VI. References...................................................

Magnolia Petroleum Company, Field Research Laboratories, Dallas, Texas
stituents into useful products receives little
attention. On the other hand, although the role
The microbiologist within the past decade h
joined the maay other technologists serving the of microorganisms in petroleum genesis (the
petroleum 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 gepurpose of this review is to indicate the scope of ologists for more than twenty years, this subject
petroleum microbiology. In time, certain aspects has yet to pass beyond the realm of speculation.
Exploration for petroleum deposits was
within this scope will likely be pursued with
much greater intensity of effort. Today, the pioneered by the rank wildcatter who was
microbial conversion of certain petroleum con- followed and surpassed by the geologist. The
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I. Introduction .......................................................................
. .......
II. Petroleum genesis
. . . 216
A. Modification of organic marine sedimentary material
1. Oxidative processes ................216
...................... 217
2. Formation of hydrocarbons in marine sediments
. . . 218
B. Factors which affect bacterial activity in sedimentary rock
................................... 218
1. Depletion of nutrients
............................... 219
2. Thermodynamic considerations
................................. 219
3. Temperature and pressure
. . . 220
C. Evidence regarding biogenesis of petroleum
1. Constitution of crude oil as opposed to known bacterial hydrocarbon products ....... 220
..................... 221
2. Observations concerning bacteria in reservoir rock
3. Comparison of petroleum genesis with coal formation
III. Petroleum exploration .................................................................... 221
. . . 221
A. Geomicrobiological prospecting for petroleum
1. Soil microorganisms as indirect indices of petroliferous emanations ............. 221
.................. 223
2. Bacterial products as indices of petroliferous emanations
. . . 224
B. Microbial activity as related to geochemical prospecting for petroleum
IV. Production of petroleum .................................................................. 225
A. Bacterial corrosion of iron and steel ................................................... 225
1. Bacteria concerned...................................................................225
2. Mechanism of anaerobic bacterial corrosion ......................................... 227
3. Importance of bacterial corrosion in drilling and production of oil ............. 227
.............................. 228
4. Remedies for bacterial corrosion
. . . 229
B. Microbial decomposition of organic drilling fluid additives
1. Fermentation of starch and other natural carbohydrates
...................... 229
2. Decomposition of sodium carboxymethylcellulose
. . . 230
C. Microbiological plugging of injection wells
1. Mechanisms ........................................................................ 230
2. Organisms .......................................................................
3. Remedies .......................................................................
. . . 231
D. Oil release from petroleum bearing rocks by bacterial action
. . . . 233
V. Refining and manufacturing of petroleum products
A. Deterioration of petroleum products .
................................................... 233
B. Bacterial desulfurization and denitrogenization of crude oil and petroleum products .... 233
C. Petroleum as a substrate for the industrial manufacture of chemicals ................... 234
VI. References ........................................................................
formation which must, at present, come principally from the academic laboratories, while in
the petroleum industry microbiologists pursue
information of a more applied nature. As time
pass, more microbiologists should swell the
thin ranks of those employed in the petroleum
industry, and thus permit more fundamental
work to be done, with results of mutual benefit to
science and the petroleum industry.
Because of developments of possible competitive advantage in this little-known field, individual petroleum companies have restricted
the publication of their research findings until
they can be adequately protected by patents.
Since patents require from two to five years to
issue, many developments in petroleum microbiology are undoubtedly being retained in the
confidential files of oil companies. The eventual
publication of this material should immediately
make certain aspects of this review obsolete.
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geophysicist followed the geologist and added
fruitful physical techniques. On the heels of the
geophysicist came the geochemist, who, in turn,
is followed by the geomicrobiologist. While the
geochemist searches for chemical evidences of
petroleum in the surface soils, the geomicrobiologist investigates the effects of microbial
activity upon these chemicals and, in addition,
looks for specific microorganisms which feed
upon hydrocarbons emanating from petroleum
Petroleum production, by which is meant
drilling for petroleum and recovering the product
as economically as possible, was, in the early
days, a crude and wasteful process. Later improvements in technology made by petroleum and
mechanical engineers resulted in large increases in
efficiency and in great increases in the yield of oil
from a given reservoir. Still later, the need for
scientific understanding of the physical principles of petroleum production led to the employment of research engineers, physicists,
chemists, and mathematicians, resulting in
further improvements in its technology. The
microbiologist has now joined these other
technologists and finds a fruitful field for research
in problems of bacterial corrosion, microbial
plugging of oil reservoir formations, fermentation
of drilling fluid additives, and even in attempts to
increase oil recovery by bacterial action within
petroleum reservoirs.
Petroleum products are routinely stored in
tanks over water and are subject to microbial
attack and modification at the oil-water interface, which may lead to deterioration of the
product. Microorganisms which attack paraffinic hydrocarbons, in particular, are many and
varied. Although the mechanism of hydrocarbon
oxidation is virtually an unexplored field, the
methodology for such investigations is little
different from that used in other intermediary
metabolism studies.
At least one university laboratory is engaged
in such studies under a grant from a petroleum
company, and it is hoped that other academic
microbiologists will be attracted to this field in
the future. An opportunity is here for fruitful
fundamental research, which could provide a
basis for applications in the refining and manufacturing of petroleum products. Although the
petroleum companies do a certain amount of
fundamental research, this is the type of in-
[VOL. 18
We shall first present a critical analysis of
present views regarding the role of bacteria in the
actual formation of petroleum. No attempt has
been made here to compile an exhaustive review
including a multitude of observations or statements, many of which would appear to be
irrelevant based on present knowledge. Practically all geologists agree that petroleum has an
organic marine sedimentary origin, but the mode
of its formation is not known. Bacterial activity
has undoubtedly been involved in petroleum
genesis, but the extent to which bacteria have
contributed to the formation of petroleum is
debatable. Attempts to demonstrate hydro-
carbon formation by bacteria under highly
artificial conditions have yielded only small
amounts of paraffinic hydrocarbons other than
methane and practically none of the other
myriad compounds present in petroleum. The
conservative viewpoint is that bacterial action is
limited to producing reduced organic matter
more closely resembling petroleum than the
original material and that the final stages of
petroleum genesis are physicochemical.
Modification of Organic Marine
Sedimentary Material
1. Oxidative processes. It is axiomatic that
bacteria will oxidize sedimentary organic matter
for the purpose of gaining energy as long as
Marine sediments are somewhat analogous to
soil in the sense that the bacterial flora and consequently bacterial activity are regulated by the
type of organic material available and the
conditions existing at a given time. The bacteria
function in both soil and marine sediments as a
biochemical means of regenerating the elements
concerned with the carbon, nitrogen, sulfur, and
phosphorus cycles of nature, thereby prohibiting
the accumulation of dead organic matter on the
soil surface as well as on the ocean floor.
2. Formtion of hydrocarbnm8 in marine sedimente. The formation of petroleum hydrocarbons
in recent marine sediments by bacteria has not
been demonstrated although it is known that the
sediments do contain methane producing
bacteria (83), and certain bacteria found in
sediments contain minute amounts of hydrocarbon as a part of their cell substance (75, 100).
Trask and Wu (85) were unable to detect liquid
hydrocarbons in sediments twenty years ago but
reported small amounts of solid hydrocarbons.
Smith (69) recently has detected small amounts
of hydrocarbons in marine sediments using
chromatographic methods. Smith extracted
sediments of the Gulf of Mexico with fat solvents
and obtained about 0.031 per cent extractables
which contained from 16 to 25 per cent paraffin
hydrocarbons besides other hydrocarbons. Trask
and Wu extracted sediments of the Florida Bay
and obtained 0.062 per cent extractables which
contained 8.9 per cent "paraffinaceous" material,
and another of their sediment samples yielded
0.087 per cent extractable material containing 27
per cent paraffin. Trask and Wu apparently were
looking for liquid petroleum in the sediments and
did not attach much significance to their findings.
Smith, on the other hand, with the modern
methods of chromatography has been able to
study the characteristics of the sediment extracts
and has found them actually to resemble petroleum, although, admittedly, not identical
with it. The role of bacteria in the formation of
these hydrocarbons is not known, but it is known
that bacterial cells contain very small amounts of
hydrocarbons. In Stone's laboratory (75) 400
grams of one bacterial cell mass yielded 0.25
per cent hydrocarbons, but analysis of 10
kilograms of another mass of bacteria revealed
only 0.03 per cent hydrocarbons. ZoBell in
1951 (99) reported an "oily" material produced
by the anaerobe De&dfovibrio, but so little of the
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physicochemical conditions permit. The most
efficient means of gaining energy from organic
compounds is for the bacteria to oxidize them in
the presence of oxygen, the carbon compounds
becoming completely oxidized to carbon dioxide
and water, thus yielding the maximum of
energy. Such oxidation can take place only at the
surface of marine sediments since below the
first few centimeters most sediments rich in
organic matter are depleted of oxygen. The
bacteria which are active in the oxidation of
sedimentary organic material in the presence of
free oxygen are common forms found in soil and
fresh water, usually facultative anaerobes such as
Peeudmonas, Achromobacter, Flavobacterium, and
Spirillum (75).
