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Applied Microbiology and Biotechnology (v.52, #1)
Biological processing of coal by D. E. A. Catcheside; J. P. Ralph (pp. 16-24).
The irregular chemical structure of coal makes it an improbable substrate for bioconversion. Nevertheless, work completed in the last 20 years makes likely the development of practicable biological processes for beneficiation of low-rank coal and the conversion of low-rank coal to specific low-molecular-mass organic molecules and novel fluid fuels.
Biotechnology and microbiology of coal degradation by R. M. Fakoussa; M. Hofrichter (pp. 25-40).
For several years it has been known that fungi and bacteria can attack and even liquefy low rank coals. This review covers the progress in coal biotechnology and microbiology, mainly during the last decade, from describing the first effects to elucidating the mechanisms used by the microorganisms. More than one mechanism is responsible for microbial coal degradation/liquefaction: oxidative enzymes (peroxidases, laccases), hydrolytic enzymes (esterases), alkaline metabolites and natural chelators. Due to the heterogeneous structure of coal, which is described in one section, and for economic reasons the review focuses on the enzymatic depolymerization of brown coal. Approaches which seem not so promising are discussed (anaerobic, reductive pathways, chemical pretreatment). Finally the possible applications and products in this field are summarized, as lignite with a worldwide production of about 940 million tons a year will continue to play an important economic role in the future.
Fungal biosolubilization of Rhenish brown coal monitored by Curie-point pyrolysis/gas chromatography/mass spectrometry using tetraethylammonium hydroxide by G. K. E. Götz; R. M. Fakoussa (pp. 41-48).
Residues and coal fractions that remained after the biosolubilization of Rhenish brown coal by strains of Lentinula edodes and Trametes versicolor have been studied by Curie-point pyrolysis/gas chromatography/mass spectrometry using tetraethylammonium hydroxide (NEt4OH) at 610 °C. To differentiate methyl derivatives of esters and ethers from free or bound hydroxyl and carboxyl groups NEt4OH was used in the thermochemolysis experiments instead the commonly used tetramethylammonium hydroxide. A comparison of humic acid fractions before and after fungal attack shows considerable alteration of the soluble macromolecules of coal. Depending on the coal fraction studied and the fungi used, the assortment of fatty acid esters released during the pyrolysis varies significantly. Furthermore, dicarbonic acid ethyl diesters as well as ethyl derivatives of aromatic ethers and acids yield information about humic acid structure and the biosolubilization of brown coal. Variations in the mixture produced are possibly caused by differences in the pattern of extracellular enzymes secreted that attack the macromolecular structural elements of brown coal. Therefore pyrolysis of native and microbiologically altered geomacromolecules using NEt4OH allows one to differentiate between free hydroxyl groups as well as substances that are attached to humic substances via ester or ether bridges, and their methylated counterparts.
Processes of liquefaction/solubilization of Spanish coals by microorganisms by F. Laborda; I. F. Monistrol; N. Luna; M. Fernández (pp. 49-56).
Several fundamental aspects of microbial coal liquefaction/solubilization were studied. The liquefied/solubilized products from coal by microorganisms were analysed. The liquid products analysed by IR titration and UV/visible spectrometry showed some alterations with regard to the original coal. Humic acids extracted from the liquefied lignite showed a reduction in the average molecular weight and a increase in the condensation index, probably due to depolymerization caused by microorganisms. The mechanisms implicated in coal biosolubilization by two fungal strains, M2 (Trichoderma sp.) and M4 (Penicillium sp.) were also studied. Extracellular peroxidase, esterase and phenoloxidase enzymes appear to be involved in coal solubilization.
Mechanisms of coal solubilization by the deuteromycetes Trichoderma atroviride and Fusarium oxysporum by U. Hölker; S. Ludwig; T. Scheel; M. Höfer (pp. 57-59).
Three different mechanisms can be envisaged that are used by fungi to solubilize coal: the production of alkaline substances, the extrusion of chelators and, of special interest in the scope of biotechnology, the action of enzymes. Whether these mechanisms are operating separately or in various combinations has not yet been finally assessed. The two deuteromycetes Fusarium oxysporum and Trichoderma atroviride solubilize coal by synergistic effects of various different mechanisms depending on the cell metabolism. F. oxysporum seems to solubilize coal by increasing the pH of the mycelial surroundings and by the action of chelators induced during growth in glutamate-containing media (without involvement of enzymes). T. atroviride, on the other hand, appears to use, in addition to an alkaline pH and a high chelator activity, at least two classes of enzyme activity to attack coal: hydrolytic activity for coal solubilization and ligninolytic activity for degradation of humic acids.
