Adhesion of anaerobic microorganisms to solid surfaces and the effect of sequential attachment on adhesion characteristics

The attachment of three anaerobic microorganisms, Desulfomonile tiedjei, Syntrophomonas wolfei, and Desulfovibrio sp. strain G11, was investigated to determine if the presence of one species could influence the adhesion of another species to glass surfaces. The results indicated that the numbers and distribution of attached cells of one species could be influenced considerably by the presence of another species and the order in which the test species were exposed to the surface. D. tiedjei was found to detach readily from surfaces when it was not the primary colonizer. The attachment of Desulfovibrio G11 as the primary colonizer appeared to be stabilized by exposure to another test species. Under certain experimental conditions the test organisms formed close associations with each other on the surfaces. These findings demonstrate that the characteristics of anaerobic community biofilms can be determined by both the adhesion characteristics of the individual species and the interactions among those microorganisms.     

Adhesion of Biodegradative Anaerobic Bacteria to Solid Surfaces

In order to exploit the ability of anaerobic bacteria to degrade certain contaminants for bioremediation of polluted subsurface environments, we need to understand the mechanisms by which such bacteria partition between aqueous and solid phases, as well as the environmental conditions that influence partitioning. We studied four strictly anaerobic bacteria, Desulfomonile tiedjei, Syntrophomonas wolfei, Syntrophobacter wolinii, and Desulfovibrio sp. strain G11, which theoretically together can constitute a tetrachloroethylene- and trichloroethylene-dechlorinating consortium. Adhesion of these organisms was evaluated by microscopic determination of the numbers of cells that attached to glass coverslips exposed to cell suspensions under anaerobic conditions. We studied the effects of the growth phase of the organisms on adhesion, as well as the influence of electrostatic and hydrophobic properties of the substratum. Results indicate that S. wolfei adheres in considerably higher numbers to glass surfaces than the other three organisms. Starvation greatly decreases adhesion of S. wolfei and Desulfovibrio sp. strain G11 but seems to have less of an effect on the adhesion of the other bacteria. The presence of Fe³⁺ on the substratum, which would be electropositive, significantly increased the adhesion of S. wolfei, whereas the presence of silicon hydrophobic groups decreased the numbers of attached cells of all species. Measurements of transport of cells through hydrophobic-interaction and electrostatic-interaction columns indicated that all four species had negatively charged cell surfaces and that D. tiedjei and Desulfovibrio sp. strain G11 possessed some hydrophobic cell surface properties. These findings are an early step toward understanding the dynamic attachment of anaerobic bacteria in anoxic environments.     

Syntrophic association of a butyrate-degrading bacterium and methanosarcina enriched from bovine rumen fluid.

An anaerobic butyrate-degrading bacterium, morphologically similar to Syntrophomonas wolfei, was isolated in coculture with Desulfovibrio strain G11 from an enrichment of bovine rumen fluid. A Methanosarcina species was the major H2-using organism in the enrichment. The results are discussed in relationship to the absence of Methanospirillum hungatei, the H2-using methanogen usually found in association with S. wolfei, and the finding of Methanosarcina rather than Methanobrevibacter ruminantium as the major H2-using bacterium in the enrichments. The finding of butyrate degraders in the rumen suggests that, if the retention time of the rumen contents becomes more prolonged, butyrate and longer-chained fatty acids might be significantly degraded.     

Adhesion of anaerobic microorganisms to solid surfaces and the effect of sequential attachment on adhesion characteristics

The attachment of three anaerobic microorganisms, Desulfomonile tiedjei, Syntrophomonas wolfei, and Desulfovibrio sp. strain G11, was investigated to determine if the presence of one species could influence the adhesion of another species to glass surfaces. The results indicated that the numbers and distribution of attached cells of one species could be influenced considerably by the presence of another species and the order in which the test species were exposed to the surface. D. tiedjei was found to detach readily from surfaces when it was not the primary colonizer. The attachment of Desulfovibrio G11 as the primary colonizer appeared to be stabilized by exposure to another test species. Under certain experimental conditions the test organisms formed close associations with each other on the surfaces. These findings demonstrate that the characteristics of anaerobic community biofilms can be determined by both the adhesion characteristics of the individual species and the interactions among those microorganisms.     

