First Metagenomic Survey of the Microbial Diversity in Bioaerosols Emitted in Waste Sorting Plants

TLDR: The NGS biodiversity measurements revealed a higher biodiversity bioaerosols that previously reported for WSP in studies carried out using culture methods followed by identification of microorganisms, providing the first survey about taxonomic biodiversity in bio aerosols from WSPs using high-throughput sequencing.

Abstract: Waste sorting activities are source of occupational bioaerosol exposures that are associated with several health disorders. New analytical tools, based on next generation sequencing (NGS) technologies, provide powerful methods to assess the microbial composition of bioaerosols. The objectives of the study were (1) to assess the feasibility and the repeatability of NGS based biodiversity measurements and (2) to study the microbial biodiversity using NGS in bioaerosols emitted in a waste sorting plant (WSP). Three stationary parallel samples were collected in a sorting cabin using closed-face cassettes equipped with polycarbonate membranes. Bacterial and fungal diversity was assessed by sequencing 16S and 18S rDNA genes using either Illumina sequencing or 454 pyrosequencing methods. At sampling point, airborne bacteria were dominated by Proteobacteria, Firmicutes and Actinobacteria with prevailing genera assigned to unclassified Enterobacteriaceae, Staphylococcus, Acinetobacter, Leuconostoc, Pseudomonas and Lactobacillus. Airborne fungi were dominated by Ascomycota with prevailing genera assigned to Penicillium, Aspergillus, Rhizopus,

Figures

Figure 2: Bacterial biodiversity at the family rank in bioaerosols from the WSP. Bioaerosol samples were collected in the Sorting Cabin (SC) and in the Outdoor Reference (OR). Data are given for the 20 dominant families found in SC samples. For practical reasons, only the mean of sequencing data from SC1, SC2 and SC3 are presented. *: corresponds to Bacteroidetes.

Figure 1 : Bacterial biodiversity at the phylum Rank in bioaerosols from the WSP. Bioaerosol samples were collected in the Sorting Cabin (SC) and in the Outdoor Reference (OR). For practical reasons, only the mean of sequencing data from SC1, SC2 and SC3 are presented.

Table 1: Overview of high-throughput sequencing data obtained from bioaerosol samples taken in the WSP. Bioaerosol samples were collected in the Sorting Cabin (SC) and in the Outdoor Reference (OR). a : Illumina sequencing; b : 454 pyrosequencing.

Figure 3: Fungal biodiversity at the genus rank in bioaerosols from the WSP. Bioaerosol samples were collected A: in the Sorting Cabin and B: in the Outdoor Reference. For practical reasons, only the mean of sequencing data from SC1, SC2 and SC3 are presented.

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First metagenomic survey of the microbial diversity in
bioaerosols emitted in waste sorting plants
Jodelle Degois, Frédéric Clerc, Xavier Simon, Cyril Bontemps, Pierre Leblond,
Philippe Duquenne
To cite this version:
Jodelle Degois, Frédéric Clerc, Xavier Simon, Cyril Bontemps, Pierre Leblond, et al.. First metage-
nomic survey of the microbial diversity in bioaerosols emitted in waste sorting plants. Annals of
Work Exposures and Health, Oxford University Press, 2017, 61 (9), pp.1076 - 1086. �10.1093/an-
nweh/wxx075�. �hal-01655252�

1
First metagenomic survey of the microbial
diversity in bioaerosols emitted in waste
sorting plants
Jodelle Degois
1
, Frederic Clerc
1
, Xavier Simon
1
, Cyril Bontemps
2
, Pierre
Leblond
2
, Philippe Duquenne
1
*
1
Department of pollutant metrology - Institut National de Recherche et de Sécurité (INRS),
Vandœuvre-lès Nancy, 54500, France
2
Université de Lorraine, Institut National de la Recherche Agronomique, Dynamique des
Génomes et Adaptation Microbienne (DynAMic), UMR INRA 1128, Nancy, 54000, France
Abstract
Waste sorting activities are source of occupational bioaerosol exposures that are associated
with several health disorders. New analytical tools, based on next generation sequencing
(NGS) technologies, provide powerful methods to assess the microbial composition of
bioaerosols. The objectives of the study were (1) to assess the feasibility and the repeatability
of NGS based biodiversity measurements and (2) to study the microbial biodiversity using
NGS in bioaerosols emitted in a waste sorting plant (WSP). Three stationary parallel samples
were collected in a sorting cabin using closed-face cassettes equipped with polycarbonate
membranes. Bacterial and fungal diversity was assessed by sequencing 16S and 18S rDNA
genes using either Illumina sequencing or 454 pyrosequencing methods. At sampling point,
airborne bacteria were dominated by Proteobacteria, Firmicutes and Actinobacteria with
prevailing genera assigned to unclassified Enterobacteriaceae, Staphylococcus,
Acinetobacter, Leuconostoc, Pseudomonas and Lactobacillus. Airborne fungi were dominated
by Ascomycota with prevailing genera assigned to Penicillium, Aspergillus, Rhizopus,