In vivo-decolorization of coal-derived humic acids by laccase-excreting fungus Trametes versicolor by R. M. Fakoussa; P. J. Frost (pp. 60-65).
Lignite (brown coal) can be liquefied/solubilized with several fungi by different mechanisms. When applied industrially, only catalytic mechanisms can compete with chemical methods. The well-known fungal ligninolytic peroxidases are at a disadvantage, in that the relatively expensive hydrogen peroxide must be used as a cofactor. Comparing several fungal strains, we observed that the fungus Trametes versicolor is able to decolorize coal-derived humic acids, producing a considerable amount of laccase in the process. During this reaction the amount of humic acids decreases whilst that of fulvic acids increases; this was verified by optical density measurement and GPC after the two substance classes had been separated.
Evidence for and expression of a laccase gene in three basidiomycetes degrading humic acids by T. Scheel; U. Hölker; S. Ludwig; M. Höfer (pp. 66-69).
The majority of lignin-degrading basidiomycetes are able to depolymerize humic acids. In this presentation the relationship and possible similarities between enzymes involved in lignin degradation and humic acid depolymerization were examined on the genetic level. We have cloned fragments of the gene encoding the extracellular ligninolytic enzyme laccase from Clitocybula dusenii, Nematoloma frowardii and a fungal strain designated i63-2, and compared the three sequences with those of several other published laccase genes. The sequenced fragments displayed a high homology both on the DNA (97%–77%) and amino acid (100%–85%) level. Furthermore, the expression of this gene in the above-mentioned fungi was demonstrated by a nested polymerase chain reaction with cDNA as template.
Transformation of macromolecules from a brown coal by lignin peroxidase by J. P. Ralph; D. E. A. Catcheside (pp. 70-77).
Indirect evidence has suggested that lignin peroxidase (LiP) of the white-rot fungus Phanerochaete chrysosporium catalyses oxidative decolourisation and depolymerisation of macromolecules from brown coal in vivo. In this study we show that LiP catalyses these transformations in vitro. Unmethylated (USC45 coal) and methylated (MWSC6 coal) fractions of solubilised macromolecules (M r > 30 000) from a brown coal were treated with a semi-purified preparation of LiP isozymes from P. chrysosporium. Both coal fractions were decolourised, losing between 26% and 39% of their absorbance at both 280 nm and 400 nm, in reactions that had an absolute requirement for H2O2 and veratryl alcohol. Neither coal fraction was transformed when the enzyme was heat-inactivated or in the presence of the LiP inhibitor metavanadate. Gel-permeation chromatography showed that MWSC6 coal but not USC45 was depolymerised and yielded low-molecular-mass (M r < 30 000) fragments. Nine monomeric products were identified by GC-MS.
Degradation of lignite (low-rank coal) by ligninolytic basidiomycetes and their manganese peroxidase system by M. Hofrichter; D. Ziegenhagen; S. Sorge; R. Ullrich; F. Bublitz; W. Fritsche (pp. 78-84).
Ligninolytic basidiomycetes (wood and leaf-litter-decaying fungi) have the ability to degrade low-rank coal (lignite). Extracellular manganese peroxidase is the crucial enzyme in the depolymerization process of both coal-derived humic substances and native coal. The depolymerization of coal by Mn peroxidase is catalysed via chelated Mn(III) acting as a diffusible mediator with a high redox potential and can be enhanced in the presence of additional mediating agents (e.g. glutathione). The depolymerization process results in the formation of a complex mixture of lower-molecular-mass fulvic-acid-like compounds. Experiments using a synthetic 14C-labeled humic acid demonstrated that the Mn peroxidase-catalyzed depolymerization of humic substances was accompanied by a substantial release of carbon dioxide (17%–50% of the initially added radioactivity was released as 14CO2). Mn peroxidase was found to be a highly stable enzyme that remained active for several weeks under reaction conditions in a liquid reaction mixture and even persisted in sterile and native soil from an opencast mining area for some days.
