A bioremediation approach using natural transformation in pure-culture and mixed-population biofilms

Bacterial transformation by naked DNA is thought to contribute to gene transfer and microbial evolution within natural environments. In nature many microbial communities exist as complex assemblages known as biofilms where genetic exchange is facilitated. It may be possible to take advantage of natural transformation processes to modify the phenotypes of biofilm communities giving them specific and desirable functions. Work described here shows that biofilms composed of either pure cultures or mixed populations can be transformed with specific catabolic genes such that the communities acquire the ability to degrade a particular xenobiotic compound. Biofilms were transformed by plasmids bearing genes encoding green fluorescent protein (mut2) and/or atrazine chlorohydrolase (atzA). Confocal microscopy was used to quantify the number of transformants expressing mut2 in the biofilms. Degradation of atrazine by expressed atzA was quantified by tandem mass spectrometry. PCR analysis was performed to confirm the presence of atzA in transformed biofilms. These results indicate that it should be possible to use natural transformation to enhance bioremediation processes performed by biofilms.     

The composition and persistence of faecal coliforms and enterococcal populations in sewage treatment plants

AIMS: The changes in structure and composition of faecal coliforms and enterococcal populations in sewage from different treatment plants, and the elimination of vancomycin- and erythromycin-resistant enterococci (VRE and ERE, respectively) in these treatment plants was analysed to determine any selective reduction. METHODS AND RESULTS: Faecal coliforms, enterococci, VRE, ERE and spores of sulphite-reducing bacteria were enumerated using standard methods. Samples were enriched where necessary in order to isolate antibiotic resistant strains. The structure and composition of these bacterial populations were determined by biochemical fingerprinting and clustering analysis. High diversity and similarity indexes were detected among all the bacterial populations in raw and treated sewage, independently of their origin and the treatment processes employed. Antibiotic resistant strains were detected in all sewage tested and no selective reduction was observed. CONCLUSIONS: The faecal coliforms and enterococci populations did not differ in the sewage samples studied. The vancomycin and erythromycin resistances of the enterococcal populations were similar in the sewage samples. Resistance to both antibiotics persisted after the treatment process independently of raw sewage flow, faecal origin or size of the human population contributing to sewage. However, sewage of mixed origin (human and animal) presented a lower similarity index for the two bacterial populations compared with that of the other human sewage analysed. SIGNIFICANCE AND IMPACT OF THE STUDY: Although a significant reduction in bacterial populations was observed, the persistence of VRE and ERE strains in the same proportions in sewage suggests that there is no selective elimination of bacterial populations during the treatment processes. The ability of antibiotic resistance strains to survive sewage treatment systems should be considered in certain water reuse programmes.     

Effect of seawater storage on coliforms, faecal coliforms and Escherichia coli.

The effect of refrigeration of seawater samples for 24 h prior to assaying coliforms, faecal coliforms and Escherichia coli was assessed. When the initial coliform counts were low, the amount of bacteria in refrigerated samples decreased. When the number of initial total coliforms was high, there was an increase following cold storage. E. coli counts also decreased. Faecal coliforms, when in the initial 200-500 count range, showed a decrease during cold storage but with good correlation (r = 0.9). In all other groups, no correlation between counts of before versus after storage was found. Assessment of seawater pollution following 24 h cold storage should not be made.     

The effects of fine-scale substratum roughness on diatom community structure in estuarine biofilms

Benthic diatoms are a major component of biofilms that form on surfaces submerged in marine environments. Roughness of the underlying substratum affects the settlement of both diatoms and subsequent macrofouling colonizers. This study reports the effects of roughness on estuarine diatom communities established in situ in the Indian River Lagoon, FL, USA. Natural communities were established on acrylic panels with a range of surface roughnesses. Smoother substrata exhibited higher cell density, species richness, and diversity. Twenty-three of 58 species were found either exclusively or more abundantly on the smooth surfaces compared to one or both roughened treatments. The results suggest a greater ability of benthic diatoms to recruit and colonize smooth surfaces, which is probably explained by a higher degree of contact between the cells and the surface.     

