The primary objective of conventional drinking water treatment and distribution is to deliver to the consumer water that is both aesthetically pleasing and does not constitute a human health risk. To achieve this, water utilities employ a range of physical (i.e. sand and membrane filtration) and chemical (i.e. flocculation and disinfection) barriers in order to reduce the numbers of microorganisms as well as the nutrients that may support their growth within biofilms. In this thesis, biofilms and microbial barriers in water treatment and distribution were therefore examined. The development of biofilms within artificial recharge was investigated in pilot column at Norsborg waterworks in Stockholm. The proportion of active bacteria, measured as numbers of EUB338-positive cells relative to the total number of bacteria enumerated by total direct counts, decreased with time. Through the addition of nutrients however, two to three times more bacteria were able to be active (measured by increase in activity after activation with additional nutrients). By extracting the recalcitrant hydrophilic and hydrophobic fractions of humic substances it was possible to assess the microbiological response to those compounds. It was shown that bacteria more firmly attached to the sand grains preferred the hydrophobic fraction whilst more loosely-associated bacteria preferred the hydrophilic one. The amount of easily degradable matter in raw water (measured as assimilable organic carbon) was generally low. Biofilms were investigated by two different methods for extraction and analysis of microorganisms. Glass slides introduced into the sand material were dominated by α-Proteobacteria, and underestimated loosely-associated bacteria whilst extracts from sand were dominated by γ-Proteobacteria, and also caused variations due to the extraction method employed The barrier function of biofilms was investigated in biofilters, also fed with raw water from Gothenburg. The focus here was on particle removal in size-intervals of 1-15 µm (protozoa) and 0.4 - 1 µm (bacteria). In both size fractions, autofluorescent microalgae, which were naturally-occurring in raw water, were also enumerated in parallel. Their removal was 60-90%. In parallel, defined amounts of fluorescent hydrophilic and hydrophobic microspheres (1 µm) were added. They showed a reduction of hydrophobic spheres by 98% and hydrophilic ones by 86%. Removal of viruses was determined by adding a defined dose of bacteriophages and gave lower reduction values of 40 - 61%. Both naturally-occurring particles in defined size intervals and added particles or organisms were shown to provide a clearer picture of barrier function than usually performed measurements of turbidity. The efficiency of chemical treatment against viruses was also measured in a pilot-plant in Gothenburg. It was shown that commonly-used MS-2 bacteriophages were much more sensitive than φX174 bacteriophages. Reduction of MS-2 over the entire chemical step (when added after dosing of chemicals) was 5-log10 whilst φX174 was reduced by 1-log10. The latter was shown to be a more conservative model for virus removal. The effects of different steps in the chemical precipitation showed that the primary dosage of chemicals and the development of flocs had great importance for the assessment of removal efficacy. When added before the dosing of chemicals, reduction of φX174 and MS-2 was 3.8-log10 and 6.2-log10, respectively. The establishment of biofilm within a distribution system was followed in a 1000 metre long pilot-plant (with parallel lines) at Lovö waterworks as well as in two of Stockholm's main distribution systems (Nockeby and Hässelby). The pilot-plant was shown to satisfactorily represent processes within the distribution systems. The development of biofilms was slow, producing thin biofilms over a one to two month periods. Numbers of bacteria were generally in the range of 104 - 105 per cm2, which is lower than shown in other earlier investigations. The implementation of primary ultra viloet (UV)-treatment in place of chlorination (both being chloraminated prior to distribution) did not considerably change the numbers of bacteria in biofilms. No significant difference could be seen between the system that had UV-treatment as a primary treatment step, and the system that was chlorinated over the whole period. Chlorine residuals were generally low at the distal parts of the distribution systems. Naturally-occurring protozoa were present in distribution systems in numbers ranging from 280 - 3500 protozoa per cm-2. Protozoa may play a significant role as predators of biofilm bacteria, however they can also act as protection for bacteria against external influences i.e. disinfection. Should sudden contamination of a distribution system occur, biofilm can provide protection and act as a site for potential regrowth of introduced microorganisms. Biofilms developed in the pilot-scale that represented water from different distances from waterworks were exposed to fluorescent microspheres, (hydrophobic and hydrophilic, 1 µm) legionellae (as a model for opportunistic bacterial pathogens) and bacteriophages (human enteric virus model) in order to determine their accumulation and persistence within the biofilm, and release to the bulk water. It was shown that introduced model organisms were released continuously, primarily through desorption, and additionally through the influence of disinfection and activity of protozoa. Desorption was also assessed in a laboratory experiment under laminar and turbulent flow. Laminar flow conditions that were representative of a distribution system gave a slow and continual release of individual cells, whilst turbulent conditions detached larger aggregates. In conclusion, based on this work an increased understanding was gained both of barrier functions at the different steps of water treatment, their effects on overall biofilm dynamics and structure and the role that biofilm plays within the drinking water system itself.

