Optimization of lignin peroxidase production and stability by <Emphasis Type="Italic">Phanerochaete chrysosporium</Emphasis> using sewage-treatment-plant sludge as substrate in a stirred-tank bioreactor

A laboratory-scale study was carried out to produce lignin peroxidase (ligninase) by white rot fungus (Phanerochaete chrysosporium) using sewage-treatment-plant (STP) sludge as the major substrate. The optimization was done using full-factorial design (FFD) with agitation and aeration as the two parameters. Nine experiments indicated by the FFD were fermented in a stirred-tank bioreactor for 3 days. A second-order quadratic model was developed using the regression analysis of the experimental results with the linear, quadratic, and interaction effects of the parameters. Analysis of variance (ANOVA) showed a high coefficient of determination (R ²) value of 0.972, thus indicating a satisfactory fit of the quadratic model with the experimental data. Using statistical analysis, the optimum aeration and agitation rates were determined to be 2.0 vvm and 200 rpm, respectively, with a maximum activity of 225 U l⁻¹ in the first 3 days of fermentation. The validation experiment showed the maximum activity of lignin peroxidase was 744 U l⁻¹ after 5 days of fermentation. The results for the tests of the stability of lignin peroxidase showed that the activity was more than 80% of the maximum for the first 12 h of incubation at an optimum pH of 5 and temperature of 55°C.     

Lignocellulose-degrading enzyme production by white-rot <Emphasis Type="Italic">Basidiomycetes</Emphasis> isolated from the forests of Georgia

The production of lignocellulolytic enzymes by eleven basidiomycetes species isolated from two ecosystems of Georgia was investigated for the first time under submerged (SF) and solid-state fermentation (SSF) of lignocellulosic by-products. Notable intergeneric and intrageneric differences were revealed with regard to the extent of hydrolase and oxidase activity. Several fungi produced laccase along with hydrolases in parallel with growth during the trophophase, showing that the synthesis of this enzyme is not connected with secondary metabolism. The lignocellulosic substrate type had the greatest impact on enzyme secretion. Some of the substrates significantly stimulated lignocellulolytic enzyme synthesis without supplementation of the culture medium with specific inducers. Exceptionally high carboxymethyl cellulase (CMCase, 122 U ml⁻¹) and xylanase (195 U ml⁻¹) activities were revealed in SF of mandarin peelings by Pseudotremella gibbosa IBB 22 and of residue after ethanol production (REP) by Fomes fomentarius IBB 38, respectively. The SSF of REP by T. pubescens IBB 11 ensured the highest level of laccase activity (24,690 U l⁻¹), whereas the SSF of wheat bran and SF of mandarin peels provided the highest manganese peroxidase activity (570–620 U l⁻¹) of Trichaptum biforme IBB 117. Moreover, the variation of lignocellulosic growth substrate provides an opportunity to obtain enzyme preparations containing different ratios of individual enzymes.     

Effects of Cultivation Parameters on the Morphology of Rhizopus arrhizus and the Lactic Acid Production in a Bubble Column Reactor

The use of filamentous Rhizopus for lactic acid production is facing a challenge due to its low yield mainly caused by the difficulty to control its morphology in submerged fermentation processes. This study was aimed at investigating the impacts of cultivation parameters on the morphology of Rhizopus arrhizus DAR 36017 and lactic acid production using waste potato starch in a laboratory scale bubble column reactor (BCR). The fungal morphology was significantly influenced by carbon sources, process pH, starch concentrations, sparger designs and aeration rates. The favorable morphology for lactic acid production was a freely dispersed small pellet, which was achieved under operation conditions at pH 5.0–6.0, starch concentrations of 60–120 g/L and aeration rates of 0.2–0.8 vvm using a sintered stainless steel disc sparger. Optimal cultivation conditions at pH 6.0 and an aeration rate of 0.4 vvm resulted in the formation of freely dispersed small pellets and 103.8 g/L lactic acid with a yield of 87 % from 120 g/L liquefied potato starch in 48 h. The overall results in terms of lactic acid yield and productivity are comparable to those reported in previous studies using immobilized Rhizopus cells in batch fermentations.     

