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     

Extracellular laccase production during hyphal interactions between Trichoderma sp. and Shiitake, Lentinula edodes

Lentinula edodes (Berk.) Pegler was cultivated in liquid media containing malt and yeast extract. Extracellular laccase activity, measured in the culture fluids, was 5–18 times higher in cultures incubated for 29 days than in cultures incubated for 24 days. The addition of water-soluble lignin derivatives or Trichoderma sp. in cultures of L. edodes incubated for 11 days increased laccase activity 3- to 20 fold. The higher response was obtained with live mycelium of Trichoderma sp., but cell-free culture fluids of Trichoderma sp. in pure cultures were also effective. Trichoderma sp. induced changes in the laccase isoenzyme pattern as a result of the alteration of laccases secreted by L. edodes and not the induction of new isoforms. Received: 3 November 1997 /  Received revision: 19 January 1998 /  Accepted: 24 January 1998     

Production of ketocarotenoids by microalgae

Among the highly valued ketocarotenoids employed for food coloration, astaxanthin is probably the most important. This carotenoid may be produced biotechnologically by a number of microorganisms, and the most promising seems to be the freshwater flagellate Haematococcus pluvialis (Chlorophyceae), which accumulate astaxanthin in their aplanospores. Many physiological aspects of the transition of the flagellate into aplanospores have been described. Mixotrophic cultivation and suitable irradiance may result in fairly good yields (up to 40 mg/l; 43 mg/g cell dry weight) within a reasonable time, under laboratory conditions. In order to compete with synthetic astaxanthin, suitable scaling-up is required. However, large-scale production in open ponds has proved unsatisfactory because of severe contamination problems. A selective medium might overcome this difficulty. Further research for the development of suitable strains is thus warranted. Received: 8 July 1998 / Received revision: 12 November 1998 / Accepted: 14 November 1998     

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     

Mechanisms of coal solubilization by the deuteromycetes Trichoderma atroviride and Fusarium oxysporum

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. Received: 3 February 1998 / Received revision: 31 August 1998 / Accepted: 3 September 1998     

Production of methoxyphenol-type natural aroma chemicals by biotransformation of eugenol with a new Pseudomonas sp.

In our screening program for microorganisms that are able to metabolize eugenol, the main component of the essential oil of the clove tree Syzigium aromaticum (sy. Eugenia cariophyllus), we found a new Pseudomonas sp. that produces several substituted methoxyphenols when eugenol is fed to the culture. A taxonomic characterization of this new organism has been performed. Examples of the biotransformation products, produced in high amounts, were vanillic acid with 3.25 g/l within 99 h, ferulic acid with 5.8 g/l within 75 h and coniferyl alcohol with 3.22 g/l within 47.5 h. By changing the culture conditions the ratio of the different metabolites could be varied. Based on these results a scheme for the degradation of eugenol by this strain has been established. Received: 1 April 1996 / Received revision: 24 June 1996 / Accepted: 1 July 1996     

Bioassays for risk assessment of coal conversion products

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. Received: 17 June 1998 / Received revison: 21 October 1998 / Accepted: 24 October 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     

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     

Chemically defined media for commercial fermentations

The use of chemically defined media is gaining popularity in some commercial fermentations, particularly for the preparation of biological products. Although these media are still not frequently developed for industrial processes, they do exhibit favorable characteristics at large scale that are not observed with traditional complex media. This review focuses on the application, development, and practical considerations, especially process economics, of fermentations in chemically defined media in an industrial environment. Received: 3 August 1998 / Received revision: 16 November 1998 / Accepted: 21 November 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     

Conversion of chlorinated propanes by Methylosinus trichosporium OB3b expressing soluble methane monooxygenase

