Quantifying mold biomass on gypsum board: comparison of ergosterol and beta-N-acetylhexosaminidase as mold biomass parameters

Two mold species, Stachybotrys chartarum and Aspergillus versicolor, were inoculated onto agar overlaid with cellophane, allowing determination of a direct measurement of biomass density by weighing. Biomass density, ergosterol content, and beta-N-acetylhexosaminidase (3.2.1.52) activity were monitored from inoculation to stationary phase. Regression analysis showed a good linear correlation to biomass density for both ergosterol content and beta-N-acetylhexosaminidase activity. The same two mold species were inoculated onto wallpapered gypsum board, from which a direct biomass measurement was not possible. Growth was measured as an increase in ergosterol content and beta-N-acetylhexosaminidase activity. A good linear correlation was seen between ergosterol content and beta-N-acetylhexosaminidase activity. From the experiments performed on agar medium, conversion factors (CFs) for estimating biomass density from ergosterol content and beta-N-acetylhexosaminidase activity were determined. The CFs were used to estimate the biomass density of the molds grown on gypsum board. The biomass densities estimated from ergosterol content and beta-N-acetylhexosaminidase activity data gave similar results, showing significantly slower growth and lower stationary-phase biomass density on gypsum board than on agar.     

Effect of Culture Conditions on Ergosterol as an Indicator of Biomass in the Aquatic Hyphomycetes

Ergosterol is a membrane component specific to fungi that can be used to estimate fungal biomass using appropriate factors of conversion. Our objectives were to determine the limits of use of ergosterol content as a measure of biomass for aquatic hyphomycetes, and to evaluate a previously established ergosterol-to-biomass conversion factor. We varied inoculum quality, growth medium, and degree of shaking of four aquatic hyphomycete species. In cultures inoculated with homogenized mycelium, we found a significant effect of shaking condition and culture age on ergosterol content. In liquid cultures with defined medium, ergosterol content reached 10 to 11 μg/mg of mycelium (dry mass) and varied by factors of 2.2 during exponential growth and 1.3 during stationary phase. The increase in ergosterol content during exponential phase could be attributed, at least in part, to rapid depletion of glucose. Oxygen availability to internal hyphae within the mycelial mass is also responsible for the differences found between culture conditions. Ergosterol concentration ranged from 0.8 to 1.6 μg/mg in static cultures inoculated with agar plugs. Ergosterol content varied by a factor of 4 in two media of different richnesses. For different combinations of these parameters, strong (r² = 0.83 to 0.98) and highly significant (P 0.001) linear relationships between ergosterol and mycelial dry mass (up to 110 mg) were observed. Overall, the ergosterol content varied by a factor of 14 (0.8 to 11 mg/g). These results suggest that care must be taken when the ergosterol content is used to compare data generated in different field environments.     

Biological Control of Olive Green Mold in Agaricus bisporus Cultivation

Successful methods to control the damaging weed mold Chaetomium olivaceum (olive green mold) in mushroom beds are not presently known. An attempt was made to control C. olivaceum by biological means. A thermophilic Bacillus sp. which showed dramatic activity against C. olivaceum on Trypticase soy agar (BBL Microbiology Systems)-0.4% yeast extract agar plates was isolated from commercial mushroom compost (phase I). When inoculated into conventional and hydroponic mushroom beds, the bacillus not only provided a significant degree of protection from C. olivaceum, but also increased yields of Agaricus bisporus.     

