Biotechnology and microbiology of coal degradation

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. Received: 19 October 1998 / Received revision: 16 December 1998 / Accepted: 21 December 1998     

Biosynthesis of polyhydroxyalkanoates from low-rank coal liquefaction products by Pseudomonas oleovorans and Rhodococcus ruber

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. Received: 13 May 1998 / Received revision: 11 August 1998 / Accepted: 12 August 1998     

Solubilization of low-rank coal by Trichoderma atroviride: Evidence for the involvement of hydrolytic and oxidative enzymes by using 14C-labelled lignite

The deuteromycete Trichoderma atroviride is able to solubilize lignite in dependence on a given carbon source for growth. When cultivated on media containing glutamate, this mold excreted a set of different enzymes with hydrolytic activity. Addition of lignite to the growth media induced the synthesis of extracellular lignite-specific esterase activity but no evidence has been provided for its direct involvement in the process of lignite solubilization. Hence, the basic capability of T. atroviride enzymes to degrade a variety of ester and ether bonds at the surface or within the bulky lignite structure was tested using coal following its direct labelling with ¹⁴C-alkyl iodide. The participation of hydrolytic and oxidative enzymes in lignite degradation was assessed by measuring the release of ¹⁴C radioactivity from selectively alkylated carboxylic and phenolic OH groups. T. atroviride cleaved both carboxylic esters using esterases and the phenolic ether bonds by using oxidative enzymes, most likely laccases. Journal of Industrial Microbiology & Biotechnology (2002) 28, 207–212 DOI: 10.1038/sj/jim/7000232 Received 05 July 2001/ Accepted in revised form 08 December 2001     

Processes of liquefaction/solubilization of Spanish coals by microorganisms

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. Received: 15 June 1998 / Received revision: 23 November 1998 / Accepted: 29 November 1998     

Cloning and characterization of an endo-β-1,3(4)glucanase and an aspartic protease from Phaffia rhodozyma CBS 6938

We describe the identification and expression cloning of two novel enzymes, a β-glucanase and an aspartic protease, secreted from the basidiomycetous yeast Phaffia rhodozyma. A cDNA library from P. rhodozyma CBS 6938 was constructed, and full-length cDNA encoding an endo-1,3(4)-β-glucanase (bg1) and an aspartic protease (pr1) were cloned by expression cloning in Saccharomyces cerevisiae W3124. The bg1 cDNA encodes a 424-residue precursor protein with a putative signal peptide. The pr1 cDNA encodes a 405-residue prepropolypeptide with an 81-residue leader peptide. The aspartic protease was purified and characterized. It has a molecular mass of 36 kDa, an isoelectric point of pH 7.5, a pH activity optimum at 4.0–6.0, and a temperature activity optimum around 40 °C. Both enzymes show only low sequence identity to other known enzymes. Received: 6 August 1998 / Received revision: 29 October 1998 / Accepted: 30 October 1998     

Effect of enclosing rocks and aeration on methanogenesis from coals

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. Received: 15 January 1998 / Received revision: 2 October 1998 / Accepted: 2 October 1998     

Production and characterization of ferulic acid esterase activity in crude extracts by Streptomyces avermitilis CECT 3339

Streptomyces avermitilis CECT 3339 produces extracellular ferulic acid esterase (FAE) activity during growth on a range of lignocellulose substrates. Maximal levels of FAE activity were detected in culture filtrates from S. avermitilis CECT 3339 grown in media containing wheat bran and yeast extract as carbon and nitrogen sources respectively. Biochemical characterization of this enzyme activity revealed that it was 100-fold higher when wheat bran was pretreated with Celluclast (a mix of hydrolytic enzymes). FAE was found to be end-product-inhibited. Characterization of the properties of the enzyme showed that FAE exhibited an activity optimum pH at 6 with pH stability between pH 6 and 8. The optimum temperature was 50 °C while the temperature stability was between 30 °C and 40 °C, with rapid inactivation at 60 °C and above. The characteristics and stability of FAE from S. avermitilis CECT 3339 suggest a potential role for this enzyme in combination with endoxylanases for the upgrading of plant-residue silage and for biopulping. Received: 17 November 1997 / Received revision: 13 March 1998 / Accepted: 13 April 1998     