Degradation of alicyclic molecules by Rhodococcus ruber CD4 by J. D. Schumacher; R. M. Fakoussa (pp. 85-90).
The present work describes investigations on the bacterial degradation of the alicyclic molecule cyclododecane. It represents a structure where the initial degradative steps have to be similar to a “subterminal” attack as there is no “terminal” part of the molecule. We were able to show that the gram-positive bacterium Rhodococcus ruber CD4 DSM 44394 oxidizes cyclododecane to the corresponding alcohol and ketone, the latter being subject to ring fission by a Baeyer-Villiger oxygenase. This key enzyme is an NADPH- and O2-dependent flavoprotein with a substrate specificity for bigger rings. The further metabolism of the resulting lactone gives rise to an ω-hydroxyalkanoic acid that is susceptible to common β-oxidation. Due to its alicyclic character and its ring size, cyclododecane is comparable to aliphatic bridge components that are an important element in the coal texture. They contribute to the three-dimensional coal structure and thus could serve as a valuable target for the oxidative abilities of R. ruber CD4 to reduce the molecular mass of coal.
Biosynthesis of polyhydroxyalkanoates from low-rank coal liquefaction products by Pseudomonas oleovorans and Rhodococcus ruber by B. Füchtenbusch; A. Steinbüchel (pp. 91-95).
A screening identified several bacteria that were able to use chemically heterogeneous low-rank coal liquefaction products as complex carbon sources for growth. Pseudomonas oleovorans and Rhodococcus ruber accumulated polyhydroxyalkanoic acids (PHA) amounting to 2%–8% of the cell dry weight when the cells were cultivated on these liquefaction products in the absence of any other carbon source. R. ruber accumulated, in addition to PHA, small amounts of triacylglycerols. The accumulated PHA consisted of 3-hydroxyhexanoate, 3-hydroxydecanoate, and 3-hydroxydodecanoate (P. oleovorans) or 3-hydroxybutyric acid and 3-hydroxyvaleric acid (R. ruber). Low-rank coal liquefaction products obtained from Trichoderma atroviride were better substrates for P. oleovorans than chemically produced fulvic acids.
EPR study and structural aspects of ferredoxins obtained from Thiobacillus ferrooxidans by A. B. Więckowski; G. P. Słowik; J. A. Gąsiorek; P. Gąsiorek; F. Domka; A. Perkowska (pp. 96-98).
A comparison of iron-sulfur proteins obtained from Thiobacillus ferrooxidans was carried out. The microorganisms were grown on iron(II)- or sulfur-containing nutrients. In both cases different, broad elctron paramagnetic resonance (EPR) lines, originating from an iron(III) compound, were detected. Additional EPR lines of tetrahedral iron(III) and free radicals were observed. The UV spectra of these compounds also differ.
Effect of enclosing rocks and aeration on methanogenesis from coals by S. Shumkov; S. Terekhova; K. Laurinavichius (pp. 99-103).
Two coals of different rank, mined in Russia, were treated by an anaerobic methanogenic enrichment culture. The addition of alkaline enclosing rock to the lower-rank coal increased the pH of the incubation medium and methane production above that of the higher-rank coal with addition of its enclosing rock. This effect was accompanied by the leaching of cations from the incubation medium. The coal was processed without a preliminary chemical treatment in a two-stage aerobic/anaerobic bioreactor containing an anaerobic methanogenic granulated enrichment culture.
Intermediary sulfur compounds in pyrite oxidation: implications for bioleaching and biodepyritization of coal by A. Schippers; T. Rohwerder; W. Sand (pp. 104-110).
Accumulation of elemental sulfur during pyrite oxidation lowers the efficiency of coal desulfurization and bioleaching. In the case of pyrite bioleaching by Leptospirillum ferrooxidans, an iron(II)-ion-oxidizing organism without sulfur-oxidizing capacity, from the pyritic sulfur moiety about 10% elemental sulfur, 2% pentathionate, and 1% tetrathionate accumulated by a recently described cyclic pyrite oxidation mechanism. In the case of pure cultures of Thiobacillus ferrooxidans and mixed cultures of L. ferrooxidans and T. thiooxidans, pyrite was nearly completely oxidized to sulfate because of the capacity of these cultures to oxidize both iron(II) ions and sulfur compounds. Pyrite oxidation in acidic solutions, mediated chemically by iron(III) ion, resulted in an accumulation of similar amounts of sulfur compounds as obtained with L. ferrooxidans. Changes of pH to values below 2 or in the iron ion concentration are not decisive for diverting the flux of sulfur compounds. The literature on pyrite bioleaching is in agreement with the findings indicating that the chemistry of direct and indirect pyrite leaching is identical.