Ergosterol Content in Various Fungal Species and Biocontaminated Building Materials

This paper reports the ergosterol content for microbial cultures of six filamentous fungi, three yeast species, and one actinomycete and the ergosterol levels in 40 samples of building materials (wood chip, gypsum board, and glass wool) contaminated by microorganisms. The samples were hydrolyzed in alkaline methanol, and sterols were silylated and analyzed by gas chromatography-mass spectrometry. The average ergosterol content varied widely among the fungal species over the range of 2.6 to 42 μg/ml of dry mass or 0.00011 to 17 pg/spore or cell. Ergosterol could not be detected in the actinomycete culture. The results for both the fungal cultures and building material samples supported the idea that the ergosterol content reflects the concentration of filamentous fungi but it underestimates the occurrence of yeast cells. The ergosterol content in building material samples ranged from 0.017 to 68 μg/g of dry mass of material. A good agreement between the ergosterol concentration and viable fungal concentrations was detected in the wood chip (r > 0.66, P ≤ 0.009) and gypsum board samples (r > 0.48, P ≤ 0.059), whereas no relationship between these factors was observed in the glass wool samples. For the pooled data of the building materials, the ergosterol content correlated significantly with the viable fungal levels (r > 0.63, P < 0.0001). In conclusion, the ergosterol concentration could be a suitable marker for estimation of fungal concentrations in contaminated building materials with certain reservations, including the underestimation of yeast concentrations.     

Effect of Plasterboard Composition on Stachybotrys chartarum Growth and Biological Activity of Spores

The effects of plasterboard composition on the growth and sporulation of Stachybotrys chartarum as well as on the inflammatory potential of the spores were studied. S. chartarum was grown on 13 modified plasterboards under saturated humidity conditions. The biomass was estimated by measuring the ergosterol content of the S. chartarum culture while the spore-induced cytotoxicity and production of nitric oxide (NO), tumor necrosis factor alpha (TNF-α), and interleukin-6 in mouse macrophages was used to illustrate the bioactivity of spores. The ergosterol content of S. chartarum correlated with the number of spores collected from plasterboards. The growth and sporulation decreased compared to that of the reference board in those cases where (i) the liner was treated with biocide, (ii) starch was removed from the plasterboard, or (iii) desulfurization gypsum was used in the core. Spores collected from all the plasterboards were toxic to the macrophages. The biocide added to the core did not reduce the growth; in fact, the spores collected from that board evoked the highest cytotoxicity. The conventional additives used in the core had inhibitory effects on growth. Recycled plasterboards used in the core and the board lacking the starch triggered spore-induced TNF-α production in macrophages. In summary, this study shows that the growth of a strain of S. chartarum on plasterboard and the subsequent bioactivity of spores were affected by minor changes to the composition of the core or liners, but it could not be totally prevented without resorting to the use of biocides. However, incomplete prevention of microbial growth by biocides even increased the cytotoxic potential of the spores.     

Ergosterol concentration and variability in genotype-by-pathogen interaction for grain mold resistance in sorghum

A lack of understanding of host-by-pathogen relations can hinder the success of breeding for resistance to a major disease. Fungal strain pathogenicity has to be understood from the virulence it can cause on susceptible genotypes and host resistance indicates which genotypes have resistance genes. Where the two worlds meet lies the place where researchers match the prevalent pathogen in the area of production with resistant varieties. This paper uses ergosterol concentration analysis as a measure of fungal biomass accumulation to assess levels of resistance in host genotypes. 11 sorghum genotypes were inoculated with 5 strains of fungi that are known to be associated with grain mold disease of sorghum. The resulting interaction was analyzed using GGE Biplot analysis and Cluster analysis which showed that none of the genotypes were resistant to Phoma sorghina and Curvularia lunata. Three genotypes were resistant to Fusarium thapsinum. One fungal strain (Alternaria alternata) does not contribute any significant damage in the grain mold disease. Fusarium graminearum causes very little grain mold disease. There was no correlation between the fungal strains. Visual scoring did not correlate with ergosterol accumulation. Resistance to grain mold in sorghum is shown to be due to vertical or specific resistance genes. Sorghum breeders should, therefore, identify predominant fungal strains in their localities and then locate and tag these resistance genes in their germplasm and pyramid them in commercial varieties.     

Intracellular and extracellular beta-N-acetylhexosaminidases from Physarum polycephalum. Comparison of vegetative and spherulation enzymes.