Characterization of Campylobacter jejuni biofilms under defined growth conditions

Campylobacter jejuni is a major cause of human diarrheal disease in many industrialized countries and is a source of public health and economic burden. C. jejuni, present as normal flora in the intestinal tract of commercial broiler chickens and other livestock, is probably the main source of human infections. The presence of C. jejuni in biofilms found in animal production watering systems may play a role in the colonization of these animals. We have determined that C. jejuni can form biofilms on a variety of abiotic surfaces commonly used in watering systems, such as acrylonitrile butadiene styrene and polyvinyl chloride plastics. Furthermore, C. jejuni biofilm formation was inhibited by growth in nutrient-rich media or high osmolarity, and thermophilic and microaerophilic conditions enhanced biofilm formation. Thus, nutritional and environmental conditions affect the formation of C. jejuni biofilms. Both flagella and quorum sensing appear to be required for maximal biofilm formation, as C. jejuni flaAB and luxS mutants were significantly reduced in their ability to form biofilms compared to the wild-type strain.     

Rimsulfuron in soil: Effect of persistence on growth and activity of microbial biomass at varying environmental conditions

The research was carried out toascertain the effect of rimsulfuron, a solfonylureaherbicide, on soil microbial biomass growth andactivity. Laboratory experiments were performed in asilty clay loam soil to relate changes of soilmicrobial biomass-C content and global hydrolyticactivity to the rimsulfuron persistence underdifferent conditions of temperature and soil humidity.The results showed that rimsulfuron persistencedepended significantly on temperature, while itremained almost unchanged by humidity changes. A rangeof half-life values from 3.5 to 14.8 days was found ina temperature range from 10 °C to 25 °C,with lower half-lives at higher temperature.Persistence data were processed with the VARLEACHmodel, in order to predict rimsulfuron persistenceunder different environmental conditions. On comparingtreated soils with untreated soil samples, decreasesin the microbial biomass-C content and increases inthe global hydrolytic activity were found to beconnected with rimsulfuron persistence at the variousexperimental conditions. These effects persisted fora short time and, they were evident earlier at highertemperature and more persistent at lower humidity.This behaviour is discussed in terms of rimsulfurontoxicity, with the consequent release of endocellularhydrolytic enzymes from the dead microorganisms. Anequation was derived to calculate the microbialbiomass-C content in response to the variation ofrimsulfuron persistence.     

Exopolysaccharide production in biofilms: substratum activation of alginate gene expression by Pseudomonas aeruginosa.

Reporter gene technology was employed to detect the activity of an alginate promoter of Pseudomonas aeruginosa when the organism was grown as a biofilm on a Teflon mesh substratum and as planktonic cells in liquid medium. Alginate biosynthetic activity was determined with a mucoid cell line derived from a cystic fibrosis isolate and containing an alginate algC promoter fused to a lacZ reporter gene. Reporter activity was demonstrated with chromogenic and fluorogenic substrates for beta-galactosidase. Expression of algC was shown to be upregulated in biofilm cells compared with planktonic cells in liquid medium. Gene up-expression correlated with alginate biosynthesis as measured by Fourier transform infrared spectroscopy, uronic acid accumulation, and alginate-specific enzyme-linked immunosorbent assay. The algC promoter was shown to have maximum activity in planktonic cultures during the late lag and early log phases of the cell growth cycle. During a time course experiment, biofilm algC activity exceeded planktonic activity except during the period immediately following inoculation into fresh medium. In continuous-culture experiments, conversion of lacZ substrate was demonstrated microscopically in individual cells by epifluorescence microscopy.     

Sodium dodecyl sulfate allows the persistence and recovery of biofilms of Pseudomonas fluorescens formed under different hydrodynamic conditions

The effect of the anionic surfactant sodium dodecyl sulfate (SDS) on Pseudomonas fluorescens biofilms was investigated using flow cell reactors with stainless steel substrata, under turbulent (Re = 5200) and laminar (Re = 2000) flow. Steady-state biofilms were exposed to SDS in single doses (0.5, 1, 3 and 7 mM) and biofilm respiratory activity and mass measured at 0, 3, 7 and 12 h after the SDS application. The effect of SDS on biofilm mechanical stability was assessed using a rotating bioreactor. Whilst high concentrations (7 mM) of SDS promoted significant biofilm inactivation, it did not significantly reduce biofouling. Turbulent and laminar flow-generated biofilms had comparable susceptibility to SDS application. Following SDS exposure, biofilms rapidly recovered over the following 12 h, achieving higher respiratory activity values than before treatment. This phenomenon of post-treatment recovery was more pronounced for turbulent flow-generated biofilms, with an increase in SDS concentration. The mechanical stability of the biofilms increased with surfactant application, except for SDS concentrations near the critical micellar concentration, as measured by biofilm removal due to an increase in external shear stress forces. The data suggest that although SDS exerts antimicrobial action against P. fluorescens biofilms, even if only partial and reversible, it had only limited antifouling efficacy, increasing biofilm mechanical stability at low concentrations and allowing significant and rapid recovery of turbulent flow-generated biofilms.     

