineral salts media containingiron, utilizing cathodic hydrogen as their soleenergy source and reducing sulfate. Analysis ofthe corrosion products has confirmed the presenceof ferrous sulfide and ferrous hydroxide. Iron keptin a sterile medium did not corrode under theconditions of neutral pH and absence of oxygenmaintained in this experiment. It is well known,however, that corrosion proceeds in the absenceof bacteria in the presence of either acids oroxygen, or under the influence of electricalcurrents.

3. Importance of bacterial corrosion in drillingand production of oil. Anaerobic bacterial corro-sion is a common problem in the drilling for and

production of petroleum. Sulfate reducing bac-teria are peculiarly well adapted to growth insubsurface oil bearing formations, and have beenfrequently isolated from depths up to 3,090 ft(3, 4, 27, 28). Gahl and Anderson (27) foundthat pure cultures isolated from the deepest,highest temperature wells had the highestoptimum and maximum temperatures for growth(37 to 50 C) and that the cultures exhibited anoptimum salt concentration for growth whichshowed some correlation with the salt concentra-tion of the brine from the well from which theculture was isolated. These findings suggestthat the bacteria found were actually multiplyingin the oil producing formation. It is also possiblethat they were introduced during drilling opera-tions, and might have been multiplying in thewell casing or tubing, using cathodic hydrogen asan energy source. ZoBell (97) reported the isola-tion of sulfate reducing bacteria from cores ofLouisiana sulfur-limestone-anhydrite formationfrom a depth of 1,560 ft under experimentalconditions which render extraneous contamina-tion unlikely.Our observations on 162 core samples of oil

bearing rocks from Texas and New Mexicoshowed sulfate reducing bacteria in 26 samplesand facultative organisms in three (89). Manysamples appeared sterile, as they gave no growthin the media used. It may be concluded thatancient sediments ordinarily contain very fewviable bacteria but may contain appreciablenumbers of specialized types, particularly sulfatereducing bacteria, in certain localized environ-ments, such as in porous, oil containing rockswhich also contain interstitial water with thenecessary mineral nutrients. Sulfate reducingbacteria are found in most produced oil wellbrines, and in water supplies used for the sec-ondary recovery of oil by water flooding (aprocess for recovering additional oil from areservoir after all the oil economically recoverableby flowing and pumping has been produced),and for primary pressure maintenance. When aclosed system is used in the presence of ironpipes and sulfate, anaerobic corrosion is generallyfound (6, 41). In water supplies for water floodingor primary pressure maintenance, such bacterialaction is usually accompanied by the formation ofa turbid water containing bacterial cells andprecipitated iron sulfide, which clogs the pores ofthe formation rock and lowers the injection rate

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(13, 54). Both of these processes are costly to thepetroleum industry.Doig and Wachter (24) have described a suc-

cesion of oil well casing failures in a Californiafield. The casing corroded in localized areas,producing holes in the pipe, at depths from 900 to7,000 feet beneath the surface. The Y inchthick steel pipe corroded through in an averagetime of four years. It was necessary to cementthe casing to seal the hole, and then drill throughthe cement plug in each case. This exampleof bacterial corrosion is similar to the pipe linecorrosion extensively studied by Hadley (31, 32),Bunker (16), and Starkey and Wight (74), inwhich the bacteria attack the outside of the pipe.This type of corrosion is severe only where thesoil conditions are anaerobic, sulfate mineralsare present, and the pH is near neutrality, withoutside limits of 5.5 to 9.5 (74). Cast iron pipeundergoes graphitisation in which the iron iscorroded to ferrous sulfide and hydroxide, leavinga pipe which still retains its outward appearancebecause of the graphite present in the cast iron,but which is so soft that it can be easily cut by aknife. The papers of Hadley, an electrical engineer,established the importance of bacterial corrosionof pipe lines. A survey of pipe lines in Pennsyl-vania, Ohio, and New York revealed that from20 to 97 per cent of the pipe lines, depending onthe terrain, were attacked by anaerobic corrosion.A simple test for anaerobic bacterial corrosion,consisting of the release of H2S upon treatingthe corrosion products on the pipe with HCl, wasshown to correlate well with the presence ofsulfate reducing bacteria determined by cultiva-tion in lactate medium. Hadley concluded thatthis type of corrosion was severe only where thesoil was ordinarily water-saturated, and betweenpH 6.2 and 7.8. In the swamps and lowlands ofOhio, six inch welded pipelines lasted only sevenyears, on the average, because of bacterialcorrosion. It was concluded that anaerobicbacterial corrosion is second only to stray-currentelectrolysis as a cause of pipeline failure.

