An overview of the bacteria and archaea involved in removal of inorganic and organic sulfur compounds from coal

FUEL
PROCESSING
TECHNOLOGY
ELSEVIER

Fuel Processing Technology 40 (1994) 167-182

An overview of the bacteria and archaea involved in removal
of inorganic and organic sulfur compounds from coal
G.I. Karavaiko*,

L.B. L o b y r e v a

Institute of Microbiology, Russian Academy of Sciences, Prospect 60 Let(va Ok(vabrya 7, bldg. 2,
117811 Moscow, Russian Federation
Received 13 January 1994; accepted in revised form 24 May 1994

Abstract
Of special importance for biohydrometallurgy are acidophilic chemolithotrophic bacteria
from a number of different taxonomic groups, namely: the genera of Thiobacillus and Leptospirillum, moderately thermophilic bacteria which we combined into the group Sulfobacillus Alicyclobacillus, and archaea of the genera Sulfolobus, Acidianus, Metallosphaera, and

Sulfurococcus.
These bacteria are able to oxidize one or more of the following compounds Fe + 2, S O and
sulfide minerals and to grow under extreme environmental conditions. Growth pH varies in the
range from 1 to 5, growth temprature - from 2 to 90°C. They can tolerate high concentration of
metal ions. They possess a great physiological, biochemical and genetic variability. Some of
them are important for removal of inorganic sulfur compounds from coals.
Some types of coals and oils contain aromatic heterocyclic compounds with the C-S bond.
Although a wide range of mostly heterotrophic and some chemolithotrophic bacteria, from
bacteria and archaea to eucaryotes, participate in its transformation, only certain organisms
have a unique capability of splitting this bond, which is impossible to be done by chemical

means. They can remove organic sulfur-containing compounds from coal.
The possibilities of application of bacteria in biological processing of coals is discussed.

Keywords: Chemolithotrophs; Complex sulfur organic compounds; Sulfide minerals

1. Introduction
Sulfur c o n t e n t in coals is k n o w n to v a r y widely from 0.5% to 11%. I n o r g a n i c sulfur
is p r e s e n t m a i n l y in the form of p y r i t e (FeS2) and, to a lesser extent, as elemental sulfur
a n d sulfides of o t h e r metals. P y r i t e occurs as concretia, lenses a n d finely dispersed
intrusions.
* Corresponding author. Tel.: 7-095-135-03-20. Fax: 7-095-135-65-30.
0378-3820/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved
SSDI 0 3 7 8 - 3 8 2 0 ( 9 4 ) 0 0 0 8 8 - B


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G.L Karavaiko, L.B. Lobyreva/Fuel Processing Technology 40 (1994) 167-182

Organic sulfur present in coals is integrated into the structural matrix in the form of
thiol, sulfide and thiophene compounds. So its removal must involve splitting
a covalent C-S bond, which, is resistant to chemical treatment.
The role of microorganisms in the oxidation of pyrite and several analogs of
complex organic sulfur-containing compounds, for example, dibenzothiophene
(DBT), is actively studied and has been reviewed by Klein et al. [1].
It has been shown that microorganisms can in principle be used for coal desulfurization, but many differences between removal of organic and inorganic sulfur have not
yet been resolved.
Inorganic sulfur removal is technically feasible, but economically not clear yet.
The aim of this work is to give an overview of diversity of a main bacteria which is
able to involve in oxidation of inorganic sulfur and possible organic sulfur in coal

desulfurization process.

2. The diversity of chemolithotrophic bacteria and the evolution of their functions
Chemolithotrophic bacteria belong to phylogenetically different groups of organisms, namely to gram-negative, gram-positive bacteria and archaea (Fig. 1). It appears
that chemolithotrophic bacteria have evolved by independent evolutionary patways.
They differ in morphology, cell wall type, nutrition type, metabolism of inorganic and
organic substrates and temperature characteristics of growth (Table 1). Their important feature, however, is the ability to oxidize Fe 2 +, So and sulfide minerals and grow

D

e

s

/

u

l

f

u

r

~

'S /1\\

!.+ Sulfol~_
/

~
lhiobacillus
/ I'~'c°llI Spirochaeta
#tlJ

~ethanobacterium [ - i ~

/

//

/

/

Fig. 1. Schematicrepresentationof the phylogenetictree of bacteria.