Influence of Substratum Characteristics on the Attachment of a Marine Pseudomonad to Solid Surfaces

The attachment of a marine Pseudomonas sp. to a variety of surfaces was investigated, and the number of bacteria which became attached was related to the surface charge and degree of hydrophobicity of the substratum. Large numbers of bacteria attached to hydrophobic plastics with little or no surface charge [Teflon, polyethylene, polystyrene, poly(ethylene terephthalate)]; moderate numbers attached to hydrophilic metals with a positive (platinum) or neutral (germanium) surface charge; and very few attached to hydrophilic, negatively charged substrata (glass, mica, oxidized plastics). The results suggest that both electrostatic and hydrophobic interactions are involved in bacterial attachment.     

The effect of a range of biological polymers and synthetic surfactants on the adhesion of a marine Pseudomonas sp. strain NCMB 2021 to hydrophilic and hydrophobic surfaces

Abstract The effect of a range of biological polymers and synthetic surfactants on the adhesion of a marine Pseudomonas sp. strain NCMB2021 to hydrophilic glass and hydrophobic polystyrene has been investigated. Brij 56 (polyethylene oxide (10) cetyl ether) was the only compound that had a significant effect, almost totally inhibiting the adhesion of Pseudomonas sp. NCMB2021 to hydrophobic polystyrene, but having little or no effect on hydrophilic glass. The surfactant was demonstrated to be effective both when present in the bacterial suspension at low concentrations (approx. 5 ppm), and when pre-adsorbed onto the substratum. Brij 56 was shown to prevent the adhesion of a range of marine and fresh-water bacteria to polystyrene.
It is proposed that on a hydrophobic substratum Brij 56 is adsorbed via its hydrophobe in such a way that the polyethylene glycol chains are pointing outwards into the aqueous phase giving a surface with a high density of uncharged, highly hydrated hydrophilic chains, forming a steric barrier which inhibits the adhesion of bacteria.     

Syntrophic growth on formate: a new microbial niche in anoxic environments

Anaerobic syntrophic associations of fermentative bacteria and methanogenic archaea operate at the thermodynamic limits of life. The interspecies transfer of electrons from formate or hydrogen as a substrate for the methanogens is key. Contrary requirements of syntrophs and methanogens for growth-sustaining product and substrate concentrations keep the formate and hydrogen concentrations low and within a narrow range. Since formate is a direct substrate for methanogens, a niche for microorganisms that grow by the conversion of formate to hydrogen plus bicarbonate--or vice versa--may seem unlikely. Here we report experimental evidence for growth on formate by syntrophic communities of (i) Moorella sp. strain AMP in coculture with a thermophilic hydrogen-consuming Methanothermobacter species and of (ii) Desulfovibrio sp. strain G11 in coculture with a mesophilic hydrogen consumer, Methanobrevibacter arboriphilus AZ. In pure culture, neither Moorella sp. strain AMP, nor Desulfovibrio sp. strain G11, nor the methanogens grow on formate alone. These results imply the existence of a previously unrecognized microbial niche in anoxic environments.     

Alterations in Adhesion, Transport, and Membrane Characteristics in an Adhesion-Deficient Pseudomonad

A stable adhesion-deficient mutant of Burkholderia cepacia G4, a soil pseudomonad, was selected in a sand column assay. This mutant (ENV435) was compared to the wild-type strain by examining the adhesion of the organisms to silica sand and their transport through two aquifer sediments that differed in their sand, silt, and clay contents. We compared the longitudinal transport of the wild type and the adhesion mutant to the transport of a conservative chloride tracer in 25-cm-long glass columns. The transport of the wild-type strain was severely retarded compared to the transport of the conservative tracer in a variety of aquifer sediments, while the adhesion mutant and the conservative tracer traveled at similar rates. An intact sediment core study produced similar results; ENV435 was transported at a faster rate and in much greater numbers than G4. The results of hydrophobic interaction chromatography revealed that G4 was significantly more hydrophobic than ENV435, and polyacrylamide gel electrophoresis revealed significant differences in the lipopolysaccharide O-antigens of the adhesion mutant and the wild type. Differences in this cell surface polymer may explain the decreased adhesion of strain ENV435.     

