J.Mycopathol.Res. 62(2) : 393-400, 2024, ISSN : 0971-3719 (Print), 2583-6315 (Online)© Indian Mycological Society, Department of Botany,
University of Calcutta, Kolkata 700 019, India

10.57023/JMycR.62.2.2024.393

Aeromycological studies of libraries in Purulia district of West Bengal
and screening for potential cellulolytic fungi
SOURAV GORAI AND SUBRATA RAHA*
Department of Botany, Sidho-Kanho-Birsha University, Purulia 723104 West Bengal

Received : 23.12.2023

Accepted : 20.04.2024

Published : 24.06.2024

Aeromycological survey of the indoor atmosphere of six libraries in the Purulia district of W est Bengal was
conducted for one year to determine intramural fungal load inside the libraries. The seasonal variation of thefungi in the air of the indoor environment in the stack rooms of libraries was also investigated. Twenty-one
fungi of 17 genera were isolated during the study period. Aspergillus niger was found to be dominant,followed by Aspergillus flavus, Chaetomium globosum and Fusarium oxysporum. The fungal count was
found to change significantly with the changing of the indoor atmosphere during different seasons. The
highest counts of fungal colonies appeared in monsoon followed by post-monsoon, winter and summer. The
colony count was found to be significantly low in summer in comparison to monsoon and post-monsoon. The
cellulolytic potential of dominant mycoflora was evaluated by determining the endoglucanase activity of thecellulase enzyme. The maximum enzyme activity in all four dominant isolates was found in pH 6 at 30ºC in
peptone as a nitrogen source during the eighth day except Aspergillus niger which showed maximum
activity on day four.
Keywords: Airborne fungi, cellulase, Aspergillus niger, Aspergillus flavus, Chaetomium globosum, Fusarium
oxysporum

INTRODUCTION
Fungi are microorganisms present everywhere
and contribute significantly to the biologicaldiversity of this world. Fungi are one of the major
bio-pollutants existing in the atmosphere anddisseminated through the air. The fungal
concentration in the air of a certain locality
depends upon the vegetational types, presenceof organic and decomposing substrates,
agricultural lands, local markets and livestock
farms. The sampling of fungal spores in an indoor
environment recently draw attention as these
spores contaminate the environment and affectthe health of residents (Shrikhandia and Sumbali,2015). Aeromycological study refers to the studyof fungal load in a particular environment. Libraries
are the main backbone of the educational system*Correspondence: subrata-raha@skbu.ac.in

and the centre for the collection of resources likebooks, newspapers, magazines and journals(Ankush and Bhajbhuje, 2014). These collections
can cover a wide array of topics and subjects,
catering to the diverse interests and needs of theirpatrons. Additionally, libraries are important
institutions that play a crucial role in society byproviding access to a wide range of information,knowledge, and resources. Libraries play a majorrole in knowledge and information dissemination,
especially in rural localities. Libraries promote
literacy and education by offering reading
programs, workshops, and access to educational
materials. The physical conditions like relative
humidity, temperature, and inorganic and organic
substrates inf luence the load of indoor
aeromycoflora. The collection of air sampling dataalso provides air quality information in theintramural environment of libraries (Swapna and
Lalchand, 2016). The fungal spores that remainin the atmosphere of the libraries can cause
serious health risks like allergic asthma, rhinitis,394

