Spatial variation and comprehensive health risk assessment of microbial indoor air quality in microenvironments of North Delhi


 The high airborne microbial concentration of indoor areas may be responsible for adverse indoor air quality linked with adverse respiratory and general health effects in the form of Sick building syndromes. The current study aimed to isolate and characterize seasonal (winter and spring) levels of culturable bio-aerosols from indoor air and their impact on human health by using an epidemiological health survey. After culturing, microorganisms were identified by standard macro and microbiological protocols followed by biochemical testing and molecular techniques. Sampling results show that bacterial aerosol concentration in the winter season varies from (300–3650 CFU/m) and fugal aerosols (300–4150 CFU/m3) in different microenvironments. However, in spring bacterial aerosols ranged (450–5150 CFU/m3) and fungi (350–5070 CFU/m3). Aspergillus and Cladosporium were major recorded fungi however, Staphylococcus, Streptobacillus, and Micrococcus were major bacterial genera among all the sites. Analysis of the questionnaire survey represented that headache (28%) and allergies (20%) were major indoor health problems followed by others. Major outcomes of the current study suggested that the elevated levels of bioaerosols are a major risk factor and causing various respiratory health problems. The observed dynamics of this study will help the inhabitants to take precautionary actions to avoid hazardous bioaerosols.

2012; Kumari et al., 2016;Park et al., 2016). The presence of AM in prenursery schools may also affect the respiratory and pediatric health and well-being of the children (Madamarandawala et al., 2019). Aspergillus, Curvularia, Penicillium, and Rhizopus were the main fungal genera record from the different indoor areas in Kolkata, India (Karmakar et al., 2020). Presence of mycotoxin-producing micromycetes indoor buildings plays a crucial role in causing SBS in occupants (Švajlenka et al., 2017). Hospitals are one of the most important indoor microenvironments spread and propagation of the aero-micro ora (Chirca, 2019). An air sampling study conducted in a Hematology hospital by Cho and co-workers reported that the Aspergillus and Penicillium were the most dominant fungal genera in the outdoor and indoor side of buildings (Cho et al., 2019). A questionnaire survey is an e cient method for analyzing the health effects of indoor air quality (Wu et al., 2018). A signi cant cross-sectional survey was conducted by Herr and co-workers in 2003, represented that waking up due to coughing, coughing, bronchitis, etc.
were major recorded problems due to microbial contaminated outdoor air (Herr et al., 2003). Another cross-sectional study conducted in Australia suggested that non-biological activities such as overcrowding, dust, water supply, etc. are associated with the health issues such as respiratory problems/asthma, skin problems, skin problems, etc. in residents (Melody et al., 2016).
Delhi, the national capital city of India is the most polluted city of the country thus attracts more attention from the government and the public. Few studies were conducted in a different part of Delhi to assess the total bacterial and fungal concentration in residential areas and waste dumping sites (Gupta et al., 1993;Ghosh et al., 2013;Kumar et al., 2013). However, previous studies were unable to indicate the possible association between indoor air quality and health issues in different seasons. Questionnaire proforma helped to know the exact health problems in subjects. Seasonal isolation and characterization by using Anderson impactor will help to evaluate the qualitative and quantitative measurement of biocontaminants in suspended particulate matter. Increased or decreased level of a particular biocontaminant and symptoms will help to correlate how they interact with each other and data will be helpful to form new guidelines for the exposure of indoor bio-contaminants.
The objective of this study was to determine the pattern of morbidity among people living in industrial, commercial, and residential due to poor air quality. Another important aspect of the research was to corelate the biological sampling data with health effects and set guidelines to improve problem of ambient air pollution.

Methodology:
2.1. Location and characterization of sampling sites: Total culturable bacterial and fungal counts were measured in the common residential microenvironments situated in North Delhi from December 2018-May 2019. Delhi is the metropolis and capital of India having a total population of around 19 million. Sampling areas are situated near (around 5 km) to the Alipur dumping Yard which spreads biological pollutants by aerosolization of biological waste. Residential houses, College classrooms, lifts, academies, and laboratories of the nearby area of Satyawati College were selected for the aerobiological sampling. Sampling localities were divided into three categories residential, commercial, and industrial ( Fig. 1).

