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Article

Towards Zoo Sustainability: Assessment of Indoor and Outdoor Bacterial Air Contamination Levels and Their Correlations with Microclimate Parameters

by
Mario Ostović
1,*,
Kristina Matković
1,*,
Anamaria Ekert Kabalin
2,
Sven Menčik
2,
Željko Pavičić
1,
Nevenka Rudan
3,
Danijela Horvatek Tomić
4,
Dijana Beneta
5 and
Ingeborg Bata
5
1
Department of Animal Hygiene, Behavior and Welfare, Faculty of Veterinary Medicine, University of Zagreb, 10000 Zagreb, Croatia
2
Department of Animal Breeding and Livestock Production, Faculty of Veterinary Medicine, University of Zagreb, 10000 Zagreb, Croatia
3
Department of Microbiology and Infectious Diseases with Clinic, Faculty of Veterinary Medicine, University of Zagreb, 10000 Zagreb, Croatia
4
Department of Poultry Diseases with Clinic, Faculty of Veterinary Medicine, University of Zagreb, 10000 Zagreb, Croatia
5
Zoological Garden of Zagreb, 10000 Zagreb, Croatia
*
Authors to whom correspondence should be addressed.
Sustainability 2025, 17(23), 10517; https://doi.org/10.3390/su172310517
Submission received: 6 October 2025 / Revised: 11 November 2025 / Accepted: 21 November 2025 / Published: 24 November 2025
(This article belongs to the Special Issue Towards Sustainability: Advanced Research on Environmental Analysis and Air Pollution)
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Abstract

Air quality in zoo premises is insufficiently investigated, yet scientific interest is growing. In these places, air pollutants such as microorganisms may represent health risk for both animals and exposed people. Thus, maintaining good air quality is crucial for ensuring long-term sustainability of zoo operations. The present study aimed to assess bacterial air contamination level and microclimate conditions in Zagreb Zoo. Measurements were performed in the area for visitors and employees inside four premises, i.e., Monkey House, Tropical House, Rainy Africa, and Bird House, as well as outside the premises in the summer–autumn period. Total number of bacteria in the premises ranged from 4.50 × 102 to 3.70 × 104 CFU/m3, and number of Gram-negative bacteria ranged from 0 to 5.50 × 102 CFU/m3. Total number of bacteria in outdoor air ranged from 50 to 8.50 × 102 CFU/m3, and number of Gram-negative bacteria ranged from 0 to 50 CFU/m3. Total number of bacteria was significantly higher in the Rainy Africa and Bird House as compared to the Monkey House and Tropical House, yielding a significant positive correlation with the number of Gram-negative bacteria in the premises. Total number of bacteria in outdoor air was significantly lower as compared to all investigated premises, except for the Monkey House, yielding a significant positive correlation with the number of Gram-negative bacteria outdoors. Air temperature showed a significant negative correlation with both total number of bacteria and number of Gram-negative bacteria, and airflow rate showed a significant negative correlation with total number of bacteria in the premises. Air temperature showed a significant positive correlation and relative humidity significant negative correlation with total number of bacteria outdoors. Study results can serve in the development of air quality standards in zoos, contributing to finding effective strategies to mitigate health risk from bioaerosols, with implications for occupational and public health, and overall zoo sustainability.

