Crude oil (petroleum) is a highly complex mixture of organic compounds of which some
1.3 million litres enters the environment each year. More then anything else, the
numerous oil‐shipping disasters, such as of the Exxon Valdez (1989), the Erika (1999)
and the Prestige (2003), have captured the public attention to this environmental
problem (Fig. 1). However, these accidents account for only a small part of the annual
global release of crude oil, as most enters the environment from deliberate discharge
and processing sites. Around three million tons of oil enters the sea each year, of
which about 20% originates from oil‐pumping operations, transport and refining activities
and 25% from non‐tanker shipping and natural seepages. More than half (55%) originates
from illegal activities that include the dumping of ballast water and oil residues
as well as accidents (Golyshin et al., 2003). Hydrocarbons are also produced continuously
by living cells as natural oils and fats (de Lorenzo, 2006). The observation that
the oceans are not covered with an oily layer is a testimony to the activity of the
hydrocarbon‐degrading microorganisms (Head et al., 2006). Several bacteria are even
known to feed exclusively on hydrocarbons (Yakimov et al., 2007). For these (facultative)
hydrocarbon degraders the occasional supertanker oil spill forms an occasional carbon
banquet. They play an important role in the clean‐up after an oil spill and form the
biological basis for the natural oil‐degrading capacity of the ecosystem. Studies
have focused on identifying and characterizing these oil‐eating microbes, as well
as how they cope with the oil/water interface, and how to improve this capacity. Here
we highlight some of the recent genomics advances in this field.
Figure 1
Left: Oil spill in the ocean (http://www.pacificariptide.com/pacifica_riptide/oil_spill/).
Right: Shipping disaster of Exxon Valdez (http://symonsez.wordpress.com/2009/03/24/blame‐exxon‐valdez‐on‐captain‐bligh‐midwest‐storms/).
Degradation of hydrocarbons
Over 17 000 organic compounds have been identified in crude oil, and subdivided into
four main classes: the saturates, aromatics, asphaltenes and resins (Marshall and
Rodgers, 2004). The susceptibility of hydrocarbons to microbial degradation can be
generally ranked as follows: linear alkanes > branched alkanes > small aromatics > cyclic
alkanes (Leahy and Colwell, 1990). The bioremediation efforts of the Exxon Valdez
oil spill have indeed shown that the (light) alkanes are depleted first and that some
compounds, such as the high‐molecular weight polycyclic aromatic hydrocarbons (PAHs),
may not be degraded at all (Atlas and Bragg, 2009).
Many environmental factors influence the breakdown of carbohydrates by microorganisms.
Specifically in marine environments, low phosphorous and nitrogen levels may limit
growth of oil‐degrading microorganisms and thus rapid oil consumption. There have
been attempts to ‘fertilize’ oil‐spill areas to create a more optimal C : N : P balance
of 100:10:1 (referred to as biostimulation) (Nikolopoulou and Kalogerakis, 2008).
In open‐sea environments, dilution of soluble nutrients quickly occurs, and the administration
of insoluble or hydrophobic (oil‐soluble) fertilizers is thought to enhance biostimulation
effectiveness. Biosurfactants seem another promising form of biostimulation. Biosurfactants
increase the oil‐surface area and with that the amount of oil actually available for
attack by bacteria (Nikolopoulou and Kalogerakis, 2009). When oil washes up on beaches
and is sequestered in the sediments, the bio‐availabilty can be severely reduced,
significantly slowing down or even preventing biodegradation. Depending on the particular
local circumstances, tilling of beaches may be applied to expose the sequestered oil.
However, tilling itself is disruptive for many coastal plants and animals and, as
the oil was beyond the reach of the biota in the first place, may do more harm than
good. Local conditions, in general, have a big effect on the efficiency of oil breakdown,
such as temperature, waves (mixing), availability of oxygen, and of course the composition
of the spilled oil. These should be taken into account when devising an intervention
strategy.
Diversity and metabolism of oil‐degrading bacteria
Notwithstanding all the factors that influence the oil‐biodegrading capacity, it all
comes down to the metabolic veracity of bacteria that do not mind to get their ‘hands’
dirty (or greasy to be more precise). A great number of bacteria have been identified
that help clean up the hydrocarbon compounds in the aftermath of oil spills. But a
significant part of the hydrocarbon content in seawater has a biological origin. Lipids
and fatty acids from plants, animals and microbes and the products of their conversion
in anoxic zones are ubiquitous. Evolution has created some bacteria that dine exclusively
on hydrocarbons, including obligate hydrocarbon degraders of the genera Oleispira,
Oleiphilus, Thalassolituus, Alcanivorax and Cycloclasticus (Fig. 2).