Vegetative microplasmodia of the slime mold, Physarum polycephalum, produce an intracellular β-N-acetylhexosaminidase enzyme when grown on a medium containing 1% glucose, 0.15% yeast extract, and 1% peptone. When early log-phase microplasmodia are induced to differentiate to spherules by starvation in a salts medium, they excrete an extracellular β-N-acetylhexosaminidase. Both of these enzymes have been purified to apparent homogeneity. Characterization studies showed that the extracellular enzyme was nonidentical to the preexisting, vegetative enzyme and the enzyme in completed spherules. Evidence demonstrating dissimilarities between the two proteins included marked differences in (i) specificities for several natural and synthetic substrates, (ii) various kinetic parameters, (iii) relative net charges as evidenced by different elution behavior from similar DE-52 cellulose chromatography columns, (iv) carbohydrate contents, and (v) subunit polypeptide molecular weights. Conclusive evidence for their nonidentity was shown in their respective amino acid compositions and divergent immunological properties. The extracellular β-N-acetylhexosaminidase demonstrated a subunit molecular weight of 25,300; the intracellular enzyme subunit molecular weight was 40,500. The extracellular enzyme, with the smaller polypeptide subunit, contained 1.79 times as many aromatic amino acid residues in tyrosine, phenylalanine, and tryptophan as the intracellular enzyme. Thus, the extracellular enzyme could not have been comprised of subunits derived from limited proteolytic hydrolysis of the larger subunits of the intracellular enzyme. Rabbit antisera prepared against each purified β-N-acetylhexosaminidase failed to yield precipitin bands with the heterologous antigen in immunodiffusion tests. Thus, apparently distinct structural genes code for these two enzymes and they may serve different, but unidentified, physiological functions.     

Effect of culture conditions on ergosterol as an indicator of biomass in the aquatic hyphomycetes

Ergosterol is a membrane component specific to fungi that can be used to estimate fungal biomass using appropriate factors of conversion. Our objectives were to determine the limits of use of ergosterol content as a measure of biomass for aquatic hyphomycetes, and to evaluate a previously established ergosterol-to-biomass conversion factor. We varied inoculum quality, growth medium, and degree of shaking of four aquatic hyphomycete species. In cultures inoculated with homogenized mycelium, we found a significant effect of shaking condition and culture age on ergosterol content. In liquid cultures with defined medium, ergosterol content reached 10 to 11 microg/mg of mycelium (dry mass) and varied by factors of 2.2 during exponential growth and 1.3 during stationary phase. The increase in ergosterol content during exponential phase could be attributed, at least in part, to rapid depletion of glucose. Oxygen availability to internal hyphae within the mycelial mass is also responsible for the differences found between culture conditions. Ergosterol concentration ranged from 0.8 to 1.6 microg/mg in static cultures inoculated with agar plugs. Ergosterol content varied by a factor of 4 in two media of different richnesses. For different combinations of these parameters, strong (r(2) = 0.83 to 0.98) and highly significant (P < 0.001) linear relationships between ergosterol and mycelial dry mass (up to 110 mg) were observed. Overall, the ergosterol content varied by a factor of 14 (0.8 to 11 mg/g). These results suggest that care must be taken when the ergosterol content is used to compare data generated in different field environments.     

Biomass estimation in solid state fermentation I. Manual biochemical methods

Summary We examined the changes in three biomass constituents (glucosamine, ergosterol, total sugar), sucrose consumption and conidia formation, during cultivation of Beauveria bassiana on agar plates or clay granules. We showed that glucosamine can be considered a good biomass indicator on condition that the media had the same constituents but not necessarily the same C/N ratio. Total sugar was not constant during the different growth phases and cannot accurately represent biomass changes. The ergosterol amounts changed during the different growth phases but, for a fixed process, it can be a good indicator of conidiation. Good correlations were obtained between glucosamine and sucrose consumption allowing biomass yield calculations.Offprint requests to: C. Desgranges     

Isolation and Identification of Aspergillus fumigatus Mycotoxins on Growth Medium and Some Building Materials

Genotoxic and cytotoxic compounds were isolated and purified from the culture medium of an indoor air mold, Aspergillus fumigatus. One of these compounds was identified as gliotoxin, a known fungal secondary metabolite. Growth of A. fumigatus and gliotoxin production on some building materials were also studied. Strong growth of the mold and the presence of gliotoxin were detected on spruce wood, gypsum board, and chipboard under saturation conditions.     

