Handbook of Hydrocarbon and Lipid Microbiology || Hydrocarbon-Degradation by Acidophilic Microorganisms

23 Hydrocarbon-Degradation byAcidophilic Microorganisms

K. N. Timm# Springer

W. F. M. RolingDepartment of Molecular Cell Physiology, VU University Amsterdam,

Amsterdam, The [email protected]

1
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1924
2
Taxonomy and Phylogeny of Hydrocarbon-Degrading Acidophiles . . . . . . . . . . . . . . . . 1924
3
Physico-Chemical Limits for Growth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1926
4
Physiology of Acidophilic Hydrocarbon Degraders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1926
5
Biochemistry and Genetics of Acidic Hydrocarbon Degradation . . . . . . . . . . . . . . . . . . 1927
6
Ecology of Hydrocarbon Degradation under Acidic Conditions . . . . . . . . . . . . . . . . . . . 1927
7
Cultivation and Maintenance of Hydrocarbon-Degrading Acidophiles . . . . . . . . . . . 1928
8
Research Needs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1929
is (ed.), Handbook of Hydrocarbon and Lipid Microbiology, DOI 10.1007/978-3-540-77587-4_140,

-Verlag Berlin Heidelberg, 2010

mailto:[email protected]

1924 23 Hydrocarbon-Degradation by Acidophilic Microorganisms

Abstract: Bio-filtration of volatile hydrocarbons and mineral oxidation in association with

coal mining and oil seepages can lead to the simultaneous occurrence of strongly acidic

conditions (pH < 4) and hydrocarbons. This chapter describes the current knowledge on

the taxonomy, ecology, physiology, biochemistry and genetics of hydrocarbon-degrading

acidophiles. Knowledge is still sparse, so far only five bacterial and nine fungal acidophilic

hydrocarbon-degraders have been described, in limited detail. Most bacterial hydrocarbon-

degraders are tolerant to high concentrations of heavy metals. The limited knowledge appears

to relate to difficulties in obtaining pure cultures and the low effort so far put in micro-

biological characterization of hydrocarbon-containing acidic environments.

1 Introduction

Oxidation of minerals, such as pyrite, is often accompanied by soil acidification, and can be

due to natural (e.g., volcanic activity) and human-activity associated processes (e.g., mineral

and coal mining). Mineral oxidation in association with coal mining and oil seepages can lead

to the presence of hydrocarbons in acidic environments and the requirement for bioremedia-

tion. This chapter describes the taxonomy, physiology, biochemistry, genetics and ecology of

acidophilic hydrocarbon degraders. Acidophiles are microorganisms metabolically active in

strongly acidic environments (pH< 2.0). As the number of described microorganisms capable

of degrading hydrocarbons at a pH below 2.0 is very limited, this chapter will also include

microorganisms capable of hydrocarbon-degradation at a pH lower than 4.0.

2 Taxonomy and Phylogeny of Hydrocarbon-DegradingAcidophiles

Acidophilic hydrocarbon-degraders are found among Bacteria and Eukarya, so far no

hydrocarbon-degrading acidophilic Archaea have been isolated. While microorganisms have

been isolated from hydrocarbon containing acidic environments, often the isolation condi-

tions (pH, hydrocarbons as source of carbon) were not representative for the in situ condi-

tions. For example, Dore et al. (2003) isolated five naphthalene-degrading strains from an

acidic coal pile, of which only one was capable of growing at a pH below 4.

Only a few bacterial isolates are currently known to grow with hydrocarbons below a

pH of 4. 16S rRNA gene based phylogenetic information is available for five bacterial isolates

(> Table 1). Gemmell and Knowles (2000) obtained 23 acidophilic, hydrocarbon-degrading

isolates, however the 16S rRNA gene was sequenced for only two of these isolates (Acidocella

sp. WJB-3 and Acidocella sp. LGS-3; >Table 1). The five bacterial isolates are associated with

two phyla, four with the Proteobacteria and one with the Actinobacteria. The Proteobacteria

isolates all belong to the Acetobacteraceae (Rhodospirillales, Alphaproteobacteria), which con-

tains several acidophilic, heterotrophic genera, such as the genus Acidiphilium. While for the

genus Acidiphilium no hydrocarbon-degrading isolates have been obtained so far, it has been

shown that at least one species, Acidiphilium cryptum, can acquire plasmids and functionally

express genes for phenol degradation (Quentmeier and Friedrich, 1994). Alkane-degrading

Acidisphaera sp. C197 colonies have a distinctive salmon-pink color, possibly due to caroten-

oid production (Hamamura et al., 2005).

