


































Energy and Earth Science 
Vol. 5, No. 4, 2022 

www.scholink.org/ojs/index.php/ees 

ISSN 2578-1359 (Print)   ISSN 2578-1367 (Online) 

29 

Original Paper 

Assessment of Microbial Air Quality of Nashik City with 

Particular Reference to Mucorales Fungi, and in Vitro 

Evaluation of Two Triazole Antifungal Drugs against the 

Prevalent Mucor Species 

Borkar, S. G.
1*

, Ajayasree, T. S.
1
 & Riddhi Damale

1
 

1
 Soil, Seed, Plant Disease Diagnostic and Research Centre, Endeavour Scientific Agriculture, 103, 

Prestige Point, In front of Nashik Road Police Station, Nashik 422 101, India 

*
 Borkar, S. G., E-mail: borkarsg@yahoo.co.in 

 

Received: September 30, 2022  Accepted: October 31, 2022   Online Published: November 24, 2022 

doi:10.22158/ees.v5n4p29                    URL: http://dx.doi.org/10.22158/ees.v5n4p29 

 

Abstract 

Air pollution particularly that of particulate matter (PM 2.5, PM 2.10), carbon monoxide, ozone, 

nitrogen dioxide, sulfur dioxide, ammonia, lead, and air microbial contaminants, has serious 

consequences on human health. Air pollution in metros and cities around the world is measured for the 

above parameters except for the microbial air contaminants. However, microbial air contaminants are 

important sources of microbial infection in humans and particularly airborne fungi are known to cause 

diseases like Aspergillosis and Mucormycosis in immunocompromised patients which are about 160 

million in the world. 

In the year 2021, Mucormycosis disease was reported as a post-covid infection in several states of 

India as a fatal disease caused by a black fungus (Mucor) prevalent in the atmospheric air. In the 

present study, we assessed the microbial air quality (colony forming unit of microbes/m
3
 of air) of 

Nashik city air, in India, for its microbial contaminant, particularly Mucor sp., and further the 

prevalent Mucor sp. was evaluated for its reaction to two triazole antifungal drugs viz. Itraconazole 

and Fluconazole available in medical stores. 

The air quality index of 90 CFU/tidal volume for Mucor species was regarded as safe, based on the 

studies. Both the triazole drugs at their active ingredient concentration (1000 µg/mL) were unable to 

check the growth of Mucor fungi. The paper discussed in detail the methods for enumeration of 

microbial contaminant/m
3
 of air and in tidal volume.  



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Keywords 

microbial air pollutant, Mucor, mucormycosis, triazole antifungal drugs, microbial CFU/tidal volume, 

microbial AQI 

 

1. Introduction 

Mucormycosis, as a post-covid infection was a serious health issue in many Indian states in 2021 

(Borkar, 2021). The fungus responsible for this disease was a species of Mucor, which is mainly 

present in the air. Based on the anatomic localization of infection by Mucor, mucormycosis can be 

classified as one of 6 forms viz. rhinocerebral, pulmonary, cutaneous, gastrointestinal, disseminated, 

and uncommon presentation (Petrikkos et al., 2012). The infection of mucormycosis has serious 

implications with permanent disabilities like debridement of the eyes and jaws of the patients, and even 

death due to invasive infections (Neilstone et al., 2021). Generally, the infection of mucormycosis was 

observed in post-covid patients, with lowered immunity, or in immunocompromised patients (Pak et al., 

2008). The specific immunocompromised conditions include: 1) severe immunocompromised 

(non-HIV)-active leukemia, lymphoma, generalized malignancy, aplastic anemia, graft versus host 

disease, congenital immunodeficiency, solid open transplant or bone marrow transplant within 2 years 

of transplantation, or persons whose transplants are of longer duration but who are still taking 

immunosuppressive drugs. 2) Chronic diseases with limited immune deficits-asplenia, chronic renal 

disease, chronic hepatic diseases (cirrhosis and alcoholism), diabetes, nutritional deficiencies, and 

people affected by pandemic diseases like covid-19 (Monica & Chandraprabha, 2022). 

At present 2% of the world population, i.e., about 160 million people are reported to be 

immunocompromised in the world (Anonymous, 2022) and probably the targeted population for the 

diseases like mucormycosis caused by Mucor fungus present in the atmospheric air.  

