Publications by authors named "Ladislav Homolka"

10 Publications

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Long term storage of Pleurotus ostreatus and Trametes versicolor isolates using different cryopreservation techniques and its impact on laccase activity.

Fungal Biol 2015 Dec 22;119(12):1345-1353. Epub 2015 Oct 22.

Institute of Microbiology, Academy of Sciences of the Czech Republic (ASCR), v.v.i., Vídeňská 1083, 142 20 Prague 4, Czech Republic.

The strain Pleurotus ostreatus Florida f6, its 45 basidiospore-derived isolates (both monokaryons and dikaryons prepared in our laboratory), Trametes versicolor strain CCBAS 614 and 22 other T. versicolor isolates obtained from the sporocarps collected in distant localities were successfully preserved for 12 y using perlite and straw cryopreservation protocols. All tested isolates survived a 12-year storage in liquid nitrogen (LN) and their laccase production and Poly B411 decolorization capacity was preserved. Also mycelium extension rate and the types of colony appearance of individual isolates remained unchanged. Different cryopreservation techniques were also tested for the short time (24 h) and the long time (6 m) storage of the culture liquid with extracellular laccase produced by T. versicolor strain CCBAS 614. The results showed that 10 % glycerol was the most suitable cryopreservant. The absence of the cryopreservant did not cause high loss of laccase activity in the samples; the presence of DMSO (5 or 10 %) in LN-stored samples caused mostly a decrease of laccase activity. For the preservation of laccase activity in the liquid culture the storage in the freezer at -80 °C is more convenient than the storage in liquid nitrogen.
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http://dx.doi.org/10.1016/j.funbio.2015.10.004DOI Listing
December 2015

Preservation of live cultures of basidiomycetes - recent methods.

Authors:
Ladislav Homolka

Fungal Biol 2014 Feb 17;118(2):107-25. Epub 2013 Dec 17.

Laboratory of Environmental Microbiology, Institute of Microbiology of the Academy of Sciences of the Czech Republic, v. v. i., Vídeňská 1083, 142 20 Prague 4, Czech Republic. Electronic address:

Basidiomycetes are used in industrial processes, in basic or applied research, teaching, systematic and biodiversity studies. Efficient work with basidiomycete cultures requires their reliable source, which is ensured by their safe long-term storage. Repeated subculturing, frequently used for the preservation, is time-consuming, prone to contamination, and does not prevent genetic and physiological changes during long-term maintenance. Various storage methods have been developed in order to eliminate these disadvantages. Besides lyophilization (unsuitable for the majority of basidiomycetes), cryopreservation at low temperatures seems to be a very efficient way to attain this goal. Besides survival, another requirement for successful maintenance of fungal strains is the ability to preserve their features unchanged. An ideal method has not been created so far. Therefore it is highly desirable to develop new or improve the current preservation methods, combining advantages and eliminate disadvantages of individual techniques. Many reviews on preservation of microorganisms including basidiomycetes have been published, but the progress in the field requires an update. Although herbaria specimens of fungi (and of basidiomycetes in particular) are very important for taxonomic and especially typological studies, this review is limited to live fungal cultures.
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http://dx.doi.org/10.1016/j.funbio.2013.12.002DOI Listing
February 2014

Biodegradation of tetrabromobisphenol A by oxidases in basidiomycetous fungi and estrogenic activity of the biotransformation products.

Bioresour Technol 2011 Oct 26;102(20):9409-15. Epub 2011 Jul 26.

Institute of Microbiology, Laboratory of Biotransformation, Academy of Sciences of the Czech Republic, Vídeňská 1083, 142 20 Prague 4, Czech Republic.

