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Environ Sci Pollut Res (2016) 23:3747–3757

DOI 10.1007/s11356-015-5585-1

RESEARCH ARTICLE

Airborne microorganisms cultivable on naturally ventilated


document repositories of the National Archive of Cuba
Sofía Borrego 1 & Ivette Perdomo 1

Received: 15 June 2015 / Accepted: 7 October 2015 / Published online: 24 October 2015
# Springer-Verlag Berlin Heidelberg 2015

Abstract The quality of the indoor air can provide very use- Keywords Archive . Aspergillus . Bacteria Gram-positive .
ful information for the artwork conservation. The aim of the Fungi . Indoor environment . Microbial indoor air quality .
study was to evaluate the microbial concentration inside six Natural cross-ventilation system
document repositories of the National Archive of the Republic
of Cuba in two months of 1 year. The repositories are large,
high, and have a natural cross-ventilation system. The micro-
bial sampling was done in July 2010 (summer or rainy month) Introduction
and February 2011 (winter or dry month) using the SAS Super
100 biocollector at 100 L/min. An appropriate selective cul- Libraries and archives have been used for centuries to preserve
ture media were used to isolate fungi and bacteria. A high total a wide variety of materials and to store precious information
microbial concentration on the north side of the building in (Cappitelli et al. 2009; Lavin et al. 2014). Documentary heri-
two studied months was observed. The fungal concentrations tage materials, such as books, maps, and drawings, as well as
were significantly higher in July 2010 in all repositories, while other paper or parchment documents preserved in indoor envi-
the bacterial concentrations were significantly higher mostly ronments, such as libraries, archives, and museums, are subject
in February 2011 only in repositories located on the first and to deterioration caused by physical, chemical, and biological
second floor of the building. Eight fungal genera in the indoor agents and can result in heavy economic and cultural losses.
air of all environments were isolated. Regardless of the side of Particularly, materials made of organic compounds can be de-
the analyzed building, Penicillium, Aspergillus, and teriorated for bacteria, fungi, insects, and rodent (Mandrioli
Cladosporium were the predominant genera. Aspergillus et al. 2003; Rakotonirainy et al. 2007; Trovão et al. 2013).
flavus and Aspergillus niger were the species isolated in al- Numerous studies have been carried out to assess the com-
most all of the analyzed repositories in the studied months. plexity of chemical, physical, and biological risks in archives,
Gram-positive bacteria prevailed among bacterial groups iso- libraries, museums, and all environments, where documents
lated from indoor air repositories, and some percentages and artworks are exhibited and stored (Nugari and Roccardi
corresponded to the genera Bacillus and Streptomyces. In 2001; Saiz-Jimenez and Gonzalez 2007; Zielińska-Jankiewicz
Cuba, the temperature and relative humidity are high during et al. 2008; Harkawy et al. 2011; Pinheiro et al. 2011; Roussell
the whole year but the natural ventilation plays an important et al. 2012; Grbić et al. 2013; Nunes et al. 2013; Guiamet et al.
role in retarding microbial growth on materials. 2011; Lavin et al. 2014). The main goal of any preventive
approach should be to limit the biodeterioration of cultural
Responsible editor: Robert Duran heritage and to prevent diseases in operators and visitors.
The quality of the indoor air can provide very useful infor-
* Sofía Borrego mation for the conservation of artworks (Nunes et al. 2013;
sofy.borrego@gmail.com Ruga et al. 2015). Aerobiological investigations have made it
possible to identify source, access flow, and areas of major
1
Laboratory of Preventive Conservation, National Archive of the accumulation of the airborne microorganisms, other than their
Republic of Cuba, Havana, Cuba daily and seasonal variation (Nugari and Roccardi 2001).
3748 Environ Sci Pollut Res (2016) 23:3747–3757

