Derivation of a growth rate equation describing microbial surface colonization

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 (C_i) containing either one cell, two cells, four cells, etc, as shown below. $$\mu = \frac{{\ln (\frac{N}{{C_i }} + 1)}}{t}$$ The attachment rate (A) is given by the following relationship: $$A = \mu C_i $$ 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.     

A computer simulation of surface microcolony formation during microbial colonization

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 (C_i) reaches a constant value (C_max) 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 C_max. It was shown that C_i approaches C_max asymptotically. The time required is dependent solely upon the growth rate and size of microcolonies. The number of one-celled microcolonies reaches 95% of C_max after 4.3 generations. At low growth rates, a relatively long incubation period is required. Alternate methods that shorten the incubation time are considered.     

Chemostat and in‐situ colonization kinetics of thermothrix thiopara on calcite and pyrite surfaces

Growth and attachment rates of Thermothrix thiopara on calcite and pyrite were quantitated in a thiosulfate‐limited chemostat and in the thermal spring where the organism is found in nature. Surface growth rates were quantitated by using the surface colonization and exponential growth equations. These two models were compared as means of determining surface growth rates. In the chemostat, T. thiopara cells colonizing calcite and pyrite surfaces grew at approximately one‐third the rate of suspended cells. However, T. thiopara attached to pyrite faster than to calcite. In the thermal spring, growth and attachment rates were equal on calcite and pyrite. It was concluded that the exponential growth equation overestimates in‐situ surface growth rates and that T. thiopara grows more slowly when colonizing mineral surfaces than when growing in suspension. Lower growth rates on surfaces may be due to a reduced cell surface area for nutrient uptake or an increased specific maintenance rate.     

Evaluation of surface colonization kinetics in continuous culture

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: $$N = (A/\mu )e^{\mu t} - A/\mu $$ where N=number of cells on surface (cells field^?1); A=attachment rate (cells field^?1h^?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 (C_i) will reach a constant value (C_max) which is equal to A divided byμ: $$\mu = \frac{{\ln \left( {\frac{N}{{C_i }} + 1} \right)}}{t}$$ Both equations gave similar results and the time required to approximate C_max 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.     

Evaluation of a proposed surface colonization equation usingThermothrix thiopara as a model organism

The colonization equation shown below was evaluated usingThermothrix thiopara as a model organism. $$N = (A/\mu )e^{\mu t} - A/\mu $$ 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%.     

Behavior of bacterial stream populations within the hydrodynamic boundary layers of surface microenvironments

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.     

Listeria monocytogenes LO28: surface physicochemical properties and ability to form biofilms at different temperatures and growth phases

The surface physicochemical properties of Listeria monocytogenes LO28 under different conditions (temperature and growth phase) were determined by use of microelectrophoresis and microbial adhesion to solvents. The effect of these parameters on adhesion and biofilm formation by L. monocytogenes LO28 on hydrophilic (stainless steel) and hydrophobic (polytetrafluoroethylene [PTFE]) surfaces was assessed. The bacterial cells were always negatively charged and possessed hydrophilic surface properties, which were negatively correlated with growth temperature. The colonization of the two surfaces, monitored by scanning electron microscopy, epifluorescence microscopy, and cell enumeration, showed that the strain had a great capacity to colonize both surfaces whatever the incubation temperature. However, biofilm formation was faster on the hydrophilic substratum. After 5 days at 37 or 20 degrees C, the biofilm structure was composed of aggregates with a three-dimensional shape, but significant detachment took place on PTFE at 37 degrees C. At 8 degrees C, only a bacterial monolayer was visible on stainless steel, while no growth was observed on PTFE. The growth phase of bacteria used to inoculate surfaces had a significant effect only in some cases during the first steps of biofilm formation. The surface physicochemical properties of the strain are correlated with adhesion and surface colonization.     

A theoretical derivation of the Contois equation for kinetic modeling of the microbial degradation of insoluble substrates

Although developed as an empirical model to describe microbial growth on soluble substrates, the Contois equation has been widely accepted for kinetic modeling of insoluble substrate degradation. Yet, the mechanistic basis underlining these successful applications remains unanswered. Unlike soluble substrates that mainly cultivate suspended cultures, microbes cultivated on insoluble substrates have the populations that attach to the substrate surface or remain suspended in the bulk solution, while those attached usually grow faster than those suspended due to their proximity to food resources. This imbalanced growth provides a plausible explanation to the inverse relationship between microbial concentration and their specific growth rate as conveyed in the Contois equation. Based on a theoretical derivation, this study revealed that the Contois equation holds true only when attached microbes substantially obstruct the access of food to their suspended counterparts. On the other hand, when plentiful insoluble substrate surfaces are exposed for cell attachment, the Contois equation will be reduced back to the classic Monod equation.     

