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Scanning confocal laser microscopy was used to directly visualize accumulation of the herbicide diclofop methyl and its breakdown products by a degradative biofilm community, cultivated in continuous-flow cell cultures. Some bacterial cells accumulated these compounds. However, most accumulation occurred in cell capsules and certain regions of the exopolymer matrix. Mass spectroscopic analysis of the biofilm material confirmed accumulation of the parent compound and its breakdown products in the biofilms. Lower molecular weight degradation products were found in the effluent, indicating mineralization of diclofop by the flow cell cultures. Grazing protozoa feeding on the biofilms nonselectively ingested cell capsules and exopolymers, suggesting direct transfer and accumulation of the contaminants in protozoa. These findings demonstrated that microbial exopolymers can play an important role in the bioaccumulation of contaminants in natural systems. [ABSTRACT FROM AUTHOR]

Bacterial biovolumes of hypertrophic Humboldt Lake (total dissolved solids = 3.3 g liter(-1); 6 m deep) and oligotrophic Redberry Lake (total dissolved solids = 20.9 g liter(-1); 17 m deep), Saskatchewan, were measured concurrently with a variety of environmental variables to identify the major factors correlated with volume changes. There was no difference (P > 0.05) in mean bacterial volume between Redberry Lake (0.084 ± 0.034 μm(3) SD) and Humboldt Lake (0.083 ± 0.021 μm(3) SD). Statistical analyses suggested there were marked differences in the factors associated with the pronounced seasonality of bacterial cell volumes in these two lakes. Variance in bacterial volume in the epilimnion of Redberry Lake was best explained by a multivariate regression model which included ciliate abundance and chlorophyll concentration (r (2) = 0.96). The model accounting for changes in hypolimnetic bacterial volume included ciliate numbers and primary production (r (2) = 0.94), of the measured variables. Bacterial volume in Humboldt Lake was most highly correlated with primary production (r (2) = 0.59). Bacterial production (estimated as the rate of thymidine incorporation into DNA) and growth (thymidine incorporation rate normalized to cell numbers) were not correlated to cell volume, with the exception of cocci volume in Humboldt Lake.

Several models of microbial surface colonization have been devised to quantitate growth and attachment rates on surfaces. One of these, the surface growth rate equation, is based on the assumption that the number of microcolonies of a given size (Ci) reaches a constant value (Cmax) that is equal to the attachment rate (A) divided by the specific growth rate (Μ). In this study, a computer simulation was used to determine the time required to reach Cmax. It was shown that Ci approaches Cmax asymptotically. The time required is dependent solely upon the growth rate and size of microcolonies. The number of one-celled microcolonies reaches 95% of Cmax after 4.3 generations. At low growth rates, a relatively long incubation period is required. Alternate methods that shorten the incubation time are considered.

Computer-enhanced microscopy (CEM) was used to study the growth kinetics of bacterial microcolonies attached to the wall of a continuous-flow slide culture. Image processing increased effective microscope resolution and quantitated colony growth at 10 min intervals. Three growth parameters were used to determine growth rate: the time required for cell fission, the specific rate of increase in cell number, and the specific rate of increase in cell area. Growth rate was initially constant regardless of colony size, as assumed previously in deriving colonization kinetics. However, at low substrate concentrations growth rate varied depending on laminar flow velocity. Growth was flow-dependent at a glucose concentration of 100 mg/liter and flow-independent at a concentration of 1 g/liter. This indicated that the surface microenvironment became substrate-depleted in the absence of sufficient laminar flow velocities and that glucose rather than oxygen was rate limiting.