Chlorinated propanes are important pollutants that may show persistent behaviour in the environment. The biotransformation of 1-chloropropane, 1,2-dichloropropane, 1,3-dichloropropane and 1,2,3-trichloropropane was studied using resting cell suspensions of Methylosinus trichosporium OB3b expressing soluble methane monooxygenase. The transformation followed first-order kinetics. The rate constants were in the order 1-chloropropane > 1,3-dichloropropane > 1,2-dichloropropane > 1,2,3-trichloropropane, and varied from 0.07 to 1.03 ml min⁻¹ mg of cells⁻¹ for 1,2,3-trichloropropane and 1-chloropropane respectively. Turnover-dependent inactivation occurred for all of the chloropropanes tested. The inactivation constants were lower for 1-chloropropane and 1,2-dichloropropane than for 1,2,3-trichloropropane and 1,3-dichloropropane. Not all the chloride was released during cometabolic transformation of the chlorinated propanes and production of monochlorinated- and dichlorinated propanols was found by gas chromatography. The reaction pathway of 1,2,3-trichloropropane conversion was studied by mass spectrometric analysis of products formed in ²H₂O, which indicated that 1,2,3-trichloropropane was initially oxidized to 2,3-dichloropropionaldehyde and 1,3-dichloroacetone, depending on whether oxygen insertion occurred on the C-3 or C-2 carbon of 1,2,3,-trichloropropane, followed by reduction to the corresponding propanols. The results show that chloropropanes are susceptible to cometabolic oxidation by methanotrophs, but that the transformation kinetics is worse than with cometabolic conversion of trichloroethylene. Received: 27 November 1997 / Received revision: 27 February 1998 / Accepted: 27 February 1998     

Fungal laccase grafts acrylamide onto lignin in presence of peroxides

Laccase (EC 1.10.3.2) from the white-rot basidomycete Trametes versicolor in the presence of organic peroxides, particularly dioxane peroxide, tetrahydrofuran peroxide and t-butylhydroperoxide, initiated free-radical copolymerization of acrylamide and lignin. Hydrogen peroxide showed no such effect. Both the type of peroxide and the catalytic efficiency of the enzyme were important to ensure a significant yield of copolymerisate and a high rate of acrylamide incorporation into a lignin backbone. The mechanism of the enzymatic grafting is discussed. Received: 12 August 1998 / Received revision: 18 November 1998 / Accepted: 21 November 1998     

EPR study and structural aspects of ferredoxins obtained from Thiobacillus ferrooxidans

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. Received: 15 July 1998 / Received revision: 8 October 1998 / Accepted: 16 October 1998     

Influence of culture parameters on extracellular peroxidase activity and transformation of low-rank coal by Phanerochaete chrysosporium

The white-rot fungus Phanerochaete chrysosporium can degrade macromolecules in low-rank coal, offering the potential for converting coal to specific products. We investigated the influence of temperature, veratryl alcohol and oxygen on transformation of a solubilised fraction of Morwell brown coal (SWC6 coal) and on the activity of lignin peroxidase and manganese (Mn) peroxidase in N-limited cultures of P. chrysosporium. After 20 days, the mass and A ₄₀₀ of SWC6 coal recovered from cultures containing 0.03% SWC6 coal, incubated at 28 °C under hyperbaric oxygen, were reduced by over 95%. The modal apparent molecular mass of the residuum was reduced by 50%. Addition of 2 mM veratryl alcohol had little effect on the transformation of SWC6 coal. The extent of transformation was reduced in cultures incubated at 37 °C or under air. In cultures under air, coal molecules were transiently polymerised. Decolourisation of SWC6 coal reflects conversion to products that cannot be recovered from the medium, not the destruction of chromophores within recoverable material. The activity of lignin peroxidase, measured in cultures free of SWC6 coal to avoid interference with the assay, correlates directly with the degradation of SWC6 coal as measured by the decline in A ₄₀₀. The data suggest that lignin peroxidase is more important than Mn peroxidase in converting SWC6 coal to products that are assimilated by cells. Received: 16 July 1997 / Received revision: 14 November 1997 / Accepted: 18 November 1997     

High-level expression of an endoxylanase gene from Bacillus sp. in Bacillus subtilis DB104 for the production of xylobiose from xylan