Microbial colonization in the seagrass Posidonia spp. roots

The pattern of colonization by microorganisms on root surfaces from three species of seagrass belonging to the genus Posidonia was assessed. Microbial abundance on roots was measured by two electronic microscope techniques. Trends in microbial colonization between species and root order were defined. In addition, eutrophication status of the sampling sites and physiological status of Posidonia oceanica (L.) Delile roots have been taken into account. Our results show high microbial abundance in the Mediterranean species P. oceanica, in comparison with the low rates of colonization found in the Australian species P. australis Hook f. and P. sinuosa Cambridge et Kuo. Microbial density tended to decrease as root order increased, and living roots always showed higher microbial abundance than dead ones. Colonization of P. oceanica roots at the three sites with different environmental status follows different trends according to root order. It is suggested that root age influences the rate of microbial colonization of seagrass roots and that colonization of root surface by microorganisms is associated with organic exudates from the roots rather than with decaying root tissues.     

Effect of mineral composition on bacterial attachment to submerged rock surfaces

A direct microscopic count technique employing fluorescein isothiocyanate stain was used to compare microbial colonization on the exposed surfaces of rocks and minerals suspended in several ponds for various time intervals. Hematitic sandstone was never colonized at a rate greater than limestone, but quartz was always colonized more rapidly than calcite. The use of single-crystal minerals (quartz and calcite) in a nested factor experiment showed that the effect of the minerals on colonization was statistically significant, but that differences among the immersion sites were also significant. Sandstone samples placed in a pond outflow accumulated microbial colonizers more rapidly than those placed in the still waters of the same pond. The results indicate that the composition of the mineral substrate, in concert with the immersion environment, controls the formation of primary slime layers in aquatic systems.     

Listeria monocytogenes LO28: Surface Physicochemical Properties and Ability To Form Biofilms at Different Temperatures and Growth Phases

The surface physicochemical properties of Listeria monocytogenes LO28 under different conditions (temperature and growth phase) were determined by use of microelectrophoresis and microbial adhesion to solvents. The effect of these parameters on adhesion and biofilm formation by L. monocytogenes LO28 on hydrophilic (stainless steel) and hydrophobic (polytetrafluoroethylene [PTFE]) surfaces was assessed. The bacterial cells were always negatively charged and possessed hydrophilic surface properties, which were negatively correlated with growth temperature. The colonization of the two surfaces, monitored by scanning electron microscopy, epifluorescence microscopy, and cell enumeration, showed that the strain had a great capacity to colonize both surfaces whatever the incubation temperature. However, biofilm formation was faster on the hydrophilic substratum. After 5 days at 37 or 20°C, the biofilm structure was composed of aggregates with a three-dimensional shape, but significant detachment took place on PTFE at 37°C. At 8°C, only a bacterial monolayer was visible on stainless steel, while no growth was observed on PTFE. The growth phase of bacteria used to inoculate surfaces had a significant effect only in some cases during the first steps of biofilm formation. The surface physicochemical properties of the strain are correlated with adhesion and surface colonization.     

Numerical dominance and phylotype diversity of marine Rhodobacter species during early colonization of submerged surfaces in coastal marine waters as determined by 16S ribosomal DNA sequence analysis and fluorescence in situ hybridization

Early stages of surface colonization in coastal marine waters appear to be dominated by the marine Rhodobacter group of the alpha subdivision of the division Proteobacteria (alpha-Proteobacteria). However, the quantitative contribution of this group to primary surface colonization has not been determined. In this study, glass microscope slides were incubated in a salt marsh tidal creek for 3 or 6 days. Colonizing bacteria on the slides were examined by fluorescence in situ hybridization by employing DNA probes targeting 16S or 23S rRNA to identify specific phylogenetic groups. Confocal laser scanning microscopy was then used to quantify and track the dynamics of bacterial primary colonists during the early stages of surface colonization and growth. More than 60% of the surface-colonizing bacteria detectable by fluorescence staining (Yo-Pro-1) could also be detected with the Bacteria domain probe EUB338. Archaea were not detected on the surfaces and did not appear to participate in surface colonization. Of the three subdivisions of the Proteobacteria examined, the alpha-Proteobacteria were the most abundant surface-colonizing organisms. More than 28% of the total bacterial cells and more than 40% of the cells detected by EUB338 on the surfaces were affiliated with the marine Rhodobacter group. Bacterial abundance increased significantly on the surfaces during short-term incubation, mainly due to the growth of the marine Rhodobacter group organisms. These results demonstrated the quantitative importance of the marine Rhodobacter group in colonization of surfaces in salt marsh waters and confirmed that at least during the early stages of colonization, this group dominated the surface-colonizing bacterial assemblage.     