Genus Thiobacillus

Rods
Typical of gramnegative bacteria
Strict and facultative
autotroph
Fe 2÷, S°, sulfide minerals
and organic compounds
2-40

1.2-5.0

Characteristic

Form of cells

Cell wall

Type of nutrition

Source of energy

Temperature, °C

pH

1.0-5.0

2-40

Fe 2 ÷, FeS2

Strict autotroph

Typical of gramnegative bacteria

Vibrions

Genus Leptospirillum

Table 1
Characteristics of different groups of chemolithotrophic bacteria

1.1-5.0

20-60

Fe 2 ÷, S°, sulfide minerals

Facultative autotroph

Typical of grampositive bacteria

Rods

Genus Sulfobacillus
and other strains

1.0 5.8

40-90

Fe 2 +, S °, sulfide minerals

Facultative autotroph

gram-negative bacteria

?

¢~

t~

Metallosphaera
Spherical

9~

Genera Acidianus Sulfurococcus


170

G.L Karavaiko, L.B. Lobyreva/Fuel Processing Technology 40 (1994) 167-182

at low pH values under aerobic conditions. T.ferrooxidans can grow anaerobically in
the presence of ferric iron and elemental sulfur [2]. Within different taxonomic groups
of such bacteria there is also a wide diversity of species and strains.
According to nucleotide composition of the DNA~ the group of thiobacilli could
be divided into two subgroups: T. ferrooxidans (with the G + C content of DNA
55.0-57.4 mol%) and T. thiooxidans (50.0-53.0 mol%) and other species with a higher G + C content of DNA (62-69 mol%). Both subgroups include species which are
able to oxidize only S o (Table 2).
Mesophilic leptospirilli are close in the G + C content (Table 2) while the G + C
content of a moderately thermophilic strain of leptospirilli is higher.
On the basis of the analyses of the primary and secondary structure of the 16s
rRNA, we placed moderately thermophilic bacteria of the genus Sulfobacillus into the
group Sulfobacillu~Alicyclobacillus [-4]. These bacteria and related unidentified organisms could also be divided into three subgroups according to their nucleotide
composition of the DNA (Table 3). Strain BC 1 is, probably, close to the bacteria of the
genus Sulfobacillus. Other strains might represent novel species.

Thermoacidophilic archaea, according to nucleotide composition of the DNA,
could be divided into two groups of organisms (Table 4). A. brierleyi has a more lower
G + C content of DNA.

3. The variability of strains of chemolithotrophic bacteria
A great diversity of strains with different physiological and biochemical capacities is
known to occur among chemolithotrophic bacteria [-24-29]. Chemolithotrophic
bacteria in nature and experiment have to adapt to different factors: ions of metals,
pH, substrate concentration and even, over a certain range, to temperature. Biochemical variability is connected with changes in molecular biology of the cell and activity
of enzymes caused by environmental changes.
The induction of the synthesis of three proteins, rusticyanin, 32 000 Da protein and
92000 Da glycoprotein was shown for T. ferrooxidans transferred from a sulfur to
ferrous iron medium [30]. The induction of the synthesis of two proteins with
molecular weight 47 000 Da and 55 000 Da was shown for T.ferrooxidans transferred
from a medium with Fe 2+ to one with S O or $ 2 0 2 [,31]. According to Mjoli and
Kulpa [30], at least the glycoprotein with molecular weight 92 000 Da could be an
integral component of the membrane iron-oxidizing system of T.ferrooxidans. Osorio
et al. [32] found several types of acidosl~able proteins in both types of cells. Proteins
(60000, 30000, 25000, 17000 and 12000 main bands) found in ferrous iron-grown
cells are similar to acid-stable heme proteins described previously for ferrous-iron
grown cells [33]. The same proteins were found in cells grown on the medium with S °.
In this case, however, 60 000, 30 000, 25 000, rusticyanin (R) and 12 000 protein bands
were highly reduced in size compared to those of cells grown with ferrous iron. Two
acid-stable proteins of molecular weight 20 000 and approximately 10000 Da were
present only in sulfur-grown cells. Amaro et al. [34] reported changes in the general