To produce xylobiose from xylan, high-level expression of an endoxylanase gene from Bacillus sp. was carried out in Bacillus subtilis DB104. A 1.62-kb SmaI DNA fragment, coding for an endoxylanase of Bacillus sp., was ligated into the Escherichia coli/B. subtilis shuttle vector pJH27Δ88, producing pJHKJ4, which was subsequently transformed into B. subtilis DB104. A maximum endoxylanase activity of 105 U/ml was obtained from the supernatant of B. subtilis DB104 harboring pJHKJ4. The endoxylanase was purified to homogeneity by ion-exchange chromatography and the production profile of xylooligosaccharides from xylan by the endoxylanase was examined by HPLC with a carbohydrate analysis column. Xylobiose was the major product from xylan at 40 °C and its proportion in the xylan hydrolyzates increased with the reaction time; at 12 h, over 60% of the reaction products was xylobiose. These results suggest that xylobiose, which has a stimulatory effect on the selective growth of the intestinal bacterium Bifidobacterium, can be mass-produced effectively by the endoxylanase of Bacillus sp. cloned in B. subtilis. Received: 2 January 1998 / Received revision: 4 March 1998 / Accepted: 4 March 1998     

Biotransformation of citronellol by the basidiomycete Cystoderma carcharias in an aerated-membrane bioreactor

The basidiomycete Cystoderma carcharias transformed citronellol into 3,7-dimethyl-1,6,7-octanetriol as the main product. 3,7-Dimethyl-6,7-epoxy-1-octanol was identified as important intermediary product of the biotransformation, and the allylic diols 2,6-dimethyl-2-octene-1,8-diol, 3,7-dimethyl-5-octene-1,7-diol and 3,7-dimethyl-7-octene-1,6-diol were found to be minor products. Microbial formation of rose oxide, a flavour-impact component, was observed for the first time. The formation of the main products was inhibited by 70% after addition of 0.1 mmol l⁻¹ cytochrome monooxygenase inhibitors. Formation of 3,7-dimethyl-1,6,7-octanetriol was effective in a bioreactor with aeration over a coil of a hydrophobic microporous polypropene capillary membrane. Production rates of up to 150 mg l⁻¹ day⁻¹ were reached and led to a product concentration of 866 mg l⁻¹ (conversion rate: 52%). The total loss of the added volatile substrate via the exhaust air was 4.5% when this aeration method was used. Received: 30 July 1998 / Received revision: 2 November 1998 / Accepted: 7 November 1998     

Cometabolic biodegradation of methyl t-butyl ether by Pseudomonas aeruginosa grown on pentane

A bacterial strain identified as Pseudomonas aeruginosa was isolated from a soil consortium able to mineralize pentane. P. aeruginosa could metabolize methyl t-butyl ether (MTBE) in the presence of pentane as the sole carbon and energy source. The carbon balance for this strain, grown on pentane, was established in order to determine the fate of pentane and the growth yield (0.9 g biomass/g pentane). An inhibition model for P. aeruginosa grown on pentane was proposed. Pentane had an inhibitory effect on growth of P. aeruginosa, even at a concentration as low as 85 μg/l. This resulted in the calculation of the following kinetic parameters (μ_max = 0.19 h⁻¹, K _s = 2.9 μg/l, K _i = 3.5 mg/l). Finally a simple model of MTBE degradation was derived in order to predict the quantity of MTBE able to be degraded in batch culture in the presence of pentane. This model depends only on two parameters: the concentrations of pentane and MTBE. Received: 16 July 1998 / Received revision: 11 November 1998 / Accepted 31 November 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     

Cryopreservation of in vitro-grown axillary shoot-tip meristems of mint (Mentha spicata L.) by encapsulation vitrification

 Alginate-coated meristems from in vitro-grown axillary buds of mint (Mentha spicata L.) were successfully cryopreserved by vitrification. Excised meristems from nodal segments cold hardened at 4  °C for 3 weeks were encapsulated and osmoprotected by a mixture of 2 M glycerol plus 0.4 M sucrose. These meristems were dehydrated with a highly concentrated vitrification solution (PVS2 solution) for 3 h at 0  °C prior to a plunge into liquid nitrogen. Successfully encapsulated vitrified meristems developed shoots within a week after plating without intermediary callus formation. The average rate of shoot formation amounted to nearly 90%. This procedure was successfully applied to other Mentha species. It was also confirmed that encapsulated vitrified meristems produced a much higher rate of shoot formation than the encapsulated dried meristems. Thus, this revised encapsulation vitrification method appears promising for the cryopreservation of mint and other germplasm. Received: 24 November 1998 / Revision received: 8 February 1999 / Accepted: 26 February 1999