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

2, 117811 Moscow, Russian Federation Received 13 January 1994; accepted in revised form 24 May 1994 Abstract Of special importance for biohydrometallurgy are acidophilic chemolithotr

FUEL PROCESSING TECHNOLOGY

E L S E V I E R 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 K a r a v a i k o * , 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 Lepto- spirillum, moderately thermophilic bacteria which we combined into the group Sulfobacil- lus 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 c o a l s is k n o w n t o v a r y w i d e l y f r o m 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 f o r m o f p y r i t e (FeS2) a n d , to a lesser extent, as e l e m e n t a l sulfur

a n d sulfides o f o t h e r m e t a l s P y r i t e o c c u r s as c o n c r e t i a , lenses a n d finely d i s p e r s e d

i n t r u s i o n s

* 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

168 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 desulfuriza- tion, 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 organ- isms, 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 impor- tant feature, however, is the ability to oxidize Fe 2 +, S o and sulfide minerals and grow

/ 'S / 1 \ \

J /

Fig 1 Schematic representation of the phylogenetic tree of bacteria

Table

9~ t~ ¢~

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 high-

er G + C content of D N A (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 or- ganisms 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 Biochemi- cal 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

Table 2

Bacteria species of the genera Thiobacillus and Leptospirillurn

171

content (mol%)

Thiobacillusferrooxidians [3, 4] Fe z+, S °, Hz and sulfide minerals 55.0-57.4

and ores

Thiohacillus prosperus [7] Fe 2+, S °, sulfide ores 63.0 64.0

Thiobacillus acidophilus [9] S °, organic compounds 62.9-63.2

Leptospirillum-like bacteria [11, 12] Fe z +, FeS2

BU-I

ALV

BC

CH

LAM

Leptospirillum thermoferrooxidans [13] Fe z+

55.6 50.6 55.2 55.1

nd 65.2

Table 3

Bacteria of genus Sulfobacillus and not classified

content (mol%l

Sulfohacillus thermosulfidooxidans [14] Fe 2+, S °, sulfide minerals and organic 47.2

compounds

Fe 2+, S °, sulfide minerals and organic 45.5 compounds

S thermosulfidooxidans subsp

asporogenes [ 15]

S thermotolerans [16]

Other bacteria [17 20]

BC 1

ALV

NAL

2b

N

TH3

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

49.3

48

56

nd

nd

nd 68.5

Table 4

Thermoacidophilic archaea

content (mol%)

Acidianus brierleyi [21] Fe 2 +, S °, sulfide minerals and some 30-33

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 sub- strates

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 a m o u n t [38]

Variations in cell protein synthesis were also observed with a change of autotrophic growth to a heterotrophic growth F o r 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 condi- tions 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 D N A 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 D N A restriction or by a change in their size

A study of restrictive samples of the chromosomal D N A 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 D N A and is inducible

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

Strains of T ferrooxidans adapted to A s 3+ contained amplificated fragments of chromosomal D N A 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 diffi- cult-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 commu- nities 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 P o r t o 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 F r o m Table 5 it can be seen that representa- tives of different groups - from bacteria and archaea to eukaryotes are present among heterotrophic bacteria capable of oxidation, to a various degree, of diben- zothiophene 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

2

OH C-H I! O

3-hydroxy-2-formyl-benzotiophene ~

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

DBT-5-oxide DBT3-5-sulfone

C-H II O

3-hydroxy-2-formyl- benzothiophene

II O

3-hydroxy-2-formyl- benzothiophene

(11I)

II O

DBT-5-oxide DBT-5-sulfone so~- so~

~ 2-hydroxybiphenyl

1,2-dihydroxy-l,2-hydro- benzothiophene

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

Some microorganisms utilize D B T 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 D B T 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 D B T 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 D B T 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 D B T oxidation [51] The authors associate the ability to oxidize D B T 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 D B T to different degrees [52] More profound genetic studies are necessary not only for understanding the D B T 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 differ- ent pathways of primary reactions occuring on the cell surface are possible: (i)

homogenization and subsequent transfer of aromatic compounds into cells; (ii) cleav- age 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 c o m p o u n d with crystallic structure In the second case, microorganisms have to possess the necessary exoenzymes The accumulation of benzpyrene predomi- nantly 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 diben- zothiophene 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 Oxy- genases are known to be inducible enzymes and are either cytochrom-P-450- or flavin-dependent [71]