The hydrocarbon-degrading Actinobacterium is aMycobacterium montefiorense-like bacte-

rium. It belongs to the Mycobacteraceae for which many hydrocarbon degrading isolates are

. Table 1

Taxonomic and physiological characteristics of acidophilic hydrocarbon-degrading

microorganisms

Species Deposition

Genbank

accession

pH

range

Hydrocarbon

use Reference

BACTERIA

Acidocella sp. IS10 ATCC BAA-

585

AF531477a 3–6 Naph Dore et al. (2003)

Acidocella sp. WJB-3 AF253412a 2.5–5 (Dd), Hd Gemmell and

Knowles (2000)

Acidocella sp. LGS-3 AF253413a ND (Dd), Hd Gemmell and

Knowles (2000)

Acidosphaera sp.

C197

AY678225a ND Dd, Hd Hamamura et al.

(2005)AY817739b

Mycobacterium

montefiorense

AM085774a ND Phen, Pyr Uyttebroek et al.

(2007)

FUNGI

Cladosporium resinae ATCC 34066 3.5–6.5 T, E Qi et al. (2002)

Cladosporium

sphaerospermum

ATCC

200384

3.5–6.5 T,E, (B, Styr) Qi et al. (2002)

Exophiala lecanni-

corni

CBS 102400 3.5–6.5 T,E, (B, Styr) Qi et al. (2002)

Exophiala jeanselmei 1.5–8 Styr Cox et al. (1997)

Exophiala

oligosperma

CBS 113408 3.9–6.9 T Estevez et al. (2005)

Cladophialosphora

sp. T1

ND T,E, Styr, (Dc) Prenafeta-Boldu

et al. (2001)

Cladophialosphora

sp. T2

ND T, (Dc, Hx) Prenafeta-Boldu

et al. (2001)

Paecilomyces variotii CBS 113409 3.9–6.9 T Estevez et al. (2005)

Phanerocheate

chrysosporium

ATCC 24725 3.5–6.5 T Qi et al. (2002)

a16S rRNA gene sequencebalkB gene sequence

ND not determined; B benzene; Dc decane; Dd dodecane; E ethylbenzene; Hd hexadecane; Hx hexane; Naph

naphthalene; Phen phenanthrene; Pyr pyrene; T toluene; Styr styrene; a letter between brackets indicate that only

minor growth occurs with the particular hydrocarbon

Hydrocarbon-Degradation by Acidophilic Microorganisms 23 1925

known, that usually functioning at near-neutral pH (Uyttebroek et al., 2007). This bacterium

could not be isolated on solid medium. Inspection of acidic, pyrene-degrading enrichments by

microscopy and molecular techniques suggested the presence of a single Mycobacterium-like

morphotype and a single 16S rRNA gene sequence (Uyttebroek et al., 2007), hence it is most

likely that this bacterium is solely present and responsible for PAH degradation in the

enrichments.

Remarkably, all described acidophilic hydrocarbon-degrading fungi were isolated from air

biofilters or have been identified as being capable of hydrocarbon degradation at low pH by


testing isolates from strain collections. Nine acidophilic, hydrocarbon degrading fungi have

been classified based on taxonomy (> Table 1). Except for the Basidomycete white rot fungus

Phanerochaete chrysosporium, all belong to the Ascomycetes.

3 Physico-Chemical Limits for Growth

The physico-chemical limits for growth and activity have especially focused on tolerance for

low pH (> Table 1). Alphaproteobacteria grow between pH 2.5 and 6, with optimal pH around 4

(Dore et al., 2003; Gemmell and Knowles, 2000). The usual pH range for fungal growth with

hydrocarbons is between 3.5 and 6.8 with an optimum pH around 6. The black yeast Exophiala

jeanselmei showed activity at pH 1.5 (Cox et al., 1997).

