Ligninolytic ability and potential biotechnology applications of the South American FungusPleurotus laciniatocrenatus

The extracellular ligninolytic enzyme system of Pleurotus laciniatocrenatus, grown under different culture conditions, was characterized and the ability of this strain to degrade different components of Eucalyptus globulus wood was determined. In shaken liquid cultures grown on a C-limited medium supplemented with yeast extract (0.1%) and peptone (0.5%), the fungus produced extracellular aryl-alcohol oxidase (Aao), laccase (Lac), manganese-dependent peroxidase (MnP) and manganese-independent peroxidase (MiP) activities, their maximum levels being, respectively, about 600, 50, 1360, and 920 pkat/mL. The supplementation of 1 mmol/L vanillic acid and 150 micromol/L CuSO4 produced an increase of Lac activity levels up to 4-fold and 68.3-fold, respectively. No significant differences were found in the levels of the other ligninolytic enzyme activities when compared to the basal medium. Solid-state fermentation cultures on E. globulus wood chips revealed Lac and MiP activities. These cultures showed degradative activity on lignin and lipophilic wood extractives.     

Embryogenic suspensions of adult cork oak: the first step towards mass propagation

Protocols have been established to clone adult cork oak trees by somatic embryogenesis using semisolid medium. However, for economically viable mass propagation, embryogenic cultures in liquid medium need to be developed. In this study, suspension cultures were initiated from embryo clusters obtained by secondary embryogenesis on a gelled medium lacking plant growth regulators. After 6 days of culture, these embryo clusters generated high cell density suspensions that also contained small organized structures (embryos and embryogenic clumps). As the culture duration increased, tissue necrosis and fewer embryogenic structures were observed and the establishment of suspension cultures failed. An alternative method was found adequate for initiation of embryogenic suspensions: embryo clusters from gelled medium were briefly shaken in liquid medium and detached cells and embryogenic masses of 41–800 μm were used as inoculum. Maintenance of embryogenic suspensions was achieved using a low-density inoculum (43 mg l^?1) by subculturing four embryogenic clumps of 0.8–1.2 mm per 70 ml of medium. Proliferation ability was maintained for almost 1 year through ten consecutive subcultures. The initiation and maintenance protocols first developed for a single genotype were effective when tested on 11 cork oak genotypes.     

Forest farming of shiitake mushrooms: An integrated evaluation of management practices

Two outdoor shiitake (Lentinula edodes) cultivation experiments, established in Missouri USA in 1999 and 2000, produced mushrooms in 2000–2005. We examined shiitake production in response to substrate species, inoculum form, inoculum strain, and inoculation timing, using total mushroom weight per log as the primary response variable with log characteristics as covariates. The significantly greater mushroom weight produced by sugar maple logs compared with white or northern red oak was attributable to the higher proportion of undiscolored wood volume in the maple logs, rather than to bark thickness or log diameter. The “wide temperature range” shiitake strain produced significantly greater yield compared with the “warm” or “cold” weather strains. Both the wide-range and warm-weather strains were stimulated to fruit by significant rain events, while the cold-weather strain was responsive to temperature. Inoculation with sawdust spawn gave significantly greater yield than colonized wooden dowels or pre-packaged “thimble” plug inoculum. The second and third full years following inoculation were the most productive.     

Growth of Pleurotus ostreatus on wheat straw and wheat-grain-based media: biochemical aspects and preparation of mushroom inoculum

Mycelial growth, intracellular activity of proteases, laccases and β-1,3-glucanases, and cytoplasmic protein were evaluated in the vegetative phase of Pleurotus ostreatus grown on wheat straw and in wheat-grain-based media in Petri dishes and in bottles. The productivity of the wheat straw and wheat-grain-based spawn in cylindrical polyethylene bags containing 5 kg of chopped straw was also determined. We observed high activity of proteases and high content of intracellular protein in cultures grown on wheat straw. This suggests that the proteases are not secreted into the medium and that the protein is an important cellular reserve. On the contrary, cultures grown on wheat straw secreted laccases into the medium, which could be induced by this substrate. P. ostreatus grown on media prepared with a combination of wheat straw and wheat grain showed a high radial growth rate in Petri dishes and a high level of mycelial growth in bottles. The productivities of wheat straw and wheat-grain-based spawn were similar. Our results show that cheaper and more productive mushroom spawn can be prepared by developing the mycelium on wheat straw and wheat-grain-based substrates.     