1. Introduction

Air quality refers to the level of pollutants present in the air [1]. The air in farm animal facilities usually contains lower or higher concentrations of pollutants, depending on animal species, age, weight and activity, stocking density, housing system, type of ventilation, bedding and feeding, season and microclimate conditions, hygiene management, and other factors [2,3,4,5]. In this regard, studies have shown that the concentration of microorganisms is highest in the air of poultry houses [6,7,8]. Poor air quality affects not only the health of animals but also of farmworkers [9,10]. Human exposure to bioaerosols is most commonly associated with respiratory system problems such as rhinitis, asthma, bronchitis, and sinusitis, followed by gastrointestinal disorders, fatigue, weakness, and headache [11]. In addition, poor air quality in farm animal facilities contributes to the pollution of outdoor environment [5,12].
As compared to the farm animal facilities, very few studies have investigated air quality in animal premises of zoological gardens, possibly due to the lack of relevant standards which the results obtained can be related to, although in recent years, this research area is gaining interest, e.g., [13,14,15,16,17,18,19,20,21,22]. Zoos can house thousands of animals and hundreds of species, cared for by their keepers who are directly exposed to bioaerosols originating from the animals and their feces, bedding, and feed. This also applies to other employees such as veterinarians. Moreover, zoos are places of mass gathering of people of different age groups, with poor air quality posing a particular health risk to children [14]. Animals in zoos may also differ in their ability to cope with air quality components and changes, whether indoor or outdoor; thus, some species may be at a greater risk of respiratory diseases [23].
Nowadays, zoos are balancing numerous challenges, including a changing economy, increased sensitivity to environmental impacts, and the need to ensure a stable workplace that attracts and keeps good employees, all while providing meaningful services to their visitors. So, aside from the core mission of preserving the animals and their natural habitats, zoos are increasingly expected to demonstrate that their operations can be managed in a highly sustainable way [24]. Accordingly, air contamination in zoos is a sustainability challenge due to the potential health risks from bioaerosols [25], highlighting the relevance of the One Health approach, which recognizes the interconnectedness and interdependence among human, animal, and environmental health to address these risks and promote global health and sustainability [26,27,28].
Previous studies assessing bacterial air contamination in zoos were conducted predominantly by Polish researchers. Grzyb and Lenart-Boroń [14] performed a study in new (giraffes and ostriches) and old animal enclosures (monkeys and pheasants) of the Kraków Zoo during autumn, winter, and spring. The study revealed that the concentration of different groups of airborne bacteria, expressed as the number of colony-forming units per m3 of air (CFU/m3), varied among the enclosures. Total number of bacteria ranged from 8.21 × 102 CFU/m3 (pheasants) to 6.88 × 103 CFU/m3 (monkeys), and total number of Gram-negative bacteria ranged from 0 CFU/m3 (ostriches) to 1.55 × 102 CFU/m3 (pheasants). The study demonstrated that the concentration of bacterial aerosol could be affected not only by the animals themselves but also by the age of enclosures. The levels of bacteria in enclosures did not exceed limit values for occupational exposure provided by Polish guidelines, i.e., 1.00 × 105 CFU/m3 for total number of bacteria and 2.00 × 104 CFU/m3 for total number of Gram-negative bacteria [29]. However, the average proportion of respirable fraction in total concentration of bacterial aerosol was quite high, with values of total number of bacteria ranging from 61.1% (giraffes) to 89.3% (monkeys) and of Gram-negative bacteria from 12.8% (monkeys) to 86.4% (pheasants), suggesting a potential threat to the health of exposed people. The authors concluded that attention should be paid to proper cleaning of animals and their enclosures, and maintaining suitable microclimate conditions in the enclosures.
In their study, Grzyb and Pawlak [17] analyzed the concentration of airborne bacteria in the rooms for giraffes, camels, elephants, kangaroos, and colobinae (monkeys) in Chorzów Zoo throughout the year. The lowest total concentration of bacterial aerosol was recorded in the rooms for colobinae (8.47 × 102 CFU/m3) and highest in the rooms for elephants (1.06 × 105 CFU/m3), both in spring. The level of bacterial aerosol was most strongly affected by the size of animals and type of bedding in the rooms. The study indicated that ventilation in animal rooms should be modified in order to improve the conditions for both animals and exposed people.
In another study by Grzyb and Pawlak [18], also conducted in the Chorzów Zoo, the concentrations of airborne bacteria in animal rooms were particularly high in spring and autumn, being related to animals shedding fur. This study showed that the average value of total number of fecal bacteria in the air was lowest in the rooms for colobinae (47 CFU/m3) and highest in the rooms for elephants (4.35 × 102 CFU/m3). The authors also identified antibiotic-resistant species of staphylococci in the air, suggesting periodic disinfection of the rooms in addition to standard cleaning. A similar conclusion was reached by Jesse Joel et al. [22], who investigated the prevalence of bacterial aerosol within animal enclosures of the Thrissur State Zoo, Thrissur, Kerala, India.
Therefore, the results of the conducted studies are important for gaining knowledge about concentrations of airborne bacteria in zoo premises and factors influencing their levels, as well as for possible monitoring and reduction strategies, and finally for establishment of the air quality standards in these places, which is in line with their sustainable development. However, earlier research concerning bacterial air quality in zoos has focused on animal enclosures themselves.
The aim of this study was to assess bacterial air contamination level in indoor areas for visitors and workers of the zoo premises. The study also included investigation on bacterial contamination of outdoor air. Finally, the study assessed correlations between indoor and outdoor concentrations of airborne bacteria and microclimate parameters.