Enhanced continuous production of lovastatin using pellets and siran supported growth of Aspergillus terreus in an airlift reactor

Lovastatin, a hypocholesterolemic agent, is a secondary metabolite produced by filamentous microorganism Aspergillus terreus in submerged batch cultivation. Lovastatin production by pellets and immobilized siran cells was investigated in an airlift reactor. The process was carried out by submerged cultivation in continuous mode with the objective of increasing productivity using pellet and siran supported growth of A terreus. The continuous mode of fermentation improves the rate of lovastatin production. The effect of dilution rate and aeration rate were studied in continuous culture. The optimum dilution rate for pellet was 0.02 h⁻¹ and for siran carrier was 0.025 h⁻¹. Lovastatin productivity using immobilized siran carrier (0.0255 g/L/h) was found to be greater than pellets (0.022 g/L/h). The productivity by both modes of fermentation was found higher than that of batch process which suggests that continuous cultivation is a promising strategy for lovastatin production.     

Production of xylanolytic enzymes by <Emphasis Type="Italic">Aspergillus terricola</Emphasis> in stirred tank and airlift tower loop bioreactors

Fungi producing high xylanase levels have attracted considerable attention because of their potential industrial applications. Batch cultivations of Aspergillus terricola fungus were evaluated in stirred tank and airlift bioreactors, by using wheat bran particles suspended in the cultivation medium as substrate for xylanase and β-xylosidase production. In the stirred tank bioreactor, in physical conditions of 30°C, 300 rpm, and aeration of 1 vvm (1 l min⁻¹), with direct inoculation of fungal spores, 7,475 U l⁻¹ xylanase was obtained after 36 h of operation, remaining constant after 24 h. In the absence of air injection in the stirred tank reactor, limited xylanase production was observed (final concentration 740 U l⁻¹). When the fermentation process was realized in the airlift bioreactor, xylanase production was higher than that observed in the stirred tank bioreactor, being 9,265 U l⁻¹ at 0.07 vvm (0.4 l min⁻¹) and 12,845 U l⁻¹ at 0.17 vvm (1 l min⁻¹) aeration rate.     

Effect of growth substrate,method of fermentation,and nitrogen source on lignocellulose-degrading enzymes production by white-rot basidiomycetes

The exploration of seven physiologically different white rot fungi potential to produce cellulase, xylanase, laccase, and manganese peroxidase (MnP) showed that the enzyme yield and their ratio in enzyme preparations significantly depends on the fungus species, lignocellulosic growth substrate, and cultivation method. The fruit residues were appropriate growth substrates for the production of hydrolytic enzymes and laccase. The highest endoglucanase (111 U ml⁻¹) and xylanase (135 U ml⁻¹) activities were revealed in submerged fermentation (SF) of banana peels by Pycnoporus coccineus. In the same cultivation conditions Cerrena maxima accumulated the highest level of laccase activity (7,620 U l⁻¹). The lignified materials (wheat straw and tree leaves) appeared to be appropriate for the MnP secretion by majority basidiomycetes. With few exceptions, SF favored to hydrolases and laccase production by fungi tested whereas SSF was appropriate for the MnP accumulation. Thus, the Coriolopsis polyzona hydrolases activity increased more than threefold, while laccase yield increased 15-fold when tree leaves were undergone to SF instead SSF. The supplementation of nitrogen to the control medium seemed to have a negative effect on all enzyme production in SSF of wheat straw and tree leaves by Pleurotus ostreatus. In SF peptone and ammonium containing salts significantly increased C. polyzona and Trametes versicolor hydrolases and laccase yields. However, in most cases the supplementation of media with additional nitrogen lowered the fungi specific enzyme activities. Especially strong repression of T. versicolor MnP production was revealed.     

Effect of soya lecithin on the enzymatic system of the white-rot fungi Anthracophyllum discolor

The present work optimized the initial pH of the medium and the incubation temperature for ligninolytic enzymes produced by the white-rot fungus Anthracophyllum discolor. Additionally, the effect of soya lecithin on mycelial growth and the production of ligninolytic enzymes in static batch cultures were evaluated. The critical micelle concentration of soya lecithin was also studied by conductivity. The effects of the initial pH (3, 4, and 5) and incubation temperature (20, 25, and 30°C) on different enzymatic activities revealed that the optimum conditions to maximize ligninolytic activity were 26°C and pH 5.5 for laccase and manganese peroxidase (MnP) and 30°C and pH 5.5 for manganese-independent peroxidase (MiP). Under these culture conditions, the maximum enzyme production was 10.16, 484.46, and 112.50 U L⁻¹ for laccase, MnP, and manganese-independent peroxidase MiP, respectively. During the study of the effect of soya lecithin on A. discolor, we found that the increase in soya lecithin concentration from 0 to 10 g L⁻¹ caused an increase in mycelial growth. On the other hand, in the presence of soya lecithin, A. discolor produced mainly MnP, which reached a maximum concentration of 30.64 ± 4.61 U L⁻¹ after 25 days of incubation with 1 g L⁻¹ of the surfactant. The other enzymes were produced but to a lesser extent. The enzymatic activity of A. discolor was decreased when Tween 80 was used as a surfactant. The critical micelle concentration of soya lecithin calculated in our study was 0.61 g L⁻¹.     

