Anaerobic Growth Ability and Alcohol Fermentation Activity of Microscopic Fungi

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1 ISSN , Applied Biochemistry and Microbiology, 2011, Vol. 47, No. 2, pp Pleiades Publishing, Inc., Original Russian Text A.V. Kurakov, K.S. Khidirov, V. S. Sadykova, D.G. Zvyagintsev, 2011, published in Prikladnaya Biokhimiya i Mikrobiologiya, 2011, Vol. 47, No. 2, pp Anaerobic Growth Ability and Alcohol Fermentation Activity of Microscopic Fungi A. V. Kurakov a, K. S. Khidirov b, V. S. Sadykova c, and D. G. Zvyagintsev b a International Biotechnological Center, Moscow State University, Moscow, Russia kurakov57@mail.ru b Faculty of Soil Science, Moscow State University, Moscow, Russia c Faculty of Biology, Moscow State University, Moscow, Russia Received April 12, 2010 Abstract The method proposed in this study was used to isolate fungi grown under anaerobic conditions and to reveal distinctions in their abundance and species composition in different habitats. The ability of micromycetes of different taxa to grow under anaerobic conditions and ensure alcohol fermentation was determined for a representative sample (344 strains belonging to more than 60 species). The group of fungi growing under anaerobic conditions included species with high, moderate, and low fermentation activity. The ability for anaerobic growth and fermentation depended on the taxonomic affiliation of fungi. In some cases, the expression of these characteristics depended on the habitat from which the strain was isolated. The maximum level of ethanol accumulation in culture liquid ( %) was detected for Absidia spinosa, Aspergillus sp. of group flavus, Aspergillus terreus, Acremonium sp., Mucor circinelloides, Mucor sp., Fusarium oxysporum, F. solani, F. sambucinum, Rhizopus arrhizus var. arrhizus, Trichoderma atroviride, and Trichoderma sp. DOI: /S INTRODUCTION Ethanol manufacturing is based on the use of selected races of yeast (Saccharomyces cerevisiae, Candida scottii, and C. tropicales) and bacteria [1 3]. Much less attention was given to studying the ability of microscopic fungi for fermentation, because they were conventionally regarded as aerobic organisms. However, in nature, these fungi often occur in habitats with a limited oxygen supply. A considerable part of fungal mycelium retains its viability in soils incubated for a long time under reducing conditions as well as in waterlogged bog soils and raised bogs [4 6]. Recent data describe the ability of micromycetes to grow under anaerobic conditions and to ensure alcohol fermentation [6, 7]. Notably, this ability was described not only for members of Mucor, Rhyzopus, and Fusarium genera, for which it had been reported earlier [8 11]. Microscopic fungi are of special interest for obtaining ethanol from plant polymeric substrates, because many of them are producers of active hydrolases. Such fungal strains could be used for processing of plant material, hydrolysis of polymeric substrates under aerobic conditions, and fermentation of saccharides under oxygen deficiency conditions. The goal of this work was to assess the activity of alcohol fermentation in microscopic mycelial fungi of different taxa isolated from different habitats. MATERIALS AND METHODS Study objects. In search for fungal isolates capable of alcohol fermentation, we proceeded from the assumption that they should grow well under anaerobic conditions in media containing simple sugars. For this study, we selected 344 strains of microscopic fungi of different taxonomic groups. Pure micromycete cultures were isolated from soil samples collected in different regions and ecotopes, such as sod podzol soil, leached chernozem, rice field soil, plant remains, bottom silts, inundated soils along water bodies, fruit bodies of macromycetes, seeds of conifers and cereals, high-moor peat, roots of bog plants and plants growing on hydromorphous meadows, buried soils, occupation layers from diggings in the antique town of Fanagoria on the Black Sea, vermicompost, digestive tract of earthworms, and capricorn beetle larvae. The majority (320 of 344) of the selected strains were isolated by standard seeding on Petri dishes containing Czapek s medium and wort agar and culturing in an air atmosphere at 25 C. Twenty-four strains were selected from cultures isolated from natural samples directly incubated under anaerobic conditions. Anaerobic isolation of fungi. Fungi were isolated under anaerobic conditions by plating freshly collected samples of modern soils, occupation layers, and buried soils, as well as fragments of roots, plant materials, and the contents of the digestive tract of invertebrates, on a glucose peptone medium containing 169