2
Wallemia and Hemicarpenteles. The NGS biodiversity measurements revealed a higher
biodiversity bioaerosols that previously reported for WSP in studies carried out using culture
methods followed by identification of microorganisms. These results provide the first survey
about taxonomic biodiversity in bioaerosols from WSPs using high-throughput sequencing.
Keywords: Waste sorting plant, Bioaerosol, Occupational exposure, Biodiversity, Next-
generation sequencing, 16S rDNA Illumina sequencing, 18S rDNA 454 pyrosequencing

3
Introduction
Household waste sorting activities are source of occupational exposure to bioaerosols
including bacteria, fungi, endotoxins, (1,3)-β-D-glucans as well as inhalable dust (Breum et
al., 1999 ; Gladding et al., 2003 ; Hebisch and Linsel, 2012 ; Schlosser et al., 2015). Studies
carried out in waste sorting plants (WSP) reported health disorders such as non-allergic
symptoms (chronic bronchitis and mucous membrane irritations) as well as allergic symptoms
(allergic asthma, allergic rhinitis and hypersensitivity pneumonitis) among the population of
WSP workers exposed to bioaerosols (Gladding et al., 2003; Ivens et al., 1997; Perez et al.,
2006).
Bacteria, fungi and viruses found in bioaerosols at the workplaces, as well as the associated
microbial compounds such as endotoxins, (1,3)-β-D-glucans, mycotoxins and allergens, were
presented as the possible causative agents of occupational diseases (Douwes et al., 2003 ;
Eduard et al., 2012). However, the complex composition of bioaerosols still prevents
identifying the specific microbial agents responsible for health effects as well as their precise
role in symptoms. As a consequence, the dose-response relationships have not yet been
established for most of non-infectious airborne microorganisms and no occupational exposure
limit values are available. Thus, defining the precise composition of the bioaerosols to which
employees are exposed to would provide a better understanding of biological risks at the
workplaces.
So far, the microbial biodiversity data published in (WSP) were obtained by cultivation of
microorganisms on nutrient media prior to identification. The corresponding studies found
that the dominant fungi belonged to Penicillium sp., Aspergillus sp., Cladopsorium sp.
(Breum et al., 1999; Lehtinen et al., 2013; Tolvanen, 2001; Viegas et al., 2014). However, the
culture method underestimates the total number of microorganisms in samples as dead cells

4
and microorganisms at viable but non cultivable (VBNC) state due to the selecting effect of
culture media and cultivation conditions (Amann et al., 1995; Heidelberg et al., 1997). Thus,
the cultured based biodiversity measurements previously performed in WSP surely have
underestimated the real microbial taxon richness.
Advances in molecular biology and Next-generation sequencing (NGS) provided new
powerful methods for biodiversity studies. These molecular methods were already used in
bioaerosol studies carried out in different occupational environments such as composting
facilities (Partanen et al., 2010), wastewater treatment plants (Lin et al., 2014), and swine
houses (Kumari and Choi, 2015). NGS overcame the limitations of culture based methods and
provided useful data (greater observed biodiversity, detection of non-preponderant taxa etc.)
for a better understanding of the microbial communities to which workers may be exposed.
However, the NGS have also some limitations due to the DNA extraction efficiency, PCR
biases or the short size of the amplified DNA fragments which do not allow the identification
at the species level. Nevertheless, the NGS could provide additional information as compared
to culture-based methods (Yoo et al., 2017). To date, metagenomics methods were not
applied to investigate the microbial biodiversity found in WSP. The aims of the present study
were (1) to assess the feasibility and the repeatability of NGS based biodiversity
measurements in WSP and (2) to apply this method in order to study the bioaerosol microbial
biodiversity in bioaerosols emitted in a WSP.
Materials and methods
Description of the WSP
Bioaerosol biodiversity was assessed in July 2014 in a French WSP sorting journal
newspapers, papers, cardboards, food packaging and other sorting wastes (4000 tons per

Frequently asked questions

Q1. What contributions have the authors mentioned in the paper "First metagenomic survey of the microbial diversity in bioaerosols emitted in waste sorting plants" ?

The objectives of the study were ( 1 ) to assess the feasibility and the repeatability of NGS based biodiversity measurements and ( 2 ) to study the microbial biodiversity using NGS in bioaerosols emitted in a waste sorting plant ( WSP ). The NGS biodiversity measurements revealed a higher biodiversity bioaerosols that previously reported for WSP in studies carried out using culture methods followed by identification of microorganisms. 