During the last decades, a strong correlation between aero- ago. A digital thermo-hygrometer (model 8705; Bresciani,
biology and biodeterioration of artworks has been claimed, Italy) was used to measure these indoor environment param-
due to the fact that the air is the main vehicle for the dispersion eters at the moment of sampling.
of microorganisms (Saiz-Jimenez and Gonzalez 2007).
Several lists of airborne microorganisms and their counter- Microbiological sampling of air
parts in artworks have been published (Mesquita et al. 2009;
Cappitelli and Sorlini 2010; Niesler et al. 2010; Harkawy et al. Two samplings were done in two different months of 1 year.
2011; Borrego et al. 2010, 2012; Nunes et al. 2013; Borrego The first sampling was done in July of 2010 (summer or rainy
and Perdomo 2014; Guiamet et al. 2011; Lavin et al. 2014; season) and the second one in February of 2011 (winter or dry
Fekadu and Melaku 2014; Skóra et al. 2015; Micheluz et al. season), using the SAS Super 100 sampler at 100 L/min
2015). Therefore, monitoring the microbiological contamina- placed at a height of approximately 1.5 m. Five zones were
tion of air is essential to assess pollution levels and to evaluate analyzed by triplicate (Fig. 1).
any potential risk to the preservation of the cultural heritage Petri dishes with Malt Extract Agar (MEA) (BIOCEN,
and represents the first step to a successful preventive strategy Cuba) supplemented with NaCl (7.5 %) (Rojas et al. 2002;
(Ruga et al. 2015). Borrego and Perdomo 2012) were used for isolating fungi and
For years, the National Archive of the Republic of Cuba nutrient agar (BIOCEN, Cuba) for bacteria. The culture me-
(NARC) carried out studies of microbial quality of the indoor dium MEA supplemented with NaCl facilitates the growth of
environment of their repositories to establish comparative stud- halophilic fungi and some xerophilic species; also, it is used to
ies and to characterize their environmental behavior. Therefore, limit the colonies growth of Mucorales.
the aim of this study was to evaluate the microbial prevalence The MEA plates were incubated for 7 days at 30 °C, and
inside six repositories of the NARC in two months of 1 year. the nutrient agar plates were incubated at 30 °C for 72 h. Then,
the colony count was performed and the necessary calcula-
tions of air were made in order to determine the microbial
Materials and methods concentration expressed in colony forming units per cubic
meter (CFU/m3).
Description of the repositories of archive Relative density (RD) was conducted according to Smith
(1980) where
The NARC is located in old urban area of Havana City known
as Old Havana, at 174 m of Havana Harbor. This institution Number of colonies of the genus or species
RD ¼  100
has 30 repositories located on three floors. The repositories Total number of colonies of all genera or species
have holes that cross over the outer walls in an angle of 45°, Relative frequency (RF) was determined according to
and they are protected by metallic meshes. These holes were Esquivel et al. (2003) where
made at different heights (1.5 and 4.5 m, approximately) to
facilitate the inflow of outside air into the repositories and to Times a genus is detected
RF ¼  100
secure a good natural cross-ventilation system (Fig. 1). The Total number of sampling realized
repositories were randomly selected and distributed across the
three floors of the building and on both sides of the same
(north and south side). Six repositories were selected for this Identification of the microbial isolates
research. The identification numbers are as follows: 4 and 6
(premises of the lower-ground floor), 14 and 20 (premises of Cultural and morphological characteristics of fungal colonies
the first floor), and 24 and 28 (premises of the second floor). were observed, and the identification was performed accord-
Repositories 4, 14, and 24 are located on the north side, ing to different manuals (Barnett and Hunter 2003; Klich and
whereas 6, 20, and 28 are on the south side of the building. Pitt 1994). Bacteria were grouped according to the morpho-
They are characterized by their large size, and their dimen- logical characteristics of the colonies and were typified by the
sions (length, width, and height) are as follows: for repository Gram stain. However, some determinations were made to
4, 15.2 m×6.2 m×2.5 m; for repositories 14 and 24, 15.2 m× identify the Streptomyces and Bacillus genera according to
6.2 m×5 m; for repository 6, 25.3 m×6.2 m×2.5 m; and for biochemical tests described in Bergey’s Manual of
repositories 20 and 28, 25.3 m×6.2 m×5 m. Systematic Bacteriology (Holt 1984).

Thermo-hygrometric sampling Statistical analysis

Temperature (T) and relative humidity (RH) are measured Student’s t test was used to evaluate differences in the fungal
daily in all the repositories of the NARC since many years and bacterial concentrations obtained in the same repository
Environ Sci Pollut Res (2016) 23:3747–3757 3749

