Anaerobic Degradation of Oil Hydrocarbons by Bacteria

Anaerobic Degradation of Oil Hydrocarbons by Bacteria

Anaerobic degradation of oil hydrocarbons focuses on the roles of sulfate-reducing and nitrate-reducing bacteria in the breakdown of complex hydrocarbon mixtures. The research highlights the mechanisms by which these bacteria metabolize hydrocarbons under anoxic conditions, contributing to bioremediation efforts in oil-contaminated environments. Key findings include the specific consumption of alkylbenzenes and n-alkanes, revealing insights into microbial processes in oil reservoirs. This study is essential for environmental scientists and microbiologists interested in anaerobic microbial ecology and the biogeochemical cycles of hydrocarbons.

Key Points

  • Explores the role of sulfate-reducing bacteria in anaerobic hydrocarbon degradation.
  • Details the specific consumption of alkylbenzenes and n-alkanes by bacteria.
  • Investigates microbial processes in oil reservoirs and their implications for bioremediation.
  • Summarizes historical and recent findings on anaerobic hydrocarbon oxidation mechanisms.
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Microbial Ecology of Oil Fields
Microbial Biosystems: New Frontiers
Proceedings of the 8
th
International Symposium on Microbial Ecology
Bell CR, Brylinsky M, Johnson-Green P (ed)
Atlantic Canada Society for Microbial Ecology, Halifax, Canada, 1999.
Anaerobic degradation of oil hydrocarbons by sulfate-reducing
and nitrate-reducing bacteria
Manabu Fukui
1**
, Gerda Harms
1
, Ralf Rabus
1
, Andreas Schramm
1
,
Friedrich Widdel
1
*, Karsten Zengler
1
, Chris Boreham
2
, Heinz Wilkes
3
1
Max-Planck-Institut für Marine Mikrobiologie, Celsiusstr. 1, D-28359 Bremen, Germany.
2
Australian Geological Survey Organization, Canberra A.C.T., Australia 2601.
3
Forschungszentrum Jülich GmbH, ICG-4, D-52425 Jülich, Germany.
* Corresponding author.
** Present address: Department of Biology, Faculty of Science, Tokyo Metropolitan University,
Minami-ohsawa 1-1, Hachio-ji, Tokyo 192-0397, Japan.
ABSTRACT
Crude oil is a complex mixture mainly composed of various saturated and aromatic
hydrocarbons. Whereas degradation of hydrocarbons by oxygen-respiring microorganisms
has been known for nearly one century, utilization of hydrocarbons under anoxic conditions
has been investigated only during the past ten years. The present paper summarizes
investigations into the anaerobic degradability of crude oil as a complex mixture of
hydrocarbons. Anaerobic growth on crude oil was observed in enrichment cultures and pure
cultures of sulfate-reducing bacteria. Several alkylbenzenes and n-alkanes were specifically
consumed with concomitant reduction of sulfate to sulfide; consumed hydrocarbons
together accounted for up to approximately 10% of the crude oil. Incompletely consumed
alkylbenzenes exhibited an enrichment in
13
C versus
12
C. Sulfate-reduction with oil
hydrocarbons may offer an explanation for ancient microbial processes in oil reservoirs
where reduced sulfur species are present, and for the undesirable sulfide formation in oil
production plants. Furthermore, an anaerobic consumption of alkylbenzenes and n-alkanes
from crude oil in enrichment cultures and pure cultures of denitrifying bacteria could be
demonstrated.
Introduction
Most crude oils are composed of more than 75% of aliphatic and aromatic hydrocarbons
[54, 57]. Oil hydrocarbons belong to the large global fraction of organic carbon that has
been preserved from an ancient biosphere due to burial followed by diagenetic and
catagenetic transformation processes. The withdrawal of photosynthetically fixed carbon
from reoxidation and recycling into the inorganic pool by bacteria and other organisms gave
rise to our oxic atmosphere. The total mass of O
2
on our planet amounts to 1,200 Tt (1,185
Tt in the atmosphere and around 12 Tt dissolved in water reservoirs; values calculated from
data summarized by Greenwood and Earnshaw [28]. One Tt = 10
12
t). Assuming that for
each molecule of O
2
generated by water cleavage one atom of carbon (C) is fixed from
CO
2
, according to the net equation of photosynthesis, 450 Tt organic C must have been
Microbial Ecology of Oil Fields
deposited (C-loss due to diagenetic decarboxylation processes not considered). The real
amount of biologically fixed organic C may be even higher since some O
2
has been
consumed by reaction with originally reduced inorganic compounds such as ferrous
minerals. Living organisms and dead biomass (in soil and water) on our planet contain 0.8
and 2.8 Tt C, respectively, which is 0.18% and 0.62%, respectively, of the total organic C
(summarized from [13]). The estimated amounts in accessible fuel reservoirs are 0.23 Tt C
in oil harboured in conventional reservoirs as well as in shales and sands, 0.044 Tt C in gas,
0.22 Tt C in lignite and 0.95 Tt C in coal (calculated from data summarized by [54, 57]).
The sum is 1.4 Tt accessible fuel-C, which is 0.32% of the total organic C. Hence, one has
to postulate that around 445 Tt, i.e. around 99% of photosynthetically produced and
presently preserved organic C is distributed in sediments of various ages where its
inaccessibility to biological reoxidation processes guarantees the maintenance of our oxic
biosphere. The hydrocarbon content of this organic material is unknown. But even if one
assumes that this enormous reservoir of organic carbon consists on the average of no more
than 1% of hydrocarbons, as the less maturated kerogens (the diagenetically transformed
biomass that gave rise to oil), it would add around 4 Tt hydrocarbon-C to the 1.4 Tt
organic C in accessible fuel reservoirs. Hence, studying the biological degradation or
degradability of hydrocarbons means directing our research interest not only to an