G.L Karavaiko, L.B. Lobyreva/Fuel Processing Technology 40 (1994) 167-182

171

Table 2
Bacteria species of the genera Thiobacillus and Leptospirillurn
Bacteria

Source of energy

The DNA G + C
content (mol%)

Thiobacillusferrooxidians [3, 4]

Fe z+, S °, Hz and sulfide minerals
and ores
So
S °, sulfide ores
Fe 2+, S °, sulfide ores
PbS, H2S, H2
S °, organic compounds
Fe 2+, FeS2
Fe z +, FeS2

55.0-57.4

Thiobacillus thiooxidans [5]
Thiobacillus cuprinus [6]
Thiohacillus prosperus [7]
Thiobacillus plumbophilus [8]
Thiobacillus acidophilus [9]
Leptospirillumferrooxidans [10]

Leptospirillum-like bacteria [11, 12]
BU-I
ALV
BC
CH
LAM

Leptospirillum thermoferrooxidans [13]

Fe z+

50.0-53.0
66.0 69.0
63.0 64.0
66.0
62.9-63.2
50.3 51.7
55.6
50.6
55.2
55.1
nd
65.2

Table 3
Bacteria of genus Sulfobacillus and not classified
Bacteria

Source of energy

The DNA G + C
content (mol%l

Sulfohacillus thermosulfidooxidans [14]

Fe 2+, S °, sulfide minerals and organic
compounds
Fe 2+, S °, sulfide minerals and organic
compounds

47.2

S. thermosulfidooxidans subsp.
asporogenes [ 15]
S. thermotolerans [16]
Other bacteria [17 20]
BC 1
ALV
NAL
2b
N
TH3

45.5
49.3

Fe 2+, S °, sulfide minerals and organic
compounds

48

56
nd
nd
nd
68.5

Table 4

Thermoacidophilic archaea
Bacteria

Source of energy

The DNA G + C
content (mol%)

Acidianus brierleyi [21]
Metallosphaera sedula [22]
Sulfurococcus yellowstonii [23]

Fe 2 +, S °, sulfide minerals and some
organic compounds

30-33
43.7-46.2
44.6


172

G.I. Karavaiko, L.B. Lohyreva/Fuel Processing Technology 40 (1994) 167 182

protein synthesis pattern which involved significant stimulation of the synthesis
of the 3.6 kDa protein when cells of T.ferrooxidans grown at pH 1.5 were transferred
into the medium with pH 3.5. Vestal et al. [35] observed qualitative and quantiative
differences in lipopolysaccharides of cells of T. ferrooxidans grown on various substrates.
The synthesis of rusticyanin, one of the major cell proteins of T. ferrooxidans, is
induced by Fe 2+ and suppressed in a medium with reduced sulfur compounds
[36, 37]. On a medium with Fe E+, the content of this protein in cells of T.ferrooxidans
amounts to 5% of the total cell protein, whereas in sulfur medium it is present at only
20% of this amount [38].
Variations in cell protein synthesis were also observed with a change of autotrophic
growth to a heterotrophic growth. For example, only chemolithotrophically grown
cells of T. cuprinus contained proteins with molecular weight about 43 kDa. Also, the
18 amino acid sequence of the N-terminal and of a protein was obtained which is
expressed only under heterotrophic growth conditions [39].
The main part of fixed carbon of 14CO2 (about 50 80%) is incorporated into
bacterial cells, while the rest (20-50%) is released into the medium as organic
substrates [40-42]. The composition of exometabolites produced by thiobacilli
depends on the substrate oxidized as well as on other factors.
These results suggest that a reorganization of the entire program of biosynthesis in
cells of chemolithotrophic bacteria may occur under changed growth conditions.
The variability mechanism so far has not been sufficiently investigated. Genetically
stable mutants are apparently produced both in nature and under laboratory conditions. Thus, the occurrence frequency of T. ferrooxidans mutants tolerant to 1.0 and
1.5 m M UO2 was approximately one per 1.3 × 10 6 and 9.0 × l0 s cells, respectively, but
could be increased by the addition of 15-150 m M of Zn, Ni and Mn [43]. It is not
clear, however, what intercellular changes took place in these cases.
A number of works published in 1981 1992 reported the presence of one or
more plasmids with a size from 2 to 30 kb in the majority of cells of T.ferrooxidans
strains isolated from different habitats. However, the investigation of plasmids in