Fig. 1 Location of the natural


cross-ventilation system in the
repositories (picture above).
Microbiological sampling zones
in each repository analyzed (A,
B). Repositories 4, 14, and 24 are
located in A or north side, and
repositories 6, 20, and 28 are
located in B or south side

but in July of 2010 and February of 2011. This test was also approximately 20 %. July is between the months of intense
used to compare the relative density of Penicillium, summer in the tropics (May–November), and the temperature
Aspergillus, and Cladosporium genera detected in the indoor levels are always above 30 °C. Also, this season is character-
environment in the repositories located in the same floor of the ized by heavy rain and hurricanes during which the relative
building and between the two months studied. humidity is significantly high, reaching values higher than
The ANOVA-1 and Duncan tests were used to compare the 90 %; for this reason, in Cuba, this month is representative
concentration of total microorganisms located on both sides of of the rainy season. February, located among the months of the
the building. A P value smaller or equal to 0.05 was consid- cold season (December–April) and characterized by scarce
ered statistically significant. rains, is representative of the drought season.
The first microbiological sampling was performed in
July 2010 (summer or rainy month), and at that time, the mean
Results values of T and RH for all repositories were 28.7 °C and 74 %,
respectively. The second analysis was made in February 2011
Regarding the temperature (T), it is observed that most of the (winter or dry month), and again, the average values of tem-
values obtained in repositories overlap with each other perature and the relative humidity values were lower (Tmean =
(Fig. 2). The mean T values obtained in repositories 6, 14, 24.4 °C, RHmean =67.6 %).
and 20 were lower than the monthly average during the year The fungal and bacterial concentrations obtained in the
under review. However, the mean values obtained in reposi- repositories show that in July 2010, the average fungal con-
tories 4, 24, and 28 were higher than the monthly average. The centration was 516 CFU/m3 while the average bacterial con-
difference of the T values between the months studied centration was 805 CFU/m3. In February 2011, the mean fun-
(July 2010 and February 2011) was approximately 6 °C. gal concentration was 228 CFU/m3 whereas the mean bacte-
The mean values of RH in repositories 20, 24, and 28 were rial concentration was 1397 CFU/m3 (Fig. 3). Also, this figure
lower than the monthly average, whereas the values of repos- shows that regardless of the location of the repositories in the
itories 4, 6, and 14 were higher than this average (Fig. 2). The building, the fungal concentration was significantly higher in
difference of the RH between the months studied was July 2010 in all of them whereas the bacterial concentrations
3750 Environ Sci Pollut Res (2016) 23:3747–3757

34.0 100.0
Fig. 2 Monthly average of
temperature (T) and relative 32.0 90.0
humidity (RH) are registered
since July 2010 to February 2011 30.0 80.0

for each repository analyzed of

Relative humidity (%)


28.0 70.0
the NARC. The arrows show the

Temperature (ºC)
months studied (July 2010: 26.0 60.0
Tmean =28.7 °C, RHmean =74 %;
February 2011: Tmean =24.4 °C, 24.0 50.0

RHmean =67.6 %)
22.0 40.0

20.0 30.0
4 6 14 20 24 28 MONTHLY AVERAGE 4 6 14 20 24 28 MONTHLY AVERAGE

18.0 20.0

MAY (2010)

JUNE

JULY

AUG.

SEPT.

OCT.

NOV.

DEC.

JAN (2011)

FEB.

MAY (2010)

JUNE

JULY

AUG.

SEPT.

OCT.

NOV.

DEC.

JAN (2011)

FEB.
were significantly higher mostly in February 2011 only in the In Fig. 4, a similar tendency was detected. Although sig-
repositories located on the first and second floors of the build- nificant differences were obtained between total concentra-
ing (identified as 14, 20, 24, and 28). In addition, it can be tions of microorganisms in both studied months, it was ob-
noticed that the highest fungal and bacterial concentrations served that in both months, the highest microbial concentra-
were obtained in the repositories located on the north side. tions were obtained on the north side of the building. In gen-
Regardless of the month, bacterial concentrations obtained eral, the highest concentrations of the total microorganisms
by plant differ significantly and the trend is increasing accord- were obtained in February 2011 in the repositories located
ing to the floor height; however, the fungal concentrations on on the first and second floors of the building.
the first floor (repositories 14 and 20) were significantly lower In this study, a total of eight genera of filamentous fungi in
than those obtained on the lower-ground part and second floor the indoor air of the repositories were isolated and identified. In
of the building. Meanwhile, fungal concentrations are signif- July 2010, regardless of the side of the building analyzed,
icantly higher in the repositories located on the lower-ground Penicillium Link., Aspergillus Nees ex Fr., and Cladosporium
floor although the RH is not much higher than the values Link ex Fr. predominated over other genera; hence, they were
obtained on the other floors, probably due to the low existing considered as abundant fungal genera (Table 1). Nevertheless, a
ventilation in those repositories. When fungal and bacterial low percentage (1 %) of the genera Epicoccum Link ex
concentrations obtained in the two months are compared be- Schlecht. and Fusarium Link ex Fr. non-sporing isolates and
tween each other, it can be seen that fungal concentrations yeast (2 and 4 %, respectively) were obtained. Therefore, these
were significantly lower in February whereas the bacterial genera were classified ecologically as rare or occasional genera
concentrations were higher in all repositories. according to the relative frequency percentages detected.