Purification and characterization of a glucokinase from young tomato (Lycopersicon esculentum L. Mill.) fruit

In order to clearly establish the properties of the enzymes responsible for hexose phosphorylation we have undertaken the separation and characterization of these enzymes present in tomato fruit (Martinez-Barajas and Randall 1996). This report describes the partial purification and characterization of glucokinase (EC. 2.7.1.1) from young green tomato fruit. The procedure yielded a 360-fold enrichment of glucokinase. Tomato fruit glucokinase is a monomer with a molecular mass of 53 kDa. Glucokinase activity was optimal between pH 7.5 and 8.5, preferred ATP as the phosphate donor (K _m = 0.223 mM) and exhibited low activity with GTP or UTP. The tomato fruit glucokinase showed highest affinity for glucose (K _m = 65 μM). Activity observed with glucose was 4-fold greater than with mannose and 50-fold greater than with fructose. The tomato fruit glucokinase was sensitive to product inhibition by ADP (K _i = 36 μM). Little inhibition was observed with glucose 6-phosphate (up to 15 mM) at pH 8.0; however, at pH 7.0 glucokinase activity was inhibited 30–50% by physiological concentrations of glucose 6-phosphate. Received: 4 October 1997 / Accepted: 10 January 1998     

Cloning of a new endoglucanase gene from Bacillus sp. BP-23 and characterisation of the enzyme. Performance in paper manufacture from cereal straw

The gene celA, encoding an endoglucanase from the strain Bacillus sp. BP-23, was cloned and expressed in Escherichia coli. The nucleotide sequence of a 1867-bp DNA fragment containing the celA gene was determined, revealing an open reading frame of 1200 nucleotides that encodes a protein of 44 803 Da. The deduced amino acid sequence of the encoded enzyme shows high homology to those of enzymes belonging to subtype 4 of the family-A cellulases. The celA gene product synthesized in E. coli showed activity on carboxymethylcellulose and lichenan but no activity was found on Avicel. Activity was enhanced in the presence of 10 mM Mg²⁺ and Ca²⁺ and showed its maximum at 40 °C and pH 4.0. Study of the performance of CelA on paper manufacture from agricultural fibres showed that treatment with the enzyme improved the properties of the pulp and the quality of paper. CelA treatment enhanced the physical properties (stretch and tensile index) of paper from wheat straw, while dewatering properties were slightly diminished. Electron-microscope analysis showed that the surface of straw fibres was modified by CelA. Received: 11 February 1998 / Received revision: 20 March 1998 / Accepted: 20 March 1998     

d-Xylose isomerases from a newly isolated strain, Paenibacillus sp., and from Alcaligenes ruhlandii : isolation, characterization and immobilisation to solid supports

d-Xylose/d-glucose isomerases from two strains, a newly isolated strain, Paenibacillus sp., and from Alcaligenes ruhlandii are described herein. The enzymes were purified to apparent homogeneity. Both of these d-xylose isomerases are homotetramers with relative subunit molecular masses of 45 000 and 53 000, respectively, as estimated by sodium dodecylsulphate-polyacrylamide gel electrophoresis. The native molecular masses determined on Superose 12 gel chromatography are 181 kDa for the enzyme from Paenibacillus sp. and 199 kDa for that from A. ruhlandii. The activity of both enzymes shows a requirement for divalent metal ions; the d-xylose isomerase from Paenibacillus sp. has the highest activity with Mn²⁺, while the enzyme from A. ruhlandii prefers Mg²⁺. Both enzymes also accept Co²⁺ with a somewhat lower efficiency, while Cu²⁺ inhibits the enzyme reaction. The binding of the metal ions obeys a biphasic characteristic, indicating the presence of two non-identical binding sites per subunit. d-Glucose is converted to d-fructose at a rate that is two- to three-fold slower than for the d-xylose isomerisation. d-Xylitol and d-lyxose are competitive inhibitors of both enzymes. Both enzymes have a pH optimum between 6.5 and 7.0, and they are active up to 60 °C. The enzyme from Paenibacillus sp. retained 50% of its activity after 4 days at 55 °C, whereas that from A. ruhlandii still retained 50% of its activity after 6 days at 55 °C. Polyacrylamide entrapment and immobilisation to both controlled pore glass and cyanogen-bromide-activated Sepharose were achieved for both enzymes with high efficiency. Received: 14 May 1998 / Received last revision: 29 July 1998 / Accepted: 29 July 1998     

