Enlisting Aero-algal Flora and Algal Allergens Using Fan Dust Sampling Method from Pune, Maharashtra

Algae in the atmosphere are one of the bio-components of the environment. Despite the fact that they may have a negative impact on human health, they are the least studied organism in the field of aerobiology. Airborne algae continue to be viable in the atmosphere in the form of spores and filaments. In order to investigate the intramural diversity of airborne algae in Pune, eight residences were chosen, from which 16 fan dust samples were collected over a ten-day period. Total (108) aero-algal forms were recorded. Out of which 107 were Cyanophyta, and only one was Chlorophyta. The dominance of Cyanophyta members is attributed to the presence of a thick mucilaginous sheath around them, which allows them to tolerate the dryness. Overall, filamentous cyanophytes (71) outnumber coccoid cyanophytes (36). However, both fan dust slide samples and culture slide samples show variance. Coccoid cyanophytes (53) outnumber filamentous cyanophytes (11) in direct slide scans, although filamentous cyanophytes (25) were somewhat more numerous in culture samples than coccoid Cyanophyta (19). This increase in filamentous forms might be because of hormogonia of filamentous algae, which remained unidentified in direct slide scan and upon inoculation underwent reproduction increasing number of filamentous algae in culture slides. Chrooccocus, Gloeocapsa, Lyngbya, Nostoc, and Stigonema were among the forms found on the first and tenth days of fan dust sampling, indicating that they were a common inhabitant of fan dust during the study and that the fan, as an aero sampler, was able to capture them, demonstrating its potency as an aero sampler. Soil can be a substantial factor in the creation of fan dust, as these genera were frequently separated from the soil. Gloeocapsa, Phormidium, Lyngbya, Schizothrix, Cylindrospermum, Nostoc, Anabaena, Scytonema, Calothrix, and Scenedesmus were among the allergic aero-algal genera discovered during the investigation.

Dust is omani present and found on different objects like tables, bookshelf, beds, floors etc. Bacteria, protozoa, fungus, pollen grain, and other organisms were discovered in dust from diverse locations. One of its most essential components was microalgae. Ehrenberg (1844) 1 was the first to report viable algae derived from sea dust. Since then, scientists have experimented with a variety of techniques to capture dust from various locations. Pandkar (2011) 2 classified a few of these approaches. Luty et al. (1964) 3 investigate aerospora in the Santa Catalina Mountains and the Tucson area at various heights. At San Antonia, Texas, Fulton et al. (1966) 4 used aircraft to collect air samples. Harold E. Schlichting, Jr. (1971) 5 studied algae and protozoa using seafoam as a sample. Burge et al. (1982) 6 investigated indoor allergens using a battery-powered rotarod sampler. Lee et al. (1989) 7 successfully trapped marine cyanobacteria in an air sample. Ahmed D. Gamal (2008) 8 employed the Petri plate exposure method in Cairo and discovered Nostoc spp. as a common isolate. Nuhu and Ahmad (2008) 9 in Saudi Arabia employed biofilm from the cooling pad of a greenhouse evaporative cooling system to evaluate cyanobacteria fouling. In their review article, Stephen Ambu 15 investigated air, water, and soil samples and discovered that algae react differently to climate variations, resulting in seasonal variance. N.K.   16 used a tilak rotarod sampler and a Petri plate exposure method to record thirty-four airborne algae in which Cyanophyta was found to be prevalent at the height of 2.5 metres. N.K. Sharma et al. (2011) 17 suggested that the abundance of terrestrial algae in the atmosphere could be due to their adaptation to dryness. Dubey 25 and Sunita Jawale et al. (2017) 26 used the Petri plate exposure method to investigate the aero algae of Aurangabad. A miniature Lakhanpal and Nair aero sampler was mounted on the mudguard of a Vespa scooter by   27 to determine the presence of allergenic algae at human breathing level in Nagpur. Chougule et al. (2016) 28 collected house dust from patients with nasobronchial allergies and discovered that Cyanophyta was the most common isolate in those homes.
Balkrishna and Gunale (1980) 29 published the first study on the occurrence of airborne algae in Pune's environment. Pandkar (2010) 30 conducted extramural rotarod sampling in Pune and found Cyanophyta to be the dominant group. V.S. Patil et al. (2014) 31 employed the Petri plate exposure method to identify 24 algal allergens in the Pune atmosphere. Pandkar (2011, 2017) 2,32 made the first attempt to employ the fan as an aero sampler in Nagpur and Guhagar. However, there were no reports on intramural air sampling, except for a few extramural reports from Pune. As a result, this research was carried out to investigate intramural aero algae employing the fan as an aero sampler.
For this investigation, the fan was employed as an aero sampler to collect samples from eight homes in and around Pune. The study focuses on a preliminary survey to validate the existence of aero algae in fan dust samples, the source of the algae, and the algal flora acquired, and to report allergic aero algae from fan dust samples collected.

