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.     

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.     

Mathematical models of mycelium growth and ergosterol synthesis in stationary mould culture

Aims:  To develop mathematical models for mycelium growth and ergosterol formation in conditions of periodic stationary culture; to verify possibilities of applying a model describing the relationship between ergosterol content and mycelium quantity.
Methods and Results:  The mould growth and ergosterol formation models covering all phases of mould growth are described using a modified logistic equation with the addition of an exponential function. The correlation between ergosterol and mycelium biomass depended on the growth phase of mould. Meaningful relation was obtained for initial two phases, when both parameters were growing equally. The quadratic function for estimation of the biomass based on ergosterol content was formulated. The error resulting from the application of this function in initial phases of moulds growth was small at 5–7%, in the following phases it was at 11–31%.
Conclusions:  Mycelium biomass can be precisely determined basing on the ergosterol content, when we know the moulds growth phase. In natural environments, without the information about growth phases, it will be possible, but with the higher error.
Significance and Impact of the Study:  Presented model based on the ergosterol content making possible to estimate the quantity of mycelium in moulds cultures and natural environments.     

Comparison of methods for estimating the biomass of three food-borne fungi with different growth patterns.

To evaluate the effectiveness of steps taken to reduce the growth of molds in food and feed, methods that can accurately quantify the degree of fungal contamination of solid substrates are needed. In this study, the ergosterol assay has been evaluated by comparing the results of this assay with spore counts and hyphal length measurements made with a microscope and with CFU counts. Three fungi with different growth patterns during cultivation on a synthetic agar substrate were used in these experiments. For the nonsporulating Fusarium culmorum, there was good agreement between changes in hyphal length, CFU, and ergosterol content. Penicillium rugulosum and Rhizopus stolonifer produced many spores, and the production of spores coincided with large increases in CFU but not with increases in hyphal length or ergosterol content. Spores constituted between 3 and 5% of the total fungal mass. Changes in ergosterol level were closely related to changes in hyphal length. It was concluded that ergosterol level is a suitable marker for use in quantitatively monitoring fungal growth in solid substrates.     

Proposal for an integrated approach to microbial environmental monitoring in cultural heritage: experience at the Correggio exhibition in Parma

Microbial environmental monitoring represents one of the most useful methods to assess potential risks related to the integrity of cultural heritage and people’s health. The monitoring plan described in the present work is based on standardized techniques for measuring microbial air and surface contamination. Air contamination is assessed through both active and passive samplings, measuring the concentration of microbes in air (in colony forming units per cubic metre, CFU/m³) and the rate at which microorganisms settle on surfaces (expressed by the Index of Microbial Air Contamination, IMA, CFU/dm²/h). For surface contamination, two parameters are measured using nitrocellulose membranes: the Microbial Buildup (MB, the total number of microorganisms accumulated on a surface in an unknown period of time prior to the sampling) and the Hourly Microbial Fallout (HMF, the number of microorganisms that settle on a specific surface during 1 h). The monitoring plan was implemented at the Pilotta Palace in Parma, Italy, during the Correggio exhibition in 2009. Samplings were taken before and during opening times. Some microbial contamination was already detected before the arrival of visitors: air contamination mean values of 99.1 CFU/m³ and 5.2 CFU/dm²/h were recorded, while MB and HMF mean values for surfaces were 92 and 7 CFU/dm², respectively. A significant increase was recorded in air contamination during opening times, with mean values of 323.7 CFU/m³ and 19.4 CFU/dm²/h; surface contamination values increased as well. This monitoring plan represents a contribution towards the definition of a much needed standardized methodology.     

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.     

Preliminary survey of indoor and outdoor airborne microfungi at coastal buildings in Egypt