Enzymes produced by <Emphasis Type="Italic">Ganoderma australe</Emphasis> growing on wood and in submerged cultures

Enzymes produced by Ganoderma australe in solid-state fermentation and submerged cultures were evaluated. Strain A464 produced laccase activity in liquid medium and in solid-state cultures containing Drimys winteri or Eucalyptus globulus wood chips, while MnP and LiP activities were not detected. On the other hand, strain A272 cultured for 75 days on E. globulus presented MnP activity of 719 IU/kg of wood. The suitability of D. winteri wood as a substrate enabling MnP production was checked with a well-documented MnP-producing basidiomycete, Ceriporiopsis subvermispora, which produced MnP activity of 327 IU/kg of wood in 9-day-old cultures. Data from two different G. australe strains (A272 and A464) indicated that MnP secretion depended on strain origin as well as on culture conditions.     

Variability in maturation and germination from white spruce somatic embryos,as affected by age and use of solid or liquid culture

Summary The yield of morphologically normal Stage 3 somatic embryos of white spruce [Picea glauca (Moench) Voss], and subsequent germinability, was affected by culture age and use of solid and/or liquid culture growth conditions. Of the conditions that were compared, best results were obtained with cultures up to 3 yr old that had been continuously grown in liquid medium. Such material yielded up to 374 morphologically normal Stage 3 embryos per g f. wt. inoculum, when routinely pretreated using a 1 wk 2,4-dichlorophenoxyacetic acid-free period before maturation. By comparison the continual use of solid culture conditions resulted in lower yields (5/g f. wt. inoculum), and the use of solid medium in combination with liquid medium showed a greater affect of age on the production of normal Stage 3 embryos (348/g f. wt at 1.5 yr down to 19/g f. wt. at 3 yr) over the age range tested. In the absence of culture pretreatment, the oldest liquid cultures yielded only 44 normal Stage 3 embryos/g f. wt. inoculum, and the comparable solid to liquid cultures yielded 1.3/g f. wt. inoculum. The number of aberrant Stage 3 embryos in older cultures was reduced as a result of culture pretreatment; for example, in the oldest liquid cultures these represented 83% of the Stage 3 embryo population without pretreatment and 45% with pretreatment. Normal Stage 3 somatic embryo yield and germination characteristics (radicle and epicotyl development) were informative in distinguishing among the conditions studied. Germination characteristics were especially important when maturation responses were incapable of distinguishing among age classes. NRCC Contribution no. 38462.     

Study of a factor produced by suspension-cultured Pinus radiata cells which enhances cell growth at low inoculum densities

Pinus radiata cells in suspension culture abruptly lose their growth capacity when diluted below a critical inoculum density. This threshold density can be lowered by adding the supernatant (conditioned medium) from healthy cultures which have been grown separately at high densities. Fresh medium is conditioned rapidly indicating that the factor responsible is either potent or produced rapidly. Activity-response curves increase progressively with concentration indicating that still greater effect may be obtained if the factor can be concentrated following separation from other medium components. The effect is not mimicked by a number of candidate compounds (including auxins, cytokinins, polyamines and vitamins). Partial characterisation studies indicate that the factor is relatively small (<1 000 dalton) and possibly an oligosaccharide. It is considered that the factor is an essential structural component of the walls of expanding cells where it is reversibly-bound.Abbreviations 2,4-D dichlorophenoxyacetic acid - LIGF Low Inoculum Growth Factor - NAA naphthalene acetic acid     