Figure 2
A phylogenetic tree illustrating the diversity of aerobic hydrocarbon‐degrading bacteria.
Organisms shown in blue can degrade saturated hydrocarbons, whereas those in red can
degrade polycyclic aromatic hydrocarbons. The organisms shown in black do not degrade
hydrocarbons. Reprinted from Head and colleagues (2006) by permission from Macmillan
Publishers Ltd, copyright 2006.
In typical carbon utilization tests, these bacteria only catabolize hydrocarbons such
as Tween 40 and 80, leaving other carbon sources, including sugars and acids, untouched
(Yakimov et al., 2007). Current interest in the metabolism of (obligate) hydrocarbon‐degrading
bacteria has spurred on various genome sequencing projects (Table 1 and Fig. 3).
Table 1
Genome sequencing projects of oil/hydrocarbon‐degrading bacteria (adapted from the
GOLD database: http://www.genomesonline.org).
Species
Genome size (kb)
GC%
Habitat
goldstampa/Reference
Completed
Alcanivorax borkumensis SK2
3120
54.7
Marine
Gc00411/b
Marinobacter hydrocarbonoclasticus VT8
4326
57.3
Marine
Gc00504/c
Arthrobacter sp. FB24
4698
65.5
Soil
Gc00445
Geobacillus thermodenitrificans NG80‐2
3607
49
Fresh water, oil fields
Gc00532/d
Ongoing
Alcanivorax sp. DG881
3789
58
Marine
Gi01400
Cycloclasticus pugetii PS‐1
Marine, sediment
Gi03249/e
Cycloclasticus sp.TUi26
2300
Fresh water
Gi02200
Marinobacter hydrocarbonoclasticus ATCC 49840
Fresh water
Gi01519
Marinobacter algicola DG893
4413
57
Marine
Gi01420
Methylomicrobium album BG8
Soil
Gi02102
Oceanicaulis alexandrii HTCC2633
3166
64
Marine
Gi00864
Oleispira antarctica RB‐8
4400
43
Marine
Gi02491/f
Rhodococcus opacus B4 PD630
Soil
Gi03264
a.
Links to sequence information and genome database can be retrieved with the goldstamp
ID.
b.
Golyshin et al. (2003).
c.
Huu et al. (1999).
d.
Feng et al. (2007).
e.
Dyksterhouse et al. (1995).
f.
Yakimov et al. (2003).
Figure 3
Geographic distribution of isolates of retrieved 16S rRNA sequences of marine obligate
hydrocarbonoclastic γ‐proteobacteria. Reprinted from Yakimov and colleagues (2007)
with permission from Elsevier.
The genome of the obligate hydrocarbon‐degrading bacterium (or hydro‐carbonoclastic
bacterium) Alcanivorax borkumensis was recently discussed in some detail (Schneiker
et al., 2006). This bacterium is of particular interest as it is a cosmopolitan marine
bacterium that blooms in oil‐contaminated areas, where it can constitute up to 80%
of the bacterial population. The features of its genome reveal how this bacterium
grows efficiently on alkanes. As hydrocarbons are poorly soluble in water, this bacterium
encodes extensive exopolyssacharide production and pili through which it can attach
to the oil‐water interface. Biosurfactants can be produced that emulsify the alkanes
and increase the oil‐water surface area. The genome contains several regions of atypical
GC content, one of which carries a complete gene set for alkane degradation (alkSB1GJH),
and more genes were found that encode proteins putatively involved in alkane degradation,
such as alkK, alkL and alkN (Fig. 4). These encode proteins such as alkane hydroxylases,
alcohol dehydrogenases, oxidoeductases, P450 cytochromes, rubredoxin and a rubredoxin
reductase (rubA and rubB). As expected for an obligate hydrocarbon degrader, functional
genes that encode typical sugar uptake mechanisms such as PEP‐dependent sugar/phosphotransferase
systems are not found in the genome. The marine origin of A. borkumensis is evident,
as many genes encode efficient scavenging systems, such as high‐affinity ABC‐transporters,
to sequester essential nutrients (Fe, Zn, Co, Mg, Mn and Mo). Furthermore, the Na+‐dependent
NADH‐quinone dehydrogenase (encoded by nqrABCDEF) and a variety of H+/Na+ antiporters
(encoded by mnhABCDEFG, nhaD, nhaB, nhaP and nhaC) enables many transport processes
to use a sodium gradient.
