Effects of DNP on the cell surface properties of marine bacteria and its implication for adhesion to surfaces

The effect of 2, 4-dinitrophenol (DNP) on the extracelluar polysaccharides (EPS), cell surface charge, and the hydrophobicity of six marine bacterial cultures was studied, and its influence on attachment of these bacteria to glass and polystyrene was evaluated. DNP treatment did not influence cell surface charge and EPS production, but had a significant effect on hydrophobicity of both hydrophilic (p = 0.05) and hydrophobic (p = 0.01) cultures. Significant reduction in the attachment of all the six cultures to glass (p = 0.02) and polystyrene (p = 0.03) was observed after DNP treatment. Moreover, hydrophobicity but not the cell surface charge or EPS production influenced bacterial cell attachment to glass and polystyrene. From this study, it was evident that DNP treatment influenced bacterial cell surface hydrophobicity, which in turn, reduced bacterial adhesion to surfaces.     

Effects of DNP on the cell surface properties of marine bacteria and its implication for adhesion to surfaces

Abstract

The effect of 2, 4-dinitrophenol (DNP) on the extracelluar polysaccharides (EPS), cell surface charge, and the hydrophobicity of six marine bacterial cultures was studied, and its influence on attachment of these bacteria to glass and polystyrene was evaluated. DNP treatment did not influence cell surface charge and EPS production, but had a significant effect on hydrophobicity of both hydrophilic (p = 0.05) and hydrophobic (p = 0.01) cultures. Significant reduction in the attachment of all the six cultures to glass (p = 0.02) and polystyrene (p = 0.03) was observed after DNP treatment. Moreover, hydrophobicity but not the cell surface charge or EPS production influenced bacterial cell attachment to glass and polystyrene. From this study, it was evident that DNP treatment influenced bacterial cell surface hydrophobicity, which in turn, reduced bacterial adhesion to surfaces.     

Dynamics in bacterial surface properties of a natural bacterial community in the coastal North Sea during a spring phytoplankton bloom

The hydrophilic and hydrophobic properties of single cells of natural bacterioplankton communities were determined using a recently developed staining method combined with confocal laser scanning microscopy and advanced image analysis. On an average, about 50% of the bacterial cell area was covered by hydrophobic and only 16% by hydrophilic properties, while about 72% was covered by the genome. However, the size of these properties was independent of the bacterial cell size. Bacterial hydrophobicity was positively correlated with ambient NH(4)(+) concentrations and negatively correlated with overall bacterial abundance. The expression of hydrophilicity was more dynamic. Over the spring phytoplankton bloom, the bacterioplankton ratio(phil/phob) repeatedly reached highest values shortly before peaks in bacterioplankton abundance were observed, indicating a direct and fast response of bacterial surface properties, especially hydrophilicity, to changing environmental conditions. Compared to bacterial strains, recently studied with the same method, cells of marine bacterioplankton communities are much smaller and less frequently covered by hydrophobic or hydrophilic properties. While the percentage area covered by the genome is essentially the same, the percentage area covered by hydrophobic or hydrophilic properties is much smaller.     

Sequential hydrophobic partitioning of cells of Pseudomonas aeruginosa gives rise to variants of increasing cell-surface hydrophobicity

The partitioning of bacterial cells in a dual aqueous-solvent phase system leads to separation into 'hydrophilic' and hydrophobic functions. Sequential multistep partitioning, accompanied by successive enrichment, gives rise to several cycles of hydrophobic and hydrophilic cell populations which possess different cell-surface hydrophobicity characteristics. Characterization of the cell-surface hydrophobicity by several methods (salting-out aggregation test, bacterial adherence to hydrocarbon, polystyrene binding and hydrophobic interaction chromatography) was carried out. The cell-surface hydrophobicity varied in the order: hydrophilic fraction < parental strain < first cycle hydrophobic variant < second cycle hydrophobic variant < third cycle hydrophobic variant. Electron microscopy showed that the most hydrophobic variant was densely covered by hydrophobic structures - fimbriae - whereas the parental strain was covered by a mixture of surface structures. The hydrophilic variant was covered by an amorphous exopolymeric substance, which is a polysaccharide, shown by its reaction with Alcian blue.     