Effect of cultural factors on cellulase activity and protein production by Aspergillus terreus

Protein production by Aspergillus terreus GN1 grown on 1.0% alkali-treated bagasse was studied under various cultural conditions. The maximum biomass protein content of 20.1% and protein recovery of 11.2% was obtained with an initial pH of 4.0, with 1/5 (v/v) inoculum in continuously shaken cultures grown for seven days. Protein content of the alkali-treated bagasse was 3.0%. Highest crude protein percent also corresponded with highest carboxymethyl cellulase and filter paper enzyme activities.     

Predicting vegetative inoculum performance to maximize phytase production in solid-state fermentation using response surface methodology

Microbial phytase is used to reduce the environmental loading of phosphorus from animal production facilities. The limiting factors in the use of this enzyme in animal feeds can be overcome by solid-state fermentation (SSF), which is a promising technology for commercial enzyme production with lower production costs. Inoculum quality and the influence of inoculum quality on phytase production are important factors which need in-depth investigation before scaling-up of high-yielding fermentation process. A full factorial experimental design for 240 h with sampling at every 24 h was used to determine the effects of the treatments, inoculum age (plate and liquid culture), media composition and the duration of SSF on the production of fungal biomass and phytase in SSF systems using Aspergillus niger. The optimal treatment combination for maximal phytase production was determined by statistically comparing all treatments at each sampling time. Both 7- and 14-day plate cultures and M1+ medium composition with 72-h-old liquid inoculum treatments resulted in optimal phytase production at 144 h of SSF, which was the shortest duration observed for maximal phytase production. This resulted in maximal phytase production with a mean of 884±121 U/g substrate, while the maximal phytase production observed at 216 h of SSF (mean phytase activity of 1008±121 U/g substrate), with the same treatment combinations, was not statistically significant from that at 144 h of SSF. Phytase production was strongly growth-associated with younger inocula. The significant treatment variables, age of liquid inoculum and the duration of SSF, were used to predict the system response for phytase production using response surface methodology. From the response surface model, the optimal response of the experiment was predicted and the reliability of the prediction was checked with the verification experiment. Journal of Industrial Microbiology & Biotechnology (2001) 26, 161–170. Received 06 June 2000/ Accepted in revised form 14 October 2000     

Plant regeneration from somatic embryos in black pepper

Embryogenic callus derived from zygotic embryos of black pepper (Piper nigrum Linn.) were induced to form somatic embryos on solid and liquid Schenk and Hildebrandt basal medium. Callus proliferation, somatic embryo-genesis and germination of embryos were achieved in about 8 months in static cultures while it took only 8 weeks in liquid suspension cultures. The highest number of embryos and plantlets was produced from cells grown as suspension cultures raised in half-strength medium without growth regulators and sucrose level reduced from 3% to 1.5%. Regenerated plants were established in soil.     

Immunogold-cytochemical labelling of β-glucosidase in the white-rot fungus Coriolus versicolor

Summary Immunogold cytochemical labelling of hyphal sections of Coriolus versicolor showed that -glucosidase was localised in the extracellular mucilage, cell wall layers and cell interior in hyphae grown on glucose-rich malt extract medium whereas in hyphae grown with carboxymethylcellulose (CMC) as sole carbon source, most labelling was in the cell wall layers and cell interior. Little mucilage was visible around hyphae from these cultures. Hyphae from beechwood cultures showed gold labelling of -glucosidase in mucilage and fungal cell walls with some intracellular labelling. Biochemical studies of enzyme activity showed that similar amounts of enzyme were detected in the growth medium when cultures were grown on CMC medium, in agitated liquid cultures or in stationary cultures. In agitated cultures grown on glucose-rich malt extract, the activity of -glucosidase in the medium was 100 times less than that detected in stationary cultures on the same medium. However activity in the hyphae of stationary CMC-grown cultures was similar to that in hyphae from stationary glucose-rich cultures. These data confirm the patterns of gold labelling observed in hyphae from stationary cultures on glucose-rich malt extract when -glucosidase was immobilised in the extracellular mucilage layer around the hyphae. In this paper we propose that a primary function of the extracellular mucilage produced by hyphae of C. versicolor in vivo is to serve as a matrix for immobilisation of -glucosidase. Its substrate, cellobiose, which is released as a result of endo-and exoglucanase hydrolysis of cellulose, is absorbed and retained by the gel filtration properties of the mucilage, so encountering the immobilised -glucosidase. Glucose produced by this reaction is retained within the mucilage matrix around the hyphae before intracellular absorption.Offprint requests to: C. S. Evans     

