Kinetic studies of laccase enzyme of Coriolus versicolor MTCC 138 in an inexpensive culture medium

An attempt was made to use cyanobacterial biomass of water bloom, groundnut shell (GNS) and dye effluent as culture medium for laccase enzyme production by Coriolus versicolor. Laccase production was found to be 10.15 ± 2.21 U/ml in the medium containing groundnut shell and cyanobacterial bloom in a ratio of 9:1 (dry weight basis) in submerged fermentation at initial pH 5.0 and 28 ± 2 °C temperature. Half life of enzyme was found to be 74 min at 60 °C. Kinetic analysis of laccase when made with substrate ABTS, K_m and V_max were found to be 0.29 mM and 9.49 μmol/min respectively. Azide and hydroxylamine were found to exert significant inhibition on thermostable laccase. Inhibitor constant (k_i) for azide and hydroxylamine were 1.33 and 0.18 mM respectively. This study forms the first report on the potential application of waste water cyanobacterial bloom and dyeing effluent as a medium for laccase production by C. versicolor MTCC138.     

Optimization of biomass production with enhanced glucan and dietary fibres content by Pleurotus ostreatus ATHUM 4438 under submerged culture

This work was aimed at optimizing biomass production by the edible basidiomycete Pleurotus ostreatus ATHUM 4438 in a submerged process with enhanced glucan and dietary fibres content. β-Glucan from Pleurotus sp. (pleuran) has been used as food supplements due to its immunosuppressive activity. Like other dietary fibre components, oyster mushroom polysaccharides can stimulate the growth of colon microorganisms (probiotics), i.e. act as prebiotics. We used the FF MicroPlate for substrate utilization and growth monitoring. The pattern of substrate catabolism forms a substrate assimilation fingerprint which is useful in selecting media components for media optimization of maximum biomass production. Different carbon sources (95) were used and then 8 of them were tested in shake flask cultures. The effect of various organic and complex nitrogen sources on biomass production was also examined and response surface methodology based on central composite design was applied to explore the optimal medium composition. When the optimized culture medium was tested in a 20-L stirred tank bioreactor, using 57 g L⁻¹ xylose and 37 g L⁻¹ corn steep liquor, high yields (39.2 g L⁻¹) of dry biomass was obtained. The yield coefficients for total glucan and dietary fibres on mycelial biomass formed were 140 ± 4 and 625 ± 9 mg g⁻¹ mycelium dry weight, respectively.     

Overproduction of Trametes versicolor laccase by making glucose starvation using yeast

In the present paper, overproduction of laccase by microbe interaction was studied. When Trametes versicolor was co-cultured with Candida sp. HSD07A in submerged fermentation, laccase activity could be improved significantly and reached 10500 ± 160 U/l, 11.8 times more than that of the contrast group. Fermentation tests of the yeast indicated that it could produce amylase and cellulase, but couldn’t excrete laccase and the overproductive laccase was produced by T. versicolor; the interaction mechanism between T. versicolor and Candida sp. HSD07A was investigated and the results showed that amylase and cellulose could hydrolyze cell walls of T. versicolor; however, the degree of hydrolysis was at a very low level, could not lead to overproduction of laccase; glucose starvation state made by the yeast was the real reason why T. versicolor could overproduce laccase; moreover, this study also proved that making glucose starvation using the yeast was a novel and effective method.     

Cellulase production and oil palm empty fruit bunch saccharification by a new isolate of Trichoderma koningii D-64

A new strain Trichoderma koningii D-64 was isolated from Singapore soil samples. It produced cellulase, xylanase, and laccase on a variety of carbon sources. Enzyme activities of 3.8 ± 0.3, 40.3 ± 5.1, 6.6 ± 0.3 and 98.8 ± 10.3 U/mL were respectively obtained for FPase, CMCase, β-glucosidase and xylanase in a mixture of 1% cellulose and 2% wheat bran. About 70–95% saccharification efficiency of oil palm empty fruit bunch was obtained using T. koningii D-64 enzymes alone without the supplement of any other commercial enzymes. Strain T. koningii D-64 is therefore a potential cellulase producer for the efficient lignocellulosic biomass conversion to fermentable sugars.     

