Appl Microbiol Biotechnol (2012) 93:1005–1020 Functional screening It is the ability to detect, isolate, and characterize expressed genes in a metagenomic library which determines the success of any function-based metagenomic assessment. A broad array of screening methods can be used (summarized in Fig. 1). Among these, three general detection strategies are distinguished (Simon and Daniel 2009): (1) Phenotypic insert detection (PID), where the expres- sion of a particular trait is used to identify positive clones (2) Modulated detection (MD), a strategy that relies on the production of a gene product that is necessary for growth under selective conditions (3) Substrate induction, a strategy that is based on the induced expression of cloned genes via a specific substrate Although a distinction is made between these three detection strategies, categorizing them into separate groups would be incorrect. Often, a combination of them is used in order to perform and optimize the screening. For instance, phenotypic detection through a GFP reporter gene can be combined with substrate-induced expression of the insert gene (Uchiyama et al. 2005). PID PID is the most commonly used approach for the functional screening of metagenomic libraries. The screen is based on the detection of specific phenotypic traits. The intensity (level) of gene expression is an important issue here since faint expression signals can be easily missed in high-throughput screenings. A possible aid is offered by microfluidic approaches using nanoliter volumes. These can offer increasing sensitivity of the assay since less gene product is required to yield a detectable phenotype (Taupp et al. 2011). Specific phenotypic traits may be detected in multiple ways. The first way is based on direct expression, for instance, by detecting pigmentation or colony morphology (Brady 2007), both of which may directly result from the expressed inserted gene (Craig et al. 2010; LeCleir et al. 2007). Another way is the (indirect) reaction or interaction of an added substance with the expressed gene product or a product that is a consequence of this expression. Lastly, detection can be based on co- expression of a reporter gene which is linked to the target gene in the library. A high diversity of methods has been developed based on these three strategies, of which the most prominent ones will be discussed below. Direct detection Visual detection is a phenotypic screening method that is relatively straightforward and “low-tech”. 1011 However, it is also quite labor-intensive. This screening method works by positive clones displaying a trait (as the result of the expression of a library gene) which is directly observable. Examples of such observable traits are colony pigmentation, irregular colony morphology, or halo forma- tion on plate overlays. Coupling this direct detection method with high-throughput technologies, such as that offered by 384-well plates, colony picking robots and microplate readers, not only shortens processing time but also enhances the reliability and comparability of screenings performed on different clones. A disadvantage of this method is its rather low resolution or sensitivity. For example, if expression is low in a certain positive clone, a phenotypic trait might not be readily detectable, resulting in an incorrect rejection of “sub-threshold” positive clones. Furthermore, the method does not allow direction to be given to the screen. In a recent study (Craig et al. 2010), clones were screened in high-throughput in multiple hosts based on the three phenotypic traits mentioned above. These traits were chosen based on the fact that they are commonly associated with small-molecule production (Craig et al. 2010). This illustrates the fact that these methods are more suited to a broad-range exploration of the metagenome for pleiotrophic traits than to directed searches for a specific pathway or metabolite. Indicator medium The use of indicator medium constitutes a direct way to detect particular small molecules, chemical reactions, or metabolic, catabolic, or antibiotic capabilities of a clone. It is a popular detection method given its suitability for high-throughput application, as well as its amenability to many experiments, from broad screenings of diverse gene products to the isolation of very specific metabolic capabilities (De Vasconcellos et al. 2010; Fan et al. 2011; Morohoshi et al. 2011; Tirawongsaroj et al. 2008). Furthermore, the relative sensitivity of this method allows it to detect changes in, e.g., pH at moderate expression levels, especially when combined with droplet-based microfluidics, where detectable concentrations are easily reached due to the small volume in use. A nice example of the use of indicator medium in screenings is the isolation of novel metallo-proteases from metagenomic libraries using milk-infused plates. The screen was based on detection of proteolytic activity in the E. coli clones, which confer the ability to hydrolyze milk proteins. The library clones were incubated on skimmed milk-containing agar plates and proteolytic activity was detected by the formation of clear haloes on the plates (Waschkowitz et al. 2009). The use of indicator media holds great promise as the successful expression of foreign genes in the host can be readily monitored in high throughput. Relying on the suc- cessful expression of foreign genes for detection might yield