environmentally and technologically important, but also to a globally rather dominant group
of organic compounds.
Brief historical overview
Biological hydrocarbon oxidation was first demonstrated around the turn of the century in
fungal and bacterial cultures (for summary see [15]). The biochemical mechanisms of
hydrocarbon oxidation have been elucidated [14, 15]. The only aerobic activation
mechanism of saturated hydrocarbons (open-chain and cyclic alkanes) that has been
substantiated thus far is the monooxygenase reaction. Aromatic hydrocarbons are initially
attacked by monooxygenases or dioxygenases, depending on the type of alkyl side chain or
the type of microorganism [26].
During the 1940s, and again twenty years later, there were reports of an anaerobic
oxidation of alkanes by sulfate-reducing bacteria of the genus
Desulfovibrio
[19, 42, 51].
However, cultures have not been preserved, or attempts to reproduce hydrocarbon
oxidation by
Desulfovibrio
strains failed [1, 2]. Interest in possible hydrocarbon oxidation
by sulfate-reducing bacteria arose from their frequent presence in oil production plants
where these bacteria produce sulfide with its many undesirable effects (for overview see
[17, 43, 45]. Even though sulfate-reducing bacteria were recognized as the source of sulfide
produced in oil fields in the 1920s [7], their electron donor and carbon substrates in these
habitats remained a matter of discussion for several decades. In his review written in 1958,
ZoBell supposed that crude oil itself, provided it is dispersed in mineral solution, supports
growth of certain sulfate-reducing bacteria; but at that time he expressed certain doubts
about an anaerobic utilization of hydrocarbons as reported before [59]. Furthermore,
geochemical studies invoked an interest in possible hydrocarbon oxidation by sulfate-
reducing bacteria. Investigations into the genesis of sulfur deposits led to the assumption
that hydrocarbons from oil formerly served as electron donors for sulfate-reducing bacteria
and yielded sulfide that was subsequently oxidized with oxygen to sulfur [52].
Microbial Ecology of Oil Fields
The first hydrocarbons for which an anaerobic degradation could be unequivocally
shown were alkylbenzenes; degradation of these, especially of toluene, was shown in
enriched bacterial populations [27, 29, 33, 53] and in pure cultures of iron-reducing [37],
denitrifying [4, 5, 20, 23, 24, 48, 55] and sulfate-reducing [10, 47] bacteria. In the
meantime, several details of the biochemistry and underlying genes of anaerobic toluene
activation have become known [9, 12, 18, 34, 46]. Toluene is activated by condensation
with fumarate yielding benzylsuccinate. This is further oxidized by reactions somewhat
resembling β-oxidation of fatty acids and yielding benzoyl-CoA and succinyl-CoA.
Furthermore, anaerobic degradation of the unsubstituted aromatic hydrocarbons, benzene
and naphthalene could be measured in enriched bacterial communities [21, 36, 58].
Consumption of an
n
-alkane as the only organic growth substrate under anoxic
conditions was demonstrated in quantitative experiments with newly isolated, mesophilic
types of sulfate-reducing bacteria under strict exclusion of air [2, 53]. Furthermore, three
strains of denitrifying bacteria have been isolated and shown to grow anaerobically on
defined
n
-alkanes [22; Behrends, Harder, Rainey, Widdel, unpublished]. The mechanism of
anaerobic alkane oxidation is still insufficiently understood. Fatty acid analyses after
anaerobic growth on
n
-alkanes suggested that in one type of sulfate-reducing bacterium the
carbon chain of the alkane is altered at the end by one carbon atom during activation; one
possibility would be the terminal addition of a C
1
-unit. This mechanism may not occur in
other species [3].
Growth of sulfate-reducing bacteria on crude oil
In addition to individual hydrocarbons, crude oil as a natural, complex mixture of
hydrocarbons was also tested as growth substrate for sulfate-reducing bacteria. A
mesophilic enrichment culture from an oil tank was shown to utilize alkylbenzenes from
crude oil added as the only source of organic compounds to defined anoxic mineral medium
[53]. Whole-cell hybridization with fluorescently labelled 16S rRNA-targeted
oligonucleotide probes revealed that more than 95% of the enriched population were
members of the suggested family of the
Desulfobacteriaceae
[45]. Members of this family
of sulfate-reducing bacteria are distinctive from
Desulfovibrio
and
Desulfomicrobium
species, for which the family
Desulfovibrionaceae
has been suggested. This observation is
in agreement with the finding that many sulfate-reducing bacteria that degrade aromatic
compounds are members of the
Desulfobacteriaceae
. Subsequent attempts to isolate the
microorganisms responsible for depletion of alkylbenzenes from crude oil in the enrichment
culture yielded two types of novel sulfate-reducing bacteria. One strain oxidized
o
-xylene,
o
-ethyltoluene and toluene, the other strain oxidized
m
-xylene,
m
-ethyltoluene,
m
-isopropyltoluene and toluene [30]. The anaerobic consumption of alkylbenzenes by the
enrichment culture caused an isotopic discrimination, as obvious from analysis of the
remaining part of the respective hydrocarbons (Table 1).
Furthermore, utilization of
n
-alkanes from crude oil by sulfate-reducing bacteria has been
demonstrated. A moderately thermophilic sulfate-reducing bacterium (optimum around 60
Û& LVRODWHG RQ
n-decane consumed n-alkanes from crude oil especially in the range from C
8
to C
11
[53]. Furthermore, an enrichment culture exhibited sulfate-dependent consumption
of n-alkanes from oil [16].
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FAQs of Anaerobic Degradation of Oil Hydrocarbons by Bacteria