Effect of Substrate and Cell Surface Hydrophobicity on Phosphate Utilization in Bacteria

We measured the rates of utilization of hydrophobic and hydrophilic phosphate compounds in gram-negative bacteria with different surface hydrophobicities, isolated from wetland habitats. Three hydrophobic and two hydrophilic bacterial species were selected for study by measuring cell adherence to hydrocarbons. The bacteria were grown under phosphorus-limited conditions with P(infi), hydrophilic (beta)-glycerophosphate, or hydrophobic phosphatidic acid as the phosphate source. Hydrophilic bacteria grew most rapidly on P(infi), followed by (beta)-glycerophosphate. Phosphatidic acid did not support growth or did so at a much later time (40 h) than did the other phosphate treatments. Although all hydrophobic species grew well on these substrates, the rate of growth of two Acinetobacter baumannii isolates on phosphatidic acid exceeded the rate of growth on phosphate or (beta)-glycerophosphate. A membrane phospholipid and lipopolysaccharide were used as a source of phosphorus by hydrophobic species, whereas hydrophilic species could not use the membrane phospholipids and used lipopolysaccharide to a lesser extent. Besides hydrophobic interaction between cells and substrate, phosphatase activity, which was cell bound in hydrophilic species but 30 to 50% unbound in hydrophobic species, affected cell growth. Dialyzed culture supernatant containing phosphatase from hydrophobic species increased the phosphate availability to hydrophilic species. Additionally, cellular extracts from a hydrophilic species, when added to hydrophilic cells, permitted growth on hydrophobic phosphate sources. Naturally occurring amphiphilic humic acids affected the utilization of P(infi) and (beta)-glycerophosphate in bacteria with hydrophilic surfaces but did not affect hydrophobic bacteria. Our results indicate that hydrophobic phosphate sources can be used by bacteria isolated from aquatic environments as the sole phosphorus source for growth. This utilization, in part, appears to be related to cell surface hydrophobicity and extracellular enzyme production.     

Determination of bacterial cell surface hydrophobicity of single cells in cultures and in wastewater in situ

Bacterial cell surface hydrophobicity is one of the most important factors that influence bacterial adhesion. A new method, microsphere adhesion to cells, for measuring bacterial cell surface hydrophobicity was developed. Microsphere adhesion to cells is based on microscopic enumeration of hydrophobic, fluorescent microspheres attaching to the bacterial surface. Cell surface hydrophobicity estimated by microsphere adhesion to cells correlates well with adhesion of bacteria to hydrocarbons or hydrophobic interaction chromatography for a set of hydrophilic and hydrophobic bacteria (linear correlation coefficients, R², were 0.845 and 0.981 respectively). We also used microsphere adhesion to cells to investigate the in situ properties of individual free-living bacteria directly in activated sludge. Results showed that the majority of the bacteria were hydrophilic, indicating the importance of cell surface hydrophobicity for bacterial adhesion in sludge, and for the overall success of the wastewater treatment process.     

Release of the extracellular matrix from conidia ofBlumeria graminis in relation to germination

The release of extracellular matrix (ECM) and the emergence of germ tubes from conidia ofBlumeria graminis were studied by light microscopy and micromanipulation. More prompt and frequent ECM release was confirmed on an artificial hydrophobic substratum than on an artificial hydrophilic substratum. Conidia initially incubated on the hydrophilic substratum were transferred by micromanipulation to either the hydrophobic or the hydrophilic substrata. Immediately after transfer onto the hydrophobic substratum, 75% of conidia released ECM, whereas only 16% did so upon transfer to the hydrophilic substratum. Conidia transferred onto the hydrophobic substratum produced a primary germ tube (PGT) more promptly and frequently than those transferred to the hydrophilic substratum. Thus, conidia recognize and respond to substratum hydrophobicity perhaps immediately after contact. When inoculated onto either isolated barley cuticle or the hydrophobic artificial substratum, 2/3 of the conidia produced a PGT from their polar regions. By contrast, on the hydrophilic substratum 2/3 of conidia did so from the side region. These results show that substratum hydrophobicity affects the location of PGT emergence from conidia. Furthermore, the study indicates that very rapid recognition of surface hydrophobicity by conidia promotes ECM release and this in turn may influence the location of PGT emergence.     

