Zebrafish, Danio rerio, are able to survive exposure to extreme acidity (pH 4). Because previous studies demonstrated that disruption of ionic balance during exposure to acidic water is the major cause of mortality in acid-sensitive freshwater species, the focus of this thesis was to characterize the molecular mechanisms enabling zebrafish to maintain their Na+ homeostasis following exposure to acidic water. Initial findings (Chapter 2) demonstrated that branchial mRNA expression of selected isoforms of claudins, major components of tight junctions, are altered in an isoform-dependent manner, suggesting the potential regulation of epithelial permeability to minimize ion loss. Concurrently, a marked stimulation of Na+ uptake was observed in adults and larvae following acid-exposure. Because of the uniqueness of this response (increasing Na+ uptake in acidic water) among freshwater teleosts, the mechanisms related to Na+ uptake and its stimulation were investigated further (Chapters 3 - 7). Pharmacological treatments and gene knockdown approaches revealed that a functional metabolon consisting of an apically expressed Na+-H+-exchanger (NHE3b) in association with an apically expressed ammonia-conducting channel (Rhcg1), enables Na+ uptake in acidic water. During chronic (>1 day) exposure to acidic water, cortisol (via glucocorticoid receptors) and catecholamines (via β-adrenergic receptors) are involved in stimulating Na+ uptake. Although catecholamines may act on both NHE3b and Na+-Cl- co-transporter (NCC), the effects of cortisol on Na+ uptake are mediated primarily by activation of NHE3b. On the other hand, during acute (<3 h) exposure to acidic water, cortisol does not appear to affect Na+ uptake; rather, the stimulation of Na+ uptake appears to be mediated by angiotensin II and catecholamines. Cyclic AMP (cAMP), a signalling molecule synthesized following the activation of β-adrenergic receptors, is critically involved in stimulating Na+ uptake, likely via activation of NHE3b and NCC. In agreement with this idea, ionocytes that express NHE3b also express high levels of β-adrenergic receptor (propranolol binding sites) as well as trans-membrane adenylyl cyclase (forskolin binding sites). Taken together, the results of this thesis provide fresh insight into the mechanisms of osmoregulation in freshwater (FW) fish. In particular, the data reveal the presence of complex pathways regulating Na+ uptake in zebrafish exposed to acidic water. The relative importance of the various pathways depends in part on the duration of exposure; acute versus chronic.

Zebrafish, Danio rerio, are able to survive exposure to extreme acidity (pH 4). Because previous studies demonstrated that disruption of ionic balance during exposure to acidic water is the major cause of mortality in acid-sensitive freshwater species, the focus of this thesis was to characterize the molecular mechanisms enabling zebrafish to maintain their Na+ homeostasis following exposure to acidic water. Initial findings (Chapter 2) demonstrated that branchial mRNA expression of selected isoforms of claudins, major components of tight junctions, are altered in an isoform-dependent manner, suggesting the potential regulation of epithelial permeability to minimize ion loss. Concurrently, a marked stimulation of Na+ uptake was observed in adults and larvae following acid-exposure. Because of the uniqueness of this response (increasing Na+ uptake in acidic water) among freshwater teleosts, the mechanisms related to Na+ uptake and its stimulation were investigated further (Chapters 3 - 7). Pharmacological treatments and gene knockdown approaches revealed that a functional metabolon consisting of an apically expressed Na+-H+-exchanger (NHE3b) in association with an apically expressed ammonia-conducting channel (Rhcg1), enables Na+ uptake in acidic water. During chronic (>1 day) exposure to acidic water, cortisol (via glucocorticoid receptors) and catecholamines (via β-adrenergic receptors) are involved in stimulating Na+ uptake. Although catecholamines may act on both NHE3b and Na+-Cl- co-transporter (NCC), the effects of cortisol on Na+ uptake are mediated primarily by activation of NHE3b. On the other hand, during acute (<3 h) exposure to acidic water, cortisol does not appear to affect Na+ uptake; rather, the stimulation of Na+ uptake appears to be mediated by angiotensin II and catecholamines. Cyclic AMP (cAMP), a signalling molecule synthesized following the activation of β-adrenergic receptors, is critically involved in stimulating Na+ uptake, likely via activation of NHE3b and NCC. In agreement with this idea, ionocytes that express NHE3b also express high levels of β-adrenergic receptor (propranolol binding sites) as well as trans-membrane adenylyl cyclase (forskolin binding sites). Taken together, the results of this thesis provide fresh insight into the mechanisms of osmoregulation in freshwater (FW) fish. In particular, the data reveal the presence of complex pathways regulating Na+ uptake in zebrafish exposed to acidic water. The relative importance of the various pathways depends in part on the duration of exposure; acute versus chronic.

