Jostein Goksøyr

Total bacterial diversity in soil and sediment communities : a review

Advances in microbial methods have demonstrated that microorganisms globally are the dominating organisms both concerning biomass and diversity. Their functional and genetic potential may exceed that of higher organisms. Studies of bacterial diversity have been hampered by their dependence on phenotypic characterization of bacterial isolates. Molecular techniques have provided the tools for analyzing the entire bacterial community including those which we are not able to grow in the laboratory. Reassociation analysis of DNA isolated directly from the bacteria in pristine soil and marine sediment samples revealed that such environments contained in the order of 10 000 bacterial types. The diversity of the total bacterial community was approximately 170 times higher than the diversity of the collection of bacterial isolates from the same soil. The culturing conditions therefore select for a small and probably skewed fraction of the organisms present in the environment. Environmental stress and agricultural management reduce the bacterial diversity. With the reassociation technique it was demonstrated that in heavily polluted fish farm sediments the diversity was reduced by a factor of 200 as compared to pristine sediments. Here we discuss some molecular mechanisms and environmental factors controlling the bacterial diversity in soil and sediments.

Bacterial and fungal activities in soil: Separation of bacteria and fungi by a rapid fractionated centrifugation technique

Abstract With three natural soils from Western and Central Norway it has been shown that by homogenization and fractionated centrifugations, two fractions could be obtained, one containing 50–80% of the bacteria, and the other all the fungi together with soil debris and the rest of the bacteria. When the separation was carried out in the cold and lasting less than 3–4 h, the fractions if used immediately had constant respiratory rates for 1–2 h. The sum of the rates agreed well with that of an unfractionated soil homogenate, making it possible to calculate the ratio between bacterial and fungal respiration. Assuming that the much higher respiration in a homogenate than in intact soil is due to inhibitory conditions in the latter and that these can be analysed by separate experiments, the technique will permit estimates of the ratio between bacterial and fungal respiration in in situ soil under given conditions. The soils used had very great differences between plate and microscopic count values for bacteria. When based on plate counts, the calculated bacterial respiratory intensities became impossibly high, while they were within biologically possible limits when microscopic count values were used. The respiratory intensities for fungi when calculated from hyphal lengths and diameter measurements, were biologically possible.

Respiratory burst after freezing and thawing of soil: Experiments with soil bacteria

Abstract The respiratory burst after freezing and thawing of soil has its maximum immediately after thawing. The reduction in bacterial plate counts after a freeze-thaw cycle was moderate, and so was also the subsequent increase. Experiments with isolated bacteria from the same soil showed that there was a similar burst when bacteria were frozen and thawed in the stationary phase. Between different strains, there was a positive correlation between the killing effect of the freeze-thaw treatment and the respiratory increase per surviving bacterium. The increased O 2 uptake during the first 11 h after thawing corresponded to a C consumption 5–15% of that in the killed bacteria. A brief respiratory increase was observed when the supernatant from a frozen-thawed culture was added to bacteria in the stationary state. The burst was more persistent when frozen-thawed bacteria were centrifuged and then resuspended in supernatants from stationary cultures of unfrozen and frozen bacteria, or even in a mineral salts solution. The optical difference spectrum between the supernatants from frozen and unfrozen cultures demonstrated leakage of cellular material after freezing and thawing. Monod kinetics for growth shows that if the substrate concentration is substantially lower than K s , and the biomass concentration relatively high, the respiratory rate is at maximum at the time when the substrate is added. The respiratory burst following freezing and thawing can thus be explained on the basis of simple growth kinetics.

Determination of bacterial DNA in soil

Abstract Two methods for the determination of DNA have been adapted for use on the bacterial fraction of an organic soil, obtained by fractionated centrifugation. The. soil contained about 1.1 × 10 10 bacteria g −1 dry weight when counted by fluorescence microscopy. One method was based on the reaction with 3,5-diaminobenzoic acid 2HCl, and the other on the specific reaction of the antibiotic mithramycin with double-stranded DNA. For the first method, about 8 × 10 8 cells were required and for the second, about 4 × 10 9 . The analytical results with the two methods agreed well. As an average of several determinations, an amount of 8.4 fg (10 −15 g) DNA per bacterial cell was found when the cells were counted by fluorescence microscopy. This is in the upper size range of bacterial genomes. showing that virtually all microscopically counted bacteria in this soil contain DNA. The total amount of DNA was about 90μg g −1 soil dry weight.

Effects of water fluctuations on microbial mass and activity in soil

When previously dried soil was remoistened, a series of microbial events occurred. The bacterial plate count population increased rapidly, with a doubling time of 4–5 h. The length of fungal hyphae and microscopic counts of bacteria increased more slowly. The microscopically counted bacterial population was estimated to have a doubling time of about 90 h. The respiratory burst occurring after 2–3 days coincided with the maximal growth rate of the bacterial plate count population. From the respiratory data, plate count bacteria were estimated to have a cell mass of 0.4 pg dry weight, whereas the mass of microscopically counted bacteria was only 10% of this. Changes in bacterial DNA content corresponded to changes in the microscopic count, whereas changes in soil catalase activity mainly corresponded to changes in the fungal biomass, which was dominant.It is suggested that bacterial plate counts and microscopic counts represent two distinct populations of bacteria, which for practical purposes may be termed zymogenous and autochthonous, respectively.

Phenotypical divergences between populations of soil bacteria isolated on different media

Bacterial strains were randomly isolated from soil using three different media with glucose (TG), Tryptone Soya Broth (TTS), and succinate (TS) as carbon sources. Plate counts obtained were 12.0×107, 4.5 ×107, and 1.5×107 g−1 soil dry weight, respectively. The strains were characterized phenotypically by the API 20B test system. A cluster analysis of all isolates revealed 40 biotypes at 80% similarity, 23 in TG, 29 in TTS, and 27 in TS. Each of the 10 most common biotypes contained 10 to 2.5% of the isolates, and 17 biotypes contained one or two isolates. The common biotypes were unevenly distributed among the isolates from the different media. About 20% of the isolates from TG and TTS were unique for the particular medium, whereas among the isolates from TS, about 60% were unique. Thirty percent of the isolates belonged to biotypes that were common to all three populations. All media gave approximately the same high diversity measured as Shannon index and Equitability, indicating no direct correlation between plate count and diversity.

A Warburg apparatus with automatic recording of respiratory 14CO2

A radiorespirometer, constructed as a modified Warburg apparatus, is described. The radioactivity is determined directly in the alkali cup, which is fastened on to a small G-M tube inserted in the cover of the Warburg flask. The contents of both the main compartment and the alkali cup are agitated by magnetic stirring. The G-M tubes from eacll flask are connected via a time switch to a rate meter and a recorder. The setup makes it possible to work with four flasks with G-M tubes simultaneously. For respiratory measurements, Warburgs direct method is used, with a corresponding number of flasks without alkali and G-M tube. Curves are shown to demonstrate the kinetics of C/sulp 14/O/sub 2/ absorption in the alkali cup and the registration of radioactivity. (auth)