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.