4. Remedies for bacterial corrosion. Anaerobicbacterial corrosion has proved a difficult processto combat. Posible methods of eliminating itinclude all means of elimiting the growth ofwlfate reducing bacteria: germicides, inhibitors,exclusion of sulfate, change in pH to a valueunfavorable for growth, prevention of anerobiosisby aeration, and removal of water from contact

with the iron. Protective coatings on the iron,corrosion resistant alloys, and cathodic protectionare other posibilities.

Germicides and inhibitors have been widelyused in the oil fields to eliminate or decreaseanaerobic corrosion. Formaldehyde was recom-mended by Menaul and Dunn (46) and by Latter(41) for reducing hydrogen sulfide corrosion inoil well equipment, particularly in the casing,tubing, rods, and pumps in producing oil wells.From one-half to two quarts per day of 37 percent USP formalin was injected into the annulusbetween the casing and the tubing. Menaul andDunn (46) found that KCN was also effectivealthough six other relatively nongermicidal com-pounds were tested and found to be ineffective.Although these authors attributed the protectiveeffect to a chemical film of undetermined com-position on the surface of the metal, the mainbenefit of the treatment may have been caused bythe inhibition of sulfate reducing bacteria.

Laboratory tests have shown that formaldehydeis an effective inhibitor of sulfate reducingbacteria and the associated corrosion at levelsof 10 to 50 parts per million of water (8, 54).Sodium cyanide is similarly effective at 10 partsper million (89). Quaternary ammonium com-pounds have been widely used as inhibitors ofvarious types of corrosion, including that causedby sulfate reducing bacteria. Breston andBarton (14) found that from two to four parts permillion of rosinamine acetate reduced thecorrosivity of water used for oil-field floodingfrom between 50 to 85 per cent, and also reducedthe count of both aerobic and anaerobic bacteria.

Field tests by Heck, Barton and Howell (35)showed that all of three quaternary compoundstested, Pur-O-San (alkyl dimethyl benzyl am-monium chloride), Arquad S (alkyl trimethylammonium chloride), and rosinamine acetate,gave good protection against acid corrosion.These inhibitors exert at least part of their effectby forming a film on the surface of the metalwhich brings about a high degree of resistance toattack, even by strong acids. Their effectivenessagainst corrosion by sulfate reducing bacteriahas not been adequately evaluated althoughBreston found them to be good agents forpreventing bacterial growth in flooding waters,and Latter (41) reported that Pur-O-San was aneffective agent for inhibiting bacteria and algaein flooding waters. Chromate ion, which has long

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been used to inhibit corrosion by dissolved oxygen,was found to be a good inhibitor of sulfate re-ducing bacteria and anaerobic bacterial corrosion.Since it retains its effectivenes over a long periodof time, it has been used in dilling mud aroundthe outside of oil well casing to provide long-term protection (89). It should be mentionedin passing that chlorine, long used to kill bacteriain water, is relatively ineffective against anaerobicbacterial corrosion because the sulfides producedby sulfate reducing bacteria react with and re-move the chlorine.

Other approaches to the control of bacterialcorrosion are applicable at times. The control ofpH in a range outside the growth range forsulfate reducing bacteria is feasible in drillingmuds and for certain flooding waters. Hunteret al. (38) reported that a pH above 9.0 effectivelyinhibited sulfate reducing bacteria. Many drillingmuds are highly alkaline (pH 10 to 13), and thusinhibit sulfate reducing bacteria in the vicinityof the drill pipe, casing, and equipment used withthem. The alkali is introduced primarily becauseit imparts desirable physicochemical character-istics to the mud, and bacterial control is co-incidental. The chemical research laboratory atTeddington, England, has been active in theinvestigation of preventive measures for anaerobiccorrosion since 1934 (20) and has evaluated manytypes of protective coatings for pipe lines.Standard coal tar enamels and hessian wrappings,even when dipped in bitumen, are relativelyineffective. A thick bitumen coating, when com-pletely covering the pipe, and special plasticcoatings show promise. Gravel packing, sur-rounding the pipe, is also good, probably becausethe gravel allows acce to air and preventsanaerobic conditions. Corrosion-resistant alloysand plastic pipe are good, but expensive. Ascosts are reduced, plastic and plastic-impreg-nated fiber-glass pipe may come into wide use inthe oil fields.