Effect of heterogeneous structure in mechanically unstressed biofilms on overall growth

In contrast to their name, biofilms are not always flat and homogeneous but instead often exhibit complex structural heterogeneity. It has been suggested that nonhomogeneous geometry is selected in order to increase biofilm growth rate. A previous study (Dockery and Klapper (2002) SIAM J. Appl. Math., 62, 853–869) of a model biofilm system in a static bulk fluid demonstrated that under some circumstances a flat biofilm-bulk fluid interface is linearly unstable to perturbation due to growth induced forces. Computations indicated that subsequent nonlinear evolution results in fingers and mushrooms of biofilm similar to structures observed in actual biofilms. However, the important complementary issue of biological functionality was not considered. Here a weakly nonlinear analysis of the simple growing biofilm layer model in Dockery and Klapper (2002, SIAM J. Appl. Math., 62, 853–869) is presented. It is argued that, at least in the case of biofilms free of external mechanical stress, overall growth is in fact generally inhibited by the presence of growing perturbations in the linear stage. Hence a more complex explanation of function is necessary.     

Effects of growth conditions on the lipid composition of Bifidobacterium bifidum subsp. pennsylvanicum

Lipid-phosphorus and lipid-galactose content and phospholipid and fatty acid composition of Bifidobacterium bifidum subsp. pennsylvanicum were examined under a wide variety of growth conditions. Cells from 29-C cultures contained less lipid-phosphorus than did cells from 37-C cultures, but their lipid-galactose content and phospholipid composition did not differ. At both temperatures, the growth phase influenced the lipid composition similarly.Phosphate, Mg²⁺ and K⁺ concentrations in the medium did neither significantly change the cellular lipid-phosphorus content nor the phospholipid composition. Only Mg²⁺-deficiency markedly reduced growth and lowered the content of cellular lipid-galactose.Omission of Tween 80 from the medium did not affect growth, but lowered the content of lipid-galactose and augmented those of lipid-phosphorus and diphosphatidylglycerol in the cell. Increased osmolarity and substitution of other Tweens for Tween 80 caused the same changes in lipid composition, and besides inhibited growth. Omitting Tween 80 and replacing it by other Tweens dramatically reduced the percentage of unsaturated fatty acids. C₁₂- and C₁₄-fatty acids made up about 50% of total fatty acids in cells from Tween 20 cultures and 12–14% in cells from Tween 40 and Tween 60 cultures. The differences in the decline of unsaturated fatty acids and in the degree of replacement of these acids by C₁₂- and C₁₄-fatty acids may be related to the variations in growth in cultures with various Tweens by way of changes in the physical state of the membrane lipids.The present investigations have been carried out with financial support from the Netherlands Organization for the Advancement of Pure Research (ZWO) through the Netherlands Foundation for Chemical Research (SON).     

Effects of growth conditions on the ion composition of Bifidobacterium bifidum subsp. pennsylvanicum

The cation content of Bifidobacterium bifidum subsp. pennsylvanicum was markedly influenced by the washing procedure of the cells, by the growth phase and the temperature, and by the composition of the culture medium. Optimal retention of cations was achieved by washing with 0.25 M MgCl₂ at 20 C. The intracellular Na⁺ concentration rose during growth in normal medium to a constant value in the stationary phase, the K⁺ concentration rose in the exponential phase, but fell in the stationary phase. Cells from 29-C cultures contained more Na⁺ and less K⁺ in the stationary phase than did cells from 37-C cultures, but the total cation content was the same at 29 and 37 C.Intracellular Na⁺ and K⁺ concentrations were dependent on the concentrations in the medium and on its osmolarity. The intracellular Na⁺/K⁺ ratio varied from 0.04 to 2.3. The concentrations of Na⁺, K⁺ and phosphate in the medium hardly affected growth. Mg²⁺-deficiency of the medium markedly decreased the concentration of Mg²⁺ within the cell; its concentration in the cell sap was greatly affected, but the amount of sedimentable, bound Mg²⁺ only slightly. The content of K⁺ within the cell decreased in Mg²⁺-deficient medium, but the concentration of Na⁺ did not. Omission of Tween 80 as well as its substitution by Tween 20 caused a decrease of intracellular K⁺. Cells from Tween 40 and Tween 60 cultures additionally contained markedly less Na⁺.The present investigations have been carried out with financial support from the Netherlands Organization for the Advancement of Pure Research (ZWO) through the Netherlands Foundation for Chemical Research (SON).     