Aeromycological studies of libraries

and atopic dermatitis to the visitors, readers andlibrary staff. Bio-deterioration of materials present
in libraries is a global problem as it damages
unique books and manuscripts in the library. The
cellulosic ingredients of papers are a good sourceof nutrients for fungi. Environmental factors likehumidity, light, and temperature along with biotic
factors like fungi, bacteria and insects
cumulatively accelerate the deterioration process(Bhattacharya et al. 2001). D-glucose units arelinked by -1,4 bonds to form homopolymer
cellulose (Islam and Roy, 2018). Cellulose is theutmost abundant natural biopolymer present in
the world. Cellulose degradation can be achievedby employing chemical methods, enzymatic
methods or both (Reddy et al. 2014). Filamentous
fungi often colonize on paper and degrade
cellulosic fibres by releasing cellulolytic enzymes(El Bergadi et al. 2014). These fungi are also
responsible for the alteration of the aesthetic andvisual of paper by secreting organic acids orpigments, a phenomenon known as foxing (Zotti
et al. 2011). Papers contain little proportion ofhemicelluloses, lignin, and some additives likefillers and pigments in addition to the major
component cellulose (El Bergadi et al. 2014). The
cellulase enzyme catalyzes the hydrolysis ofhomopolymer cellulose to glucose by thesynergistic action of three enzymes
endoglucanase, cellobiohydrolase, and glucosidase (Sherief et al. 2010). Traces of waterin a storage environment accelerate the hydrolytic
degradation of cellulosic fibres (Meehnian et al.
2016).

[J.Mycopathol.Res :Agar, and Corn Meal Agar with Chloramphenicol
were used for the isolation of indoor aeromycota.
The exposed plates were kept in an incubator at
28°C for 5 days. The fungal samples fromdeveloping colonies were mounted in slides withlactophenol cotton blue solution for microscopic
observation. The identification was done basedon microscopic observation and colonymorphology from standard literature (Nagamani
et al. 2006; Watanabe, 2010).
Screening of cellulolytic potential
The screening of cellulose enzyme was basedon carboxymethyl cellulose (CMC) assay (Legodi
et al. 2019). CMC agar with Congo Red (CR) wasused as a medium for the determination ofcellulolytic activity. The media was composed of
CMC (2g), KH2PO4(0.5g), MgSO4 (0.25g), gelatin(2g), Congo Red (0.2g), agar (15g), distilled water(1l) and pH was maintained at 6.8 (Gupta et al.
2012). The most prevalent fungal isolates were
inoculated on CMC agar plate and incubated for
5 days at 28°C. A yellow halo zone around thefungal colony appears due to the release of boundCongo Red after hydrolysis of CMC, confirming
the cellulolytic activity of the isolated fungi.

MATERIALS AND METHODS
Aeromycological study
Purulia is the westernmost district of the stateWest Bengal and is very close to the Jharkhand
state. The aeromycological sampling was
conducted in six different libraries present in bothrural and urban areas in the Purulia district. The
survey and isolation of airborne fungi wereconducted over one year, from June 2021 to May
2022. The fungi present in the atmosphere of theintramural environment of libraries were isolatedusing the Petriplates exposure method (Gorai etal. 2022). Potato Dextrose Agar with
Chloramphenicol, Rose Bengal ChloramphenicolFig.1. Effect of the incubation period on endoglucanase activity

Fig.2. Effect of pH on the endoglucanase activity

62(2) June, 2024]

Sourav Gorai and Subrata Raha

395

Enzyme Production

Statistical analysis of airspora

Carboxymethyl cellulose broth was prepared bydissolving CMC (2g), KH2PO4 (0.5g), MgSO 4(0.25g), and peptone (2g) in 1l distilled water todetect enzyme production. The media was
autoclaved and inoculated with the fungal isolates.
The broths containing fungal mycelia were
subjected to centrifugation at 4°C at 5000 rpm
for 15 min. The supernatant was collected andstored in the sterilized vials at 4°C for further
assays of the cellulase enzyme.

One-way ANOVA test was performed to analyze
the effect of seasonal variation in the mean colony
counts during the study duration. Post-hoc test
was done to identify exactly which seasonalcounts significantly differ from each other. The
significance was determined at the level of 0.05.

