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, allowingdetermination of a direct measurement of biomass density byweighing. Biomass density, ergosterol content, and beta-N-acetylhexosaminidase(3.2.1.52) activity were monitored from inoculation to stationaryphase. Regression analysis showed a good linear correlationto biomass density for both ergosterol content and beta-N-acetylhexosaminidaseactivity. The same two mold species were inoculated onto wallpaperedgypsum board, from which a direct biomass measurement was notpossible. Growth was measured as an increase in ergosterol contentand beta-N-acetylhexosaminidase activity. A good linear correlationwas seen between ergosterol content and beta-N-acetylhexosaminidaseactivity. From the experiments performed on agar medium, conversionfactors (CFs) for estimating biomass density from ergosterolcontent and beta-N-acetylhexosaminidase activity were determined.The CFs were used to estimate the biomass density of the moldsgrown on gypsum board. The biomass densities estimated fromergosterol content and beta-N-acetylhexosaminidase activitydata gave similar results, showing significantly slower growthand lower stationary-phase biomass density on gypsum board thanon agar.

INTRODUCTION

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Introduction

In moist buildings mold growth is suspected to cause healthproblems. Methods allowing quantification of mold on buildingmaterials are therefore important when evaluating mold damagein buildings and the quality of the remediation efficacy. Determinationof the ergosterol content is commonly used to estimate fungalbiomass in various environments (1, 4, 10, 11) and has beenshown to be a suitable marker for estimation of fungal concentrationson building materials contaminated by mold (7). However, determinationof the ergosterol content is rarely used in practice for investigationsof building materials as it requires advanced equipment andhighly trained personnel. Instead, cultivation methods and microscopyof tape lift samples with counts of fungal propagules (conidia,hyphae, conidiophores) are used. Quantification of mold biomassby microscopy may be influenced by a high degree of observersubjectivity (5, 12) and interference due to debris and fibersand can only be performed by trained microbiologists. Cultivationof mold from building materials usually takes 4 to 7 days andis not well suited for quantification of fungal biomass sinceit has been shown to be mainly a measure of sporulation andsince spores only constitute 0 to 5% of the total fungal biomass(10).

Beta-N-acetylhexosaminidase activity was shown to correlatewith the fungal index molecules ergosterol and the phospholipidfatty acid 18:2 ${omega}$ 6 in soil samples (4). A method for detectionand quantification of mold biomass on building materials basedupon fluorogenic detection of beta-N-acetylhexosaminidase activityhas been developed (the MycoMeter test) (9). The method is muchsimpler to perform than ergosterol detection and can be performedon-site.

This study compares measurements of fungal biomass growing onagar and on gypsum plates, measured as both biomass dry weightand ergosterol and enzyme activity.

MATERIALS AND METHODS

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Materials and Methods

Fungal strains.
Stachybotrys chartarum (IBT 9695) and Aspergillus versicolor(IBT 16000) were obtained from the culture collection of Biocentrum,Technical University of Denmark.

Substrate and inoculation.
The inoculum cultures were grown for 6 weeks on Czapek yeast(autolysate) extract agar medium. Suspensions of spores fromthese cultures were made by adding 10 ml of sterile tap waterwith 0.01 ml of Tween 80 to an agar plate and stirring witha Drigalski spatula. The spore suspensions were then filteredthrough a nylon filter (41-µm pore size), and the filtratewas diluted to a final volume of 40 to 80 ml.

A V8 agar medium (2) was used for the experiments on agar plates(9- by 9-cm square). Sterilized cellophane membranes were placedon top of the agar medium (8). The spore solution (0.2 ml) wasthen added to the agar plates and the spores were distributedevenly by stirring with a Drigalski spatula.

Gypsum board with wallpaper was cut into discs with a diameterof 11.5 cm. These discs were placed in 16-cm-diameter plasticcontainers, and 45 ml of sterile tap water was added. After24 h the water had been absorbed by the gypsum discs. A totalof 12 discs were inoculated with each mold species. The discswere then inoculated using the same procedure as described forthe agar plates except that 0.25 ml of the suspension was usedand the use of cellophane membranes was omitted. After inoculationthe discs were incubated at 25°C in a glass desiccator (innerdiameter, 30 cm) to which 500 ml of sterilized distilled waterhad been added to maintain humid conditions.