T. ferrooxidans failed to link them to organisms resistant to metal ions. Most of the
plasmids appeared to be cryptic. Later, the study of the chromosomal part of the
genome of T. ferrooxidans was started. Specifically, Shiratori et al. [44] showed that
the gene of mercury tolerance was localized in the chromosomal DNA. Also, the CO2
fixation and synthesis of rusticyanin and Fe z +-oxidase were shown to be governed at
the gene level [38, 45-47].
The polymorphism of chromosomal DNA in different strains of T. ferrooxidans
was studied by Karavaiko et al. [48]. Individual patterns of chromosomal DNA
restriction in these strains supported the assumption of high geneic variability of
T.ferrooxidans in response to environmental factors. It was found that, in a number of
cases, the adaptation process was accompanied by the appearance of amplificated
fragments in samples with chromosomal DNA restriction or by a change in their size.
A study of restrictive samples of the chromosomal DNA showed that, in strains
actively oxidizing Fe 2 + in the presence of Zn (70 g/l), the gene of zinc tolerance was
probably located in the 98 kb fragment of the chromosomal DNA and is inducible.


G.1. Karavaiko, L.B. Lobyreva /Fuel Processing Technology 40 (1994) 167-182

173

Strains of T. ferrooxidans adapted to A s 3+ contained amplificated fragments of
chromosomal DNA 28 kb in size. This suggests that metal tolerance genes are, in fact,
localized at different sites of DNA. An increased tolerance of T. ferrooxidans to Zn
and As arises from amplification of tolerance genes and, therefore, has to do with their
increased activity.
Genetic characteristics of other chemolithotrophic bacteria are little known. The
question of possibility of practical application of chemolithotrophic bacteria for coal
desulfurization seems to be more complicated. It is also closely connected with
tchnological aspects of coal utilization. Inorganic forms of sulfur, present in finely

graded coal could be probably oxidized by means of such bacteria as T.ferrooxidans.
Their successful utilization of nonferrous metals leaching and of processing of difficult-to-dress gold and silver containing concentrates in dense pulps could be used as
an example. Others thiobacilli (see Table 2) are either absent in the pulp, or present in
the comparatively low concentration (102-103 cells/ml). That does not allow to
consider them as significant for the intensive leaching processes in reactors. Regarding
coal desulfurization, special attention should be paid to leptospirilli and their communities with sulfur-oxidizing thiobacilli, which are able to oxidize pyrite and Fe + 2 at
lower pH values than T.ferrooxidans ([11, 49] our unpublished data). The adaptation
of bacteria to concrete types of coal is essential for their application in technological
processes, as the coals contain different types of pyrites. Acidic pilot plant in Porto
Torres (Italy) or coal treatment allows to obtain necessary data for the economical
evaluation of this technology [50].
The attempts to utilize moderately thermophilic bacteria and several archaea, such
as S. yellowstonii, [23] did not lead to the intensification of pyrite oxidation process.
These bacteria are more complicated for utilization; the processes require more energy
and reactors should be made of highly resistant materials.

