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    Resistance of environmental bacteria to heavy metals and antibiotics in selected New Zealand soils : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Health Sciences at Massey University, Wellington, New Zealand
    (Massey University, 2020) Heydari, Ali
    The usage of superphosphate fertilizers, animal remedies and other material containing heavy metals (HMs) in agriculture and horticulture is a problematic issue resulting in the accumulation of HMs in the soil. The presence of these HMs in the soil leads to the induction of resistance of environmental bacteria to these heavy metals and may co-select for resistance against a broad range of antibiotics (Abs). This co-selection may increase the health risk for both humans and livestock because of resistance to the wide range of Abs. As well as direct health effects, an increase in Ab resistance may impose a significant burden on the livestock industry and primary production, leading to potential for immense social and financial losses. The current project was aimed to investigate the resistance of soil-borne bacteria sampled from selected regions of New Zealand. Genetic diversity of these bacteria and molecular aspects of horizontal transfer of HM and Ab resistance genes to other bacterial hosts was also investigated. Soil samples with a different history of usage, including pastoral and arable with high levels of HMs (e.g. cadmium (Cd) and zinc (Zn)) were collected from the Waikato region (WR), as well as soil from an area of native bush (background) as control. Waikato Region (WR) is one of the regions in New Zealand with high levels of HMs in soil, due to the regular use of HM-containing superphosphate fertilizers and animal remedies. Belmont Regional Park (BRP) airstrip soil was used as a novel site to explore bacterial communities’ resistance to HMs, and any co-selected Ab resistance. A comprehensive investigation was performed to simulate the soil environment contaminated with various levels of HMs to interrogate induced resistance to HMs and Abs in soil bacterial communities using microcosms with 6 weeks and 6 months incubation. The experiments carried out to investigate soil bacterial resistance to HMs and Abs were divided into two different categories, including physiological and molecular experiments. The physiological tests included plate culturing with a range of HMs and Abs concentrations and Pollution Induced Community Tolerance (PICT) analysis. Molecular investigations using Terminal Restricted Fragments Lengths Polymorphism (TRFLP) and Next Generation 16s rDNA were conducted to determine the probable changes in bacterial community structures induced by selection pressure of HMs presence in soil samples. Cd resistance genes were detected in individual bacterial isolates using specific oligomeric DNA primers via the polymerase chain reaction (PCR). Horizontal transfer of these genes to new bacterial recipients was investigated. Finally, Cd resistant bacterial isolates involved in Horizontal Gene Transfer (HGT) were identified using 16s rDNA Sanger Sequencing. Results clearly showed that there were significant differences between the levels of resistance to HMs and Abs in bacterial isolates from WR’s pastoral and arable soils compared to background soil (native bush). Differences between BRP soil samples with higher levels of HMs compared to those with lower HMs concentrations, and also microcosms’ with a range of HM levels showed there were significantly greater number of bacterial isolates resistant to HMs and Abs in soils with the higher initial levels of HMs. Pollution Induced Community Tolerance (PICT) analysis provided complementary results in concordance with the results of plate culturing experiments and showed the higher levels of bacterial resistance to HMs and Abs in soils with the higher initial levels of HMs. Terminal Restriction Fragment Length Polymorphism (TRFLP) and 16s rDNA Next Generation Sequencing experiments investigated HM-induced bacterial communities' structure changes and revealed significant differences among the bacterial community structures in the selected BRP and microcosms soil samples. The HGT experiments revealed the horizontal transfer of Cd resistance genes from donor isolates (from WR, BRP, and microcosms soils) to a characterised recipient bacterial strain in vitro, suggesting these genes were carried by mobile genetic elements. Overall, the result of the current project showed that there were higher levels of bacterial resistance to HM and also to Ab occurred while different levels of HMs were present in the soil. In addition, higher levels of HM and Ab resistance induction occurred in the presence of specific concentrations of HMs in microcosms’ soils. The bacterial community structures were changed in the presence of various levels of HM in soil. The investigation of bacterial community structures changes in microcosms containing background soil samples were greater compared to the microcosms containing pastoral soils; it is concluded in higher changes in bacterial communities in soils in presence of selection pressure of HMs. Cd resistance genes located on mobile genetic elements were able to be transferred horizontally form donor bacterial strains to recipients and the transconjugants showed resistance not only to Cd, Zn and/or Hg, but also to a range of Abs; it showed the possibility of spread of these HM resistance genes to the new bacteria in soil and conferring HM and subsequent Ab resistance in recipients.