<p>Life is cellular. Cells grow and divide to give two new cells; this process is called the cell cycle. The chromosome in a bacterium is replicated into two identical copies before the cell divides. DNA replication is a fundamental process common to all forms of life.</p><p>In my thesis, I have studied control of chromosome and plasmid replication in <i>Escherichia coli</i>, a rod-shaped bacterium. Plasmids are extrachromosomal autonomously replicating DNA molecules. </p><p>I have combined the classical Meselson-Stahl density-shift and DNA hybridisation with theoretical analysis of DNA replication. The minimal time between two successive replications of the same molecule, the eclipse, was determined for both plasmid and chromosome.</p><p>The aim was to investigate the processes ensuring the precise timing of chromosome replication in the cell cycle. In wild-type strains, the chromosomal eclipse was long. Mutations affecting the so-called sequestration process, the superhelicity of the DNA, and the initiation protein, DnaA, reduced the eclipse.</p><p>Fast-growing <i>E. coli</i> has overlapping replicative phases with synchronous initiation from multiple initiation sites, <i>oriC</i>. I have investigated the complex interplay between different control processes by measuring the length of the eclipse and the degree of asynchronous initiation in various mutants.</p><p>I have measured the eclipse period of plasmid R1 during up- and down-shifts in plasmid copy number. The length of the eclipse was found to be determined by structural events as well as by the properties of the copy-number-control system.</p><p>During downshift from very high copy numbers, the rate of plasmid replication started very slowly and gradually increased until the normal copy number was achieved, in accordance with the +<i>n</i> model.</p><p>The CopB system of plasmid R1 was shown to be a rescue system preventing cells with few plasmid copies from losing the plasmid in some of the daughter cells.</p>

Life is cellular. Cells grow and divide to give two new cells; this process is called the cell cycle. The chromosome in a bacterium is replicated into two identical copies before the cell divides. DNA replication is a fundamental process common to all forms of life. In my thesis, I have studied control of chromosome and plasmid replication in Escherichia coli, a rod-shaped bacterium. Plasmids are extrachromosomal autonomously replicating DNA molecules. I have combined the classical Meselson-Stahl density-shift and DNA hybridisation with theoretical analysis of DNA replication. The minimal time between two successive replications of the same molecule, the eclipse, was determined for both plasmid and chromosome. The aim was to investigate the processes ensuring the precise timing of chromosome replication in the cell cycle. In wild-type strains, the chromosomal eclipse was long. Mutations affecting the so-called sequestration process, the superhelicity of the DNA, and the initiation protein, DnaA, reduced the eclipse. Fast-growing E. coli has overlapping replicative phases with synchronous initiation from multiple initiation sites, oriC. I have investigated the complex interplay between different control processes by measuring the length of the eclipse and the degree of asynchronous initiation in various mutants. I have measured the eclipse period of plasmid R1 during up- and down-shifts in plasmid copy number. The length of the eclipse was found to be determined by structural events as well as by the properties of the copy-number-control system. During downshift from very high copy numbers, the rate of plasmid replication started very slowly and gradually increased until the normal copy number was achieved, in accordance with the +n model. The CopB system of plasmid R1 was shown to be a rescue system preventing cells with few plasmid copies from losing the plasmid in some of the daughter cells.