Other growth limits are less well investigated, and often not at low pH or with a

hydrocarbon as carbon source. All currently described bacterial and fungal isolates grow at

mesophilic conditions. The optimal temperatures for growth at pH 5.9 are 30 and 37�C for the

toluene-degrading fungi Cladophialophora sp. T1 and sp. T2, respectively (Prenafeta-Boldu

et al., 2001). Both isolates grew at all temperatures tested (20–37�C). The fungi P. variotii

and E. oligosperma shows fastest growth at 30�C and grows at least in the temperature range

23–40�C in media with pH 7 (Estevez et al., 2005).

Acidic conditions are often the result of dissolution of metal-containing minerals. A

characteristic of many acidophilic bacteria is their high tolerance to heavy metals. Indeed, a

high tolerance to mercury (60 mg/ml) is observed for hydrocarbon-degrading Acidocella sp.

IS10 (Dore et al., 2003), while 10 mM of Fe2+, Ni2+, Zn2+, Cr3+, Co2+or Cd2+ is tolerated by

Acidocella sp. WJB-3 (Gemmell and Knowles, 2000). Acidocella sp. LGS-3 tolerates 2 mM of

these metals but was not tested for its tolerance of higher concentrations (Gemmell and

Knowles, 2000).

4 Physiology of Acidophilic Hydrocarbon Degraders

Acidophilic hydrocarbon degrading bacteria consume many classes of hydrocarbons: mono-

cyclic and polycyclic aromatic hydrocarbons (PAH) and alkanes (> Table 1). The degree of

mineralization varies considerably; the M. montefiorense-containing enrichment mineralized

73% of pyrene to carbon dioxide (Uyttebroek et al., 2007). In contrast, aerobic mineralization

of hydrocarbons to carbon dioxide by Acidosphaera and Acidocella strains was limited: in

experiments with radiolabeled substrate only up to 10% of the radiolabel was recovered as14CO2 (Dore et al., 2003; Hamamura et al., 2005). Bacterial isolates have only been tested for

utilization of one particular type of hydrocarbon, with oxygen as terminal electron acceptor.

They have not been checked for consumption of other classes of hydrocarbons or anaerobic

growth. The Acidocella and Acidosphaera strains also use non-hydrocarbons under aerobic

conditions, such as carboxylic derivatives of hydrocarbons and common substrates like

alcohol and acetate (Dore et al., 2003; Gemmell and Knowles, 2000). These strains are closely

related to the genus Acidiphilium, members of this genus are capable of iron-reduction under

acidic, aerobic conditions (Johnson and Bridge, 2002).

Hydrocarbon-degrading, acidophilic fungi have mainly been isolated from biofilters

cleaning gases, and therefore primarily the utilization of volatile aromatic hydrocarbons

under aerobic conditions has been investigated. Volatile aromatic hydrocarbons are completely

mineralized to carbon dioxide. Sugars and oxygenated aromatic compounds such as phenol and


cresol are also degraded (Estevez et al., 2005; Prenafeta-Boldu et al., 2001; Qi et al., 2002).

Cladophialophora strains grow also with the alkane decane, although poorly (Prenafeta-Boldu

et al., 2001).

5 Biochemistry and Genetics of Acidic HydrocarbonDegradation

Acidophiles require a circumneutral intracellular pH and must maintain a pH gradient of

several pH units across the cellular membrane in order to grow at low pH. Distinctive

features in their pH homeostasis are a reversed membrane potential, a highly impermeable

cell membrane and predominance of secondary cation transporters. Even though a circum-

neutral pH is maintained, intracellular enzymes have been found to be functional at

much lower pHs (Baker-Austin and Dopson, 2007). If and how these adaptations affect

uptake and metabolism of hydrocarbons at low pH is still unknown. Organic acids are

common intermediates in aerobic oil degradation. Acidophiles are generally capable of

organic acid degradation which will help to avoid uncoupling of the respiratory chain by

diffusion of protonated forms into the cell followed by dissociation of a proton (Baker-Austin

and Dopson, 2007).