3000
900
I July (2010) February (2011) July (2010) February (2011) II
A j
800 H *f *
2500
700
k
2000 *
CFU/m3

600
B J
I
500
CFU.m-3

1500 g
D
400 d * h
K a * * i
300 1000
E *
C C b e
200 F *
500
F
100 c c

0 0
4 6 14 20 24 28 4 6 14 20 24 28
North South North South North South
North South North South North South
Basement First floor Second floor
Basement First floor Second floor

Fig. 3 Mean values of fungal (I) and bacterial (II) concentrations (CFU/ Student’s t test (P≤0.05) on comparing the fungal concentrations (I)
m3) in the indoor air of the repositories according to the location in the isolated in February between repositories located in the same floor. a, b,
building of NARC. Asterisk indicates significant differences according to d, e, h, i Significant differences according to Student’s t test (P≤0.05) on
Student’s t test (P≤0.05) on comparing the bacterial concentrations (II) comparing the bacterial concentrations (II) isolated in July between
obtained in the same repository but in July of 2010 (rainy month) and repositories located in the same floor. c, f, g, j, k Significant differences
February of 2011 (drought month). A, B, D, E, H, I Significant differences or not according to Student’s t test (P≤0.05) on comparing the bacterial
according to Student’s t test (P ≤ 0.05) on comparing the fungal concentrations (II) isolated in February between repositories located in
concentrations (I) isolated in July between repositories located in the the same floor. The microbial determination in each repository was made
same floor. C, F, J, K Significant differences or not according to in five zones by triplicate and the data averaged (n=15)
Environ Sci Pollut Res (2016) 23:3747–3757 3751

2000 3500
c* July/2010 February/2011
1800 i*
a* 3000
1600 h*
b* 2500
1400 k
f
d

CFU/m3
1200

CFU/m3
2000
1000 j
e 1500
800
600 1000
400 g* g
500
200
0 0
4 6 14 20 24 28 4 6 14 20 24 28
North South North South North South North South North South North South
Basement First floor Second floor Basement First floor Second floor
Fungy Bacteria Fungy Bacteria

Fig. 4 Behavior of the total microbial concentrations in the indoor air of comparing the total microbial concentrations of the repositories located
the repositories according to the location in the building of NARC. to the north of the building. d–f Significant differences according to
Asterisk indicates significant differences according to Student’s t test Duncan’s test (P≤0.05) on comparing the total microbial concentrations
(P≤0.05) on comparing the total microbial concentrations between the of the repositories located to the south of the building. The microbial
repositories located to the north and the south of the building. a–c determination in each repository was made in five zones by triplicate
Significant differences according to Duncan’s test (P ≤ 0.05) on and the data averaged (n=15)

Table 1 Relative density (RD), relative frequency (RF), and ecological classification (EC) of the fungal genera isolated from the indoor air from the six
repositories of the NARC

Genera Repositories

Basement First floor Second floor

North South North South North South


4 6 14 20 24 28
RD (%) RF (%) EC

July 2010 (summer or rainy season)


Penicillium Link. 90* a 40 c 84* e 10 g 39 h 56* j 100 A
Aspergillus Nees ex Fr. 3l 56* n 6p 47* r 14 t 22 v 100 A
Cladosporium Link ex Fr. 3a – 10 c 43* e 47* f 19 g 83 A
Epicoccum Link ex Schlecht. – – – – – 1 17 R
Fusarium Link ex Fr. 1 – – – – 1 33 O
Non-sporing isolates 1 – – – – 1 33 O
Yeast 2 4 – – – – 33 O
February 2011 (winter or dry season)
Penicillium Link. 11 b 18 d 12 f 6g 11 i 10 k 100 A
Aspergillus Nees ex Fr. 37 m 31 ñ 30 q 28 s 30 u 38 w 100 A
Cladosporium Link ex Fr. 46 b 38 51 d 50 e 43 f 45 h 100 A
Alternaria Nees ex Fr. 2 3 – 5 3 2 83 A
Curvularia Boedijn 1 – 2 2 3 1 83 A
Mucor Mich. Ex St.-Am. – 5 3 2 2 1 83 A
Fusarium Link ex Fr. 2 3 – 3 2 – 67 F
Non-sporing isolates – – 2 3 5 3 67 F
Yeast 1 2 – 1 1 – 67 F