Developments in destructive and non-destructive pathways for selective desulfurizations in oil-biorefining processes by L. Setti; P. Farinelli; S. Di Martino; S. Frassinetti; G. Lanzarini; P. G. Pifferi (pp. 111-117).
Biocatalytic desulfurization is still not a commercial technology, but conceptual engineering and sensitivity analyses have shown that the approach is very promising. The purpose of this paper is to investigate further some aspects of the biodesulphurization pathways, discussing the non-destructive pathway with the well-known Rhodococcus rhodochrous IGTS8. Findings revealed byproducts, such as 2′-hydroxybiphenyl (HBP), sulfite and sulfate, obtained by the desulfurization of dibenzothiophene (DBT), to exert an inhibiting effect. The results suggest that IGTS8 may follow two different metabolic pathways in stationary-growth-phase cells or under growing conditions. The first pathway is characterized by oxidative steps, which convert DBT to DBT sulfoxide and to DBT sulfone. The sulfone is transformed to 2-(2′-hydroxyphenyl)benzene sulfinate and then to HBP and sulfite by a sulfinic acid hydrolase. In the second pathway the sulfone is further oxidized to 2-(2′-hydroxyphenyl)benzene sulfonate and then to HBP and sulfate by a sulfonic acid hydrolase. Experiments using benzene sulfonic acid suggest that the sulfonic acid hydrolase is an induced enzyme.
Bioremoval of organic and inorganic sulphur from coal samples by F. Gómez; R. Amils; I. Marín (pp. 118-121).
The microbial ecology of different Spanish coal samples has been studied. Several bacteria have been isolated from enrichment cultures and characterised and their biodesulphurization abilities evaluated. Using morphological and physiological properties, different isolates have been related to species of the Xanthomonas, Pseudomonas, Chryseomonas and Moraxella genera. Some of the isolates, B(30)15 and T(30)10, gave important levels of organic desulphurization, close to 70%. Other isolates, B(30)7 and B(30)8, were able to remove inorganic sulphur with high efficiencies, over 67%. One of the isolates, B(30)10, metabolically related to Xanthomonas maltophila, was able to remove both organic and inorganic sulphur at neutral pH, with efficiencies of 69% and 68% respectively. The results obtained underline the potential use of some of these strains for industrial coal desulphurization processes.
Manipulation of the DNA coding for the desulphurizing activity in a new isolate of Arthrobacter sp. by L. Serbolisca; F. de Ferra; I. Margarit (pp. 122-126).
A new bacterial strain able to cleave CS bonds from organosulphur heterocyclic compounds through the 4-S pathway and tentatively classified as Arthrobacter sp. was recently isolated. In the present short article we describe the cloning and the characterization of the DNA encoding the enzymes responsible for desulphurization in this microorganism, referred to as Arthrobacter sp. DS7. The desulphurization operon was found to be located in a large plasmid that also bears the genes conferring cadmium and arsenic resistance. By shortening this plasmid, a new cloning vector was prepared and used to obtain a recombinant derivative strain that desulphurizes dibenzothiophene despite of the presence of inorganic sulphur in the growth medium.
Bioassays for risk assessment of coal conversion products by S. Schacht; C. Sinder; F. Pfeifer; J. Klein (pp. 127-130).
Traditional as well as biotechnological processing of coal leads to complex mixtures of products. Besides chemical and physical characterization, which provides the information for product application, there is a need for bioassays to monitor properties that are probably toxic, mutagenic or cancerogenic. Investigations carried out focused on the selection, adaptation and validation of bioassays for the sensitive estimation of toxic effects. Organisms like bacteria, Daphnia magna and Scenedesmus subspicatus, representing different complexities in the biosphere, were selected as test systems for ecotoxicological and mutagenicity studies. The results obtained indicate that bioassays are, in principle, suitable tools for characterization and evaluation of coal-derived substances and bioconversion products. Using coal products, coal-relevant model compounds and bioconversion products, data for risk assessment are presented.