Figure 4
Ubiquity of gene clusters for the degradation of (A) aliphatic and (B) (poly) aromatic
fractions of oil.
A. Organization of genes homologous to the A. borkumensis alk gene
cluster in the hydrocarbon‐degrading marine proteobacteria. Homologous genes are highlighted
by shaded areas: sequences predicted to code for LuxR‐type transcriptional activators
of the alkane genes, AlkS, are marked in green, genes for the alkane degradation pathway
are indicated in blue. Percentages of protein identity/similarity of polypeptides
from A. borkumensis with those of M. aqaeolei and O. alexandrii are shown. Gene designations:
alkB1, alkane monooxygenase; alkG, rubredoxin; alkJ, alcohol dehydrogenase; alkH,
aldehyde dehydrogenase.
B. Organization of gene clusters of polycyclic aromatic hydrocarbons
(PAH) degradation pathways of Cycloclasticus sp. A5 and PAH‐degrading α‐proteobacteria.
pdxA genes predicted to code for the pyridoxal‐phosphate biosynthesis enzyme are colored
in green, PAH degradation genes are in blue. Percentages of protein identity/similarity
of the polypeptides from Cycloclasticus sp. A5 with those of the PAH‐degrading α‐proteobacteria
are shown. Gene designations: phnA1, iron‐sulfur protein (ISP) α subunit of PAH dioxygenase;
phnA2, ISP β‐subunit of PAH dioxygenase; phnA3, Rieske‐type [2Fe‐2S] ferredoxin, phnA4,
NADH‐ferredoxin oxidoreductase; phnC, extradiol [3,4‐dihydrophenanthrene] dioxygenase;
phnD, 2‐hydroxy‐2H‐benzo[h]chromene‐2‐carboxylate isomerase. Reprinted from Yakimov
and colleagues (2007) with permission from Elsevier.
Biodegradation of crude oil
The PAHs found in oil are particularly resistant to microbial degradation, by the
intrinsic stability of the aromatic ring. Polycyclic aromatic hydrocarbons form a
great health hazard as many are toxic or carcinogenic and persist in the oil‐polluted
environments long after the (linear) alkanes are degraded. Luckily, PAHs also have
biological origins and are, for example, formed by the combustion of organic matter
in forest fires. A natural occurrence of compounds usually means that some bacteria
can be found in Nature that can feed on them. Polycyclic aromatic hydrocarbons comprise
of two or more fused aromatic rings with a diverse range of branching types and aromatic
groups. Not surprisingly therefore, effective PAH breakdown involves whole communities
of both bacteria and eukaryotes. Within these communities the PAH‐degrading capabilities
of Arthrobacter, Burkholderia, Mycobacterium, Pseudomonas, Sphingomonas and Rhodococcus
are best studied, which is supplemented by sequencing efforts (Table 2) (Seo et al.,
2007; Vandermeer and Daugulis, 2007; Haritash and Kaushik, 2009; Martinkova et al.,
2009).
Table 2
Genome sequencing projects of PAH‐degrading bacteria (adapted from the GOLD database:
http://www.genomesonline.org).
PAH degraders
Genome size (kb)
GC%
Habitat
goldstampa/Reference
Completed
Arthrobacter chlorophenolicus A6
4395
65.9
Soil
Gc00930
Mycobacterium flavenscens PYR‐GCK
5619
67
Soil
Gc00534
Mycobacterium sp. JLS
6048
68
Soil
Gc00516
Mycobacterium sp. MCS
5705
68
Soil
Gc00392
Mycobacterium vanbaalenii PYR‐1
6491
67.8
Sediment
Gc00479
Polaromonas naphthalenivorans CJ2
4410
62.5
Fresh water
Gc00486/c
Rhodococcus erythropolis PR4
6516
62
Marine
Gc00980
Rhodococcus opacus B4
7913
67
Gc00982
Sphingomonas wittichii RW1
5382
67
Fresh water
Gc00571
Pseudomonas sp.b
Ongoing
Arthrobacter phenanthrenovorans Sphe3
Soil
Gi01675
Burkholderia sp. Ch1‐1
3789
58
Soil
Gi03275
Burkholderia sp. Cs1‐4
Rhizosphere
Gi03276
Cycloclasticus sp. TU126
2300
Fresh water
Gi02200
Cycloclasticus pugetii PS‐1
Marine, sediment
Gi03249
Mycobacterium sp. Spyr1
66
Soil
Gi02013
a.