Since acidophiles, like neutralophiles, require a circumneutral intracellular pH, one may

possible expect similar degradation pathways as in microorganisms growing under circum-

neutral conditions. Screening of naphthalene-degrading Acidocella sp. IS10 with probes for

genes involved in naphthalene degradation (nahAc, nahAd, phnAc, nahH and xylE) did not

reveal the presence of these genes (Dore et al., 2003). Since this isolate does not mineralize

naphthalene completely, Acidocella sp. IS10 likely does not contain genes that encode enzymes

responsible for the later steps of naphthalene mineralization. It may contain analogues of

genes encoding enzymes involved in the early steps that are fairly similar to those tested but

sufficient different to remain undetected by the applied probes and primers. Alternatively, the

metabolic pathway might be very different to those operating at circumneutral pH.

The alkane-degrading Acidisphaera sp. C197 contains a putative gene (alkB) for alkane

hydroxylase that is 92.5% similar to that of Xanthobacter flavus and 89.7% to Alcanivorax

borkumensis (Hamamura et al., 2005).

White rot fungi produce extracellular peroxidises such as lignin peroxidise and manganese

peroxidise to degrade lignin compounds. These enzymes are not specific and also attack

aromatic pollutants with a structure similar to lignin regions. Non-ligninolytic fungi have

intracellular mechanisms for PAH degradation that lead to dihydrodioal and hydroxyl PAH

metabolites (Kennes and Veiga, 2004). Whether these mechanisms are functional under acidic

conditions is unknown. The acidophilic black yeast Exophiala jeanselmei (> Table 1) starts

degradation with an attack on the side chains of mono-aromatics, the metabolic reactions

include hydroxylation and carboxylation steps (Kennes and Veiga, 2004).

6 Ecology of Hydrocarbon Degradation under AcidicConditions

Environments in which acidophilic hydrocarbon degraders have been encountered are air

biofilters treating volatile hydrocarbons (Estevez et al., 2005; Kennes and Veiga, 2004),

biodegraded oil seepages (Hamamura et al., 2005; Roling et al., 2006) and coal mine runoff


and coal tar pits, as coal contains hydrocarbons and sulphur compounds which are leached by

rain and exposure to air (Stapleton et al., 1998; Uyttebroek et al., 2007).

First evidence for degradation of aromatic hydrocarbons under extreme acidic conditions

(pH 2.0) was obtained in 1998 for soil samples and enrichments derived from a coal storage

pile in the USA (Stapleton et al., 1998). An essential role for eukaryotes in degradation of

toluene and naphthalene was proposed based on the inhibition of hydrocarbon degradation

after treatment with cycloheximide. Acidiphilium and Acidocella strains isolated from the same

enrichments degraded only oxygenated derivates of mono-aromates (e.g., catechol). Similar

microorganisms were also observed in natural hydrocarbon seeps on two different continents

Yellowstone, USA, and Dorset, UK; (Hamamura et al., 2005; Roling et al., 2006). The Dorset

seep was characterized by strongly degraded oil, while oil in the acidic Yellowstone soils

showed hardly any sign of biodegradation. Despite these differences, both seeps were domi-

nated by members of the genera Acidisphaera, Acidiphilium and Acidocella, according to

cultivation-independent 16S rRNA gene surveys. The sequences were closely related to isolated

hydrocarbon degraders. Community fingerprints of microbial community in the natural

hydrocarbon seeps in Yellowstone Park revealed a dominant band whose sequence was

identical to alkane-degrading Acidosphaera strain C197, which was isolated from the same

seepage (Hamamura et al., 2005). These observations suggest that hydrocarbon degradation at

this location is not dependent on eukaryotes. However, oil-degrading enrichments from the

strongly degraded Dorset seep only contain fungi and no bacteria (Roling et al., 2006).

No evidence for the association of Alphaproteobacteria with hydrocarbon degradation was

obtained for an acidic coal pyrolysis site in Belgium (Uyttebroek et al., 2007). In stead,

Mycobacteria appeared important in degradation. A single Mycobacterium species dominated

in situ, its sequence was identical to the slow-growing M. montefiorense related species that

dominated pyrene-degrading enrichment cultures from the same location. This Mycobacteri-

um also significantly contributed to the in situ bacterial community.