Bioconversion of lignocellulosic palm byproducts into enzymes and lipid by newly isolated oleaginous fungi

Direct conversion of palm pressed fiber (PPF) and palm empty fruit bunches (EFB) into enzymes and lipid by oleaginous fungi were performed through solid-state fermentation (SSF). Among the strains tested, TSIP9 converted PPF and EFB into lipid with the highest yield of 31.1 ± 1.7 mg/gram dry substrate (gds) and 37.5 ± 2.2 mg/gds, respectively. It also produced high activity of cellulolytic enzymes. It was identified as Aspergillus tubingensis. The similar fatty acids of its lipid to those of plant oil indicate its suitable use as biodiesel feedstock. The cellulase and xylanase production by this strain was improved when EFB was pretreated with alkaline. When alkaline-pretreated EFB was added with palm kernel cake (PK) as an alternative nitrogen source and the culture conditions were optimized through response surface methodology (RSM), the production of lipid, cellulase and xylanase were increased up to 88.5 ± 4.9 mg/gds, 26.1 ± 0.1 U/gds and 59.3 ± 0.3 U/gds, respectively. This study reveals the potential use of in situ cellulolytic enzymes producing fungi and the optimal conditions for direct conversion of lignocellulosic biomass into lipid.     

A novel membrane-surface liquid co-culture to improve the production of laccase from Ganoderma lucidum

In this work, a laccase producer, Ganoderma lucidum, was separated and identified according to its morphological characteristics and phylogenetic data. A 4000 U/l and 8500 U/l of laccase activity was obtained in 500 ml flask by submerged culture and biomembrane-surface liquid culture (BSLC), respectively. Furthermore, the novel biomembrane-surface liquid co-culture (BSLCc) was developed by adding Saccharomyces cerevisiae to reactor in order to shorten the fermentation period and improve laccase production. Laccase activity obtained by BSLCc, 23 000 U/l, is 5.8 and 2.7 times of that obtained by submerged culture and BSLC, respectively. In addition, laccase production by BSLCc was successfully scaled-up to 100 l reactor, and 38 000 U/l of laccase activity was obtained on day 8. The mechanism of overproducing laccase by BSLCc was investigated by metabolism pathway analysis of glucose. The results show glucose limitation in fermentation broth induces the secretion of laccase. The addition of S. cerevisiae, on one hand, leads to an earlier occurrence of glucose limitation state, and thus shortens the fermentation time; on the other hand, it also results in the appearance of a series of metabolites of the yeast including organic acids, ethanol, glycerol and so forth in fermentation broth, and both polyacrylamide gel electrophoresis analysis and enzyme activity detection of laccase show that these metabolites contribute to the improvement of laccase activity.     

Use of Saccharum spontaneum (wild sugarcane) as biomaterial for cell immobilization and modulated ethanol production by thermotolerant Saccharomyces cerevisiae VS3

Saccharum spontaneum is a wasteland weed consists of 45.10 ± 0.35% cellulose and 22.75 ± 0.28% of hemicellulose on dry solid (DS) basis. Aqueous ammonia delignified S. spontaneum yielded total reducing sugars, 53.91 ± 0.44 g/L (539.10 ± 0.55 mg/g of substrate) with a hydrolytic efficiency of 77.85 ± 0.45%. The enzymes required for hydrolysis were prepared from culture supernatants of Aspergillus oryzae MTCC 1846. A maximum of 0.85 ± 0.07 IU/mL of filter paperase (FPase), 1.25 ± 0.04 IU/mL of carboxy methyl cellulase (CMCase) and 55.56 ± 0.52 IU/mL of xylanase activity was obtained after 7 days of incubation at 28 ± 0.5 °C using delignified S. spontaneum as carbon source under submerged fermentation conditions. Enzymatic hydrolysate of S. spontaneum was then tested for ethanol production under batch and repeated batch production system using “in-situ” entrapped Saccharomyces cerevisiae VS₃ cells in S. spontaneum stalks (1 cm × 1 cm) size. Immobilization was confirmed by the scanning electron microscopy (SEM). Batch fermentation of VS₃ free cells and immobilized cells showed ethanol production, 19.45 ± 0.55 g/L (yield, 0.410 ± 0.010 g/g) and 21.66 ± 0.62 g/L (yield, 0.434 ± 0.021 g/g), respectively. Immobilized VS₃ cells showed maximum ethanol production (22.85 ± 0.44 g/L, yield, 0.45 ± 0.04 g/g) up to 8th cycle during repeated batch fermentation followed by a gradual reduction in subsequent cycles of fermentation.     