Optimization of production of caffeine demethylase by <Emphasis Type="Italic">Pseudomonas</Emphasis> sp. in a bioreactor

The effect of pH, aeration rate, and agitation rate on specific productivity of caffeine demethylase from Pseudomonas sp. was studied in a bioreactor. Maximum specific productivity of caffeine demethylase of 2,214 U g cell dry weight⁻¹ h⁻¹ was obtained at 0.27 vvm, 700 rpm, and pH 7.0. Under these conditions, volumetric oxygen transfer coefficient was 74.2 h⁻¹, indicating that caffeine demethylase production by Pseudomonas sp. was highly oxygen-dependent. Different metabolite formation at different agitation and aeration rates can be used as a strategy for recovery of pharmaceutically important metabolites from caffeine by manipulation of conditions in a bacterial culture. This is the first report on production of high levels of caffeine demethylase in bioreactors.     

Degradation of phenanthrene by the endophytic fungus <Emphasis Type="Italic">Ceratobasidum stevensii</Emphasis> found in <Emphasis Type="Italic">Bischofia polycarpa</Emphasis>

Some strains of white rot fungi, non-lignolytic fungi and litter-decomposing basidiomycetes have been recognized as PAH degraders. The purpose of our research was to enlarge the scope of PAH-degrading fungi and explore the huge endophytic microorganism resource for bioremediation of PAHs. In this study, phenanthrene was used as a model PAHs compound. Nine strains of endophytic fungi isolated from four kinds of plant from Eupharbiaceae were screened for degradation of phenanthrene. The endophytic fungus Ceratobasidum stevensii (strain B6) isolated from Bischofia polycarpam showed high degradation efficiency and was selected for further studies. Into the fungal culture, 100 mg l⁻¹ phenanthrene was added, and after 10 days of incubation, about 89.51% of the phenanthrene was removed by strain B6. Extracellular ligninolytic enzyme activities of strain B6 were tested. The results showed that manganese peroxidase [MnP] was the predominant ligninolytic enzyme and that its production was greatly induced by the presence of phenanthrene. To confirm the involvement of MnP in phenanthrene degradation, promotion and inhibition studies on MnP in different concentration level of Mn²⁺ and NaN₃ were performed. Additionally, fungal mycelium-free and resuspended experiments were carried out. The results showed no apparent correlation between MnP activity and phenanthrene degradation. The mycelium and fresh medium were the crucial factors affecting the degradation of phenanthrene. To date, this is the first report on PAH degradation by Ceratobasidum stevensii. This study suggests that endophytic fungi might be a novel and important resource for microorganisms that have PAH-degrading capabilities.     

Enzymatic characterization of Chilean native wood-rotting fungi for potential use in the bioremediation of polluted environments with chlorophenols