Extracellular polymeric substances responsible for bacterial adhesion onto solid surface

The influence of extracellular polymeric substances (EPS) on bacterial cell adhesion onto solid surfaces was investigated using 27 heterotrophic bacterial strains isolated from a wastewater treatment reactor. Cell adhesion onto glass beads was carried out by the packed-bed method and the results were discussed in terms of the amount of each EPS component produced and cell surface characteristics such as zeta potential and hydrophobicity. Protein and polysaccharides accounted for 75-89% of the EPS composition, indicating that they are the major EPS components. Among the polysaccharides, the amounts of hexose, hexosamine and ketose were relatively high in EPS-rich strains. For EPS-poor strains, the efficiency of cell adhesion onto glass beads increased as the absolute values of zeta potential decreased, suggesting that electrostatic interaction suppresses cell adhesion efficiency. On the other hand, the amounts of hexose and pentose exhibited good correlations with cell adhesiveness for EPS-rich strains, indicating that polymeric interaction due to the EPS covering on the cell surface promoted cell adhesion. It was concluded that, if the EPS amount is relatively small, cell adhesion onto solid surfaces is inhibited by electrostatic interaction, and if it is relatively large, cell adhesion is enhanced by polymeric interaction.     

The partitioning of cholesterol oxidase in Triton X-114-based aqueous two-phase systems.

Cholesterol oxidase from various bacterial sources (membrane-bound and extracellular) was studied in Triton X-114R solutions above the cloud point. The influence of temperature, salt, enzyme concentration and source, and pH on phase equilibrium and enzyme partitioning was investigated in this detergent-based aqueous two-phase system. The method combines remarkable recovery (over 70% and 90% in the detergent-rich phase for the extracellular and membrane-bound forms, respectively) and 10 to 20-fold concentration of the enzyme in just one purification step. The results from cholesterol oxidase are compared with other proteins, both hydrophobic and hydrophilic. The system shows considerable promise for selectively partitioning proteins based on their surface hydrophobicity.     

Hydrophobic features of EPS extracted from anaerobic granular sludge: an investigation based on DAX-8 resin fractionation and size exclusion chromatography

The hydrophobic fractionation of extracellular polymeric substances (EPS) extracted from anaerobic granular sludge was performed on the DAX-8 resin (two elution pH conditions, i.e., pH 2 and pH 5 were tested). The impact of seven different EPS extraction methods on EPS hydrophobicity features was assessed. The results showed that the extraction methods and bulk solution pH influenced dramatically the biochemical composition of the EPS, and in turn, the hydrophobicity determined. Besides, EPS extracting reagents i.e., formaldehyde, ethanol, sodium dodecyl sulfate (SDS), and Tween 20 not only introduced extra carbon content in the total organic carbon (TOC) measurement but also interacted with the DAX-8 resin. By comparing the apparent molecular weight (aMW) distribution of untreated and pH-adjusted EPS samples, more complete EPS aMW information was preserved at pH 5. Thus, elution at pH 5 was preferred in this study for the qualitative analysis of EPS hydrophobic features. The hydrophobic fraction of EPS retained by the resin at pH 5 was ascribed to a wide aMW range, ranging from >440 to 0.3 kDa. Within this range, EPS molecules ranging from 175 to 31 kDa were mostly retained by the DAX-8 resin, which indicates that these EPS molecules are highly hydrophobic.