Measurements of growth.
A 9-cm² sample was cut from the agar plates overlaid with cellophane,and the mold was gently washed off the cellophane onto a preweighedmembrane filter and dried at 80°C for 24 h for determinationof biomass dry weight (6). A 1-cm² sample was cut for determinationof enzyme activity and ergosterol content. Three replicateswere used for determination of biomass dry weight, enzyme activity,and ergosterol content. On days 0, 1, and 2 the three replicateswere pooled for detection of biomass dry weight.

Circular plugs (diameter, 1 cm; thickness, 1 to 2 mm) were sampledfrom the gypsum plates with a cork bore. The plugs containedthe wallpaper, the paper from the gypsum plate, and a smallamount of attached gypsum. The plugs were transferred into a5-ml vial. Enzyme activity and ergosterol content were determinedfrom the same plug. At each sampling time three replicates wereused for determination of ergosterol content and enzyme activity.The replicates were sampled from three different gypsum discs.The experiments on both agar and gypsum board have been conductedtwice, with similar results.

The beta-N-acetylhexosaminidase activity was measured, typicallywithin an hour after sampling, by use of a modified versionof the MycoMeter protocol (MycoMeter handbook, September 2001).One milliliter (enough to cover the plug) of the fluorogenicenzyme substrate solution was added to each container. After30 min of incubation at ambient temperature, the enzyme activitywas quantified by measuring the fluorescence on a Picofluorfluorometer (Turner Designs, Sunnyvale, Calif.) according tothe instructions given in the MycoMeter protocol. After determinationof the enzyme activity, the samples were stored at -20°Cfor ergosterol quantification.

Ergosterol content was determined by a slightly modified versionof the method previously described (6). A mixture of 4.0 mlof methanol, 1.0 ml of 2.0 M NaOH, and 100 µl of 1.0-µl/ml[4-²H₂]ergosterol was added to each vial. The samples were hydrolyzedat 85°C for 90 min. After cooling to room temperature, theergosterol was extracted twice with 0.8 ml of pentane evaporatedto dryness, and the sample was derivatized using pyridine-N,O-bis(trimethylsilyl)trifluoroacetamide(BSTFA) (1:1). Samples were then redissolved in 50 µlof toluene and analyzed using gas chromatography-tandem massspectrometry (GC-MS/MS) (Finnigan GCQ). Separation was performedon a 0.10-µm diameter, 30-m long HP-5 Trace column (Hewlett-Packard,Avondale, Pa,). m/z 363 and 365 were used as parent ions andm/z 157 and 159 were the daughter ions for ergosterol and [4-²H₂]ergosterol,respectively.

RESULTS

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Results

S. chartarum was inoculated onto agar covered with cellophane,which allowed for determination of biomass density. Figure 1shows biomass density, ergosterol content, and beta-N-acetylhexosaminidaseactivity monitored over a 7-day period. Approximately 5 daysafter inoculation, the biomass density reached a maximum (stationaryphase) of 1.7 mg per cm². Linear regression analysis of datafrom days 1 to 7 showed that the biomass density correlatedwith both ergosterol content (r² = 0.968; P < 0.001) andenzyme activity (r² = 0.935; P < 0.001). Experiments withA. versicolor gave similar results with respect to both maximumbiomass density (1.4 mg per cm²) and correlation to ergosterolcontent (r² = 0.922; P < 0.001) and enzyme activity (r² =0.968; P < 0.01). The coefficients of variation were 15 to37% for ergosterol content and 21 to 28% for enzyme activity.Conversion factors (CF) for both ergosterol content and enzymeactivity were calculated from the slope of the linear regression(Table 1) and used for estimating the biomass dry weight densityof molds on the gypsum board.

