Cultivation-success of rare soil bacteria is not influenced by incubation time and growth medium

Abstract

Rare bacterial species have recently attracted interest due to their many potential beneficial functions. However, only little is known about their cultivability. In this study we test the hypotheses that the use of flow cell-sorting for cultivation results in a high proportion of rare soil bacterial isolates relative to bacterial taxa that are abundant in soil. Moreover, we investigate whether different oligotrophic cultivation media and a prolonged incubation time increase the number of cultivated rare species. In a cultivation study we used flow cell sorting to select for small cells and to separate single cells, and grew bacteria on different oligotrophic media with prolonged incubation times. The abundance of the isolates in the field was assessed by comparing them to a 454-sequencing dataset from the same soil. Consequentially, all bacterial isolates were classified as either rare (<0.01% relative abundance) or abundant (>0.01% relative abundance) in the field soil. We found more bacterial taxa among the isolates that were abundant in soil than would be expected by the proportion of abundant species in the field. Neither incubation time nor growth medium had an influence on the recovery of rare species. However, we did find differences in time until visible growth on the plate between different phylogenetic classes of the isolates. These results indicate that rare cultivable species are active and not more likely to be dormant than abundant species, as has been suggested as a reason for their rarity. Moreover, future studies should be aware of the influence incubation time might have on the phylogenetic composition of the isolate collection.

Fig 1. Species abundance distribution of the field community.

Species abundance distribution of all OTUs in the field community (black) displayed as log-transformed relative abundance vs. species rank in the community; OTUs that were matched to a bacterial isolate are marked in red.

Fig 2. Phylogenetic composition of the bacterial field and isolated communities.

Percentage of the classes Alpha-, Beta- and Gammaproteobacteria, Actinobacteria and Bacilli in the field community including all other 80 classes that were not isolated in this study as “Other” and the percentage of the 5 isolated classes only in the field community, as well as the percentages of the 5 classes in the set of isolated OTUs (n = 43).

Fig 3. Species abundance distribution of bacterial classes.

Species abundance distributions of the OTUs in the field community belonging to the 5 different classes isolated in this study displayed as log-transformed relative abundance vs. species rank in the community; OTUs that were matched to a bacterial isolate are marked in colour.

Fig 4. Time of first bacterial growth on the plates.

Density plot of number of bacterial isolates showing visible growth on the plate along incubation time; lines represent densities of the different phylogenetic classes.