2 170 KURAKOV et al. 1.0 g/l KH 2 PO 4, 0.5 g/l KCl, 0.5 g/l MgSO 4, 0.01 g/l FeSO 4, 2.0 g/l (NH 4 ) 2 SO 4, 15.0 g/l agar, 5.0 g/l peptone, 5.0 g/l glucose, and 0.5 g/l yeast extract. The medium also contained a microelement solution (1 ml/l) of the following composition: 500 mg/l EDTA, 200 mg/l FeCl 2 7H 2 O, 10 mg/l ZnCl 2 7H 2 O, 3 mg/l MgCl 2 4H 2 O, 30 mg/l H 3 BO 4, 2 mg/l CoCl 2 6H 2 O, 1 mg/l CuCl 2 2H 2 O, 3 mg/l Na 2 MoO 4, and 2 mg/l NiCl 2 6H 2 O. To suppress bacterial growth, antibiotics streptomycin and chloramphenicol were added to sterile melted medium to a final concentration of 200 mg/l. Petri dishes with the seeded material were placed into special plastic boxes (BioMerieux, France) and incubated under anaerobic conditions at 25 C for 7 10 days. Anaerobic conditions were created with the use of GENbox anaer anaerobic gas packets (BioMerieux, France). Oxygen was consumed and the level of carbon dioxide in boxes increased for 2.5 h. Anaerobiosis in boxes was monitored by indicators based on rezazurin (Oxoid Ltd., England). This approach for creating an oxygen-free atmosphere was used earlier for anaerobic cultivation of bacteria [12, 13]. In this paper, it was used to check the ability for anaerobic growth of pure fungal cultures and to isolate them from natural samples under anaerobic conditions. In this case, it is necessary to use media that are optimal for fungal culturing, supplemented with yeast extract and/or vitamins, microelements, and increased concentrations of antibacterial antibiotics. Incubation should be performed at a lower temperature (25 C) for one or two weeks. At least ten samples from each habitat were analyzed. Identification of fungal isolates. Microscopic fungi were identified by culture morphological traits using identification guides to this taxonomic group [14 18]. Thirty isolates, which were classified into different taxonomic groups on the basis of culture morphological traits, were identified by PCR amplification; amplicons were sequenced and analyzed (GenBank Data system: de). The purity of strains (i.e., the absence of satellite bacteria and bacterial contamination) was monitored by microscopic analysis and plating on cycloheximide-containing media. Assessment of fungal growth under anaerobic conditions. The strains isolated from the materials that were incubated under aerobic conditions were tested for the ability to grow under anaerobic conditions in glucose mineral medium or on Czapek s agar medium supplemented with yeast extract and microelements (ph 6.5). The media were supplemented with a microelement solution (1 ml/l) containing 0.8 g/l Na 2 B 4 O 7 10H 2 O, 0.8 g/l NaMoO 4 2H 2 O, 0.4 g/l FeSO 4 2H 2 O, 0.4 g/l CuSO 4 5H 2 O, 0.8 g/l MnSO 4 2H 2 O, and 0.8 g/l ZnSO 4 7H 2 O. Petri dishes were placed in air-tight plastic boxes with anaerobic atmosphere generators and anaerobiosis indicators. Seeded material was incubated at 25 C for 7 14 days. In dishes where fungal growth was detected, the size of colonies was measured and the radial growth rate was calculated [19]. Assessment of alcohol fermentation activity of strains under anaerobic conditions. The alcohol fermentation activity of fungal strains was assessed by ethanol accumulation in culture liquid under anaerobic conditions. Mycelium was preliminarily growth in liquid Czapek s medium for 4 6 days, washed with sterile distilled water, and transferred to 100-ml flasks containing 50 ml of mineral medium supplemented with glucose (1, 4, and 5%). Ethanol production by the most active cultures was determined at higher glucose concentrations (10 and 20%). Flasks were closed with rubber plugs, and air was substituted with N 2 by blowing with nitrogen for 1 min. Then, the flasks were incubated at 25 С for one week. The content of ethanol was determined in filtered culture liquid on day 4 and 7 using a Chrom 3700 gas liquid chromatograph (Moscow Experimental Plant, Russia) equipped with a flame ionization detector and a SOVPOL column (1.5 m). Chromatography was performed under the following conditions: argon carrier gas, 160 С column temperature 240 С evaporator temperature, 250 С detector temperature. To calculate the specific ethanol production activity, fungal biomass in flasks was determined after the experiment was completed. Mycelium was washed with distilled water and dried at С to a constant weight [19]. The efficiency of glucose fermentation to ethanol was calculated as well. The endoglucanase activity of fungi was determined