Forty six species and two sterile fungi and yeast species were isolated from samples collected both indoors and outdoors of coastal buildings located in an Egyptian coastal city. Twenty flats from ten buildings were investigated; children living in these buildings have been reported to suffer from respiratory illnesses. Samples were taken using a New Brunswick sampler (model STA-101) operating for 3.0 min at a flow rate of 6.0 l/min. Most of the species isolated have been associated with symptoms of respiratory allergies. Indoors the total culturable fungal count was 1548 CFU/m³; outdoors, it was 1452 CFU/m³. Indoor values of culturable fungal count, total spores count and ergosterol content ranged from 52 to 124 CFU/m³, 100 to 400 spore/m³ and 5 to 27.7 mg/m³, respectively, whereas outdoor levels typically varied between 25 and 222 CFU/m³, 110 and 900 spore/m³ and 3.3 and 67.2 mg/m³, respectively. The maxima for these parameters were detected indoors in house no. 6 and outdoors, outside of house no. 7. The most abundant species were primarily mitosporic (2832 CFU/m³). The most frequent species in both the indoor and outdoor samples were Cladosporium cladosporioides followed by Alternaria alternata and Penicillium chrysogenum,with inside:outside ratios of 1.4, 1.8 and 1.9, respectively. The patterns of fungal abundance were influenced to some extent by changes in the relative humidity and temperature. Other factors, such as type of culture media, rate of sedimentation, size, survival rates of spore and species competition,also affected fungal counts and should be taken into consideration during any analysis of bioaerosol data.     

Effect of culture conditions on the biomass determination by ergosterol of <Emphasis Type="Italic">Lentinus crinitus</Emphasis> and <Emphasis Type="Italic">Psilocybe castanella</Emphasis>

Estimating fungal growth is important in processes of soil bioremediation. It has been demonstrated that ergosterol is a good indicator of fungal biomass in solid substrata. In the present study were evaluated the effects upon the ergosterol rate of Lentinus crinitus Berk. and Psilocybe castanella Peck through the culture conditions of these fungi, which are evaluated for the bioremediation of soils contaminated by organochlorates. A good correlation between fungal biomass and ergosterol was observed for both species. The culture conditions did not influence the ergosterol rate of L. crinitus. Yet the ergosterol rate of P. castanella was influenced from 35 days of culture and when grown in the presence of 15.00 g hexachlorobenzene l⁻¹ of culture medium. So it is possible to estimate growth of both species using ergosterol as indicator in processes of soil bioremediation since the influences observed in the ergosterol rate of P. castanella are considered.     

Towards the design of an optimal strategy for the production of ergosterol from Saccharomyces cerevisiae yeasts

The total yield of ergosterol produced by the fermentation of the yeast Saccharomyces cerevisiae depends on the final amount of yeast biomass and the ergosterol content in the cells. At the same time ergosterol purity—defined as percentage of ergosterol in the total sterols in the yeast—is equally important for efficient downstream processing. This study investigated the development of both the ergosterol content and ergosterol purity in different physiological (metabolic) states of the microorganism S. cerevisiae with the aim of reaching maximal ergosterol productivity. To expose the yeast culture to different physiological states during fermentation an on‐line inference of the current physiological state of the culture was used. The results achieved made it possible to design a new production strategy, which consists of two preferable metabolic states, oxidative‐fermentative growth on glucose followed by oxidative growth on glucose and ethanol simultaneously. Experimental application of this strategy achieved a value of the total efficiency of ergosterol production (defined as product of ergosterol yield coefficient and volumetric productivity), 103.84 × 10^?6 g L^?1h^?1, more than three times higher than with standard baker's yeast fed‐batch cultivations, which attained in average 32.14 × 10^?6 g L^?1h^?1. At the same time the final content of ergosterol in dry biomass was 2.43%, with a purity 86%. These results make the product obtained by the proposed control strategy suitable for effective down‐stream processing. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:838–848, 2017     

Some observations on air filtration

Summary 1. A method has been developed for testing the filtration efficiency of some filter materials. For each of the materials investigated — cotton wool, stillite and carbon — a suitable filter has been devised.2. The filtered air was analyzed as to its germ content with the aid of a set of 3 capillary impingers.3. The cotton wool filter gave on the whole satisfactory results provided that due attention was given to the packing of the filter and its sterilisation. Clear indications were obtained that the degree of the contamination of the air was of vital importance.4. The stillite filter proved to have the advantage of combining a high filtration efficiency with a low resistance to the passing air. Also for the stillite filter a critical degree of contamination of the air was established; on surpassing this degree the filtration effect was endangered.5. The carbon filter proved to be most efficient, but had a relatively low specific filtering capacity. It was found that the filtration result was depending on the height of the carbon column and on the velocity and the degree of contamination of the air.6. It should be stressed that in all experiments artificially contaminated air was used, and that the number of germs present in the air to be filtered was in all cases many times larger than that usually occurring in normal air.     