In vivo-decolorization of coal-derived humic acids by laccase-excreting fungus Trametes versicolor

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. Received: 27 August 1998 / Received revision: 4 November 1998 / Accepted: 7 November 1998     

Transformation of macromolecules from a brown coal by lignin peroxidase

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. Received: 20 March 1998 / Received revision: 3 September 1998 / Accepted: 3 September 1998     

Detection of ferulic acid esterase production by Bacillus spp. and lactobacilli

The production of feruloyl esterase activity by Bacillus spp. and lactobacilli can be detected in an agar-plate assay. The assay involves the substitution of the main carbon source in specific agar with ethyl ferulate. A number of Bacillus spp., predominantly B. subtilis strains, were found to exhibit feruloyl esterase activity by this method. Of the examined lactobacilli, Lb. fermentum (NCFB 1751) showed the highest level of ferulic acid esterase activity. The enzyme was released from harvested cells by sonication and showed pH and temperature optima of 6.5 and 30 °C respectively. Received: 2 February 1998 / Received revision: 20 April 1998 / Accepted: 27 April 1998     

Production of d-hydantoinase by halophilic Pseudomonas sp. NCIM 5109

About 1000 bacterial colonies isolated from sea water were screened for their ability to convert dl-5-phenylhydantoin to d(−)N-carbamoylphenylglycine as a criterion for the determination of hydantoinase activity. The strain M-1, out of 11 hydantoinase-producing strains, exhibited the maximum ability to convert dl-5-phenylhydantoin to d(−)N-carbamoylphenylglycine. The strain M-1 appeared to be a halophilic Pseudomonas sp. according to morphological and physiological characteristics. Optimization of the growth parameters revealed that nutrient broth with 2% NaCl was the preferred medium for both biomass and enzyme production. d-Hydantoinase of strain M-1 was not found to be inducible by the addition of uracil, dihydrouracil, β-alanine etc. The optimum temperature for enzyme production was about 25 °C and the organism showed a broad pH optimum (pH 6.5–9.0) for both biomass and hydantoinase production. The organism seems to have a strict requirement of NaCl for both growth and enzyme production. The optimum pH and temperature of enzyme activity were 9–9.5 and 30 °C respectively. The biotransformation under the alkaline conditions allowed the conversion of 80 g l⁻¹ dl-5-phenylhydantoin to 82 g l⁻¹ d(−)N-carbamoylphenylglycine within 24 h with a molar yield of 93%. Received: 15 September 1997 / Received revision: 5 January 1998 / Accepted: 6 January 1998     

Fungal biosolubilization of Rhenish brown coal monitored by Curie-point pyrolysis/gas chromatography/mass spectrometry using tetraethylammonium hydroxide

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. Received: 13 July 1998 / Received revision: 12 October 1998 / Accepted: 16 October 1998     

Bioremoval of organic and inorganic sulphur from coal samples

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. Received: 26 June 1998 / Received revised: 1 October 1998 / Accepted: 2 October 1998     

Production of (R )-3-pentyn-2-ol through stereospecific hydrolysis of racemic 3-pentyn-2-ol esters with microbial enzymes

Microorganisms and commercial enzymes were screened for their ability to produce (R)-3-pentyn-2-ol from racemic 3-pentyn-2-ol esters through stereospecific hydrolysis. Among the esters formed with acetic acid, propionic acid, hexanoic acid and benzoic acid, the acetate was most effectively hydrolyzed by microbial cells and commercial lipases with high stereospecificity. Rhodococcus rubropertinctus AKU NOC082 was a good catalyst for (R)-3-pentyn-2-ol production through the hydrolytic resolution of racemic 3-pentyn-2-yl acetate. With 15%, 25% and 50% (v/v) racemic 3-pentyn-2-yl acetate as the substrate, 42.6%, 40.8% and 40.0% was hydrolyzed in 5 h, 10 h and 98 h respectively, under the optimized conditions (pH 7.0, 30 °C, 7.5% wet cell concentration), the (R) enantiomer of 3-pentyn-2-ol being formed with an optical purity of 97.8%, 98.0% and 94.2% respectively. Received: 2 June 1998 / Received revision: 3 August 1998 / Accepted: 3 September 1998     