<p>The primary objective of conventional drinking water treatment and distribution is to deliver to the consumer water that is both aesthetically pleasing and does not constitute a human health risk. To achieve this, water utilities employ a range of physical (i.e. sand and membrane filtration) and chemical (i.e. flocculation and disinfection) barriers in order to reduce the numbers of microorganisms as well as the nutrients that may support their growth within biofilms. In this thesis, biofilms and microbial barriers in water treatment and distribution were therefore examined. The development of biofilms within artificial recharge was investigated in pilot column at Norsborg waterworks in Stockholm. The proportion of active bacteria, measured as numbers of EUB338-positive cells relative to the total number of bacteria enumerated by total direct counts, decreased with time. Through the addition of nutrients however, two to three times more bacteria were able to be active (measured by increase in activity after activation with additional nutrients). By extracting the recalcitrant hydrophilic and hydrophobic fractions of humic substances it was possible to assess the microbiological response to those compounds. It was shown that bacteria more firmly attached to the sand grains preferred the hydrophobic fraction whilst more loosely-associated bacteria preferred the hydrophilic one. The amount of easily degradable matter in raw water (measured as assimilable organic carbon) was generally low. Biofilms were investigated by two different methods for extraction and analysis of microorganisms. Glass slides introduced into the sand material were dominated by α-Proteobacteria, and underestimated loosely-associated bacteria whilst extracts from sand were dominated by γ-Proteobacteria, and also caused variations due to the extraction method employed </p><p>The barrier function of biofilms was investigated in biofilters, also fed with raw water from Gothenburg. The focus here was on particle removal in size-intervals of 1-15 µm (protozoa) and 0.4 - 1 µm (bacteria). In both size fractions, autofluorescent microalgae, which were naturally-occurring in raw water, were also enumerated in parallel. Their removal was 60-90%. In parallel, defined amounts of fluorescent hydrophilic and hydrophobic microspheres (1 µm) were added. They showed a reduction of hydrophobic spheres by 98% and hydrophilic ones by 86%. Removal of viruses was determined by adding a defined dose of bacteriophages and gave lower reduction values of 40 - 61%. Both naturally-occurring particles in defined size intervals and added particles or organisms were shown to provide a clearer picture of barrier function than usually performed measurements of turbidity. </p><p>The efficiency of chemical treatment against viruses was also measured in a pilot-plant in Gothenburg. It was shown that commonly-used MS-2 bacteriophages were much more sensitive than φX174 bacteriophages. Reduction of MS-2 over the entire chemical step (when added after dosing of chemicals) was 5-log10 whilst φX174 was reduced by 1-log10. The latter was shown to be a more conservative model for virus removal. The effects of different steps in the chemical precipitation showed that the primary dosage of chemicals and the development of flocs had great importance for the assessment of removal efficacy. When added before the dosing of chemicals, reduction of φX174 and MS-2 was 3.8-log10 and 6.2-log10, respectively. </p><p>The establishment of biofilm within a distribution system was followed in a 1000 metre long pilot-plant (with parallel lines) at Lovö waterworks as well as in two of Stockholm's main distribution systems (Nockeby and Hässelby). The pilot-plant was shown to satisfactorily represent processes within the distribution systems. The development of biofilms was slow, producing thin biofilms over a one to two month periods. Numbers of bacteria were generally in the range of 104 - 105 per cm2, which is lower than shown in other earlier investigations. The implementation of primary ultra viloet (UV)-treatment in place of chlorination (both being chloraminated prior to distribution) did not considerably change the numbers of bacteria in biofilms. No significant difference could be seen between the system that had UV-treatment as a primary treatment step, and the system that was chlorinated over the whole period. Chlorine residuals were generally low at the distal parts of the distribution systems. Naturally-occurring protozoa were present in distribution systems in numbers ranging from 280 - 3500 protozoa per cm-2. Protozoa may play a significant role as predators of biofilm bacteria, however they can also act as protection for bacteria against external influences i.e. disinfection. Should sudden contamination of a distribution system occur, biofilm can provide protection and act as a site for potential regrowth of introduced microorganisms. Biofilms developed in the pilot-scale that represented water from different distances from waterworks were exposed to fluorescent microspheres, (hydrophobic and hydrophilic, 1 µm) legionellae (as a model for opportunistic bacterial pathogens) and bacteriophages (human enteric virus model) in order to determine their accumulation and persistence within the biofilm, and release to the bulk water. It was shown that introduced model organisms were released continuously, primarily through desorption, and additionally through the influence of disinfection and activity of protozoa. </p><p>Desorption was also assessed in a laboratory experiment under laminar and turbulent flow. Laminar flow conditions that were representative of a distribution system gave a slow and continual release of individual cells, whilst turbulent conditions detached larger aggregates. In conclusion, based on this work an increased understanding was gained both of barrier functions at the different steps of water treatment, their effects on overall biofilm dynamics and structure and the role that biofilm plays within the drinking water system itself.</p>