A microautoradiographic study of the activity of attached and free-living bacteria

Microautoradiography, combined with epifluorescent microscopy, was used to evaluate the uptake of tritiated amino acids by a marine Pseudomonas sp. A comparison was made between the activity of bacteria free-living in the medium and bacteria which were attached to glass, polyethylene or polystyrene substrata. The proportion of active bacteria was lower for free-living cells (53–82%) and those attached to polystyrene (53–76%) than for those attached to glass (77–99%) or polyethylene (73–96%). For bacteria attached to glass, assimilated labelled substrate was retained within the cell over 3 h, whereas with polyethylene, labelled material was released from the cells and adsorbed on the surrounding substratum. Hence the physiological activity of attached bacteria depended on the chemical composition of the substratum.     

Amino Acid Assimilation and Electron Transport System Activity in Attached and Free-Living Marine Bacteria

Amino acid assimilation and electron transport system activity of a marine Pseudomonas sp. was evaluated to determine whether the activity of bacteria attached to solid surfaces differed from that of free-living bacteria or bacteria which had been attached but subsequently desorbed from the substratum (detached bacteria). Bacteria were allowed to attach to glass and to a range of plastic surfaces (Thermanox, polyvinylidene fluoride, polyethylene, polytetrafluoroethylene). Microautoradiography and staining with a tetrazolium salt to demonstrate electron transport system activity were used to compare the activity of these organisms with that of free-living or detached cells. The water-wettability of the surfaces was evaluated by measuring the advancing contact angle (θ_A) of water on each surface, to determine whether there was a relationship between activity and substratum hydrophilicity. There was an increase in the proportion of leucine-assimilating attached bacteria and in the proportion of attached cells demonstrating electron transport system activity with an increase in substratum θ_A, but the relationship between activity of attached and free-living cells depended on the substratum. Activity appeared to promote firm attachment, and detached bacteria assimilated fewer amino acids than did attached cells. There was no general effect of surfaces on attached bacterial activity, and attached cells may be more, or less, active than free-living cells, depending on the amino acid, its concentration, and substratum properties.     

Analysis of bacterial detachment from substratum surfaces by the passage of air-liquid interfaces

A theoretical analysis of the detachment of bacteria adhering to substratum surfaces upon the passage of an air-liquid interface is given, together with experimental results for bacterial detachment in the absence and presence of a conditioning film on different substratum surfaces. Bacteria (Streptococcus sobrinus HG1025, Streptococcus oralis J22, Actinomyces naeslundii T14V-J1, Bacteroides fragilis 793E, and Pseudomonas aeruginosa 974K) were first allowed to adhere to hydrophilic glass and hydrophobic dimethyldichlorosilane (DDS)-coated glass in a parallel-plate flow chamber until a density of 4 x 10(6) cells cm(-2) was reached. For S. sobrinus HG1025, S. oralis J22, and A. naeslundii T14V-J1, the conditioning film consisted of adsorbed salivary components, while for B. fragilis 793E and P. aeruginosa 974K, the film consisted of adsorbed human plasma components. Subsequently, air bubbles were passed through the flow chamber and the bacterial detachment percentages were measured. For some experimental conditions, like with P. aeruginosa 974K adhering to DDS-coated glass and an air bubble moving at high velocity (i.e., 13.6 mm s(-1)), no bacteria detached upon passage of an air-liquid interface, while for others, detachment percentages between 80 and 90% were observed. The detachment percentage increased when the velocity of the passing air bubble decreased, regardless of the bacterial strain and substratum surface hydrophobicity involved. However, the variation in percentages of detachment by a passing air bubble depended greatly upon the strain and substratum surface involved. At low air bubble velocities the hydrophobicity of the substratum had no influence on the detachment, but at high air bubble velocities all bacterial strains were more efficiently detached from hydrophilic glass substrata. Furthermore, the presence of a conditioning film could either inhibit or stimulate detachment. The shape of the bacterial cell played a major role in detachment at high air bubble velocities, and spherical strains (i.e., streptococci) detached more efficiently than rod-shaped organisms. The present results demonstrate that methodologies to study bacterial adhesion which include contact with a moving air-liquid interface (i.e., rinsing and dipping) yield detachment of an unpredictable number of adhering microorganisms. Hence, results of studies based on such methodologies should be referred as "bacterial retention" rather than "bacterial adhesion".     