Effect of the composition of the nutrient media and culture conditions on the growth of strains of Campylobacter jejuni and C. coli

The influence of the composition of culture media and cultivation conditions on the growth of Campylobacter strains has been studied. Blood-containing media have been found to ensure the most favorable growth conditions. No differences between the use of RCT supplement (rifampicin, cephalothin and trimethoprim in amounts of 10, 15 and 5 mg/l respectively) and DC supplement (0.1% of sodium deoxycholate and 15 mg/l of cephalothin), as well as between different methods used for the creation of microaerophilic conditions, have been established.     

Effect of substrate type on bacterial community composition in biofilms from the Great Barrier Reef

Natural and anthropogenic impacts such as terrestrial runoff, influence the water quality along the coast of the Great Barrier Reef (GBR) and may in turn affect coral reef communities. Associated bacterial biofilms respond rapidly to environmental conditions and are potential bioindicators for changes in water quality. As a prerequisite to study the effects of water quality on biofilm communities, appropriate biofilm substrates for deployment in the field must be developed and evaluated. This study investigates the effect of different settlement substrates (i.e. glass slides, ceramic tiles, coral skeletons and reef sediments) on bacterial biofilm communities grown in situ for 48 days at two locations in the Whitsunday Island Group (Central GBR) during two sampling times. Bacterial communities associated with the biofilms were analysed using terminal restriction fragment length polymorphism (T-RFLP) and clone library analyses of 16S rRNA genes. Findings revealed that substrate type had little influence on bacterial community composition. Of particular relevance, glass slides and coral skeletons exhibited very similar communities during both sampling times, suggesting the suitability of standardized glass slides for long-term biofilm indicator studies in tropical coral reef ecosystems.     

Content and composition of hopanoids in Zymomonas mobilis under various growth conditions.

By using a new method for quantification of the different hopanoid derivatives, a total hopanoid content of about 30 mg/g (dry cell weight) was observed in Zymomonas mobilis. This value is the highest reported for bacteria so far. The major hopanoids in Z. mobilis were the ether and glycosidic derivatives of tetrahydroxy-bacteriohopane, constituting about 41 and 49% of the total hopanoids. Tetrahydroxybacteriohopane itself, diplopterol, and hopene made up about 6, 3, and 1%, respectively. Only minor changes in hopanoid composition were observed with changes in growth conditions. Earlier reports on a correlation between hopanoid content and ethanol concentration in the medium could not be confirmed. Over a wide range of ethanol concentrations (5 to 60 g/liter), growth rates (0.08 to 0.25 h-1), and temperatures (25 to 37 degrees C), the molar ratio of hopanoids to phospholipids in the cells amounted to about 0.7. Only at growth rates of greater than 0.30 h-1 did the molar ratio increase to about 1.     

Effect of UV light disinfection on antibiotic-resistant coliforms in wastewater effluents.

Total coliforms and total coliforms resistant to streptomycin, tetracycline, or chloramphenicol were isolated from filtered activated sludge effluents before and after UV light irradiation. Although the UV irradiation effectively disinfected the wastewater effluent, the percentage of the total surviving coliform population resistant to tetracycline or chloramphenicol was significantly higher than the percentage of the total coliform population resistant to those antibiotics before UV irradiation. This finding was attributed to the mechanism of R-factor-mediated resistance to tetracycline. No significant difference was noted for the percentage of the surviving total coliform population resistant to streptomycin before or after UV irradiation. Multiple drug resistance patterns of 300 total coliform isolates revealed that 82% were resistant to two or more antibiotics. Furthermore, 46% of these isolates were capable of transferring antibiotic resistance to a sensitive strain of Escherichia coli.