Biological air Sampling:
Air samples were collected from (July September 2019) by using the conventional settle plate method (passive gravitational method) ( Moldoveanu A M 2015). Petri plates containing mycological medium (Sabourd Dextrose agar with Chloramphenicol and Tryptic Soy agar supplemented with cycloheximide). All the samples were collected in the triplicate form and one set of agar plates were also exposed in the open-air environment as control. Samples were collected at the active hours' time so that the highest bioaerosols could be collected. Sampling was performed aseptically in each room at an average height of 1.5 m (Human breathing height). The fall season was selected for the study because many experimental and epidemiological studies of microbial air quality showed that the highest fungal spores and allergic reactions are caused in this season.

Enumeration and Identi cation of the culturable bioaerosols:
Culturable bioaerosols from all the microenvironments were cultured on optimum conditions. The plate count method was used for counting the airborne microbes. After sampling samples were transferred to the laboratory aseptically and incubated for cultivation at 37℃ for 24-36 hours in case of bacterial samples. For fungal samples the incubation was done at 26-28℃ for 7-14 days Indoor & Outdoor Temperature and Humidity were also recorded during the assessment using the Digital thermometer (Hanna Instruments USA). After incubation, the bacterial and fungal colonies were analyzed by standard procedures. Bacterial colonies were counted by using a colony counter. Microbial growth on agar plates was initially identi ed by morphology and appearance of colonies, furthermore, Gram's staining was performed followed by Biochemical and Molecular characterization in the case of bacteria (Bergey's manual 2008). Lactophenol Cotton Blue stain was used to visualize the fungal samples (Sutton and Fothergill, 1997 . Ethics Committee con rmed and approved the research, con rm that all research was performed in accordance with relevant guidelines/regulations. All authors con rm that informed consent was obtained from all participants and/or their legal guardians. Research was performed in accordance with the Declaration of Helsinki. All authors also con rm that human participants' names and other HIPAA identi ers were removed from all sections of the manuscript, including supplementary information. To achieve its form is divided into four parts, in the very rst part, demographic data like name, address, gender, age, and informed consent form containing general information about the study was included. The next part of the form includes volunteer agreement and declaration of witness. The next sector of the form is the most important part (Indoor air quality Questionnaire) which also contains general information in starting and later on, it has 26 questions related to allergy, general health effects, allergens medication history, and feedback for improving air quality. In the last part, Environmental factors like temperature and humidity were measured Moreover, it also has general health parameters e.g., Sugar, Blood pressure of respondents.
2.6 Data Analysis: Minimum, maximum, mean, and standard deviation of the bioaerosols were recorded and calculated. Statical analysis and entry of questionnaires were performed using Statical Package for Social Sciences (SPSS 24) and Microsoft Excel 2019. Single way Analysis of Variance (ANOVA) was used to show the correlation between different groups (eg. Outdoor and indoor). The signi cance of data (p-value) and odds ratio was also analyzed by using the same software. P-value was maintained at 0.05 throughout the study.
3. Results And Discussion: 3.1. Demographics of the enrolled subjects in the study: Total 223 subjects (144 (64%) male, 64 (29%) female, and 15(7%) children less than 18 years) of different age groups were enrolled in this study. The proportion of male subjects were around two-third of the total enrolled subjects. Subjects were classi ed on basis of age, sex, and residential area (Table 1). Participants were randomly selected from colleges, localities, coaching centres, and other indoor places.
Participants were explained about the scope, consent form, and questionnaire survey. Information and consent were taken from their parents in case of children below 18 years. All the subjects involved in the survey signed the consent designed by the Department of Environmental Studies, Satyawati College, University of Delhi. Subjects involved in the study were also informed about the biological sampling in different seasons in their houses.
3.2 Health effects of microbial and indoor air quality on subjects: ). An epidemiological study conducted in southern India on effects of indoor air pollutants, socioeconomic, and housing characteristics on well being of children and women recorded that these factors signi cantly in uence respiratory health and may increase the burden of respiratory illness (Rumchev et al., 2017). Findings of the pilot study conducted in Finland to assess the indoor air problems suggested that irritated, stuffy, or runny nose (20%), itching, burning, or irritation of the eyes (17%), and fatigue (16%) were the most common symptoms in the workers. The proportion of indoor air problems in women than men similar to our present study which indicates the signi cance of the current study (Reijula et al 2004