Bacterial adhesion to hydroxyl- and methyl-terminated alkanethiol self-assembled monolayers.

The attachment of bacteria to solid surfaces is influenced by substratum chemistry, but to determine the mechanistic basis of this relationship, homogeneous, well-defined substrata are required. Self-assembled monolayers (SAMs) were constructed from alkanethiols to produce a range of substrata with different exposed functional groups, i.e., methyl and hydroxyl groups and a series of mixtures of the two. Percentages of hydroxyl groups in the SAMs and substratum wettability were measured by X-ray photoelectron spectroscopy and contact angles of water and hexadecane, respectively. SAMs exhibited various substratum compositions and wettabilities, ranging from hydrophilic, hydroxyl-terminated monolayers to hydrophobic, methyl-terminated monolayers. The kinetics of attachment of an estuarine bacterium to these surfaces in a laminar flow chamber were measured over periods of 120 min. The initial rate of net adhesion, the number of cells attached after 120 min, the percentage of attached cells that adsorbed or desorbed between successive measurements, and the residence times of attached cells were quantified by phase-contrast microscopy and digital image processing. The greatest numbers of attached cells occurred on hydrophobic surfaces, because (i) the initial rates of adhesion and the mean numbers of cells that attached after 120 min increased with the methyl content of the SAM and the contact angle of water and (ii) the percentage of cells that desorbed between successive measurements (ca. 2 min) decreased with increasing substratum hydrophobicity. With all surfaces, 60 to 80% of the cells that desorbed during the 120-min exposure period had residence times of less than 10 min, suggesting that establishment of firm adhesion occurred quickly on all of the test surfaces.     

一株硫酸盐还原菌的分离鉴定和系统发育分析

从处理硫酸盐废水的厌氧折流板反应器中分离得到一株硫酸盐还原菌D11, 该菌株革兰氏反应阴性, 无芽孢, 菌体杆状稍有弯曲, 宽度在0.6 μm~0.8 μm, 长度在1.8 μm~3.3 μm之间, 有极生单鞭毛, 能运动, 接触酶阳性, 氧化酶阴性。菌株生长的pH范围介于6.0~8.0之间, 最适pH为7.0, 生长温度范围为25°C~37°C, 最适温度为30°C。能够以葡萄糖、蔗糖、乙酸、乳酸、乙醇和丙二醇为唯一碳源生长, 不能利用丙三醇、丁醇、琥珀酸和苹果酸。菌株DNA的G+C含量为62.7 mo     

Staphylococcus aureus accessory regulators: expression within biofilms and effect on adhesion.