B. Microbi Decomposition of Organic DriUingFluid Additives

Oil wells are now almost always drilled withsome form of drilling fluid in the bore hole.The liquid suspensions used vary widely incomposition and may have either a water base,an oil base, or a mixture of the two comprisingan emulsion base. A wide variety of substances,both organic and inorganic, may be added to

drilling fluids to impart desired characteristics,and different types of fluids are used to overcomespecial problems encountered in drilling differenttypes of formations. The major functions of adrilling fluid are: (a) to lubricate the drill bit,(b) to cool the bit, (c) to carry away chips ofrock cut by the bit, (d) to plaster the walLs ofthe hole, thus preventing caving-in of looseformations and minimizing filtration intopermeable beds, (e) to apply hydrostatic pressureto the formation in order to prevent loss of oiland gas from the strata.

1. Fermentation of starch and other naturalcarbohydrates. Perhaps the most common type ofdrilling fluid is a "mud" comprised of a dispersionof clay in water. Various organic colloids arecommonly added to such muds to reduce therate of filtration of water through the mud cakelaid down on the walls of the borehole. The mostcommon of these water-loss reducing agents aregelatinized starch and sodium carboxymethyl-cellulose. Both are subject to microbial attack.Starch is rapidly decomposed by a wide varietyof microorganisms, including aerobic, facultative,and anaerobic forms. Some muds, particularlylime base muds, have a pH above 10.5 and aretherefore practically immune to microbial attack.Others of lower pH may support heavy bacterialgrowth. Thus, starch fermentation has become aserious problem, sometimes resulting in loss ofthe entire mud supply, and involving the risk ofserious damage to the well. At least two mudservice companies have developed highly effec-tive bacterial inhibitors, composed primarily ofparaformaldehyde, for preventing such fermenta-tions. Research toward the development ofimproved inhibitors appears desirable althoughpresent products are effective and fairly moderatein cost.

2. Decompositon of sodium carboxymethyl-cellulose. Sodium carboxymethylcellulose (CMC)has proved, in practice, to be relatively resistantto microbial deterioration in drilling muds.However, studies by Reese, Sui, and Levinson(59) have shown that molds, actinomycetes, andbacteria produce enzymes capable of attackingand partially hydrolyzing commercial grades ofsodium carboxymethylcellulose. The extent ofattack of enzymes on sodium carboxymethyl-cellulose was found to decrease as the degree ofsubstitution of the cellulose with sodium carboxy-methyl groups increased. The authors postulated

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that the enzymes tested would not attack sodiumcarboxymethylcellulose in which every anhydro-glucose unit in the cellulose molecule is sub-stituted with at least one sodium carboxymethylgroup. Recent investigations (89) have shownthat CMC degraded to the maximum extentpossible by the action of certain bacteria issuperior to the original CMC for use in certainmud systems; it confers approximately the samereduction in water loss, while it produces a mudwith better viscometric properties because of itslower average molecular weight. In other mudsystems, the bacterially treated product is lesseffective as its molecular size is too small to be ofmaidmum effectiveness as a water-loss reducingagent where the clay particles are larger becauseof partial flocculation of the clay.

C. Microbiological Plugging of Injection WellsPresent practice in the oil industry involves the

injection of large volumes of water into manydeep wells. Water flooding and primary pressuremaintenance are carried on by injecting waterinto oil reservoirs for the purpose of increasingthe recovery of oil. Salt water produced from oilreservoirs is frequently disposed of by injectingit into wells drilled into the same reservoir, oranother suitable porous formation. In all thesetypes of water injection, microorganisms presentin the water have frequently given rise to partialplugging of the injection well, thus decreasing theinjection rate, sometimes to the point at whichthe well becomes useless.

1. Mechanism. The cause of this plugging isfamiliar to every microbiologist who has em-ployed filters to remove microorganisms fromaqueous suspensions. Just as the pores of abacteriological filter becoming clogged with cellsresult in a decreasing filtration rate, the pores ofreservoir rocks may clog with microorganismscontained in the injected water. Where porousfiltration media are of uniform pore size, correla-tion of pore size with the size of microorganismswhich will plug the filter is relatively simple.The pore entry diameter must generally be atleast twice the diameter of the microbial cells forthe cells to pass through without serious plugging.When cells are spiral or elongated, the pore entrydiameter must be even larger, relative to thecell diameter, to prevent plugging.