In the absence of free oxygen strictly anaerobic
bacteria are active as well as the facultative
anaerobes. Certain anaerobic bacteria such as the
Deoulfovibrio have been given much attention
regarding their role in petroleum genesis, especially by ZoBell (104). These bacteria oxidize
organic compounds in sediments and concomitantly reduce oxidized forms of sulfur, using
them as hydrogen acceptors. This process takes
place in the absence of oxygen resulting in
oxidized compounds, energy for the Detdfovibrio
and hydrogen sulfide. Because hydrogen sulfide
reacts with metals to give a black sulfide precipitate, the blackening of organic sediments is
usually an indication of the activities of Desulfovibrio. Other anaerobic bacteria may be
active in sediments, but little attention has been
given them. Anaerobes other than Desufovibrio
oxidize organic compounds in the absence of
oxygen by using other organic compounds as
hydrogen acceptors rather than sulfur compounds.
The hydrolysis products of protein and
carbohydrate materials are the most rapidly
metabolized compounds, yielding C02, NH3,
H2S, CHI and fatty acids depending upon the
bacteria and the conditions involved (75).
Other materials such as chitin and lignin are
more slowly decomposed by bacterial action and
form the basis for the accumulation of marine
humus (92). Marine humus, like soil humus, is
chemically ill-defined and may be described
simply as a colloidal residual of undecomposed
organic matter which because of its resistance to
oxidation very slowly succumbs to bacterial
decomposition processes.
requirement for nitrogen, would be expected to
attack preferentially the nitrogenous compounds;
the sediments, therefore, become progressively
less rich in nitrogenous compounds with time and
depth of burial (33). Trask in his extensive work
(86) showed that ancient sediments contain a
carbon/nitrogen ratio of about 14 whereas this
ratio for recent sediments is 8.5. These observations may be considered as circumstantial
evidence for bacterial activity, but the formation
of petroleum by bacteria under adequately
simulated or actual geological conditions has yet
to be observed.
Treibs (87), who has studied organically rich
recent deposits such as are found in the Black
Sea, is of the opinion that oil is generated from
the nonlipid organic constituents in the sediments
as well as from the lipid constituents. Treibs
calculated petroleum generally to be 85.7 per
cent carbon and 14.3 per cent hydrogen. The
atom ratios are thus 7.15 to 14.3, and the empirical formula can be considered (CH2)1 for
all practical purposes. Organic matter was calculated by Treibs to be 55 per cent carbon, 7 per
cent hydrogen, 5 per cent oxygen and 3 per cent
nitrogen (based upon a logical mixture of carbohydrate, protein and fat of which living things
are composed). The atom ratios of carbon, hydrogen and oxygen then are 4.6-7-2.2. If one
assumes that carbon dioxide is the most logical
decomposition product of this organic mixture,
the organic material thus becomes depleted of
oxygen, and the ratio of carbon to hydrogen
becomes 3.5 to 7 or (CH2)1, the same as the
empirical formula for petroleum. It may be concluded with regard to bacterial action that it
defnitely can and does remove carbon dioxide
from dead organic matter under anaerobic conditions and thereby contributes to its ultimate
reduction making it more like petroleum.
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material was available that an accurate identification of it was imposible. In 1952, Dr. Hanson
of the Mellon Institute examined a small amount
of unsaponifiable material (67 milligras)
submitted to him by ZoBell, which was described
as having been produced by Deulfoibrio as it
grew autotrophically in a synthetic medium
consisting of carbonate, sulfate and other mineral
alts in a hydrogen gas atmosphere. Dr. Hanson
remarked: "Although it was necessary to forego
some of the usual techniques employed in handling materials of this type because of the small
amount available, some information on the
chemical constitution of this oily extract was
obtained. Chromatography made possible the
separation of the total mateil into five distinct
fractions. Although the first of these fractions
could not be analyzed further, it seems likely that
it was composed entirely of hydrocarbon material.
The second fraction, as shown by infrared absorption and the elementary analysis, is largely
hydrocarbon of paraffinic character, and if any
non-hydrocarbon components are present, they
must make up a very small part of the cut. The
third chromatographic fraction was the first to
contain any amount of non-hydrocarbon constituents and these were largely oxygen-containing substances. Unfortunately, the remainder of
the fractions could not be studied further
because of the small amounts, but they are
undoubtedly composed of non-hydrocarbon
materials. If any nitrogen or sulfur components
were present in the original sample, they must
have been concentrated in the last fractions"
(100). Thus, the material was apparently, in
part, the hydrocarbon fraction of the bacterial
cells, similar to that of the bacteria examined by
Stone (75).
This hydrocarbon material is synthesized by
bacteria as part of the baeterial cell and, as such,
very probably exists in sediments a bacterially
produced constituent of the hydrocarbon found
there. Furthermore, bacterial flora under the
reduced conditions of recent marine sediments
would have a tendency to attack the more
oxidized constituents of the sediments, thus
preserving the more reduced organic material
such as the lipid fracoion including the hydrocarbons. Smith (69) recently has shown that the
percentage of less polar (reduced) compounds
increases with the depth of sediments; therefore,
with time. Bacteria, because of their growth
[VOL. 18
B. Factors Which Affect Bactrial Actiiy in
Sedimentary Rock
1. Depletion of nutrients. The first limiting
factor of bacterial activity in organic sedimentary
material is a lack of free oxygen. The oxygen
demand of the sediments is apparently great
enough to deplete free oxygen at an early stage in
sedimentation (26). Lack of free oxygen results
in the accumulation of sedimentary orgaic
matter which otherwise would be oxidized (or
mineralized) ultimately to carbon dioxide,
reduced compounds is possible under anaerobic
conditions. Furthermore, Stadtman and Barker
(72) and Buswell and Mueller (17) have elucidated two mechansms for bacterially formed
methane dependent upon the bacteria involved.
One mechanism involves a reduction of carbon
dioxide, the other a reduction of the methyl
group of methanol or acetic acid. Thus, it is
conceivable that still other, longer alkyl radicals
can be reduced to corresponding paraffinic
hydrocarbons by anaerobic bacteria. While
most attempts to demonstrate this have failed(17, 83), recently Davis and Squires (23) found
other gaseous hydrocarbons, including ethane,
in the order of a few parts per million in methane
As organic matter becomes more reduced in
the sediments, presumably because of hydrogen
transfer resulting from anaerobic oxidations, it
becomes progressively more difficult to oxidize
because it is less susceptible to activation from a
thermodynamic standpoint. The anaerobic conversion of compounds such as tyrosine to yield
phenol or cresol, the alleged production of even
benzene (33), and the already mentioned methane
formation from fatty acids indicate a bacteriological means of carrying organic matter to a
state as reduced as petroleum; but these observations are not indicative of anaerobic bacterial
activity in general or of such activity in sedimentary rock. There is a tendency for highly
reduced organic matter to resist bacterial
decomposition or modification under anaerobic
conditions. Experimental work designed to
subject sedimentary material in various stages
of petroleogenesis to anaerobic bacterial action
should serve to elucidate the affect of such
action. Various ways of accelerating bacterial
activity may be used, such as adjustment of the
mineral concentration, temperature, pH, moisture
and bacterial flora. Under optimal conditions for
anaerobic bacterial activity a reasonable estimate
of their potential function at various stages of
petroleogenesis may be made, provided the data
are extrapolated as realistically as possible to
geological conditions. The foregoing is no easy
task, but approaches in the past have made
realistic extrapolation of data impossible due to a
distinct separation of the bacterial system being
studied from the sedimentary system being
form of lipids and hydrocarbons, as already men- considered.
3. Temperature and pressure. In 1946, Cox (21)
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mineraLs and water (93). Bacterial decomposition
under anerobic conditions proceeds at a relatively slow rate, the hydrogen from the decomposable (oxidizable) organic compounds being
transferred through the bacterial enzyme system
to hydrogen acceptors such as oxidized organic
compounds or to forms of oxidized sulfur. Thus,
general anaerobic bacterial activity ultimately
leads to an accumulation of more reduced organic material and hydrogen sulfide. As pointed
out earlier, the activities of the sulfate reducing
bacteria (De8ulfovibrio spp.) have received a
a great deal of attention (104) whereas other
anaerobic bacteria which may be active in marine
sediments have received little. Desulfovibrio,
because of its peculiar metabolism, primarily
reduces oxidized forms of sulfur rather than
organic matter.' If sulfate becomes limiting in
the environs, activity of De8ulfovibrio spp.
ostensibly ceases. Connate waters associated with
petroleum reservoirs are notably low in sulfate
although there are many exceptions (29).
Nitrogen in available form must be present in
order for bacterial activity to proceed. As the
bacteria incorporate nitrogen into their celLs, it is
largely converted into protein. Upon death of
the cell and its subsequent decomposition the
protein nitrogen is converted into ammonia and
is therefore susceptible to dissipation. In this way
the sediments could become depleted of available nitrogen, and the consequence would be a
decrease in bacterial activity.
Actually very little is known about the bacterial activity that ensues in recent marine
sediments, and practically nothing is known of
such activity in source beds productive of
petroleum as we know it. The various stages of
petroleum formation have yet to be clearly defined, and the bacterial flora, bactei activity,
or the nutritive factors influencing such activity
have not been determined.
2. Thermodynamic conierations. It can be
demonstrated in the laboratory that anaerobic
bacteria convert fatty acids into methane although the production of significant amounts of
higher paraffin homologs has not been accomplished (17, 83). This indicates that a bacteriological reduction of already relatively
I There are small amounts of reduced organic
matter in the Desulfovbrio bacterial cells in the
specific bacterial flora and activities involved.
