Microbial Interaction is Among the Key Factors for Isolation of Previous Uncultured Microbes.

Pure cultivation of microbes is still limited by the challenges of microbial uncultivability, with most microbial strains unable to be cultivated under standard laboratory conditions. The experience accumulated from advanced techniques such as in situ cultivation has identified that microbial interactions exist in natural habitats but are absent in laboratory cultures. These microbial interactions are likely one of the key factors in isolating previously uncultured microbes. The need for better knowledge of the mechanisms operating in microbial interactions has led to various experiments that have utilized microbial interactions in different approaches to microbial cultivation. These new attempts to understand microbial interactions not only present a new perspective on microbial uncultivability but also provide an opportunity to access uncultured phylogenetically novel microbes with their potential biotechnology applications. In this review, we focus on studies of the mechanisms of microbial interaction where the growth of other microbes is affected. Additionally, we review some successful applications of microbial interactions in cultivation methods, an approach that can play an important role in the bioprospecting of untapped microbial resources.

A targeted liquid cultivation method for previously uncultured non-colony forming microbes.

A large number of microbes are not able to form colonies using agar-plating methods, which is one of the reasons that cultivation based on solid media leaves the majority of microbial diversity in the environment inaccessible. We developed a new Non-Colony-Forming Liquid Cultivation method (NCFLC) that can selectively isolate non-colony-forming microbes that exclusively grow in liquid culture. The NCFLC method involves physically separating cells using dilution-to-extinction (DTE) cultivation and then selecting those that could not grow on a solid medium. The NCFLC was applied to marine samples from a coastal intertidal zone and soil samples from a forest area, and the results were compared with those from the standard direct plating method (SDP). The NCFLC yielded fastidious bacteria from marine samples such as Acidobacteriota, Epsilonproteobacteria, Oligoflexia, and Verrucomicrobiota. Furthermore, 62% of the isolated strains were potential new species, whereas only 10% were novel species from SDP. From soil samples, isolates belonging to Acidobacteriota and Armatimonadota (which are known as rare species among identified isolates) were exclusively isolated by NCFLC. Colony formation capabilities of isolates cultivated by NCFLC were tested using solid agar plates, among which approximately one-third of the isolates were non-colony-forming, approximately half-formed micro-colonies, and only a minority could form ordinary size colonies. This indicates that the majority of the strains cultivated by NCFLC were previously uncultured microbial species unavailable using the SDP method. The NCFCL method described here can serve as a new approach to accessing the hidden microbial dark matter.

Accessing previously uncultured marine microbial resources by a combination of alternative cultivation methods.

Few microbes can grow under laboratory conditions, highlighting the fact that the majority of microbes in environment are still uncultured and untapped resources. This study used alternative cultivation methods, diffusion chambers (DC), dilution-to-extinction culture (DTE) and modified agar preparation step (PS media) to cultivate previously uncultured marine bacterial species. These methods were applied to samples from a coastal intertidal zone, and the results were compared with those from standard direct plating (SDP) cultivation. Among the strains isolated with DC, DTE and PS media methods, 28%, 48% and 33% were novel species, respectively, while the SDP method resulted in the isolation of only 9% of novel species. Most isolates were unique to the method used for their cultivation. This implies that each method is selective in its own way, which is different from SDP, thus able to access species that are difficult to obtain using conventional approaches. Comparing the diversity showed that 75 genera were recovered by the alternative methods, 2.7 times higher than that of the SDP cultivation, which constituted 45% of total diversity from culture-independent sequencing. We conclude that combining alternative cultivation methods represents a highly promising key for accessing 'microbial dark matter'.

Application of the filter plate microbial trap (FPMT), for cultivating thermophilic bacteria from thermal springs in Barguzin area, eastern Baikal, Russia.

Hot springs are regarded as treasury of valuable thermophiles. Like other bacteria, thermophiles are not easily cultivated using conventional culture methods. We used an advanced cultivation method, the filter plate microbial trap (FPMT), to isolate bacteria from thermal springs. In total, 184 isolates were obtained from five thermal springs using the FPMT and standard agar plate method, and their 16S rRNA gene sequences were analyzed. FPMT allowed us to obtain a culture collection that was larger, richer, and more novel than that obtained by standard cultivation. Seven novel species were obtained using the FPMT technique, whereas only one was isolated using a standard cultivation. We also found clear differences in the patterns of phylogenetic diversity and physiological properties between isolates from two cultivation methods. The results have encouraged us to apply the FPMT method in other extreme environments and offer further support for fostering the development of new cultivation methods.

Export of non-point source suspended sediment, nitrogen, and phosphorus from sloping highland agricultural fields in the East Asian monsoon region.

Excess sediment and nutrient export from agricultural fields with steep slopes is a major concern linked to surface water quality in Korea. In this study, the export of suspended sediment (SS), total nitrogen (TN), and total phosphorus (TP) and their event mean concentrations (EMCs) in surface runoff from a highland mixed land use (61% forested, 30% cropped, 9% other) watershed were quantified. In 2007, the Korean Ministry of Environment (MoE) declared the study area as a priority region for non-point source (NPS) pollution management and initiated various best management practices (BMPs) in the study watershed. SS, TN, and TP concentrations in Mandae Stream were monitored for 5 years (2009-2013) to evaluate the effectiveness of BMPs. Average EMCs for SS, TN, and TP were as high as 986, 3.4 and 0.8 mg/L, respectively. The agricultural export coefficients of agricultural land in the study watershed for SS, TN, and TP were 5611, 171, and 6.83 kg/ha/year, respectively. A comparison with results from other studies shows that both EMCs and agricultural export coefficients in the study watershed were much higher than most of the results reported for watersheds in other regions. The results show that sediment and nutrient export from intensive agriculture areas with steep slopes continue to be a major concern for the downstream reservoir, Lake Soyang. Remedial strategies should be directed towards controlling sources of SS, TN, and TP to improve downstream water quality in sloping highland agricultural areas in Korea.