Biomass of pelagic fungi in Baltic rivers

Concentrations of pelagic fungal biomass, determined as the content of ergosterol in particulate matter, were measured in 49 Baltic rivers during summer 1999. The ergosterol concentration varied 12-fold, from 12.6 to 152.5 ng l⁻¹ (average of 56.4 ng l⁻¹) and correlated positively with concentrations of dissolved organic matter and inorganic nutrients as well as with spectral DOM properties indicative of terrestrial sources. The fungal biomass was 12- to 100-fold lower than the biomass of pelagic bacteria, suggesting that fungi in the water column of the rivers probably were of minor importance in the riverine ecosystems at the sampling time. Handling editor: J. Padisak     

The Ability of Plant Compost Leachates to Control Black Mold (Aspergillus niger) and to Induce the Accumulation of Antifungal Compounds in Onion Following Seed Treatment

Onion seeds treated with leachates of composts prepared from alfalfa and sunflower stalks, at the dosages of 10% and 20% respectively, were inoculated with Aspergillus niger van Tieghem, causal agent of onion black mold disease. The ability of the leachates to induce the production of antifungal compounds and to control black mold were tested at seedling and set stages. Leachates from both composts were able to reduce disease incidence in sets, but not disease severity in onion seedlings. Extracts from treated seedlings and sets were fractionated by thin layer chromatography for their content of antifungal compounds. There were no significant differences between the fractions of alfalfa and sunflower compost leachates in the inhibition of the mycelium growth of A. niger, with the exception of one fraction. The presence of fluorescent pseudomonads and Pantoae agglomerans [synonym: Erwinia herbicola (Löhnis)] bacteria was determined in both leachates. The population of P. agglomerans was higher in the sunflower compost leachate compared to the alfalfa leachate. The tested strains of both bacteria were able to inhibit mycelium growth of the fungal pathogen in agar tests. This study suggests the possible role of beneficial bacteria in the induction of antifungal compounds in onion against A. niger during seedling and set stages.     

Rhizopus delemar菌固态发酵生物量测定及其脂肪酶合成特征

研究了Rhizopus delemar菌固态发酵曲中麦角固醇的提取及其高效液相色谱（HPLC）测定方法。结果表明,固态曲中麦角固醇分离提取以1：25（w/v)的丙酮回流浸提2.0h为最佳,HPLC测定麦角固醇的条件为：HypersilC-8柱。甲醇/水（85/15,v/v)为流动相,流速为1.0mL/min,紫外检测波长为282nm。柱温为30℃。依据HPLC的分析测定,确定了麦角固醇与菌丝体间的定量关系,并以此为基础。测定了Rhizopus delemar菌固态发酵过程中的生物量变化。得到了生长曲线,发现当发酵培养至60h时固态曲中的生物量达到最大值为0.18g菌丝体/g干曲,而该菌所合成的脂肪酶的活力在48h达到最大值。     

Elaboration and application of mathematical model for estimation of mould contamination of some building materials based on ergosterol content determination

The study presents a mathematical function describing a correlation between the amount of ergosterol and the number of colony-forming units (CFU) of mould contaminating selected building materials such as: a block of cellular concrete, gypsum—carton board and gypsum—carton board covered with emulsion paint. The dependence obtained for a particular material as well as an average dependence for all the investigated materials has been described by means of an exponential equation. It has been found out that there is high, statistically significant correlation between ergosterol content and CFU number of mould in all of the building materials. The correlation coefficients have ranged from r=0.790 to 0.933. The elaborated equation describing the above dependence can be applied to estimate mould contamination by means of culture methods within the range 10³–10⁸ CFU/m² of the surface. In addition, the estimated level of ergosterol in these materials has been shown to be the criterion by which to evaluate the degree of filamentous fungal contamination. It has been assessed that an ergosterol content exceeding the level of 3.96 mg/m² indicates the active development of mould. This criterion has been applied to evaluate several building materials i.e.: concrete, gypsum board, emulsion coatings, brick, plaster, wallpaper, glass wool, mineral wool and wood. No statistically significant differences have been observed between CFU number of mould calculated from a model equation on the basis of the ergosterol content and CFU number of mould experimentally determined by traditional methods. The results presented in this paper show that the elaborated equation of correlation between the ergosterol content and CFU number of mould can be applied to estimate mould contamination of different building materials, based on the determination of ergosterol.     

