**3.2 Impingement**

*Extremophilic Microbes and Metabolites - Diversity, Bioprospecting and Biotechnological...*

estimated to range between 104 and 106 m<sup>−</sup><sup>3</sup>

**3. Microbial sampling**

found in the oceans, soils, and subsurface. Between approximately 18 and 50 km above sea level (stratosphere), temperature, oxygen, and humidity decrease and with them the number of cells. Above the ozone layer (between 18 and 35 km into stratosphere), ultraviolet (UV) and cosmic radiation become lethal factors. Once in the mesosphere (above 50 km), life is difficult to imagine; however microorganisms of terrestrial origin could arrive to the stratosphere from lower layers via different phenomena (human activity, thunderstorms, dust storms, or volcanic activity), and bacteria have been found isolated up to 41 km or in dust samples from the International Space Station (**Figure 1**) [6, 29]. Therefore, airborne microbes are always present in the atmosphere [11, 30, 31], and their permanence is dynamic, resulting in an environment with enormous variability. Estimates calculate that over 1021 cells are lifted into the atmosphere every year, leading to considerable transport and dispersal around the atmosphere, with a large portion of these cells returning to the surface due to different atmospheric events as part of a feedback cycle. Undoubtedly, airborne microbes play an important role in meteorological processes. They have been linked to the nucleation phenomena that lead to the formation of clouds, rain, and snow and to the alteration of precipitation events [32–34]. Their presence is essential to understand long-range dispersal of plant and potential pathogens [7, 35, 36] and maintain diversity in ground systems and could interfere with the productivity of natural ecosystems [17, 18]. On the other hand, airborne bacteria can have important effects on human health, being responsible for different phenomena such as seasonal allergies and respiratory diseases. Based on data from terrestrial environments, the global abundance of airborne bacteria has been

incorporating direct counting by microscopy or quantitative PCR have provided more accurate estimates of the number of airborne microbes, which apparently

There is a great variety of airborne microorganism sampling systems, allowing us to select the most suitable one depending on our objectives [42]. On the other hand, no standardized protocols exist, which is a major pitfall when developing our objectives. This fact has led some authors to propose the creation of consortiums of interested parties for establishing standardized protocol reproducibility [20], as well as the need to establish global networks of aerobiological studies [11]. Two approaches are proposed: particles or cells can be collected passively or directly from the atmosphere. Passive media usually involves decanting [43] and collecting particles over snow [44] or through the collection of atmospheric water [45]. On the other hand, active methodologies entail three major approaches: filtration, impaction, and liquid impingement. All three approaches are very efficient when developing culture-dependent techniques. In contrast, culture-independent approaches produce some serious problems that make the work difficult: the high variability of the system and the low biomass mean that sampling campaigns are, in many cases, extremely inefficient [20]. Lastly, the use of airborne platforms is not very extended, but they represent a good opportunity to

Filtration is a simple and cheap method that is often efficient. It involves pumping air through a filter where the mineral and biological particles are trapped. Filters of different materials and porosity are available made of cellulose, nylon, polycarbonate

point to a higher number of cells present in the atmosphere [38–41].

conduct a more direct study of the atmosphere [5, 19, 31].

[37]. However, more recent studies

**44**

**3.1 Filtration**

In impingement, particles are collected in a liquid matrix [20]. Normally a buffer is used such as phosphate buffer saline (PBS) that helps maintain the viability of the cells. One of the more widely used liquid impingers is BioSampler SKC (**Figure 2B**). In this case, the tangential movement of the particles inside the flow impinger retains the particles in the collecting liquid. The suspension obtained could be used for culturing or for molecular ecology assays [20]. One of the advantages of impingement collection is that it facilitates quantitative techniques such as flow cytometry or in situ hybridization [47].