Inulinase overproduction by a mutant of the marine yeast Pichia guilliermondii using surface response methodology and inulin hydrolysis

In this study, in order to isolate inulinase overproducers from the marine yeast Pichia guilliermondii, its cells were treated by using UV light and LiCl. The mutant M-30 with enhanced inulinase production was obtained and was found to be stable after cultivation for 20 generations. Response surface methodology (RSM) was used to optimize the medium compositions and cultivation conditions for inulinase production by the mutant M-30 in liquid fermentation. Inulin, yeast extract, NaCl, temperature, pH for maximum inulinase production by the mutant M-30 were found to be 20.0 g/l, 5.0 g/l, 20.0 g/l, 28 °C and 6.5, respectively. Under the optimized conditions, 127.7 U/ml of inulinase activity was reached in the liquid culture of the mutant M-30 whereas the predicted maximum inulinase activity of 129.8 U/ml was derived from RSM regression. Under the same conditions, its parent strain only produced 48.1 U/ml of inulinase activity. This is the highest inulinase activity produced by the yeast strains reported so far. We also found that inulin could be actively converted into monosaccharides by the crude inulinase.     

Novel biotransformation process of podophyllotoxin to produce podophyllic acid and picropodophyllotoxin by Pseudomonas aeruginosa CCTCC AB93066, Part II: Process optimization

This work optimized the novel biotransformation process of podophyllotoxin to produce podophyllic acid by Pseudomonas aeruginosa CCTCC AB93066. Firstly, the biotransformation process was significantly affected by medium composition. 5 g/l of yeast extract and 5 g/l of peptone were favorable for podophyllic acid production (i.e. 25.3 ± 3.7 mg/l), while not beneficial for the cell growth of P. aeruginosa. This indicated that the accumulation of podophyllic acid was not corresponded well to the cell growth of P. aeruginosa. 0 g/l of sucrose was beneficial for podophyllic acid production (i.e. 34.3 ± 3.9 mg/l), which led to high podophyllotoxin conversion (i.e. 98.2 ± 0.1%). 1 g/l of NaCl was the best for podophyllic acid production (i.e. 47.6 ± 4.0 mg/l). Secondly, the production of podophyllic acid was significantly enhanced by fed-batch biotransformation. When each 100 mg/l of podophyllotoxin was added to the biotransformation system after 4, 10 and 25 h of culture, respectively, podophyllic acid concentration reached 99.9 ± 12.3 mg/l, enhanced by 284% comparing to one-time addition (i.e. 26.0 ± 2.1 mg/l). The fundamental information obtained in this study provides a simple and efficient way to produce podophyllic acid.     

Antioxidative,antimicrobial and cytotoxic effects of the phenolics of Leea indica leaf extract

This study investigated the phytochemical, antioxidative, antimicrobial and cytotoxic effects of Leea indica leaf ethanol extract. Phytochemical values namely total phenolic and flavonoid contents, total antioxidant capacity, DPPH radical scavenging effect, FeCl₃ reducing power, DMSO superoxide scavenging effect and Iron chelating effects were studied by established methods. Antibacterial, antifungal and cytotoxic effects were screened by disk diffusion technique, food poison technique and brine shrimp bioassay, respectively. Results showed the total phenolic content 24.00 ± 0.81 g GAE/100 g, total flavonoid content 194.68 ± 2.43 g quercetin/100 g and total antioxidant capacity 106.61 ± 1.84 g AA/100 g dry extract. Significant (P < 0.05) IC₅₀ values compared to respective standards were recorded in DPPH radical scavenging (139.83 ± 1.40 μg/ml), FeCl₃ reduction (16.48 ± 0.64 μg/ml), DMSO superoxide scavenging (676.08 ± 5.80 μg/ml) and Iron chelating (519.33 ± 16.96 μg/ml) methods. In antibacterial screening, the extract showed significant (P < 0.05) zone of inhibitions compared to positive controls Ampicillin and Tetracycline against Gram positive Bacillus subtilis, Bacillus cereus, Bacillus megaterium, and Staphylococcus aureus and Gram negative Salmonella typhi, Salmonella paratyphi, Pseudomonas aeroginosa, Shigella dysenteriae, Vibrio cholerae, and Escherichia coli. Significant minimum inhibitory concentrations compared to tetracycline were obtained against the above organisms. In antifungal assay, the extract inhibited the growth of Aspergillus flavus, Candida albicans and Fusarium equisetii by 38.09 ± 0.59, 22.58 ± 2.22, and 22.58 ± 2.22%, respectively. The extract showed a significant LC₅₀ value compared to vincristine sulfate in cytotoxic assay. The results evidenced the potential antioxidative, antimicrobial and cytotoxic capacities of Leea inidica leaf extract to be processed for pharmaceutical use.     