Phase, darkfield, and computer-enhanced microscopy were used to observe the surface microenvironment of flow cells during bacterial colonization. Microbial behavior was consistent with the assumptions used previously to derive surface colonization kinetics and to calculate surface growth and attachment rates from cell number and distribution. Surface microcolonies consisted of closely packed cells. Each colony contained 2(n) cells, where n is the number of cell divisions following attachment. Initially, cells were freely motile while attached, performing circular looping movements within the plane of the solid-liquid interface. Subsequently, cells attached apically, maintained a fixed position on the surface, and rotated. This type of attachment was reversible and did not necessarily lead to the formation of microcolonies. Cells became irreversibly attached by progressing from apical to longitudinal attachment. Longitudinally attached cells increased in length, then divided, separated, moved apart laterally, and slid next to one another. This resulted in tight cell packing and permitted simultaneous growth and adherence. After approximately 4 generations, individual cells emigrated from developing microcolonies to recolonize the surface at new locations. Surface colonization byPseudomonas fluorescens can thus be subdivided into the following sequential colonization phases: motile attachment phase, reversible attachment phase, irreversible attachment phase, growth phase, and recolonization phase.

Phase and computer-enhanced microscopy were used to observe the surface microenvironment of continuous-flow slide cultures during microbial colonization and to document the diversity of bacterial colonization maneuvers among natural stream populations. Surface colonization involved 4 discrete types of cell movement, which were designated as packing, spreading, shedding, and rolling maneuvers. Each maneuver appeared to be associated with a specific species population within the community. The packing maneuver resulted in the formation of a monolayer of contiguous cells, while spreading maneuvers resulted in a monolayer of adjacent cells. During the shedding maneuver, cells attached perpendicular to the surface and the daughter cells were released. The rate of growth of new daughter cells gradually decreased as the attached mother cell aged. During the rolling maneuver, cells were loosely attached and continuously somersaulted across the surface as they grew and divided. Only those populations with a packing maneuver conformed fully to the assumptions of kinetics used previously to calculate growth and attachment rates from cell number and distribution. Consequently, these kinetics are not applicable to stream communities unless fluorescent antisera are used to study specific species populations within natural communities. Virtually all of the cells that attached to the surface were viable and underwent cell division. The abundance of unicells on surfaces incubated in situ was thus primarily the consequence of bacterial colonization behavior (shedding and spreading maneuvers) rather than the adhesion of dead or moribund cells.

A surface growth rate equation is derived which describes simultaneous growth and attachment during microbial surface colonization. The equation simplifies determination of attachment and growth rate, and does not require a computer program for solution. This rate equation gives the specific growth rate (Μ) as a function of the number of cells on the surface (N), the incubation period (t), and the number of colonies (Ci) containing either one cell, two cells, four cells, etc, as shown below.[Formula: see text] The attachment rate (A) is given by the following relationship:[Formula: see text] The proposed colonization kinetics are compared with exponential growth kinetics using 3-dimensional computer plots. Colonization kinetics diverged most from exponential kinetics when the growth rate was low or the attachment rate was high. Using these kinetics, it is possible to isolate the effects of growth and attachment on microbial surface colonization.

Computer-enhanced microscopy (CEM) was used to monitor bacteria colonizing the inner surfaces of a 1×3 mm glass flow cell. Image analysis provided a rapid and reliable means of measuring microcolony count, microcolony area, and cell motility. The kinetics of motile and nonmotilePseudomonas fluorescens surface colonization were compared at flow velocities above (120μm sec(-1)) and below (8μm sec(-1)) the strain's maximum motility rate (85μm sec(-1)). A direct attachment assay confirmed that flagellated cells undergo initial attachment more rapidly than nonflagellated cells at high and low flow. During continuous-flow slide culture, neither the rate of growth nor the timing of recolonization (cell redistribution within surface microenvironments) were influenced by flow rate or motility. However, the amount of reattachment of recolonizing cells was both flow and motility dependent. At 8μm sec(-1) flow, motility increased reattachment sixfold, whereas at 120μm sec(-1) flow, motility increased reattachment fourfold. The spatial distribution of recolonizing cells was also influenced by motility. Motile cells dispersed over surfaces more uniformly (mean distance to the nearest neighbor was 47.0μm) than nonmotile cells (mean distance was 14.2μm) allowing uniform biofilm development through more effective redistribution of cells over the surface during recolonization. In addition, motile cell backgrowth (where cells colonize against laminar flow) occurred four times more rapidly than nonmotile cell backgrowth at low flow (where rate of motility exceeded flow), and twice as rapidly at high flow (where flow exceeded the rate of motility). The observed backgrowth of Mot(+) cells against high flow could only have occurred as the result of motile attachment behavior. These results confirm the importance of motility as a behavioral mechanism in colonization and provides an explanation for enhanced colonization by motile cells in environments lacking concentration gradients necessary for chemotactic behavior.

