Spatial and Temporal Variations in Chitinolytic Gene Expression and Bacterial Biomass Production during Chitin Degradation

Growth of the chitin-degrading marine bacterium S91 on solid surfaces under oligotrophic conditions was accompanied by the displacement of a large fraction of the surface-derived bacterial production into the flowing bulk aqueous phase, irrespective of the value of the surface as a nutrient source. Over a 200-h period of surface colonization, 97 and 75% of the bacterial biomass generated on biodegradable chitin and a nonnutritional silicon surface, respectively, detached to become part of the free-living population in the bulk aqueous phase. Specific surface-associated growth rates that included the cells that subsequently detached from the substrata varied depending on the nutritional value of the substratum and during the period of surface colonization. Specific growth rates of 3.79 and 2.83 day⁻¹ were obtained when cells first began to proliferate on a pure chitin film and a silicon surface, respectively. Later, when cell densities on the surface and detached cells as CFU in the bulk aqueous phase achieved a quasi-steady state, specific growth rates decreased to 1.08 and 0.79 day⁻¹ on the chitin and silicon surfaces, respectively. Virtually all of the cells that detached from either the chitin or the silicon surfaces and the majority of cells associated with the chitin surface over the 200-h period of surface colonization displayed no detectable expression of the chitin-degrading genes chiA and chiB. Cells displaying high levels of chiA-chiB expression were detected only on the chitin surface and then only clustered in discrete areas of the surface. Surface-associated, differential gene expression and displacement of bacterial production from surfaces represent adaptations at the population level that promote efficient utilization of limited resources and dispersal of progeny to maximize access to new sources of energy and maintenance of the population.     

Differentiation of chitinase-active and non-chitinase-active subpopulations of a marine bacterium during chitin degradation

The ability of marine bacteria to adhere to detrital particulate organic matter and rapidly switch on metabolic genes in an effort to reproduce is an important response for bacterial survival in the pelagic marine environment. The goal of this investigation was to evaluate the relationship between chitinolytic gene expression and extracellular chitinase activity in individual cells of the marine bacterium Pseudoalteromonas sp. strain S91 attached to solid chitin. A green fluorescent protein reporter gene under the control of the chiA promoter was used to evaluate chiA gene expression, and a precipitating enzyme-linked fluorescent probe, ELF-97-N-acetyl-beta-D-glucosaminide, was used to evaluate extracellular chitinase activity among cells in the bacterial population. Evaluation of chiA expression and ELF-97 crystal location at the single-cell level revealed two physiologically distinct subpopulations of S91 on the chitin surface: one that was chitinase active and remained associated with the surface and another that was non-chitinase active and released daughter cells into the bulk aqueous phase. It is hypothesized that the surface-associated, non-chitinase-active population is utilizing chitin degradation products that were released by the adjacent chitinase-active population for cell replication and dissemination into the bulk aqueous phase.     

Effect of Motility on Surface Colonization and Reproductive Success of Pseudomonas fluorescens in Dual-Dilution Continuous Culture and Batch Culture Systems

The colonization of glass surfaces by motile and nonmotile strains of Pseudomonas fluorescens was evaluated by using dual-dilution continuous culture (DDCC), competitive and noncompetitive attachment assays, and continuous-flow slide culture. Both strains possessed identical growth rates whether in the attached or planktonic state. Results of attachment assays using radiolabeled bacteria indicated that both strains obeyed first-order (monolayer) adsorption kinetics in pure culture. However, the motile strain attached about four times more rapidly and achieved higher final cell densities on surfaces than did the nonmotile strain (2.03 × 10⁸ versus 5.57 × 10⁷ cells vial^-1) whether evaluated alone or in cocultures containing motile and nonmotile P. fluorescens. These kinetics were attributed to the increased transport of motile cells from the bulk aqueous phase to the hydrodynamic boundary layer where bacterial attachment, growth, and recolonization could occur. First-order attachment kinetics were also observed for both strains by using continuous-flow slide culture assays analyzed by image analysis. The DDCC system contained both aqueous and particulate phases which could be diluted independently. DDCC results indicated that when cocultures containing motile and nonmotile P. fluorescens colonized solid particles, the motile strain replaced the nonmotile strain in the system over time. Increasing the aqueous-phase rates of dilution decreased the time required for extinction of the nonmotile strain while concurrently decreasing the overall carrying capacity of the DDCC system for both strains. These results confirmed that bacterial motility conveyed a selective advantage during surface colonization even in aqueous-phase systems not dominated by laminar flow.     

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.     

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.     