The induction of mycelial development inAureobasidium pullulans (IMI 45533) by yeast extract

Germ tube and subsequent mycelial development from yeast-like and swollen cells ofAureobasidium pullulans (IMI 45533) was induced by yeast extract in defined liquid medium. This morphogenetic transition was dependent on inoculum size; pH effects were not involved and once mycelial development was induced in the cells it continued even in the absence of yeast extract. The progeny of mycelium and future generations were unaffected by yeast extract. Cessation of germination was not due to any obvious medium changes but appeared to be partly due to the production of a germination inhibitor, which could also be produced by control cells grown in the absence of yeast extract.     

Enhanced Production of 2,3-Butanediol by Klebsiella pneumoniae Grown on High Sugar Concentrations in the Presence of Acetic Acid

The bioconversion of sugars present in wood hemicellulose to 2,3-butanediol (hereafter referred to as butanediol) by Klebsiella pneumoniae grown on high initial concentrations (up to 10%) of sugars was investigated. Initial fermentation studies with a chemically defined medium suggested that sugar levels in excess of 2% could not be utlized even when a higher inoculum size (5 to 10%) was used. The addition of nutrient supplements, viz., yeast extract, urea, ammonium sulfate, and trace elements resulted in a 10 to 50% increase in butanediol yields, although sugar utilization remained incomplete. The concentration of end products normally found at the termination of fermentation was shown to be noninhibitory to growth and substrate utilization. Acetic acid was inhibitory at concentrations above 1%, although growth and butanediol yield were stimulated in cultures supplemented with lower levels of acetic acid. The efficient utilization of 4% substrate concentrations of d-glucose and d-xylose was achieved, resulting in butanediol yields of 19.6 and 22.0 g/liter, respectively.     

Doublet cells inTetrahymena as indicators of culture media composition

Stomatogenesis in ciliates is a complex and carefully orchestrated event. The exo⁻ mutant SB255 ofTetrahymena thermophila has defects in mucocyst formation and docking and can also have one or two mouths. Three common culture media (proteose peptone, Medium 357, and yeast extract) were analyzed for total C, N, and inorganic elements and then tested for their effect on the number of mouths present in SB255. Cultures of SB255 grown in Medium 357 consisted of a mixed population of cells with either two mouths (doublet) or one mouth. Cultures from the same original stock grown in Medium 357 (SBm) and in 1% proteose peptone (SBpp) had different percentages of doublet cells in 1-, 2-, 3-, and 4-d-old cultures. When transferred to and grown in 1% yeast medium, both SBpp and SBm cultures had increased percentages of doublets over a 4-d culture period. When grown in 0.1, 0.5, or 1% yeast medium for 2 d, both SBpp and SBm cultures had more doublets in 1% than in either 0.1 or 0.5% yeast medium. Cultures of SBm grown in Medium 357. or 1% yeast medium for 2 d had a 10-fold increase in doublet cells compared to the inoculum. After 2 d in 1% proteose peptone, SBm cultures had percentages of doublet cells almost equal to that of the inoculum. Immunofluorescence and scanning electron microscopy (SEM) were used to examine cellular morphology of the doublet cells. These findings suggest that enriched media promote the growth of doublet cells. Furthermore, these doublets could prove to be a useful model system for the study of biological roles of trace elements.     