Acidophilic, hydrocarbon-degrading Archaea have not been reported so far, even though

Archaea are supposed to be more common for and better adapted to extreme environments

than Bacteria and Eukarya. Cultivation-independent analysis of a natural, biodegraded hydro-

carbon seepage in Dorset, UK, did not reveal the presence of Archaea while Bacteria and

Eukarya were encountered (Roling et al., 2006). Archaea do not appear to have been targeted

in other molecular studies.

Cultivation-independent analysis of hydrocarbon-containing acidic environments so far

mainly targeted ribosomal RNA genes, with the exception of the study by Stapleton et al.

(1998). They attempted to hybridize isolated DNA with molecular probes targeting genes

commonly associated with toluene and naphthalene degradation at neutral pH (nahA, nahH,

nahG, todC1C2, tomA), but did not observe hybridization despite that mineralization of

aromatic compounds at pH 2.0 occurred.

7 Cultivation and Maintenance of Hydrocarbon-DegradingAcidophiles

Enrichment, isolation and maintenance of acidophilic, hydrocarbon-degrading microorgan-

isms is mainly performed using defined mineral medium with a hydrocarbon as sole source of

carbon at low pH. Fungal isolates have so far been directly isolated from environmental

samples, bacteria are generally enriched prior to isolation.


Obtaining pure acidophilic, hydrocarbon-degrading isolates appears sensitive to type of

samples used and enrichment conditions. In their study, Uyttebroek et al. (2007) obtained

enrichments capable of degrading pyrene and phenanthrene at low pH (<4) only for acidic

PAH-contaminated soils and not for PAH-contaminated soils with pH 8. These enrichment

cultures in mineral medium often failed to resume growth on phenanthrene or pyrene after

being transferred to fresh medium. No colony forming units could be obtained on plate from

enrichments that were successfully transferred (Uyttebroek et al., 2007). Stapleton et al. (1998)

made comparable observations for hydrocarbon-degrading enrichments obtained from an

acidic coal storage pile. Acidiphilum, Acidocella strains, a fungus and a yeast strain were

isolated from these enrichments but neither pure isolates nor defined mixed cultures of

these isolates were capable of hydrocarbon degradation.

Tolerance to low pHmight be lost depending on initial enrichments conditions (Uyttebroek

et al., 2007). Cultures enriched from acidic soil at pH 7 grew during subsequent cultivation on

pyrene at various pH values not so well at low pH as cultures originally enriched at lower

pH (3 or 5), even though community analysis suggested that in these cultures the same

M. montefiorense strain is involved in pyrene degradation.

These observations suggest that great care should be taken to use appropriate conditions

(e.g., sample selection, low pH) in the enrichment, isolation and maintenance of acidophilic

hydrocarbon degraders.

8 Research Needs

A challenge in the bioremediation of hydrocarbon contaminated soils is that these soils are

often also polluted with other types of pollutants, such as metals. As acidophilic organisms are

generally tolerant to high levels of heavy metals, these organisms might be good candidates for

such sites. While evidence for the occurrence of microbial degradation of hydrocarbon

degradation under acidic conditions is accumulating (Hamamura et al., 2005; Stapleton

et al., 1998; Uyttebroek et al., 2007), the microorganisms involved and the physiology,

biochemistry and genetics of acidiphilic biodegradation have remained largely unexplored.

Furthermore, isolations from acidic environments contaminated with hydrocarbons have

mainly concerned bacteria, even though in some cases degradation appears to dependent on

eukaryotes (Roling et al., 2006; Stapleton et al., 1998). More emphasis should be given to the

isolation and characterization of acidophilic bacteria and fungi, guided by information from

cultivation-independent characterization of who degrades what (by e.g., stable isotope prob-

ing and micro-autoradiography combined with fluorescence in situ hybridization).

Another important research direction is to establish whether adaptations to life at low pH,

such as low membrane permeability, affect the biochemistry of hydrocarbon degradation.

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Handbook of Hydrocarbon and Lipid Microbiology || Hydrocarbon-Degradation by Acidophilic Microorganisms