4. The diversity of heterotrophs and chemolithotrophs oxidizing complex organic
substrates
Microbial transformations of complex organic substrates are usually studied with
dibenzothiophene as a model compound. From Table 5 it can be seen that representatives of different groups - from bacteria and archaea to eukaryotes are present
among heterotrophic bacteria capable of oxidation, to a various degree, of dibenzothiophene. The pathways of dibenzothiophene oxidation are also very diverse.
Some microorganisms (I) are able to oxidize only the peripheral aromatic ring of
DBT, forming water-soluble products. Other microorganisms, along with aromatic
ring oxidation, can oxidize the sulfur heteroatom without its abstraction from the
carbonaceous structure (II and III). Fungi, however, do not interact with the aromatic
ring of DBT. Complete oxidation of dibenzothiophene to SO 2-, which involves
splitting of the C-S bond, was shown only for a limited number of prokaryotes:
Sulfolobus acidocaldarius, Brevibacterium sp. and a mutant strain Pseudomonas sp.
CB1.

(II) Oxidation of dibenzothiophene to
its corresponding DBT-5-oxide,
DBT-5-sulfone with and without
oxidative degradation of aromatic
ring

Pseudomonas alkaligenes [51]
Ps. stutzeri [51]
Ps. putida [52]

(I) Deoxigenation of peripheric
aromatic ring resulted in the
formation of water soluble
compounds, the thiophene
nucleus is still intact

Ps. putida [57, 58]

Ps. jianii [54, 55]
Rhizobium sp.,
Acinetobaeter sp. [56]

Pseudomonas abikonensis [-54, 55]

Ps. aeruyinosa ERC-8 [53]

Microorganisms

Pathways of oxidation

Table 5
Metabolic pathways for the microbial degradation of dibenzothiophene

OH

CQQ H

DBT3-5-sulfone

O
DBT-5-oxide

4-[2-(3hydroxy)thionaphtenyl]2-oxo-3buthenoic acid

~

C-H
I!
O
3-hydroxy-2-formyl-benzotiophene

~

Products of oxidation

II
O
3-hydroxy-2-formylbenzothiophene

C-H
II
O
3-hydroxy-2-formylbenzothiophene

5

t",,

P~

2.


(11I) Oxidationof the sulfuratom
of dibenzothiopheneinto sulfate
so~so~ -

SO~- and ~
2-hydroxybiphenyl
SO42-, CO2, H20

Corynebacterium sp. SY1 [64]

Brevibacterium sp. DO [65]

DBT-5-sulfone

II
O

DBT-5-oxide

Pseudomonas sp. CB1 (mutant) [62]
Sulfolobus acidocaldarius [63]

Cunninqhamella elegans [60]
Rhizopus arrhizus Mortierella
isabellina [61]

Beyerinckia sp. [59]

1,2-dihydroxy-l,2-hydrobenzothiophene

t,o

4t~
Z~


176

G.L Karavaiko, L.B. Lobyreva/Fuel Processing Technologv 40 (1994) 167-182

Some microorganisms utilize DBT as the sole source of sulfur, carbon and energy
for growth. Other bacteria require additional substrates (cosubstrates) or growth
factors.
It is well known that sulfur-organic compounds incorporated in coal are hardly
available for bacteria. Thus the achievements obtained in the experiments with DBT
should not be extrapolated on coals. These data demonstrate mainly the general
ability of bateria to oxidize complex sulfur-organic compounds.