2. Materials and Methods

2.1. Study Area and Zoo Management

This study was carried out in the Zoological Garden of Zagreb founded in 1925. The Zoo covers 7 ha of Maksimir Park, housing approximately 7000 animals and 400 species [30]. The conditions for accommodation and care of animals are regulated by the national Animal Protection Act [31] and Ordinance on the Requirements for the Establishment and Operation of Zoos [32]. The Zagreb Zoo is a member of the European Association of Zoos and Aquaria, World Association of Zoos and Aquariums, and International Zoo Educators Association [30].
All measurements were performed in the summer–autumn period, from August to October 2024. Indoor measurements took place in the area for visitors and employees of the following Zoo premises: Monkey House, Tropical House (with crocodiles and caimans, numerous species of snakes, lizards, turtles, amphibians, and fish, and a few species of birds and mammals), Rainy Africa (with pygmy hippopotamuses, greater vasa parrots, a couple of species of snakes and lizards, and fish) and Bird House as a part of the Vivarium (with different species of parrots and songbirds) (Figure 1). Selection of the premises was based on their convenient accessibility, with their description being given in Table 1. The premises were cleaned in the period from 8 a.m. to 11 a.m. every day, and the animals were fed five to seven times a day, depending on the species, from 8 a.m. to 7 p.m. Animals in the Monkey House and Bird House, and pygmy hippopotamuses in the Rainy Africa had access to outdoor enclosures nearly to the end of the study period. Outdoor measurements took place at a distance of about 10 m from one of the selected premises near the Zoo entrance, which served as a control site (Figure 1), as suggested previously, e.g., [14,15].

2.2. Data Collection

The investigated parameters were measured at weekly intervals (n = 12 weeks) from 9.00 a.m. to 12 p.m. after the Zoo was opened for visitors, and after cleaning the premises and feeding the animals. On each occasion, measurement of microclimate parameters and microbial air sampling were performed at the same sites, under the same conditions, at a height of 1.5 m above ground level, in the human breathing zone, with both sets of measurements inside the premises and outdoors including five replicates.
The values of microclimate parameters, air temperature (°C), relative humidity (%), and airflow rate (m/s), were determined as instantaneous readings using portable digital devices (Testo SE & Co. KGaA, Lenzkirch, Germany). Air temperature and relative humidity were measured using Testo 625 thermohygrometer, with an accuracy of ±0.5 °C and ±2.5% (5 to 95%), respectively, while Testo 425 thermal anemometer was used to measure airflow rate, with accuracy of ±(0.03 m/s + 5% of measuring value).
Air was sampled with a portable SAS Super 100TM device (PBI International, Milan, Italy), with an airflow rate of the device of 100 L/min. For each sample, 20 L of air was collected. Air was aspired through a 219-hole sampling head onto an agar surface in a Petri dish. The sampling head was sterilized before each sampling and disinfected between measurements using cotton balls immersed in 70% ethanol. Tryptic Soy Agar (Biolife, Milan, Italy) and Mac Conkey Agar (Biolife, Milan, Italy) were used for determination of total number of mesophilic bacteria and number of Gram-negative bacteria, respectively. All plates were incubated in aerobic conditions at 37 °C for 48 h. The grown colonies were counted by use of a digital colony counter (J.P. Selecta, Barcelona, Spain), and the results were adjusted using the following formula: X = (Pr × 1000)/V, where X is CFU/m3, Pr is probable number of bacterial colonies obtained by positive hole correction (Pr is read from the device manufacturer’s table on the basis of the number of counted colonies), and V is volume of sampled air (L), with 1000 L = 1 m3.

2.3. Statistical Analysis

Statistical analyses were carried out by Statistica v. 14.1.0.8 reference software (Cloud Software Group, Inc., Palo Alto, CA, USA, 2023, Data Science Workbench, http://tibco.com, accessed on 13 December 2024). Data were processed by standard procedures of descriptive statistics. The normality of data distribution was assessed with the Kolmogorov–Smirnov test. Kruskal–Wallis test was used to test the significance of differences in the average number of airborne bacteria, while the significance of differences in the average values of microclimate parameters among the premises, including outdoor air (control), was tested by one-way ANOVA followed by post hoc Tukey HSD test. Spearman rank order correlation was used to determine the correlations among investigated parameters. A value of p < 0.05 was considered statistically significant in all analyses.