C. Eviden Regarding Biogenesis of Petroleum
1. Constitution of crude oil as opposed to known
bacterial hydrocarbon products. Van Nes and Van
Westen (91) point out that it is logical to assume
crude oil to contain cyclic compounds similar in
basic structure to those which occur in living
organisms. Terpenes, sesquiterpenes, and polyterpenes which appear to be polymerized iso-
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proposed a "geological fence" secured to "posts",
namely, organic matter, marine environment,
temperature, pressure and time, within which the
herd of facts pertaining to petroleum formation
should be brought. Observations relative to
bacterial activity should logically be considered in
the light of known temperature and pressure
ranges existent in sedimentary rock. Definite
ranges of temperature and pressure exist beyond
which bacteria are no longer physically stable nor
biochemically active.
Cox points out that petroleum is probably
formed in sedimentary sections not exceeding
5,000 ft in thicknes. The minimum temperature
expected would be about 65 C and the maximum
would be slightly higher than 100 C. Maximum
pressure due to an overburden of 5,000 ft would
be about 5,000 lb/sq in, hydrostatic head would
be 2,000 psi. Certain bacteria can metabolize at
temperatures of 55 to 75 C, and some sporeforming bacteria can resist temperature up to 100 C
(55). Furthermore, certain bacteria which do not
even form spores can apparently withstand a
mechanical pressure of 75,000 psi. However,
definite changes in bacterial activity can be
observed under the influence of 3,000 psi. ZoBell
and Johnson (106) give data to show that certain
bacteria including sporeformers are killed at
pressures of 7,500 and 9,000 psi in 48 hours.
Isolated observations of bacterial resistance to
relatively high temperature and pressure are
insufficient evidence of potential bacterial
activity related to petroleum formation under
geological conditions. The term "barophilic"
has been coined by ZoBell and Johnson (106) to
describe certain bacterial strains (some of
marine origin) that grow at a pressure of 9,000
psi. Careful scrutiny of their data reveals that no
marked differences exist in the pressure tolerances
of some terrestrial bacteria as compared with the
marine bacteria. The interesting feature of their
experiments was the concomitant increase in
pressure tolerance with temperature over the
ranges of 1-600 atmospheres and 20-40 C.
While bacterial activity may not be completely
prevented by geological conditions of temperature
and pressure as we know them, we have no
knowledge as yet concerning such activity under
these conditions. What knowledge is available
pertains to very recent sediments which have no
great amount of overburden, and even this
knowledge is extremely limited regarding the
[VOL. 18
prene units occur abundantly in nature (especially in plants), and these type compounds are
amply represented in petroleum. Furthermore,
the sulfur, nitrogen and oxygen containing compounds of petroleum very likely are similar to
compounds found in living nature although little
pertinent information regarding this is available.
Bacteria could hardly be responsible for the
biosynthesis of the myriad compounds in crude
oil, e.g., the hydrocarbon components which
make up about 95 per cent of petroleums consisting of varying amounts of paraffinic, naphthenic and aromatic groups. While the constitution of the hydrocarbon fraction of bacterial
cells is not known in detail (75, 100), it is certainly
not analogous with crude oil. Methane is the
only hydrocarbon known to be produced extracellularly in any quantity by bacteria. It
appears, therefore, that their function in petroleogenesis is confined to some modification of the
precursor organic material rather than actual
conversion of this material into crude oil.
Another possible assumption, which seems
farfetched, is that bacteria utilize all protopetroleum, converting it into their own cell substance (containing small amounts of hydrocarbon), the nonhydrocarbon fraction of which
is reconverted again by other bacteria into cell
substance containing small amounts of hydro-
carbon, and so ad infinitum. The result,
ostensibly, is an eventual accumulation of
hydrocarbons, a disappearance in proto-petroleum and a small residual bacterial flora. It
would follow however, that the hydrocarbon
fraction of bacterial cells very closely resembles
petroleum, while actually it appears to be
almost exclusively paraffinic (75, 100).
It is difficult to visualize the process of events
just described for many reasons, among them
being the observation that crude oil contains
many compounds, including chlorophyll porphyrin (87), which could not be formed by
matter and converting it into "humus". The
conversion of peat into lignite, then bituminous
coal, and finally anthracitic coal is conceded to be
due to physicochemical changes brought on by
compaction and heat during geologic time. Coal
formation certainly is largely an in situ process,
and the observed fossil imprints of leaves and
other organized plant structures, e'ven in the
advanced bituminous statie of coal, point to its
origin. It is assumed that while bacterial action
has had some part in the modification of coal in
the peat state, such action could not be responsible for the later changes in physicochemical
composition which result in lignite, bituminous
and anthracitic coal.
Petroleum formation, on the other hand, is not
so well outlined. Without regard to a discussion
of the differences in source material leading to
either coal or petroleum, suffice it to say that
petroleum may or may not be formed in situ
and modification of it may actually take place
during migration. The organic source material
of petroleum has very probably undergone some
modification by bacteria, just as has coal in the
peat state, but a most important distinction
exists in the respective environments of the
source materials during petroleum and coal
genesis. As mentioned above, coal originates from
organic accumulations which contain relatively
little inorganic matter, e.g., as in swamp conditions; therefore little inorganic surface is in
contact with the organic matter. Petroleum in its
various stages of formation is presumed to have
been constantly in intimate contact with a large
inorganic surface as a result of its marine sedimentary origin (15). The catalytic action of
surface might influence a conversion into petroleum of the trapped organic matter which
escapes bacterial decomposition. Brooks (15)
discusses the possible role of active surface
minerals in petroleum formation at the moderate
temperatures prevailing in oil producing reservoirs.
A. Geomicrobiological Prospecting for Petroleum
1. Soil microorganims as indirect indices of
petroliferous emanations. Sohngen, one of the
first bacteriologists to become interested in
hydrocarbon oxidizing microorganisms, in 1906
described an enrichment method of isolating
methane oxidizing bacteria from soil (70).
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bacterial synthesis after sedimentation. Bacterial
action must at least be limited to the formation
in sediments of those compounds which are
conceivably formed by bacteria, regardless of the
time which bacteria are active in the sediments.
2. Observation concerning bacteria in reservoir
rock. In 1952, Schwartz and Mueller (66) reported anaerobic bacteria in oil bearing sands in
Western Germany where "oil is recovered by
mining". While they failed to find aerobic
microbial forms such as mold fungi, actinomycetes or strictly aerobic bacteria, they think
that the anaerobes could have invaded the oil
fields "after opening of the mines". The authors
referred to the observations made by certain
USSR and USA scientists regarding bacteria in
crude oil and associated brines. They maintained
that a discrepancy exists between the presence of
so many kinds or species of bacteria in reservoir
fluids taken from oil wells and the presence of
only a small number of strictly anaerobic forms
in marine source beds. Schwartz and Mueller
think this may be caused by a secondary invasion of the oil reservoirs during drilling operations. Drilling muds sometimes contain many
millions of bacteria per milliliter.
Ekzertzev (25) in 1951 described observations
made of the bacterial flora in oil reservoirs near
Vtoroi Baku in Russia. The depth of the samples
ranged from 1,000 to 6,000 ft. He reported finding
12 to 117 million bacterial cells per gram of dry
sample in oil bearing rock, but no bacteria from
horizons devoid of oil. Ekzertzev mentioned
technical difficulties in making the microscopic
bacterial counts and gave no descriptions of the
bacteria observed. It is conceivable that bacterial cells would be difficultly differentiated from
oil globules in the oil bearing rock sample preparation. Microscopic examination by bacteriologists of oil reservoir rock from other regions
would be of interest.
3. Comparison of petroleum genesis with coal
formation. Plant materials consisting primarily of
lignin and cellulose, which have accumulated
under conditions adverse to microbial decomposition, appear to be the source of coal (36).
One outstanding feature of these accumulations
is the preponderance of organic matter relative
to inorganic matter. The most accepted mechanism for coal formation is through the peat state
where microbial action, though slow, operates
over long periods of time modifying the organic
oxidizing bacteria and cellulose decomposing bacteria (ostensibly methane forming bacteria).
Particularly significant were those samples which
contained methane oxidizers, in the absence of
cellulose decomposers. The ceilulose decomposers
were detected by observation of paper decomposition in a mineral salts medium together with
the soil samples during a prescribed incubation
period. The determination of methane oxidizing
bacteria was likewise qualitative. Samples of soil
were added to test tubes with a mineral salts
medium and the tubes placed under a bell jar.
A water seal was used through which methane
was introduced in admixture with oxygen. Incubation at 34-35 C lasted for 12-14 days, and
methane oxidizing bacteria, when present, characteristically formed a pellicle on the surface of
the mineral medium.
In spite of its simplicity, use of the method
resulted in detecting anomalies of methane
oxidizing bacteria in the subsoil which were
asociated with gas and oil producing areas.
Some of these bacterial surveys preceded drilling
operations. Mogilevskii (50) concluded the
method had promise, but that a development of
a quantitative interpretation was desired. A
study of bacterial indicators for higher hydrocarbons was suggested as well as a determination
of the optimum depths for soil
Later Russian workers followed the lead of
Mogilevskii. In 1947 Bokova et al. (11) and
Subbota (79) described experiments and field
surveys involving methane oxidizing bacteria as
well as other gaseous hydrocarbon oxidizing
bacteria. Subbota continued to compare the
cellulose decomposing bacterial flora with the
methane oxidizing bacterial flora as had Mogilevskii. Bokova and co-workers isolated not only
methane oxidizing bacteria from the soil but
also ethane and propane oxidizing bacteria.
