Two-Dimensional Cell Separation: a High-Throughput Approach to Enhance the Culturability of Bacterial Cells from Environmental Samples

In the manuscript, we have developed a novel yet simple and innovative approach to improving the cultivability of natural microorganisms without sophisticated instrumentation. The method used gradient centrifugation combined with serial dilution (two-dimensional cell separation) to improve taxum recovery from samples. Culture-independent sequence data from various environmental samples have revealed an immense microbial diversity of environmental, clinical, and industrial importance that has not yet been cultured. Cultivation is imperative to validate findings emerging from cultivation-independent molecular data and exploit the isolated organisms for biotechnological purposes. Efforts have been made to boost the cultivability of microbes from environmental samples by use of a range of techniques and instrumentation. The manuscript presents a novel yet simple and innovative approach to improving the cultivability of natural microorganisms without sophisticated instrumentation. By employing gradient centrifugation combined with serial dilution ("two-dimensional cell separation"), significantly higher numbers of genera (>2-fold higher) and species (>3-fold higher) were isolated from environmental samples, including soil, anaerobic sludge, and landfill leachate, than from using serial dilution alone. This simple and robust protocol can be modified for any environment and culture medium and provides access to untapped microbial diversity. IMPORTANCE In the manuscript, we have developed a novel yet simple and innovative approach to improving the cultivability of natural microorganisms without sophisticated instrumentation. The method used gradient centrifugation combined with serial dilution (two-dimensional cell separation) to improve taxum recovery from samples. This simple and robust protocol can be modified for any environment and culture medium and provides access to untapped microbial diversity. This approach can be incorporated with less labor and complexity in laboratories with minimal instrumentation. As cultivation is a workflow that is well suited to lower-resource microbiology labs, we believe improvements in cultivability can increase opportunities for scientific collaborations between low-resource labs and groups focused on high-resource cultivation-independent methodologies.