Acid protease from Trichoderma reesei: limited proteolysis of fungal carbohydrases

Mechanisms regulating post-secretory limited proteolysis, carried out by the acid protease from Trichoderma reesei, were studied by following the release of α-galactosidase and multiple forms of cellobiohydrolase from this species. Both the rate of the proteolysis and the mode of action of the protease were affected by the pH of the culture medium, and only weakly depended on the amount of the enzyme. At pH between 2.7 and 3.5 the proteolytic reaction was limited, while at lower pH proteins were completely digested. Proteolysis depended on the degree of glycosylation of secreted enzymes. Inhibition of post-secretory deglycosylation decreased the rate of limited proteolysis in the culture medium in the course of fungal growth. Glucose and cellobiose, the main products of cellulose degradation carried out by the fungal cellulolytic complex, inhibited the proteolysis of the cellobiohydrolase in a concentration-dependent manner. A 32-kDa aspartic protease (EC 3.4.23.18) secreted by T. reesei was purified to homogeneity. The acid protease cleaved α-galactosidase and cellobiohydrolase into the same proteolytic fragments that had been isolated from the culture medium. Received: 4 December 1998 / Received revision: 22 February 1999 / Accepted: 5 March 1999     

Pectin methylesterases from poplar cambium and inner bark: localization, properties and seasonal changes

In the course of a study on the early events of cambial derivative differentiation in Populus × euramericana, seasonal changes in the pattern of pectin methylesterase (PME, EC 3.1.1.11) isoforms were followed. During the resting season, cell wall extracts contained mainly alkaline isoforms with an M_r around 55 kDa and optimal pH between 5.6 and 6.0. Neutral isoforms with an M_r around 35 kDa and optimal pH between 6.0 and 6.6 predominated in the extracts during the period of high meristematic activity. In the active cambial initials and in their immediate derivatives, the enzymes were immunolocalized exclusively in the dictyosomes. In older cells, they were present both in dictyosomes and in wall junctions. These results indicate that exportation of neutral PMEs towards the walls might be considered as an early marker of differentiation in cambial derivatives. Received: 17 May 1996 / Accepted: 5 November 1996     

Purification and properties of a novel raw starch degrading cyclomaltodextrin glucanotransferase from Bacillus firmus

A novel raw starch degrading cyclomaltodextrin glucanotransferase (CGTase; E.C. 2.4.1.19), produced by Bacillus firmus, was purified to homogeneity by ultrafiltration, affinity and gel filtration chromatography. The molecular weight of the pure protein was estimated to be 78 000 and 82 000 Da, by SDS-PAGE and gel filtration, respectively. The pure enzyme had a pH optimum in the range 5.5–8.5. It was stable over the pH range 7–11 at 10 °C, and at pH 7.0 at 60 °C. The optimum temperature for enzyme activity was 65 °C. In the absence of substrate, the enzyme rapidly lost its activity above 30 °C. K _m and k _cat for the pure enzyme were 1.21 mg/ml and 145.17 μM/mg per minute respectively, with soluble starch as the substrate. For cyclodextrin production, tapioca starch was the best substrate used when gelatinized, while wheat starch was the best substrate used when raw. This CGTase could degrade raw wheat starch very efficiently; up to 50% conversion to cyclodextrins was obtained from 150 g/l starch without using any additives. The enzyme produced α-, β- and γ-cyclodextrins in the ratio of 0.2:9.2:0.6 and 0.2:8.6:1.2 from gelatinized tapioca starch and raw wheat starch with 150 g/l concentration respectively, after 18 h incubation. Received: 25 September 1998 / Received revision: 15 December 1998 / Accepted: 21 December 1998