Variation in seasonal microbial concentration in different residential places:
Data obtained from microbial air sampling of selected indoor sites are represented in Fig. 3. The average concentration of culturable microorganisms (bacteria and fungi) in three months of December (2019-Feb 2020). By comparison of collected air samples data from different indoor sites (enrolled subjects) during spring and winter season shows microbial concentration is less in winter and high in the spring season than winter. In the winter season, bacterial concentration was highest in academies and the fungal load was highest in gas agencies. In the winter season, the highest bacterial concentration of 2366 ± 725 CFU/m3 was monitored in houses and the fungal count was highest in gas agencies 2983 ± 1106 followed by 2961 ± 567 CFU/m3 in coaching academies ( Fig. 3.1). Due to overcrowding and industries spring season in Delhi is hotter and more humid than in other nearby cities. Average bacterial load ranged around 2600 CFU/m3 in all microenvironments except laboratories and peaked in houses 2719 ± 1168 CFU/m3. In the case of fungi, more variation was observed between different environments and the highest concentration was observed in gas agencies with 3713 ± 1665 CFU/m3 (Fig. 3.2). This is supposed that due to high activities of students, staff in academic places the bacterial and fungal load was high in comparison to other sites. The lowest concentration of microorganisms in all seasons was in college laboratories in the case of bacteria and as well as fungi. Microbial concentrations observed in each sampling site were compared with the other sites and with background by using one-way ANOVA and signi cant statical difference (p > 0.05) was observed. However, bacterial and Fungal counts in college lifts did not show any signi cant variation (p > 0.05) between background and sampling site. Similar results were recorded in the study conducted in a hospital for the spring and winter seasons in Pakistan (Asif et al., 2018). Previous studies reported that airborne biotic concentration of more than 1000 CFU/m3 shows microbial contamination in the environment (Golo t-Szymczak and Gorny 2010).
Outdoor areas of sampling sites were used as controls in the study. Variation in microbial concentration was recorded in control samples i.e., it was higher than site samples in a few cases and lower in others. Goyer and co-workers also reported that exposure to various bacterial concentrations may affect human  Table 2 shows dominant culturable bacterial and fungal isolates from different sampling sites. More genera of bacteria and fungi were recorded from residential houses than other sampling sites (i.e., College, gas agencies, etc). The identi cation of fungal genera from different sites shows that Aspergillus spp., Penicillium, Rhizopus, Cladosporium, Alternaria, and Candida, were present predominantly in residential houses, and Cladosporium, Alternaria & Aspergillus were observed in almost all the sampling sites. Aspergillus spp. and Cladosporium were common throughout all sampling sites. Staphylococcus, Micrococcus, and Streptobacillus spp. were the most dominant genera of bacteria in all the selected sites and E. coli, G + cocci, Pseudomonas also recorded in few locations. Figure 4 shows  Culturable bacterial spore count of indoor areas exceeds outdoor counts except for laboratories. In the case of fungi, indoor bioaerosols were higher than outdoors in houses, academies, and gas agencies. A polish study shows the positive correlation between the coarse particulate matter with bioaerosol concentration but it was also observed that temperature has different effects in each season (Bragoszewska et al., 2018c). Microbial exposures are signi cantly affected by seasons which are in uenced by temperature, relative humidity, and exchange rates (Frankel et al., 2012).

Identi cation of major airborne Bacteria and Fungi present in the selected Indoor Microenvironments
Isolation, evaluation, and characterization are very necessary for controlling microbial contamination in indoor environments. Human activities, poor ventilation, and degradation of waste products are major sources of these contaminants (Karmakar et al., 2020). In this study, we have particularly focused on the biological sampling of the microenvironments which are crucial for the spread and propagation of airborne diseases but the least attention is paid on these. Current data represents that the microbial load of indoor sites changes season to season and time to time on behalf of different conditions. Collected data has been also compared with the previous studies and found as presented by different authors in the past (

Conclusion:
The major culturable bioaerosols isolated and identi ed in the current study belong to the opportunistic pathogens category. High concentrations of the microbes were observed in indoor areas in comparison to outdoors, which indicates the relation of respiratory health problems with these. Headaches are allergies are the major symptoms observed in inhabitants. To date, only limited studies are available about seasonal bioaerosol monitoring in Delhi. Hence, sampling data collected from indoor sites in different seasons will help to know more about the seasonal health problems. Data generated by this study will help to give knowledge about the bio-contaminants in indoor areas responsible for adverse health effects. Data generated from this study may contribute to the promotion and implementation of preventative public health programs and the formulation of recommendations aimed at providing healthier outdoor environments. It will also help to standardize protocols so that how we can reduce the level of biological pollution.