in a medium with microcrystalline cellulose (type 100; Sigma, United States), which contained 1.0 g/l KH 2 PO 4, 0.5 g/l MgSO 4, 1.0 g/l KCl, 2.0 g/l NaNO 3, 0.01 g/l FeSO 4, 0.04 g/l CaCl 2 (CaCO 3 ), 0.02 g/l yeast extract, and 8.0 g/l microcrystalline cellulose. The medium was prepared in tap water. Flasks (250 ml) containing 100 ml of sterile liquid medium were inoculated with mycelium and spores and cultured on a shaker (120 rpm) at 28 C for 7 days. Culture liquid was collected to 2-ml plastic tubes; mycelium and remained cellulose were removed by centrifugation at g for 2 min. Endoglucanase and xylanase activities in samples were determined by accumulation of reducing sugars according to Somogyi Nelson using glucose as a standard and 1% solutions of carboxymethylcellulose (CMC) and birch xylan (Sigma, United States) as substrates, respectively. Enzymatic activities were determined at ph 5.0 and 50 С for 5 min in the case of CMC and for 10 min in the case of xylan [20]. The activities of 1,4-β-endoglucanase and xylanase were expressed in international units (IU/ml). Statistical data processing. All experiments were performed in triplicate. The mean values, standard deviations, and variation coefficients were calculated.

3 ANAEROBIC GROWTH ABILITY AND ALCOHOL FERMENTATION ACTIVITY 171 Table 1. Microscopic fungi isolated from different habitats under anaerobic conditions Genus/species Soil Buried soil and occupation layers High-moor peat and true mosses Roots and plant substrates Habitats associated with invertebrates Acremonium sp. + Aspergillus niger Aspergillus sp. gr. flavus + + Clonostachys rosea f. rosea Cunningamella elegans + Fusarium oxysporum F. solani + + Fusarium spp Mucor circinelloides + Mucor hiemalis + + Mucor spp Rhizopus arrhizus var. arrhizus + Paecylomyces sp. + + Torrubiella confrasa + Trichoderma aureoviride + T. harzianum + + Trichoderma spp Zygorrhynchus heterogamus + Zygorrhynchus sp Mycelia sterilia (light-colored) Mycelia sterilia (dark-colored) + RESULTS AND DISCUSSION Members of 23 species and several isolates in the form of mycelia sterilia were isolated from materials obtained from different habitats, which were incubated under anaerobic conditions (Table 1). Differences in the composition and dominance of species in samples from soils, high-moor peat, plant remains, roots, buried soils and occupation layers, as well as ecological niches associated with invertebrates were found. The most diverse spectrum of species was found in seeded materials from soils, which was incubated under anaerobic conditions. It included members of Acremonium sp., Aspergillus sp. group flavus, Aspergillus niger, Clonostachys rosea f. rosea, Cunningamela elegans, Fusarium oxysporum, F. solani, Fusarium spp., Mucor circinelloides, M. hiemalis, Mucor spp., Rhizopus arrhizus var. arrhizus, several species of the genus Trichoderma, Zygorhunchus heterogamus, Zygorhunchus sp., and organisms represented by light- and dark-colored mycelia sterilia. In general, this species composition was similar to that revealed using Hangate s method modified for anaerobic isolation of micromycetes from different types of soils [6]. Thus, the proposed approach can be used to isolate microscopic fungi under anaerobic conditions from soils and other objects as well as for assessment of the ability for anaerobic growth of their pure cultures. A much smaller number of species was isolated from buried soils and occupation layers that were incubated under anaerobic conditions. They were dominated by species of the genera Fusarium, Mucor, Trichoderma as well as by Clonostachys rosea f. rosea, Aspergillus niger, and Aspergillus sp. group flavus. These fungal taxa are often isolated from soil samples from southern taiga and steppe zones, which are incubated under anaerobic conditions [6]. Seeded materials of roots and various plant remains often contained species of genera Fusarium (Fusarium oxysporum), Trichoderma, and Clonostachys, as well as members of Mucorales (Mucor spp., Cunningamela elegans, and Zygorhinchus sp.). Samples from high-moor peat were dominated by species of genera Trichoderma and Zygorhunchus. Fungi isolated from seeded samples of the digestive tract of earthworms and capricorn beetle larvae, as well as from the habitats associated with them (coprolites, vermicompost, and processed wood), which were incubated under anaerobic conditions, included Mucor circinelloides, M. hiemalis, Rhyzopus arrhizus var. arrhizus, Zygorhunchus sp., Fusarium oxysporum, F. solani, Aspergillus niger, Clonostachys rosea f. rosea, and Trichoderma harzianum.

