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Poult Sci 2007. 86:2095-2100
© 2007 Poultry Science Association
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ENVIRONMENT, WELL-BEING, AND BEHAVIOR

The Effectiveness of Various Biofiltration Substrates in Removing Bacteria, Endotoxins, and Dust from Ventilation System Exhaust from a Chicken Hatchery

L. Tymczyna1, A. Chmielowiec-Korzeniowska and A. Drabik

Department of Animal Hygiene and Environment, Faculty of Biology and Animal Breeding, University of Agriculture in Lublin, 20–950 Lublin, Poland

1 Corresponding author: leszek.tymczyna{at}ar.lublin.pl


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
The objective of this study was to evaluate the effectiveness of various organic and organic-mineral biofilter media in purifying ventilation exhaust from a chicken hatchery room. Three different substrates were tested. Efficiency levels for the removal of dust, gram-negative bacteria, and bacterial endotoxin were recorded. The microbiological properties of the substrates were also studied. All of the biofilter substrates were highly effective in removing gram-negative bacteria, moderately effective in reducing dust levels, and only slightly effective in removing endotoxin. The substrate that was most efficient in retaining bioaerosols was the organic-mineral medium containing 20% halloysite, 40% compost, and 40% peat, which generally had at least satisfactory efficiency values for removing all of the contaminants tested.

Key Words: biofiltration • endotoxin • gram-negative bacteria • dust • chicken hatchery


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
In hatchery rooms, as in poultry barns, the main components of dust are feed residue, feces, feathers, and epiderm (Whyte, 1993; González-Miret at al., 2006). Poultry dust also contains viruses, bacteria, bacterial toxins, fungi, fungal spores, parasites, and protein antigens. Therefore, workplace and environmental contamination with microorganism and microbial toxins is a major problem associated with poultry production.

The biological agents that pose a hazard for those who work in this industry include the following: viruses, such as Newcastle virus; bacteria and bacterial endotoxins; Chlamydia psttaci; fungi, such as Aspergillus spp.; parasites, such as Toxoplasma gondii; and protein allergens.

A wide range of bacteria can pose a hazard to hatchery workers, including Alcaligenes faecalis, Bordetella spp., Acinetobacter calcoaceticus, Salmonella spp., Yersinia spp., and Listeria monocytogenes (Dutkiewicz et al., 2002).

All of these agents can be carried through the air throughout the workspace in aerosol or particulate form. They can even be carried outdoors through the ventilation system, where they can pose a serious risk to public health. Some poultry pathogens have been reported to cause illness, especially respiratory tract disease, even up to 3 km from their source (Seedorf et al., 1998; Wathes, 1998; Schlegelmilch et al., 2005).

Disinfection of the hatchery facility is not enough to protect the health of hatchery employees and the community at large. Microbial toxins and allergens can be released even from dead microbial material. One method that has been used to prevent the release of airborne biological agents is biofiltration, in which an inexpensive filter system is used to capture airborne droplets and particulates.

The objective of this study was to evaluate the effectivity of various organic and organic-mineral biofilter media in removing microbiological contaminants from ventilation exhaust from a chicken hatchery.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 REFERENCES
 
The study was conducted at the Poultry Hatchery in De bówka, 20 km south of Warsaw, Poland. The hatchery has an annual output of 20 to 25 million Cobb and Ross meat hens, which represents 4% of the national production.

A biofilter was installed in the ventilation outlet of the hatching room (Figure 1Go), which was equipped with 8 hatchers (AS-4H, Petersime, Zulte, Belgium) and 12 incubators (AS-4S, Petersime) with an input of 115,000 eggs. The biofilter measured 2.0 x 1.8 x 1.8 m and included the following components: a high-pressure fan with a maximum capacity of 1,500 m3/h, an air humidifier, and a biofiltration chamber (constructed by the present authors).


Figure 1
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  		Figure 1. Experimental setup: 1 = incubator and hatchery; 2 = humidifier; 3 = air distribution; 4 = biofilter media.

 
The biofiltration chamber was divided into 3 independent parts to facilitate the simultaneous assessment of biofiltration properties of 3 different fillings – beds. The depth of the filter medium was from 1.2 to 1.4 m.

