Assessment of soil bacterial communities with emphasis on the phylum Acidobacteria

The seasonal culturability (February, April, August) of bacterial cells from a microbial community of an alpine calcareous soil was assessed employing the MicroDrop technique using different laboratory media with humic acid analogs (HA), a mixture of polymers (POL), artificial root exudates (RO), nutrient broth, or soil extract as carbon and energy sources. Thereby, the summer August sample showed the highest culturability value in media supplemented with soil extract (13.5%). Since only 81 wells of a total number of 1008 individual growth tests were overgrown with the February soil sample, the cultivation success was the lowest for the winter environment (0.16%). The major aim of the present study, however, was to assess the cultivation success for cells even exposed to extreme environmental conditions by using defined media. Therefore, subsequent analysis focused on the cultures obtained from the February sample and in media supplemented with RO. It was shown that the monomeric organic carbon of RO proved to be superior to POL and HA for the optimization of the cultivation success (i.e., 71 of the total number of 81 cultures). The quantitative PCR approach confirmed the high coverage of the present analysis since the target groups (Firmicutes, Actinobacteria, Bacteroidetes, Alphaproteobacteria, Betaproteobacteria, Acidobacteria) constituted 73.6% of all eubacteria in the sample whereas the major part was composed of Alphaproteobacteria (49.2%) and Acidobacteria (20.1%). A total of 251 bacteria were analyzed representing 53 distinct phylotypes of which 73% are previously unknown. The majority of the cultured fraction was closely related to the Alphaproteobacteria with the largest number of different phylotypes and the highest evenness value. Although this phylum dominated the cultivated fraction, its cultivation success was hundredfold lower than its abundance in the natural community (0.4% of total cell numbers). Also the Bacteroidetes were most frequently cultured but were dominated by one phylotype (Sphingoterrabacterium pocheensis). The relative culturability of the Bacteroidetes was the highest of all groups and reached 25% of the numbers detected by real-time PCR. The lowest culturability was assessed for the Acidobacteria with only one single cultivated phylotype using media with POL supplemented with signal compounds. However, this phylotype represents a novel, previously unknown acidobacterium, strain Jbg-1. The phylum Acidobacteria mostly consists of environmental 16S rRNA gene sequences and so far comprises only the four validly described species Holophaga foetida, Geothrix fermentans, Acidobacterium capsulatum and Terriglobus roseus. In the present thesis two different novel strains of acidobacteria were isolated. Strain Jbg-1 and the second strain Wbg-1, which was recovered from a coculture with a methanotrophic bacterium established from calcareous forest soil. Both strains represent members of subdivision 1 of the phylum Acidobacteria and are closely related to each other (98.0 % 16S rRNA gene sequence similarity). At a sequence similarity of 93.8-94.7%, strains Jbg-1 and Wbg-1 are only distantly related to the closest described relative, Terriglobus roseus, and accordingly are described as members of the novel genus Edaphobacter gen. nov. Based on the DNA-DNA-similarity between strains Jbg-1 and Wbg-1 of 11.5-13.6% and their chemotaxonomic and phenotypic characteristics, the two strains are assigned to two separate species, Edaphobacter modestus sp. nov. with strain Jbg-1T (= ATCC BAA-1329T = DSM 18101T) as the type strain, and E. aggregans sp. nov. with strain Wbg-1T (= ATCC BAA-1497T = DSM 19364T) as the type strain. The two novel species are adapted to low carbon concentrations and to neutral to slightly acidic conditions. It was shown that strain Jbg-1 was also well adapted to long-term survival and to higher carbon concentrations after subcultivation. Unexpectedly, a high percentage of interspecific interaction was obtained for the cultivation approach of the February alpine soil (75% cocultures), which represented the major reason for the low cultivation success. Only 16 out of 71 cultures with RO consisted of single cultivated strains. Due to the frequent occurrence of different bacteria in the same cultures, the actual cultivation success was 4.9 fold higher than the value calculated from the abundance of positive cultures. For subsequent analysis, the effect of different treatments during the cultivation approach on the number and composition of bacteria cultured was investigated. In order to differentiate between free-living and attached cells, bacteria were detached from soil particles and used to set up parallel incubations. The detachment from soil particles prior to inoculation had no effect on the total cultivation success and on co-cultivation. Furthermore, signal compounds (cyclic AMP and N-butyryl homoserine lactone), however, increased the cultivation success and co-culturability. Addition of signal compounds yielded different types of activated bacteria and enhanced the total number of phylotypes per co-culture towards 4, 5, 6, and 7 different bacteria. The major part of the single cultivated strains represented a single phylotype, which was related to Sphingoterrabacterium pocheensis. In contrast, most co-cultures contained members of the Alpha- and Betaproteobacteria whereas relatives of Phyllobacterium brassicacearum, Rhodospirillum rubrum, Inqulinus ginsengisoli, Delftia tsuruhatensis, and Rhodocyclus tenuis were the most abundant ones. In conclusion, it is supposed that cell-to-cell interaction routinely occurs between different species of microorganisms, although the way, how these aerobic microorganisms beneficially interact remained to be shown. The elucidation of such interactions seems to be the most successful approach to enhance the culturability of interesting soil bacteria to promote their growth in pure or defined co-cultures.