Pseudomonas sp. strain B13 and Pseudomonas putida OUS82 were genetically tagged with the green fluorescent protein and the Discosoma sp. red fluorescent protein, and the development and dynamics occurring in flow chamber-grown two-colored monospecies or mixed-species biofilms were investigated by the use of confocal scanning laser microscopy. Separate red or green fluorescent microcolonies were formed initially, suggesting that the initial small microcolonies were formed simply by growth of substratum attached cells and not by cell aggregation. Red fluorescent microcolonies containing a few green fluorescent cells and green fluorescent microcolonies containing a few red fluorescent cells were frequently observed in both monospecies and two-species biofilms, suggesting that the bacteria moved between the microcolonies. Rapid movement of P. putida OUS82 bacteria inside microcolonies was observed before a transition from compact microcolonies to loose irregularly shaped protruding structures occurred. Experiments involving a nonflagellated P. putida OUS82 mutant suggested that the movements between and inside microcolonies were flagellum driven. The results are discussed in relation to the prevailing hypothesis that biofilm bacteria are in a physiological state different from planktonic bacteria.Surface adhesion of bacteria and subsequent cell binary fission and exopolymer production lead to the formation of bacterial biofilms (see reference 6 for a review). Application of confocal scanning laser microscopy (CSLM) has led to the suggestion that microcolonies are the basic structural units in sessile communities (4). It was found that biofilms are highly hydrated open structures containing a high fraction of exopolymers and large void spaces between the microcolonies (18). Mushroom-shaped microcolonies, separated by channels and voids, were observed in Pseudomonas fluorescens biofilms (15, 16). Investigations of multispecies biofilm communities on granular activated carbon in fluidized-bed reactors revealed that growth occurred as discrete microcolony structures separated by channel boundaries (19). Microbial biofilms in river water were shown to have a ridged structure, with microcolonies forming ridges parallel to the direction of flow (23).Evidence is now emerging that motility may play a role in structure formation in biofilms. After the initial attachment to the substratum, Pseudomonas aeruginosa evidently moves on the substratum by means of twitching motility, and it has been suggested that the initial microcolonies are formed by aggregation of bacteria (25). Furthermore, it has been suggested that the initial microcolonies in Vibrio cholerae El Tor biofilms are formed via flagellar motility of the cells along the substratum (32). In addition, Wolfaardt et al. (34) and Nielsen et al. (24) observed structural changes in biofilms in response to changing environments, suggesting that established biofilms in some cases may display dynamic behavior. Since it appears that structures in both young and mature biofil...

We examined the harmful side effects on indigenous soil microorganisms of two organic solvents, acetone and dichloromethane, that are normally used for spiking of soil with polycyclic aromatic hydrocarbons for experimental purposes. The solvents were applied in two contamination protocols to either the whole soil sample or 25% of the soil volume, which was subsequently mixed with 75% untreated soil. For dichloromethane, we included a third protocol, which involved application to 80% of the soil volume with or without phenanthrene and introduction of Pseudomonas fluorescens VKI171 SJ132 genetically tagged with luxAB::Tn5. For both solvents, application to the whole sample resulted in severe side effects on both indigenous protozoa and bacteria. Application of dichloromethane to the whole soil volume immediately reduced the number of protozoa to below the detection limit. In one of the soils, the protozoan population was able to recover to the initial level within 2 weeks, in terms of numbers of protozoa; protozoan diversity, however, remained low. In soil spiked with dichloromethane with or without phenanthrene, the introduced P. fluorescens VKI171 SJ132 was able to grow to a density 1,000-fold higher than in control soil, probably due mainly to release of predation from indigenous protozoa. In order to minimize solvent effects on indigenous soil microorganisms when spiking native soil samples with compounds having a low water solubility, we propose a common protocol in which the contaminant dissolved in acetone is added to 25% of the soil sample, followed by evaporation of the solvent and mixing with the remaining 75% of the soil sample.

Pesticides applied to agricultural soils are subject to environmental concerns because leaching to groundwater reservoirs and aquatic habitats may occur. Knowledge of field variation of pesticide-related parameters is required to evaluate the vulnerability of pesticide leaching. The mineralization and sorption of the pesticides glyphosate and metribuzin and the pesticide degradation product triazinamin in a field were measured and compared with the field-scale variation of geochemical and microbiological parameters. We focused on the soil parameters clay and organic carbon (C) content and on soil respiratory and enzymatic processes and microbial biomass. These parameters were measured in soil samples taken at two depths (Ap and Bs horizon) in 51 sampling points from a 4-ha agricultural fine sandy soil field. The results indicated that the spatial variation of the soil parameters, and in particular the content of organic C, had a major influence on the variability of the microbial parameters and on sorption and pesticide mineralization in the soil. For glyphosate, with a co-metabolic pathway for degradation, the mineralization was increased in soils with high microbial activity. The spatial variability, expressed as the CV, was about five times higher in the Bs horizon than in the Ap horizon, and the local-scale variation within 100 m(2) areas were two to three times lower than the field-scale variation within the entire field of about 4 ha.

No abstract

The phenoxyacetic acid herbicide MCPA (2-methyl-4-chlorophenoxyacetic acid) is frequently detected in groundwater beneath Danish agricultural fields. We investigated spatial variation in microbial MCPA mineralization potential in a flat agricultural field of fine sandy soil (USDA classification: Humic Dystrudept) located on the Yoldia plains of Northern Jutland, Denmark. Samples for determination of MCPA mineralization and sorption were collected from the Ap and Bs horizons at 51 sampling sites located in a 200 x 220 m grid. Spatial variation in sorption was low in both horizons (distribution coefficient, 0.36-4.16 L kg(-1)). Sorption correlated strongly with soil organic carbon content in both horizons (CV, 93 and 83%, respectively) and negatively with soil pH. [Ring-(14)C]-MCPA mineralized readily in the Ap horizon, with 49 to 62% of the (14)C-MCPA being converted to (14)CO(2) during the 67-d incubation period. With the subsoil, mineralization of (14)C-MCPA varied considerably between samples (0.5-72.8%). At neither depth was there correlation between (14)C-MCPA mineralization and sorption, soil pH, organic carbon content, clay content, number of colony-forming units (CFU), pseudomonad CFU, or any of the four microbial activity parameters measured. The presence of microbial genes encoding for the TfdA enzyme was quantified using real-time polymerase chain reaction. No correlation was found between MCPA mineralization potential and the natural background number of tfdA genes present in the soil samples. The degradation kinetics suggests that the high (14)C-MCPA mineralization rate detected in soil samples was linked to growth of the MCPA-degrading soil microbial community.