Optimization of culture conditions forcellulase activity

Fungal spores in the air of libraries play asignificant role in the biodeterioration of books andother cellulosic materials. Moreover, theimportance of the study of fungal airspora in theindoor atmosphere is increasing due to the
manifestation of fungal infection and allergy, as
the spores and mycelial fragments enter into our
respiratory system during breathing while handlingor reading the books in library. The petriplate
exposure is the most appropriate method for the
study of intramural mycobiota (Lanjewar andSharma, 2014). The air in the indoor libraryenvironment was found to contain a good numberof fungi along with their spores for the entire year.
A total of 3121 fungal colonies belonging to 21
species under 17 genera along with some sterile
mycelial forms were isolated during this one yearof aeromycological survey of libraries (Table 1).
The most dominant fungal isolate was found to
be Aspergillus niger (15.76%) and the otherdominant fungal isolates were Aspergillus flavus(11.15%), Chaetomium globosum (9.45%) andFusarium oxysporum (9.42%). These four
dominant fungi contributed about 46% of total
fungal counts throughout the year and the genus
Aspergillus alone contributed 28% of the total
fungal load. Curvularia lunata (6.89%),
Cladosporium cladosporioides (5.64%), Alternaria
alternata (5.96%), and Rhizopus stolonifer (5.06%)
were the other significant fungal species traced
during the air sampling. Three species ofAspergillus viz. Aspergillus niger, Aspergillus
flavus, and Aspergillus fumigatus were detectedin the intramural environment of libraries, among
which Aspergillus niger was the dominant
species. The predominance of Aspergillus in theindoor library environment was reported at
Universities such as University of Michigan,

In CMC broth media the effects of different culture
parameters like incubation period, temperature,
pH and nitrogen source on cellulase production
by the dominant fungal isolates were determined.
The effects of all the parameters on enzyme
activity were assayed by determining the cellulase
activity at different temperatures (20ºC, 30ºC, and40°C), incubation periods (up to 16 days at 3-day
intervals), pH (5, 6, 7, and 8), and nitrogen sources(gelatin and peptone).
Assay of Endo-  -1,4-glucanase by DNS
methodThe activity of Endo--1,4-glucanase of cellulase
was determined through the DNS (3,5Dinitrosalicylic acid) method by measuring thequantity of reducing sugar released by thehydrolytic activity of the enzyme (Miller, 1959). The
substrate i.e., CMC solution (1%) was preparedusing 1N citrate buffer (pH 5.0). Crude enzyme(100 l) along with 1 ml citrate buffer was mixed
with 1 ml CMC solution and the mixture was kept
at 45°C for 45 min. The reaction was stopped byadding a DNS solution (Islam and Roy, 2018). The
DNS-treated samples were then placed in aboiling water bath for 10 minutes followed bycooling for colour development and stabilization.
The absorbance was taken at 540 nm by UV-Vis
spectrophotometer (Shimadzu UV1800). One
unit of enzyme activity was expressed as theamount of enzyme that could hydrolyze CMC todischarge 1mol of glucose within 1 min ofreaction time (Shanmugapriya et al. 2012).

RESULTS AND DISCUSSION
Aeromycological investigation

396

Aeromycological studies of libraries[J.Mycopathol.Res :Table.1: Monthly count of fungal colonies in the indoor environment of libraries

Spore types

Month

Total

%

Jun

Jul

Aug

Sep

Oct

Nov

Dec

Jan

Feb

Mar

Apr

May

Curvularia lunata

12

17

20

11

17

21

27

31

18

16

16

09

215

6.89

Cladosporium
cladosporioides

-

-

-

-

06

17

32

50

34

18

11

08

176

5.64

Aspergillus niger

42

45

49

43

30

25

34

45

36

54

48

41

492

15.76

Aspergillus flavus

34

48

42

42

34

31

28

25

18

15

12

19

348

11.15

Aspergillus
fumigatus

16

12

08

02

-

-

-

-

-

-

-

05

43

1.38

Penicillium
chrysogenum

-

-

05

07

07

16

02

08

06

02

-

-

93

2.98

Fusarium
oxysporum

32

40

42

37

32

28

23

17

16

08

07

12

294

9.42

Drechslera sp.