In this study, the following were used: organic medium containing 50% compost and 50% peat (OM); organic-mineral medium containing 20% bentonite, 40% compost, and 40% peat (BM); organic-mineral medium containing 20% halloysite, 40% compost, and 40% peat (HM).

Five series of experiments were carried out during the 6-mo course of the study. In each series of experiments, 5 air samples were collected: 2 in the air intake duct of the biofilter (in the hatchery room) and 3 at the air outlet duct (i.e., 1 at each biofiltration chamber).

The air samples were collected with the help of a stationary aspirator (AS-50, Twomet, Zgierz, Poland) fitted with a polyvinyl chloride filter. The aspirator was constructed so that 1.5 m3 of air was passed directly onto the filter.

The filters were weighed before and after sample collection. The total dust content was then calculated according to the following formula:


Formula

where M1 = the mass of the filter before sampling; M2 = the mass of the filter after sampling; and V = the volume of the sample. Sample volume was calculated as the product of the volumetric flow rate and the sampling time. Results were recorded in terms of milligrams per cubic meter of air.

After sampling, the filters were extracted for 1 h in sterile 0.85% NaCl with 0.05% Tween 80 added as an emulsifier. Serial 10-fold dilutions were prepared. Serial dilutions for bacterial analysis were prepared using sterile physiological saline. Serial dilutions for endotoxin analysis were prepared using pyrogen-free water.

For each sample, 0.1 mL of the initial filter wash and of each of the serial dilutions was inoculated onto Petri plates containing eosin and methyl blue agar. Two replicates were plated for each dilution. The cultures were incubated for either 1 d at 37°C, 3 d at 22°C, or 3 d at 4°C.

After incubation, the microbial colonies on each plate were macroscopically examined in terms of size, shape, structure, color, and other distinguishing characteristics. Bacteria from each colony type were then gram-stained and examined under a microscope. The bacteria were categorized into morphological classes. The bacteria in each class were then quantified in terms of colony-forming units per cubic meter of air (Dutkiewicz, 1978).

Bacterial isolates were then identified. Enterobacteriaceae were identified using the API-20E test strip, and nonfermenters were identified using the API-20NF test strip (bioMèrieux, Marcy l’Étoile, France).

Endotoxin concentration was measured using the Limulus test kit (bioMèrieux), and 0.1 mL of the initial filter wash and of each of the serial dilutions was mixed with 0.1 mL of Limulus reagent. A standard serial dilution was prepared using Escherichia coli endotoxin (Associates of Cape Code Inc., East Falmouth, MA). A negative control was prepared using pyrogen-free distilled water. The samples were incubated in a water bath for 1 h at 37°C. The reaction was then read by inverting the sample tubes. A sample was considered to be positive if a stable gel had formed that stayed in place when the sample tube was inverted (Levin and Bang, 1964).

The temperature of the filter material was determined using an electronic thermometer. The pH was measured using a pH meter (CP-104, Elmetron, Zabrze, Poland). Moisture content was determined gravimetrically.

Samples of the filter material were taken with a disinfected soil sampler, transferred to sterile containers, and shaken to ensure sample uniformity. Microorganisms were then quantified by serially diluting 1.0-g aliquots of each sample.

Bacterial, actinomycete, and fungal counts were determined by inoculating 0.1 mL of each of the serial dilutions onto petri plates containing the appropriate medium. Two replicates were plated for each dilution. The plates were then incubated.

Mesophilic bacteria were cultured on grown agar at 37°C for 24 h. Psychrophilic bacteria were cultured on grown agar at 22°C for 72 h. Proteolytic bacteria were cultured on Frazier medium at 26°C for 7 d. Actinomycetes were cultured on actinomycetes agar containing 5 g/L nystatin at 26°C for 7 d. Fungi were cultured on Sabouraud agar at 26°C for 5 d.

Bacterial, actinomycete, and fungal counts were then calculated and recorded in terms of colony-forming units per cubic meter of air.