Since petroleum reservoir rocks ordinarily

exhfibit a wide range of pore sizes, the problem isgreatly complicated. The pore size distribution ofreservoir rocks may be estimated by measuringthe volume of mercury injected into a clean, drysample by increasing increments of pressure. Theresults are plotted as a curve expressing thefractional part of the total pore volume filled bymercury as a function of pore entry diameter.Empirically it has been found that reservoirrocks containing an appreciable fraction of poreslarger than three microns will pass large numbersof sulfate reducing bacteria up to 0.6 Ju in diam-eter and 3 Ju long without serious plugging (89).Many reservoir rocks contain, principally, poresof larger diameter than this and are not seriouslyplugged by small bacteria. Others that contain alarge proportion of smaller pores may be plugged.

2. Organism. The potential plugging micro-organisms in injection water vary with theconditions under which the water is stored.Water kept in open pits exposed to sunlight maycontain algae and photosynthetic bacteria, as wellas autotrophic and heterotrophic aerobic andfacultative bacteria. Beck (8) found algae andspecies of Crenothrix, Beggiatoa, and Pseudo-monas in Pennsylvania flooding waters insufficient numbers to make the water turbid.Storage of injection waters in the dark willeliminate photosynthetic microorganisms, butnot the others. The use of a closed system, inwhich water is pumped from deep wells into theinjection wells, without exposure to air, elimi-nates aerobic but not anaerobic bacteria. Undersuch conditions anaerobic bacteria, particularlysulfate reducing bacteria, may cause plugging.

3. Remedies. Many different methods oftreatment have been developed to render waterfit for injection. The East Texas Salt WaterDisposal Company employs an elaborate purifica-tion system comprising skimming off the oil,aerating the water, allowing sediments to separatein settling tanks, filtering, and finally chlorinatingto eliminate further microbial growth. Eventhis procedure is not always effective. Beck (8)found that 10 parts per million of formaldehydewas effective against sulfate reducing bacteriain laboratory tests. Heck, Barton, and Howell(35) showed that from two to ten parts permillion of any of several quaternary ammoniumcompounds were effective, in field tests, inreducing bacterial numbers in flooding waters.

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D. Oil Release from Petroleum Bearing Rocks byBaterial Action

In 1944, C. E. ZoBell, the Director of anAmerican Petroleum Institute research projecton the role of bacteria in the origin of petroleum,applied for a patent on a bacteriological processfor treatment of fluid bearing earth formations.The patent, issued in 1946, was dedicated tothe public by the American Petroleum Institute(95). Briefly, the principle involved is thetreatment of a petroleum bearing formation withhydrocarbon oxidizing, sulfate reducing bacteriafor the purpose of bringing about chemical andphysical changes in the reservoir which wouldresult in increased production of oil. The bacteriawere designated by ZoBell as Desulfovibrio hydro-carbonoclasticus and D. halohydrocarbonoclasticus.A recent patent by ZoBell (103) extends thecoverage on release of oil by sulfate reducingbacteria to hydrogen utilizing, sulfate reducingbacteria.Many mechanisms were discussed by means of

which the bacteria could increase the recoveryof oil. The bacteria were stated to utilize certainhydrocarbons present in crude oil as an energysource, although the attack was slow and in-complete, and to produce acids, and probablycarbon dioxide, from these hydrocarbons. Theacids were then postulated to react with cal-careous minerals such as limestone and dolomitein the reservoir, thus dissolving them and libera-ting additional carbon dioxide. Sulfate reducingbacteria also dissolve gypsum, converting thecalcium sulfate to more soluble calcium sulfide.The solution of the minerals was expected toresult in an increase in the porosity and permea-bility of the formation, making oil recoveryeasier and more complete. The carbon dioxide,to the extent to which it did not dissolve in thereservoir fluids, would increase gas pressure in thereservoir thus tending to increase recovery. Thebacteria might also produce methane and hy-drogen which would have a similar effect. Thesolution in the oil of any produced carbon dioxideand methane would reduce the viscosity of theoil, which should also tend to increase recovery.The bacteria were shown to produce surface activeagents which should reduce interfacial tensionsin the reservoir, again presumed to be a favorableeffect. The growth of the bacteria attached tosolid surfaces in the reservoir should also have a