Tetrabromobisphenol A (TBBPA) degradation was investigated using white rot fungi and their oxidative enzymes. Strains of the Trametes, Pleurotus, Bjerkandera and Dichomitus genera eliminated almost 1 mM TBBPA within 4 days. Laccase, whose role in TBBPA degradation was demonstrated in fungal cultures, was applied to TBBPA degradation alone and in combination with cellobiose dehydrogenase from Sclerotium rolfsii. Purified laccase from Trametes versicolor degraded approximately 2 mM TBBPA within 5 h, while the addition of cellobiose dehydrogenase increased the degradation rate to almost 2.5 mM within 3 h. Laccase was used to prepare TBBPA metabolites 2,6-dibromo-4-(2-hydroxypropane-2-yl) phenol (1), 2,6-dibromo-4-(2-methoxypropane-2-yl) phenol (2) and 1-(3,5-dibromo-4-hydroxyphen-1-yl)-2,2',6,6'-tetrabromo-4,4'-isopropylidene diphenol (3). As compounds 1 and 3 were identical to the TBBPA metabolites prepared by using rat and human liver fractions (Zalko et al., 2006), laccase can provide a simple means of preparing these metabolites for toxicity studies. Products 1 and 2 exhibited estrogenic effects, unlike TBBPA, but lower cell toxicity.
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http://dx.doi.org/10.1016/j.biortech.2011.07.036DOI Listing
October 2011

Effect of long-term preservation of basidiomycetes on perlite in liquid nitrogen on their growth, morphological, enzymatic and genetic characteristics.

Fungal Biol 2010 Nov-Dec;114(11-12):929-35. Epub 2010 Sep 17.

Institute of Microbiology, Academy of Sciences of the Czech Republic (ASCR), v.v.i., Vídeňská 1083, 142 20 Prague 4, Czech Republic.

The macro- and micro-morphological features, mycelial extension rate, enzymatic activities and possible genetic changes were studied in 30 selected strains of basidiomycetes after 10-year cryopreservation on perlite in liquid nitrogen (LN). Comparisons with the same strains preserved by serial transfers on nutrient media at 4°C were also conducted. Production of ligninolytic enzymes and hydrogen peroxide was studied by quantitative spectrophotometric methods, whereas semiquantitative API ZYM testing was used to compare the levels of a wide range of hydrolytic enzymes. Our results show that cryopreservation in LN did not cause morphological changes in any isolate. The vitality of all fungi was successfully preserved and none of the physiological features were lost, even though the extension rate and enzyme activity were slightly affected. Moreover, sequence analysis of eight strains did not detect any changes in their genetic features after cryopreservation. These findings suggest that the perlite-based freezing protocol is suitable for long-term preservation of large numbers of basidiomycetes.
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http://dx.doi.org/10.1016/j.funbio.2010.08.009DOI Listing
February 2011

Decolorization of Orange G and Remazol Brilliant Blue R by the white rot fungus Dichomitus squalens: toxicological evaluation and morphological study.

Chemosphere 2007 Oct 29;69(5):795-802. Epub 2007 Jun 29.

Institute of Microbiology AS CR, Vídenská 1083, 142 20 Prague 4, Czech Republic.

Dichomitus squalens efficiently decolorized both Orange G and Remazol Brilliant Blue R (RBBR) at concentrations of 0.5gl(-1) and 3gl(-1) in static and shaken culture and also on solid medium within 14d. The presence of the dyes in the culture medium mostly caused a decrease in biomass production and in growth rate, which was more significant in the case of RBBR. After 14d of cultivation, electron microscopy showed substantial morphological changes in mycelia of D. squalens growing in media containing dyes. The hyphae deformations were more intensively manifested in solid media than in liquid culture. In all the cases, the morphological changes were more prominent in the presence of RBBR. Higher concentrations of both dyes brought about more intensive changes. The toxicity of synthetic dyes Orange G and RBBR was tested using a bioassay based on the growth inhibition of duckweed Lemna minor. Two endpoints such as the number of fronds and their weight were studied during the bioassay. The results showed higher toxicity of RBBR than that of Orange G. The toxicity of both dyes decreased after the decolorization process.
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http://dx.doi.org/10.1016/j.chemosphere.2007.04.083DOI Listing
October 2007

The influence of extracellular H2O2 production on decolorization ability in fungi.

J Basic Microbiol 2006 ;46(6):449-55

Institute of Microbiology AS CR, Vídenská 1083, 142 20 Prague 4, Czech Republic.

A set of 50 randomly chosen fungal strains belonging to different basidiomycete species was tested for H2O2 and ligninolytic enzyme production and for decolorization of synthetic dyes Orange G and Remazol Brilliant Blue R. The decolorization capacity of individual strains was influenced by the level of H2O2 and laccase activity. The strains producing H2O2 at a concentration of 1.0-1.5 microM exhibited the most efficient decolorization; higher or lower H2O2 concentration reduced this ability. None of the strains without a detectable laccase activity was able to decolorize the tested dyes.
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http://dx.doi.org/10.1002/jobm.200610064DOI Listing
February 2007

Basidiomycete cultures on perlite survive successfully repeated freezing and thawing in cryovials without subculturing.