Giardia intestinalis is one of the leading causes of diarrheal diseases, affecting about 280 million people every year. By characterizing the virulence factors of G. intestinalis, new drug targets can be found to treat giardiasis. In addition to the adhesive disc and the variant surface proteins, cysteine proteases are some of the most interesting virulence factors in Giardia. In this project, one of the major secreted cysteine proteases, CP10217, was studied. The intent was to study the structure by modelling and to characterize CP10217 by expressing it in yeast cells and purifying the supernatant by using Immobilized Metal Affinity Chromatography (IMAC). The molecular modelling showed that the Giardia CP can be modelled on the structures of human and Trypanosoma brucei CPs. The process of expressing and purifying CP10217 in this manner proved difficult. The protease seemed to be very active when expressed, probably resulting in self-cleavage into its active form and later digestion of the whole protein, leading to a low protein yield from the purification. Two approaches were tested in order to increase the protein yield. First, expression at different pH ranges, and secondly, by re-cloning CP10217 with an extra 108 bp sequence at the 5’ end. While changes in pH did not seem to affect the yield, sequencing results of the new vector showed that the cloning worked. More work on this new vector is needed to further analyse, and possibly characterize CP10217.

Bacteria from the genus Acidithiobacillus are often associated with biominingand acid mine drainage. Biomining utilises acidophilic, sulphur and ironoxidising microorganisms for recovery of metals from sulphidic low grade oresand concentrates. Acid mine drainage results in acidification and contaminationwith metals of soil and water emanating from the dissolution of metal sulphidesfrom deposits and mine waste storage. Acidophilic microorganisms play acentral role in these processes by catalysing aerobic oxidation of sulphides.Acceleration of mineral solubilisation is a positive aspect in biomining whereas,in acid mine drainage it is undesirable and accordingly, microbial iron andsulphur oxidation is promoted in the first case and measures are taken to inhibitit in the second case. In this thesis, several approaches were taken in order tounderstand and increase oxidation efficiency in biomining and to gain an insightinto the biochemical reactions taking place in these environments. A laboratoryscale bioreactor was designed and tested allowing simulation of bioleaching inheaps of mine tailings at different aeration, irrigation and particle size conditions(Paper I). A new psychrotolerant strain of Acidithiobacillus ferrooxidans wascharacterised that has an application in boreal heap bioleaching. Iron, reducedinorganic sulphur compound oxidation and bioleaching of various ores by thisstrain was studied as well as gene expression during oxidation of tetrathionateand/or ferrous iron (Papers III &amp; IV). Expression and regulation of atetrathionate hydrolase from Acidithiobacillus caldus, a key enzyme in reducedinorganic sulphur compound metabolism of this bacterium was investigated andthe presence of this enzyme in a bioleaching mixed culture was shown. The genecluster that harbours the gene coding for tetrathionate hydrolase (tetH) wasdescribed for the first time (Paper II).

Diss. (sammanfattning) Uppsala : Uppsala universitet, 2006. / Härtill 5 uppsatser.

Diss. (sammanfattning) Linköping : Univ., 2003. / Härtill 6 uppsatser.

Diss. (sammanfattning) Uppsala : Uppsala universitet, 2006. / Härtill 4 uppsatser.

Plasmids are small, circular, extrachromosomal genetic elements that replicate independently of the bacterial chromosome. Plasmids play a crucial role in the dissemination of antibiotic resistance genes through conjugation. Sub minimal concentrations of antibiotics have been shown to select for antibiotic resistance, but their effect on conjugation remains poorly understood. To study this, we constructed a Cre/lox system using a Cre-expressing plasmid and a chromosomal floxed BFP. We found that the system was not functioning as designed. Instead of expressing BFP upon conjugation, cells expressed BFP before conjugation and lost BFP expression upon recombination. We were able to locate the problem to the loxP site present after recombination, which interfered with the expression of BFP through two start codons present in the loxP spacer region. Both deletion and inversion of the loxP site present after recombination restored BFP expression to expected levels, providing strong evidence that the loxP sites were responsible for the opposite BFP expression observed.