favorable effect by crowding oil away fromsurfaces to which it might be attached. It wasfurther suggested that the bacteria mightsplit high molecular weight compounds in thecrude oil into fragments of lower molecularweight, thus decreasing the viscosity of the oil.The liberation of oil from solid surfaces wasnoticed by ZoBeli in experiments designed tocompare the effectiveness of various inert ab-sorbents for dispersing hydrocarbons in bacterialcultures for growth experiments (98). Inoculatedcultures in mineral salts solution developed afilm of oil on the surface, while sterile controls didnot. A repetition of the experiments with oilsoaked beach sand, Athabaska tar sand, and oilcontaining shales yielded similar results. Experi-ments on cores of oil bearing sand from NewYork and Pennsylvania oil fields, immersed injars of nutrient medium, gave conflicting resultsin that oil was released from only about half theinoculated samples. ZoBell (98) emphasized themany problems to be overcome before large-scalefield applications could have any hope of success,and concluded that bacterial oil release constitutesa promising field for future research by micro-biologists in cooperation with petroleum en-gineers. ZoBeli (101) believes that, regardiessof whether sulfate reducing bacteria can be usedin the secondary recovery of oil, they haveperformed an important role in the concentrationand migration of oil leading to petroleum de-posits over millions of years of geologic time. Theevidence for this belief is cited above.Beck (7) investigated the possibility of applying

the foregoing method to oil recovery in theBradford, Pennsylvania, field using Desulfovibriocultures obtained from ZoBell, and others whichhe isolated himself. His methods were similar toZoBell's but were refined by the quantitativemeasurement of released oil. He was unable todemonstrate the release of Bradford crude oil,either from artificial mixtures of oil and sand, orfrom crushed cores of Bradford sand. Thebacteria would not penetrate the fine pores ofconsolidated Bradford sandstone nor would theygrow to a measurable extent using Bradfordcrude oil as the sole carbon source. Mackenzie(43) published a brief abstract covering results ofexperiments on oil release from cores by sulfatereducing bacteria. Encouraging results wereobtained on inoculating enrichment cultures into

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oil bearing sand cores treated with mineralsolutions containing phosphate buffers. Methane,hydrogen and hydrogen sulfide were evolved,and oil analyses on the cores after incubationshowed that some of the oil had been removedby the bacterial treatment. The author empha-sized the importance of phosphate as a mineralnutrient.

In order for bacteria to release oil from apetroleum reservoir by any of the mechanismslisted, they must penetrate the pores of thereservoir rock throughout a substantial part ofthe reservoir, and multiply therein. Several typesof reservoirs are therefore immediately ruledout of consideration. Reservoirs of extremelysmall pore size will not permit the bacteria topenetrate. Those of high temperature, above80 C, wirl probably not permit the bacteria tomultiply although it is conceivable that sulfatereducing bacteria may be found which willmultiply at higher temperatures. Many reservoirsare available, however, which are well within therange of pore size distribution and temperaturefor successful growth of the bacteria.

In addition to the cited requirements, themineral requirements, growth factor require-ments, and energy source requirements of thebacteria must be met. ZoBell (98) has indicatedthat many oil, formation waters, when mixedwith crude oil, provide all these. Our own ex-perience indicates little or no growth of sulfatereducing bacteria under such conditions, nor isthe growth improved by any of the usual mineralnutrients (ions of ammonium, calcium, mag-nesium, potassium, iron, sulfate, and phosphate).Updegraff and Wren (88) found little or nogrowth under such conditions and suggested theuse of a nutrient such as molasses. Many oilfield waters do support good growth of sulfatereducing bacteria when a readily availableenergy source such as lactate or glucose is added.Others are deficient in phosphate or availablenitrogen compounds. It would probably benecessary to introduce such an energy sourceinto the formation, along with any mineralnutrients which may be deficient in the formationwater, to obtain satisfactory growth within theformation.The literature contains several references

purporting to demonstrate the oxidation of manyparaffin hydrocarbons by sulfate reducing bac-teria (52, 63, 81). Crude oil is primarily a mixture

of paraffinic and naphthenic hydrocarbons. Yetmany crude oiLs fail to support the growth ofsulfate reducing bacteria. Our own results (90)and those of Beck (7) have been negative in thisrespect. Kuznetsov (40) presented the interestingobservation that only one of three samples ofRussian crude oil tested supported any growth ofsulfate reducing bacteria although heptane wasslowly utilized. He concluded that the process ofsulfate reduction at the expense of the organicmatter in petroleum proceeds extremely slowly,and depends on the chemical composition of thepetroleum.O'Bryan and Ling (53) succeeded in growing

sulfate reducing bacteria in cores of Edwardslimestone from an outcrop in Texas, using lactatemedium both with and without oil. The bacterialtreatment lowered the permeability slightly,showing some plugging by the bacteria.