     

Function of quorum sensing and cell signaling in the formation of aerobic granular sludge

Aerobic granular sludge (AGS) has recently attracted attention because of its excellent settling ability and treatment efficiency compared with traditional activated sludge. This review provides recent advances on the formation process of AGS and mainly analyzes the function of quorum sensing (QS) and cell signaling during AGS formation. QS and cell signaling play important roles in the formation of AGS. QS can accelerate the synthesis of extracellular polymeric substance (EPS) and increase microbial adhesion to the surface of AGS. Cell signaling can also promote the secretion of EPS and influence biofilm formation. Cyclic diguanylate (c-di-GMP), as a second messenger, acts an important role in granulation. C-di-GMP causes bacteria to adhere to each other and form a biofilm. Adding Ca²⁺ benefits bacterial growth and promotes c-di-GMP secretion. Adding Mn²⁺ reduces c-di-GMP content and triggers AGS disintegration. Finally, the review discusses the possible trends of AGS: QS and cell signaling can lay a theoretical foundation for the formation mechanism of AGS and would be of practical significance for its application in the future.     

Efficient treatment of garbage slurry in methanogenic bioreactor packed by fibrous sponge with high porosity

Adding a supporting material to a methanogenic bioreactor treating garbage slurry can improve efficiency of methane production. However, little is known on how characteristics (e.g., porosity and hydrophobicity) of the supporting material affect the bioreactor degrading garbage slurry. We describe the reactor performances and microbial communities in bioreactors containing hydrophilic or hydrophobic sheets, or fibrous hydrophilic or hydrophobic sponges. The porosity affected the efficiency of methane production and solid waste removal more than the hydrophilic or hydrophobic nature of the supporting material. When the terminal restriction fragment length polymorphism technique was used at a lower organic loading rate (OLR), microbial diversities in the suspended fraction were retained on the hydrophobic, but not the hydrophilic, sheets. Moreover, real-time quantitative polymerase chain reaction (PCR) performed at a higher OLR revealed that the excellent performance of reactors containing fibrous sponges with high porosity (98%) was supported by a clear increase in the numbers of methanogens on these sponges, resulting in larger total numbers of methanogens in the reactors. In addition, the bacterial communities in fractions retained on both the hydrophobic and hydrophilic fibrous sponges differed from those in the suspended fraction, thus increasing bacterial diversity in the reactor. Thus, higher porosity of the supporting material improves the bioreactor performance by increasing the amount of methanogens and bacterial diversity; surface hydrophobicity contributes to maintaining the suspended microbial community.     

Contact Angle Measurement and Cell Hydrophobicity of Granular Sludge from Upflow Anaerobic Sludge Bed Reactors

The contact angle, which is generally used to evaluate the hydrophobicities of pure bacterial strains and solid surfaces, was used to study mixed cell cultures of bacteria involved in anaerobic digestion. Previously published data and data from this study showed that most acidogens are hydrophilic (contact angle, <45(deg)) but most of the acetogens and methanogens isolated from granular sludge are hydrophobic (contact angle, >45(deg)). The hydrophobicities of mixtures of hydrophilic and hydrophobic cells were found to be linearly correlated with the cell mixing ratio. The hydrophobicities of cells present in effluents from upflow anaerobic sludge bed reactors which were treating different types of substrates were different depending on the reactor conditions. When the reactor liquid had a high surface tension, cells sloughing off from sludge granules, as well as cells present on the outer surfaces of the granules, were hydrophobic. Short-term batch enrichment cultures revealed that proteins selected for highly hydrophilic cells. Long-term in-reactor enrichment cultures revealed that sugars selected for hydrophilic acidogens on the surfaces of the granules, while fatty acids tended to enrich for hydrophobic methanogens. When linear alkylbenzenesulfonate was added, the cells on the surfaces of granules became more hydrophilic. Control tests performed with pure cultures revealed that there was no change in the surface properties due to linear alkylbenzenesulfonate; hence, the changes in the wash-out observed probably reflect changes in the species composition of the microbial association. A surface layer with moderate hydrophobicity, a middle layer with extremely high hydrophobicity, and a core with high hydrophobicity could be distinguished in the grey granules which we studied.     