The following statistical parameters were calculated for all of the results collected: number of observations, arithmetic mean, SD and arithmetic mean error, and CV. The mean levels of dust, gram-negative bacteria, and bacterial endotoxin in the hatching room air were compared with the mean contamination levels in the air after bio-treatment using Tukey’s and Dunett’s tests. Series-specific and sampling site-specific coefficients for filter media efficiency were calculated using the Kruskal-Wallis non-parametric tests. The dependence of the degree of pollutant reduction on the properties of the filter material was analyzed by calculating the correlation coefficient. Calculations were carried out with the help of the SAS version 9 (SAS Institute Inc., Cary, NC) and Statistica version 6.0 (StatSoft Inc., Tulsa, OK) software packages.


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
REFERENCES
 
Technological progress in the design of hatching facilities has brought about a dramatic reduction in air contamination, especially by particulates (Skórska, 1986). The dust level measured in the hatchery room in this study was relatively low, 0.95 mg/m3 (Table 1Go). This is lower than dust levels encountered in farm buildings (Wathes, 1998; Baykov and Stoyanov, 1999; Chang et al., 2001; Zucker et al., 2000; Kalingan at al., 2004). Because employees in modern hatcheries are exposed to lower dust levels, the risk of respiratory tract disease should be lower, at least theoretically. The risk to the environment and the community at large should also be lower.


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  		Table 1. Mean levels of dust, gram-negative bacteria, and bacterial endotoxin in hatching room air and in the air after biotreatment
 
In this study, the level of bacteria in the hatchery room was 4,051.8 cfu/m3. Gram-negative bacteria made up 16% (651 cfu/m3) of the total bacterial population (Table 1Go). The following gram-negative bacteria were isolated: Acinetobacter baumanii, Acinetobacter genospecies, A. faecalis, Citrobacter youngae, Enterobacter cloacae, E. coli, Flavobacterium meninogosepticum, Flavobacterium tirrenicum, Flavobacterium breve, Klebsiella sp., Klebsiella pneumoniae, Pseudomonas putida, Pseudomonas stutzeri, Pseudomonas diminuta, Pseudomonas sp., Pseudomonas pseudoalcaligenes, Leclercia adecarboxylata, Sphingomonas paucimobilis, Sphingomonas multivorum, Xanthomonas maltophila, Agrobacterium radiobacter, and Pantoea spp.

Endotoxin makes up about 70% of the outer cell membrane in gram-negative bacteria, where it protects the bacterium from host defense mechanisms, bile acids, and hydrophobic antibiotics.

Long-term inhalation of air polluted with organic dust alters immunological reactivity in an exposed organism (Brook et al., 2004). Bacterial endotoxin lipopolysaccharides appear to be an antigenic, which can provoke an immunopathogenic reaction (Chang et al., 2001). Endotoxin also plays a key role in sepsis and septic shock and can cause respiratory problems in workers exposed to aerosols and particulates containing gram-negative bacteria.

Endotoxin is released into the environment by both living and dead bacteria. Therefore, disinfection does not effectively reduce the level of endotoxin in the workplace. For example, in a study on air contamination in a tobacco factory, there was no correlation between the numbers of viable gram-negative bacteria and the concentration of endotoxin in the air (Reiman and Uitti, 2000).

In this study, the mean concentration of gram-negative bacteria in the hatchery room was 651 cfu/m3, and the mean concentration of bacterial endotoxin was 11.15 ng/ m3 (Table 1Go). Even at this level, endotoxin could induce respiratory system problems in humans (Rylander, 1977). The Dutch Expert Committee on Occupational Standards has set the permissible limit for endotoxin at 4.5 ng/m3 in areas where workers work an 8-h shift (Heederick and Douwes, 1997). However, based on our results, it may be difficult, or even impossible, to reduce the concentration of endotoxin to this level in hatcheries.

Throughout this study, air from the hatching room was passed through the ventilation system to the biofilter, where it was biologically treated. The air exiting the bio-filter contained less aerosols and particulates than the air entering it. However the extent to which the filter was effective in reducing airborne contaminants depended on which filter media was used (Table 1Go).