Low water pH is an ionoregulatory challenge to freshwater teleosts. Larval zebrafish (Danio rerio) exposed to pH 4 water experience increased loss of Na⁺ and respond with increases in ionocyte abundance and whole-body concentrations of cortisol. Because cortisol plays a role in regulating early development, particularly of the stress axis, the present study asked whether the increase in cortisol in embryos exposed to pH 4 water causes dysregulation of the stress axis in later life. Baseline whole-body cortisol levels measured at 4, 6, and 15 days post-fertilization (dpf) did not differ between pH 4-exposed and control fish. At 6 dpf, pH 4-exposed fish had higher concentrations of cortisol compared to control fish following a stressor, but no difference was detected at 15 dpf. In addition, transcript abundances for key genes of the stress axis did not differ between control and pH 4-exposed fish. Based on these results, exposure to pH 4 water in early life does not influence the stress axis or cortisol responses later in life. Increases in ionocyte abundance in response to low pH have the potential to alter gill morphology, thereby impairing gas transfer, a trade-off known as the osmorespiratory compromise. The present study tested the hypothesis that zebrafish reared in pH 4 water have reduced gas transfer capacity in accordance with the osmorespiratory compromise. Indicators of gas transfer and ionoregulation were measured at 6, 15, 30 and 90 dpf. Across all ages examined, fish reared in pH 4 water had significantly higher whole-body concentrations of Na⁺, higher ionocyte abundances and thicker gills than control fish. These differences were accompanied by higher ventilation frequencies and higher critical PO₂ (Pcrit) values. Additionally, adult fish raised in low pH had a significantly higher rate of oxygen consumption compared to control fish. These results support the hypothesis that development in water of low pH impairs gas transfer, as predicted by the osmorespiratory compromise.

SiO₂ is the most abundant oxide in the earth and its properties, behaviors and interactions are of immense scientific and technological importance. Of particular importance are the interactions of silica with aqueous fluids because these fluids are present in nearly every natural setting. The dissolution of silica and glass by HF plays a very important role in technology and is widely used for the etching of silica and silicate glasses in the glass industry, in the flint industry, in surface micromachining, in etching of glass fibers for near-field optical probes, in the creation of frosted surfaces for decorative applications like frosted glass and cosmetic vials. I performed 57 batch reactor experiments in acidic fluoride solutions to measure the dissolution rate of quartz. Quartz dissolution rate data from other published studies were combined with the rate data from my experiments and these 75 data were analyzed using multiple linear regression to produce an empirical rate law for quartz r_qz = 10^-4.53 (e^-18932/RT) a_HF^1.18 a_H⁺^-0.39 where -5.13 < a_HF < 1.60, -0.28 < pH < 7.18, and 25 < T < 100 °C. Similarly, 97 amorphous silica dissolution rate data from published studies were analyzed using multiple linear regression to develop an empirical rate law for amorphous silica r_as = 10^0.48 (e^-34243/RT) a_HF^1.50 a_H⁺^-0.46 where -5.13 < a_HF < 1.60, -0.28 < pH < 7.18 and 25 < T < 70 °C. An examination of the empirical rate laws suggests that the rate-determining step in the reaction mechanism involves a coordinated attack of HF and H⁺ on the Si-O bond where the H⁺ ion, acting as a Lewis acid, attacks the bridging O atom, while the F end of a HF molecule, acting as a Lewis base, attacks the Si atom. This allows a redistribution of electrons from the Si-O bond to form a O-H and a Si-FH bond, thus "breaking" the Si-O bond. In order to quantify the effect of fluoride on the dissolution of silica, I also performed a series of 81 quartz dissolution and 20 amorphous silica dissolution experiments in batch reactors over a pH range of 0 to 7 to investigate the effect of H⁺ on silica dissolution rates. Between pH 3.5 and 7 silica dissolution rates are independent of pH, but they increase significantly below pH 3.5, so that the dissolution rate of both quartz and amorphous silica at pH 0 is more than an order magnitude faster than the dissolution rate at pH 3.5. I found that the empirical rate law for the dissolution of the"disturbed surface" of quartz in the pH range of 0 to 3.5 is r_qz,pH = 10^-0.23 (e^-59392/RT) a_H⁺^0.28 where 0 < pH < 3.5 and 25 < T < 55°C. The empirical rate law for amorphous silica dissolution in the pH range 0 to 3.5 is r_qz,pH = 10^0.56 (e^-64754/RT) a_H⁺^0.40 where 0 < pH < 3.5 and 25 < T < 55°C. Based on the empirical rate laws I suggest that the rate-determining step in the reaction mechanism involves a coordinated attack of H₃O⁺, acting as a Lewis acid reacts, on a bridging O atom and the O end of a H₂O, acting as a Lewis base, on the Si atom. This results in a redistribution of electrons from the Si-O bridging bond to form two Si-OH surface species. / Ph. D.

