Evaluation of Microbial Growth and Survival on Construction materials treated with Anabec NewBuild 30

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1 Evaluation of Microbial Growth and Survival on Construction materials treated with Anabec NewBuild 30 Absar Alum, Ph.D. Department of Civil and Environmental Engineering Arizona State University Tempe, AZ Final Report Prepared for Anabec Corporation April 8, 2005

2 INTRODUCTION The Anabec Corporation, of Clarence New York is marketing NewBuild 30, a proprietary product, to control microbial growth in residential and commercial buildings. A study was performed to evaluate the product against some of the most common microorganisms detected in indoor air samples in water-damaged building materials. The test microorganisms included three types of bacteria (Escherichia, Bacillus, and Salmonella) and two types of fungi (Penicillium and Stachybotrys). The test and control samples for this study were provided by Anabec Corporation. All the test material was treated with NewBuild 30 by Anabec. The microbial challenge studies were conducted at the National Science Foundation Water Quality Center at Arizona State University. The study was planned to evaluate the potential for microbial growth on construction materials inoculated with selected microorganisms before and after treatment with NewBuild 30 agent. Mycelial fragments, fungal spores, and bacterial cells are commonly detected in indoor air samples and on waterdamaged building materials. These facts were considered in the experimental plan. For the mold challenge study, separate set of experiments were performed to test the NewBuild 30 agent against fungal mycelium and fungal spores. Experiments with bacterial agents were performed using actively growing bacterial cells. Research was conducted in controlled environmental chambers, and temperature and relative humidity were monitored regularly. Experiments were designed to determine the ability of the NewBuild 30 to both deflect microbial growth. The objective of this study was to verify the proof of concept for microbial inactivation capabilities of the NewBuild 30 agent on drywall and pinewood. MATERIAL AND METHODS Test Microorganisms The pure cultures of E. coli (ATCC 25922), Bacillus subtilis (ATCC 27689), Salmonella typhimurium (ATCC ), Penicillium chrysogenum (ATCC 10134) and Stachybotrys chartarum (ATCC 18541) were obtained from American Type Culture Collection (ATCC Manassas, VA). The standard procedures described in the Standard Methods for the Examination of Water and Wastewater, 19th Edition were followed for bacterial culture maintenance, propagation, and samples assays. The fungal cultures were maintained on culture media recommended by ATCC using standards methods (Table1). Test Material The material tested in this study was provided by Anabec. The test material consisted 3x3 coupons of pinewood and drywall thoroughly treated with NewBuild 30 agent. In this study, non-treated coupons of drywall and pinewood were included as control samples. The surface area of each coupon was divided into six inoculation zones (Figure1). Each inoculating zone was seeded with specified number of microorganisms. Inoculation Strategy For the mold challenge study, two strategies were used to inoculate the test and control samples. The first strategy included the use of actively growing fungal mycelium on agar media. One-centimeter diameter fungal-mycelial-mats were cut from actively growing pure cultures of Penicillium and Stachybotrys. The fungal mats were placed on samples in inverted

3 position. In these positions, fungal mycelia were cushioned between samples surface and nutrient medium, which provided the best possible scenario for initiation to initiate fungi on samples. This scenario never happens in real world; however, the technique was employed to provide the worst test conditions. The second strategy included the use of fungal spores to inoculate test material. Pure cultures of Penicillium and Stachybotrys were grown in petri dishes and maintained until full sporulation was achieved. Fungal spores were transferred onto coupons by direct contact methods. Approximately, 10 million spores were deposited on each sample points in all samples. This inoculation method represents the natural scenario where airborne spores land on building materials and initiate fungal growth under favorable conditions. For bacterial challenge study, drywall and pinewood coupons treated with NewBuild 30 agent were inoculated with high numbers (~10 8 ) of each of the bacterial species. Control samples for drywall and pinewood materials were also inoculated with same number of test bacterial species. Samples were incubated for 15 days. Test and control samples were assayed from the respective bacterial species at specified time interval. Test Conditions In order to test the ability of NewBuild 30 agent to deflect microbial growth, construction materials was treated with the NewBuild 30 agent and inoculated with high titer stocks ( ) of the test microorganisms. Control sample were also inoculated with same numbers of test microorganisms. Inoculated samples were placed in tightly sealed polyethylene containers. The samples inoculated with fungal mycelium and bacterial cell were incubated under >90% relative humidity. Whereas, samples inoculated with fungal spores were incubated under <30% relative humidity. The inoculated samples were incubated at 37 C and 28 C. Treated material was aged in the environmental chambers for a designated period of time (8 weeks for fungi and 2 weeks for bacteria). Samples were periodically removed from incubation chambers and analyzed for microbial growth and viability. Microbial Growth and Viability Assay The samples inoculated with fungal mat (mycelium) were monitored every other day for visible mold growth (Figures 2, 3, and 4). The percent surface area covered by the growth of Penicillium and Stachybotrys was visually appreciated. The samples inoculated with fungal spores were assayed for spore viability after 30, 60 and 75 days of incubation at specified temperatures. Fungal spores were recovered using glycine elution buffer and spores were tested for viability using hanging drop method. Briefly, the recovered spores were suspended in a drop of water and deposited on a glass slide. The glass slide was incubated in humidity chamber in inverted position. The number of germinated spores were counted using microscope. The samples inoculated with bacterial cells were assayed for bacterial cell viability on day 0, 1, 2, 3, 4, 5, 7, 9, 11, 13, and 15. Bacterial cells were recovered using glycine elution buffer and were tested for viability using membrane filtration on selective medium (Figure 5). RESULTS The mold growth data for samples inoculated with fungal mycelium is presented in Figures 6 and 7. Untreated drywall and pinewood coupons incubated at 37 C and 28 C, showed profuse growth of Penicillium and Stachybotrys after 75 days of incubation, resulting