This work presents a preliminary report of a series of studies on the ability of several indigenous wood-rotting fungi from Chile to produce hydrolytic and ligninolytic enzymes and the evaluation of these native microorganism to future research on potential applications in bioremediation programs. Wood-rotting Basidiomycete fungi were collected from indigenous hardwood forest in the South of Chile. Twenty-eight strains were identified and qualitative enzymatic tests for peroxidases, laccase, tyrosinase, xylanase and cellulase production were performed in solid medium. Eleven selected strains were evaluated in liquid medium to quantify their ligninolytic enzyme production and their capacity to grow in solid medium supplemented with 2,4-dichlorophenol (2,4-DCF), 2,4,6-trichlorophenol (2,4,6-TCF) and pentachlorophenol (PCP). PCP degradation and ligninolytic enzymes production were also evaluated in liquid medium. Results showed that laccase was present in 28 of the selected strains (≈73%). Peroxidase was present in 40% and cellulase in 37% of the strains. Xilanase and tyrosinase were obtained in a smaller percentage in the strains (28% and 7%, respectively). The 11 selected strains showed high concentrations of lignin peroxidase (Lip) and manganese peroxidase (MnP). Anthracophyllum discolor (Sp4), produced LiP and MnP at 90.3 and MnP 125.5 U L⁻¹ respectively, compared to the control fungus Phanerochaete chrysosporium CECT-2798 that produced 58.1 and 118.4 U L⁻¹ of LiP and MnP. Tolerance test showed that native Chilean fungi did not present high tolerance to 2,4,6-TCF and PCP but were quite tolerant to 25 and 50 mg L⁻¹ of 2,4-DCF. However, pre-acclimatization in 2,4-DCP notably improved the growth in medium with 2,4,6-TCP and PCP. PCP in liquid medium was efficiently degraded by the fungi Anthracophyllum discolor, Lenzites betulina (Ru-30) and Galerina patagónica (Sp3), and the major MnP activity was produced by A. discolor (Sp4) (67 U L⁻¹).     

Concentration of fungal ligninolytic enzymes by ultrafiltration and their use in distillery effluent decolorization

Extracellular ligninolytic enzymes secreted by seven different fungi grown under solid-state fermentation using agro-residue as substrate were extracted through successive extractions. In general, most of the enzymes were recovered during first and second extractions. These extractants were then subjected to ultrafiltration using a 10 kDa membrane for further concentration. The permeates collected after every filtration and the final retentate were analyzed for protein and enzymes. In all the isolates, enzyme concentration was maximum in first retentate, which reduced significantly in second, third, and fourth retentate. Maximum per unit laccase (14.44 U g⁻¹) and MnP production (142.2 U g⁻¹) was observed in Fusarium verticillioides TERIDB16 while maximum LiP production (137.42 U g⁻¹) was in Alternaria gaisen TERIDB6. The retentate was further used for checking its decolorization efficiency of undiluted distillery effluent. The maximum decolorization (37%) was obtained using the enzyme extract of Pleurotus florida EM1303.     

Construction,expression and characterization of fusion enzyme from <Emphasis Type="Italic">Arthrobacter oxydans</Emphasis> dextranase and <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> amylase

An artificial fusion protein of Arthrobacter oxydans dextranase and Klebsiella pneumoniae α-amylase was constructed and expressed in Escherichia coli. Most of the expressed protein existed as an insoluble fraction, which was solubilized with urea. The purified fusion enzyme electrophoretically migrated as a single protein band; M = 137 kDa, and exhibited activities of both dextranase (10.8 U mg⁻¹) and amylase (7.1 U mg⁻¹), which were lower than that of reference dextranase (13.3 U mg⁻¹) and α-amylase (103 U mg⁻¹). The fusion enzyme displayed bifunctional enzyme activity at pH 5–7 at 37°C. These attributes potentially make the fusion enzyme more convenient for use in sugar processing than a two-enzyme system.     

Production and optimization of thermophilic alkaline protease in solid-state fermentation by <Emphasis Type="Italic">Streptomyces</Emphasis> sp. CN902

The purpose of the present research is to study the production of thermophilic alkaline protease by a local isolate, Streptomyces sp. CN902, under solid state fermentation (SSF). Optimum SSF parameters for enzyme production have been determined. Various locally available agro-industrial residues have been screened individually or as mixtures for alkaline protease production in SSF. The combination of wheat bran (WB) with chopped date stones (CDS) (5:5) proved to be an efficient mixture for protease production as it gave the highest enzyme activity (90.50 U g⁻¹) when compared to individual WB (74.50 U g⁻¹) or CDS (69.50 U g⁻¹) substrates. This mixed solid substrate was used for the production of protease from Streptomyces sp. CN902 under SSF. Maximal protease production (220.50 U g⁻¹) was obtained with an initial moisture content of 60%, an inoculum level of 1 × 10⁸ (spore g⁻¹ substrate) when incubated at 45°C for 5 days. Supplementation of WB and CDS mixtures with yeast extract as a nitrogen source further increased protease production to 245.50 U g⁻¹ under SSF. Our data demonstrated the usefulness of solid-state fermentation in the production of alkaline protease using WB and CDS mixtures as substrate. Moreover, this approach offered significant benefits due to abundant agro-industrial substrate availability and cheaper cost.     

