Control of Growth of Disease-causing Microorganisms and Greenhouse Gas Emissions from Swine Operations using Zinc Oxide Nanoparticles

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1 The Canadian Society for Bioengineering The Canadian society for engineering in agricultural, food, environmental, and biological systems. La Société Canadienne de Génie Agroalimentaire et de Bioingénierie La société canadienne de génie agroalimentaire, de la bioingénierie et de l environnement Paper No. CSBE Control of Growth of Disease-causing Microorganisms and Greenhouse Gas Emissions from Swine Operations using Zinc Oxide Nanoparticles Alvin C. Alvarado Prairie Swine Centre Inc., Saskatoon, Saskatchewan; alvin.alvarado@usask.ca Bernardo Z. Predicala Prairie Swine Centre Inc., Saskatoon, Saskatchewan; bernardo.predicala@usask.ca Written for presentation at the CSBE/SCGAB 2013 Annual Conference University of Saskatechewan, Saskatoon, Saskatchewan 7-10 July 2013 ABSTRACT The effectiveness of zinc oxide (ZnO) nanoparticles in reducing the levels of diseasecausing microorganisms and greenhouse gas (carbon dioxide and methane) emissions in swine production operations was investigated in two fully instrumented and identical environmental chambers at the Prairie Swine Centre barn facility in Saskatoon. Each chamber housed 6 growerfinisher pigs. A ventilation air recirculation system was installed in each chamber; one chamber had a filter loaded with ZnO nanoparticles installed in the recirculation duct while the other chamber had filter pad only (not loaded with nanoparticles). Throughout the 15-day trial, the effect of ZnO nanoparticles on bioaerosols, greenhouse gases and pig performance was assessed. Results indicated that partial filtration of air in the chamber with a filter with zinc oxide nanoparticles in the ventilation recirculation system achieved reduction in bioaerosol concentrations at the exhaust stream and animal-occupied zone. In addition, the installation of air filtration system with ZnO nanoparticles in the chamber did not have any beneficial or adverse impact on carbon dioxide and methane emissions as well as on average daily gain and feed intake of pigs. Further studies using other deployment techniques will be conducted to provide a more comprehensive evaluation of the feasibility of nanoparticle application in swine facilities. Keywords: Nanoparticles, zinc oxide, bioaerosols, greenhouse gases, swine production Papers presented before CSBE/SCGAB meetings are considered the property of the Society. In general, the Society reserves the right of first publication of such papers, in complete form; however, CSBE/SCGAB has no objections to publication, in condensed form, with credit to the Society and the author, in other publications prior to use in Society publications. Permission to publish a paper in full may be requested from the CSBE/SCGAB Secretary, 2028 Calico Crescent, Orleans, ON, K4A 4L7 or contact secretary@bioeng.ca. The Society is not responsible for statements or opinions advanced in papers or discussions at its meetings.