Links to sequence information and genome database can be retrieved with the goldstamp
ID.
b.
There are genome sequencing projects of over 50 Pseudomonas species/strains, of which
17 are complete, many of which are capable of PAH degradation.
c.
Yagi et al. (2009).
These microbes involved in the second‐phase clean‐up require different approaches
to biostimulation. For example, although aeration and availability of water remain
crucial, the addition of fertilizers actually had a negative impact on the PAH degradation
(Vinas et al., 2005). The study of PAH‐degrading bacteria may provide insights how
to get a more complete oil clean‐up (Lafortune et al., 2009). The metabolism required
for efficient breakdown of PAHs has been best studied in species of Pseudomonas with
relatively simple model PAHs, such as napthalenes, phenanthrene and anthracene (Habe
and Omori, 2003). In Pseudomonas species, the genes that are required for the breakdown
of PAHs (nah, pah, dox phn, phd and nag operons) (Fig. 4) can often be found on plasmids,
facilitating genetic transfer and acquisition in oil‐polluted environments.
In light of the ever‐increasing world demand for efficient oil‐cleanup measures, we
should mention the ‘flip‐side of the coin’ of oil‐degrading bacteria. The growth of
hydrocarbon‐degrading bacteria in oil fields and oil tanks in general affects not
so much the quantity, but certainly the quality of oil. Oil fields occur typically
at several thousand metres below ground and, at temperatures of 60–90°C, are considered
sterile. However, as soon as oil is extracted, hydrocarbon‐degrading bacteria capable
of oxidizing hydrocarbons start to grow (Yemashova et al., 2007). Moreover, when the
self‐flow of oil stops, a common practice to extract the oil is to flood the stratum
with (non‐sterile) water. Bacteria (and fungi) that grow in the oil fields produce
peroxides, pitch acids and in some cases H2S that decrease the thermostability and
volatility of the fuel. Although many different species are involved, a notorius group
of bacteria in this regard are the sulfate‐reducing bacteria. These bacteria are able
to use the sulfate in the oil as alternative to oxygen and produce H2S. Moreover,
they stimulate growth of other bacteria that can subsequently use the H2S (Muyzer
and Stams, 2008). Their ability to use sulfate combined with their significant ecological
(and economic) impact has made them the focus of current research and several sulfate‐reducing
bacteria have been completely sequenced (and published) in the last few years. These
include the Desulfovibrio vulgaris Hildenborough, the arctic Desulfotalea psychrophila
and the archeal Archaeoglobus fulgidus (Klenk et al., 1997; Heidelberg et al., 2004;
Rabus et al., 2004). Their ability to degrade oil in anaerobic conditions may prove
useful for the natural oil‐degrading capacity in certain types of ecosystems. Moreover,
the lessons learnt on biostimulation can be applied (in reverse) to control microbial
deterioration of oil, such as removal of water, oxygen and addition of biocides.
Future perspectives
In the last few years, considerable progress has been made in getting to know the
bacteria that eat oil and how they do it. The next step is to actually use all this
information to enhance and control the capability of oil degradation by bio‐augmentation.
Bio‐augmentation, the addition of specific microorganisms, could considerably speed‐up
the natural biodegradation progress. Ideally, as alkanes are relatively quickly degraded,
bio‐augmentation should focus on microbes that are specifically engineered to tackle
compounds that are still resistant to microbial attack. The search for such a super‐bug
has culminated in the first ever patent of a living organism, an engineered Pseudomonas
sp., capable of degrading camphor, octane, salicylate and naphthalene (US Patent 4259444).
When such bacteria are introduced into the environment, they typically quickly disappear.
Their introduction into already colonized niches is very difficult, as most fall prey
to protozoa (Cases and de Lorenzo, 2005). In summary, the oil spills demonstrate the
immense adaptive capabilities of the bacterial community to their environment. In
time, like all natural hydrocarbons, crude oil is completely degraded by the microorganisms
or made inaccessible. Ironically, when speeding up the biodegradation of oil‐spill
sites via bacteria, we should probably take most care not to do disturb the environment,
again.