Development of rhizoremediation as a treatment technology in the removal of Polycyclic aromatic Hydrocarbons (PAHs) from the environment

Numerous classes and types of chemicals, which contaminate soil, complicate the removal of many toxic compounds from the environment. For example, many soils are contaminated with one or more metals, radioactive and inorganic compounds. Large areas are polluted with recalcitrant organic substances that pose environmental problems due to their toxicity and tendency to disperse through wind and water errosion. Polycyclic aromatic Hydrocarbons (PAHs) are ubiquitous pollutants found in soil at wood preservation plants and gasworks. PAHs are chemical compounds that persist in the environment and thus cause pollution worldwide. Their persistence in the environment is due to their low water solubility. The major source of PAHs is from the combustion of organic material such as coal, tar, wood and rubber. PAHs have been detected in a wide variety of environmental samples including air, soil, sediments, water, oils, tars and foodstuff. Most people are exposed to PAHs when they breathe smoke, autoemissions or industrial fumes. Because PAHs are toxic, mutagenic and carcinogenic to humans and animals, their elimination from the environment is of paramount importance. The estimated costs for the clean up of PAHs contaminated sites with conventional techniques such as incineration and landfilling are enormous. The search for alternative methods to restore polluted sites in a less expensive, less labour intensive, safe and environmentally friendly way is required. Such an alternative method is rhizoremediation, which is defined as the use of plants in association with microorganisms to degrade environmental pollutants such as PAHs. Microbial communities exposed to hydrocarbons become adapted, exhibiting selective enrichment and genetic changes resulting in an increased proportion of hydrocarbon degrading bacteria and bacteria plasmid encoding hydrocarbon catabolic genes. Adapted microbial communities have higher proportions of hydrocarbon degraders that can respond to the presence of hydrocarbon pollutants. The aim of this study was to identify bacteria isolated from the rhizosphere of Elusine coracana, Biddens pilosa, Brantha serratia and Cyperus esculentus grown in polluted and unpolluted soil and to evaluate the potential of bacteria isolated from the rhizosphere of these plants grown in polluted soil for their ability to bioremediate Polycyclic aromatic Hydrocarbons (PAHs). Different concentrations (1%, 3% and 5%) of naphthalene and acenaphthene (PAHs) were made and added to 500ml Bacteriological agar.1000µl of bacterial suspensions were spread onto the surface of naphthalene and acenaphthene-based agar plates and incubated for 48h at 37OC. Results showed that Brevibacillus brevis, Brevindimonas versicularis, Vibrio vulnificus, Chryseo indologenes, Micrococcus spp, Bacillus stearothermophilus, Pseudomonas putida and Pseudomonas spinosa showed excellent growth in all concentrations in naphthalene based agar medium. However, Micrococcus spp and Pseudomonas spinosa showed limited growth in acenaphthene based agar medium. Amongst all these bacteria tested for their potential in utilizing PAHs, B. brevis, V. vulnificus, C. indologenes, B. stearothermophilus, and P. putida were the most promising for biodegradation of PAHs, since none of them were affected by any change in concentration either in naphthalene or acenaphthene.Dissertation (MSc (Microbiology))--University of Pretoria, 2007.Microbiology and Plant Pathologyunrestricte

