Abstract
The isolation and cultivation of heterotrophic marine bacteria
opens possibilities to study their physiology and genomes with respect to
their function in the marine environment. In the pelagic marine realm
bacteria remineralize more than half of the photosynthetically produced
biomass, and thus play an important role in the biogeochemical cycling of
elements.
Flavobacteria are abundant of up to 30% in the North Sea. In previous
studies marine Gammaproteobacteria, Alphaproteobacteria and Actinobacteria
were predominantly cultivated, but cultures of Flavobacteria
were infrequently obtained. This thesis addresses the isolation of phylogenetically
diverse marine Flavobacteria using three new approaches.
First, samples were retrieved from various pelagic and benthic
habitats of the North Sea. Second, a new marine artificial seawater
HaHa medium was developed to facilitate the growth of Flavobacteria.
This medium was supplemented with saccharides and proteins as
carbon sources at a concentration of 2 g/L. Third, a specific 16S rRNA
gene PCR assay was applied to identify Flavobacteria-Cytophagia
among the colonies. The molecular screen was preferred over the identification
by cell and colony morphology, since the latter has predominantly
resulted in the isolation of strains of the genera Arenibacter, Cellulophaga
and Maribacter. The 375 Flavobacteriaceae strains isolated on agar
plates comprised (i) seven presumably novel genera, (ii) 42 presumably
novel species in 22 validly described Flavobacteriaceae genera and (iii)
many isolates that were so far not distinguishable from 37 type strains in
16 genera. Thus, in contrast to previous studies, we could show that phylogenetically
diverse Flavobacteria from the North Sea can be cultivated on
solid medium.
The isolation of representative strains of the genera Formosa,
Polaribacter, and Reinekea from the North Sea was attempted. In
previous studies these bacterial populations were proposed to be of importance
during coastal diatom-dominated phytoplankton blooms, based
on their high abundance of 15% to 25% of the bacterioplankton and their
potential capability to decompose algae derived polysaccharides. A new
medium was devised which had the same composition as the marine HaHa
medium, but with environmental-like micromolar carbon, nitrogen, and
phosphate concentrations. Aerobic dilution cultivation in the HaHa
medium led to a high culturability of 35% of the bacterioplankton
in spring 2010 and 27% of the bacterioplankton in summer 2010. 23
strains of Flavobacteria, Alphaproteobacteria, Gammaproteobacteria,
and Actinobacteria were obtained directly by dilution cultivation
of single cell inocula. One strain that belonged to the genus Reinekea
was isolated by generating co-cultivatures of randomly mixed bacterial
populations which potentially had a positive effect on the growth
of Reinekea. Strains that affiliated with Polaribacter , Formosa, Gillisia
(Flavobacteria), the Roseobacter clade associated (RCA) lineage (Alphaproteobacteria),
Reinekea, and the OM182 clade (Gammaproteobacteria) had
16S rRNA gene sequence identities of >99.9% with 16S rRNA clones
of the bacterioplankton from the North Sea in spring 2009. In addition,
draft genomes of Formosa, Polaribacter , and Reinekea strains were used
to recruit reads of metagenomes of the bacterioplankton in spring 2009.
Thereby, reads of >95% nucleotide identity covered the draft genomes
of the Formosa clade B strain to 94%, of Reinekea sp. to 90% and of
Polaribacter sp. to 50%. Based on these results we argue that the novel
species of Formosa, Polaribacter , and Reinekea are representatives of
ecologically relevant clades catalyzing the remineralization of coastal
diatom-dominated phytoplankton biomass.
The physiological characteristics of the strains were investigated focusing
on the growth on different mono- and polysaccharides, to provide
further evidence that Formosa, Polaribacter and Reinekea species
could prevail in different ecological niches during algae decay. Interestingly,
Polaribacter strains grew heterotrophically on all tested sulfated (e.g.
agar, carrageenan) and non-sulfated polysaccharides (e.g. cellulose, laminarin),
whereas Formosa strains grew only on non-sulfated polysaccharides.
In contrast, Reinekea sp. did not grow on polysaccharides but
on all tested mono-, di-, and trisaccharides including N-acetylneuraminic
acid. Finally, I proposed for these novel species the names ’Formosa
flavarachnoidea’, ’Formosa forsetii ’, ’Polaribacter forsetii ’, ’Polaribacter
frigidimaris’, ’Polaribacter adhaesivus’, and ’Reinekea forsetii ’.