Many of the genes encoding the virulence factors for Staphylococcus aureus are controlled by the accessory gene regulator (agr) and staphylococcal accessory regulator (sar). This regulation may be affected by the environment in which the organisms are grown. In the majority of ecosystems, bacteria grow attached to surfaces and form biofilms. We used S. aureus strains containing mutations inactivating agr and sar to determine whether the presence of these genes influences the attachment of the bacterium to a surface. We also used strains harbouring reporter constructs of the agr and sar operons to determine their expression in biofilms. The attachment study results showed that the sarA mutant strain adhered better to glass than did the agrA mutant or the wild type. There was an increased adherence to fibronectin-coated glass for all three strains compared to glass. Thus, these adhesion studies demonstrate that agr and sar have pleiotrophic effects on the surface expression of molecules responsible for binding to different substrata. In the biofilms higher numbers of bacteria and the greatest expression were observed at the base, but there were no observable differences between the reporter constructs. Expression of the agr and sar reporter fusions was significantly higher in the deepest layers of the biofilms where the greatest numbers of bacteria were also observed, perhaps as one might expect for genes that are regulated in a cell density dependent fashion.     

Quantification of syntrophic fatty acid-beta-oxidizing bacteria in a mesophilic biogas reactor by oligonucleotide probe hybridization.

Small-subunit rRNA sequences were obtained for two saturated fatty acid-beta-oxidizing syntrophic bacteria, Syntrophomonas sapovorans and Syntrophomonas wolfei LYB, and sequence analysis confirmed their classification as members of the family Syntrophomonadaceae. S. wolfei LYB was closely related to S. wolfei subsp. wolfei, but S. sapovorans did not cluster with the other members of the genus Syntrophomonas. Five oligonucleotide probes targeting the small-subunit rRNA of different groups within the family Syntrophomonadaceae, which contains all currently known saturated fatty acid-beta-oxidizing syntrophic bacteria, were developed and characterized. The probes were designed to be specific at the family, genus, and species levels and were characterized by temperature-of-dissociation and specificity studies. To demonstrate the usefulness of the probes for the detection and quantification of saturated fatty acid-beta-oxidizing syntrophic bacteria in methanogenic environments, the microbial community structure of a sample from a full-scale biogas plant was determined. Hybridization results with probes for syntrophic bacteria and methanogens were compared to specific methanogenic activities and microbial numbers determined with most-probable-number estimates. Most of the methanogenic rRNA was comprised of Methanomicrobiales rRNA, suggesting that members of this order served as the main hydrogen-utilizing microorganisms. Between 0.2 and 1% of the rRNA was attributed to the Syntrophomonadaceae, of which the majority was accounted for by the genus Syntrophomonas.     

Influence of Substratum Wettability on Attachment of Freshwater Bacteria to Solid Surfaces

We studied the attachment of a number of freshwater bacteria from River Sowe, Coventry, England, to test substrata. The attachment of each organism to hydrophobic and hydrophilic surfaces was evaluated, and further studies evaluated the attachment of selected isolates to a number of substrata with a range of water wettabilities. The wettability of each substratum was determined by contact angle measurements and was expressed as the work of adhesion (W_A). No generic pattern of attachment to the test surfaces was found, although the majority of the organisms isolated showed a preference for the hydrophobic surface. A more detailed study of selected isolates showed a relationship between W_A and number of attached cells. Each bacterium attached in maximum numbers to a surface that was characteristic of that organism and that had a W_A between 75 and 105 mJ m⁻².     

Sulfate reduction and possible aerobic metabolism of the sulfate-reducing bacterium Desulfovibrio oxyclinae in a chemostat coculture with Marinobacter sp. Strain MB under exposure to increasing oxygen concentrations