3. Results and Discussion

Total number of airborne bacteria inside the premises varied on average between 1.50 × 103 and 5.80 × 103 CFU/m3 (range 4.50 × 102–3.70 × 104 CFU/m3) (Figure 2), being consistent with the values reported in previous studies conducted in animal enclosures of Poland zoos [14,17]. As compared to Polish recommendations regarding permissible total number of bacteria in the air of working premises (1.00 × 105 CFU/m3) [29], the air inside all premises in our study should not be considered as contaminated, but in comparison with these recommendations for permissible total number of bacteria in the air of residential and public utility premises (5.00 × 103 CFU/m3) [29], as well as other sources [33,34,35], the air in the Rainy Africa and Bird House can be considered as even highly contaminated. There are no globally accepted threshold limit values concerning levels of airborne microorganisms in indoor environments. The Commission of the European Communities expert group suggests that non-industrial indoor environments with concentrations of airborne bacteria above 2.00 × 103 CFU/m3 are very highly contaminated [33]. The World Health Organization expert group recommends that total concentration of microorganisms in indoor air should not exceed 1.00 × 103 CFU/m3 [34], which is in line with the report of the US government agency, Occupational Safety and Health Administration, suggesting that the number of airborne microorganisms above this value may point to indoor contamination, but biodiversity of bioaerosol should also be considered when assessing health hazards [35].
As shown in Figure 2, total number of airborne bacteria was significantly higher (p < 0.001) in the Rainy Africa and Bird House than in the Monkey House and Tropical House. In comparison, Grzyb and Lenart-Boroń [14] found a significantly higher total number of airborne bacteria in enclosures for monkeys than in those for giraffes, pheasants, and ostriches. However, it should be held in mind that these authors measured the concentrations of airborne bacteria in animal enclosures themselves. In addition, many factors can affect the bacterial aerosol levels in animal premises including the age of the premises and the number of animals per unit area. The Monkey House and Bird House where we conducted the measurements are both old premises, but the number of birds in our study, although smaller in size, was notably higher, and the respective area smaller than in the study by Grzyb and Lenart-Boroń [14], while monkey cages are additionally protected by glass, which could result in lower emissions of bacteria into the area for visitors and employees. The lower total concentration of bacteria in the Tropical House as compared to the Bird House and Rainy Africa can also be explained by the glass walls of animal rooms facing the area for visitors and workers. Unlike that, the Bird House has a wire mesh fixed on the door windows to the rooms, which were open during the measurements, and the indoor enclosure for pygmy hippopotamuses in the Rainy Africa has a glass fence, not a glass wall like the room for greater vasa parrots towards the area for visitors and workers. Grzyb and Pawlak [17] found the lowest total concentration of airborne bacteria in the premises for monkeys, about three times lower than Grzyb and Lenart-Boroń [14], attributing it to the lack of bedding in the premises. The enclosures in the Zagreb Monkey House have no bedding either. Thus, the higher total number of bacteria in the Rainy Africa can also be linked to floor covering with shredded bark in the visitor area, as well as to the size of the animals (pygmy hippopotamuses). The highest total number of bacteria determined in the Bird House can be explained by their activity along with the use of natural ventilation in the rooms. In farm environments, poultry houses are well known to generate excessive dust, a carrier of microorganisms, which can originate from feed, dried fecal matter, and feather particles [36].
Total number of airborne bacteria in outdoor air was, on an average, 3.50 × 102 CFU/m3 (range 50–8.50 × 102 CFU/m3) (Figure 2), being in accordance with previous reports [14,17]. Total number of bacteria outdoors was significantly lower (p < 0.001) than inside all premises (Figure 2), with the indoor to outdoor value ratio (I/O) ranging from 4.3 (Monkey House) to 16.6 (Rainy Africa), suggesting the existence of an internal source of bacterial emissions if I/O is >1 [37]. In the study by Grzyb and Pawlak [17], the I/O for total concentration of airborne bacteria in summer and autumn for enclosures for colobinae was 6 and 4, respectively, which is in line with this study, and for enclosures for elephants even 25 and 34, respectively. However, the