The microbial determination in all cases was made in five points of each repository by triplicate and the data averaged (n=15). Letters a–k indicate
significant differences or not according to Student’s t test (P≤0.05) on comparing the relative density of the Penicillium genus between July 2010 and
February 2011; letters l–n, ñ, and p–w indicate significant differences according to Student’s t test (P≤0.05) on comparing the relative density of the
Aspergillus genus between July 2010 and February 2011; and superscript letters a–h indicate significant differences or not according to Student’s t test
(P≤0.05) on comparing the relative density of the Cladosporium genus between July 2010 and February 2011
EC ecological classification according to the percentages of RF, A abundant (81–100 %), C common (61–80 %), F frequent (41–60 %), O occasional
(21–40 %), R rare (0.1–20 %)
*P≤0.05, significant differences according to Student’s t test on comparing the relative density of Penicillium, Aspergillus, and Cladosporium obtained
in the repositories located in the same floor of building (north and south side) in July 2010
3752 Environ Sci Pollut Res (2016) 23:3747–3757

In February 2011, the relationship among the genera varied


significantly because the predominant genus was
Cladosporium followed by Aspergillus spp. and Penicillium
spp. in all repositories. Also, other genera, such as Fusarium
(detected in July in repositories 4 and 28), Alternaria Nees ex
Fr., Curvularia Boedijn, and Mucor Mich. Ex St.-Am., were
detected in lower relative densities. These genera appear in
almost all repositories and were not isolated in July 2010. As
their relative frequency percentages are greater than 81 %, they
are ecologically classified as abundant genera, similar to the
genera Aspergillus, Cladosporium, and Penicillium. On this
occasion, non-sporing isolates and yeast were isolated in high
percentages too; hence, the ecological category changed from
rare (July 2010) to frequent (February 2011).
The fact that Aspergillus was one of the predominant gen-
era and is a potentially pathogenic species was important to
make the taxonomic identification to species isolated. Figure 5
shows the Aspergillus species detected in the two sampling
months. Only seven species were detected in this study. In the
first isolation performed (July 2010), the greatest diversity of
Aspergillus species was obtained in repositories located on the
south side of the building.
In July 2010, six species were detected in repositories 6 and
20 located on the south side of the building while only three
species were detected in repository 28 (located in the south
too). Also, it can observed that the percentages of the species
Aspergillus flavus, Aspergillus niger, Aspergillus ochraceus,
Aspergillus oryzae, Aspergillus terreus, and Aspergillus
versicolor were higher than 4 % in repositories 6 and 20; this
represents that their concentrations ranged from approximate- Fig. 5 Relative density (RD) of the Aspergillus species detected in the
ly 14 to 43 CFU/m3 in repository 6 while the concentrations in repositories in the two months studied
repository 20 ranged from approximately 5 to 12 CFU/m3.
In February 2011, a total of seven species were detected.
On this occasion, A. niger and A. flavus were identified in all Discussion
repositories in the highest concentrations; for that reason,
these species are classified ecologically as abundant species. During the last decades, a strong correlation between aerobi-
Regarding bacterial groups, it can be observed that in all ology and biodeterioration of artworks has been claimed, due
repositories regardless of the building floor and month studied, to the fact that the air is the main vehicle for the dispersion of
the Gram-positive bacteria (between cocci and bacilli) microorganisms (Saiz-Jimenez and Gonzalez 2007; Ruga
prevailed as much in July 2010 as in February 2011 and the et al. 2015). Generally, airborne microorganisms were similar
bacilli forms prevailed too (Table 2). Some percentages obtain- or identical to those isolated from biodeteriorated artworks
ed among sporulated Gram-positive bacilli correspond to the (archive, library, mural paintings, museum collections, etc.)
genus Bacillus which varied according to the building floor and, in many cases, a direct effect was inferred (Nugari and
because the greater the height of the floor, the greater the con- Roccardi 2001; Canhoto et al. 2004; Saiz-Jimenez and
centration. This genus was the most abundant Gram-positive Gonzalez 2007; Zielińska-Jankiewicz et al. 2008; Cappitelli
bacilli isolated in repositories located on the lower-ground floor et al. 2009; Borrego et al. 2010; Pinzari et al. 2010; Harkawy
and the first floor. The percentages obtained were equivalent to et al. 2011; Pinheiro et al. 2011; Pasquarella et al. 2011, 2012;
concentrations ranging from 91.2 to 340.2 CFU/m3. Grbić et al. 2013; Borrego and Perdomo 2014; Borrego and
The Streptomyces genus had similar behavior, and different Molina 2014; Molina and Borrego 2014).
percentages (2 to 12 %) were obtained in the repositories Fungal colonization causes significant disfigurement of
located on the north side of the building. The percentages documentary materials, especially books and documents in
obtained were equivalent to concentrations ranging from 2.3 paper or their derivatives. Apart from the undesirable aesthetic
to 198.8 CFU/m3. aspects, the presence of some microorganisms can lead to the
Environ Sci Pollut Res (2016) 23:3747–3757 3753