2 INTRODUCTION In recent years, the rapidly developing field of nanotechnology has produced new nanomaterials that were proven effective in environmental remediation applications such as air purification (Kim et al., 2006; Nonami et al., 2004), and groundwater and wastewater treatments (Hu et al., 2005; Varadhi et al., 2005; Elliott and Zhang, 2001). While some of these nanoparticle applications in other industries are already in commercial stage, there has been no effort to take advantage of the advances in nanotechnology to address sustainability issues in the swine industry regarding emissions. This study examined the application of nanoparticles as an effective, safe, and viable means for reducing levels of hazardous gases and disease-causing microorganisms in swine production environments. Results from a previous project at the Prairie Swine Centre (PSCI) demonstrated that the use of nanoparticles can effectively reduce the levels of ammonia and hydrogen sulfide emitted from swine manure slurry (Asis and Predicala, 2006). However, our previous work also highlighted other significant aspects that still need to be investigated before this technology can be widely applied to the livestock industry. It would be useful to determine the effectiveness of these nanoparticles on greenhouse gas and bioaerosol emissions. Greenhouse gas and bioaerosol emissions are important concerns not only for swine operations but also to other types of livestock operations such as dairy and poultry. Bioaerosols are airborne particles of biological origin, including viruses, bacteria, fungi, and all varieties of living microorganisms; these are typically involved in the causation of diseases for both animals and workers. Knowing that one important property of nanoparticles is its ability to control growth of microorganisms (Sunada et al., 1998), it is then logical to evaluate whether nanoparticles can be used in controlling the airborne transmission of certain diseases within or between livestock barns. A series of laboratory-scale experiments were conducted to determine the impact of different types of nanoparticles on the growth and population of microorganisms commonly encountered in swine barn environments. Results showed that zinc oxide (ZnO) nanoparticles had the most effective antimicrobial activity among all the nanoparticles tested. In addition, ZnO nanoparticles have proven effective in reducing hazardous gases such as hydrogen sulphide and ammonia in swine barns based from previous related study (Alvarado, 2011). Thus, ZnO nanoparticles were selected for this present study. The goal of this study was to investigate the use of nanoparticles for controlling bioaerosol and greenhouse gas (CO 2 and CH 4 ) emissions from livestock operations, and to assess its impact on pig performance through average daily gain and average daily feed intake. MATERIALS AND METHODS Description of facilities and experimental set-up The effectiveness of the use of zinc oxide (ZnO) nanoparticles in reducing greenhouse gas emissions and levels of microorganisms was evaluated in two fully instrumented and identical environmental chambers at the PSCI barn facility. Each chamber has inside dimensions of 4.2 m x 3.6 m x 2.7 m. Ceiling and internal walls were fully covered with stainless steel sheets to eliminate emissions from these surfaces. Figure 1-A shows the pen layout of one chamber which was a mirror image of the other. The pen area has a slatted concrete floor near the chamber door and a solid floor extending towards the opposite end of the pen. The solid floor has a slope of 8% towards the slatted floor. A commercial feeder and cup-type water drinker were installed on one side of the plastic penning. The pen was surrounded with plastic matrix flooring for easy access to the collection tubs underneath the slatted floor. The tubs in the two chambers were almost identical in size with dimensions of approximately 2 m x 1.25 m x 0.3 m. 2

3 Each chamber was operated on a negative pressure ventilation system. Fresh air was forced through a filtration unit (Circul-Aire USA-H204-B, Dectron International, Roswell, GA, USA) by a 0.6-m diameter centrifugal fan (Delhi BIDI-20, Delhi Industries Inc., Delhi, ON, Canada) before entering the chambers through an actuated ceiling inlet. The room air was exhausted from the chamber through a sidewall exhaust fan (H18, Del-Air Systems Inc., Humboldt, SK, Canada) and into the exhaust duct with a flow measuring device to monitor the airflow rate. To condition the entering air at desired settings, a 5-ton air conditioning unit (Raka-060 CAZ, Setra Systems, Boxborough, MA, USA) and a 10-kW electric heater (Chromalox, Dimplex North America Ltd., Cambridge, ON, Canada) were used. Except for the air conditioning unit, all these equipment were controlled with a Rapid Control System (Del-Air Systems Inc., Humboldt, SK, Canada). In evaluating the effectiveness of the ZnO nanoparticle treatment during swine production operations, a ventilation air recirculation system was installed in each chamber (Figure 1-A). As shown in Figure 1-B, the system was operated at negative pressure relative to the chamber air space. The axial fan (Godro Multifan, Vostermans BV, IL, USA) drew in contaminated air within the chamber and passed through the 36-centimetre diameter galvanized sheet duct where the filter housing was installed. The filter housing was square-shaped with 38 cm x 38 cm dimension for ease in installation and to meet the required face velocity of the test filter and air flow rate output from the fan. Treated air was then distributed back to the chamber through the 30-centimetre diameter duct connected to the downstream side of the fan. The duct has 8 equally-spaced 3.8- centimetre diameter holes which served as outlets for the treated air to flow back in the chamber airspace. Fan speed was controlled using a manual speed controller (Phason Model MSC-4, Winnipeg, Canada). Experimental procedure At the start of each trial, 6 grower pigs with initial weights of about 55 kg to 60 kg were brought into each chamber; the difference on the average initial weights of pigs in both chambers was within ± 1 kg of each other. The pigs were fed with standard grow-finish diets (from Grower 2 to Finisher 1 feed) which were weighed before putting into the feeders. Air temperature in each chamber was set to 18ºC on the first week and then 17ºC on the second week following standard temperature guidelines for grower pigs (PSCI, 2000). Two replicate trials were conducted; each trial lasted for 15 days. On the 5 th day of each trial, filters were installed in the test rooms. The treatment room had a filter loaded with ZnO nanoparticles. With a fluidized bed filter design and loading amount of 0.28 g/cm 2 of filter cross-sectional area (determined from the previous related study), the test filter was loaded with a total of 405 g of nanoparticles. The control chamber, on the other hand, had a filter pad only (not loaded with nanoparticles). Both filter systems had a dimension of 38 cm (L) x 38 cm (W) x 3.2 cm (T) and made out of a plastic styrene material with honeycomb structure. The upstream and downstream faces of the plastic structure were covered with a commercially-available filter pad material (Model HPE30621 Electrostatic Hammock filter pad, True Blue Company, LaPorte, Indiana, USA) to keep the nanoparticles within the filter. 3