These workers were particularly interested in
specificity relative to the particular hydrocarbons
which could be utilized by the different bacteria.
They reported that all methane oxidizing bacteria isolated failed to utilize ethane or propane.
These they classified as Methanomonas methanica
despite former reports, e.g., of Tausz and Donath
(82), that this organism was capable of utilizing these hydrocarbons. Bokova and coworkers also reported the isolation of an ethane
oxidizing bacterium which could not utilize
methane, and a propane oxidizing bacterium
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Methods for determining the presence of hydrocarbon oxdizing bacteria in soil have since been
patented (34, 78) and assigned to petroleum
companies. The premise is that detection of
hydrocarbon oxidizers will serve as an index of
hydrocarbons in the soil. Gaseous hydrocarbons
are believed to emanate from subsurface petroleum reservoirs into the soil.
In 1943, Hassler obtained the first U. S.
patent (34), and in 1954, Strawinski obtained the
latest U. S. patent (78) describing methods of
prospecting for oil based upon measuring gas
uptake by hydrocarbon oxidizing bacteria in
systems containing soil, gaseous hydrocarbons
and oxygen. Russian workers, particularly the
geologist Mogilevskii, had proposed in 1940 the
utilization of data obtained in bacteriological
studies of the subsoil for the purpose of detecting
and contouring gas emanating areas (50).
Bacterial surveys of oil and gas fields were made
by Mogilevskii and co-workers during the years
1937-1939 in conjunction with gas surveys. The
Russian microbiologist, V. S. Butkevich, head of
the Microbiology Department of the Timiryazev
Agricultural Academy, participated in this
work in which a total of more than 3,000 soil
samples was studied. Gas surveys previously
carried out by the Russians had established that
only negligible concentrations of gaseous hydrocarbons could be found in the soil, even over
known gas deposits, and they questioned whether
these gases could serve as a medium for bacteria.
Furthermore, as pointed out by Mogilevskii, the
bacterial surveys, like the gas surveys, were
complicated by the presence of mete in the
surface soi layers, the result of organic matter
decomposition rather than seepage from crude oil
and gas reservoirs.
Some of the physiological properties of the
methane oxidizing bacteria (found in the subsoil
layers at a depth of two to three meters) were
studied under the direction of Professor
Butkevich. The bacteria were capable of developing in an atmosphere containing methane
and oxygen in the presence of moisture and
mineral salts. Hence, it was concluded that a low
concentration of methane, in a steady supply, is
the determining factor making it possible for
methane oxidizing bacteria to grow in the subsoil.
At the suggestion of Butkevich, Mogilevskii
had the soil samples analyzed for both methane
[VOL. 18
tionships totally unrelated to petroliferous
emanations (1). Anomalies in the abundance of
methane oxidizing bacteria in the soil must therefore be scrutinized carefully before they are given
significance as an index of petroleum-gas emanation. The adaptive ability of bacteria to utilize
organic compounds, including hydrocarbons,
must likewise be considered. Therefore, the
detection in the soil of bacteria which can oxidize
the various hydrocarbons in natural gas is not
necessarily an index of natural gas emanation.
Seasonal fluctuations in the soil bacterial flora,
including the hydrocarbon oxidizing flora, must
likewise be considered, as pointed out by Subbota (79).
2. Bactrial products as indices of petroliferous
emanations. In 1942, Blau (9) described a method
for detecting a "color change" in the soil as an
index of bacterial action upon hydrocarbon gases
emanating from subterranean petroleum deposits. The best reagent used for this purpose was
reported to be sodium peroxide although a
variety of reagents were employed. According to
Blau, the "color change" resulted with soil
samples containing hydrocarbon consuming
bacteria which converted hydrocarbons into
polymerized and oxidized compounds of high
molecular weight that appeared to be carboxylic
acids. He intimated that bacterial cells themselves
could account for the color reaction, described
as "deep red to light yellow", depending upon
the reagent employed.
In 1943, he pointed out further that these
"bodies of high molecular weight" apparently
fluoresce under the influence of ultraviolet light
(10). Slavina (68) more recently studied the
fluorescence of certain soil bacteria includng
hydrocarbon oxidizers. Bacterium aliphaticum
liquef which utilized pentane, hexanes,
and heptane fluoresced brilliant green, while
Methanomonas methanica reportedly active on
methane, ethane, and propane did not fluoresce
in ultraviolet light. Evidence of practical success
utilizing the above methods as prospecting
parameters of petroliferous emanation apparently has not been published.
A manifestation of surface soils, described as
"paraffin dirt", has long been associated with
certain oil and gas producing areas by petroleum
2 Ethane has been found by Rosaire (60) to be geologists. One assumption prevailed that a
in the order of a few parts per billion in soils over deposition of high concentrations of paraffing
oil and gas fields.
emanating from petroleum deposits resulted in
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which could not utilize ethane or methane.
Subbota (79) pointed out that the bacterial
method of oil prospecting proposed by geologist
Mogilevskii recently came into use in oil exploration in Russia, in conjunction with gas
surveys. It was also used independently by a
specialized office of the Central Department of
Eastern Oil Exploration and All-Union Scientific
Research Institute of Hydraulic Geology and
Geological Engineering. Bokova and his associates (11) reported the discovery of a gas field in
Stavropol Kavkaz and an oil pool in Ikhta, the
results of drilling to check bacteriological prospecting data.
German workers, Schwartz and Mueller, have
likewise reported that bacteriological prospecting
for petroleum has promise and claim some success
using a quantitative dilution method. In a review (66) they refer only briefly to their own
unpublished observations, and no details are
Another approach toward exploitation of
bacteria in petroleum prospecting has been
proposed by Sanderson (64), namely, the planting
of hydrocarbon oxidizing bacteria in the soil and
observing their growth in response to emanating
hydrocarbons. He maintained that it was preferable to bury pure cultures at a depth of four or
five feet between sterile layers of permeable
material (e.g., asbestos) and keep them out of
contact with the soil. Technical difficulties of such
a procedure, particularly in view of the slow rate
of growth of the bacteria in the presence of the
minute amounts of emanating hydrocarbons,
would be anticipated.' Varying. water level in the
soil because of unpredictable seasonal rains
would likely inundate planted bacterial cultures
in many areas where such a method is employed.
Practical success in detecting hydrocarbon gas
emnation by the Sanderson method has not
been reported in the scientific literature. For that
matter, success in geomicrobiological prospecting
for petroleum on a commercial basis has not been
reported in scientific journals apparently, except
by the Russians already referred to.
Several factors influence results of soil analysis
for hydrocarbon oxidizing bacteria. Methane oxidizing bacteria, particularly, have been observed
and their function described in ecological rela
B. Microbial Acivity as Related to Geochemical
Prospecting for Petroleum
Visible seepages of hydrocarbon gases and
crude oil at the surface of the earth have served
man as an index of subsurface accumulation of
petroleum for many years. Practically all of such
seepages have been observed by this time, at
least in this country. Invisible seepages which
also may serve as a means of finding oil must be
detected by technical means. Sokolov (71) and
Laubmeyer (42) were among the first to investigate methods of soil gas surveying as a means
of geochemical prospecting for petroleum.
Sokolov, in about 1930, began investigating gas
surveying in Russia. Soil gas was assayed for
gaseous hydrocarbons, including methane, using
an intricate hot filament (combustion) means of
measurement. Over known oil and gas deposits
the range of hydrocarbons found was from
0.0001-0.2 per cent of the soil gas. It is significant
that among Sokolov's collaborators was Mogilevskii who later, in 1937, proposed that bacterial
surveys be made as a means of prospecting for
petroleum. While Sokolov appreciated the fact
that anaerobic bacteria in the soil produced
methane which could mask the micro appearances
of petroleum gases coming from subsurface
reservoirs, it was his associate, Mogilevskii, who
maintained that due to the preponderance of
methane in natural gas, anomalies in methane
oxidizing bacteria were significant if observed at
depths ordinarily below organic matter decomposition in the soil (50).
American investigators (37, 45, 61) became
interested in the observations of Sokolov and of
Laubmeyer and began their own geochemical
surveys. Rosaire (61) in particular was an active
proponent of geochemical prospecting based upon
soil analyses. He was especially interested in
hydrocarbon gases, such as ethane, propane,
and butane, which may be considered "direct"
indices of petroleum because of their practically
unique origin. He showed further interest in
secondary products arising from the oxidation
and polymerization of these emanating gases.
Rosaire points out that these secondary products
(called "soil waxes") resemble hydrocarbons but
chemically they are not true hydrocarbons. Their
molecular composition, mode or rate of formation has not been clarified. It is interesting that
Rosaire, Horvitz (37), and McDermott (45)
along with others (62), in discussing factors
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the waxy appearing nature of the soil. However,
Milner (49) gave a good description of this
peculiar material and pointed out its low hydrocarbon content more than twenty-five years ago.
Recent studies by Davis (22) on a "paraffin
dirt" bed in Texas confirmed suspicions of other
observers that microorganisms were responsible
for a conversion of hydrocarbon gases into
microbial cell material, thus accounting for the
waxy appearance of the soil in a localized area.
