Background

The metagenomic analysis has shown an overwhelming number of bacterial populations which are beyond the cultivation in the normal laboratory conditions (Gans et al., 2005; Pham and Kim, 2012). These bacteria which are not-yet cultured and not able to grow on synthetic media are known as unculturable bacteria. The “unculturable” does not imply that these bacteria can never be cultured, but instead, it indicates the absence of information on their natural habitat and growth requirements (Stewart, 2012; Chaudhary et al., 2019). The molecular analysis has reported that uncultured bacteria possess novel biological and chemical properties, produce secondary metabolites, and comprise a diverse functional role (Ferrari et al., 2005; Vartoukian et al., 2010; van Dorst et al., 2016). The secondary metabolites such as novel antibiotics, enzymes, toxins, alkaloids have potential use for human welfares. Due to these beneficial aspects, several studies have attempted to explore and cultivate the hidden uncultured bacteria from the environments (Sait et al., 2002; Ferrari et al., 2005; Davis et al., 2005; George et al., 2011; van Dorst et al., 2016).

There are several difficulties in cultivating unculturable species in the laboratory setups. The unculturable bacteria fail to replicate in laboratory conditions due to lack of natural growth environment and inappropriate incubation time, temperature, and nutrients (Stewart, 2012). Next, traditional approach of cultivation is mainly based on nutrient-rich media which favors only rapid growers (Chaudhary et al., 2019). In the natural habitat, microbe-microbe interaction and their metabolism are crucial aspects for survival and growth. These microbial behaviors depend on several physiological parameters which include pH, osmotic conditions, temperature, and nutrients (Sait et al., 2002). Therefore, without addressing above-mentioned limitations, it is not possible to cultivate missing bacterial species. Strategies such as simulating natural habitat, modifying nutrients composition, optimizing growth conditions, co-culturing with helper agents, and prolonging the incubation time during cultivation of uncultured domains have reported good results (Kaberlein et al., 2002; Joseph et al., 2003; Pham et al., 2012; Stewart, 2012; Chaudhary et al., 2019). Some recent advancement has been made in cultivation techniques using diffusion chambers, transwell plates, microbioreactors, optical tweezers and laser microdissection (Pham et al., 2012). However, the available techniques are still inadequate to address the limitation of cultivation of unculturable bacteria. Any attempt to develop novel cultivation technique for unculturable bacteria accounts a great significance in this field.

Enrichment cultivation utilizing diffusion bioreactor is a promising strategy to cultivate previously uncultured microbes. It helps to enrich microbial species by creating natural environment and allows the microbes with the access to growth components and signaling compounds (Bollmann et al., 2007). In addition, it also provides environment for bacterial interaction (Stewart, 2012; Chaudhary et al., 2019). Diffusion bioreactors are also suitable to cultivate various microbial species from varieties of samples such as soil, sediments, and water samples (Kakumanu et al., 2012; Chaudhary et al., 2019). This paper describes a novel cultivation technique for uncultured soil bacteria using newly designed diffusion bioreactor. Diffusion bioreactor permits the passage of essential elements from the soil into the inner chamber mimicking the natural environments (Kaberlein et al., 2002; Bollmann et al., 2007; Kakumanu and Williams, 2012). Here, we present an effective protocol for experimental setup using diffusion bioreactors to isolate and cultivate the unculturable soil bacteria.