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    Biosorption and leaching of heavy metals from activated sludge applied to soil : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Technology at Massey University, Palmerston North, New Zealand
    (Massey University, 2002) Mahenthiran, Pushpalatha
    Accumulation of heavy metals in sewage sludge and soil and their subsequent movement to ground water and surface water are major environmental issues. Cadmium (Cd), copper (Cu), zinc (Zn), nickel (Ni) and chromium (Cr) are the most commonly occurring sludge-borne heavy metals in New Zealand. The potential toxicity of these heavy metals depends more on their availability and mobility than on their total content. This study examined the adsorption-desorption and potential leachability of these heavy metals in sewage sludge and a volcanic soil. Results of adsorption - desorption experiments using Cd, Cu, Zn and Ni showed that activated sewage sludge sorbed Cd, Cu and Zn more effectively than Ni. Adsorption capacities of Cd, Cu and Zn were 35.7-44.8, 14.1-26.4 and 57.5-59.5 mg/g biomass, respectively. The affinity of activated sewage sludge with Ni was very low thereby no further isotherm study was carried in Ni. Biosorption increased with increases in pH. Adsorption capacity also increased with increases in initial metal ion concentration but the adsorption yield decreased. Chloride ion concentration (0.145 N) had a more significant effect on the reduction of adsorption of Cd than on the reduction of the adsorption of either Cu or Zn. A desorption study was carried out using deionized water, 0.1 N Na2SO4,0.1 N K2SO4,0.1 M Na citrate, and 0.1 M Na2CO3 solutions and the results showed that Zn desorbed more in every desorbing agent. Results of the study of the adsorption behaviour of Zn in volcanic Egmont soil in the presence of phosphate showed an increase in adsorption of Zn and the presence of nitrate did not show any significant difference in adsorption. Both 500 and 1000 mg/kg phosphate levels reduced the water-soluble Zn in volcanic Egmont soil remarkably. The desorption study showed that more Zn was desorbed with 0.1 M KNO3 than with deionized water and 0.1 M KH2PO4. An in situ leaching study was carried out in volcanic Egmont soil using Zn amended sewage sludge and inorganic Zn as Zn sources and soil columns were pretreated with nitrate and phosphate anions. More Zn leached from inorganic Zn applied soil columns than from Zn amended sewage sludge applied soil columns. There was no substantial difference in the amount of Zn leached between nitrate and phosphate treated columns. Determination of total acid digestible Zn in sewage sludge and inorganic Zn applied soils showed a greater accumulation of Zn in 0-10 cm depth. More Zn moved to the lowest (25-32 cm) depth in the nitrate treated inorganic Zn applied soil column and less Zn moved to the lowest (25-32 cm) soil depth in the phosphate treated sewage sludge applied column. Fractionation of Zn in Zn amended sewage sludge showed that most of the fractions of Zn were in water-soluble and exchangeable, followed by carbonate and organically complexed forms. However, fractionation of Zn in control volcanic Egmont soil showed that most of the Zn was in oxide and residual forms. After the application of both Zn amended sewage sludge and inorganic Zn, the overall percentages of water-soluble and the exchangeable, carbonate and organically complexed forms of total Zn increased. All the fractions of Zn in both sludge and inorganic Zn applied columns decreased with the increase in soil depthFractionation of Zn in inorganic Zn applied soil showed that the increase in the exchangeable and oxide forms of Zn was higher in the phosphate than in the nitrate treatment. The overall percentage of the water-soluble and the exchangeable and the carbonate forms of total Zn increased except the organic, the oxide and the residual form in inorganic Zn applied soil columns. The results of this study suggest that activated sewage sludge has a high affinity for Cd, Zn and Cu. Zn desorbed from sewage sludge more easily than Cu indicating that the Zn from the sewage sludge may be more reactive than Cu in soil. Ex situ and in situ studies showed that phosphate remarkably limited the Zn movement in both sewage sludge and inorganic Zn applied soils but nitrate did not have any significant impact on the movement of Zn.
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    Application of sodium deoxycholate for separation of heavy metals : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Environmental Engineering at Massey University, Palmerston North, New Zealand
    (Massey University, 2003) Sikder, Swapan Kumar
    Sodium deoxycholate (NaDC) and sodium taurodeoxycholate (NaTDC) have been used to bind and subsequently remove nickel, copper and zinc ions from aqueous streams by ultrafiltration. The mixture of metal and NaDC solutions forms a precipitate of insoluble metal deoxycholate. This precipitate can be removed from the solution in most cases by conventional techniques such as filtration and centrifugation. Ultrafiltration membranes made of polyethersulphone can also retain the precipitate effectively, producing an environmentally accepted effluent. The exception is the high copper and NaDC mixture where none of the operations works at room temperature because of the gelatinous nature of copper deoxycholate. The removal of metal ions by precipitating them as metal deoxycholate is affected by such parameters as equilibration time, surfactant-to-metal (S/M) ratio, feed concentration, temperature and pH. An equilibration time of 3 hours or greater is required to have a major part of precipitation completed. A S/M ratio of 2.5 is sufficient, except in the case of low nickel concentration when a higher S/M ratio is necessary. A higher temperature (e.g., >40°C) does not significantly affect the metal removal, but increases the process flux markedly. The high copper and NaDC mixture can also be operated with reasonable flux at a higher temperature. The use of NaDC to precipitate metal ions is inappropriate below or above the neutral pH value because DCA starts to precipitate as the pH is lowered to around 6, and metal ions precipitate as metal hydroxide at a high pH. Because different metal ions have a differential affinity for the deoxycholate ions, NaDC can potentially separate one metal from mixtures of metals. The separation of individual metals from copper/nickel mixture is good compared to poor separations for copper/zinc, zinc/nickel and copper/zinc/nickel mixtures. This is evident from the molar ratios of two metals of 1:67, 1:2.5 and 1:7 respectively in the respective permeates. The mixture of metal and NaTDC solutions is homogeneous and metals are removed by micellar-enhanced ultrafiltration (MEUF). Since NaTDC is expensive and difficult to recover, its application may not be economically feasible. Based on the present research, two NaDC-mediated processes are proposed: (1) a process for removing metal ions from single-metal systems, and (2) a process for separating copper/nickel mixtures.