Weak rolling adhesion enhances bacterial surface colonization

Bacterial adhesion to and subsequent colonization of surfaces are the first steps toward forming biofilms, which are a major concern for implanted medical devices and in many diseases. It has generally been assumed that strong irreversible adhesion is a necessary step for biofilm formation. However, some bacteria, such as Escherichia coli when binding to mannosylated surfaces via the adhesive protein FimH, adhere weakly in a mode that allows them to roll across the surface. Since single-point mutations or even increased shear stress can switch this FimH-mediated adhesion to a strong stationary mode, the FimH system offers a unique opportunity to investigate the role of the strength of adhesion independently from the many other factors that may affect surface colonization. Here we compare levels of surface colonization by E. coli strains that differ in the strength of adhesion as a result of flow conditions or point mutations in FimH. We show that the weak rolling mode of surface adhesion can allow a more rapid spreading during growth on a surface in the presence of fluid flow. Indeed, an attempt to inhibit the adhesion of strongly adherent bacteria by blocking mannose receptors with a soluble inhibitor actually increased the rate of surface colonization by allowing the bacteria to roll. This work suggests that (i) a physiological advantage to the weak adhesion demonstrated by commensal variants of FimH bacteria may be to allow rapid surface colonization and (ii) antiadhesive therapies intended to prevent biofilm formation can have the unintended effect of enhancing the rate of surface colonization.     

Numerical Dominance and Phylotype Diversity of Marine Rhodobacter Species during Early Colonization of Submerged Surfaces in Coastal Marine Waters as Determined by 16S Ribosomal DNA Sequence Analysis and Fluorescence In Situ Hybridization

Early stages of surface colonization in coastal marine waters appear to be dominated by the marine Rhodobacter group of the α subdivision of the division Proteobacteria (α-Proteobacteria). However, the quantitative contribution of this group to primary surface colonization has not been determined. In this study, glass microscope slides were incubated in a salt marsh tidal creek for 3 or 6 days. Colonizing bacteria on the slides were examined by fluorescence in situ hybridization by employing DNA probes targeting 16S or 23S rRNA to identify specific phylogenetic groups. Confocal laser scanning microscopy was then used to quantify and track the dynamics of bacterial primary colonists during the early stages of surface colonization and growth. More than 60% of the surface-colonizing bacteria detectable by fluorescence staining (Yo-Pro-1) could also be detected with the Bacteria domain probe EUB338. Archaea were not detected on the surfaces and did not appear to participate in surface colonization. Of the three subdivisions of the Proteobacteria examined, the α-Proteobacteria were the most abundant surface-colonizing organisms. More than 28% of the total bacterial cells and more than 40% of the cells detected by EUB338 on the surfaces were affiliated with the marine Rhodobacter group. Bacterial abundance increased significantly on the surfaces during short-term incubation, mainly due to the growth of the marine Rhodobacter group organisms. These results demonstrated the quantitative importance of the marine Rhodobacter group in colonization of surfaces in salt marsh waters and confirmed that at least during the early stages of colonization, this group dominated the surface-colonizing bacterial assemblage.     

Kinetics of microbial cell growth

As a rate equation of microbial cell growth, the Monod equation is widely used. However, this equation cannot fully correspond to real courses of microbial cell growth in many batch cultivations. Especially, predicted values based on this equation do not agree with observed values in many continuous cultivations. In this paper, which introduces new concepts of critical concentration and coefficient of consumption activity, the growth rate equation which corresponds to the whole period including lag period is newly derived and characteristics of microbial cell growth in batch cultivation are clarified. Further, applying the new rate equation to continuous cultivation, a general equation with which to calculate cell concentration is derived and characteristics of microbial cell growth in continuous cultivation are clarified. The calculated values of cell concentration based on the new theory showed quite good agreement with the observed values in both batch and continuous cultivation.     

Microbial selectivity on mineral surfaces: possible implications for weathering processes

In order to understand how microorganisms influence mineral surface processes, a better assessment of how microorganisms colonise mineral surfaces in situ is necessary. A crucial question in understanding mineral–microbial processes is whether colonization by microbial cells on mineral surfaces is a random process or whether it follows a selective pattern related primarily to the chemical composition of the mineral.     

Colonization rates and community structure of diatoms on three different rock substrata in a lotic system

Diatom assemblages were monitored at weekly intervals over a 5 week period on Verde limestone, Supai sandstone, and Andesitic basalt substrata in a mountain stream in northern Arizona, U.S.A. Density, Shannon-Weiner diversity, evenness, and community similarity (SIMI) were used to compare colonization patterns and community structure between individual substratum types. Average standing crop values were nearly two-fold higher on sandstone than on either basalt or limestone substrata after the first week of the study. It is proposed that differences in micro-surface features between substrata and possibly the rate of substratum solubilization may cause these differences in density early in the colonization period. Following the initial week, standing crop and community structure were significantly similar on all substrata for the remainder of the study period. Maximum densities were attained by the third week and remained relatively constant on all substrata for the remainder of the study.