Published Article / The development of tolerance to low pH levels and the existence of cross-resistance may promote survival of bacteria in acidic foodstuff and in acidic environments such as the human stomach, in so doing escalating the probability of food poisoning. Similar to antimicrobial resistance developing, there is growing concern that effectiveness of organic acids may decrease as a result of the emergence of acid-tolerant food-borne pathogens. The objectives of this study were to determine the development of acid tolerance in selected food-borne pathogenic bacteria and to explore the activity of organic acids against acid tolerant pathogens. Bacterial strains were screened for acid-tolerance and susceptible strains were induced through exposure to increasing concentrations of an inorganic acid, as well as acidic foodstuffs. Susceptibility to six organic acids at various pH levels was assessed in order to evaluate the possible relationship between altered antimicrobial activity and acid tolerance. Salmonella enterica sv. Enteritidis ATCC 13076 and Escherichia coli ATCC 25922 were found to rapidly develop acid tolerance, while intrinsic acid tolerance was noted in Salmonella enterica sv. Typhimurium ATCC 14028. Pseudomonas aeruginosa ATCC 27853 demonstrated intermediate intrinsic acid tolerance. As expected, pH played a significant role in inhibitory activity of the organic acids as these compounds exhibit optimum antimicrobial activity at a lower pH (pH ≤5). It is, however, necessary to further elucidate the two-way role of pH in foodstuff concomitant to the addition of an organic acid.

Marine sulfide – bearing sediments that oxidize when in contact with oxygen and leach outelements in high concentrations to small watercourses have been a problem for many years allover the world especially around the Bothnian Bay. The purpose of this study was to furtherinvestigate the sulfur mineralogy present in acid sulfate soils in the area of Luleå, Sweden. Asecondary aim was to see if elements leach out and accumulate in an acid sulfate soil closer tothe recipient. Samples were taken in two profiles (one oxidized and one waterlogged) fromfour sites (sites A-D) and were analyzed for whole rock geochemistry. Two sites were furtherinvestigated for mineralogy in polished samples with an optical microscope, Ramanspectroscopy and SEM-EDS. Each profile consisted of three layers: oxidation zone, transitionzone and reduced zone. The oxidation zone above the groundwater table was light grey withbrown iron hydroxide staining. Parts that lied under the water table were dark grey-black within general strong odor (“rotten eggs”) due to its sulfur content. It was usually straightforwardto distinguish and separate the layers from each other directly in the field, however in somecases pH was needed for confirmation.A general feature of investigated polished samples is the presence of abundant framboidalpyrites that are common in reduced marine sediments. The transition zone was formed in suboxicconditions and this feature is reflected by the mineralogy. Many morphologies of theframboidal pyrite were observed in this layer and signs of both dissolution and formationoccur. In the sample from site C one could observe elemental sulfur in form of large (up to 50μm) euhedral crystals. In the samples with pH&lt;4, no sulfides occur as they have been replacedby jarosite (site B). Site C lacks these sulfur-bearing hydroxides which is thought to be due toa sulfur concentration of &lt;0.2 %. Sulfur shows extensive leaching at most sites but at site B andD1, it accumulates in the transition zone. Elements like cobalt (Co), nickel (Ni) and zinc (Zn)are leached out or are accumulated further down in the profile. Elements that could have beentransported and have accumulated in the waterlogged profiles are Co, Ni, Zn and chromium(Cr) and in some profiles also copper (Cu) and vanadium (V). / Coastal watercourses in Bottenviken: Method development and ecological restoration- A cross-border Swedish-Finnish cooperation project

