Soil is the foundation of all terrestrial environments; it can harbor billions of microbes per gram [1] and plays critical roles in nutrient availability, biogeochemical cycling, and bioremediation processes. Despite its importance, the soil is perhaps the least understood part of the environment [2]. Characterizing its physical and chemical properties has been essential for agriculture for thousands of years. However, soil’s biological component has generally been overlooked until the past few centuries, and even then, scientists were usually limited to studying macrofauna like earthworms and small insects. The invention of optical microscopes powerful enough to resolve microorganisms enabled the visual study of microbes [3]. Only recently, with advances in sequencing and “meta-omics” technologies, have scientists been able to investigate soil microbes and their communities comprehensively. Today, there are many ways to determine various physical and chemical properties of soil and several metrics to evaluate soil quality as a whole. However, many soil quality metrics do not consider microbial community factors [4] despite their noted importance [4, 5]. The omission of community factors in these metrics is partly due to the difficulties of biological characterization. Determining what microorganisms are present is possible through next-generation sequencing (NGS), and though these methods have advantages over traditional culturing methods, NGS is not without its drawbacks. Moreover, knowing the microbes’ functions is often essential, requiring different data with separate analysis methods. It can be challenging to interpret the large amounts of data generated by NGS, particularly when gaps remain in the databases used for analysis. Because of these challenges, many papers have called for developing novel techniques to complement NGS methods [6,7,8,9,10,11], and biosensing may offer an excellent suite of tools to do just that.

Biosensing has been defined as “a device that uses specific biochemical reactions mediated by isolated enzymes, immunosystems, tissues, organelles, or whole cells to detect chemical compounds usually by electrical, thermal or optical signals” by the International Union of Pure and Applied Chemistry (IUPAC) [12]. In essence, biosensing detects a target compound or condition using some biological component. Such devices are often used in biomedical applications [13,14,15,16,17,18] and ecotoxicology [19,20,21]. Many biosensor studies use entire organisms to sense the target compound or condition [16, 19, 22,23,24,25,26,27,28], but other studies instead use only specific molecules without the surrounding cellular structure [11, 14, 15, 29, 30]. These molecules, or bioreceptors, are essential components in biosensing devices and are often the focal points of research.

Numerous research works and commercialization efforts have been made in the past couple of decades for biosensors, most notably toward reducing the device size, device cost, assay time, operation cost, and operation complexity while maintaining specificity and sensitivity comparable to more standard laboratory instruments. Such biosensing methods and biosensor devices may identify microbial species and their makeup in the soil, significantly reducing the cost and time associated with NGS methods. However, biosensors are inherently limited in specificity compared to NGS in identifying species. Substantial cross-binding can occur with antibody- or aptamer-based bacterial biosensing. In addition, biosensing may not be optimal for identifying a large number of species simultaneously. For example, detecting 100 microbial species could require 100 different bioreceptors pre-loaded on a biosensing platform, significantly augmenting the assay complexity and limiting its usefulness compared to current NGS technology.

This correspondence paper aims to summarize current methods and techniques for soil microbiome characterization and then to summarize and provide insights and recommendations for future opportunities with biosensing technologies. See Fig. 1 and Fig. 2 for visual overviews of these two technologies. These steps are described in detail in the following sections.

Overview of next-generation sequencing (NGS) process. Amplicon-based methods include the amplification step, while shotgun methods instead include DNA fragmentation. Figure created with BioRender.com

Overview of biosensing process as potentially applied to soil microbiome characterization. Figure created with BioRender.com

In its entirety, the microbiome encompasses all the parameters that make up a community of microbes. This includes physical, chemical, and biological characteristics, but we will focus on the biological characteristics, like the microbial community dynamics and structure, for this correspondence paper.

Intrinsically, soil is a difficult medium to work with because of its physical properties. Soil is an opaque mixture, which significantly limits the use of optical microscopy techniques. Soil is also very spatially heterogeneous. Depth significantly affects parameters like moisture and the carbon–nitrogen ratio [31]. Also, roots create a unique environment rich in metabolites and signaling molecules; this environment is called the rhizosphere and is a critical region of plant–microbe interaction and symbiosis [23, 32,33,34]. There is spatial heterogeneity even at the microscale, such as an uneven distribution of nutrients, organisms, and microclimate conditions across millimeters or shorter distances [35]. This heterogeneity complicates soil analyses because average properties could be misleading for understanding microscale interactions [8]; however, measuring microscale properties is technically challenging. Lastly, soil properties can be time-dependent. Not only can soil parameters change across seasons, but the day-night fluctuations can be significant, along with the short-term disturbance or perturbation [36,37,38]. In addition, many of its components influence each other; no soil properties are genuinely independent [39]. There is also no “standard” or baseline for soil properties, complicating soil comparison studies [7]. Nonetheless, scientists can make loose comparisons within a soil type, which is characterized by physical parameters and geological history. Finally, most soil testing is destructive, so measuring any parameter over time for the same soil sample is generally impossible. This limits data to be snap-shots instead of continuous [40].

The obstacles listed above are mostly matrix effects, so the first option when characterizing microbes from the soil is to try to remove those effects as much as possible. Many studies have done this when working with soil or other complex matrices like feces [19, 24, 41,42,43,44,45]. Liquid extraction of soil is a common method to remove matrix effects. The sample is usually sieved through a 2-mm sieve to remove larger dirt particles and small rocks or leaves [46,47,48,49]. Then, the soil is incubated in a liquid medium, and the supernatant is extracted for further analysis. However, liquid extraction does not extract all of the microbes, and moreover, the precise method of transport to the lab and any pre-treatments can significantly affect the microbes detected by NGS [50, 51]. The sample could also be digested via chemical and mechanical means to create a homogeneous solution; there are many commercial kits available for this purpose that have been widely used in research for many years [4,5,6, 24, 25, 30, 33, 34, 36, 37, 44, 46, 47, 50, 52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71]. There are many different brands and products available, including FastDNA SPIN kits from MP Biomedicals, EZNA DNA kits from Omega Bio-tek, DNeasy Power kits from Qiagen, NuceloSpin kits from Macherey–Nagel, ZymoBIOMICS DNA kits from Zymbo Research, and INTEST.pro kits from BIOMES. Specific products are usually tailored to detect particular types of organisms from particular matrices or environments. Even with these kits, soil remains a difficult medium to work with. Until there are standardized procedures for soil analysis, various kits should be considered for different soil samples because each has its biases, pros, and cons [50, 72,73,74].

After dealing with matrix effects and generating a sample for further analysis, the microbes can begin to be characterized, and culturing in growth media is the traditional method for this. Different colony morphologies or absorbance characteristics can be detected for characterization. In addition, culturing allows for functional or biofilm assays in the selected presence or absence of various metabolites or conditions [4, 55, 64,