Comparison of the fouling release properties of hydrophobic fluorinated and hydrophilic PEGylated block copolymer surfaces: attachment strength of the diatom Navicula and the green alga Ulva

To understand the role of surface wettability in adhesion of cells, the attachment of two different marine algae was studied on hydrophobic and hydrophilic polymer surfaces. Adhesion of cells of the diatom Navicula and sporelings (young plants) of the green macroalga Ulva to an underwater surface is mainly by interactions between the surface and the adhesive exopolymers, which the cells secrete upon settlement and during subsequent colonization and growth. Two types of block copolymers, one with poly(ethylene glycol) side-chains and the other with liquid crystalline, fluorinated side-chains, were used to prepare the hydrophilic and hydrophobic surfaces, respectively. The formation of a liquid crystalline smectic phase in the latter inhibited molecular reorganization at the surface, which is generally an issue when a highly hydrophobic surface is in contact with water. The adhesion strength was assessed by the fraction of settled cells (Navicula) or biomass (Ulva) that detached from the surface in a water flow channel with a wall shear stress of 53 Pa. The two species exhibited opposite adhesion behavior on the same sets of surfaces. While Navicula cells released more easily from hydrophilic surfaces, Ulva sporelings showed higher removal from hydrophobic surfaces. This highlights the importance of differences in cell-surface interactions in determining the strength of adhesion of cells to substrates.     

Detachment ofPseudomonas fluorescens from biofilms on glass surfaces in response to nutrient stress

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.Paper contribution number 128, Centre de Rechcrches Alimentaires de St Hyacinthe.     

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.     

Behavior ofPseudomonas fluorescens within the hydrodynamic boundary layers of surface microenvironments

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 2ⁿ 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.     

Characterization of initial events in bacterial surface colonization by two Pseudomonas species using image analysis

The processes leading to bacterial colonization on solidwater interfaces are adsorption, desorption, growth, and erosion. These processes have been measured individually in situ in a flowing system in real time using image analysis. Four different substrata (copper, silicon, 316 stainless-steel and glass) and 2 different bacterial species (Pseudomonas aeruginosa and Pseudomonas fluorescens) were used in the experiments. The flow was laminar (Re = 1.4) and the shear stress was kept constant during all experiments at 0.75 N m(-2). The surface roughness varied among the substrata from 0.002 mum (for silicon) to 0.015 mum (for copper). Surface free energies varied from 25.1 dynes cm(-1) for silicon to 31.2 dynes cm(-1) for copper. Cell curface hydrophobicity, reported as hydrocarbon partitioning values, ranged from 0.67 for Ps. fluorescens to 0.97 for Ps. aeruginosa.The adsorption rate coefficient varried by as much as a factor of 10 among the combinations of bacterial strain and substratum material, and was positively correlated with surface free energy, the surface roughness of the substratum, and the hydrophobicity of the cells. The probability of desorption decreased with increasing surface free energy and surface roughness of the substratum. Cell growth was inhibited on copper, but replication of cells overlying an initial cell layer was observed with increased exposure time to the cell-containing bulk water. A mathematical model describing cell accumulation on a substratum is presented.     

Bacterial adsorption to smooth surfaces: Rate, extent, and spatial pattern

The influence of bulk-water bacterial cell concentration and specific growth rate history on bacterial adsorption rates to surfaces was investigated using response surface analysis. A pure culture of Pseudomonas sp. 224S was grown in a chemostat and pumped into a continuous flow reactor where the bacteria were exposed to clean, glass surfaces under turbulent flow conditions for a period of six hours. Adsorption rate decreased approximately linearly with increasing specific growth rate history. Glass surfaces became saturated with 224S at ca. 0.1% coverage and the resulting spatial pattern of the adsorbed cells deviated from random in the direction of uniformity.     

Virulence and the environment: a novel role for Vibrio cholerae toxin-coregulated pili in biofilm formation on chitin

The toxin-coregulated pilus (TCP) of Vibrio cholerae is required for intestinal colonization and cholera toxin acquisition. Here we report that TCP mediates bacterial interactions required for biofilm differentiation on chitinaceous surfaces. We also show that undifferentiated TCP- biofilms have reduced ecological fitness and, thus, that chitin colonization may represent an ecological setting outside the host in which selection for a host colonization factor may take place.     

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.     