present study showed no significant correlation between total number of bacteria, or between the number of Gram-negative bacteria inside and outside the premises (Table 2), indicating that bacteria in the external environment were substantially diluted by air and/or that they were subjected to various physico-chemical parameters of the atmosphere [38], which affected their concentrations outdoors. Studies performed on industrial farms have shown that animal facilities are a source of significant emissions of microorganisms into the atmospheric air, poultry houses in particular [39,40]. However, the bacterial aerosol concentrations recorded on large-scale farms are significantly higher in comparison with zoos [14,17], as also confirmed by our study. In farm conditions, the number of airborne bacteria in animal facilities generally ranges up to 108 CFU/m3 [41,42,43]. For instance, the study by Górny et al. [43] demonstrated that the concentrations of microbial air pollutants significantly decreased at a distance of 500 m from a poultry house.
Gram-negative bacteria are among the most significant public health problems worldwide due to their high resistance to antibiotics [44,45,46]. Farm environments are a well-established source of these bacteria, posing a high risk of respiratory diseases in both animals and humans [47,48]. In livestock and poultry houses, most microorganisms in the air are non-pathogenic, and Gram-negative bacteria form less than 8% of the total number of bacteria [49,50]. In this study, the number of Gram-negative bacteria in the air inside the premises varied on an average between 0 and 50 CFU/m3 (range 0–5.50 × 102 CFU/m3) (Figure 3), which is in line with previous studies [14,18]. According to Polish recommendations for permissible total number of Gram-negative bacteria in the air of working premises (2.00 × 104 CFU/m3) [29], the air inside all investigated premises should not be considered as contaminated. Even regarding permissible total number of Gram-negative bacteria in the air of residential and public utility premises (2.00 × 102 CFU/m3) [29], their number inside the premises was around the recommended value. However, like the total number of bacteria, the number of Gram-negative bacteria in indoor air significantly differed (p < 0.05) among particular premises, with the highest values being recorded in the Rainy Africa and Bird House (Figure 3). As shown in Table 2, there was a moderate positive correlation between total number of bacteria and number of Gram-negative bacteria in indoor air (r = 0.527, p < 0.05). Grzyb and Lenart-Boroń [14] did not find significant differences in the total number of Gram-negative bacteria among enclosures for giraffes, monkeys, pheasants, and ostriches, although total number of airborne bacteria in their study, as mentioned earlier, was significantly higher in the enclosures for monkeys than in other enclosures. In the study by Grzyb and Pawlak [18], total number of fecal bacteria in the air was on an average lowest in the rooms for monkeys, which is in line with our study. In terms of Gram-negative bacteria, there have been reports on Coxiella burnetii infection in zoo animals, a zoonotic pathogen easily spread by aerosol, highlighting the need of continuous monitoring and effective strategies of prevention and control of this pathogen [51,52,53]. In their study, Jesse Joel et al. [22] identified various bacteria in the air from animal enclosures of a zoo, which may pose a health threat, including zoonotic Gram-negative bacterium Pseudomonas aeruginosa that showed exceptional resistance to a spectrum of antibiotics tested. The study underscored the need for effective management strategies regarding the spread of antibiotic-resistant bacteria in zoo enclosures, and of reasonable use of antibiotics to preserve both animal and human health.
The number of Gram-negative bacteria in outdoor air was, on an average, 0 CFU/m3 (range 0–50 CFU/m3) being significantly lower (p < 0.05) than inside all premises except for the Monkey House (Figure 3), which could be related to the lowest total number of bacteria found in the Monkey House (Figure 2). As shown in Table 2, a weak but significant positive correlation was found between total number of bacteria and number of Gram-negative bacteria in outdoor air (r = 0.209; p < 0.05). In the study by Grzyb and Pawlak [18], the number of airborne bacteria in the rooms for colobinae was also most similar to that recorded in the outdoor environment, whereas Grzyb and Lenart-Boroń [14] did not detect Gram-negative bacteria in the zoo outdoor air. In this sense, it has been demonstrated that Gram-positive bacteria are more resistant to environmental conditions due to their thick cell wall [6].