Updegraff and Wren (90) studied the processof secondary recovery of oil by sulfate reducingbacteria using various types of porous mediaand crude oils (typical apparatus shown infigure 2). Cultures of bacteria obtained fromZoBell were employed, including some whichwere also used by Beck (7), as well as severalstrains isolated from oil well brines, limestonecores, and mud. The experimental work wascarried on in cooperation with persons experiencedin petroleum reservoir engineering, and wasconcerned primarily with the most fundamentalrequirement of any proposed oil recovery method;that is, the demonstration of whether or not theprocess has any favorable effect on the rateand/or amount of oil recovery from porousmedia. Many different media, all containingadequate minerals, with and without addedorganic nutrients, were employed. The sulfatereducing bacteria always grew well in inoculatedmaterials, and penetrated the sand packs andcores at rates of one to two inches per day,but none of these (more than 50 packs of sandand crushed limestone, or consolidated sandstoneand limestone samples) showed consistent effecton released or residual oil attributable to theDesulfovibrio cultures used although certainexperiments gave data suggesting bacterial oilrelease. Sterile controls, subjected to identicaltreatment, produced the same amount of oil,within experimental error, except where mercuricchloride was present. This chemical was foundto inhibit oil recovery from porous media because

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it reacted with sulfur compounds in the crude oil,producing gummy, solid precipitates. Thus, anytests in which mercuric chloride treated controlsare employed are meaningless. Similarly, re-frigerated controls, as employed in certainexperiments reported by ZoBell (98), would beinvalid for a similar reason since low temperatureincreases the viscosity of the oil, and gravityseparation of oil from oil sands decreases asviscosity increases.A study of the mechanism by which bacteria

might release oil, in the light of present knowledgeof petroleum reservoir engineering, led to thefollowing conclusions:

1. The dissolution of limestone or othercalcareous minerals by sulfate reducing bacteriawas so slow and incomplete, even in the presenceof a readily available energy source, that it couldnot be expected to release appreciable amountsof oil in a reasonable length of time.

2. Gas pressure can move oil through porousmedia, but it has not been demonstrated thatsufficient gas is produced by De&ulfovibrio toexert this effect.

3. The literature on petroleum productionengineering contains conflicting evidence onwhether detergents can increase oil recovery.Some detergents appear to be effective, andothers ineffective. The traces of surface activeagents produced by sulfate reducing bacteriawould not be expected to influence oil recoverywithin reasonable time limits.

4. Tenacious adherence of the bacteria tosolid surfaces may crowd oil off these surfaces,but no evidence was obtained that this processhad any effect in recovering oil from oil bearingsands or rocks.

5. Reduction of the viscosity of crude oil,either by direct bacterial action on the oil, or bysolution of bacterially produced gases in the oil,was not observede Large changes in viscosity areordinarily required to obtain significant increasesin oil recovery. It is doubtful that Desulfovibriocan be applied successfully in the field forrecovery of oil in commercially attractivequantities.

Sanderson (65) was issued a patent on amethod for recovering oil from kerogen typeshale, comprising treatment of the shale withClostridium sporogenes, C. histolyticum, C.kntoputrescens, or Pseudomona fluorescens ormixtures of these bacteria.

V. REFINING AND MANUFACTuIRING OFPET!ROLEUM PRODUJCTS

A. Deterioration of Petroleum ProductsThe literature on the decomposition of hydro-

carbons and petroleum products has beencomprehensively reviewed by ZoBell (96). It isclear that virtually all petroleum products, whenstored in the presence of water, may undergosome deterioration as a result of the activities ofhydrocarbon oxidizing microorganisms. Thaysen(84) described an interesting case of spontaneousignition in a tank of purified kerosene stored overriver water. An organism was isolated whichfermented kerosene and gave methane, acetal-dehyde, lactic acid, and acetic acid as products.Nitrate was an essential hydrogen acceptor.The spontaneous ignition was believed to havebeen caused by the ignition of methane liberatedin the fermentation. Steel tanks were also shownto support the growth of sulfate reducing bacteriawhich contaminated the stored petroleum prod-ucts with hydrogen sulfide. Allen (2) showedthat bacterial action at the interface betweengasoline and water in storage tanks may produceperoxides and gums and precipitate lead tetra-ethyl, leading to deterioration of the gasoline.