Table 2 Relative density of different bacterial groups isolated from the air of the repositories studied at the National Archive of the Republic of Cuba

Localization Repository Bacterial groups July 2010 February 2011


(summer or rainy season) (winter or dry season)
Relative density (%)

Lower-ground floor North 4 Gram-positive cocci 20.3 2.7


Gram-negative cocci 5.2 –
Non-sporulated Gram-positive bacilli 35.1 56.8a
Sporulated Gram-positive bacilli 25.8b 32.5b
Gram-negative bacilli 13.6 8.0
South 6 Gram-positive cocci 14.4 35.5
Gram-negative cocci 10.5 10.0
Non-sporulated Gram-positive bacilli 30.3 46.3a
Sporulated Gram-positive bacilli 20.5b 3.1c
Gram-negative bacilli 24.3 5.1
First floor North 14 Gram-positive cocci 4.6 5.3
Gram-negative cocci 15.8 –
Non-sporulated Gram-positive bacilli 48.9a 57.7a
Sporulated Gram-positive bacilli 20.4b 30.0b
Gram-negative bacilli 10.3 7.0
South 20 Gram-positive cocci 27.6 13.0
Gram-negative cocci – 8.1
Non-sporulated Gram-positive bacilli 46.2a 50.5
Sporulated Gram-positive bacilli 10.8b 23.0b
Gram-negative bacilli 15.4 5.4
Second floor North 24 Gram-positive cocci 30.1 11.6
Gram-negative cocci 1.7 2.5
Non-sporulated Gram-positive bacilli 46.6a 60.1a
Sporulated Gram-positive bacilli 8.3c 20.8b
Gram-negative bacilli 13.3 5.0
South 28 Gram-positive cocci 10.0 11.3
Gram-negative cocci 5.3 3.8
Non-sporulated Gram-positive bacilli 48.5a 66.8a
Sporulated Gram-positive bacilli 25.7b 10.1c
Gram-negative bacilli 10.5 8.0
a
Indicates that it included a percentage of Streptomyces sp. strains. The levels are variable and tend to be higher in July 2010
b
Indicates that part of this percentage belongs to the Bacillus genus
c
Indicates that this percentage belongs only to the Bacillus genus

