Resuscitation-promoting factor assists single-cell isolation for screening potential low-temperature biphenyl-degrading bacteria in Qinghai-Tibet Plateau

The mining of microbial resources has always been the focus of researchers. In the traditional microbial isolation method, due to the competition among dominant bacteria and the limitation of culture conditions, many microorganisms are unculturable, which are referred to as "microbial dark matter". Isolating bacteria before they begin to grow can eliminate interspecific competition and provide a conductive environment for subsequent cultivation. However, this approach does not alleviate the growth limitations caused by laboratory culture conditions on many microorganisms. Resuscitationpromoting factors can resuscitate and promote the growth of many bacteria. It has been widely used by researchers to isolate functional strains. Therefore, addition of the resuscitation-promoting factor can improve the isolation efficiency of low-activity and slow-growing microorganisms. However, no study has investigated the performance of the combination of these two methods. The Qinghai-Tibet Plateau harbors a huge reserve of microbial resources. The recent industrial development has accelerated resource exploitation in cold area in recent years. The fragile ecological environment of the Qinghai-Tibet Plateau is threatened by several factors including the crude oil and persistent organic pollutants. Given the prolonged low temperature environment in the plateau, digging indigenous microbial resources is of great significance to cold environment pollution remediation. In this study, we combined single-cell isolation and resuscitation-promoting factor addition. Droplet microfluidic was used to isolate single cells and resuscitation-promoting factor was added during the culture process. In this way, potentially low-temperature biphenyl-degrading strains were isolated from the surface soil samples at different altitudes of the Qinghai-Tibet Plateau. We also investigated the response of isolated strains to resuscitation-promoting factor. A total of 24 strains that could grow at low temperature (15 degrees C) with biphenyl as the sole carbon source were obtained, among which 18 strains were from the resuscitation-promoting factor addition group. Resuscitation-promoting factor addition significantly improved the efficiency of functional bacteria screen. The obtained strains belonged to 9 genera including Microbacterium. Most of the obtained strains showed obvious response to resuscitation-promoting factor, and some strains showed marked resuscitation-promoting factor dependence indicating that they almost did not grow without resuscitation-promoting factor addition. This demonstrates the mechanism by which the resuscitation-promoting factor improves single-cell isolation for functional bacteria. The successful isolation of potential low-temperature biphenyl-degrading bacteria from the Qinghai-Tibet Plateau provides the microbial resources for the subsequent prevention and control of biphenyl pollution in the Qinghai-Tibet Plateau and other cold areas. This study presents a new method for isolating and screening of functional bacteria through the combination of resuscitation-promoting factor and single-cell isolation. This method has high efficiency, low cost, and a broad application spectrum. It is a new method for the mining of "microbial dark matter".