Statistical analysis, nevertheless, did not reveal any significant differences in the levels of these contaminants among the biofiltration media used due to the high variation among sample values within each of the media treatment groups. The mean dust level of air exiting the bio-filter chambers ranged from 0.12 mg/m3 with the HM medium to 0.17 mg/m3 with the OM medium.

The air exiting the biofilter was contaminated with gram-negative bacteria only with the OM medium. With the BM and HM media, no gram-negative bacteria were detected.

Endotoxin concentration in the air exiting the biofilter was 9.9 ng/m3 with the OM medium, 9.3 ng/m3 with the BM medium, and 5.4 ng/m3 with the HM medium.

Mean values for decontamination efficiency across the 3 substrates and the 5 different treatment times are presented in Table 2Go, and mean values for decontamination efficiency for each substrate across the 5 different treatment times are presented in Table 3Go. The efficiency values observed in this study ranged from satisfactory to very high. The efficiency value for dust removal averaged about 85%. The efficiency value for removing gram-negative bacteria was almost 100% with all of the media tested (Table 2Go). Efficiency values for removing endotoxin were significantly lower and ranged from 11% with the OM medium to over 51% with the HM medium, with a mean value of 26.4%.


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  		Table 2. Mean efficiency values for the removal of dust, gram-negative bacteria, and bacterial endotoxin obtained from all experimental series and media (%)
 

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  		Table 3. Mean efficiency values for the removal of dust, gram-negative bacteria, and bacterial endotoxin for biofilter media obtained from all experimental series (%)
 
Although the HM medium was numerically most efficient in removing dust and gram-negative bacteria, with an efficiency value of 81.6% for dust and 99.6% for gram-negative bacteria, statistical analysis did not reveal any significant differences in efficiency values among the 5 series of experiments conducted in terms of biofilter efficiency. There were also no significant differences among the different treatment times tested.

Efficiency values for the removal of dust and gram-negative bacteria remained about the same throughout the study. Efficiency values for the removal of dust never fell below 50%, and efficiency values for the removal of gram-negative bacteria never fell below 99% (Figure 2Go).


Figure 2
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  		Figure 2. Efficiency values for the removal of dust, gram-negative bacteria, and bacterial endotoxin for the 5 series of experiments conducted.

 
However, there was a level of variation in the efficiency values for the removal of endotoxin. Efficiency values were highest in the first series of experiments and lowest (exceeded –98%) in the second and fifth series of experiments. In the second and fifth series of experiments, endotoxin concentrations in the air exiting the biofilter were actually higher than in the intake air.

Biofiltration relies predominantly on the activity of the bacterial flora living in the filter medium. Based on the microbiological analyses presented in Table 4Go, the media that provided the best conditions for the growth of the resident bacterial flora were the OM and HM media. The OM medium provided the best conditions for psychrophilic and proteolytic bacteria, and the HM medium provided the best conditions for mesophilic bacteria and actinomycetes.


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  		Table 4. Microbiological and physiochemical properties of the biofilter media tested throughout the experiment
 
The OM medium provided the least favorable conditions for gram-negative bacteria. However, the OM medium was the least effective in removing endotoxin. This suggests that the components of the filter medium may have been a source of endotoxin. On the other hand, the number of viable gram-negative bacteria was low in the air exiting the biofilter with the OM substrate. This suggests that gram-negative bacteria flowing through the OM substrate were killed and not simply trapped in the filter substrate as was the case with the HM medium. This would explain the low efficiency value for the removal of endotoxin seen with the OM medium. These observations are based only on the comparison of the mean values of contamination levels not supported with the statistical analysis (low number of replications). They still should be mentioned.

There was no statistically significant correlation between efficiency values for dust and endotoxin removal or the microbiological properties of the media tested (Table 5Go). There was, however, a negative correlation between efficiency values for the removal of gram-negative bacteria and the amount of psychrophilic bacteria living on the media tested (r = –0.77).


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  		Table 5. Correlation coefficient between reduction of gram-negative bacteria, bacterial endotoxin, and dust and media properties
 
Biotreatment of air contaminated with chemical pollutants has been intensively studied, both in Poland and Germany, Spain, China, Taiwan (Kalingan et al., 2004; Prado et al., 2004; Tymczyna et al., 2004; Chmielowiec-Korzeniowska et al., 2005; van Groenestijn and Kraakman, 2005). However, little attention has been paid to the use of biotreatment in removing microbiological contaminants. All of the studies carried out to date have shown that biofilter performance depends on the choice of filter material, regardless of which contaminant is to be removed.