Dynamics of microbial communities on marine snow aggregates: colonization,growth, detachment,and grazing mortality of attached bacteria

We studied the dynamics of microbial communities attached to model aggregates (4-mm-diameter agar spheres) and the component processes of colonization, detachment, growth, and grazing mortality. Agar spheres incubated in raw seawater were rapidly colonized by bacteria, followed by flagellates and ciliates. Colonization can be described as a diffusion process, and encounter volume rates were estimated at about 0.01 and 0.1 cm(3) h(-1) for bacteria and flagellates, respectively. After initial colonization, the abundances of flagellates and ciliates remained approximately constant at 10(3) to 10(4) and approximately 10(2) cells sphere(-1), respectively, whereas bacterial populations increased at a declining rate to >10(7) cells sphere(-1). Attached microorganisms initially detached at high specific rates of approximately 10(-2) min(-1), but the bacteria gradually became irreversibly attached to the spheres. Bacterial growth (0 to 2 day(-1)) was density dependent and declined hyperbolically when cell density exceeded a threshold. Bacterivorous flagellates grazed on the sphere surface at an average saturated rate of 15 bacteria flagellate(-1) h(-1). At low bacterial densities, the flagellate surface clearance rate was approximately 5 x 10(-7) cm(2) min(-1), but it declined hyperbolically with increasing bacterial density. Using the experimentally estimated process rates and integrating the component processes in a simple model reproduces the main features of the observed microbial population dynamics. Differences between observed and predicted population dynamics suggest, however, that other factors, e.g., antagonistic interactions between bacteria, are of importance in shaping marine snow microbial communities.     

Chitin Degradation Proteins Produced by the Marine Bacterium Vibrio harveyi Growing on Different Forms of Chitin

Relatively little is known about the number, diversity, and function of chitinases produced by bacteria, even though chitin is one of the most abundant polymers in nature. Because of the importance of chitin, especially in marine environments, we examined chitin-degrading proteins in the marine bacterium Vibrio harveyi. This bacterium had a higher growth rate and more chitinase activity when grown on (beta)-chitin (isolated from squid pen) than on (alpha)-chitin (isolated from snow crab), probably because of the more open structure of (beta)-chitin. When exposed to different types of chitin, V. harveyi excreted several chitin-degrading proteins into the culture media. Some chitinases were present with all of the tested chitins, while others were unique to a particular chitin. We cloned and identified six separate chitinase genes from V. harveyi. These chitinases appear to be unique based on DNA restriction patterns, immunological data, and enzyme activity. This marine bacterium and probably others appear to synthesize separate chitinases for efficient utilization of different forms of chitin and chitin by-products.     

Bacterial biofilms and surface fouling

Bacteria are attracted to surfaces. Their surface adhesion, with subsequent binary fission and exopolymer production, leads to the formation of biofilms. Such biofilms consist of bacterial cells in a matrix of their own exopolysaccharide glycocalyces. In addition to the bulk fluid and the surface, biofilms constitute a third physical phase. The close proximity of the bacterial cells in the biofilm matrices assists the formation of metabolically dependent consortia. The chemical and physical activities of these microbial communities produces a heterogeneous system at the colonised surface. Metabolites, produced at specific points on the surface, can lead to the development of effective anodes and cathodes at adjoining locations on the surface. In this way the fouling of a surface by bacterial biofilm development facilitates focal attack on that surface. This pit formation is characteristic of bacterial surface activities as diverse as dental decay and metal corrosion. In this review, we examine bacterial adhesion, biofilm formation and several instances of focal bacterial attack on colonised surfaces. However, pathogenic biofilms and the fouling of biological surfaces, with the exception of caries formation, is outside the scope of this paper.     

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.     

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.     

Monolayer adsorption of a "bald" mutant of the highly adhesive and hydrophobic bacterium Acinetobacter sp. strain Tol 5 to a hydrocarbon surface

The affinity of microbial cells for hydrophobic interfaces is important because it directly affects the efficiency of various bioprocesses, including green biotechnologies. The toluene-degrading bacterium Acinetobacter sp. strain Tol 5 has filamentous appendages and a hydrophobic cell surface, shows high adhesiveness to solid surfaces, and self-agglutinates. A "bald" mutant of this bacterium, strain T1, lacks the filamentous appendages and has decreased adhesiveness but retains a hydrophobic cell surface. We investigated the interaction between T1 cells and an organic solvent dispersed in an aqueous matrix. During a microbial-adhesion-to-hydrocarbon (MATH) test, which is frequently used to measure cell surface hydrophobicity, T1 cells adhered to hexadecane droplet surfaces in a monolayer, whereas wild-type cells aggregated on the droplet surfaces. The adsorbed T1 cells on the hexadecane surfaces hindered the coalescence of the droplets formed by vortexing, stabilizing the emulsion phase. Following the replacement of the aqueous phase with fresh pure water after the MATH test, a proportion of the T1 cells that had adsorbed to the hydrocarbon surface detached during further vortexing, suggesting a reversible adsorption of T1 cells. The final ratio of the adhering cells to the total cells in the detachment test coincided with that in the MATH test. The adhesion of T1 cells to the hydrocarbon surface conformed to the Langmuir adsorption isotherm, which describes reversible monolayer adsorption. Reversible monolayer adsorption should be useful for green technologies employing two-liquid-phase partitioning systems and for bioremediation because it allows effective reaction and transport of hydrophobic substrates at oil-water interfaces.