4 172 KURAKOV et al. Table 2. Growth of microscopic fungal strains that were isolated from materials incubated under aerobic conditions and then cultured under anaerobic conditions Genus/species checked Number of strains radial growth rate, mm/h >0.10 Absidia sp. 3 3 Absidia spinosa Acremonium sp. 2 2 Actinomucor elegans 1 1 Aspergillus sp. gr. flavus 2 2 Aspergillus niger 4 4 Aspergillus terreus Aspergillus sp Clonostachys rosea f. rosea C. solani f. solani 1 1 Clonostachus sp Cunningamella echinulata 1 1 Fusarium avenacium F. dimerum 1 1 F. fusarioides 1 1 F. moniliforme F. oxysporum 7 7 F. poae 1 1 F. sambucinum 1 1 F. solani 7 7 F. sporotrichioides Fusarium spp Mucor circinelloides 2 2 Mucor hiemalis 2 2 Mucor plumbeus 1 1 Mucor spp Paecilomyces sp. 2 2 Rhizopus arrhizus var. arrhizus Sphaerostilbella aureonitens 1 1 Trichoderma asperellum T. atroviride T. citrinoviride 2 2 T. harzianum T. koningii T. viride Triñhoderma spp Zygorrhynchus heterogamus 1 1 Z. moelleri 2 2 Zygorrhynchus sp The results of testing the ability of strains of different species isolated from conventional seeded material that were incubated under aerobic conditions to grow under anaerobic conditions are summarized in Table 2. Fungal colonies grown under anaerobic conditions on solid media formed small quantities of air mycelium or mycelium spreading over the surface of the medium. The radial growth rate of fungal colonies under anaerobic conditions was times lower that that determined under aerobic conditions, and the fungal biomass accumulated 20 times more slowly [6]. Micromycetes of different taxonomic affiliation significantly differed in the radial growth rate and size of colonies formed under anaerobic conditions on nutrient medium. We found that the vast majority of strains of some species, such as F. oxysporum, F. solani, Clonostachys rosea f. rosea, Mucor circinelloides, M. hiemalis, and Rhizopus arrhizus var. arrhizus were characterized by moderate and good growth under anaerobic conditions. However, this ability markedly varied in strains of other species. In some cases, this characteristic depended on the habitat from which they were isolated. In particular, Trichoderma isolates from highmoor peat, silts, and inundated soils along water bodies showed good growth under anaerobic conditions. By contrast, the strains isolated from the surface of seeds and fruiting bodies of macromycetes were unable to grow under anaerobic conditions. Moderate and good growth under anaerobic conditions was usually observed in members of zygomycetes (Absidia, Mucor, Actinomucor, Cunningamella, Rhyzopus, and Zygorhynchus), mitotic fungi of the ascomycete affinity (Clonostachys, Fusarium, and Trichoderma), and, more rarely, in some species of genera Aspergillus, Paecylomyces, Acremonium, and some others. In addition to the species whose ability to grow under anaerobic conditions had been reported earlier [6, 9 12], anaerobic growth was demonstrated for members of Absidia spinosa, Aspergillus sp. group flavus, Clonostachys solani f. solani, Cunningamella echinulata, Fusarium avenacium, F. dimerum, F. fusarioides, F. moniliforme, F. poae, F. sambucinum, F. sporotrichioides, Trichoderma asperellum, T. atroviride, and T. citrinoviride. Very poor growth under anaerobic conditions was observed for strains of Aspergillus sp. group flavus, Aspergillus terreus, Aspergillus wentii, Aspergillus sp., Chaetomium globosum, Fusarium clamydosporum, F. sambucinum, Fusarium spp., Humicola fuscoatra, Humicola grisea, Mucor sp., Paecilomyces lilacinus, Paecilomyces marquindii, Paecilomyces sp., Penicillium funiculosum, P. islandicum, and Trichoderma citrinoviride. These micromycetes stopped to grow under anaerobic conditions after forming microcolonies not more than 1 3 mm in diameter, which could be distinguished only under a microscope. The ability for anaerobic growth was not detected when testing one or two strains of Alternaria alternata, Aphalocladium sp., Aureobasidium pullulans,