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FIG. 1. Biomass density (dry weight) (A), ergosterol content (B), and ß-N-acetylhexosaminidase activity per cm² (C) of S. chartarum grown on V8 agar overlaid with a cellophane membrane.

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TABLE 1. CFs and estimated biomass densities for growth on gypsum board

With gypsum board both ergosterol content and enzyme activitydata showed that the molds grew significantly slower, takinglonger to reach maximum biomass density (Fig. 2). The estimatedmaximum biomass density from determinations of ergosterol contentand enzyme activity on the gypsum board yielded values in thesame order of magnitude for the two species (Table 1) and wasaround a quarter to a third (on average 23% for A. versicolorand 32% for S. chartarum) of that obtained on agar.

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FIG. 2. Ergosterol content (A) and ß-N-acetylhexosaminidase activity (B) of S. chartarum grown on wallpapered gypsum board.

The detection limits were determined to be approximately 10ng for ergosterol detection and 12 arbitrary fluorescence unitsfor enzyme activity. Values above the detection limits weregenerally seen earlier with the enzymatic assay than with theergosterol content analysis.

DISCUSSION

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Discussion

The present study has shown a linear correlation between bothergosterol and ß-N-acetylhexosaminidase activity andthe actual biomass density measured by weighing the mold growingon agar plates covered with cellophane. This confirms the previouslydescribed correlation between ß-N-acetylhexosaminidaseactivity and ergosterol content in soil samples (4) as wellas demonstrates that the enzyme is growth related, representingthe fungal biomass in both the growth and the stationary phase.

The use of enzyme activity as a fungal biomass indicator haspreviously been attempted, and a good correlation was foundbetween laccase activity and the biomass of Agaricus bisporuson rye grain (3). However, not all filamentous fungi possesslaccase activity, which excludes the use of laccase activityas a general indicator of fungal biomass. Unlike laccase, ß-N-acetylhexosaminidaseactivity appears to be present in all filamentous fungi, asit has been found in each of 42 arbitrarily chosen species (unpublishedresults).

The CF for ergosterol found in the present study (5.1 to 5.4µg of ergosterol/mg of biomass dry weight) is within thesame range as was earlier reported (1, 3, 11). Seitz et al.(11) found an ergosterol content of 2.3 to 5.9 µg/mg ofdry weight in three fungi grown in liquid cultures in malt extractmedium, and Matcham et al. (3) found 2 to 2.7 µg of ergosterol/mgof dry weight in Agaricus bisporus grown in liquid cultureson malt extract medium. The average ergosterol content in 12aquatic hyphomycetes was 5.5 µg/mg of dry weight (rangingfrom 2.3 to 11.5 µg/mg of dry weight) (1). The CFs foundin the present study were established on agar medium and maynot be representative when fungi are growing on different buildingmaterials such as gypsum board. Other factors, such as interspeciesdifferences, age of the mold growth, and growth conditions ingeneral, may affect the CFs and therefore limit the accuracyof the biomass density estimates. The data suggest that theinterspecies difference may be more significant for the enzymeactivity than for the ergosterol content.

While estimation of biomass density from CFs may have limitationswhen it comes to very accurately predicting the fungal biomassdensity, the present study has shown that ergosterol contentand beta-N-acetylhexosaminidase activity measurements yieldthe same information on the time course of fungal growth andthe level of biomass density obtained. Quantification of ergosterolis used in scientific studies of mold on building materialsbut requires advanced equipment and highly trained personnel,which limits the use of ergosterol measurements in mold remediationcases. The beta-N-acetylhexosaminidase activity method can beperformed on-site with relatively simple equipment and is thereforea useful and convenient alternative for quantification of moldbiomass on building materials.

ACKNOWLEDGMENTS

This study is part of the Danish research program "Mould inBuildings." The program is supported by the Danish Governmentand private companies through the Danish Research Agency.

FOOTNOTES

* Corresponding author. Mailing address: MycoMeter ApS, Lersoe Parkallé 40, 2100 Copenhagen, Denmark. Phone: 45 39161072. Fax: 45 39161073. E-mail: mreeslev{at}mycometer.dk.

REFERENCES

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