Analyses of a representative "paraffin dirt"
sample showed the dried soil to contain 17.6
per cent organic carbon, 1.2 per cent organic
nitrogen, 0.27 per cent lipid (organic matter
soluble in CCII), and 0.0038 per cent saturated
hydrocarbons. Microscopic examination of the
soil revealed an abundance of microorganisms
including protozoa, filamentous fungi, yeasts,
actinomycetes and bacteria. Among the bacteria,
especially, were varieties capable of utilizing
methane and other gaseous hydrocarbons as
carbon sources. Mass spectrometer analysis of the
soil gas collected about six feet below the surface
of the "paraffin dirt" bed showed the presence
of 1.4 per cent methane and 0.13 part per million
of ethane. Traces of other gaseous hydrocarbons
were indicated. It is believed that the organic
matter of "paraffin dirt" consists largely of
microbial cells, living and dead.
Laboratory experiments consisting of passing
natural gas through two ordinary surface soils
for a period of months resulted in a marked increase in organic content of the soils. The number
of microorganisms also increased markedly as
the gas flow continued. Both soils acquired a
waxy, gummy appearance, and one of the soils
upon microscopic (wide field binocular) examination was indistinguishable from specimens of
"paraffin dirt" collected in the field. The other
treated soil, while similar, was not identical in
character with the field samples, primarily, it is
believed, because of an original difference in soil
The fixation of organic matter in the form of
hydrocarbon oxidizing microbial cells as they
consume the emanating hydrocarbons ostensibly
results in a food source for other microorganisms.
The latter thus feed indirectly upon hydrocarbon
emanations. "Paraffin dirt" is a misnomer since
the waxy appearance of the soil is not caused by
paraffin, as is borne out by its low lipid and
hydrocarbon content.
[VOL. 18
between anodic and cathodic areas. ZoBell (105)
pointed out many ways in which bacteria may
contribute to the corrosion of iron and steel.
He emphasized the multiplicity of interrelated
chemical, mechanical, electrical, and biological
mechanisms that combine to cause corrosion, and
concluded that the worst and most extensive
work of bacteria is of a nonspecific nature such
as producing acidic microspheres, oxygen concentration cells, surface charges, or hydrogen
sulfide. This is no doubt true of the marine
environments with which the author was primarily concerned, and the petroleum industry
has to contend with this severely corrosive
environment in its offshore drilling structures,
pipe lines, and tankers. Marine paints and
cathodic protection are the principal methods of
combatting marine corrosion. The complex
nature of this environment usually makes it
impossible to evaluate the extent to which
bacteria contribute to corrosion. This may
account for the paucity of published information
about the corrosion of iron and steel under
aerobic conditions.
The role of bacteria in the corrosion of iron
and steel under anaerobic conditions is better
understood. Although the oil and gas industries
sustain an enormous annual loss through the
anaerobic corrosion of iron and steel (31, 32, 74),
it is only recently that the role of bacteria in this
process has been appreciated by the petroleum
industry (see figure 1). As early as 1934, however,
von Wolzogen Kuhr and Van der Vlugt (39)
presented an explanation of anaerobic bacterial
corrosion which is generally accepted today.
1. Bacteria concerned. Sulfate reducing bacteria
capable of utilizing molecular and cathodic
hydrogen are the principal agents of anaerobic
bacterial corrosion. Since their discovery by
Beijerinck in 1895, investigations have revealed
that these bacteria are abundant in soil, sediments of fresh water and marine origin, sulfur
springs, and mineral waters, including oil well
waters. Starkey and Wight (74) and ZoBell and
Rittenberg (107) have reviewed this literature in
detail. The sulfate reducing bacteria are obligate
A. Bacterial Corrosion of Iron and Steel
anaerobes. Shturm (67) has reported the aerobic
Iron and steel, as well as other metals, corrode growth of sulfate reducing bacteria, but Grossman
in aqueous media principally because of electro- and Postgate (30) pointed out that Shturm's
lytic action resulting from differences in potential results may be explained by the fact that
I Davis and
Squires (23) detected ethane in reducing bacteria will grow in culture media
methane fermentations in concentrations ranging exposed to the air, provided that sufficient
from 0.1 to 7 ppm.
sulfide or other reducing agent is present. Our
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affecting geochemical prospecting, did not consider microbial activity as a possible means of
either modifying or destroying the index hydrocarbons.
Since it has long been agreed that methane in
soil may have either a biological or a petroliferous
origin, geochemists in the U. S. have had a
tendency to shun measurements of methane as
being nonsignificant. It should be pointed out,
however, that there is no knowledge of the
actual amounts of biomethane produced in
ordinary soils. Rosaire (61) likewise referred to
ethylene of biological origin (e.g., ripening fruits,
plant tissues) as a factor to be considered in
geochemical prospecting. More recently ethylene
formation by filamentous fungi has been shown
by Nickerson (51) and Williamson (94). Ethylene
and other olefins have been observed in natural
gases only rarely and in small amounts. Buswell
and Mueller (17) in 1952 reiterated that ethane
and higher hydrocarbons had not been observed
in bacterial methane fermentations and that if
present must be in concentrations less than 20
parts per million of the partially purified
methane.3 Thus, for all practical purposes one
would assume that ethane in the soil is principally
of petroleum origin and that ethylene has principally a biological origin. Interestingly enough,
McDermott (45) reported both ethane and
ethylene in concentrations of 0.02 to 0.10 ppm
by weight in the soil over oil fields.
Horvitz (37) in discussing "soil wax" indicated
that it was observed in a thousand to ten thousandfold greater concentration in soil than the
lighter constituents such as ethane, propane, and
butane. While a true knowledge of "soil wax"
was admittedly lacking, he maintained it was
"empirically significant material", implying that
it was a geochemical parameter of importance.
Knowledge of the chemical characteristics of
this organic material would be required before
either speculation or experiments could relate it
to microbial activity in soil.
[ VO)L. 18
Fig. 2
Fig. 1
Figure 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 the
right aqueous nutrient medium. The tube at the right goes to It vacuum p)ump. The entire aPparatus
may be autoclaved, and the sand pack can then be saturate(l with meassured volumes of oil and
water under aseptic conditions.
owni experience with sulfate Ireducing bacteria
from widely scattered habitats confirms the fact
that aerobic sulfate reducing bacter ia are riare
oI nonexistent (89).
Breed et al. (12) list three accepted species of
sulfate rieducing bacteria: Desulfovibrio desulfuricans, D. rubentschickii, and D. aestuarii. D.
desulfuricans and D. rubentschickii are characterized as species preferring a low salinity medium,
i.e., less than two per cent sodium chloride, while
D. aestuarii grows preferentially in sea water oI
three per cent salt media. D. rubentschickii
differs from D. desulfuricans only in being able
to utilize certain organic acids (acetic, propioinic,
and butyric) as energy sources which are not
utilized by D. desulfuricans. Starkey (73) described Sporovibrio desulfuricans, a thermophilic
sporeforming strain. ZoBell, cited in Breed et al.
(12), concludedl that sporefoimatioin is the excep-
tion rather than the rule among sulfate reducing
bacteiia, a statement with which we concur.
M\1iller has shown that both fresh water and
marine strains prooduce the greatest amount of
hydrogen sulfide in media containing about one
per cent sodium chloride. All strains teste(l
produced more than 2,000 mg of hydrogen sulfide
per liter of medium when supplie(l with essential
minerals, lactate as an energy source, and sulfate
as a hydrogen acceptor. :Miller (47) reported that
sulfate rieducing bacteria require an unknow!n
growth factor (or factors) found in yeast extract
or other natural materials. None of the known
bacterial growtth factors or amino acids could be
substituted for the natural material. Baumanii
and Denk (5) and Postgate (56) reported essentially the same results.
In nature, sulfate reducing bacteria, normally
utilize sulfate as a hydrogen acceptor, thus
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2. Mechanism of anaerobic bactrial corrosion.
The mechanism of anaerobic bacterial corrosion
proposed by von Wolzogen Kuhr and Van der
Vlugt (39) has not been amply confirmed (16,
74). The following equations summarize the
1. 4Fe -- 4Fe+ + 8e (Anodic solution of iron)
2. 8e + 8H+ -) 8H (Cathode)
3. HsSO4 + 8H n H2S + 4H20
Depolarization by the oxidation of
cathodic hydrogen by sulfate reducing
4. Fe+ + H2S ± FeS + 2H+
5. 3Fe+ +6 (OH)- ± 3Fe(OH)2 of corrosion
6. 4Fe + H2SO4 +2H20 ! FeS + 3Fe (OH)2
Equation 3, implying the oxidation of cathodic
hydrogen by sulfate reducing bacteria, was confirmed by Starkey and Wight (74) using enrichment cultures and by Butlin, Vernon, and
Whiskin (20) employing pure cultures of sulfate
reducing bacteria. In these experiments the
bacteria grew in mineral salts media containing
iron, utilizing cathodic hydrogen as their sole
energy source and reducing sulfate. Analysis of
the corrosion products has confirmed the presence
of ferrous sulfide and ferrous hydroxide. Iron kept
in a sterile medium did not corrode under the
conditions of neutral pH and absence of oxygen
maintained in this experiment. It is well known,
however, that corrosion proceeds in the absence
of bacteria in the presence of either acids or
oxygen, or under the influence of electrical
3. Importance of bacterial corrosion in drilling
and production of oil. Anaerobic bacterial corrosion is a common problem in the drilling for and
production of petroleum. Sulfate reducing bacteria are peculiarly well adapted to growth in
subsurface oil bearing formations, and have been
frequently isolated from depths up to 3,090 ft
(3, 4, 27, 28). Gahl and Anderson (27) found
that pure cultures isolated from the deepest,
highest temperature wells had the highest
optimum and maximum temperatures for growth
(37 to 50 C) and that the cultures exhibited an
optimum salt concentration for growth which
showed some correlation with the salt concentration of the brine from the well from which the
culture was isolated. These findings suggest
that the bacteria found were actually multiplying
in the oil producing formation. It is also possible
that they were introduced during drilling operations, and might have been multiplying in the
well casing or tubing, using cathodic hydrogen as
an energy source. ZoBell (97) reported the isolation of sulfate reducing bacteria from cores of
Louisiana sulfur-limestone-anhydrite formation
from a depth of 1,560 ft under experimental
conditions which render extraneous contamination unlikely.