Cutting oil emulsions, used in machine shops,support growth of many types of bacteria, in-cluding sulfate reducers, which cause deteriora-tion of the oil, and objectionable odors. Someauthorities believe that these bacteria may causedermatitis in workmen handling such oils.

B. Bacterial, Desulfuriation and Denitrogenizationof Crude Oil and Petroleum Products

Maliyantz (44) observed that certain sulfatereducing bacteria attacked Ru n crude oil,and removed part of the sulfur in the process.Our own results (89) with Mid-ContinentAmerican crude oils were different in that nochange in the sulfur content of the crude oil wasbrought about when the crude oil was treatedwith sulfate reducing bacteria in various media,both with and without the presence of sulfurcompounds other than those in the crude oil.Strawinski (76) observed a decrease of 12.5 percent in the sulfur content of an Arabian crude oilwhen the oil was mixed with a sulfur-free mediumcontaining mineral salts and glucose, and incu-bated for four days with a culture of Peudomonassp. which had been selected for its ability to

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utilize sulfur compounds present in the oil. In alater patent, Strawinski (77) disclosed an im-proved two-step process whereby the oil wasfirst treated with a culture of an aerobic bac-terium in a sulfur-free medium, thus convertingpart of the sulfur to sulfates, and then with aculture of sulfate reducing bacteria, which con-verted the sulfates to hydrogen sulfide. Thismethod was claimed to result in more completeremoval of sulfur from the crude oil. ZoBeil (102)described a general method of desulfurizingpetroleum products by means of hydrogenaseproducing bacteria acting on the oil underanaerobic conditions in the presence of hydrogen.

Bacteriological methods of desulfurizing crudeoil are not in general use in the petroleumindustry. The sulfur compounds in crude oilare mostly of high molecular weight, and our ownexperience shows them to be attacked by micro-organisms with great difficulty. Microbiologic.aldesulfurization of crude oil is not likely tocompete with chemical methods unless moreeconomical and effective methods are developed.A similar problem is the microbial denitro-

genization of petroleum. Nitrogen compoundsare also troublesome in the refining of certain oils,and might be removed microbiologically in wayssimilar to those used for sulfur. However, theliterature does not reveal any developmentstoward this goal.

C. Petroleum as a Substrate for the IndustrialManufacture of Chemicals

Another promising line of research whichappears to have been generally neglected is theuse of petroleum as a substrate for the industrialmanufacture of chemicals. Crude oil and naturalgas, pound for pound, are far cheaper than otheravailable organic substrates. Taggart (80) ob-tained a patent on a method of producing fattyacids, esters, and low-boiling alcohols by theaction of BaciUus paraffinicus on natural gasunder aerobic conditions. With natural gaspriced at 0.2 to 0.4 cents per pound of organicmatter, it does not seem out of the question toconsider the possibility of the manufacture offoodstuffs by microbial action on this substratesince microorganisms are known which convertgaseous hydrocarbons to protoplasm with ahigh degree of efficiency.

REFERENCES1. AIYERS, P. A. S. 1920 The gases of swamp

rice soils. V. A methane-oxidizing bac-terium from rice soils. Mem. Dept. Agr.India, Chem. Ser., 6, 173-180.

2. ALLEN, F. H. 1944 The effect of variousmicroorganisms on the precipitation oflead tetraethyl from aviation fuels andthe formation of gum in motor gasolines.PhD Thesis, The University of Texas,Austin, Texas.

3. BASTIN, E. S. 1926 The problem of thenatural reduction of sulphates. Bull. Am.Assoc. Petroleum Geol., 10, 1270-1299.

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Field test of corrosion inhibitor for low pHflood water. Producers Monthly, 12, no.1, 13-17.

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25. EKZERTZEV, V. A. 1951 Microscopic in-vestigations of bacterial flora in oil-bear-ing environs near Vtoroi Baku. Mikro-biologiya, 20, 324-329. English transla-tion by R. T. Beyer.

26. EMERY, K. O., AND RITTENBERG, S. C. 1952Early diagenesis of California basin sedi-ments in relation to the origin of oil. Bull.Am. Assoc. Petroleum Geol., 36, 735-805.