Comparison of N(2) Fixation and Yields in Cajanus cajan between Hydrogenase-Positive and Hydrogenase-Negative Rhizobia by In Situ Acetylene Reduction Assays and Direct N Partitioning

Pigeon peas [Cajanus cajan (L.) Millsp.] were grown in soil columns containing ¹⁵N-enriched organic matter. Seasonal N₂ fixation activity was determined by periodically assaying plants for reduction of C₂H₂. N₂ fixation rose sharply from the first assay period at 51 days after planting to a peak of activity between floral initiation and fruit set. N₂ fixation (acetylene reduction) activity dropped concomitantly with pod maturation but recovered after pod harvests. Analysis of ¹⁵N content of plant shoots revealed that approximately 91 to 94% of plant N was derived from N₂ fixation. The effect of inoculation with hydrogenase-positive and hydrogenase-negative rhizobia was examined. Pigeon peas inoculated with strain P132 (hydrogenase-positive) yielded significantly more total shoot N than other inoculated or uninoculated treatments. However, two other hydrogenase-positive strains did not yield significantly more total shoot N than a hydrogenase-negative strain. The extent of nodulation by inoculum strains compared to indigenous rhizobia was determined by typing nodules according to intrinsic antibiotic resistance of the inoculum strains. The inoculum strains were detected in almost all typed nodules of inoculated plants.

Gas samples were taken from soil columns several times during the growth cycle of the plants. H₂ was never detected, even in columns containing pigeon peas inoculated with hydrogenase-negative rhizobia. This was attributed to H₂ consumption by soil bacteria. Estimation of N₂ fixation by acetylene reduction activity was closest to the direct ¹⁵N method when ethylene concentrations in the gas headspace (between the column lid and soil surface) were extrapolated to include the soil pore space as opposed solely to measurement in the headspace. There was an 8-fold difference between the two acetylene reduction assay methods of estimation. Based on a planting density of 15,000 plants per hectare, the direct ¹⁵N fixation rates ranged from 67 (noninoculated) to 134 kilograms per hectare, while grain yields ranged from 540 to 825 kilograms per hectare. Grain yields were not increased with N fertilizer.

     

Thermoactive β-N-acetylhexosaminidase production by a soil isolate of Penicillium monoverticillium CFR 2 under solid state fermentation: parameter optimization and application for N-acetyl chitooligosaccharides preparation from chitin

Two fungal strains were evaluated for β-N-acetylhexosaminidase production by solid state fermentation using different agro-industrial residues such as commercial wheat bran (CWB) and shrimp shell chitin waste (SSCW), of which Penicillium monoverticillium CFR 2 a local soil isolate showed significantly (P ≤ 0.001) higher β-N-acetylhexosaminidase activity on CWB medium as compared with the activity of Fusarium oxysporum CFR 8. Fermentation parameters such as incubation temperature, incubation time, initial moisture content and inoculum concentration were optimized by statistically designed experiments, using 3**(4–1) fractional factorial design of Response Surface Methodology. The high R² (0.9512) observed during validation experiment showed the usefulness of the model. Highest level of enzyme activity (311.84 U/g IDS) was predicted at 75% (w/w) initial moisture content, 26 °C incubation temperature, 168 h incubation time and initial inoculum, at the highest concentration tested (2.95 ml spore suspension/5 g substrate). Statistical optimization yielded a 4.5 fold increase in β-N-acetylhexosaminidase activity. The crude β-N-acetylhexosaminidase showed optimum temperature of 57 ± 1 °C and pH of 3.6 and retained 50% activity after 1 h of incubation at 57 ± 1 °C. SDS–PAGE zymogram revealed crude enzyme was a monomer with an apparent molecular weight ~110 kDa. The crude enzyme formed 6.81 ± 0.03 mM/l of N-acetyl chitooligosaccharides from colloidal chitin in 24 h of incubation. HPLC analysis revealed hydrolysate contained 37.57% N-acetyl chitotriose and 62.43% N-acetyl chitohexose, indicating its potential for specific N-acetyl chitooligosaccharides production.     