Production and activities of chitinases and hydrophobins from Lecanicillium lecanii

The production of chitinases and hydrophobins from Lecanicillium lecanii was influenced by the cultivation method and type of carbon source. Crude enzyme obtained from solid-substrate culture presented activities of exochitinases (32 and 51 kDa), endochitinases (26 kDa), β-N-acetylhexosaminidases (61, 80, 96 and 111 kDa). Additionally, submerged cultures produced exochitinases (32 and 45 kDa), endochitinases (10 and 26 kDa) and β-N-acetylhexosaminidases (61, 96 and 111 kDa). β-N-acetylhexosaminidases activity determined in solid-substrate culture with added chitin was ca. threefold (7.58 ± 0.57 U mg⁻¹) higher than submerged culture (2.73 + 0.57 U mg⁻¹). Similarly, hydrophobins displayed higher activities in solid-substrate culture (627.3 ± 2 μg protein mL⁻¹) than the submerged one (57.4 ± 4.7 μg protein mL⁻¹). Molecular weight of hydrophobins produced in solid-substrate culture was 7.6 kDa and they displayed surface activity on Teflon.     

Mineralisation of 14C-labelled synthetic lignin and ligninolytic enzyme activities of litter-decomposing basidiomycetous fungi

Within a screening program, 27 soil litter-decomposing basidiomycetes were tested for ligninolytic enzyme activities using agar-media containing 2,2′-azinobis(3-ethylbenzthiazoline-6-sulphonate), a humic acid or Mn²⁺ ions as indicator substrates. Most active species were found within the family Strophariaceae (Agrocybe praecox, Stropharia coronilla, S. rugosoannulata) and used for mineralisation experiments with a ¹⁴C-ring-labelled synthetic lignin (¹⁴C-DHP). The fungi mineralised around 25% of the lignin to ¹⁴CO₂ within 12 weeks of incubation in a straw environment; about 20% of the lignin was converted to water-soluble fragments. Mn-peroxidase was found to be the predominant ligninolytic enzyme of all three fungi in liquid culture and its production was strongly enhanced in the presence of Mn²⁺ ions. The results of this study demonstrate that certain ubiquitous litter-decomposing basidiomycetes possess ligninolytic activities similar to the wood-decaying white-rot fungi, the most efficient lignin degraders in nature. Received: 20 April 2000 / Received revision: 12 July 2000 / Accepted: 16 July 2000     

Cellulase production by Aspergillus niger in biofilm, solid-state, and submerged fermentations

Cellulase production by Aspergillus niger was compared in three different culture systems: biofilm, solid-state, and submerged fermentation. Biofilm and solid-state fermentations were carried out on perlite as inert support, and lactose was used as a carbon source in the three culture systems. In cryo-scanning electron microscopy, biofilm and solid-state cultures gave similar morphological patterns and confirmed that both spore first attachment and hyphal adhered growth are helped by the production of an adhesive extracellular matrix. Biofilm cultures produced higher cellulase activities than those in submerged and solid-state cultures (1,768, 1,165, and 1,174 U l⁻¹, respectively). Although biofilm cultures grew less than the other cultures, they produced significantly higher cellulase yields (370, 212, and 217 U g⁻¹ lactose, respectively) and volumetric productivities (24, 16, and 16 U l⁻¹ h⁻¹, respectively). Likewise, endoglucanase and xylanase activities were higher in biofilm cultures. Under the conditions tested, it seems that fungal attached growth on perlite may favor better enzyme production. Biofilms are efficient systems for cellulase production and may replace solid-state fermentation. Biofilm fermentation holds promise for further optimization and development. The results of this work reveal that fungal biofilms may be used for the commercial production of cellulase employing the technology developed for submerged fermentation at high cell densities.     