J Microbiol Methods 2007 Jun 11;69(3):529-32. Epub 2006 Oct 11.

Institute of Microbiology AS CR, Vídenská 1083, 142 20 Prague 4, Czech Republic.

Mycelial basidiomycete cultures on perlite in cryovials survived successfully three successive cycles of freezing, storage in liquid nitrogen (LN) and thawing without noticeable changes. This indicates that using perlite as a carrier for cryopreservation could in most cases overcome difficulties caused by interrupted supply of LN or electric power during the storage. Cultures on perlite can also be reused for successive inoculations.
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http://dx.doi.org/10.1016/j.mimet.2006.09.004DOI Listing
June 2007

Basidiomycete cryopreservation on perlite: evaluation of a new method.

Cryobiology 2006 Jun;52(3):446-53

Institute of Microbiology AS CR, Prague, Czech Republic.

A new cryopreservation method using perlite as a carrier was evaluated on a large set of mycelial cultures of basidiomycetes. The viability and some other characteristics--growth, macro- and micromorphology, and laccase production--of 442 strains were tested after 48-h and then after 3-year storage in liquid nitrogen using a perlite protocol (PP). All (100%) of them survived successfully both 48-h storage and 3-year storage in liquid nitrogen without noticeable growth and morphological changes. Also laccase production was unchanged. The viability and laccase production of a part (250) of these strains were compared with those of the strains subjected to an original agar plug protocol (OP). Using OP, 144 strains (57.6%) out of 250 survived a 3-year storage in liquid nitrogen. The results indicate that the cryopreservation protocol used significantly influences survival of the strains. Markedly better results were achieved using the PP.
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http://dx.doi.org/10.1016/j.cryobiol.2006.02.003DOI Listing
June 2006

Synthetic dye decolorization capacity of white rot fungus Dichomitus squalens.

Bioresour Technol 2006 Nov 27;97(16):2153-9. Epub 2005 Oct 27.

Institute of Microbiology AS CR, Vídenská 1083, 142 20 Prague 4, Czech Republic.

The ability to decolorize eight chemically different synthetic dyes (Orange G, Amaranth, Orange I, Remazol Brilliant Blue R (RBBR), Cu-phthalocyanin, Poly R-478, Malachite Green and Crystal Violet) by the white rot fungus Dichomitus squalens was evaluated on agar plates. The fungus showed high decolorization capacity and was able to decolorize all dyes tested, but not to the same extent. Some of the dyes did not limit the decolorization capacity of the strain tested even at a concentration of 2g/l. The presence of the dyes in solid media reduced the mycelial growth rate of D. squalens; a positive correlation was found between the growth rate and the decolorization ability. Decolorization of Orange G and RBBR was studied also in liquid culture, where both dyes caused an enhancement of ligninolytic enzyme and overall hydrogen peroxide production and a decrease of biomass production. RBBR was removed to a higher extent than Orange G.
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http://dx.doi.org/10.1016/j.biortech.2005.09.014DOI Listing
November 2006

Orange G and Remazol Brilliant Blue R decolorization by white rot fungi Dichomitus squalens, Ischnoderma resinosum and Pleurotus calyptratus.

Chemosphere 2005 Jul;60(3):398-404

Institute of Microbiology AS CR, Vídenská 1083, 142 20 Prague 4, Czech Republic.

Thirty different white rot strains were screened for Orange G and Remazol Brilliant Blue R (RBBR) decolorization on agar plates. Three promising strains, Dichomitus squalens, Ischnoderma resinosum and Pleurotus calyptratus, selected on the basis of this screening, were used for decolorization study in liquid media. All three strains efficiently decolorized both Orange G and RBBR, but they differed in decolorization capacity depending on cultivation conditions and ligninolytic enzyme production. Two different decolorization patterns were found in these strains: Orange G decolorization in I. resinosum and P. calyptratus was caused mainly by laccase, while RBBR decolorization was effected by manganese peroxidase (MnP); in D. squalens laccase and MnP cooperated in the decolorization processes.
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http://dx.doi.org/10.1016/j.chemosphere.2004.12.036DOI Listing
July 2005