Q2. What are the future works in "First metagenomic survey of the microbial diversity in bioaerosols emitted in waste sorting plants" ?

However, further studies based on a larger number of samples and other targeted genes ( ITS, genes involved in microbial pathogenicity or in the expression of allergens etc. ) are needed to confirm these primary results and to investigate the uncertainty of biodiversity measurements as well as the variation of airborne microbiomes in time and in space at occupational settings. 

Q3. What primers were used for amplification of the rDNA gene?

Bacterial 16S rDNA gene (V4-V5 variable region) was amplified using the universal primers F-GTGYCAGCMGCCGCGGTA and RCCCCGYCAATTCMTTTRAGT. 

Q4. What was the main fungal phyla found in bioaerosol samples?

The three main fungal phyla found in bioaerosol samples were Ascomycota (91.1 %), an early diverging fungal lineage [Mucor and Rhizopus] (4.64 %) and Basidiomycota (3.70 %). 

Q5. What phyla were found in the OR?

The other found phyla belonged to Chloroflexi, Cyanobacteria, Deinococcus-Thermus and Planctomycetes and account individually for less that 2.5 % of the total reads. 

Q6. What were the dominant fungal genera in the SC samples?

The dominant outdoor fungal genera were Cladosporium (45.2 %), unidentified Ascomycota (8.3 %), Oligoporus (7.3 %), unidentified Basidiomycota (3.6 %) and, Penicillium (3.5 %). 

Q7. What was the common type of bacteria in the Finnish waste treatment plant?

For airborne bacteria, in a plant located in Finland where household wastes were sorted, bioaerosols were dominated by unidentified Gram-positive and Gram-negative cultivated bacteria and by bacteria belonging to Pseudomonas, Bacillus and Micrococcus (Rahkonen, 1992). 

Q8. How was the bioaerosol sample transported to the laboratory?

Bioaerosol samples were transported to the laboratory during the day of sampling using a cold box and were then stored at 4°C until analysis. 

Q9. What was the percentage of reads in the OR sample?

In the OR sample, Fungi and Plantae represented 70.0 % and 29.9 % of the reads, respectively, and the remaining ones were unidentified (0.1 %). 

Q10. What methods were used to assess the bacterial risk?

To assess the bacterial risk, others methods such as the identification of cultured bacteria using MALDI-TOF could be used in addition with the sequencing. 

Q11. How many species of bacterial species were observed in the present study?

In others studies, the authors observed from 20 bacterial genera and up to 38 bacterial species using culture-based methods (Rahkonen, 1992; Nielsen et al., 1995; Breum et al., 1999; De Vasconcelos Pinto et al., 2015; Madsen et al., 2016). 

Q12. What were the dominant fungal otus in the SC samples?

The ten dominant fungal OTUs represented 98.5 % of eukaryotic OTUs with a predominance of the Penicillium, Aspergillus and Rhizopus genera (Figure 3.a). 

Q13. What are the limitations of the NGS?

the NGS have also some limitations due to the DNA extraction efficiency, PCR biases or the short size of the amplified DNA fragments which do not allow the identification at the species level. 

Q14. What was the percentage of reads assigned to Plantae?

In SC samples, Fungi represented 99.4 % of the reads and the remaining ones were assigned to Plantae (0.12 %) or remained unidentified (0.44 %). 

Q15. How many reads were in the OR sample?

For bacterial 16S rDNA, Illumina sequencing provided about 78,600 reads in the OR sample and between 69,600 and 100,000 reads in the SC samples (Table 1). 

Q16. Why did the NGS not assess the bacterial biodiversity at the species level?

due to the limits of the sequencing methods, the bacterial biodiversity could not be assessed at the species level in the study. 

Q17. What were the samples used in the present study?

The present study was carried out with a limited number of samples and only three occupational bioaerosols and one reference were analyzed. 

Q18. How many reads were assigned to bacterial OTUs in the OR and SC samples?

For Eukaryota 18S rDNA, 454 pyrosequencing provided about 6,600 reads for in the OR sample and between 14,546 and 21,206 reads for the SC samples (Table 1). 

Q19. How many genera were assigned to microbial communities?

The results showed that the components of airborne microbial communities were assigned to 4 phyla and 112 genera for bacteria and to 3 phyla and 22 genera for fungi. 

Q20. What was the repeatability of the bioaerosol measurement process?

The repeatability of the bioaerosol measurement process was assessed on the 16S rDNA OTUs found in the three parallel replicate samples collected in the SC. 

Q21. What were the common bacterial families in the SC?

The proportion of these families was between 9.34% (Entorobacteriaceae) and 0.83% (Aerococcaceae) in the SC and accountedfor 57% of the bacterial reads.