Air temperature inside the premises varied on an average from 23.15 to 24.55 °C (range 16.00–29.20 °C), relative humidity from 62.18 to 70.05% (range 43.00–83.20%), and airflow rate from 0.08 to 0.11 m/s (range 0.01–0.40 m/s). There were no significant differences in indoor air temperature and airflow rate among the premises, whereas relative humidity was significantly higher (p < 0.01) in the Rainy Africa and Tropical House as compared to the Monkey House and Bird House (Table 3). Previous research has shown that the preferable temperature range for humans is 17–24 °C, with stress temperature thresholds being lower at higher humidity. However, prolonged exposure to temperatures above 25 °C accompanied with high humidity can cause heat stress. Even short exposure to temperatures above 35 °C with high humidity, or above 40 °C with low humidity, can be fatal for humans [54]. Regarding relative humidity, it is generally recommended to maintain the values indoors for human comfort and health between 40 and 60% [55]. The preferred airflow rate is between 0.1 and 0.2 m/s [56]. Therefore, it seems that microclimate conditions inside the investigated premises did not pose a threat to the health of exposed people, even though the values of relative humidity in the Rainy Africa and Tropical House exceeded 80%.
Air temperature outdoors (average 22.24 °C, range 6.90–32.90 °C) was significantly lower (p < 0.05) only in comparison with that inside the Monkey House, although there was a tendency for a difference (p < 0.06) also in comparison with the Tropical House and Bird House, while airflow rate outdoors (average 0.44 m/s, range 0.22–1.16 m/s) was significantly higher (p < 0.001) than inside all premises (Table 3). Moisture in the premises can originate from the animals themselves, evaporation from their skin, excrement, and exhaled air, then wet floors and walls, feed, and outdoor air [14,17]. In the present study, relative humidity outdoors (average 65.49%, range 49.80–81.40%) was not significantly different as compared to the values determined inside the premises, except for the Rainy Africa, where the air was significantly more humid (p < 0.01) than outdoor air (Table 3). The lowest air temperature and highest relative humidity in the Rainy Africa can be explained by air circulation through the entrance for visitors and the exit to the outdoor enclosure for pygmy hippopotamuses, and the presence of a pool in the indoor enclosure for hippopotamuses.
Correlations of microclimate parameters with airborne bacteria inside the premises are presented in Table 4. Air temperature showed a weak but significant negative correlation with both total number of bacteria (r = −0.141; p < 0.05) and number of Gram-negative bacteria (r = −0.164; p < 0.05), and airflow rate yielded a weak but significant negative correlation (r = −0.152; p < 0.05) with total number of bacteria. Relative humidity inside the premises showed no significant correlation with the number of bacteria. A negative correlation between the air temperature and the number of bacteria inside the premises can be explained by the animals using exclusively indoor enclosures with a decrease in the outdoor temperature, which could contribute to an increase in the number of bacteria in the premises. In their study, Grzyb and Pawlak [17] report on a significant negative correlation between airflow rate and concentration of the “thickest” bacterial aerosol fractions with a diameter above 4.7 μm, whereas Grzyb and Lenart-Boroń [14] found no significant relationship between microclimate parameters and bacterial aerosol concentration in animal rooms of the zoo, attributing it to the inconsiderable variability of microclimate parameters in the rooms.
Air temperature yielded a moderate positive correlation (r = 0.413; p < 0.05) and relative humidity moderate negative correlation (r = −0.552; p < 0.05) with total number of bacteria in outdoor air. The correlation between airflow rate and total number of bacteria in outdoor air was negative, but not significant (Table 5). No significant correlations were found between microclimate parameters and the number of Gram-negative bacteria in outdoor air either (Table 5), probably due to small variability of these bacteria outdoors.
In accordance with previous studies [14,15,17,18,22], controlling microclimate parameters and regular maintenance of hygienic conditions in zoo premises are imposed as the key sustainability practices to mitigate health risk from bioaerosols not only for animals, but also for humans visiting and working in zoos. Moreover, this study demonstrated that a glass wall protection of animal enclosures could be practical in reducing the emissions of air microorganisms into the area intended for visitors and employees of the zoo.