irreversible degradation of the paper if viable cells are not conditions for microbial growth vary depending on the spe-
detected early enough, resulting in significant loss in value cies, but few species can live under the equilibrium of relative
and quality of such material. humidity of a material with 65 % water activity (aw =0.65)
The key environmental factors which determine germina- (Pasenen et al. 2000). It has been reported that hygroscopic
tion and growth on paper-based material are the prevailing materials (paper, textiles, leather, etc.) are the most susceptible
environmental factors of relative humidity (RH), temperature to microbial growth (Green et al. 2003). Therefore, it is very
and gas composition, and the level of initial contamination of important to record the RH of the repositories daily.
the material (Canhoto et al. 2004). The fungal growth is in- In Cuba, the temperature and relative humidity are high
creased by these factors, but water is the most important in- throughout the year but the natural cross-ventilation system
gredient for the germination, growth, and development of in- plays an important role in slowing down microbial growth.
door bacterial and fungal bioaerosols. Relationships between Therefore, in this study, these hygrometric variables were tak-
indoor bioaerosols and moisture content have been established en into account in their relation to the concentration and var-
in several studies (Green et al. 2003). The most favorable iability of the microbiota of the repositories, which represents
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a potential risk of biodeterioration for documents and damage 560<high concentration <1000 CFU/m3, and very high con-
to the health of staff. centration >1000 CFU/m3. According to the authors, the in-
The analysis obtained shows that the prevalence of bacteria door air of repositories was classified from contaminated to
was high in February 2011 (dry months) in repositories locat- highly contaminated. Only in February 2011, repositories 4
ed on the first and second floors mainly, while the highest and 6 had concentrations of total microorganisms of 380 and
concentration of fungi was obtained in July 2010 (rainy sea- 327 CFU/m3, respectively; hence, only on this occasion the
son) in all repositories. The behavior obtained in February environments of these repositories were classified as
may be due to the increase of the external dust entering the moderate.
repositories through the holes of the natural cross-ventilation When comparing these results with those reported by
system. This effect is marked at the greatest height of the Borrego and Perdomo (2012), differences can be observed.
building due to the fact that penetration of dust in the reposi- Taking into account concentrations of total microorganisms
tories is facilitated by the height. A similar result was reported reported by the authors, the environments of the repositories
by Borrego and Perdomo (2012) in a similar study carried out studied are classified as follows: two were moderate, two were
in previous years. highly polluted, and two were very high polluted. This shows
Weather conditions in tropical countries as Cuba is one of the dynamic behavior of indoor environments naturally venti-
the factors responsible for the microbial behavior indoors. The lated; hence, similar studies should be carried out
high relative humidity and temperature recorded during the systematically.
rainy season especially favor the development of fungal prop- Despite that there is no international standard to determine
agules and increase the level of viable conidia in the atmo- whether an indoor environment is contaminated or not, the
sphere. However, in winter, frequent strong winds occur as a World Health Organization has suggested that an environment
result of the systematic arrival of cold fronts to the island, with a microbial prevalence of above 1000 CFU/m3 should be
causing a significant increase in the speed of northern and considered contaminated (Nevalainen and Morawaska 2009;
northeastern winds, which facilitates the inflow of bacterial Nunes et al. 2013). Thus, the microbial concentration should
cells and dust towards the building through the natural venti- be lower than 1000 CFU/m3. Other authors consider that mi-
lation systems. In summer, particularly from May until the end crobial prevalence should not exceed 750 CFU/m3; above this
of November, the rains are intense, the hurricanes occur fre- level, the environment is regarded as contaminated (Radler de
quently, and the relative humidity increases significantly. Aquino and De Góes 2000) and still other authors consider
During this period, the rains cause systematic cleaning of the that 150 CFU/m3 should be the maximum allowable limit for
atmosphere (De la Rosa et al. 2002; Sabariego et al. 2004) fungi (Cappitelli et al. 2009; Micheluz et al. 2015).
and, therefore, the bacterial concentration in the outdoor envi- Predominant fungal genera in the study were Aspergillus,
ronment tends to decrease whereas the fungi concentrations Penicillium, and Cladosporium. Also, these genera are pre-
are favored by high humidity, although the building has natu- dominant in similar environments in other countries (Lignell
ral ventilation. This effect is increased in the repositories lo- 2008; Zielińska-Jankiewicz et al. 2008; Niesler et al. 2010;
cated on the lower-ground floor of the building, although a Harkawy et al. 2011; Roussell et al. 2012; Trovão et al.
high fungal concentration was also detected in a repository 2013; Nunes et al. 2013; Guiamet et al. 2011; Lavin et al.
located on the second floor and the north side. In the study 2014; Fekadu and Melaku 2014; Skóra et al. 2015; Ruga
carried out previously by Borrego and Perdomo (2012), dif- et al. 2015; Micheluz et al. 2015).
ferent behaviors were obtained in the two repositories of the Moreover, only in July 2010, the Epicoccum genus was
second floor of the building. detected in one repository (28, located on the second floor
Nevertheless, in the repositories located on the north side of and the south side of building) but Alternaria, Curvularia,
the building, both fungal and bacterial concentrations were and Mucor were other genera isolated in the majority of the
significantly higher due to northern winds which easily pene- repositories only in the month of February 2011 (dry season).
trate into the building. This tendency was similar to the one These genera are found in soil and on the plants, and it is
reported previously (Borrego and Perdomo 2012). possible that the vegetation around the building contributes
In this study, the microbial prevalence is similar to that in to the presence of these fungal genera because they are
other studies carried out in indoor environments of Cuban transported by the air into the building.
libraries, archives, and museums (Rojas et al. 2002, 2008, Particularly, the concentrations of the Aspergillus genus in
2012; Borrego and Perdomo 2012, 2014; Borrego and the two months ranged from 23.2 to 105.8 CFU/m3. These
Molina 2014; Molina and Borrego 2014). concentrations are considered within a high concentration ac-
A scale of four ranges of concentrations to determine the cording to Holmberg (1987), who suggested that the levels of
microbial contamination level was proposed by Roussell et al. Aspergillus spp. should be lower than 50 CFU/m3 to avoid
(2012). The values indicate the following: low concentration excessive exposure to these bioaerosols and their consequent
<170 CFU/m3, 170<moderate concentration <560 CFU/m3, effects on health.
Environ Sci Pollut Res (2016) 23:3747–3757 3755