Influence of age on ergosterol content in mycelium of Aspergillus ochraceus

Ergosterol has been suggested as a paramoter for studying the content and evolution of fungal infestation in different products. Important differences in this parameter have been described for different genera and species. A study of the relationship between ergosterol content and dry mass in a strain of Aspergillus ochraceus , which is able to produce an antifungal agent, has therefore been carried out in order to use this parameter to accurately evaluate the fungal mass of this strain. We report herein our research work to determine the ergosterol content of the mycelial mass of our mould and how it changes with the fungal age.     

Ergosterol as an indicator of mould growth on wood in relation to culture age, humidity stress and nutrient level

Changes in ergosterol content in cultures of Penicillium brevicompactum and Aspergillus versicolor on wood with time, changes in humidity or addition of glucose solutions to wood were studied with HPLC. Lowering of the humidity level caused a very large decline in ergosterol content of cultures of P. brevicompactum on wood over a 10 day period, although small amounts remained after this time. After an initial increase, up to an inoculation time of 45 days, reductions were also observed in control samples maintained at 100% RH, but these were smaller. The amount of ergosterol decreased to very low levels in wood impregnated with low levels of glucose during a 93 day incubation period. Ergosterol concentration in hyphae produced in surface liquid cultures was shown to be higher in mycelia growing on media enriched with nitrogen or with more available nutrients. The concentration of ergosterol in the mycelia of P. brevicompactum in surface liquid cultures varied by a factor of 5 from 2 to 10 mg g⁻. The results clearly show that ergosterol present in solid materials in mainly related to active biomass. With certain prerequisites, ergosterol determinations could also be used for total fungal biomass estimations on wood.     

Total and Free Ergosterol in Mycelia of Saltmarsh Ascomycetes with Access to Whole Leaves or Aqueous Extracts of Leaves

Three species of saltmarsh ascomycetes were grown in the presence of all of the constituents of their natural substrate (leaves of cordgrass) or were presented only with aqueous extracts of the leaves. These two growth-condition treatments had no significant effect on total ergosterol content of the fungal mycelia, contrary to an earlier hypothesis that availability of plant lipids would lower fungal ergosterol contents. Mycelial content of free ergosterol was about twice as variable as that for total (free plus esterified) ergosterol. Total ergosterol (data pooled for all species) was strongly correlated to organic mycelial mass (r² = 0.43, P < 0.00001, and slope = 4.59 μg of ergosterol mg of organic mass^-1).     

Use of gas chromatography-mass spectrometry/solid phase microextraction for the identification of MVOCs from moldy building materials

Gas chromatography-mass spectrometry/solid phase microextraction (GC-MS/SPME) was applied to identify microbial volatile organic compounds (MVOCs) in water-damaged, mold-infested building materials (gypsum board papers (n=2), mineral wool, and masonite) and in cultivated molds (Aspergillus penicillioides, Stachybotrys chartarum, and Chaetomium globosum). Three SPME fibers (65-microm PDMS-DVB, 75-microm Carboxen-PDMS, and 70-microm Carbowax-stableflex) designed for automated injection were used of which the latter showed best performance. A number of previously reported MVOCs were detected both in the building materials and the cultivated molds. In addition, methyl benzoate was identified both in the S. chartarum and A. penicillioides cultures and in the building materials. SPME combined with GC-MS may be a useful method for the determination of MVOCs emitted from mold-infested building materials.     