Although the source of Mucor infection is through the air, which we breathe, no documentation for this 

fungus in the City Air quality Index around the world, is available on regular basis in the metros. The 

air pollution in many metros and cities around the world is measured for particulate matter (PM 2.5 and 

PM 10), Carbon monoxide (CO), and other harmful gases, but nowhere it is depicted for fungal air 

contaminants responsible for human diseases like Aspergillosis (Borkar, 2020) and Mucormycosis 

(Borkar, 2021). The lack of air quality studies for harmful air microbes can be seen as a setback to 

keeping in check the source of infection and the optimal population threshold of the microbes in the air 

for human fungal infection particularly mucormycosis. 

Therefore, in the present study, we assessed the air quality of Nashik city air, in India, for its microbial 

contaminant particularly Mucor sp. a known fungus to cause Mucormycosis in humans. The 

Mucormycosis fungi are also reported to develop resistance to the antifungal drug Amphotericin-B, the 

most commonly used drug for its control (Ellis, 2002; Asghar, 2019). Therefore, we evaluated two 

triazole antifungal drugs for their effectiveness against the Mucor fungus prevalent in the Nashik city 

air sample. 



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2. Method 

2.1 Detection of Microbial Flora in the Air Sample 

2.1.1 Growth Medium Used for Detection of Microbial Flora in Air Sample 

Potato-dextrose-agar (PDA) medium was used for the detection of microbial flora in the air sample. 

Petri plates of 7.5 cm diameter having sterilized PDA media were exposed to air breeze at 10 different 

locations in the city jurisdiction of Nashik Municipal Corporation. These 10 locations represented the 

east, west, north, south, and center locality of the city. Petri plates in 3 replicates/locations were used to 

assess the prevalence of microbes in the air at a given location. 

The air sample for microbial prevalence was assessed in the 2
nd

 week of June 2022. The maximum 

temperature, minimum temperature, and atmospheric humidity during the sampling period were 35
0
C, 

29
0
C, and 77% respectively.  

The air sample was assessed for the microbial population of fungi, bacteria, actinomycetes, yeast, etc., 

and, particular emphasis was given to the presence of Mucorales fungi in the air, as during the same 

period in 2021, the Mucorales fungi (Mucor sp. or black fungus) caused Mucormycosis disease in the 

city patients. 

2.1.2 Collection of Air Samples on Microbial Growth Media 

The microbial growth media plates were exposed to the city air of the respective location for 1-2 

minutes and covered with an upper lid. Such exposed plates for each location were numbered and 

brought to the laboratory within a period of 3 h. These plates were incubated in a BOD incubator at 

29±1
0
C temperature for the growth of airborne microbes trapped on the plates. Reading for the growth 

of bacterial colonies was taken after 48 h of incubation while reading for the presence of fungal 

colonies was noted after 3 days onwards and up to 10 days. 

2.1.3 Enumeration of Microbial Colony Forming Units (CFU)/m
3
, Trapped in the Media Plates 

To enumerate the Colony Forming Unit (CFU) of each type of microbe on the growth media in a plate 

of 7.5 cm diameter, the simplified method was followed. The 7.5 cm diameter of the plate was 

earmarked length and breadthwise, as shown in Figure 1, to calculate the area in cm
3
. 

 

 

Figure 1. Earmarking on Microbial Growth Medium Plates to Count Microbial CFU/cm
3
 

 



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The earmarked length on the plate was 5.5 cm with a breadth of 5 cm, and the height (air space 

available) on the growth medium on the plate was 1 cm, thus giving an exposed area of 27.5 cm
3
. The 

microbial colonies of respective microbes viz. bacteria, fungi (Mucor, Aspergillus, Fusarium, sterile 

mycelia, Pilobolus, etc.), actinomycetes, and yeast contained in 27.5 cm
3
 of air volume, which formed 

their respective colonies on growth media, were counted in the earmarked area. The number of colonies 

of a particular microbe obtained on this earmarked area was converted for m
3
 area by employing the 

multiplication factor of 3.63 (100 divided by 27.5=3.63). Thus, the Colony Forming Unit (CFU) of 

each microbe was calculated as CFU/m
3 
area. 

2.2 Enumeration of Microbial CFU/Breath and Assessment of Microbial Air Quality Index 

Conversion of m
3 

to liter: 1 cubic meter is equal to 1000 liter, therefore an area of 27.5 cubic cm is 

equal to 0.0275 liters or 27.5 milliliters (air). During normal breathing an adult human breath around 

500 mL air/breath which is known as tidal volume (Hallett et al., 2021), and therefore to convert 27.5 

ml of air into 500 ml of air a conversion factor of 18 (500 divided by 27.5=18) was used. Thus, the 

number of microbial colonies obtained in a 27.5 cubic centimeter area was multiplied by 18 to get the 

number of CFU present per breath of air (i.e., 500 ml air). 

