An Optimized Active Sampling Procedure for Aerobiological DNA Studies

Abstract

The Earth's atmosphere plays a critical role in transporting and dispersing biological aerosols. Nevertheless, the amount of microbial biomass in suspension in the air is so low that it is extremely difficult to monitor the changes over time in these communities. Real-time genomic studies can provide a sensitive and rapid method for monitoring changes in the composition of bioaerosols. However, the low abundance of deoxyribose nucleic acid (DNA) and proteins in the atmosphere, which is of the order of the contamination produced by operators and instruments, poses a challenge for the sampling process and the analyte extraction. In this study, we designed an optimized, portable, closed bioaerosol sampler based on membrane filters using commercial off-the-shelf components, demonstrating its end-to-end operation. This sampler can operate autonomously outdoors for a prolonged time, capturing ambient bioaerosols and avoiding user contamination. We first performed a comparative analysis in a controlled environment to select the optimal active membrane filter based on its ability to capture and extract DNA. We have designed a bioaerosol chamber for this purpose and tested three commercial DNA extraction kits. The bioaerosol sampler was tested outdoors in a representative environment and run for 24 h at 150 L/min. Our methodology suggests that a 0.22-µm polyether sulfone (PES) membrane filter can recover up to 4 ng of DNA in this period, sufficient for genomic applications. This system, along with the robust extraction protocol, can be automated for continuous environmental monitoring to gain insights into the time evolution of microbial communities within the air.

Keywords: DNA extraction; active sampling; air-filtration; bioaerosols; commercial off-the shelf (COTS).

Figure 1

(a) Components of the bioaerosol sampler; (b) core of the bioaerosol sampler with the counter fan coupled to the MAF sensor; (c) assembled components of the bioaerosol sampler inside the housing; (d) touch screen GUI showing the parameters of sampling in progress.

Figure 2

Laboratory-built portable bioaerosol chamber. This portable system consists of a filter holder that holds nucleopore filters of 47-mm diameter attached to a vacuum pump that helps bioaerosol suction. A nebulizer connected to a compressor generates the aerosols. The HEPA filter draws in clean air and stabilizes the pressure within the box.

Figure 3

Summary of the optimised DNA extraction protocol from steps 1–4. The sonication of the sampled filter in a warm bath and bead beating are the key steps in the extraction process as they significantly improve the extraction efficiency.

Figure 4

Results of bioaerosol chamber sampling experiments. The boxplot represents five biological replicates (n = 5) for each membrane filter type. Yeast cells were used as a positive control whose mass was constant with each replicate, and varying biomass of soil supernatant was used to validate the consistency of the filter efficiency. The graph was plotted using Prism 9. Error bars indicate the mean with standard deviation.

Figure 5

(a,b) Bioaerosol sampler loaded with the 47 mm diameter filter; (c) sampling in an outdoor environment; and (d) 1X PBS added to moisten the filter.

Figure 6

SEM images captured at ~5.00 K X Zoom using 10.00 KV electron beam on the post-sampled filters subjected to yeast bioaerosol generated within the constrained chamber. The oval-shaped single yeast cell can be distinctively seen captured onto the different filters. The microscale characterization of the filters using SEM imaging reveals the morphology and structure of the filters and their influence on trapping bioaerosol particles. PES (hydrophilic) and PTFE (hydrophobic) have good loading capacity due to higher surface area of entrapment. PC is a flat filter and has the least surface area for bioaerosol loading. TissueQuartz is a fibrous filter and has good loading capacity but suffers from lower efficiency compared to membrane filters.