Material and Method
Despite the numerous types of samplers listed above, an affordable, adaptable sampler is always required for fundamental qualitative analysis. One or more ceiling fans can be found in any home, workplace, or institution. Because of the electrostatic charge created on the fan blades due to friction with air, a thick coat of airborne dust collects on the edges of fan blades as they revolve. The first layer of dust absorbs moisture from the air, increasing their ability to capture more air particles and resulting in a thicker dust coat on the fan blades. Furthermore, because these fans are primarily out of human reach, they collect only dust from the air and are less susceptible to contamination. Given that fan was employed as a sampler in the current investigation, a novel strategy in aerobiological research is rarely undertaken.
From March to June 2019, 16 samples were gathered utilising a fan as a sampler in and around Pune. Initially, dust was collected from the fan's blades with the help of a spatula and stored in a clean zip lock bag. After collecting fan dust, the blades of the fan were cleaned using a soap solution and then a clean towel. For the next ten days, the fan was used in the usual manner. The dust was scraped from the edges of fan blades using a spatula on the tenth day and placed in a clean zip lock bag. Fan dust collected was processed by following two methods,

Fan dust Culture Preparation
Samples in zip lock bags were then bought to the laboratory; some of the dust from samples collected from the fan was inoculated in sterile B.G.11 media and was incubated naturally. Growth starts to appear after a month. Upon optimum growth, the slide was prepared using glycerin as mounting media.

direct Fan dust Slide Preparation
The remaining samples were utilised to prepare direct fan dust slides. These fan dust samples were macerated with dilute HCl to dissolve dust calcium and magnesium carbonates. The remaining acid was washed well with sterile D/W using a centrifuge (Pandkar 2011, Pandkar 2017) 2,32 . Few drops from the supernatant were mounted on the centre of a clean glass slide with glycerin as mounting media. The slide was then covered with a 22×60 mm coverslip and sealed with nail polish. Slides prepared were further identified and microphotographed. The identification of algae was done by using standard available literature. (Desikacharya, 1959;Prescott, 1954) 33  Cumulatively 108 algal forms were recorded. Out of (108) aero-algal forms recorded, (63) were identified up to class level, (19) up to genera level and (26) up to species level, respectively ( Table 1 and Table 2). Out of 108 algal forms, 107 were Cyanophytes pointing towards its supremacy in the indoor environment of Pune. Similar results were obtained by Gunale (1980) 29 35 at Fergusson College Pune in which samples were collected from various habitats shows Cyanophyta's dominance with the scarce presence of Chlorophyta followed Bacillariophyta.
Cyanophyta members were further divided into Cyanophyta filamentous and Cyanophyta coccoid types. Coccoid cyanophytes (53) were shown to be dominant over filamentous cyanophytes (11) in direct slide scanning, although filamentous cyanophytes (25) were marginally more numerous than coccoid (19) cyanophytes in culture samples (Graph I and Graph II). This discrepancy could be attributed to the fact that coccoid cyanophytes were in a vegetative stage or that the spore forms were not viable. In addition, coccoid cyanophytes could easily be swept away by the wind due to their circular form and lightweight. As a result, their quantity was high in direct slides of fan dust samples. On the other hand, hormogonia might be found as tiny particles in direct fan dust that remain unidentified in direct fan dust slide proliferated after being inoculated in culture media, increasing the number of filamentous algae in culture samples. It specifies that sampling should be followed by culture, which will aid in the study of latent viable spores, which are difficult to see during slide scanning.
Generic diversity obtained from culture reveals diverse aero algal filamentous cyanophytes compared to coccoid cyanophytes. In culture samples, genus Nostoc (11) was dominant with Nostoc punctiforme recorded from (7) samples followed by Nostoc verrucosum, Nostoc muscorum, and Nostoc ellipsosporum recorded from (1) sample each. It was followed by Stigonema (3) with Stigonema hormides and Stigonema   (2)  Variation among algal forms per spot in culture samples was observed. HFD I (6) shows maximum algal forms, followed by HFD III, where (5) algal forms were recorded.HFD II, HFD IV, and H.F.D. V recorded (4) algal forms each. These cultures were obtained from fan dust samples collected on the first day of sampling.
The same spots on the tenth day show a significant drop in the algal count. HFD II' recorded (3) algal forms followed by HFD I', HFD IV', and HFD III' recorded (2) algal forms each. HFD III' shows the presence of a single form. Sampling spots like HFD VI, HFD VII and HFD VIII with (2) algal forms remained unchanged on both days of sampling (Graph IV).
Chrooccocus, Gloeocapsa, Lyngbya, Nostoc, and Stigonema were among the forms found on the first and tenth days of fan dust sampling, indicating that they were a common inhabitant of fan dust during the study and that the fan, as an aero sampler, was able to capture them, demonstrating its potency. Soil can play a key role in the formation of fan dust, as these genera were commonly isolated from soil Sathish C. et al. (2020) 36 .
Fan dust, a typical component of household dust that contains a number of allergenic components, appears to be a potential cause of human exposure and sensitisation to different algae, perhaps resulting in clinical allergy disorders. However, a more thorough investigation is needed because all houses reveal the existence of aero algal forms, which could be possible allergenic algal forms capable of causing allergic reactions in immunocompromised people.

ConClUSion
A total of 108 algal species were discovered in 8 homes in and around Pune. There were 107 cyanophytes, showing that the Cyanophyta dominated the group. The existence of a thick mucilaginous coating around Cyanophyta members allowed them to endure desiccation by dryness, resulting in their supremacy. Sampling must be followed by culturing to see any latent spore forms. Gloeocapsa, Phormidium, Lyngbya, Schizothrix, Cylindrospermum, Nostoc, Anabaena, Scytonema, Calothrix, and Scenedesmus were among the allergenic aero-algae found during the study. Allergic species such as Nostoc muscorum, Nostoc punctiforme, and Phormidium molle were also discovered. However, such intramural allergenic algae represent significant health hazards, necessitating more investigation.
According to the results of the study, a ceiling fan may be employed as a cost-effective multifunctional aero sampler.