Enhanced fructooligosaccharides and inulinase production by a <Emphasis Type="Italic">Xanthomonas campestris</Emphasis> pv. <Emphasis Type="Italic">phaseoli</Emphasis> KM 24 mutant

Xanthomonas campestris pv phaseoli produced an extracellular endoinulinase (9.24 ± 0.03 U mL⁻¹) in an optimized medium comprising of 3% sucrose and 2.5% tryptone. X. campestris pv. phaseoli was further subjected to ethylmethanesulfonate mutagenesis and the resulting mutant, X. campestris pv. phaseoli KM 24 demonstrated inulinase production of 22.09 ± 0.03 U mL⁻¹ after 18 h, which was 2.4-fold higher than that of the wild type. Inulinase production by this mutant was scaled up using sucrose as a carbon source in a 5-L fermenter yielding maximum volumetric (21,865 U L⁻¹ h⁻¹) and specific (119,025 U g⁻¹ h⁻¹) productivities of inulinase after 18 h with an inulinase/invertase ratio of 2.6. A maximum FOS production of 11.9 g L⁻¹ h⁻¹ and specific productivity of 72 g g⁻¹ h⁻¹ FOS from inulin were observed in a fermenter, when the mutant was grown on medium containing 3% inulin and 2.5% tryptone. The detection of mono- and oligosaccharides in inulin hydrolysates by TLC analysis indicated the presence of an endoinulinase. This mutant has potential for large-scale production of inulinase and fructooligosaccharides.     

The morphological and physiological evolution of <Emphasis Type="Italic">Aspergillus terreus</Emphasis> mycelium in the submerged culture and its relation to the formation of secondary metabolites

The influence of the morphology and differentiation of Aspergillus terreus hyphae on the formation of mevinolinic acid (lovastatin) and (+)-geodin was tested. Lovastatin titre was the highest (above 60 mg l⁻¹) in the system with smaller pellets (diameter below 1.5 mm) and high biomass concentration (above 10 g l⁻¹ in the idiophase). These biomass features were induced by the higher initial number of spores in the preculture (above 2 × 10¹⁰ l⁻¹). At the initial number of spores below 2 × 10⁹ l⁻¹ (+)-geodin biosynthesis was the most efficient but it was rather connected with the elevated C/N ratio than with the pellet size. In order to quantify the hyphal differentiation in fungal pellets a special approach was used. The sectioning of the stained pellets together with the image analysis and calculation procedures were applied. The analysis of hyphal differentiation indicated that lovastatin formation was correlated with the fraction of the active, growing hyphae.     

Effect of mycelial morphology on ergothioneine production during liquid fermentation of Lentinula edodes

The morphological type of a microorganism generally influences its metabolite production. In the present study, we investigated the effects of the mycelial morphology of shiitake (Lentinula edodes) on the production of 2-mercaptohistidine trimethylbetaine (ergothioneine, ESH) during liquid fermentation. Analyses of the distribution of ESH in mycelial cells of different morphological types revealed that the ESH content of pellets obtained from the liquid fermentation media was much greater than the content in the free mycelia and clumps. The concentration of ESH in pellets on day 15 of liquid fermentation reached 0.79 mg/g dry weight (DW), which is approximately three times the concentration found in mycelia clumps (0.28 mg/g DW) and free mycelia (0.31 mg/g DW). Macroscopic image analysis of the development and morphological changes of the pellets during a liquid fermentation period of up to 25 days indicated that pellet growth showed a highly positive correlation with the increase in ESH concentration (r ² = 0.9851). A reduced agitation rate of 50 rpm for the culture medium was suitable for pellet formation and size enlargement. The results obtained in this work would be helpful in guiding the intentional manipulation of the distribution and enrichment of ESH in L. edodes through changes in liquid fermentation conditions.     

Evaluation of inert and organic carriers for <Emphasis Type="Italic">Verticillium lecanii</Emphasis> spore production in solid-state fermentation

Growth and sporulation of Verticillium lecanii on inert and organic carriers (sugar-cane bagasse, corncob, rice straw, polyurethane foam and activated carbon) in a solid-state fermentation process was studied. Sugar-cane bagasse and polyurethane foam produced 10¹⁰ spores g⁻¹ dry carrier whereas corncob, rice straw, and activated carbon yielded, respectively 8 × 10⁹, 4 × 10⁹, and 3 × 10⁸ spores g⁻¹. Chitinase activity of the conidia was in the following order: sugar-cane bagasse (3.3 U mg⁻¹) > wheat bran (3.0 U mg⁻¹) > polyurethane foam (2.7 U mg⁻¹). There was no significant difference (2.5–2.7 U mg⁻¹) in the proteinase activity among the conidia from the three cultures. Scanning electron microscopy shows that aerial mycelium freely penetrated into the internal area of polyurethane foam. Sugar-cane bagasse provided enough area for vegetative hyphae to attach. Of the carriers analyzed, polyurethane foams and sugar-cane bagasse were the best carriers for V. lecanii growth and spore production.