Water systems in buildings have been reported to contribute to pseudomonal infection transmission and have been associated with Legionnaires’ disease (LD) outbreaks, for they provide the perfect conditions for bacteria proliferation and biofilms formation. An overview of the problem has highlighted that the economic burden, the healthcare and mortality costs of both LD and pseudomonal infections are significant. Although critical to the safe delivery of water, pathogen control continues to remain a challenge as current hot water treatments are not always effective, are often energy intensive and require expensive maintenance. This thesis was set out to evaluate the potential use of electrochemical disinfection (ED) in controlling pathogens in hot water systems of buildings. In this project, we performed a fundamental systematic study on the effect of geometrical and operational parameters in a flask, to gather an understanding of the effect of each parameter on the rate of bacteria elimination, crucial for the design and optimization of electrolytic cells. ED prototypes were then installed in in the hot water systems of two different buildings operating at 60°C, the temperature recommended for Legionella control (HSE, 2013), and their efficacy was monitored long term. In one of the buildings, 2 to 4– log reductions in total bacteria counts was observed, while Pseudomonas species counts were reduced by 3 log. The apparent failure in the other building was due to the inadequate operation of the water system. In order to achieve the 2019 zero carbon targets for new non-domestic buildings set by the UK government, the energy demand associated with heating water needs to be addressed, but maintaining systems at such high temperatures renders difficult the use of greener technologies that could further reduce the CO2 impact of heating water. Given that ED generates disinfectants and that the Health and Safety Executive advises that if hot water is treated with biocides, water temperatures can be reduced, the efficacy of the prototype device was evaluated under laboratory conditions at temperatures between 30 and 45˚C. The prototype was found to be effective both on laboratory-grown biofilm and on planktonic Legionella pneumophila serogroup 1, with 5-log reduction on bacteria counts.

The rivers, lakes and seas are the major water sources for the animal and plant kingdom in this earth. In recent times, the usage and wastage of water have been increasing due to the uncontrolled population growth. In addition to that, the rapid industrialization over the years has led to the gradual depletion of the natural resources like water, soil and air. Some of these industries discharge contaminants like organic products and inorganic (or) toxic heavy metals without treatment into the environment, leading to its degradation. Zinc is the 24th most abundant element present in the earth crust, amounting 75 ppm (0.0075%). The concentration of zinc present in the soil and seawater is about 64 ppm and 30 ppb respectively (Emsley, 2001). Generally, the zinc is found with the base metals such as copper and lead and it has less affinity with oxides and strong affinity with sulphides. Sphalerite, a zinc sulphide ore, is majorly containing 60-62% of zinc. The other sources of zinc from the minerals are smithsonite, hemimorphite, quartzite, and hydro zincate. The major sources of zinc contamination arise from several industrial activities such as mining, coal, waste combustion and steel and iron processing. Drinking water also contains certain amounts of Zn, which may be higher when it is stored in metal tanks. The acute toxicity arises from the ingestion of excessive amounts of zinc salts, either accidentally or as dietary supplement. Vomiting, nausea and stomach cramps usually occur after the consumption of more than 500 mg of zinc sulfate. In addition to that, the higher amounts of zinc affect gastrointestinal tract, liver, bone and prostate glands. Finally, Zn can interrupt the activity in soils, as it negatively influences the activity of microorganisms and earthworms, thus retarding the breakdown of organic matter. To combat this problem, techniques such as chemical precipitation, ion exchange, reverse osmosis, etc. are adopted, but these processes result in a huge amount of secondary sludge formation, inefficient removal of metals and are not cost effective. In recent times, an innovative, eco-friendly, cost-effective method has been introduced to treat the toxic heavy metals namely bioremediation. ―Bioremediation‖ is a process of removal of organic or inorganic contaminants by using bacteria, fungi, algae and its metabolites In this research work, the potential of four bacterial strains of the Pseudomonas sp. such as P.putida, P.alcaligenes, P.aeruginosa and P.fluorescens and the extracellular proteins secreted by these four species for the bio-sorption of zinc has been investigated through batch experiments. The mechanisms of interaction between the zinc ion and the bacterial biomass as well as with the extracellular proteins have been elucidated. Additionally, a carbon paste electrode has been modified by using Pseudomonas sp. and its metabolites to develop biosensors for zinc and the lower limit of detection of zinc in aqueous solution has been determined. The major objectives of this research work are specified below: • To study the potential of Pseudomonas sp. such as P.putida, P.alcaligenes, P.aeruginosa and P.fluorescens for the bio sorption of zinc, in batch systems. • To determine the speciation of zinc with respect to pH in the growth medium