Effects of Hydrophobic and Electrostatic Cell Surface Properties of Bacteria on Feeding Rates of Heterotrophic Nanoflagellates

The influence of cell surface hydrophobicity and electrostatic charge of bacteria on grazing rates of three common species of interception-feeding nanoflagellates was examined. The hydrophobicity of bacteria isolated from freshwater plankton was assessed by using two different methods (bacterial adhesion to hydrocarbon and hydrophobic interaction chromatography). The electrostatic charge of the cell surface (measured as zeta potential) was analyzed by microelectrophoresis. Bacterial ingestion rates were determined by enumerating bacteria in food vacuoles by immunofluorescence labelling via strain-specific antibodies. Feeding rates varied about twofold for each flagellate species but showed no significant dependence on prey hydrophobicity or surface charge. Further evidence was provided by an experiment involving flagellate grazing on complex bacterial communities in a two-stage continuous culture system. The hydrophobicity values of bacteria that survived protozoan grazing were variable, but the bacteria did not tend to become more hydrophilic. We concluded that variability in bacterial cell hydrophobicity and variability in surface charge do not severely affect uptake rates of suspended bacteria or food selection by interception-feeding flagellates.     

Effects of hydrophobic and electrostatic cell surface properties of bacteria on feeding rates of heterotrophic nanoflagellates

The influence of cell surface hydrophobicity and electrostatic charge of bacteria on grazing rates of three common species of interception-feeding nanoflagellates was examined. The hydrophobicity of bacteria isolated from freshwater plankton was assessed by using two different methods (bacterial adhesion to hydrocarbon and hydrophobic interaction chromatography). The electrostatic charge of the cell surface (measured as zeta potential) was analyzed by microelectrophoresis. Bacterial ingestion rates were determined by enumerating bacteria in food vacuoles by immunofluorescence labelling via strain-specific antibodies. Feeding rates varied about twofold for each flagellate species but showed no significant dependence on prey hydrophobicity or surface charge. Further evidence was provided by an experiment involving flagellate grazing on complex bacterial communities in a two-stage continuous culture system. The hydrophobicity values of bacteria that survived protozoan grazing were variable, but the bacteria did not tend to become more hydrophilic. We concluded that variability in bacterial cell hydrophobicity and variability in surface charge do not severely affect uptake rates of suspended bacteria or food selection by interception-feeding flagellates.     

Extraction of Structural Extracellular Polymeric Substances from Aerobic Granular Sludge

To evaluate and develop methodologies for the extraction of gel-forming extracellular polymeric substances (EPS), EPS from aerobic granular sludge (AGS) was extracted using six different methods (centrifugation, sonication, ethylenediaminetetraacetic acid (EDTA), formamide with sodium hydroxide (NaOH), formaldehyde with NaOH and sodium carbonate (Na₂CO₃) with heat and constant mixing). AGS was collected from a pilot wastewater treatment reactor. The ionic gel-forming property of the extracted EPS of the six different extraction methods was tested with calcium ions (Ca²⁺). From the six extraction methods used, only the Na₂CO₃ extraction could solubilize the hydrogel matrix of AGS. The alginate-like extracellular polymers (ALE) recovered with this method formed ionic gel beads with Ca²⁺. The Ca²⁺-ALE beads were stable in EDTA, formamide with NaOH and formaldehyde with NaOH, indicating that ALE are one part of the structural polymers in EPS. It is recommended to use an extraction method that combines physical and chemical treatment to solubilize AGS and extract structural EPS.     

Role of hydrophobicity in bacterial adherence to carbon nanostructures and biofilm formation

The role of cell and surface hydrophobicity in the adherence of the waterborne bacterium Mycobacterium smegmatis to nanostructures and biofilm formation was investigated. Carbon nanostructures (CNs) were synthesized using a flame reactor and deposited on stainless steel grids and foils, and on silicon wafers that had different initial surface hydrophobicities. Surface hydrophobicity was measured as the contact angle of water droplets. The surfaces were incubated in suspensions of isogenic hydrophobic and hydrophilic strains of M. smegmatis and temporal measurements of the numbers of adherent cells were made. The hydrophobic, rough mutant of M. smegmatis adhered more readily and formed denser biofilms on all surfaces compared to its hydrophilic, smooth parent. Biofilm formation led to alterations in the hydrophobicity of the substratum surfaces, demonstrating that bacterial cells attached to CNs are capable of modifying the surface characteristics.     