16

12

06

02

-

-

-

-

-

-

-

04

40

1.28

Alternaria
alternata
Alternaria
brassicicola

19

23

27

32

27

18

12

05

-

-

08

15

186

5.96

-

06

10

12

09

09

15

11

13

12

07

-

104

3.33

Nigrospora oryzae

06

14

17

21

11

07

02

-

-

-

-

-

78

2.50

Rhizopus
nigricans
Chaetomium
globosum

08

12

20

26

19

12

15

08

06

-

-

-

126

4.04

36

48

42

37

30

23

18

15

12

10

10

14

295

9.45

Mucor mucedo

12

14

22

16

10

08

-

-

-

-

-

-

88

2.82

Rhizopus
stolonifer
Cunninghamellaechinulata

14

18

26

24

21

16

16

12

11

-

-

-

158

5.06

12

11

08

06

02

-

-

-

-

-

-

-

41

1.31

Aureobasidium sp.

10

10

07

06

03

02

-

-

-

-

-

-

38

1.22

Paecilomyces sp.

10

14

16

21

18

12

07

04

-

-

-

-

102

3.27

Stachybotrys sp.

06

09

14

08

05

02

-

-

-

-

-

-

44

1.41

Diplodia sp.

04

07

13

08

03

-

-

-

-

-

-

-

35

1.12

Phoma sp.

04

06

06

08

12

10

15

13

08

-

-

-

62

1.99

Sterile mycelia

07

08

06

08

05

05

06

04

02

03

04

05

63

2.02

Total

312

388

411

377

303

262

260

241

174

138

123

132

3121

% contribution

10.00

12.43

13.17

12.08

9.71

8.39

8.33

7.72

5.58

4.42

3.94

4.23

University of Madras, and from Nagpur by Ankush
and Bhajbhuje (2014) support this present finding.
Five fungal species viz. Aspergillus niger,
Aspergillus flavus, Fusarium oxysporum,
Chaetomium globosum and Curvularia lunata
were present in the air of the libraries throughout

the year. The common fungal constituents in theindoor atmosphere of libraries at Aurangabad are
Curvularia sp., Helminthosporium sp., Bispora
sp., Fusarium sp., Torula sp. and Cladosporium
sp. as reported by previous authors. Curvularia
lunata, Fusarium oxysporum and Cladosporium

Sourav Gorai and Subrata Raha

62(2) June, 2024]

397

Table 2 : ANOVA table for seasonal variation of colony countSource of Variation

SS

df

MS

F

Between Groups

13481.76

34493.92

3.642168

Within Groups

103644.1

841233.859

Total

117125.9

87

P-value

F crit.01*

2.713227*Significance at the level of 0.05

Table 3: Results of Post Hoc test for ANOVA

LevelsMean Difference

P-value

Monsoon - Post-monsoon

7.69

.48

Monsoon - Winter

19.82

.08

Monsoon - Summer

32.64

.004*

Post-monsoon - Winter

12.13

.25

Post-monsoon - Summer

24.95

.01*

Winter - Summer

12.82

.21

*Significance at the level of 0.05
Table 4: Seasonal variation of fungi in the indoor atmosphere of libraries

Season with fungal contribution (in %)Fungal isolates

MonsoonPost-monsoon

WinterSummerCurvularia lunata

4.54

5.21

11.18

10.43

-

2.45

17.06

9.41

Aspergillus niger

12.59

10.43

16.91

36.39

Aspergillus flavus

11.48

11.38

10.44

11.70

Aspergillus fumigatus

3.33

0.21

-

1.27

Cladosporium cladosporioides

Penicillium chrysogenum

0.46

3.19

2.35

0.51

Fusarium oxysporum

10.56

10.32

8.24

6.87

Drechslera sp.