Proteolytic and amylolytic enzymes for bacterial biofilm control

Biofilms are characterized by surface attachment, structural heterogeneity; genetic diversity; complex community interactions and an extracellular matrix of polymeric substances (EPS). Biofilms deposit and adhere to all surfaces that are immersed in aqueous environments. EPS serves many functions including: facilitation of the initial attachment of bacterial cells to a surface; formation and maintenance of the micro colony; enables the bacteria to capture nutrients; causes biofouling; cell-cell communication and enhances bacterial resistance antimicrobial agents. EPS also function as a stabilizer of the biofilm structure and as a barrier against hostile environments. Extracelullar polymeric substances are composed of a wide variety of materials including polysaccharides, proteins, nucleic acid, uronic acid, DNA, lipid and even humid substances. EPS can be hydrophilic or hydrophobic depending on the structural components making up such EPS and the environmental conditions were the biofilms are developing. The exopolysachharides (EPS) synthesized by microbial cells vary greatly in their composition and in their chemical and physical properties within the bacterial strains. Due to variety in the structural components of the bacterial EPS, removal of biofilms by compounds that have no effects on the biofilm EPS would be difficult. Enzymes are proven to be effective in degrading biofilm EPS. The manner in which enzymes degrade the biofilm EPS is through binding and hydrolysis of the EPS components (proteins and carbohydrates) molecules and converting them into smaller units that can be transported through the cell membranes and then be metabolized. The objectives of this study were to grow Pseudomonas fluorescens and mixed bacterial species biofilms in nutrient rich and nutrient limited medium conditions; to determine the EPS, protein and carbohydrate concentrations of the biofilm grown in rich and in limited nutrient conditions and to test the efficiency of protease and amylase enzymes for the degradation of the EPS and biofilm removal. In the results, there was a slight difference in the number of viable cells grown in biofilms that were fed than the cells of the unfed biofilms. As a result, the EPS, protein and carbohydrate concentrations were higher in the fed biofilms than the unfed biofilms. There are contradictory reports about the composition of EPS especially with the ratio of carbohydrate to protein. Some of these reports indicate that certain biofilms EPS have bigger proportion of proteins and some found polysaccharides to be the dominant composition of the EPS of the biofilms. Nonetheless, the quantity and the composition of the EPS produced by bacterial biofilms depend on a number of factors such as microbial species, growth phase and the type of limiting substrate. Enzymes were tested individually and in combination for the degradation of biofilm EPS. For efficient removal of biofilm, it is important that the structural components of the biofilm EPS should be known before application of the relevant enzymes. In this study, the test enzymes were effective for the degradation of the biofilm EPS except for the protease Polarzyme which had no activity. The reason for the inefficiency of Polarzyme may be due to its incompatibility with the specific protein structural components of the biofilm EPS tested in this study. The manner in which the enzymes degrade the biofilm EPS is through binding and hydrolysis of the protein and carbohydrate molecules and converting them into smaller units that can be transported through the cell membranes and then be metabolized. In addition, the mode of enzymatic action will depend on the specific EPS components and this in turn will determine its efficacy. The protease enzymes tested individually and in combination were most effective for EPS degradation. The efficiency of the proteases may be due to their broad spectrum activity in degrading a variety of proteins acting partly as the multi structural components of Pseudomonas fluorescens and mixed bacterial species biofilm EPS. On the other hand, amylase enzymes tested individually and in combination was less effective for the EPS degradation. The structures of polysaccharides synthesized by microbial cells vary. Microbial exopolysaccharides are comprised of either homopolysachharides or heteoropolysaccharides. A number of lactic acid bacteria produce heteropolysaccharides and these molecules form from repeating units of monosaccharides including D- glucose, D- galactose, L- fructose, L- rhamnose, D- glucuronic acid, L- guluronic acid and D- mannuronic acid. The type of both linkages between monosaccharides units and the branching of the chain determines the physical properties of the microbial heteropolysaccharides. Due to a wide range of linkages and the complexity of polysaccharides structures, it would therefore be difficult for the amylases to break down the bond linkages and the monomers making up polysaccharides which determine the physical and chemical structure of the EPS. It was therefore not surprising that the amylase enzymes tested for the degradation of Pseudomonas fluorescens and mixed bacterial species biofilms, were less effective than the proteases. Hence, when the amylase enzymes were tested in combination with the protease enzymes, efficiency improved. It was therefore concluded that the protease enzymes were the primary remedial compounds and the amylase enzymes were the secondary remedial compounds. Conclusion If a compound or compounds capable of destroying all the structural components of different EPS that are produced by different biofilms growing under different conditions is found then the “city of microbes” (biofilms) would be destroyed permanently. If only an enzyme or enzymatic mixture capable of shutting down or deactivating the quorum sensing systems of different biofilm EPS could be found, then there would not be any formation of biofilms. In this study, protease enzymes tested individually and in combination were the most effective in the degradation of biofilm EPS than the amylase enzymes resulting in the reduction of large population of the biofilm cells attached on the substratum. Recommendation Amylase enzymes tested individually and in combination were less efficient for the degradation of the biofilm EPS and biofilm removal. This may be due to the complex structure of the exopolysaccharides synthesized by different biofilms. Also, the bond linkages between monosaccharides units and the branching of the chain complex the structures and as a result confer in the physical properties of the microbial biofilms. Hence, when the amylase enzymes were tested in combination with the protease enzymes, activity improved. For efficient degradation of biofilm EPS, it is therefore recommended that, protease and amylase enzymes should be tested in combination. In addition, the structure of the biofilm EPS should be investigated so that relevant enzymatic mixtures are tested for biofilm removal.Thesis (PhD)--University of Pretoria, 2010.Microbiology and Plant Pathologyunrestricte

Protease and amylase enzymes for biofilm removal and degradation of extracellular polymeric substances (EPS) produced by Pseudomonas fluorescens bacteria

Removal of biofilms is difficult. In industrial settings, both the inactivation and removal of biofilms are of huge concern. If only disinfection without the removal of attached biofilms occurs, the inactivated biofilm cells may provide an ideal environment for further adhesion and growth, resulting in a complex matrix. Microbial resistance to biocides and their negative environmental impact are the main reasons for finding alternative biofilm control strategies. Enzymes may offer such an alternative. The objective of this study was to determine the effect of commercial proteases and amylases on biofilms formed by Pseudomonas fluorescens. Biofilms were grown in diluted medium containing glass wool used as the attachment surface. Extracellular polymeric substances (EPS) were extracted and EPS composition was determined. Protease (savinase, everlase and polarzyme) and amylase (Amyloglucosidase and Bacterial Amylase Novo) activity was tested on both biofilms and on extracted EPS. After testing enzymes, biofilm integrity was evaluated by scanning electron microscopy. EPS composition consisted predominantly of proteins. Everlase and Savinase were the most effective enzymatic treatments on removing biofilms and degrading the EPS