What are sulfate-reducing bacteria and their role in oil degradation?
Sulfate-reducing bacteria (SRB) are microorganisms that utilize sulfate as a terminal electron acceptor in the absence of oxygen. They play a crucial role in the anaerobic degradation of hydrocarbons by metabolizing complex oil mixtures, such as alkylbenzenes and n-alkanes. This process not only contributes to the natural attenuation of oil spills but also impacts the geochemistry of oil reservoirs. Understanding the mechanisms of SRB can inform bioremediation strategies for contaminated sites.
How do nitrate-reducing bacteria contribute to the degradation of oil hydrocarbons?
Nitrate-reducing bacteria utilize nitrate as an electron acceptor during the anaerobic degradation of hydrocarbons. This process can enhance the biodegradation of oil contaminants in environments where oxygen is limited, such as groundwater aquifers. The study highlights that these bacteria preferentially degrade specific hydrocarbons, including alkylbenzenes, during different growth phases. Their metabolic pathways provide insights into potential bioremediation techniques for oil-polluted sites.
What are the implications of anaerobic hydrocarbon degradation for bioremediation?
Anaerobic hydrocarbon degradation by bacteria is significant for bioremediation efforts, particularly in oil-contaminated environments. Understanding the metabolic capabilities of sulfate-reducing and nitrate-reducing bacteria allows for the development of targeted bioremediation strategies. These strategies can enhance the natural degradation processes, leading to more effective cleanup of oil spills. The research findings provide a framework for improving bioremediation techniques in various environmental settings.
What specific hydrocarbons are targeted by sulfate-reducing bacteria?
Sulfate-reducing bacteria specifically target alkylbenzenes and n-alkanes during the anaerobic degradation of crude oil. The study indicates that these bacteria can consume up to 10% of crude oil hydrocarbons, demonstrating their potential for bioremediation. The degradation process involves complex biochemical pathways that transform these hydrocarbons into less harmful substances. Understanding which compounds are metabolized can help in assessing the effectiveness of bioremediation strategies.
What historical context is provided regarding the study of anaerobic degradation?
The historical context of anaerobic degradation of hydrocarbons reveals that initial studies date back to the early 20th century. Early research identified the potential of sulfate-reducing bacteria to oxidize hydrocarbons, but it wasn't until recent decades that significant advancements were made in understanding these processes. The evolution of research has led to a clearer picture of how these bacteria function in oil reservoirs and their role in the biogeochemical cycling of carbon. This background is essential for appreciating the current state of knowledge in microbial ecology.

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