Effect of plasterboard composition on Stachybotrys chartarum growth and biological activity of spores

The effects of plasterboard composition on the growth and sporulation of Stachybotrys chartarum as well as on the inflammatory potential of the spores were studied. S. chartarum was grown on 13 modified plasterboards under saturated humidity conditions. The biomass was estimated by measuring the ergosterol content of the S. chartarum culture while the spore-induced cytotoxicity and production of nitric oxide (NO), tumor necrosis factor alpha (TNF-alpha), and interleukin-6 in mouse macrophages was used to illustrate the bioactivity of spores. The ergosterol content of S. chartarum correlated with the number of spores collected from plasterboards. The growth and sporulation decreased compared to that of the reference board in those cases where (i) the liner was treated with biocide, (ii) starch was removed from the plasterboard, or (iii) desulfurization gypsum was used in the core. Spores collected from all the plasterboards were toxic to the macrophages. The biocide added to the core did not reduce the growth; in fact, the spores collected from that board evoked the highest cytotoxicity. The conventional additives used in the core had inhibitory effects on growth. Recycled plasterboards used in the core and the board lacking the starch triggered spore-induced TNF-alpha production in macrophages. In summary, this study shows that the growth of a strain of S. chartarum on plasterboard and the subsequent bioactivity of spores were affected by minor changes to the composition of the core or liners, but it could not be totally prevented without resorting to the use of biocides. However, incomplete prevention of microbial growth by biocides even increased the cytotoxic potential of the spores.     

The detection of hyaluronidase on electrophoresis membranes

A method is described for the detection of hyaluronidase enzyme activity following electrophoresis on cellulose acetate membranes prior to elution of the enzyme from the support medium. It appears to be specific for endo-N-acetylhexosaminidase activity. It detects 0.005 of the least amount of enzyme detected by a miniaturized turbidity reduction test or by a standard viscosity reduction test.     

Micro-assay to Measure the Allergenicity of a Kunitz-type Soybean Trypsin Inhibitor toward Balb/c Mice by Using RBL-2H3 Cells

The allergenicity of a Kunitz-type soybean trypsin inhibitor (KSTI) was investigated by a micro-assay of β-N-acetylhexosaminidase released from RBL-2H3 cells primed with the anti-KSTI serum. KSTI stimulated the release of β-N-acetylhexosaminidase from RBL-2H3 cells primed with the antiserum. The response of RBL-2H3 cells to the reaginic activity of the mouse anti-KSTI serum correlates fairly well with that by the passive cutaneous anaphylaxis (PCA) test, the sensitivity of both assays appearing to be similar. These results suggest that measuring the β-N-acetylhexosaminidase released from RBL-2H3 is a convenient way for studying the allergen or the reaginic activity of a murine serum in place of the PCA test.     

Biomass estimation in solid state fermentation II. On-line measurements

Summary Two methods for on-line biomass measurement were tested with success: (a) infrared (IR) estimation of cell components (glucosamine and ergosterol) and medium residues (sucrose and nitrogen) directly on solid medium; (b) the CO₂ evolution rate during cultivation. These methods were very satisfactory for following biomass changes during a defined process. The IR measurements correlated well with manual methods. The correlation between CO₂ and glucosamine measurements was very satisfactory with good precision. However, they did not permit comparison between processes.Offprint requests to: C. Desgranges