Our observations on 162 core samples of oil
bearing rocks from Texas and New Mexico
showed sulfate reducing bacteria in 26 samples
and facultative organisms in three (89). Many
samples appeared sterile, as they gave no growth
in the media used. It may be concluded that
ancient sediments ordinarily contain very few
viable bacteria but may contain appreciable
numbers of specialized types, particularly sulfate
reducing bacteria, in certain localized environments, such as in porous, oil containing rocks
which also contain interstitial water with the
necessary mineral nutrients. Sulfate reducing
bacteria are found in most produced oil well
brines, and in water supplies used for the secondary recovery of oil by water flooding (a
process for recovering additional oil from a
reservoir after all the oil economically recoverable
by flowing and pumping has been produced),
and for primary pressure maintenance. When a
closed system is used in the presence of iron
pipes and sulfate, anaerobic corrosion is generally
found (6, 41). In water supplies for water flooding
or primary pressure maintenance, such bacterial
action is usually accompanied by the formation of
a turbid water containing bacterial cells and
precipitated iron sulfide, which clogs the pores of
the formation rock and lowers the injection rate
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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 reducible
sulfur compound is essential for growth, Baumann and Denk (5) reported growth of pure
cultures of DesulfoMbrio utilizing nitrate as the
only hydrogen acceptor. Postgate (58) found 8
strains of sulfate reducing bacteria of 12 tested
which required no reducible sulfur or nitrogen
compounds when grown with pyruvate. Our
own observations confirm this finding (89).
with the iron. Protective coatings on the iron,
corrosion resistant alloys, and cathodic protection
are other posibilities.
Germicides and inhibitors have been widely
used in the oil fields to eliminate or decrease
anaerobic corrosion. Formaldehyde was recommended by Menaul and Dunn (46) and by Latter
(41) for reducing hydrogen sulfide corrosion in
oil well equipment, particularly in the casing,
tubing, rods, and pumps in producing oil wells.
From one-half to two quarts per day of 37 per
cent USP formalin was injected into the annulus
between the casing and the tubing. Menaul and
Dunn (46) found that KCN was also effective
although six other relatively nongermicidal compounds were tested and found to be ineffective.
Although these authors attributed the protective
effect to a chemical film of undetermined composition on the surface of the metal, the main
benefit of the treatment may have been caused by
the inhibition of sulfate reducing bacteria.
Laboratory tests have shown that formaldehyde
is an effective inhibitor of sulfate reducing
bacteria and the associated corrosion at levels
of 10 to 50 parts per million of water (8, 54).
Sodium cyanide is similarly effective at 10 parts
per million (89). Quaternary ammonium compounds have been widely used as inhibitors of
various types of corrosion, including that caused
by sulfate reducing bacteria. Breston and
Barton (14) found that from two to four parts per
million of rosinamine acetate reduced the
corrosivity of water used for oil-field flooding
from between 50 to 85 per cent, and also reduced
the count of both aerobic and anaerobic bacteria.
Field tests by Heck, Barton and Howell (35)
showed that all of three quaternary compounds
tested, Pur-O-San (alkyl dimethyl benzyl ammonium chloride), Arquad S (alkyl trimethyl
ammonium chloride), and rosinamine acetate,
gave good protection against acid corrosion.
These inhibitors exert at least part of their effect
by forming a film on the surface of the metal
which brings about a high degree of resistance to
attack, even by strong acids. Their effectiveness
against corrosion by sulfate reducing bacteria
has not been adequately evaluated although
Breston found them to be good agents for
preventing bacterial growth in flooding waters,
and Latter (41) reported that Pur-O-San was an
effective agent for inhibiting bacteria and algae
in flooding waters. Chromate ion, which has long
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(13, 54). Both of these processes are costly to the
petroleum industry.
Doig and Wachter (24) have described a succesion of oil well casing failures in a California
field. The casing corroded in localized areas,
producing holes in the pipe, at depths from 900 to
7,000 feet beneath the surface. The Y inch
thick steel pipe corroded through in an average
time of four years. It was necessary to cement
the casing to seal the hole, and then drill through
the cement plug in each case. This example
of bacterial corrosion is similar to the pipe line
corrosion extensively studied by Hadley (31, 32),
Bunker (16), and Starkey and Wight (74), in
which the bacteria attack the outside of the pipe.
This type of corrosion is severe only where the
soil conditions are anaerobic, sulfate minerals
are present, and the pH is near neutrality, with
outside limits of 5.5 to 9.5 (74). Cast iron pipe
undergoes graphitisation in which the iron is
corroded to ferrous sulfide and hydroxide, leaving
a pipe which still retains its outward appearance
because of the graphite present in the cast iron,
but which is so soft that it can be easily cut by a
knife. The papers of Hadley, an electrical engineer,
established the importance of bacterial corrosion
of pipe lines. A survey of pipe lines in Pennsylvania, Ohio, and New York revealed that from
20 to 97 per cent of the pipe lines, depending on
the terrain, were attacked by anaerobic corrosion.
A simple test for anaerobic bacterial corrosion,
consisting of the release of H2S upon treating
the corrosion products on the pipe with HCl, was
shown to correlate well with the presence of
sulfate reducing bacteria determined by cultivation in lactate medium. Hadley concluded that
this type of corrosion was severe only where the
soil was ordinarily water-saturated, and between
pH 6.2 and 7.8. In the swamps and lowlands of
Ohio, six inch welded pipelines lasted only seven
years, on the average, because of bacterial
corrosion. It was concluded that anaerobic
bacterial corrosion is second only to stray-current
electrolysis as a cause of pipeline failure.
4. Remedies for bacterial corrosion. Anaerobic
bacterial corrosion has proved a difficult process
to combat. Posible methods of eliminating it
include all means of elimiting the growth of
wlfate reducing bacteria: germicides, inhibitors,
exclusion of sulfate, change in pH to a value
unfavorable for growth, prevention of anerobiosis
by aeration, and removal of water from contact^. 18
drilling fluids to impart desired characteristics,
and different types of fluids are used to overcome
special problems encountered in drilling different
types of formations. The major functions of a
drilling fluid are: (a) to lubricate the drill bit,
(b) to cool the bit, (c) to carry away chips of
rock cut by the bit, (d) to plaster the walLs of
the hole, thus preventing caving-in of loose
formations and minimizing filtration into
permeable beds, (e) to apply hydrostatic pressure
to the formation in order to prevent loss of oil
and gas from the strata.
1. Fermentation of starch and other natural
carbohydrates. Perhaps the most common type of
drilling fluid is a "mud" comprised of a dispersion
of clay in water. Various organic colloids are
commonly added to such muds to reduce the
rate of filtration of water through the mud cake
laid down on the walls of the borehole. The most
common of these water-loss reducing agents are
gelatinized starch and sodium carboxymethylcellulose. Both are subject to microbial attack.
Starch is rapidly decomposed by a wide variety
of microorganisms, including aerobic, facultative,
and anaerobic forms. Some muds, particularly
lime base muds, have a pH above 10.5 and are
therefore practically immune to microbial attack.
Others of lower pH may support heavy bacterial
growth. Thus, starch fermentation has become a
serious problem, sometimes resulting in loss of
the entire mud supply, and involving the risk of
serious damage to the well. At least two mud
service companies have developed highly effective bacterial inhibitors, composed primarily of
paraformaldehyde, for preventing such fermentations. Research toward the development of
improved inhibitors appears desirable although
present products are effective and fairly moderate
in cost.
2. Decompositon of sodium carboxymethylcellulose. Sodium carboxymethylcellulose (CMC)
has proved, in practice, to be relatively resistant
to microbial deterioration in drilling muds.
However, studies by Reese, Sui, and Levinson
(59) have shown that molds, actinomycetes, and
bacteria produce enzymes capable of attacking
and partially hydrolyzing commercial grades of
sodium carboxymethylcellulose. The extent of
attack of enzymes on sodium carboxymethylcellulose was found to decrease as the degree of
substitution of the cellulose with sodium carboxymethyl groups increased. The authors postulated
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been used to inhibit corrosion by dissolved oxygen,
was found to be a good inhibitor of sulfate reducing bacteria and anaerobic bacterial corrosion.
Since it retains its effectivenes over a long period
of time, it has been used in dilling mud around
the outside of oil well casing to provide longterm protection (89). It should be mentioned
in passing that chlorine, long used to kill bacteria
in water, is relatively ineffective against anaerobic
bacterial corrosion because the sulfides produced
by sulfate reducing bacteria react with and remove the chlorine.