Enhanced production of insect raw-starch-digesting alpha-amylase accompanied by high erythritol synthesis in recombinant Yarrowia lipolytica fed-batch cultures at high-cell-densities

Recently we reported on raw-starch-digesting ability of alpha-amylase from an insect Sitophilus oryzae (SoAMY) expressed in recombinant Yarrowia lipolytica cells, and demonstrated its usefulness in simultaneous saccharification and fermentation processes with industrial yeasts. In this study we applied fed-batch cultures of Y. lipolytica 4.29 strain reaching high-cell-densities (up to 70 [g_DCW/L]), to enhance SoAMY production. SoAMY activity in the medium reached the peak value of 22,979.23 ± 184 [AU/L], at volumetric productivity of 121.58 ± 1.75 [AU/L/h], and yield of 71.83 ± 3.08 [AU/g_glycerol], constituting roughly 160-fold improvement, compared to the best previous result. The cultivations were accompanied by high production of erythritol (83.58 [g/L]), at the marginal production of mannitol (5.46 [g/L]). Elementary analyses of media constituents, the enzyme and the yeast biomass gave better insight into carbon and nitrogen fluxes distribution. Due to application of genetic engineering and bioprocess engineering strategies, the insect-derived enzyme can be produced at the quantities competitive to microbial catalysts.     

Enhanced ethanol and chitosan production from wheat straw by Mucor indicus with minimal nutrient consumption

Recently, Mucor indicus was introduced as a promising ethanol producing microorganism for fermentation of lignocellulosic hydrolysates, showing a number of advantages over Saccharomyces cerevisiae. However, high nutrient requirement is the main drawback of the fungus in efficient ethanol production from lignocelluloses. In this study, application of fungal extract as a potential nutrient source replacing all required nutrients in fermentation of wheat straw by M. indicus was investigated. Wheat straw was pretreated with N-methylmorpholine-N-oxide (NMMO) at 120 °C for 1–5 h prior to enzymatic hydrolysis. Hydrolysis yield was improved at least by 6-fold for 3 h pretreated straw compared with that of untreated one. A fungal extract was produced by autolysis of M. indicus biomass, an unavoidable byproduct of fermentation. Maximum free amino nitrogen (2.04 g/L), phosphorus (1.50 g/L), and total nitrogen (4.47 g/L) as well as potassium, magnesium, and calcium in the fungal extract were obtained by autolysis of the biomass at 50 °C and pH 5.0. The fungal extract as a nutrient-rich supplement substituted yeast extract and all other required minerals in fermentation and enhanced the ethanol yield up to 92.1% of the theoretical yield. Besides, appreciate amounts of chitosan were produced as another valuable product of the autolysis.     

Characterization and kinetic properties of the purified Trematosphaeria mangrovei laccase enzyme

The properties of Trematosphaeria mangrovei laccase enzyme purified on Sephadex G-100 column were investigated. SDS–PAGE of the purified laccase enzyme showed a single band at 48 kDa. The pure laccase reached its maximal activity at temperature 65 °C, pH 4.0 with K_m equal 1.4 mM and V_max equal 184.84 U/mg protein. The substrate specificity of the purified laccase was greatly influenced by the nature and position of the substituted groups in the phenolic ring. The pure laccase was tested with some metal ions and inhibitors, FeSO₄ completely inhibited laccase enzyme and also highly affected by (NaN₃) at a concentration of 1 mM. Amino acid composition of the pure enzyme was also determined. Carbohydrate content of purified laccase enzyme was 23% of the enzyme sample. The UV absorption spectra of the purified laccase enzyme showed a single peak at 260–280 nm.     