3.15

0.21

-

1.02

Alternaria alternata

6.39

8.19

2.50

5.85

Alternaria brassicicola

1.48

3.19

5.74

4.83

Nigrospora oryzae

3.43

4.15

0.29

-

Rhizopus nigricans

3.70

6.06

4.26

-

Chaetomium globosum

11.67

9.57

6.62

8.65

Mucor mucedo

4.44

3.62

-

-

Rhizopus stolonifer

5.37

6.49

5.74

-

Cunninghamella echinulata

2.87

0.85

-

-

Aureobasidium sp.

2.50

1.17

-

-

Paecilomyces sp.

3.70

5.43

1.62

-

Stachybotrys sp.

2.69

1.60

-

-

Diplodia sp.

2.22

1.17

-

-

Phoma sp.

1.48

3.19

5.29

-

Sterile mycelia

1.94

1.91

1.76

3.05

398

Aeromycological studies of libraries

Fig.3. Effect of temperature on the endoglucanase activity

Fig. 4. Effect of nitrogen source on the endoglucanase activity

cladosporioides were also the common
constituents of the air mycoflora of libraries in the
Purulia district. Along with different species ofAspergillus, Cladosporium spp., Penicillium spp.
and several other fungi like Alternaria alternata,
Fusarium solani, Mucor hiemalis,Curvularia
lunata, and Rhizopus stolonifer were recorded
from indoor air of libraries from different areas
like Cuba (Borrego et al. 2010), Ethiopia(Hayleeyesus and Manaye, 2014), Poland(Pastuszka et al. 2000; Zielinska-Jankiewicz etal. 2008), and India ( Bhattacharya et al. 2001;
Shrikhandia and Sumbali, 2015) which
corroborate with the findings of current
investigation.The highest fungal counts were
attributed to August (13.17%) followed by July
(12.43%) and September (12.08%) and thelowest number was found in April (3.94%).
Seasonal variation of airborne fungi
The study period was divided into four seasons:monsoon (June-August), post-monsoon(September-November), winter (DecemberFebruary) and summer (March-May). The highest
fungal colony counts were observed during
monsoon (35.6%) followed by post-monsoon(30.18%), winter (21.63%), and summer[J.Mycopathol.Res :(12.59%). One-way ANOVA (Table 2) suggested
a significant variation in the seasonal colony
numbers (P<0.05). Further, post-hoc analysis(Table 3) indicated a significant difference in thecolony counts between both monsoon andsummer as well as post-monsoon and summer(P<0.05). In seasonal variations of fungal
appearance, the fungus Aspergillus niger(12.59%) was found to be dominant followed by
Chaetomium globosum (11.67%), Aspergillus
flavus (11.48%) and Fusarium oxysporum(10.56%) in monsoon. In post-monsoon
Aspergillus flavus (11.38%) was the highest in
occurrence followed by Aspergillus niger(10.43%), Fusarium oxysporum (10.32%) and
Chaetomium globosum (9.57%). In winterCladosporium cladosporioides (17.06%) was thedominant followed by Aspergillus niger (16.91%),
Curvularia lunata (11.18%), and Aspergillus flavus(10.44%). In summer Aspergillus niger (36.39%)
again appeared as the dominant fungus followedby Aspergillus flavus (17.70%), Curvularia lunata(10.43%) and Cladosporium cladosporioides(9.41%) (Table 4).
Enzymatic activity
Several fungal species can use cellulose as acarbon source for their growth and sporulation.
An extensive range of fungi were traced from theatmosphere of libraries due to the existence ofdifferent cellulosic materials in libraries which
provide a good carbon source for them. Thus, it
was felt necessary to determine the cellulolytic
potential of isolated dominant fungi as a part ofthis present investigation. The four dominant fungiviz. Aspergillus niger, Aspergillus flavus,
Chaetomium globosum and Fusarium
oxysporum were tested for cellulose utilization
by culturing in the existence of the only carbon
source cellulose. These four fungi were shown
luxuriant growth by utilizing cellulose through the
production of the enzyme cellulase. The cellulase
production was found maximum on day 4 for
Aspergillus niger, and then a gradual decrease
was observed. In the case of Aspergillus flavus,
Chaetomium globosum and Fusarium
oxysporum, the cellulase activity was boosted
with the culture duration and the highest activity
was noticed on day 8 followed by a sharp decline.
On day 8, Aspergillus flavus produced the62(2) June, 2024]