The colonization equation shown below was evaluated usingThermothrix thiopara as a model organism.[Formula: see text] where: N=number of cells on surface (cells field(-1)); A = attachment rate (cells field(-1) h(-1)); M=specific growth rate (h(-1)); t=incubation period (h).Previous studies of microbial surface colonization consider attachment and growth independently. However, the proposed colonization equation integrates the effects of simultaneous attachment and growth. Using this equation, the specific growth rate ofT. thiopara was found to be 0.38±0.3 h(-1) during in situ colonization. Estimates ofμ were independent of incubation period after 4 h (2 generations). Shorter incubations were inadequate to produce sufficient microcolonies for accurate determination of specific growth rate. Empirical data for the time course of colonization fell within the 95% confidence interval of predicted values. The attachment rate, although assumed to be constant, was found to continuously increase with time. This increase may have been an artifact due to the continuous deposition of travertine on the surface, or may indicate the need for a function to replace A in the colonization equation. Using the exponential growth equation, the progeny of cells that attach during incubation are considered to be progeny of cells that attach initially. This erroneously inflated the growth rate by 55%.

The effects of glucose and nitrogen depletion on the colonization of glass Petri plates byPseudomonas fluorescens were studied in batch culture. Colonization of the surfaces was initiated before colonization of the bulk phase, and biofilm formation was observed. This resulted in an apparent lag in the batch growth curve for the cell suspension. The lag phase was an artifact caused by the partitioning of cells between the bulk and solid phase of the culture and was not due to a reduction in the growth rate of unattached cells. The specific growth rate of the unattached cells (0.331 hour(-1)) was almost twice that determined for the total population (0.171 hour(-1)). Consequently the growth rate of biofilm-forming bacteria cannot be determined in batch culture unless the growth of both attached and unattached cells is monitored, and batch growth curves may contain artifacts due to the formation and dispersion of biofilms. The depletion of either glucose or nitrogen led to the active detachment of cells from the biofilm. An increase in the hydrophobicity of unattached cells was noted on depletion of carbon. This increase was the result of emigration of cells from the surface into the bulk phase.

An equation describing the initial phases of microbial surface colonization is presented. Simultaneous microbial attachment and growth are considered as the primary components of colonization. A table is given that permits determination of growth rate from the density and distribution of cells present on surfaces after incubation in situ. Other methods used to calculate microbial growth rate on surfaces are evaluated. The new procedure is more accurate and less time consuming than those used previously. Published data on microbial surface colonization more closely follow the proposed colonization equation than the exponential growth equation, which overestimates the growth rate.

Two equations, describing surface colonization, were evaluated and compared using suspended glass slides in a continuous culture ofPseudomonas aeruginosa. These equations were used to determine surface growth rates from the number and distribution of cells present on the surface after incubation. One of these was the colonization equation which accounts for simultaneous attachment and growth of bacteria on surfaces:[Formula: see text] where N=number of cells on surface (cells field(-1)); A=attachment rate (cells field(-1)h(-1));μ=specific growth rate (h(-1)); t=incubation period (h). The other was the surface growth rate equation which assumes that the number of colonies of a given size (Ci) will reach a constant value (Cmax) which is equal to A divided byμ:[Formula: see text] Both equations gave similar results and the time required to approximate Cmax may not be as long as was previously thought. In all cases both A andμ continuously decreased throughout the incubation period. These decreases may be due to various effects of microbial accumulation on the surface. Both equations accurately determined surface growth rates despite highly variable attachment rates. Growth rates were similar for both the liquid phase of the culture and the solid-liquid interface (0.4 h(-1)). Use of the surface growth rate equation is favored over the use of the colonization equation since the former does not require a computer to solve forμ and the counting procedure is simplified.