Pilot-scale production of mosquitocidal toxins by Bacillus thuringiensis and Lysinibacillus sphaericus under solid-state fermentation

Mosquitocidal toxins of Bacillus thuringiensis israelensis (Bti) and Lysinibacillus sphaericus 14N1 (Ls14N1) were produced under solid-state fermentation using agro-industrial wastes. Sugar beet pulp–sesame meal (1:1) and wheat germ meal–linen meal (1:1) at 9% were the efficient substrate mixtures for the growth and toxin production of Bti and Ls14N1, respectively. Bti was more active after the addition of beef extract (0.2%) or yeast extract (0.5%) to the medium. On the other hand, the addition of yeast extract (0.2%) or NYSM salts (2%) significantly enhanced the toxicity produced by Ls14N1. The optimum conditions for the maximum toxicity of Bti were at pH 7–8, 20–30% moisture, 4–10% inoculum and 7 days incubation. For Ls14N1, the best conditions were pH 6.5–7.5, 20–30% moisture, 4–10% inoculum and 5 days incubation. It was found that the best thickness of carrier-substrates in the plate (15?cm in diameter) for the maximum mosquitocidal activity was about 0.5?cm for Bti and 0.5–1?cm for Ls14N1. Pilot-scale production in aluminium trays applying the above conditions showed a decrement of toxicity of fermented cultures and some plates were contaminated. These problems were dissolved by reducing the moisture content to 15%, increasing inoculum to 10% and manual agitation of trays every-day.     

Evaluation of Methods for Reestablishment of L-Cell Suspension Cultures Directly from Liquid Nitrogen Stored Stocks

Methods were developed and evaluated for the preservation of tissue cells grown in suspension culture and the reestablishment of suspension cultures directly from inoculum stored at -175 C. The factors investigated were processing pH, temperature of processing, freezing medium, and method of inoculation of the starter suspension cultures from the frozen stock (-175 C). Three parameters, cell viability, cell size, and growth potential in suspension culture after freezing, were used to evaluate the various factors. The results indicate that cells processed at 4 C, frozen at 1 C per min to -50 C in a medium containing 5% dimethyl sulfoxide plus 10% bovine serum at concentrations of 2 x 10(7) to 4 x 10(7) cells/ml, and stored at -175 C will reestablish suspension cultures directly from frozen seed. A 1-ml amount of frozen stock inoculated into 99 ml of medium routinely produced 2 x 10(6) to 3 x 10(6) viable cells/ml (2 x 10(8) to 3 x 10(8) total cells) in suspension culture in 4 to 5 days. Inoculum preserved by this procedure grew equally well in either serum-free or serum-containing growth medium.     

Biodegradation of gossypol by the white oyster mushroom,Pleurotus florida,during culturing on rice straw growth substrate,supplemented with cottonseed powder

The capacity of the white oyster mushroom, Pleurotus florida to biodegrade gossypol was studied, when grown on rice straw supplemented with cottonseed powder. The mushroom fruiting bodies did not contain any residues of gossypol at concentrations of cottonseed powder 0.15–0.60% nitrogen contents of rice straw at the end of mycelial ramification. However, the cottonseed supplementation (at 0.30% N level itself) caused a doubling in the mushroom yield and its protein content, per unit weight straw substrate. The mushroom mycelium when grown on synthetic medium in liquid cultures was able to biodegrade gossypol. A pre-incubation period of 5 days before the addition of gossypol into the culture medium, an inoculum load 10 mg and an incubation period of 10 days at 25 °C caused the biodegradation of 100 g gossypol. Increased concentrations of gossypol required increased duration and increased inoculum levels to effect biodegradation. However, the effect was more pronounced with an increase in inoculum density. The fungal monoculture when grown in rice straw (powder) (5%) + glucose (1%) liquid culture medium, showed an increase in hexosamine content and laccase activity that produced an increased degradation of gossypol over an incubation period from 5 to 25 days. Enzymic extracts of the mycelial monoculture raised on the chopped rice straw substrate when incubated with 100 g of gossypol demonstrated its biodegradability; the increase in enzyme concentration showed enhanced gossypol degradation. This study adds to the world list of organic compounds that Pleurotus is able to biodegrade, and explains the cause of non-yellowing of the white oyster mushroom (P. florida) fruiting bodies, during culture on rice straw with supplementation of cottonseed powder for enhancing the mushroom yields.     