5 ANAEROBIC GROWTH ABILITY AND ALCOHOL FERMENTATION ACTIVITY 173 Blakslea trispora, Botrytis cinerea, Cladosporium cladosporioides, C. herbarum, Geomyces pannorum, Geotrichum candidum, Lecanicillium sp., Paecilomyces variotii, Penicillium aurantiongriseum, P. chrysogenum, P. brevicompactum, P. canescens, P. citreoviride, P. citrinum, P. diversum, Penicillium frequentans, P. glabrum, P. implicatum, P. nalgiovense, P. restrictum, P. rolfsii, P. spinulosum, P. thomii, Penicillium spp. (six strains), Sordaria fimicola, Stachybotrys sp., Talaromyces sp., Tolypocladium geodes, T. inflatum, Umbelopsis ramanniana, and Verticillium nigrescens. Anaerobic growth was not detected either for some strains of Absidia sp., Aspergillus terreus, Aspergillus spp., Fusarium avenacium, F. dimerum, F. moniliforme, F. sambucinum, F. sporotrichioides, Humicola fuscoatra, Paecilomyces marquindii,, Paecilomyces sp., Trichoderma asperellum (33 strains), T. atroviride, T. citrinoviride (16 strains), T. hamatum, T. harzianum (17 strains), T. viride (21 strains), T. koningii (five strains), and Trichoderma spp. (14 strains). Testing the ability of members of different species of the genus Penicillium that were isolated under aerobic conditions to grow under anaerobic conditions showed that only two strains (Penicillium funiculosum and P. islandicum) showed very poor growth (formed microcolonies), whereas all other members of this genus were unable to grow under anaerobic conditions. It should be noted that members of the genus Penicillium, dark-colored fungi (Alternaria alternata, Aureobasidium pullulans, Cladosporium cladosporioides, C. herbarum, and Stachybotrys sp.), and some other species (Geomyces pannorum, Sordaria fimicola, Talaromyces sp., and Botrytis cinerea), which are typical micromycetes of terrestrial biogeocenoses, were not isolated from samples of soils and other environmental samples incubated under anaerobic conditions (Table 1). Thus, the ability to grow under anaerobic conditions was determined primarily by the taxonomic affiliation of fungal strain. In some cases, this characteristic depended on the habitat from which the strain was isolated (the presence of long periods of limited oxygen supply). Alcohol fermentation activity was determined for 170 strains of microscopic mycelial fungi that showed moderate and good growth under anaerobic conditions. All strains of these facultatively anaerobic fungi were capable of alcohol fermentation in glucose-containing medium (Table 3). The highest alcohol fermentation activity in glucose-containing medium was detected for fungal strains of the following taxonomic groups: Absidia spinosa, Aspergillus sp. group flavus, Aspergillus terreus, Acremonium sp., Mucor circinelloides, Mucor sp., Fusarium oxysporum, F. solani, F. sambucinum, Rhizopus arrhizus var. arrhizus, Trichoderma atroviride, and Trichoderma sp. In most cases, the efficiency of glucose fermentation by these fungi exceeded 50%. It was somewhat lower in members of Trichoderma aureovir- Table 3. Fermentation activity of strains of microscopic mycelial fungi characterized by moderate and good growth under anaerobic conditions Genus/species Number of strains Number of active strains C 2 H 5 OH on day 4 6, % Absidia spinosa 1 1 Absidia sp. 2 2 Acremonium sp. 1 1 Actinomucor elegans 1 1 Aspergillus sp. gr. flavus Aspergillus niger Aspergillus terreus Aspergillus sp. 1 1 Clonostachys rosea 1 1 var. rosea Cunningamella elegans 1 1 Fusarium avenacium 2 2 F. clamydosporum 1 1 Fusarium fusarioides 1 1 Fusarium oxysporum Fusarium sambucinum Fusarium solani F. sporotrichioides Fusarium sp Mucor circinelloides Mucor hiemalis 1 1 Mucor spp Rhizopus arrhizus var. 1 1 arrhizus Sphaerostilbella aureonitens Trichoderma asperellum T. aureoviride T. atroviride 2 2 T. citrinoviride 2 2 T. harzianum T. hamatum 1 1 T. koningii 1 1 T. viride 2 2 Triñhoderma spp Zygorrhynchus 1 1 heterogamus Z. moelleri 1 1 Zygorrhynchus sp. 3 1