2.3 Identification of Microbial Colonies 

2.3.1 Identification of Bacteria 

The bacterial colonies were studied for their morphology, color, elevation, shape, margin, and the gram 

reaction of the bacteria (Borkar, 2017). The prevalence of bacterial density/m
3
 air was estimated. 

2.3.2 Identification of Fungal Colonies 

The fungal colonies were studied for their texture, and color and were identified under a binocular 

microscope based on the fungal spores, structures, and fruiting bodies (Funder, 1968). 

2.4 In Vitro Evaluation of Triazole Antifungal Drug on Mucor Species 

Ten isolates of Mucor species trapped on growth media from 10 locations of city air were studied for 

their reaction to two triazole antifungal drugs viz. Itraconazole (200 mg capsule) and Fluconazole (150 

mg tablet), are being used for other invasive fungal infections in humans. 

Different concentrations of these antifungal drugs viz. at 100 µg/mL; 500µg /mL and 1000 µg/mL were 

tested against the mucor species isolates obtained from 10 different locations. Sterile PDA media 

containing the above concentrations of these triazole drugs were prepared, poured into sterilized Petri 

plates, and solidified. The 8 mm disc of the Mucor fungal growth of individual isolate was placed, in an 

inverted position, on these media in Petri plates. The inoculated plates were incubated in a BOD 

incubator at 29±1
0
C temp for Mucor growth/inhibition of Mucor growth, and the reading was taken 

after 5 days of incubation. 

 

 

 

 



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3. Results  

3.1 Presence of Microbes in Nashik City Air at Different Locations 

The microbes (Table 1) present and trapped in the Nashik city air were Mucor, Aspergillus, Fusarium, 

Pilobolus, and sterile fungi among the fungal species. Besides these fungal species, Actinomycetes and 

bacterial species were also present (Figure 2). No yeast/candida species were present in the air sample. 

On the air sample plates, the bacterial colonies appeared within 48 hrs; whereas the fungal colonies 

appeared after 5 days of incubation of the microbial growth media plates. Among the fungal species, 

Mucor was dominant over other fungal species with a range of 3.63 to 18.15 Colony Forming Units 

(CFU)/M
3
 air and differed with the locations in the city (Figure 3). Maximum CFU/M

3
 of Mucor was 

present in Panchavati, Bombay Naka followed by Trimbak road areas. 

 

Table 1. Presence of Microbes in Nashik City Air at Different Locations 

Name of 

location 

Presence of microbes (CFU/M
3
) in air 

Mucor Aspergillus Fusarium Actinomycetes Bacteria Pilobolus 
Sterile 

Fungi 

Dwarka 3.63 0.0 0.0 29.04 119.79 0.0 0.0 

Deolali 3.63 3.63 0.0 3.63 7.26 0.0 0.0 

Bytco Point 3.63 3.63 3.63 3.63 29.04 0.0 0.0 

Pandav leni 3.63 0.0 0.0 3.63 23.31 0.0 0.0 

Bombay 

Naka 
18.15 0.0 0.0 7.26 166.50 0.0 0.0 

Trimbak 

road 

(papaya 

nursery) 

10.89 0.0 0.0 3.63 96.57 0.0 0.0 

Panchavati 18.15 0.0 0.0 21.78 153.18 0.0 0.0 

Adgao naka 7.26 0.0 0.0 83.49 126.54 7.26 0.0 

Nandur 

naka 
7.26 0.0 3.63 7.26 29.97 0.0 0.0 

Mahsrud 7.26 0.0 0.0 10.89 156.09 0.0 7.26 

 

The environment during air sampling: Air temp Max=35
0
C; Min=29

0
C, RH=77%, Time of 

sampling=3.00 PM. 



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Figure 2. Microbial Flora of Nashik City Air 

 

 

Figure 3. Trapping of Air Microbes at 10 Locations in Nashik City 

 



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The Mucor species trapped in the air sample exhibited the black color mycelial growth of the fungus 

(Figure 4) which was further purified and identified as Mucor sp. based on the fungal structures. The 

microscopic observations exhibited the black color sporangial fruiting bodies, sporangiospores, and 

aggregation of black-brown thick structures of mycelial masses as debris (Figure 5). 