SEM micrographs demonstrated that surfaces of submerged substrata in streams are modified after the first week by the accumulation of organic aggregates. The establishment of an “organic matrix” early in the colonization process may provide relatively similar attachment surfaces for microbial invasion. This appears to reduce the initial microtopographic differences displayed by substrata and allows for a more uniform colonization pattern.     

Factors controlling hyporheic respiration in a desert stream

1. Experimental manipulations were performed to determine the biological, chemical and physical attributes that govern sediment respiration in the hyporheic zone of Sycamore Creek, a Sonoran Desert stream. 2. Hyporheic respiration per unit volume of sediment was inversely related to diameter of sediment particles, indicating that respiration is affected by availability of substrate for microbial colonization (i.e. sediment surfaces). Respiration rate per unit surface area on sediments was positively correlated with particle diameter, indicating greater metabolic activity of microbes on larger sediments. 3. Hyporheic respiration was more than twice as high in water collected from the surface flow than from subsurface flow. Further, hyporheic respiration was highest immediately following exposure of sediments to surface water and declined over time, presumably due to exhaustion of labile organic matter. 4. Microbial activity was stimulated by addition of algal leachate; however, amendments of leaf leachate had little effect. Respiration was also elevated with dextrose and leucine amendments, but not with inorganic nitrogen additions, indicating hyporheic respiration is carbon limited. 5. Water from the stream surface is probably enriched in labile organic matter derived from algae and stimulates respiration at points of hydrologic downwelling where surface water enters hyporheic sediments. The physical structure of sediments further affects metabolism by affecting the area available for microbial attachment.     

Bacterial colonization and biofilm development on minimally processed vegetables

Bacterial biofilms have been observed and reported on food and food-processing surfaces and can contribute to increased risks for product quality and food safety. The colonization of fruit and vegetables by pectynolitic bacteria like Pseudonomas fluorescens attributable to conditions such as soft rot, can also manifest as biofilms. A developed biofilm structure can provide a protective environment for pathogens such as Listeria monocytogenes reducing the effectiveness of sanitisers and other inhibitory agents. Understanding the colonization of bacteria on leaf surfaces is essential to the development of a better understanding of the leaf ecology of vegetable products. Studies of microbial colonization of leaf surfaces have been conducted using SEM and more recently using confocal microsocpy techniques. In the current study, a Leica TCS NT laser scanning confocal microscope was used to investigate biofilm formation using vital fluorescence staining on intact vegetable leaves. Reflection contrast and fluorescence three-dimensional imaging successfully delineated bacterial and biofilm morphology without disturbing the bacterial or leaf surface structure. The results demonstrate the presence and development of biofilm on the surface of lettuce. The biofilms appeared to originate on the cuticle in distinct micro-environments such as in the natural depression of the stomata, or in the intercellular junction. Bacteria also adhered to and developed biofilm colonies within an hour of contact and with clean stainless steel surfaces. Our study investigates the progression of biofilm formation from leaf colonization, and will assist in characterising the critical mechanisms of plant/host interaction and facilitate the development of improved preservation, sanitising and packaging strategies for minimally processed vegetable products.     

Anti-biofouling property studies on carboxyl-modified multi-walled carbon nanotubes filled PDMS nanocomposites

Polydimethylsiloxane (PDMS) with exceptional fouling-release properties is extremely susceptible to the microfouling resulted from the colonization of the pioneer microorganisms in the marine environment. In this study, six carboxyl-modified multi-walled carbon nanotubes (cMWNTs) nanoparticles were incorporated into the PDMS matrix, respectively, in order to produce the cMWNTs-filled PDMS nanocomposites (CPs) with improved antifouling (AF) properties. The AF properties of the six CPs were examined via the field assays conducted in Weihai, China. The effects of the anti-biofouling potential of the CPs (i.e. the P₃ surface) on the colonization of the pioneer prokaryotic and eukaryotic microbes were investigated using the single-stranded conformation polymorphism technique via the comparison of the diversity indices. Different CPs have displayed differential and better AF properties as compared to that of the unfilled PDMS (P₀). The P₃ surface has exhibited exceptional anti-biofouling capacity compared with the other CPs surfaces, which can effectively prevent biofouling for more than 14 weeks in the field. The SSCP analysis revealed that the P₃ surface may have significant modulating effect on the pioneer microbial communities. The pioneer eukaryotic microbes seemed more susceptible than the pioneer prokaryotic microbes to be subjected to the major perturbations exerted by the P₃ surface. The dramatically reduced eukaryotic-microbial diversity may contribute to the impeding and weakening of the development and growth of the biofilm. The P₃ surface has the potential to be used for future maritime applications.