Isbister and Kobylinski have shown that a mutant strain of Pseudomonas species
CB1 capable of active DBT oxidation decreased the organic sulfur content from 18%
to 47% in different types of coals [66]. The other strain, Pseudomonas sp. CB2 capable
of diphenyl sulfide oxidation removed only up to 30% of organic sulfur of coals tested
[67].
The culture of Pseudomonas sp. CB1 was used for the desulfurization of different
types of coals in continuous culture. Depending on the coal type, the oxidation of
organic sulfur was from 19% to 57% [66]. Evidently, these results culd be improved
by the utilization of highly active bacterial strains and the optimization of the process
itself.
Control mechanisms of DBT and other organic sulfur compounds metabolism in
microorganisms are not yet studied. A plasmid with size 55 M G D was found in
a number of bacteria of the genus Pseudomonas capable of DBT oxidation [51]. The
authors associate the ability to oxidize DBT with the presence of this plasmid.
A plasmid sized from 15.4 to 17.7 M G D was discovered in 14 isolates obtained from
a mine and able to oxidize DBT to different degrees [52]. More profound genetic
studies are necessary not only for understanding the DBT oxidation mechanisms but
also for obtaining highly active strains.
The mechanisms of oxidation of complex sulfur organic compounds are not clear
either. Since heterocyclic sulfur organic compounds are not water-soluble, two different pathways of primary reactions occuring on the cell surface are possible: (i)
homogenization and subsequent transfer of aromatic compounds into cells; (ii) cleavage of the aromatic ring outside the cell and the transfer of soluble products into the
cell. The first mechanism of microbial cell interaction with an insoluble substrate
could be illustrated by oxidation of benzpyrene representing, as well as DBT, a cyclic
aromatic compound with crystallic structure. In the second case, microorganisms
have to possess the necessary exoenzymes. The accumulation of benzpyrene predominantly in free lipids was observed in Mycobacteriumflavum and in Bacillus meyaterium
(Fig. 2(a) and (b)). In bacteria, benzpyrene is accumulated mainly in cytoplasm, [68,
69] while in yeasts it is accumulated in mitochondria [70]. It was suggested by the
authors that the solution and transport of this compound into cells was conneted with
cell lipids and lipoprotein structures and that the oxidation of benzpyrene occurred on
membrane structures. This hypothesis might also be applied to oxidation of dibenzothiophene and of other complex aromatic compounds by certain microorganisms.

The cleavage of the aromatic ring in the bacterial cell occurs via its hydroxylation
by means of monooxygenases (hydroxylases) with the involvement of oxygen. Oxygenases are known to be inducible enzymes and are either cytochrom-P-450- or
flavin-dependent [71].


G.I. Karavaiko, L.B. Lobyreva/Fuel Processing Technology 40 (1994) 167-182

177

Fig. 2. Localization of benzpyrene in bacterial cells [64]. (a) Bacillus megaterium (asporogenous mutant,
× 2000); (b) Mycobacteriumflavum ( x 2000).


178

G.L Karavaiko, L.B. Lobyreva/Fuel Processing Technolo~' 40 (1994) 167 182

Unlike with bacteria, the transformation of dibenzothiophene by fungi proceeds
without aromatic ring rearrangement through sulfur atom oxidation. It could be
catalyzed by sulfoxidases similar to those of mammalian and microbial origin [71]. In
the case of fungi, the oxidation of dibenzothiophene probably also occurs within the
cell. Yet, many fungi are known to be capable of oxidizing unsoluble aromatic
compounds by means of phenoloxidases (laccases) which are exocellular inducible
enzymes [72, 73].

5. Conclusions

1. Chemolithotrophs capable of Fe 2+, So and sulfide minerals oxidation are found
in different phylogenetically remote groups of bacteria. Their limited number, till date,
has no explanation. Possibly, new species and genera of such organisms will be

described in the future.
2. The physiology, biochemistry and molecular biology of chemolithotrophic bacteria are characterized by unusually large variability which is controlled at the gene
level. As a result, the program of biosynthetic processes in the cell can be reorganized
to adapt to changing conditions of the environment.
3. Many heterotrophic microorganisms are capable of oxidizing complex organic
sulfur-containing compounds with the formation of soluble products. However, only
a few of them are capable of splitting the C-S bond. Both these processes might be of
practical value given that soluble organic sulfur-containing compounds could be
washed out of coal.
4. At least several thiobacilli and leptospirilli could be utilized for the pyrite
elimination from the coal. Scale-up testing performed by Professor G. Rossi in Porto
Torres (Italy) would allow to clear up the perspectives of this process and its
economical value.
The perspectives of practical application of heterotrophic microorganisms utilization for coal desulfurization are not clear so far, at least for the large-scale processes.
From the microbiological point of view this process is quite probable for pulps with
fine coal particles.
However, the range of microorganisms, utilized in this process, is quite narrow.
Besides, according to the review of Kilbane [-74], they are gradually losing their initial
natural activity in DBT oxidation. Thus the investigation of physiology, biochemisry
and genetics of such microorganisms should be enhanced.

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