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The effect of signal molecules on the cultivation efficiency of bacteria from the Gotland Deep in the central Baltic Sea was investigated. Numbers of cultivated cells were determined by the most-probable-number (MPN) technique. Artificial brackish water supplemented with different carbon substrates at low concentrations (200 M each) was employed as the growth medium. Compared to the results of previous studies, this approach yielded significantly higher cultivation efficiencies (up to 11% in fluid media). A further and pronounced increase in cultivation success was accomplished by the addition of cyclic AMP (cAMP), N-butyryl homoserine lactone, or N-oxohexanoyl-DL-homoserine lactone at a low concentration of 10 M. The most effective inducer was cAMP, which led to cultivation efficiencies of up to 100% of total bacterial counts. From the highest positive dilutions of these latter MPN series, several strains were isolated in pure culture and one strain (G100) was used to study the physiological effect of cAMP. Dot blot hybridization revealed, however, that strain G100 represented only a small fraction of the total bacterial community. This points towards an inherent limitation of the MPN approach, which does not necessarily recover abundant species from highly diverse communities. Bacterial cells of strain G100 that were starved for 6 weeks attained a higher growth rate and a higher biomass yield when resuscitated in the presence of cAMP instead of AMP.

High-throughput cultivation was combined with rapid and group-specific phylogenetic fingerprinting in order to recover representatives of three freshwater bacterioplankton communities. A total of 570 bacterial cultures were obtained by employing the most probable number and MicroDrop techniques. The majority of the cultured bacteria were closely related to previously uncultured bacteria and grouped with the ␣-Proteobacteria, ␤-Proteobacteria, Actinobacteria, Firmicutes, or Flavobacteria-Cytophaga lineage. Correspondingly, the natural bacterioplankton community was analyzed by high-resolution phylogenetic fingerprinting of these five bacterial lineages. 16S rRNA gene fragments were generated for each lineage and subsequently separated by denaturing gradient gel electrophoresis. By the combination of five group-specific PCR protocols, the total number of 16S rRNA gene fingerprints generated from the natural communities was increased sixfold compared to conventional (eubacterial) fingerprinting. Four of the environmental ␣-Proteobacteria 16S rRNA gene sequences obtained from the natural community were found to be identical to those of bacterial isolates. One of these phylotypes was detected in 14 different cultures and hence represented the most frequently cultured bacterium. Three of these 14 strains were characterized in detail. Their complete 16S rRNA gene sequences showed only 93% similarity to that of Sandaracinobacter sibiricus, the closest relative described so far. The novel phylotype of bacterium is a strict aerobe capable of using numerous organic carbon substrates and contains bacteriochlorophyll a bound to two different photosynthetic light-harvesting complexes. Dot blot hybridization revealed that the strains occur in lakes of different trophic status and constitute up to 2% of the microbial community.Planktonic bacteria mediate a significant proportion of the carbon turnover in freshwater lakes (17). So far, however, a more comprehensive understanding of the functions of freshwater bacteria has been hindered by the limited information on their physiology. Very few (in some cases as little as 0.001%) of the cells multiply in laboratory media (3). Even in improved, dilute culture media, the cultivation success of freshwater bacteria may not exceed 1.4% (9, 45). Culture-independent 16S rRNA-based studies indicate that the previously uncultured fraction comprises numerous unknown bacteria and entirely novel phylogenetic groups (20,23,54,58,64). In order to be able to link the structure and function of freshwater bacterioplankton, cultivation approaches thus need to be directed toward the isolation of its characteristic members. Recently, a few typical planktonic ␤-Proteobacteria and Actinobacteria could be isolated by employing novel cultivation approaches (10,24,25).High-throughput approaches for extinction culturing in small volumes of low-nutrient media have recently been established and applied to bacterioplankton samples (9,10,14). As an alternative separation method, bacterial cells have been encaps...

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Using the MicroDrop((R)) microdispenser system, a novel approach for high throughput cultivation assays for the determination of numbers of culturable bacteria, and for the isolation of bacteria in liquid media was established. The MicroDrop device works similar to an ink jet printer. Droplets of 150-200 pl are created at the nozzle of a glass micropipette by means of a computer-driven piezo transducer, and are dispensed automatically at predetermined positions with the aid of a XYZ-positioning system. The actual drop volume is highly reproducible and is determined by the pulse duration, the pulse frequency and the micropipette geometry. Culture media in 96-well microtiter plates were inoculated with constant numbers of bacteria from three different natural freshwater lakes. The number of culturable bacteria in the sample can be calculated from the frequency of wells showing bacterial growth, based on a binomial distribution of culturable cells. Our method was compared to the conventional most probable number (MPN) approach, the technique presently most often used for the determination of bacterial culturability and for the isolation of numerically dominant culturable bacteria. As opposed to the MPN technique, our approach yields data with much higher statistical significance (i.e. a 10 times lower standard deviation) due to the higher number of parallels which can be performed in each microtiter plate. The values of culturable bacteria as determined by the MPN and MicroDrop techniques were only weakly correlated (r(2)=0.570, n=42, P<0.001). Cultivation efficiencies obtained with the MicroDrop technique were systematically lower than MPN values by a factor of 2.7, indicating a significant overestimation of culturability by the latter method. The composition of the cultured bacterial fraction was determined by denaturing gradient gel electrophoresis fingerprinting of 16S rDNA fragments and sequencing. This demonstrated that phylogenetically similar bacteria were recovered by both cultivation techniques. Both methods resulted in the recovery of many previously unknown aquatic bacteria affiliated to the same taxonomic groups and, in one case, in the isolation of a numerically dominant, but not-yet-cultured beta-Proteobacterium which was ubiquitous in all three lakes.