Legionella in industrial cooling towers: monitoring and control strategies

Aims: Legionella contamination of industrial cooling towers has been identified as the cause of sporadic cases and outbreaks of legionellosis among people living nearby. To evaluate and control Legionella contamination in industrial cooling tower water, microbiological monitoring was carried out to determine the effectiveness of the following different disinfection treatments: (i) continuous chlorine concentration of 0·01 ppm and monthly chlorine shock dosing (5 ppm) on a single cooling tower; (ii) continuous chlorine concentration of 0·4 ppm and monthly shock of biocide P3 FERROCID 8580 (BKG Water Solution) on seven towers. Methods and Results: Legionella spp. and total bacterial count (TBC) were determined 3 days before and after each shock dose. Both strategies demonstrated that when chlorine was maintained at low levels, the Legionella count grew to levels above 10⁴ CFU l^?1 while TBC still remained above 10⁸ CFU l^?1. Chlorine shock dosing was able to eliminate bacterial contamination, but only for 10–15 days. Biocide shock dosing was also insufficient to control the problem when the disinfectant concentration was administered at only one point in the plant and at the concentration of 30 ppm. On the other hand, when at a biocide concentration of 30 or 50 ppm was distributed throughout a number of points, depending on the plant hydrodynamics, Legionella counts decreased significantly and often remained below the warning limit. Moreover, the contamination of water entering the plant and the presence of sediment were also important factors for Legionella growth. Conclusions: For effective decontamination of outdoor industrial cooling towers, disinfectants should be distributed in a targeted way, taking into account the possible sources of contamination. Significance and Impact of the Study: The data of the research permitted to modify the procedure of disinfection for better reduce the water and aerosol contamination and consequently the exposure risk.     

Microbiological Analysis of Food Contact Surfaces in Child Care Centers

A study of six child care centers was conducted to assess the microbiological quality of three food contact surfaces (one food serving surface and two food preparation surfaces) and one non-food contact surface (diaper changing surface) to determine the effectiveness of cleaning and sanitization procedures within the facilities. Aerobic plate counts (APCs) and Escherichia coli/coliform counts of 50-cm² areas on all surfaces were determined using standard microbiological swabbing methods. Samples were taken three times a day (preopening, lunchtime, and following final cleanup) twice per month for 8 months in each child care center (n = 288 sampling times). Mean log APCs over the survey period were 1.32, 1.71, 1.34, 1.96, 1.50, and 1.81 log CFU/50 cm² for the six centers. Mean log coliform counts were 0.15, 0.40, 0.33, 1.41, 0.28, and 1.12 CFU/50 cm² for the same centers. Coliforms were detected in 283 of 1,149 (24.7%) samples, with counts ranging from 1 to 2,000 CFU/50 cm², while E. coli was detected in 18 of 1,149 (1.6%) samples, with counts ranging from 1 to 35 CFU/50 cm². The findings of this study demonstrated that the extent of bacterial contamination was dependent on the center, time of day, and the area sampled. While no direct correlation between contamination and illness can be made, given the high risk of food-borne illness associated with children, microbial contamination of food contact or non-food contact surfaces is an aspect of food safety that requires more attention. Emphasis on training and the development of modified standard sanitation operating procedures for child care centers are needed to reduce potential hazards.     

Zeralenone and ergosterol contents in corn samples of Transylvania, Romania

70 corn samples for feed use out of the harvests of the years 2001 and 2002 were collected randomly in Transylvania, Romania and analysed for zearalenone and for ergosterol as fungal mass indicator. High ergosterol contents were found in some samples with a maximum value of 72 mg/kg, zearalenone contents showed values between 4 and 2250 μg/kg in samples positive, indicating a rather high degree of fungal contamination as well as a serious zearalenone contamination. Presented at the 25^th Mykotoxin Workshop in Giessen, Germany, May 19–21, 2003     

Mould and mycotoxin contamination of stored corn in Turkey

A total of 167 corn samples, including imported and locally grown corn, were obtained from various regions and store houses in Turkey and surveyed for mould occurrence and mycotoxin content. The mould contamination level was 10⁵ — 10⁶ colonies/g.A. flavus, A. niger, F. oxyporum, P. variable, andRhizopus spp. However, the dominant flora showed significant differences between the imported and domestic corns. Afiatoxin B₁ was found in 16 % of the samples ranging from 2–74μg/kg. Ochratoxin A and sterigmatocystin were found at minimum detection levels. Mycotoxin production characteristics of mould isolates were also determined.     

Trace Metals in Fleece Wool and Correlations with Yellowness

The presence of copper and iron in metal-doped wool has been shown previously to be associated with the production of free radicals and yellowing in photo-irradiated wool. In this study, the yellowness and trace metal content of 700 wool samples was measured to determine if photoyellowing, catalysed by metals, is a major determinant of the colour of fleece wool. Iron and copper content did not positively correlate with yellowness and yellower wool tended to have lower levels of these metals. Instead, a strong positive correlation of yellowness with the calcium, manganese and magnesium content was observed in yellow wools. High levels of calcium and magnesium is consistent with biofilm formation by Pseudomonas bacteria that have previously been associated with non-scourable staining of wool.