Submerged growth of the cultivated mushroom,Agaricus bisporus

Agaricus bisporus grew well in submerged culture in a medium containing malt extract, phosphate, and casein. Moderate growth occurred in defined media containing glucose, asparagine, phenylalanine, vitamins and minerals. Other amino acids did not stimulate growth. Growth was stimulated by vegetable oils, partly due to utilization of the oils, and partly to a more complex mechanism. Oleates had the same effect as vegetable oils; palmitates a lesser one. In shaken flasks maximum yield was reached after 22–24 days and in stirred and aerated fermentors after 8–10 days. Besides dry weight of mycelium, laccase activity was determined. The latter determination is suitable for a rapid estimation of the growth in routine experiments. The flavour of the mycelium was like that of mushrooms but weaker. It was strongest in standing liquid cultures and on solid media. The mycelium grown in submerged culture was suitable as spawn for mushroom culture. Presented at the First International Mycological Congress, Exeter, 7–16 September 1971, and at the Meeting of the Netherlands Society for Microbiology, Rotterdam, 8 December 1971. We thank the Mushroom Experiment Station, Horst, the Netherlands, for kindly supplying the compost for fructification experiments and Mr. P. Arntz, M.Sc., for advice in the fermentor work. F. IJ. Dijkstra is indebted to the Royal Netherlands Fermentation Industries (Gist-Brocades), Delft, for a research grant.     

In vitro multiplication in liquid culture of Syngonium contaminated with Bacillus spp. and Rathayibacter tritici

Contaminated Syngonium clusters were multiplied in an air lift bioreactor in liquid medium containing sucrose with the medium being circulated through a sterilizing filter. After 30 days, the culture in filtered medium produced 19.5 shoot initials per gram fresh weight of inoculum compared to 8.7 shoot initials produced in unfiltered medium. Transfer to an elongation medium with 30 mg l^-1 Rifampicin produced shoots on 67% of the clusters, while transfer to elongation medium without Rifampicin poduced shoots on 40% of the clusters. Clusters grown for three subcultures in a reactor without medium filtration had lost their multiplication ability. Clusters grown for three subcultures in a reactor with filtration, however, continued to show a two-three fold increase in fresh weight and shoot production.Abbreviations MS Murashige and Skoog     

Fed-batch approach to production of 2,3-butanediol by Klebsiella pneumoniae grown on high substrate concentrations

The bioconversion of sugars present in wood hemicellulose to 2,3-butanediol by Klebsiella pneumoniae grown on high sugar concentrations was investigated. When K. pneumoniae was grown under finite air conditions in the presence of added acetic acid, 50 g of D-glucose and D-xylose per liter could be converted to 25 and 27 g of butanediol per liter, respectively. The efficiency of bioconversion decreased with increasing sugar substrate concentrations (up to 200 g/liter). Butanediol production at low sugar substrate concentrations was less efficient when the organism was grown under aerobic conditions; however, final butanediol values were higher for cultures grown on an initial sugar concentration of 150 g/liter, particularly when the inoculum was first acclimatized to high sugar levels. When a double fed-batch approach (daily additions of sugars together with yeast extract) was used under aerobic conditions, up to 88 and 113 g of combined butanediol and acetyl methyl carbinol per liter could be obtained from the utilization of 190 g of D-xylose and 226 g of D-glucose per liter, respectively.