With regard to the Cladosporium genus, a contrary behav- potentially irritating, such as amines and sulfur compounds;
ior was reported previously by Borrego and Perdomo (2012), and some are volatile and odorous, such as geosmin (Lignell
who indicated the highest concentrations in the rainy season. 2008; Rintala et al. 2008). It has been reported that high con-
However, in this study, the highest percentage of centrations of Bacillus species in indoor air are usually indic-
Cladosporium spp. was obtained in the dry season. This could ative of water damage or lack of building maintenance (Baxter
be given by an increase on the microbial concentration in the et al. 1981). In this case, the presence of high concentration of
outdoor air, which is introduced into the building through the this bacterial genus may indicate the increase in the outdoor
natural cross-ventilation system, because although the mea- microbial contamination which is introduced into the building
surements were not performed, it is thought that the increase across the natural cross-ventilation system. However, the
in the construction of houses in areas close to archive, the cut building does not have signs of humidity in none of the repos-
of some trees in these months, and the excessive vehicular itories analyzed; therefore, it is thought that the presence of
traffic may have contributed to increased pollution. this genus in the indoor environment of repositories is due to
All genera of fungi isolated in this study may affect human its entrance from outside the building.
health, as they are allergen (Mesquita et al. 2009; Nevalainen Finally, these bacterial genera were detected previously in
and Morawaska 2009; Roussell et al. 2012). Since many spe- the repositories of NARC but in different concentrations
cies of Aspergillus are not only highly allergenic but also (Borrego et al. 2010, 2012; Borrego and Perdomo 2012).
opportunistic pathogens and, in Cuba, climate changes in win- This indicates the necessity to study systematically the behav-
ter are quick and sudden, which stimulates the onset of fre- ior of the microbial concentrations in indoor environments.
quent attacks of allergies, sneezing, and asthma, we decided to It is important to highlight that in the indoor air, microor-
perform a detailed analysis of Aspergillus species. Two spe- ganisms come and go, which is a natural phenomenon.
cies were detected in almost all of the repositories in February Ventilation and cleaning are the usual removal processes of
2011 and July 2010: A. niger and A. flavus. This result is microbes in indoor environments. The type of ventilation af-
consistent with previous reports (Pinheiro et al. 2011; Grbić fects microbial concentrations in indoor environments. It is
et al. 2013; Borrego and Perdomo 2012, 2014). reported that mechanical ventilation is more efficient than nat-
A. flavus is one of the main agents of human allergic bron- ural ventilation in filtering particles from the intake air and in
chial aspergillosis and pulmonary infection in immunocom- removing pollutants. This difference is also reflected in mi-
promised patients, whose pathology may be aggravated by the crobial concentrations, which, in naturally ventilated build-
production of mycotoxins. It also occurs in external ears and ings, can be two to seven times higher than that in buildings
may be involved in otitis, occasionally with severe complica- with mechanical ventilation (Lignell 2008). However, the fact
tions. Also, the species is one of the common agents of the that there is a natural cross-ventilation system in indoor envi-
mycotic sinusitis. A. niger is frequently isolated from human ronments in countries with hot and humid climates strongly
external ears; therefore, this species is considered as the prime limits the growth of mold on materials, despite the high levels
etiologic agent of otomycosis, producing cutaneous infec- of fungal contamination that may exist.
tions, endophthalmitis, and onychomycoses; also, the immu-
nocompromised patients are affected by this species (De Hoog
Acknowledgments The authors acknowledge the financial support re-
et al. 2000). ceived from the Assistance Program for Archives of Latin America of
Regarding the bacteria, the Gram-positive bacteria Spain, ADAI (Projects 134/2010 and 064/2012).
prevailed among the bacterial groups isolated from the six
repositories and this is consistent with other studies performed
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