 

 

Figure 4. Fungal Colony of Mucor sp. Present in Nashik Air 

 

 

Figure 5. Microscopic Fruiting Bodies (Sporangium) of Mucor with an Aggregated Thick Mass of 

Fungal Debris on Growth Media and in Broth 



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The thick-walled fungal debris was more common besides the sporangium of the fungus in both solid 

(PDA) media and liquid potato dextrose broth. This fungal debris contained the mycelial and spore 

masses (Figure 6) of the Mucor fungus. Similar fungal structures are also reported in histopathological 

studies of mucormycosis infection (Figure 7) (Choudhary & Gahlot, 2021).    

 

 

Figure 6. Spore Masses Turned into Black Thick Structures in Fungal Growth Media 

 

 

Figure 7. Aggregation of Fungal Hypha in Mucormycosis Infection 

 

Among the air microbial communities, the bacterial species was most dominant and varies from 7.26 to 

166.50 CFU/M
3
 air. The maximum population of bacterial species was trapped in areas of Bombay 

Naka, Mahsrud followed by Panchavati. The bacterial species exhibited circular, raised, opaque, white, 

or pink colonies on growth media. The bacteria were gram-negative, cocci, and seem to be species of 

Streptococcus.                   

 

6 

7 



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These fungal and bacterial species survived an atmospheric temperature of 35
0
C during the May month 

of the summer season in the Nashik city air. 

Mucor was the dominant species among the fungal microflora in the air. The fungal species load per 

tidal volume (the volume of air which we breathe in a single breath) was calculated for the city air 

(Table 2) which differed with locations in the city and for Mucor fungus, it ranged from 18 to 90. 

 

Table 2. Presence of Fungal Microbes in the Tidal Volume of Air in Nashik City at Different 

Locations 

Sr. no Name of Location 
Presence of fungal spores/breath (500mL air) in city air 

Mucor Aspergillus Fusarium Pilobolus Sterile Fungi 

1 Dwarka 18 0.0 0.0 0.0 0.0 

2 Deolali 18 18 0.0 0.0 0.0 

3 Bytco Point 18 18 18 0.0 0.0 

4 Pandav leni 18 0.0 0.0 0.0 0.0 

5 Mumbai Naka 90 0.0 0.0 0.0 0.0 

6 
Trimbal road 

( Papaya nursery) 
54 0.0 0.0 0.0 0.0 

7 Panchavati 90 0.0 0.0 0.0 0.0 

8 Adgao Naka 36 0.0 0.0 36 0.0 

9 Nandur Naka 36 0.0 18 0.0 0.0 

10 Mahsrud 36 0.0 0.0 0.0 36 

 

3.2 In Vitro Efficacy of Triazole Antifungal Drug against Mucor sp. Prevalent in Nashik City Air 

The in vitro efficacy of two triazole antifungal drugs viz. Itraconazole and Fluconazole on the growth 

of mucor species indicated (Table 3) that these drugs were ineffective against the fungus. All three 

concentrations, i.e., 100, 500, and 1000 µg/mL were ineffective to check the growth and sporulation of 

the Mucor fungi (Figure 8). However, in the presence of these drugs, the mycelium has a reduced 

growth rate on the media containing these drugs, as compared to growth on normal growth media. On 

the normal growth media, the Mucor growth at 5 days was 7.00 cm diameter as against 1.45 to 2.77 cm 

on triazole-containing growth media. The percent inhibition of growth on triazole-containing media 

was 60.42 to 79.28 percent (Table 4). 

 

 

 

 

 



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Table 3. In Vitro Efficacy of Triazole Compounds against Mucor sp. Prevalent in Nashik City Air 

Triazole 

compound 

Concent

ration 

(µg /mL) 

Mycelial growth (in cm) of location-specific 

Mucor isolate number 

Average 

growth 

Itraconazole 

(200 mg 

cap) 

containing 

PDA media 

 1 2 3 4 5 6 7 8 9 10  

100 

 
1.9 2.0 1.8 1.8 1.7 1.6 1.7 2.0 1.6 1.6 1.77 

500 

 
1.7 2.0 1.5 1.9 1.4 1.5 1.6 1.8 1.7 1.7 1.66 

1000 

 
1.2 1.5 1.5 1.7 1.4** 1.4 1.2 1.6 1.5 1.5 1.45 

Fluconazole 

(150 mg 

tab) 

containing 

PDA media 

100 

 
2.4 3.0 2.0 2.0 2.5 2.9 2.6 1.9 4.0 4.0 2.77 

500 

 
1.8 2.2 2.0 2.3 2.0* 2.0 1.8 1.8 2.6 

2.6*

** 
2.12 

1000 

 
1.6 2.6 2.0 2.1 

2.0**

* 
2.4 1.9 1.5 2.0 2.4 2.05 

Control 

PDA 

without 

Triazole 

compound 

0.0 

 
7.00 

Note. *=change in cultural characteristic; **=Fruiting structure of Mucor absent; ***=Reverse side of 

culture is pink-red as fungal growth absorbs Triazole compound to give pink-red color. 