Sourav Gorai and Subrata Raha

uppermost activity f ollowed by Fusarium
oxysporum and Chaetomium globosum (Fig.1).
pH of the medium was another parameter used
for determining the effect on enzyme production.
The fungi were cultured at different pH ranges(5.0 to 8.0) to determine the optimum pH for the
production of cellulase. All the tested isolates wereable to produce cellulase from pH 5 to 8 with the
maximum at slightly acidic conditions at pH
6,similar to earlier findings where it was reported
that the optimum pH for cellulase activity ofAspergillus niger is 6.0-7.0. The acidic pH of themedium promotes cellulase production was also
claimed by Andrade et al. (2011) and Sarkar and
Aikat (2014).The activity was significantly
decreased when the pH was increased to 8
(Fig.2). The consequence of temperature on
enzyme release was tested by incubating in threedifferent temperatures viz. 20ºC, 30ºC and 40ºC
at pH 6.0. It is interesting to note that the ideal
temperature for the production of the enzyme forall the isolates was found at 30ºC. Chaetomium
globosum was found to produce the highest
activity followed by Aspergillus niger, Fusarium
oxysporum, and Aspergillus flavus in theiroptimum temperature however, in below andabove optimum temperature tested fungi are also
found to be capable of cellulase production (Fig.3).
Sohail et al. (2009) also observed sufficient
endoglucanase production at 30ºC for Aspergillus
niger. The maximum cellulase activity was also
reported in Trichoderma reesei at 30ºC andgradually decreased with the enhancement of
temperature (Darabzadeh et al. 2019).
Temperature significantly affects the growth andmetabolism of an organism. Temperature below
the optimum was reported to decrease the
enzyme activity as a result of low penetration ofsubstrates across the cell, thus reducing
cellulase production of test fungi (Dutt and Kumar,2014). Two nitrogen sources peptone and gelatinwere used to analyze the preference of nitrogensource for cellulase enzyme production. In boththe nitrogen sources Aspergillus niger produced
the maximum activity and Fusarium oxysporum
showed the lowest activity. All the tested fungal
isolates showed higher enzymatic activity whengrown in peptone as a nitrogen source (Fig.4).
The optimum condition for the cellulolytic potentialof Aspergillus niger was determined on day 4, at
pH 6, after incubation at 30ºC with peptone as a399

nitrogen source. However, Aspergillus flavus,
Chaetomium globosum and Fusarium
oxysporum were found to reach their highest
cellulolytic potential at pH 6 and 30ºC with peptone
as a nitrogen source after incubation for 8 days.
Reddy et al. (2014) got maximum enzyme
recovery between 3 to 5 days of incubation andhigh cellulase activity for Chaetomium wasreported after 144 hours of fermentation (AlKharousiet al. 2015). Dutt and Kumar (2014)
studied the production of cellulase from
Aspergillus niger and Aspergillus flavus and got
the maximum activity at day five. Another study
conducted by Ramanathan et al. (2010) observed
the optimum cellulase production of Fusarium
oxysporum at 12 days of incubation at pH 6 at
50ºC. So, the optimum parameters for the
maximum activity of the cellulase enzyme varyfrom species to species.
ACKNOWLEDGEMENT
We express our sincere gratitude to DSTBT,
Govt. of West Bengal (552(Sanc.)/ST/P/S&T/2G5/2018) for their financial support.
DECLARATION
Conflict of interest: Authors declare no interest
conflict.
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