4 Door 2.0 m 0.3 m Collectio n tub Concrete slats 1.2 m Water drinker Plastic penning Slope direction 2.1 m Feeder Solid floor Air inlet Air outlet Recirculation system 0.6 m 1.5 m 1.5 m 0.6 m A fan test filter B Figure 1. Layout of chamber with the added ventilation air recirculation system (A). Schematic diagram (B) of the recirculation system used for the room-scale experiment. 4

5 Sample collection and analysis 1. Bioaerosols Airborne microbial concentration (bioaerosol) in the chamber was monitored according to NIOSH Method 0800 which was designed for identification and assessment of culturable microorganisms in indoor air. Specifically, bioaerosol sampling was done by impaction using an Andersen N-6 sampler. The sampler is a multi-orifice, cascade impactor with 400 holes. It was operated with a Petri dish with R2A agar. Preliminary tests showed that the animal-occupied zone, and the ventilation inlet and exhaust streams were the appropriate location in each chamber to sample bioaerosols and 10-minute duration was the appropriate bioaerosol sampling time. On each sampling day during the actual room-scale experiment, 3 samples were collected inside each chamber at an air flow rate of 28.3 L/min. This was repeated twice to obtain duplicate, consecutive samples per location. Sampling of bioaerosols was done before filter installation (day 5 of the trial) and after filter installation (days 10 and 15). All culture plates were incubated at room temperature (25ºC ± 5ºC) for 72 hr which was determined from preliminary test. After incubation, the CFUs were counted with a colony counter. 2. Greenhouse gases (CH 4 and CO 2 ) Concentrations of greenhouse gases (CH 4 and CO 2 ) in each chamber were determined using gas chromatography. Gas samples were collected using a syringe and about 20 ml sample was injected into an evacuated container. The evacuated containers were sent out to a gas chromatography laboratory at the University of Saskatchewan for analysis. Gas sampling was done before filter installation (day 5) and after filter installation (days 10 and 15 of each trial). During each sampling day, gas levels in each room were monitored near the exhaust area. 3. Pig performance (ADG and ADFI) The effect of the treatment on pig s average daily gain (ADG) and average daily feed intake (ADFI) was assessed. ADG was based on the average growth rates determined by taking the difference in pig s weight at the start and end of the test. ADFI, however, was computed by dividing the total weight of feed consumed by the product of number of pigs and number of days on feed. RESULTS AND DISCUSSION Effect on bioaerosol concentrations Figure 2 shows the total CFU concentrations in the inlet and exhaust streams as well as in the animal-occupied zones of the test chambers. Among the three sampling locations, the total CFU concentrations in the inlet zones of the test chambers regardless of the sampling time were the least. This was expected because the main source of bioaerosols were inside the chambers and besides, these air streams came directly from outside of the barn and were pre-filtered before being distributed to each chamber. In addition, the total CFU in the inlet zone of the treatment chamber ranged from CFU/m 3 to CFU/m 3 ; this was not significantly different (p>0.05) from the control chamber which ranged from CFU/m 3 to CFU/m 3 throughout the trial (Figure 2A). This implied that the two chambers had the same initial condition in terms of bioaerosol concentration. 5