4 in entire surface area covered with fungal growth. The treated drywall coupons incubated at 37 C showed very minor growth of Penicillium and Stachybotrys. The area colonized by each of fungal species accounted for less than 0.1% of the total surface area. After 75 days, approximately 1% of the coupons surface was colonized with each of the test fungus. In case of treated pinewood coupons, the samples incubated at 37 C did now show any visible growth of Penicillium and Stachybotrys however, incubation at 28 C resulted in a very minor fungal growth accounting for approximately 0.1% surface area colonization. Independent set of experiments were performed to test the viability of Penicillium and Stachybotrys spores on pinewood and drywall coupons and results are presented in Figures 8 and 9. After 75 days of incubation at 37 C and 28 C, less than 0.1% of the spores inoculated on treated pinewood and drywall samples were found viable. The data suggest that effectiveness of NewBuild 30 agent against fungal spores is not impacted by the type of construction material or temperature profile tested in this study. A series of experiments were performed to evaluate the effectiveness of NewBuild 30 agent to control E. coli, Bacillus subtilis, and Salmonella typhimurium on pinewood and drywall and results are presented in figures 10 and 11. Approximately 100 million cells of E. coli, Bacillus subtilis, and Salmonella typhimurium were inoculated on test and control coupons. After 24 hours of incubation no test bacteria were recovered from treated pinewood or drywall coupons. The treated samples remained negative for all the three test bacteria throughout the course of this study. The data suggests that E. coli, Bacillus subtilis, and Salmonella typhimurium cells are rapidly inactivated on contact with NewBuild 30 treated surfaces. It is important to note that the observations are based on small-scale experiment for proof of concept. However, for the comprehensive measurements of inactivation a detailed inactivation study needs to be performed.

5 Table1. Culture media used for propagation and assay of test microorganisms Microorganisms Propagation Assay Growth conditions ( C) E. coli, TSB m-endo agar 37 Bacillus subtilis, NB ATCC medium Salmonella typhimurium TSB NA 37 Penicillium chrysogenum PDA PDA 25 Stachybotrys chartarum RFA RFA 25 TSB: Trypticase soy agar NA: Nutrient agar NB: Nutrient broth PDA: Potato dextrose agar RFA: Rabbit food agar ATCC medium 713: Nutrient Broth + salts (see appendix1) Figure1. Schematic representation of the coupons surface area Inoculation zone

6 Figure2. Untreated drywall sample inoculated with Penicillium mycelial pads (After 60 days of incubation at 37 C) Figure3. Treated drywall sample inoculated Penicillium mycelial pads (After 75 days of incubation at 37 C) Figure4. Untreated drywall sample inoculated Penicillium mycelial pads (After 75 days of incubation at 28 C)

7 Figure5. E. coli recovered from test and control samples of drywall and wood samples after 24 hours incubation. Note: Lower row is the E. coli recovered from control samples and top row is the E. coli recovered from test samples

8 Figure6. Percent area of samples covered with Penicillium and Stachybotrys growth (Experiments performed at 37 C) Control Treated Drywall Treated Wood Figure7. Percent area of samples covered with Penicillium and Stachybotrys growth (Experiments performed at 28 C) Control Treated Drywall Treated Wood

9 Figure8. Viability of Penicillium and Stachybotrys spores on drywall and pinewood (Experiments performed at 37 C) 1.00E E E E E E E+00 Wood Control Treated Wood Drywall Control Treated Drywall 30 day 60 day 75day Figure9. Viability of Penicillium and Stachybotrys spores on drywall and pinewood (Experiments performed at 37 C) 1.00E E E E E E E+00 Wood Control Treated Wood Drywall Control Treated Drywall 30 day 60 day 75day

10 Figure10. Survival of bacterial species on treated and non-treated wallboard 1.00E+08 Survival of bacterial species on treated and non-treated wallboard Number of Bacteria 1.00E E E E E E E E+00 Day0 Day1 Day2 Day3 Day4 Day5 Day7 Day9 Day11 Day13 Day15 E.coli Bacillus Salmonella Treatment Figure11. Survival of bacterial species on treated and non-treated pinewood Survival of bacterial species on treated and non-treated wood Number of bacteria 1.00E E E E E E E E E+00 Day0 Day1 Day2 Day3 Day4 Day5 Day7 Day9 Day11 Day13 Day15 E.coli Bacillus Salmonella Treatment

11 APPENDIX-1 ATCC medium 713 Nutrient Broth g MgSO 4. 7H 2 O g MnCl mg KCl g Distilled water L Adjust ph to Autoclave at 115C for 30 minutes, Aseptically add FeSO 4 (0.001 M) ml Ca(NO 3 ) 2 (1 M) ml

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