 

 



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Figure 8. Growth of Mucor Isolates on Triazole Antifungal Drug Containing Growth Media 

(Front Side of Plate (A) and Reverse Side of Plate (B)) 

 

 

 

 

 

 

 

 

 

 

 

 

A 

B 



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Table 4. Influence of Triazole Compounds on Growth Rate (cm/d) of Mucor sp. Prevalent in the 

City Air 

Triazole compound Concentration 

(ug /mL) 

Average Mucor 

Growth * 

(in cm on 5
th

 day) 

Inhibition of 

Mucor growth 

(% inhibition) 

Inhibition of 

growth rate (cm 

/d) 

    

 Itraconazole (200 mg 

capsule) 

 

100 

 

500 

 

1000 

 

1.77 

 

1.66 

 

1.45 

 

5.23 (74.71) 

 

5.34 (76.28) 

 

5.55 (79.28) 

 

1.04 

 

1.06 

 

1.11 

  

 Fluconazole (150 mg 

tablet) 

 

100 

 

500 

 

1000 

 

2.77 

 

2.12 

 

2.05 

 

4.23 (60.42) 

 

4.88 (69.71) 

 

4.95 (70.71) 

 

0.84 

 

0.97 

 

0.99 

Control 

(No triazole 

compound) 

 

0 

                       

                 7.00 cm growth on 5
th

 day 

                  (growth rate 1.4 cm/d) 

Note. *=Average growth of 10 isolates of Mucor collected at different locations of the city, on 

particular triazole concentration. 

 

4. Discussion 

The fungus mucor is a causative agent of mucormycosis and mostly affects immunocompromised 

people. Several disease conditions like AIDS, Cancer, diabetes, malnutrition, certain genetic disorders, 

organ transplants, congenital immunodeficiency, primary immunodeficiency diseases (PDIs), 

lymphohematogenous malignancy (LHM), non-cytotoxic immunosuppression, splenectomy and 

chronic diseases with limited immune deficits are known as immunocompromised (Meidani et al., 2014) 

and may favor the infection of mucormycosis. The population of immunocompromised persons is 

estimated to be about 160 million people in the world (Anonymous, 2022) and thus this quantum of the 

population is in the risk group of mucormycosis, if the sufficient CFU of Mucor is present in a tidal 

volume to cause mucormycosis. 

The presence of up to 90 CFU of Mucor/tidal volume seemed to be insufficient to cause mucormycosis, 

since no case of mucormycosis was reported in the city during the month of May 2022 and in 

subsequent months as compared to the mucormycosis cases in the same month in 2021. The probable 



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reason seems to be the hindrance of entry of Mucor spores by the cilia in the nostrils into the nasal 

cavities and paranasal sinuses and the innate immunity of the person. Mucormycosis is mainly a 

disease of the immunocompromised person (Pandilwar et al., 2020).  

Thus, the study of microbial Air Quality Index (AQI) is an important issue while studying the air 

quality of cities and metros. The microbial air quality index can be defined as the sum of various CFU 

of microbes present in the air and their significance in causing the concern disease/diseases. This is the 

first publication on microbial AQI, mucor/tidal volume, and the methodology of its enumeration. 

It is advised to maintain the air quality index for Mucor sp, a dominant fungal species in the city air at a 

minimal level to avoid mucormycosis infection. This can be achieved by employing the air vacuum 

cleaner machinery, in the Mucor species-dominated areas of the city based on the microbial air quality 

index for Mucorales. 

 

Acknowledgment 

The authors thank Mr. Suyog Koli, Suyash Nagre, Om Diwakar, Miss. Sweta Gadekar, Mansi Adsare, 

and Sayli Diwakar, all graduating students of Biotechnology, at City College, Nashik, for collection of 

microbial air samples at different locations in Nashik city. 

 

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