Leaf production and shoot dynamics of Thalassia testudinum by a direct census method

The annual leaf growth and shoot dynamics of Thalassia testudinum were examined in a meadow located near Havana City, Cuba, using direct censuses between January 1995 and January 1996. The net rate of shoot population change, specific shoot recruitment and mortality rates were calculated as the difference between the densities of shoots (tagged or untagged) in consecutive sampling events. The leaf biomass, the daily production, the turnover rate and the rate of leaf biomass loss were also estimated. The estimated mean dry leaf biomass (124.9 ± 9.5 g m⁻²), daily dry leaf production (3.3 ± 0.2 g m⁻² day⁻¹) and turnover rate (2.7 ± 0.1% day⁻¹) were comparable to values previously reported for this species in Cuba and elsewhere. The production of leaves and shoots were higher in spring, declined towards mid summer, and showed the minimum values in January. Shoot recruitment prevailed over shoot mortality from January to March and from July to August, whereas most of the annual shoot mortality occurred between May and July and between August and October. The meadow examined was in close demographic balance along the study period. The results demonstrate that direct census provides reliable estimates of rapid shoot dynamics in T. testudinum.     

High efficiency bioethanol production from barley straw using a continuous pretreatment reactor

We developed a new pretreatment process for producing high-efficiency bioethanol from a lignocellulosic biomass. Barley straw was pretreated with sodium hydroxide in a twin-screw extruder for continuous pretreatment. The biomass to ethanol ratio (BTER) for optimal pretreatment conditions was evaluated by response surface methodology. Simultaneous saccharification and fermentation (SSF) was conducted to investigate the BTER with 30 FPU/g cellulose of enzyme and 7% (v/v) yeast (Saccharomyces cerevisiae CHY 1011) using 10% (w/v) pretreated biomass under various pretreatment conditions. The maximum BTER was 73.00% under optimal pretreatment conditions (86.61 °C, 0.58 M, and 84.79 mL/min for temperature, sodium hydroxide concentration, and solution flow rate, respectively) and the experimental BTER was 70.01 ± 0.59%. SSF was performed to investigate the optimal enzyme and biomass dosage. As a result, maximum ethanol concentration and ethanol yield were 46.00 g/L and 77.36% at a loading pretreated biomass of 20% with 30 FPU/g cellulose of the enzyme dosage for barley straw to bioethanol. These results are a significant contribution to the production of bioethanol from barley straw.     

Chemistry,antioxidant and antimicrobial potential of nutmeg (Myristica fragrans Houtt)

Antioxidant and antimicrobial activities of nutmeg (Myristica fragrans Houtt) seed extracts were evaluated. Seeds were extracted with acetone, ethanol, methanol, butanol and water. All the extracts have shown significant antioxidant and antimicrobial activities against the tested microorganisms. Among all extracts, acetone extract has shown the highest antioxidant activity. The acetone extract showed 93.12 ± 1.48 mg gallic acid equivalents (GAE)/100 g dry weight total phenolic content, DPPH scavenging activity of 63.04 ± 1.56%, chelating activity of 64.11 ± 2.21% and 74.36 ± 1.94% inhibition of β-carotene bleaching, at 1 mg/mL extract concentration. Out of all extracts, acetone extract was able to exert antimicrobial activity against all tested bacteria and fungi. Acetone extract has shown the strongest antibacterial and antifungal activity with Staphylococcus aureus (13.8 ± 0.42 mm) and Aspergillus niger (14.4 ± 0.37 mm), respectively. GC–MS analysis of acetone extract has revealed the presence of 32 compounds of extract representing 99.49%. Sabinene (28.61%) has shown the highest occurrence in the extract. β-Pinene (10.26), α-pinene (9.72), myristicin (4.30%), isoeugenol (2.72%), p-cymene (1.81%), carvacrol (1.54%), eugenol (0.89%) and β-caryophellene (0.82%) were reported as possible contributor for antioxidant and antimicrobial activity of nutmeg.     