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30. GROSSMAN, JOY P., AND POSTGATE, J. R.1953 Cultivation of sulfate-reducing bac-teria. Nature, 171, 600-602.

31. HADLEY, R. F. 1939 Microbiological an-aerobic corrosion of steel pipe lines. Oiland Gas J., 38, no. 19, 92-94.

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34. HASSLER, G. L. 1943 Apparatus for meas-uring pressure in containers. U. S. PatentNo. 2,321,292. Assigned to Shell Develop-ment Co.

35. HECK, E. T., BARTON, J. K., AND HOWELL,W. E. 1949 Further field test results onuse of corrosion inhibitors and bactericidesfor secondary flood waters. ProducersMonthly, 13, no. 6, 27-34.

36. HENDRICKS, T. A. 1945 The origin of coal,pp. 1-24. In Chemistry of coal utilization.Vol. I. John Wiley and Sons, Inc., NewYork.

37. HORVITZ, L. 1939 On geochemical pros-pecting. Geophysics, 4, 210-228.

38. HUNTER, J. B., MCCONOMY, H. F., ANDWESTON, R. F. 1948 Enviroumental pHas a factor in control of anaerobic bacterialcorrosion. Oil and Gas J., 47, no. 28, 249-250.

39. VON WOLZOGEN KtHR, C. A. H., AND VANDER VLUGT, I. S. 1934 The graphitiza-tion of cast iron as an electrobiochemicalprocess in anaerobic soils. Water, 18,147-165.

40. KUZNETSOV, S. I. 1950 Investigation of thepossibility of contemporaneous formationof methane in gas-petroleum formations inthe Saratov and Buguruslan regions.Mikrobiologiya, 19, 193-202. Englishtranslation, Associated Technical Services,East Orange, N. J.

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paratus for detecting the presence of profit-able deposits in the earth. U. S. PatentNo. 1,843,878.

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94. WILLIAMSON, C. E. 1950 Ethylene, a met-abolic product of diseased or injuredplants. Phytopathology, 40, 205-208.

95. ZoBsLL, C. E. 1946 Bacteriological process

for treatment of fluid-bearing earth forma-tions. U. S. Patent No. 2,413,278. As-signed to the American Petroleum Insti-tute.

96. ZoBELL, C. E. 1946 Action of microor-ganisms on hydrocarbons. Bacteriol.Revs., 10, 1-49.

97. ZoBELL, C. E. 1946 Functions of bacteriain the formation and accumulation ofpetroleum. Oil Weekly, 120, no. 12, 30-36.

98. ZoBELL, C. E. 1947 Bacterial release of oilfrom oil bearing materials. World Oil,I, 126, no. 13, 36-44; II, 127, no. 1, 35-40.

99. ZoBELL, C. E. 1951 Role of microor-ganisms in petroleum formation. Am.Petroleum Inst. Research Project 43A,Report No. 33.

100. ZoBELL, C. E. 1952 Role of microor-ganisms in petroleum formation. Am.Petroleum Inst. Research Project 43A, Re-port No. 39.

101. ZOBELL, C. E. 1952 Part played by bac-teria in petroleum formation. J. Sedi-ment. Petrol., 22, 42-49.

102. ZoBELL, C. E. 1953 Process of removing

1954] 237


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sulfur from petroleum hydrocarbons andapparatus. U. S. Patent No. 2,641,564.Assigned to the Texaco DevelopmentCorporation.

103. ZoBELL, C. E. 1953 Recovery of hydro-carbons. U. S. Patent No. 2,641,566. As-signed to the Texaco Development Corpo-ration.

104. ZoBELL, C. E. 1946-1952 Role of micro-organisms in petroleum formation. Am.Petroleum Inst. Research Project 43A,Annual and Quarterly Reports.

105. ZoBELL, C. E. 1954 Biological factors in

marine corrosion. Paper presented at a

conference on marine corrosion problems.The University of California, Berkeley,California, Feb. 8-9, 1954.

106. ZoBELL, C. E., AND JOHNSON, F. H. 1949Influence of hydrostatic pressure on thegrowth and viability of terrestrial andmarine bacteria. J. Bacteriol., 57, 179-189.

107. ZOBELL, C. E., AND RITmNBERG, S. C. 1948Sulfate-reducing bacteria in marine sedi-ments. J. Marine Research (Sears Foun-dation), 7, 602-617.

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