The Canadian mining industry is one of Canada’s largest industrial sectors, creating jobs and a significant contributor to the economy. However, the mining activities can be detrimental to the environment due to the release of pollutants. Mining extensively uses nitrogen-based explosives, creating explosive impacted mining wastewaters (EIMWW) that contains substantial quantities of ammonia which is toxic to fish and thus, it has serious environmental repercussions. Ion-exchange (IE) with zeolite is an effective method for ammonia removal as it is easily automated, has a rapid start-up, is not significantly impacted by cold temperature or toxicity effects. Thus, it is particularly suited for Canadian mines. However, the traditional IE regeneration approach of using high concentration NaCl solutions creates a secondary polluting stream. Chlorine regeneration of ammonia-loaded zeolite appears to be a promising option to avoid such a secondary source of contamination. An evaluation of this option and other alternative regeneration are the main focus of this thesis. This thesis includes two initiatives. The first is a set of multi-cycle column loading and regeneration tests to investigate the feasibility of a zeolite (SIR-600) column for ammonia removal from a synthetic EIMWW, containing K and Ca as competing ions, coupled with regeneration using different concentration of chlorine solutions and combined salt+chlorine regeneration solutions. NaOCl regeneration was quite effective, but it was slower than salt regeneration. During the NaOCl regeneration, the main mechanism appears to be the oxidation of ammonia to nitrogen gas and hydrogen ions, however the Na in the NaOCl solution also seems to have a role in the regeneration. This results in pH levels around 3 for approximately half the regeneration cycles. In the combined salt+chlorine regeneration, the incorporation of the salt leads to more rapid elusion of the three ions presumbaly because of the higher sodium concentration (205 meq/L Na versus 14 meq/L Na). The long-term total ammonia nitrogen (TAN) uptake of SIR-600 regenerated with a NaOCl and NaOCl-NaCl were fairly similar, they varied within a relatively small range (0.185meq/g - 0.202meq/g). Thus, the various regeneration schemes did not impact the TAN uptake. The only apparent limitation of NaOCl regeneration is that it required a longer duration. However, the NaOCl is very promising because it resulted in very similar TAN uptakes, the SIR-600 showed a higher preference for TAN over K and avoided creating an additional process waste stream. The second initiative addressed concerns regarding the long-term integrity of SIR-600 arising from its exposure to low pH solutions during the regeneration. Long-term batch tests were performed to expose SIR-600 to low pH conditions (pH=2, pH=3, pH=4) and the characteristics of this IE material were evaluated. The 3-month low batch exposure experiment showed that pH below 4 decreased the TAN uptake capacity by up to 58%. There was no considerable impact on the surface gravimetric analysis (TGA) and Powder x-ray diffraction (PXRD). The exposure to pH=2 and pH=3 led to breakdown of the outer surface of SIR-600 and the creation of fine particles. It also led to decreases in the BET surface area and a decrease in the TAN uptake proportional to the decrease in the BET surface area. Thus, the exposure to pHs below 4 impacts the durability of SIR-600, so SIR-600 may have to be replaced more frequently. However, regeneration with NaOCl solutions still seems very promising as it avoids the creation of a secondary waste stream.

A bifidobacteria mix (nine strains) was evaluated for its effect on the growth and survival of Escherichia coli O157:H7 (ATCC 43890) in 11% NFDM and MRS broth under the optimum growth conditions for E. coli O157:H7 growth (37¡ãC, aerobic). Preliminary experiments were conducted to obtain the growth curves of the 9 strains of bifidobacteria and E. coli O157:H7 and confirm the inhibitory effect of acidity on E. coli O157:H7 (pH of media adjusted to 3.8). Acidapted E. coli O157:H7 showed no difference in resistance toward bifidobacteria (P>0.05) when compared to the non-acid adapted one. Escherichia coli O157:H7 did not survive in the supernatant of the bifodbacteria mix collected after incubation (37¡ãC) with aerobic shaking (8 h). However, the pathogen was able to grow after the pH of the supernatant was adjusted to 6.50 (pH of fresh MRS broth). Results suggest that a high content of bifidobacteria has a strong inhibitory effect on E. coli O157:H7, in part due to the low pH. However, products from bifidobacteria may also exert inhibitory effects.