For example, a biofilter using coconut shavings was the most efficient biofiltration system, reducing the level of microbiological contamination by 99% (Schlegelmilch et al., 2005). Biofilter efficiency was improved when the physical structure of the material was not uniform and when the moisture content was high.

In this study, microbiological examinations showed that the biofilter media tested varied in terms of efficiency in removing bioaerosols and microorganisms. They were all highly effective in removing pathogenic bacteria from the air flowing through them. Although some microorganisms were detected in the air exiting the biofilter, they did not pose any health hazard.

In this study, all of the biofilter media were highly effective in removing gram-negative bacteria, moderately effective in reducing dust levels, and only slightly effective in removing endotoxin. The substrate that was most efficient in retaining bioaerosols was the HM medium, which generally had at least satisfactory efficiency values for removing all of the contaminants tested.


   ACKNOWLEDGMENTS
 
This study was funded by the Polish Committee for Research, program number P06 010 27.

Received for publication October 18, 2006. Accepted for publication May 24, 2007.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
Baykov, B., and M. Stoyanov. 1999. Microbial air pollution caused by intensive broiler chicken breeding. FEMS Microbiol. Ecol. 29:389–392.

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Chmielowiec-Korzeniowska, A., L. Tymczyna, A. Drabik, and H. Malec. 2005. Biofiltration of volatile organic compounds in the hatchery. Ann. Anim. Sci. 5:371–378.

Dutkiewicz, J. 1978. Exposure to dust-borne bacteria in agriculture. I. Environmental studies. Arch. Environ. Health 33:250–259.[Web of Science][Medline]

Dutkiewicz, J., R. Spiewak, and L. Jabllonski. 2002. Classification of Harmful Biological Factors. Occurrence in Environment of Work. Ad punctum, Lublin, Poland.

González-Miret, M. L., M. L. Escudero-Gilete, and F. J. Heredia. 2006. The establishment of critical control points at the washing and air chilling stages in poultry meat production using multivariate statistics. Food Control 17:935–941.[Web of Science]

Heederick, D., and J. Douwes. 1997. Towards an occupational exposure limit for endotoxins? Ann. Agric. Environ. Med. 4:17–19.

Kalingan, A. E., C. M. Liao, J. W. Chen, and S. C. Chen. 2004. Microbial degradation of livestock-generated ammonia using biofilters at typical ambient temperatures. J. Environ. Sci. Health B 39:188–198.

Levin, J., and F. B. Bang. 1964. The role of endotoxin in the extracellular coagulation of Limulus blood. Bull. Johns Hopkins Hosp. 115:265–274.[Web of Science][Medline]

Prado, O. J., M. C. Veiga, and C. Kennes. 2004. Biofiltration of waste gases contaminating a mixture of formaldehyde and methanol. Appl. Microbiol. Biotechnol. 65:235–242.[Web of Science][Medline]

Reiman, M., and J. Uitti. 2000. Exposure to microbes, endotoxins and total dust in cigarette and cigar manufacturing: An evaluation of health hazards. Ann. Occup. Hyg. 44:467–473.[Abstract/Free Full Text]

Rylander, R. 1977. Evaluation of the risks of endotoxins and humidifer disease. Clin. Allergy 14:109–112.

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Seedorf, J., J. Hartung, M. Schröder, K. H. Linkert, V. R. Phillips, M. R. Holden, R. W. Sneath, J. L. Short, R. P. White, S. Pedersen, H. Takai, J. O. Johnsen, J. H. M. Metz, P. W. G. Groot Koerkamp, G. H. Uenk, and C. M. Wathes. 1998. Concentrations and emissions of airborne endotoxins and microorganisms in livestock buildings in Northern Europe. J. Agric. Eng. Res. 70:97–109.

Skórska, C. 1986. Microflora of air in modern hatcheries. Med. Wiejska 4:211–216.

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