6 174 KURAKOV et al. ide, T. harzianum, T. koningi, T. viride, Fusarium sporotrichoides, Aspergillus niger, Clonostachys rosea f. rosea, and Zygorhynchus heterogamus. The maximum efficiency of glucose fermentation by strains of these species was 25 50%, which corresponded to accumulation of % ethanol on day 4 7 in media containing 1 2% glucose. Much lower alcohol fermentation activity was detected for strains of other species of genera Fusarium and Trichoderma, as well as for Mucor hiemalis, Zygorhynchus moelleri, and Cunningamella elegans. Ethanol accumulation either was not detected or did not exceed 0.02% in Clonostachys spp. (six strains), Fusarium clamydosporum, F. moniliforme (two strains), F. poae, F. sporotrichioides, Fusarium sp. (three strains), Humicola fuscoatra, H. grisea, Rhizopus oryzae, Trichoderma àsperellum, T. atroviride (two strains), T. citrinoviride (three strains), T. hamatum, T. harzianum (two strains), T. koningii (two strains), T. viride, Trichoderma sp. (12 strains), Zygorrhynchus moelleri, and Zygorrhynchus sp. (two strains). It was established that ethanol production by natural isolates of yeast Saccharomyces cerevisiae KBP-3781 and Hanseniaspora sp. 1R and 2KBP in medium containing 1% glucose reached and mg/ml, respectively. At this activity, Hanseniaspora sp. strains fermented as much as 39 49% of glucose to ethanol on day 4 and 62% of glucose on day 7. In Saccharomyces cerevisiae, these values were much higher: 59 90% of glucose was fermented on day 4. The specific activity of ethanol production by wild-type Hanseniaspora sp. and Saccharomyces cerevisiae strains was mg C 2 H 5 OH/h per gram dry biomass, which is times higher than that of fermenting micromycetes. Ethanol accumulation in the medium containing 1% glucose in the case of F. oxysporum, F. solani, Trichoderma harzianum, and Aspergillus terreus reached % of the level of ethanol production by Saccharomyces cerevisiae and Hanseniaspora sp. strains; in the case of Trichoderma aureoviride, this value accounted for 20 50%. Therefore, the ethanol fermentation rate of many facultatively anaerobic mycelial fungi was close to that of wild-type yeast strains. The alcohol fermentation activity of fungal strains producing % ethanol (Table 3) in media containing 4 5% glucose was determined at higher glucose concentrations (10%). The maximum level of ethanol accumulation in media for these mycelial micromycetes reached %, which is also comparable with the activity of natural yeast isolates. Determination of hydrolytic activity of fermenting micromycetes showed that some strains of Trichoderma atroviride, Aspergillus terreus, Fusarium oxysporum, F. solani, and C. rosea f. rosea exhibited fairly high endoglucanase and xylanase activities ( IU/ml culture liquid). Strains with alcohol fermentation activity comparable to that of Mucorales (saccharolytics) were also found among species with a broad spectrum of hydrolytic enzymes, namely, A. terreus, Aspergillus sp. group flavus, Trichoderma harzianum, T. atroviride, F. oxysporum, and F. solani. There is a certain relationship between the habitat where fungi were isolated and their ability to ferment glucose to ethanol. For example, Aspergillus strains isolated from buried soils and occupation layers were characterized by high fermentation activity, whereas other strains of this genus exhibited lower activity. To compare with the fungi that grew well under anaerobic conditions, the ability for alcohol fermentation was estimated for 20 micromycete strains that either showed no growth or grew very poorly under anaerobic conditions (Humicola grisea, Tolypocladium inflatum, T. geodes, Umbelopsis issabelina, Lecanicillium sp., Penicillium brevicompactum, P. citreoviride, P. frequentans, Penicillium spinulosum, P. thomii, Penicillium spp., Paecylomyces lilacinus, P. varioti, Paecylomyces sp., Alternaria alternata, Aureobasidium pullulans, and Geomyces pannorum) (Tables 2, 3). Alcohol fermentation activity in these fungi was either low or absent. Thus, similarly to yeast [21, 22], taxa characterized by different growth intensity under anaerobic conditions and different activity of alcohol fermentation can be distinguished among micromycetes: strong, moderate, and weak fermenting fungi, as well as fungi unable to ferment ethanol. 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