4. Conclusions

The present study showed that the concentrations of airborne bacteria in the area for visitors and employees of the zoo varied depending on the animals present inside the premises. The highest values of total number of bacteria and number of Gram-negative bacteria were found in the Rainy Africa and Bird House, indicating that premises with larger animals and birds, where animals are kept in indoor bedded area without a glass wall protection, may pose a significant source of bioaerosol, in particular, with the onset of a colder period when they spend all the time indoors. The obtained results can be useful in the development of air quality standards in zoos, contributing to finding effective bioaerosol reduction strategies, thus leading to good air quality in these places and their sustainability. Future research should include analysis of the qualitative composition of bioaerosols in the zoo premises and assessment of health risks for both animals and exposed people.

Author Contributions

Conceptualization, M.O.; methodology, M.O., K.M. and Ž.P.; validation, M.O. and K.M.; formal analysis, A.E.K. and S.M.; investigation, M.O., K.M., A.E.K., S.M., D.B. and I.B.; resources, M.O., K.M., A.E.K., Ž.P. and N.R.; data curation, A.E.K. and S.M.; writing—original draft preparation, M.O., K.M., A.E.K., D.B. and I.B.; writing—review and editing, S.M., Ž.P., N.R. and D.H.T.; visualization, N.R. and D.H.T.; supervision, M.O.; project administration, M.O.; funding acquisition, M.O., K.M., Ž.P. and N.R. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by support from the University of Zagreb, Zagreb, Croatia.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The original contributions presented in this study are included in the article. Further inquiries can be directed to the corresponding authors.

Conflicts of Interest

The authors declare no conflicts of interest.

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Sustainability 17 10517 g001
Figure 1. Location of the selected animal premises of Zagreb Zoological Garden. Legend: 1 Monkey House, 2 Tropical House, 3 Rainy Africa, 4 Bird House, and C Control site. The figure has been created from a Zoo map available at https://zoo.hr/english/.
Figure 1. Location of the selected animal premises of Zagreb Zoological Garden. Legend: 1 Monkey House, 2 Tropical House, 3 Rainy Africa, 4 Bird House, and C Control site. The figure has been created from a Zoo map available at https://zoo.hr/english/.
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Figure 2. Total number of airborne bacteria inside (I) and outside (O) the investigated premises of Zagreb Zoological Garden. a,b,c Different letters denote statistically significant differences at p < 0.001.
Figure 2. Total number of airborne bacteria inside (I) and outside (O) the investigated premises of Zagreb Zoological Garden. a,b,c Different letters denote statistically significant differences at p < 0.001.
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Figure 3. Number of Gram-negative bacteria inside (I) and outside (O) the investigated premises of Zagreb Zoological Garden. a,b,c,d Different letters denote statistically significant differences at p < 0.05.
Figure 3. Number of Gram-negative bacteria inside (I) and outside (O) the investigated premises of Zagreb Zoological Garden. a,b,c,d Different letters denote statistically significant differences at p < 0.05.
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Table 1. Characteristics of the selected animal premises of Zagreb Zoological Garden.
Table 1. Characteristics of the selected animal premises of Zagreb Zoological Garden.
ParameterMonkey HouseTropical HouseRainy AfricaBird House
Year of Construction1996 renovated197419991978
Indoor Area for Animals (m2)10235016242
Indoor Area for Visitors and Employees (m2)322302044
Animals (n)14~358 (crocodiles: 3; caimans: 2; other reptiles: 55; amphibians: 10; fish: ~270; birds: 4; mammals: 14)45 (pygmy hippopotamuses: 3; birds: 2; reptiles: 10; fish: 30)20
Type of VentilationAir conditioning chambers (always on)Air conditioning chambers (always on)Air conditioning chambers (always on)Natural
Type of Bedding in Animal EnclosuresNo beddingShredded bark + sandShredded bark
No bedding (pygmy hippopotamuses)		No bedding
Type of Flooring/Bedding in Area for Visitors and EmployeesCeramic tilesConcrete + stoneShredded barkConcrete
Table 2. Correlations between airborne bacteria inside the premises (I) and in outdoor air (O) of Zagreb Zoological Garden.
Table 2. Correlations between airborne bacteria inside the premises (I) and in outdoor air (O) of Zagreb Zoological Garden.
ParameterTotal Number of Bacteria (I)
(CFU/m3)		Number of Gram-Negative Bacteria (I) (CFU/m3)Total Number of Bacteria (O)
(CFU/m3)		Number of Gram-Negative Bacteria (O) (CFU/m3)
Total Number of Bacteria (I) (CFU/m3)1.0000.527 *0.0290.053
Number of Gram-Negative Bacteria (I) (CFU/m3) 1.0000.0370.011
Total Number of Bacteria (O) (CFU/m3) 1.0000.209 *
Number of Gram-Negative Bacteria (O) (CFU/m3) 1.000
* p < 0.05.
Table 3. Values of microclimate parameters inside (I) and outside (O) the investigated premises of Zagreb Zoological Garden.
Table 3. Values of microclimate parameters inside (I) and outside (O) the investigated premises of Zagreb Zoological Garden.
PremisesAir Temperature
(°C)		Relative Humidity (%)Airflow Rate
(m/s)
Mean ± SD (Min–Max)
Monkey House (I)24.55 a ± 3.00
(19.30–29.00)		62.18 a ± 6.96
(43.00–74.50)		0.10 a ± 0.05
(0.01–0.21)
Tropical House (I)24.46 a,b ± 3.59
(16.50–29.20)		67.21 b,c ± 7.18
(53.30–82.80)		0.08 a ± 0.04
(0.01–0.18)
Rainy Africa (I)23.15 a,b ± 3.83
(16.00–28.30)		70.05 c ± 6.48
(55.90–83.20)		0.11 a ± 0.07
(0.01–0.40)
Bird House (I)24.45 a,b ± 2.87
(16.10–27.70)		62.41 a ± 7.15
(46.00–74.10)		0.08 a ± 0.05
(0.01–0.25)
Control (O)22.24 b ± 7.62
(6.90–32.90)		65.49 a,b ± 10.01
(49.80–81.40)		0.44 b ± 0.18
(0.22–1.16)
a,b,c Different letters within a column denote statistically significant differences at p < 0.05 (air temperature), p < 0.01 (relative humidity), and p < 0.001 (airflow rate).
Table 4. Correlations between microclimate parameters and airborne bacteria inside the premises of Zagreb Zoological Garden.
Table 4. Correlations between microclimate parameters and airborne bacteria inside the premises of Zagreb Zoological Garden.
ParameterAir Temperature (°C)Relative Humidity (%)Airflow Rate (m/s)
Total Number of Bacteria
(CFU/m3)		−0.141 *0.050−0.152 *
Number of Gram-Negative Bacteria (CFU/m3)−0.164 *0.052−0.070
* p < 0.05.
Table 5. Correlations between microclimate parameters and airborne bacteria in outdoor air of Zagreb Zoological Garden.
Table 5. Correlations between microclimate parameters and airborne bacteria in outdoor air of Zagreb Zoological Garden.
ParameterAir Temperature (°C)Relative Humidity (%)Airflow Rate (m/s)
Total Number of Bacteria
(CFU/m3)		0.413 *−0.552 *−0.132
Number of Gram-Negative Bacteria (CFU/m3)0.126 −0.143−0.251
* p < 0.05.
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Ostović, M.; Matković, K.; Ekert Kabalin, A.; Menčik, S.; Pavičić, Ž.; Rudan, N.; Horvatek Tomić, D.; Beneta, D.; Bata, I. Towards Zoo Sustainability: Assessment of Indoor and Outdoor Bacterial Air Contamination Levels and Their Correlations with Microclimate Parameters. Sustainability 2025, 17, 10517. https://doi.org/10.3390/su172310517