Other approaches to the control of bacterial
corrosion are applicable at times. The control of
pH in a range outside the growth range for
sulfate reducing bacteria is feasible in drilling
muds and for certain flooding waters. Hunter
et al. (38) reported that a pH above 9.0 effectively
inhibited sulfate reducing bacteria. Many drilling
muds are highly alkaline (pH 10 to 13), and thus
inhibit sulfate reducing bacteria in the vicinity
of the drill pipe, casing, and equipment used with
them. The alkali is introduced primarily because
it imparts desirable physicochemical characteristics to the mud, and bacterial control is coincidental. The chemical research laboratory at
Teddington, England, has been active in the
investigation of preventive measures for anaerobic
corrosion since 1934 (20) and has evaluated many
types of protective coatings for pipe lines.
Standard coal tar enamels and hessian wrappings,
even when dipped in bitumen, are relatively
ineffective. A thick bitumen coating, when completely covering the pipe, and special plastic
coatings show promise. Gravel packing, surrounding the pipe, is also good, probably because
the gravel allows acce to air and prevents
anaerobic conditions. Corrosion-resistant alloys
and plastic pipe are good, but expensive. As
costs are reduced, plastic and plastic-impregnated fiber-glass pipe may come into wide use in
the oil fields.
B. Microbi Decomposition of Organic DriUing
Fluid Additives
Oil wells are now almost always drilled with
some form of drilling fluid in the bore hole.
The liquid suspensions used vary widely in
composition and may have either a water base,
an oil base, or a mixture of the two comprising
an emulsion base. A wide variety of substances,
both organic and inorganic, may be added to
C. Microbiological Plugging of Injection Wells
Present practice in the oil industry involves the
injection of large volumes of water into many
deep wells. Water flooding and primary pressure
maintenance are carried on by injecting water
into oil reservoirs for the purpose of increasing
the recovery of oil. Salt water produced from oil
reservoirs is frequently disposed of by injecting
it into wells drilled into the same reservoir, or
another suitable porous formation. In all these
types of water injection, microorganisms present
in the water have frequently given rise to partial
plugging of the injection well, thus decreasing the
injection rate, sometimes to the point at which
the well becomes useless.
1. Mechanism. The cause of this plugging is
familiar to every microbiologist who has employed filters to remove microorganisms from
aqueous suspensions. Just as the pores of a
bacteriological filter becoming clogged with cells
result in a decreasing filtration rate, the pores of
reservoir rocks may clog with microorganisms
contained in the injected water. Where porous
filtration media are of uniform pore size, correlation of pore size with the size of microorganisms
which will plug the filter is relatively simple.
The pore entry diameter must generally be at
least twice the diameter of the microbial cells for
the cells to pass through without serious plugging.
When cells are spiral or elongated, the pore entry
diameter must be even larger, relative to the
cell diameter, to prevent plugging.
Since petroleum reservoir rocks ordinarily
exhfibit a wide range of pore sizes, the problem is
greatly complicated. The pore size distribution of
reservoir rocks may be estimated by measuring
the volume of mercury injected into a clean, dry
sample by increasing increments of pressure. The
results are plotted as a curve expressing the
fractional part of the total pore volume filled by
mercury as a function of pore entry diameter.
Empirically it has been found that reservoir
rocks containing an appreciable fraction of pores
larger than three microns will pass large numbers
of sulfate reducing bacteria up to 0.6 Ju in diameter and 3 Ju long without serious plugging (89).
Many reservoir rocks contain, principally, pores
of larger diameter than this and are not seriously
plugged by small bacteria. Others that contain a
large proportion of smaller pores may be plugged.
2. Organism. The potential plugging microorganisms in injection water vary with the
conditions under which the water is stored.
Water kept in open pits exposed to sunlight may
contain algae and photosynthetic bacteria, as well
as autotrophic and heterotrophic aerobic and
facultative bacteria. Beck (8) found algae and
species of Crenothrix, Beggiatoa, and Pseudomonas in Pennsylvania flooding waters in
sufficient numbers to make the water turbid.
Storage of injection waters in the dark will
eliminate photosynthetic microorganisms, but
not the others. The use of a closed system, in
which water is pumped from deep wells into the
injection wells, without exposure to air, eliminates aerobic but not anaerobic bacteria. Under
such conditions anaerobic bacteria, particularly
sulfate reducing bacteria, may cause plugging.
3. Remedies. Many different methods of
treatment have been developed to render water
fit for injection. The East Texas Salt Water
Disposal Company employs an elaborate purification system comprising skimming off the oil,
aerating the water, allowing sediments to separate
in settling tanks, filtering, and finally chlorinating
to eliminate further microbial growth. Even
this procedure is not always effective. Beck (8)
found that 10 parts per million of formaldehyde
was effective against sulfate reducing bacteria
in laboratory tests. Heck, Barton, and Howell
(35) showed that from two to ten parts per
million of any of several quaternary ammonium
compounds were effective, in field tests, in
reducing bacterial numbers in flooding waters.
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that the enzymes tested would not attack sodium
carboxymethylcellulose in which every anhydroglucose unit in the cellulose molecule is substituted with at least one sodium carboxymethyl
group. Recent investigations (89) have shown
that CMC degraded to the maximum extent
possible by the action of certain bacteria is
superior to the original CMC for use in certain
mud systems; it confers approximately the same
reduction in water loss, while it produces a mud
with better viscometric properties because of its
lower average molecular weight. In other mud
systems, the bacterially treated product is less
effective as its molecular size is too small to be of
maidmum effectiveness as a water-loss reducing
agent where the clay particles are larger because
of partial flocculation of the clay.
[voL. 18
Oil Release from Petroleum Bearing Rocks by
solid surfaces in the reservoir should also have a
favorable effect by crowding oil away from
surfaces to which it might be attached. It was
further suggested that the bacteria might
split high molecular weight compounds in the
crude oil into fragments of lower molecular
weight, thus decreasing the viscosity of the oil.
The liberation of oil from solid surfaces was
noticed by ZoBeli in experiments designed to
compare the effectiveness of various inert absorbents for dispersing hydrocarbons in bacterial
cultures for growth experiments (98). Inoculated
cultures in mineral salts solution developed a
film of oil on the surface, while sterile controls did
not. A repetition of the experiments with oil
soaked beach sand, Athabaska tar sand, and oil
containing shales yielded similar results. Experiments on cores of oil bearing sand from New
York and Pennsylvania oil fields, immersed in
jars of nutrient medium, gave conflicting results
in that oil was released from only about half the
inoculated samples. ZoBell (98) emphasized the
many problems to be overcome before large-scale
field applications could have any hope of success,
and concluded that bacterial oil release constitutes
a promising field for future research by microbiologists in cooperation with petroleum engineers. ZoBeli (101) believes that, regardiess
of whether sulfate reducing bacteria can be used
in the secondary recovery of oil, they have
performed an important role in the concentration
and migration of oil leading to petroleum deposits over millions of years of geologic time. The
evidence for this belief is cited above.
Beck (7) investigated the possibility of applying
the foregoing method to oil recovery in the
Bradford, Pennsylvania, field using Desulfovibrio
cultures obtained from ZoBell, and others which
he isolated himself. His methods were similar to
ZoBell's but were refined by the quantitative
measurement of released oil. He was unable to
demonstrate the release of Bradford crude oil,
either from artificial mixtures of oil and sand, or
from crushed cores of Bradford sand. The
bacteria would not penetrate the fine pores of
consolidated Bradford sandstone nor would they
grow to a measurable extent using Bradford
crude oil as the sole carbon source. Mackenzie
(43) published a brief abstract covering results of
experiments on oil release from cores by sulfate
reducing bacteria. Encouraging results were
obtained on inoculating enrichment cultures into
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Baterial Action
In 1944, C. E. ZoBell, the Director of an
American Petroleum Institute research project
on the role of bacteria in the origin of petroleum,
applied for a patent on a bacteriological process
for treatment of fluid bearing earth formations.
The patent, issued in 1946, was dedicated to
the public by the American Petroleum Institute
(95). Briefly, the principle involved is the
treatment of a petroleum bearing formation with
hydrocarbon oxidizing, sulfate reducing bacteria
for the purpose of bringing about chemical and
physical changes in the reservoir which would
result in increased production of oil. The bacteria
were designated by ZoBell as Desulfovibrio hydrocarbonoclasticus and D. halohydrocarbonoclasticus.
A recent patent by ZoBell (103) extends the
coverage on release of oil by sulfate reducing
bacteria to hydrogen utilizing, sulfate reducing
Many mechanisms were discussed by means of
which the bacteria could increase the recovery
of oil. The bacteria were stated to utilize certain
hydrocarbons present in crude oil as an energy
source, although the attack was slow and incomplete, and to produce acids, and probably
carbon dioxide, from these hydrocarbons. The
acids were then postulated to react with calcareous minerals such as limestone and dolomite
in the reservoir, thus dissolving them and liberating additional carbon dioxide. Sulfate reducing
bacteria also dissolve gypsum, converting the
calcium sulfate to more soluble calcium sulfide.
The solution of the minerals was expected to
result in an increase in the porosity and permeability of the formation, making oil recovery
easier and more complete. The carbon dioxide,
to the extent to which it did not dissolve in the
reservoir fluids, would increase gas pressure in the
reservoir thus tending to increase recovery. The
bacteria might also produce methane and hydrogen which would have a similar effect. The
solution in the oil of any produced carbon dioxide
and methane would reduce the viscosity of the
oil, which should also tend to increase recovery.