Enhancing GDP-fucose production in recombinant Escherichia coli by metabolic pathway engineering

Guanosine 5′-diphosphate (GDP)-fucose is the indispensible donor substrate for fucosyltransferase-catalyzed synthesis of fucose-containing biomolecules, which have been found involving in various biological functions. In this work, the salvage pathway for GDP-fucose biosynthesis from Bacterioides fragilis was introduced into Escherichia coli. Besides, the biosynthesis of guanosine 5′-triphosphate (GTP), an essential substrate for GDP-fucose biosynthesis, was enhanced via overexpression of enzymes involved in the salvage pathway of GTP biosynthesis. The production capacities of metabolically engineered strains bearing different combinations of recombinant enzymes were compared. The shake flask fermentation of the strain expressing Fkp, Gpt, Gmk and Ndk obtained the maximum GDP-fucose content of 4.6 ± 0.22 μmol/g (dry cell mass), which is 4.2 fold that of the strain only expressing Fkp. Through fed-batch fermentation, the GDP-fucose content further rose to 6.6 ± 0.14 μmol/g (dry cell mass). In addition to a better productivity than previous fermentation processes based on the de novo pathway for GDP-fucose biosynthesis, the established schemes in this work also have the advantage to be a potential avenue to GDP-fucose analogs encompassing chemical modification on the fucose residue.     

Direct ethanol production from N-acetylglucosamine and chitin substrates by Mucor species

Chitin, which is a polymer of β-(1–4) linked N-acetyl-d-glucosamine (GlcNAc) residues, is one of the most abundant renewable resources in nature, after cellulose. In this study, we found some native Mucor strains, which can use GlcNAc and chitin substrates as carbon sources for growth and ethanol production. One of these strains, M. circinelloides NBRC 6746 produced 18.6 ± 0.6 g/l of ethanol from 50 g/l of GlcNAc after 72 h and the maximum ethanol production rate was 0.75 ± 0.1 g/l/h. Furthermore, M. circinelloides NBRC 4572 produced 6.00 ± 0.22 and 0.46 ± 0.04 g/l of ethanol from 50 g/l of colloidal chitin and chitin powder after 16 and 12 days, respectively. We also found an extracellular chitinolytic enzyme producing strain M. ambiguus NBRC 8092, and successfully improved ethanol productivity of NBRC 4572 from colloidal chitin using crude chitinolytic enzyme derived from NBRC 8092. The ethanol titer reached 9.44 ± 0.10 g/l after 16 days. These results were the first bioethanol production from GlcNAc and chitin substrates by native organisms, and also suggest that these Mucor strains have great potential for the simultaneous saccharification and fermentation (SSF) of chitin biomass.     

Cloning,expression, and characterization of a thermostable and pH-stable laccase from Klebsiella pneumoniae and its application to dye decolorization

A thermostable and pH-stable laccase from Klebsiella pneumoniae was cloned and expressed in Escherichia coli. The recombinant laccase (rLac) achieved a specific activity of 7.12 U/mg after purification by Ni-affinity chromatography. Optimal enzyme activity was observed at pH 4.0 and 35 °C for 2,2′-azino-bis (3-ethylbenzthiazoline sulfonic acid) (ABTS) oxidization and pH 8.0 and 70 °C for 2,6-dimethoxyphenol (2,6-DMP) oxidization. Thermostability and pH stability studies showed that the rLac was stable over the range of 30–70 °C and pH 5.0–9.0 using 2,6-DMP as substrate. Circular dichroism analysis suggested that the secondary structure of the rLac mainly consisted of α-helix that played a vital role in maintaining laccase activity and revealed the potential mechanisms for the changes in laccase activity under varying pHs (3.0–11.0) and temperatures (20–90 °C). Finally, the rLac could decolorize the tested dyes with high decolorization efficiency.     

Do spawn storage conditions influence the colonization capacity of a wheat-straw-based substrate by Agaricus subrufescens?

Storage conditions of the spawn of edible fungi are of major importance to facilitate the production of mushrooms. Here, standard storage conditions at 10 °C or 15 °C were used and the potential of colonization of standard European compost by the tropical species Agaricus subrufescens was assessed during the spawn running phase. Two lignocellulolytic activities, laccase and CMC-cellulase, were enhanced after storage compared to control as well as substrate transformation, as described by the aromaticity ratio and a humification ratio calculated from NMR data. This result indicates that mycelium growth probably occurred during storage at 10 or 15 °C, leading to a larger amount of biomass in the inoculum. Moreover, the microbial functional diversity of the substrate was favored, showing that the electivity of the substrate was maintained. Thus, these findings indicate that recommendations for the mushroom producers can be established for A. subrufescens cultivation under European standard conditions.