6 Contrary to the inlet zones, variations of CFU levels in the exhaust streams of the test chambers were observed after the test filters were installed in the recirculation ducts on day 5 as shown in Figure 2B. Prior to filter installation (day 0), CFU concentrations in the chambers were not significantly different (p>0.05) from each other. The total CFU concentrations in the treatment chamber achieved about 26% decrease on day 5 after the filter loaded with ZnO nanoparticles was installed in the chamber. However, the CFU levels in the exhaust stream of the control chamber showed increasing trends until day 10. It is important to note that both chambers had similar configurations and operations of the recirculation system; they just differed in the type of filter installed, i.e., the treatment chamber had filter with ZnO nanoparticles while the control chamber had filter pad only and not loaded with nanoparticles. Thus, the observations above indicate that the decrease in CFU concentration in the treatment chamber can be attributed to the ZnO nanoparticles loaded onto the filters. On day 5, the mean (± SD) CFU concentration in the exhaust stream of the treatment chamber was ± 97.4 CFU/m 3 while the control chamber had ± CFU/m 3. Among the three sampling locations, the animal-occupied zone of each chamber had the highest bioaerosol levels regardless of sampling times (Figure 2C). On average, the total CFU concentrations in the animal-occupied zones (about 0.5 m from pen floor) of the treated chamber were 4 times higher its inlet concentrations, while the control chamber values were 5.2 times higher its corresponding inlet concentrations. This could be due to the fact that this particular location was closer to where the manure was deposited (pen floor and the pit) compared to the other sampling locations. Comparing the treatments, a slight reduction (6%) relative to the day 0 value was observed in the treatment room 5 days after the filter with ZnO nanoparticles was installed. The CFU levels in the control chamber, however increased as the trial progressed. On day 5, the mean (± SD) CFU concentration in the animal-occupied zone of the treatment chamber was ± CFU/m 3 while the control chamber had ± CFU/m Inlet 4000 CFU/m 3 of air Day 0 Day 5 Day 10 Sampling time Treated Control A 6

7 5000 Exhaust 4000 CFU/m 3 of air Day 0 Day 5 Day 10 Sampling time Treated Control B Animal CFU/m 3 of air Day 0 Day 5 Day 10 Sampling time Treated Control C Figure 22. Mean (± SD) CFU concentration in the inlet (A) and exhaust (B) streams and in the animal-occupied zone (C) of the treated and untreated (control) chambers during the room-scale experiment. As observed in Figure 2, the filter with ZnO nanoparticles worked effectively in reducing the levels of bioaerosol in the animal-occupied zone and in the ventilation exhaust stream in the chamber at 5 days after the filter was installed in the recirculation duct; the total CFU levels, however started to increase after 5 days. In addition, it was observed that the reduction of CFU concentration at the exhaust stream in the treated chamber was higher than those at the animal-occupied zone. This could be due to the air movement in the chamber while the air recirculation system was in 7

8 operation. A portion of air in the manure pit was drawn in by the fan; the remainder may go upward through the floor slats and most likely be captured at the animal level during bioaerosol sampling. On the other hand, the recirculation duct expelled treated air that passed through the ZnO-treated filter through outlets directed towards the exhaust stream. Effect on greenhouse gas levels Actual levels of carbon dioxide and methane in each chamber are plotted in Figure 3. Variations in carbon dioxide levels in the treated and control chambers followed similar trend (Figure 3A). On day 0, mean carbon dioxide concentration in the treated chamber was ± ppm while the control chamber had ± ppm; these levels were almost unchanged until day 5. However, on day 10, mean carbon dioxide levels in the treated and control chambers increased to ± ppm and ± ppm, respectively. Opposite trend was observed for the actual concentration of methane in each chamber as shown in Figure 3B. Mean methane concentration in the treated chamber on day 0 was 4.7 ± 2.6 ppm; this level increased slightly on day 5 but decreased to 3.8 ± 3.5 ppm on day 10 after the filter with ZnO nanparticles was installed. Similar pattern was observed in the control chamber. On day 0, mean methane levels in the control chamber was 5.5 ± 0.3 ppm and dropped to 4.0 ± 3.4 ppm on day 10. Comparing the treatments, concentrations of carbon dioxide and methane in the treated chamber was not significantly different (p>0.05) from the levels in the control chamber Carbon dioxide Concentration, ppm Day 0 Day 5 Day 10 Sampling time Treated Control A 8