京都大学 / 新制・課程博士 / 博士(人間・環境学) / 甲第25389号 / 人博第1131号 / 新制||人||263(附属図書館) / 京都大学大学院人間・環境学研究科相関環境学専攻 / (主査)教授 瀬戸口 浩彰, 教授 市岡 孝朗, 教授 西川 完途, 教授 井鷺 裕司 / 学位規則第4条第1項該当 / Doctor of Human and Environmental Studies / Kyoto University / DGAM

Listeria monocytogenes is a foodborne pathogen with a high mortality rate in susceptible populations and is of great public health concern with regard to food safety. The ability to grow at refrigeration temperatures during storage and at low pH levels during food processing has enabled the species to establish and sustain growth on processed food. Some food products particularly at risk of contamination by L. monocytogenes are deli or processed meat products, seafood, processed vegetables, dairy products and other food that do not require heating or reheating before consumption. The aims of this study are therefore to investigate firstly the prevalence rates of the species in high risk food products and a food processing plant in Brisbane. Secondly, to determine whether food isolates are better than environmental isolates at surviving the stress factors in food processing environments, or if their lineage groupings are a better indicator of their survival. Thirdly, to compare the survival of food and environmental isolates under temperature stress in co-cultures. A survey of more than 100 high-risk food products at supermarkets was carried out to investigate the prevalence of L. monocytogenes and other Listeria species in food. Isolates were also obtained from a food processing plant during routine tests. This study has found a low prevalence rate (under 10%) of L. monocytogenes in the processed vegetables and meat products tested. Other products tested included processed and raw seafood and processed fresh fruit. More L. monocytogenes isolates were isolated from the food processing plant (101) than from the food survey (25). Listeria grayi (73 isolates), a non-pathogenic species, was more frequently isolated from the food survey. The characterisation of those isolates has revealed their lineage groupings and REP-PCR profiles, which did not appear to be related to their sources. A selected group of 25 isolates were also serotyped for further identification. A larger number of lineage II isolates (70) were found compared to lineage I isolates (25), and were more common in food than the environments; while some (7) produced inconclusive results in the lineage PCR. The REP-PCR did not separate isolates of different sources, lineages or serotypes. In order to investigate the survival fitness of L. monocytogenes isolates whilst under environmental stress relevant to food safety, ten isolates from the food survey and food processing plant were chosen. Five isolates each from lineages I and II were subjected to temperatures ranging from 4ºC to 30ºC and pH levels from 4.0 to 6.0 for two weeks continuously, with their growth monitored by either optical density or plate counts. It was found that the isolates were most susceptible at the combination of pH 4.0 and 4ºC, where the growth of the isolates was completely inhibited. Again no relationship was observed between the lineage or the sources and the survival fitness of the chosen isolates. Due to the frequency of L. monocytogenes being co-isolated with other Listeria species as well as other food-borne pathogens, the relative competitive fitness of four of the isolates from the survival fitness experiment were compared in co-cultures at 4ºC and 30ºC at pH 7.4 in a small-scale preliminary study. The four isolates from food and environments were grown in broth cultures in pairs with the plate counts performed on antibiotic-supplemented selective TSA agar. The isolates were distinguished on agar supplemented with tetracycline which the isolates had acquired resistance to for this purpose. No significant difference (P>0.05) was observed between the lineages or the sources and the competitive fitness of the isolates in this study. The isolates always produced slightly more colonies in the antibioticresistant form compared to the wildtype form but did not seem to relate to the competitive fitness of the isolates. It would seemed that within the scope of this study, neither the lineage, serotype nor source of the isolates indicated any isolate with a better ability of survival while at low temperatures and low pH levels in pure and mixed cultures. However, other classifying groups such as serotypes, RAPD profiles may reveal possible co-relations, as well as a wider isolate pool. Furthermore, different stress factors could be included as part of an investigation on the survival of L. monocytogenes, as this study focused on food safety during processing.