AMA Style

Ostović M, Matković K, Ekert Kabalin A, Menčik S, Pavičić Ž, Rudan N, Horvatek Tomić D, Beneta D, Bata I. Towards Zoo Sustainability: Assessment of Indoor and Outdoor Bacterial Air Contamination Levels and Their Correlations with Microclimate Parameters. Sustainability. 2025; 17(23):10517. https://doi.org/10.3390/su172310517

Chicago/Turabian Style

Ostović, Mario, Kristina Matković, Anamaria Ekert Kabalin, Sven Menčik, Željko Pavičić, Nevenka Rudan, Danijela Horvatek Tomić, Dijana Beneta, and Ingeborg Bata. 2025. "Towards Zoo Sustainability: Assessment of Indoor and Outdoor Bacterial Air Contamination Levels and Their Correlations with Microclimate Parameters" Sustainability 17, no. 23: 10517. https://doi.org/10.3390/su172310517

APA Style

Ostović, M., Matković, K., Ekert Kabalin, A., Menčik, S., Pavičić, Ž., Rudan, N., Horvatek Tomić, D., Beneta, D., & Bata, I. (2025). Towards Zoo Sustainability: Assessment of Indoor and Outdoor Bacterial Air Contamination Levels and Their Correlations with Microclimate Parameters. Sustainability, 17(23), 10517. https://doi.org/10.3390/su172310517

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