9 Concentration, ppm Methane Day 0 Day 5 Day 10 Sampling time Treated Control B Figure 3. Mean (± SD) concentration of greenhouse gases from samples collected in the untreated (control) and treated chambers. Effect on pig performance The average daily gain and average daily feed intake of the pigs in the control chamber were slightly higher than those in the treated chamber by about 0.03 kg/daypig and 0.02 kg/day-pig, respectively (Table 1). It was observed that the average daily feed intake followed the same trend as average daily gain which could be due to the positive correlation between the pig s average daily feed intake and average daily gain (NRC, 1987). However, no significant difference between the two chambers was observed for these parameters (p>0.05). Thus, the results indicated that the installation of air filter with zinc oxide nanoparticles in the recirculation system had no significant adverse or beneficial effect on pig performance. Table 1. Average daily gain and feed intake of pigs in the control and treated chambers during the room-scale experiments. Hog performance parameters Treated Control Average daily gain (ADG), kg/day-pig 0.90 ± ± 0.12 Average daily feed intake (ADFI), kg/day-pig 2.70 ± ± 0.52 CONCLUSIONS The installation of air filter loaded with zinc oxide nanoparticles in the ventilation air recirculation system achieved 26% and 6% reduction in total CFU concentrations at the exhaust stream and animal-occupied zone, respectively. The partial filtration set-up can be made more effective with better capture of air in the room to pass through the filtration system. The treatment had no significant impact on carbon dioxide and methane levels in the chamber. Also, pig performance was not adversely affected by the installation of the ZnO-treated filter in the room. 9

10 Acknowledgements. The authors would like to acknowledge the financial support provided by the Agriculture and Agri-Food Canada through the Canadian Agricultural Adaptation Program (CAAP) delivered by the Agriculture and Food Council of Alberta, and the Saskatchewan Agriculture Development Fund to this research project. The authors also acknowledge the strategic funding provided by Sask Pork, Alberta Pork, Manitoba Pork Council, Ontario Pork, and the Saskatchewan Ministry of Agriculture to Prairie Swine Centre Inc. REFERENCES Alvarado, A Control of hydrogen sulphide, ammonia and odour emissions from swine barns using zinc oxide nanoparticles. M.Sc. Thesis. Saskatoon, Saskatchewan: University of Saskatchewan, Department of Chemical and Biological Engineering. Asis, D. and B. Predicala Investigation of use of nanoparticles for reducing gas emissions from swine manure. MBSK ASABE/CSBE North Central Intersectional Meeting, Saskatoon, SK. Elliott, D. W., and W. Xian Zhang Field assessment of nanoscale bimetallic particles for groundwater treatment. Environ. Sci. Technol. 35, Hu, J., J., G. Chen, and I. M.C. Lo Removal and recovery of Cr(VI) from wastewater by maghemite nanoparticles. Water Res. 39: Kim, J. H., G. Seo, D.L. Cho, B. C. Choi, J. B. Kim, H. J. Park, M. W. Kim, S. J. Song, G. J. Kim, and S. Kato Development of air purification device through application of thin-film photocatalyst. Catalysis Today. 111: Nonami, T. H. Hase, and K. Funakoshi Apatite-coated titanium dioxide photocatalyst for air purification. Catalysis Today.96: NRC Nutrient requirements of swine, National Research Council. National Academy Press, Washington, D.C. Sunada, K., Y. Kikuchi, K. Hashimoto and A. Fujishima Bactericidal and detoxification effects of TiO2 thin film photocatalysts. Environ. Sci. Technol. 32, Varadhi, S.N., H. Gill, L. Apoldo, K. Liao, R. Blackman, and W. Wittman Full-scale nanoiron injection for treatment of groundwater contaminated with chlorinated hydrocarbons. In Proceedings of the Natural Gas Technologies Conference. Orlando, Florida. 10