The bacteria were shown to produce surface active
agents which should reduce interfacial tensions
in the reservoir, again presumed to be a favorable
effect. The growth of the bacteria attached to
of paraffinic and naphthenic hydrocarbons. Yet
many crude oiLs fail to support the growth of
sulfate reducing bacteria. Our own results (90)
and those of Beck (7) have been negative in this
respect. Kuznetsov (40) presented the interesting
observation that only one of three samples of
Russian crude oil tested supported any growth of
sulfate reducing bacteria although heptane was
slowly utilized. He concluded that the process of
sulfate reduction at the expense of the organic
matter in petroleum proceeds extremely slowly,
and depends on the chemical composition of the
O'Bryan and Ling (53) succeeded in growing
sulfate reducing bacteria in cores of Edwards
limestone from an outcrop in Texas, using lactate
medium both with and without oil. The bacterial
treatment lowered the permeability slightly,
showing some plugging by the bacteria.
Updegraff and Wren (90) studied the process
of secondary recovery of oil by sulfate reducing
bacteria using various types of porous media
and crude oils (typical apparatus shown in
figure 2). Cultures of bacteria obtained from
ZoBell were employed, including some which
were also used by Beck (7), as well as several
strains isolated from oil well brines, limestone
cores, and mud. The experimental work was
carried on in cooperation with persons experienced
in petroleum reservoir engineering, and was
concerned primarily with the most fundamental
requirement of any proposed oil recovery method;
that is, the demonstration of whether or not the
process has any favorable effect on the rate
and/or amount of oil recovery from porous
media. Many different media, all containing
adequate minerals, with and without added
organic nutrients, were employed. The sulfate
reducing bacteria always grew well in inoculated
materials, and penetrated the sand packs and
cores at rates of one to two inches per day,
but none of these (more than 50 packs of sand
and crushed limestone, or consolidated sandstone
and limestone samples) showed consistent effect
on released or residual oil attributable to the
Desulfovibrio cultures used although certain
experiments gave data suggesting bacterial oil
release. Sterile controls, subjected to identical
treatment, produced the same amount of oil,
within experimental error, except where mercuric
chloride was present. This chemical was found
to inhibit oil recovery from porous media because
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oil bearing sand cores treated with mineral
solutions containing phosphate buffers. Methane,
hydrogen and hydrogen sulfide were evolved,
and oil analyses on the cores after incubation
showed that some of the oil had been removed
by the bacterial treatment. The author emphasized the importance of phosphate as a mineral
In order for bacteria to release oil from a
petroleum reservoir by any of the mechanisms
listed, they must penetrate the pores of the
reservoir rock throughout a substantial part of
the reservoir, and multiply therein. Several types
of reservoirs are therefore immediately ruled
out of consideration. Reservoirs of extremely
small pore size will not permit the bacteria to
penetrate. Those of high temperature, above
80 C, wirl probably not permit the bacteria to
multiply although it is conceivable that sulfate
reducing bacteria may be found which will
multiply at higher temperatures. Many reservoirs
are available, however, which are well within the
range of pore size distribution and temperature
for successful growth of the bacteria.
In addition to the cited requirements, the
mineral requirements, growth factor requirements, and energy source requirements of the
bacteria must be met. ZoBell (98) has indicated
that many oil, formation waters, when mixed
with crude oil, provide all these. Our own experience indicates little or no growth of sulfate
reducing bacteria under such conditions, nor is
the growth improved by any of the usual mineral
nutrients (ions of ammonium, calcium, magnesium, potassium, iron, sulfate, and phosphate).
Updegraff and Wren (88) found little or no
growth under such conditions and suggested the
use of a nutrient such as molasses. Many oil
field waters do support good growth of sulfate
reducing bacteria when a readily available
energy source such as lactate or glucose is added.
Others are deficient in phosphate or available
nitrogen compounds. It would probably be
necessary to introduce such an energy source
into the formation, along with any mineral
nutrients which may be deficient in the formation
water, to obtain satisfactory growth within the
The literature contains several references
purporting to demonstrate the oxidation of many
paraffin hydrocarbons by sulfate reducing bacteria (52, 63, 81). Crude oil is primarily a mixture
[VoL. 18
kntoputrescens, or Pseudomona fluorescens
mixtures of these bacteria.
A. Deterioration of Petroleum Products
The literature on the decomposition of hydrocarbons and petroleum products has been
comprehensively reviewed by ZoBell (96). It is
clear that virtually all petroleum products, when
stored in the presence of water, may undergo
some deterioration as a result of the activities of
hydrocarbon oxidizing microorganisms. Thaysen
(84) described an interesting case of spontaneous
ignition in a tank of purified kerosene stored over
river water. An organism was isolated which
fermented kerosene and gave methane, acetaldehyde, lactic acid, and acetic acid as products.
Nitrate was an essential hydrogen acceptor.
The spontaneous ignition was believed to have
been caused by the ignition of methane liberated
in the fermentation. Steel tanks were also shown
to support the growth of sulfate reducing bacteria
which contaminated the stored petroleum products with hydrogen sulfide. Allen (2) showed
that bacterial action at the interface between
gasoline and water in storage tanks may produce
peroxides and gums and precipitate lead tetraethyl, leading to deterioration of the gasoline.
Cutting oil emulsions, used in machine shops,
support growth of many types of bacteria, including sulfate reducers, which cause deterioration of the oil, and objectionable odors. Some
authorities believe that these bacteria may cause
dermatitis in workmen handling such oils.
B. Bacterial, Desulfuriation and Denitrogenization
of Crude Oil and Petroleum Products
Maliyantz (44) observed that certain sulfate
reducing bacteria attacked Ru n crude oil,
and removed part of the sulfur in the process.
Our own results (89) with Mid-Continent
American crude oils were different in that no
change in the sulfur content of the crude oil was
brought about when the crude oil was treated
with sulfate reducing bacteria in various media,
both with and without the presence of sulfur
compounds other than those in the crude oil.
Strawinski (76) observed a decrease of 12.5 per
cent in the sulfur content of an Arabian crude oil
when the oil was mixed with a sulfur-free medium
containing mineral salts and glucose, and incuor bated for four days with a culture of Peudomonas
sp. which had been selected for its ability to
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it reacted with sulfur compounds in the crude oil,
producing gummy, solid precipitates. Thus, any
tests in which mercuric chloride treated controls
are employed are meaningless. Similarly, refrigerated controls, as employed in certain
experiments reported by ZoBell (98), would be
invalid for a similar reason since low temperature
increases the viscosity of the oil, and gravity
separation of oil from oil sands decreases as
viscosity increases.
A study of the mechanism by which bacteria
might release oil, in the light of present knowledge
of petroleum reservoir engineering, led to the
following conclusions:
1. The dissolution of limestone or other
calcareous minerals by sulfate reducing bacteria
was so slow and incomplete, even in the presence
of a readily available energy source, that it could
not be expected to release appreciable amounts
of oil in a reasonable length of time.
2. Gas pressure can move oil through porous
media, but it has not been demonstrated that
sufficient gas is produced by De&ulfovibrio to
exert this effect.
3. The literature on petroleum production
engineering contains conflicting evidence on
whether detergents can increase oil recovery.
Some detergents appear to be effective, and
others ineffective. The traces of surface active
agents produced by sulfate reducing bacteria
would not be expected to influence oil recovery
within reasonable time limits.
4. Tenacious adherence of the bacteria to
solid surfaces may crowd oil off these surfaces,
but no evidence was obtained that this process
had any effect in recovering oil from oil bearing
sands or rocks.
5. Reduction of the viscosity of crude oil,
either by direct bacterial action on the oil, or by
solution of bacterially produced gases in the oil,
was not observede Large changes in viscosity are
ordinarily required to obtain significant increases
in oil recovery. It is doubtful that Desulfovibrio
can be applied successfully in the field for
recovery of oil in commercially attractive
Sanderson (65) was issued a patent on a
method for recovering oil from kerogen type
shale, comprising treatment of the shale with
Clostridium sporogenes, C. histolyticum, C.
C. Petroleum as a Substrate for the Industrial
Manufacture of Chemicals
Another promising line of research which
appears to have been generally neglected is the
use of petroleum as a substrate for the industrial
manufacture of chemicals. Crude oil and natural
gas, pound for pound, are far cheaper than other
available organic substrates. Taggart (80) obtained a patent on a method of producing fatty
acids, esters, and low-boiling alcohols by the
action of BaciUus paraffinicus on natural gas
under aerobic conditions. With natural gas
priced at 0.2 to 0.4 cents per pound of organic
matter, it does not seem out of the question to
consider the possibility of the manufacture of
foodstuffs by microbial action on this substrate
since microorganisms are known which convert
gaseous hydrocarbons to protoplasm with a
high degree of efficiency.
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PhD Thesis, The University of Texas,
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utilize sulfur compounds present in the oil. In a
later patent, Strawinski (77) disclosed an improved two-step process whereby the oil was
first treated with a culture of an aerobic bacterium in a sulfur-free medium, thus converting
part of the sulfur to sulfates, and then with a
culture of sulfate reducing bacteria, which converted the sulfates to hydrogen sulfide. This
method was claimed to result in more complete
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described a general method of desulfurizing
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producing bacteria acting on the oil under
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experience shows them to be attacked by microorganisms with great difficulty.
desulfurization of crude oil is not likely to
compete with chemical methods unless more
economical and effective methods are developed.
A similar problem is the microbial denitrogenization of petroleum. Nitrogen compounds
are also troublesome in the refining of certain oils,
and might be removed microbiologically in ways
similar to those used for sulfur. However, the
literature does not reveal any developments
toward this goal.
[voL. 18
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