A chemostat coculture of the sulfate-reducing bacterium Desulfovibrio oxyclinae together with a facultative aerobe heterotroph tentatively identified as Marinobacter sp. strain MB was grown under anaerobic conditions and then exposed to a stepwise-increasing oxygen influx (0 to 20% O(2) in the incoming gas phase). The coculture consumed oxygen efficiently, and no residual oxygen was detected with an oxygen supply of up to 5%. Sulfate reduction persisted at all levels of oxygen input, even at the maximal level, when residual oxygen in the growth vessel was 87 microM. The portion of D. oxyclinae cells in the coculture decreased gradually from 92% under anaerobic conditions to 27% under aeration. Both absolute cell numbers and viable cell counts of the organism were the same as or even higher than those observed in the absence of oxygen input. The patterns of consumption of electron donors and acceptors suggest that aerobic incomplete oxidation of lactate to acetate is performed by D. oxyclinae under high oxygen input. Both organisms were isolated from the same oxic zone of a cyanobacterial mat where they have to adapt to daily shifts from oxic to anoxic conditions. This type of syntrophic association may occur in natural habitats, enabling sulfate-reducing bacteria to cope with periodic exposure to oxygen.     

Degradation of Adsorbed Protein by Attached Bacteria in Relationship to Surface Hydrophobicity

The relationships among surface energy, adsorbed organic matter, and attached bacterial growth were examined by measuring the degradation of adsorbed ribulose-1,5-bisphosphate carboxylase (a common algal protein) by attached bacteria (Pseudomonas strain S9). We found that surface energy (work of adhesion of water) determined the amount and availability of adsorbed protein and, consequently, the growth of attached bacteria. Percent degradation of adsorbed ribulose-1,5-bisphosphate carboxylase decreased with increasing hydrophobicity of the surface (decreasing work of adhesion). As a result, growth rates of attached bacteria were initially higher on hydrophilic glass than on hydrophobic polyethylene. However, during long (6-h) incubations, growth rates increased with surface hydrophobicity because of increasing amounts of adsorbed protein. Together with previous studies, these results suggest that the number of attached bacteria over time will be a complex function of surface energy. Whereas both protein adsorption and bacterial attachment decrease with increasing surface energy, availability of adsorbed protein and consequently initial bacterial growth rates increase with surface energy.     

Adhesion of algal cells to surfaces

This paper reports the cell–substratum interactions of planktonic (Chlorella vulgaris) and benthic (Botryococcus sudeticus) freshwater green algae with hydrophilic (glass) and hydrophobic (indium tin oxide) substrata to determine the critical parameters controlling the adhesion of algal cells to surfaces. The surface properties of the algae and substrata were quantified by measuring contact angle, electrophoretic mobility, and streaming potential. Using these data, the cell–substratum interactions were modeled using thermodynamic, DLVO, and XDLVO approaches. Finally, the rate of attachment and the strength of adhesion of the algal cells were quantified using a parallel-plate flow chamber. The results indicated that (1) acid–base interactions played a critical role in the adhesion of algae, (2) the hydrophobic alga attached at a higher density and with a higher strength of adhesion on both substrata, and (3) the XDLVO model was the most accurate in predicting the density of cells and their strength of adhesion. These results can be used to select substrata to promote/inhibit the adhesion of algal cells to surfaces.     

Hydrophobicity, Adhesion, and Surface-Exposed Proteins of Gliding Bacteria

The cell surface hydrophobicities of a variety of aquatic and terrestrial gliding bacteria were measured by an assay of bacterial adherence to hydrocarbons (BATH), hydrophobic interaction chromatography, and the salt aggregation test. The bacteria demonstrated a broad range of hydrophobicities. Results among the three hydrophobicity assays performed on very hydrophilic strains were quite consistent. Bacterial adhesion to glass did not correlate with any particular measure of surface hydrophobicity. Several adhesion-defective mutants of Cytophaga sp. strain U67 were found to be more hydrophilic than the wild type, particularly by the BATH assay and hydrophobic interaction chromatography. The very limited adhesion of these mutants correlated well with hydrophilicity as determined by the BATH assay. The hydrophobicities of several adhesion-competent revertants ranged between those of the wild type and the mutants. As measured by the BATH assay, starvation increased hydrophobicity of both the wild type and an adhesion-defective mutant. During filament fragmentation of Flexibacter sp. strain FS-1, marked changes in hydrophobicity and adhesion were accompanied by changes in the arrays of surface-exposed proteins as detected by an immobilized radioiodination procedure.