IR study of self-assembly of capsular exopolymers from Pseudomonas sp. NCIMB 2021 on hydrophilic and hydrophobic surfaces

Capsular exopolymers (EPS) of the bacterium Pseudomonas sp. NCIMB 2021 are allowed to self-assemble on hydrophilic and hydrophobic gold surfaces. Tapping mode atomic force microscopy confirms the differences in the surface topography between EPS adsorbed on both surfaces. Fourier-transform IR spectroscopy indicates that the EPS surface coverage is much greater on the hydrophobic surface. Furthermore, an increased contribution is observed from hydrophobic (i.e., methyl and tyrosyl residues) and electrostatic (i.e., carboxylate residues) groups at the hydrophobic surface, but there is relatively less neutral polymer compared to the hydrophilic surface. The behavior of this EPS is in agreement with the behavior of cells of Pseudomonas sp. NCIMB 2021 at hydrophilic and hydrophobic surfaces.     

Cell surface hydrophobicity and charge of Staphylococcus aureus and coagulase-negative staphylococci from bovine mastitis

The effects of seven growth media on cell surface hydrophobicity of a collection of Staphylococcus aureus and coagulase-negative staphylococci isolated from bovine mastitis were compared in the salt-aggregation test. Thirty-three per cent of Staph. aureus strains showed extremely high cell surface hydrophobicity (auto-aggregated) and 28% were moderately hydrophobic while 26% were hydrophilic after growth on horse blood agar at 37 degrees C for 18 h. There were great variations in the proportion and degree of the hydrophobicity depending on the medium used. Cultivations on/in capsule-inducing media caused a shift from a high to a low degree of hydrophobicity, although a microscopically detectable capsule or slime layer was seen in only one strain. This strain and encapsulated reference strains had a hydrophilic cell surface and migrated faster in free zone electrophoresis than cells of unencapsulated strains. Cells of strains grown on staphylococcus medium 110 agar migrated faster than those grown on horse blood agar regardless of their capsule production. Coagulase-negative staphylococci showed uniformly hydrophilic cell surface after cultivation on horse blood agar, but not when grown in tryptic soy broth or proteose peptone broth. It was concluded that most of the Staph. aureus strains from bovine mastitis under a variety of growth conditions in stationary phase culture constantly expressed hydrophobic cell surface.     

Cell surface hydrophobicity and charge of Staphylococcus aureus and coagulase-negative staphylococci from bovine mastitis

The effects of seven growth media on cell surface hydrophobicity of a collection of Staphylococcus aureus and coagulase-negative staphylococci isolated from bovine mastitis were compared in the salt-aggregation test. Thirty-three per cent of Staph. aureus strains showed extremely high cell surface hydrophobicity (auto-aggregated) and 28% were moderately hydrophobic while 26% were hydrophilic after growth on horse blood agar at 37°C for 18 h. There were great variations in the proportion and degree of the hydrophobicity depending on the medium used. Cultivations on/in capsule-inducing media caused a shift from a high to a low degree of hydrophobicity, although a microscopically detectable capsule or slime layer was seen in only one strain. This strain and encapsulated reference strains had a hydrophilic cell surface and migrated faster in free zone electrophoresis than cells of unencapsulated strains. Cells of strains grown on staphylococcus medium 110 agar migrated faster than those grown on horse blood agar regardless of their capsule production. Coagulase-negative staphylococci showed uniformly hydrophilic cell surface after cultivation on horse blood agar, but not when grown in tryptic soy broth or proteose peptone broth. It was concluded that most of the Staph. aureus strains from bovine mastitis under a variety of growth conditions in stationary phase culture constantly expressed hydrophobic cell surface.