and the maximum inhibitory effect of zinc on the growth of the four chosen Pseudomonas sp. • To isolate and characterize the extracellular proteins from the four Pseudomonas sp. such as P.putida, P.alcaligenes, P.aeruginosa and P.fluorescens. • To study the biosorption of zinc by extracellular proteins secreted by the Pseudomonas sp. • To elucidate the mechanisms involved in the biosorption of zinc at the microbe- metal interface and protein-metal ion interface for all the four systems by different characterization studies such as zeta potential, FTIR analysis and EDAX analysis. • To develop a biomass modified CPE using bacterial cells and extracellular protein to detect the concentration of zinc in aqueous solutions adopting voltammetric techniques. The significant results obtained from this research work are summarized as follows: The initial studies were concentrated on the bio sorption of zinc by using four Pseudomonas species such as P.putida, P.alcaligenes, P.aeruginosa and P.fluorescens. The various factors affecting the bio sorption of zinc by these species were investigated by varying the contact time (10-80 min), pH (2-5±0.2), biomass concentration of the four species in the range of 108- 1011 cells / mL, and the initial zinc concentration from 5 mg/L to 80 mg/L respectively, keeping other parameters such as temperature and agitation speed constant in all the experiments. From the results obtained, the maximum percentage of biosorption achieved by the P.putida, P.alcaligenes, P.aeruginosa and P.fluorescens was found to be 60%, 93%, 70% and 65% respectively for 25 mg/L at pH 5±0.2. The equilibrium time taken by the four species to achieve maximum biosorption was about 10 min and the biosorption kinetics adhered to pseudo-second order reaction and the rate constants were determined for different concentrations of zinc. The biosorption isotherm followed both the Langmuir and Freundlich isotherm models. The Gibbs free energy (ΔG) values determined from the Langmuir isotherm model for all the four systems were found to be -26, -32, -30 and -28 kJ /mole respectively. The Gibbs free energy values indicate that the biosorption of zinc ions onto the bacterial surface is a chemi-sorption process involving co-ordination, complexation or chelation. The characterization studies, namely zeta potential, FTIR analysis and SEM-EDX were also carried out on the bacterial cells before and after interaction with zinc. These studies also provide evidence in support of the complexation of zinc with the functional groups on the bacterial cell surface apart from electrostatic interaction. In the second part of the investigation, the inhibitory effect of zinc on the growth of four Pseudomonas sp. was investigated by varying the concentration of zinc from 50 mg/L to 1000 mg/L and the stability of zinc was analysed with respect to pH (2-12) with different concentrations from 50 - 1700 mg/L. It was found that in the absence of zinc the time taken to reach the exponential phase and the specific growth were almost the same for all the four systems. However, in the presence of zinc ions, the growth of the four Pseudomonas sp. was suppressed beyond 50mg/L of zinc. A control study on the stability of zinc in Luria broth medium showed that zinc was highly stable up to 200 mg/L from pH 2-8. However, the stability of zinc in the growth medium decreased beyond that concentration Additionally, studies on the biosorption of zinc were performed using extracellular proteins isolated from the four Pseudomonas sp. The amount of protein was estimated by the Bradford protein assay method at 594 nm. The biosorption experiments were carried out by varying the protein concentration from 50 to 1000µg/mL and the zinc concentration from 50-1000 mg/L and keeping other parameters fixed, namely such as pH at 5±0.2, reaction time of 20 min, temperature at 30±0.2 and the speed of rotation of 200 rpm. It was found that the maximum percentage of zinc biosorbed by the proteins isolated from P.putida was found to be 91% at 500µg/mL of protein concentration and from the other three species, it was found to be about 60% of biosorption at the same protein concentration. The biosorption isotherms of zinc for extracellular protein adhered to the Giles H1 type for all the four systems. The maximum amount of zinc biosorbed by the protein isolated from P.putida, P.alcaligenes, P.aeruginosa and P.fluorescens was found to be 35.6, 19,18.3 and 10 mg/µg respectively and the Gibbs free energy values were found to be -32, -22,-22 and -23 kJ/mole. The mechanisms involved in protein-zinc interaction were elucidated using FTIR analysis and EDX analysis. The FTIR analysis revealed, that the zinc ions were complexed with carboxylic and amine functional groups. Further, the potential of P.putida, P.alcaligenes, P.aeruginosa and P.fluorescens and their extracellular proteins of P.putida as biosensors for detecting zinc ions in aqueous solutions, using electrochemical methods such as, Cyclic Voltammetry and Differential pulse anodic stripping voltammetry, was assessed. The developed carbon paste electrode coated by the biomass showed an approximately 3-fold increase in the sensing of Zn2+ ion in comparison with the bare electrode. The lower limit of detection of the biosensor for zinc ions by Cyclic voltammetry was found to be 10-6 M, and in case of DPASV the lower limit of detection was about 10-7M. The lower limit of detection of the protein modified biosensor for zinc ions by cyclic voltammetry was found to be 10-7M and in the case of DPASV method the lower limit of detection was found to be 10-9 M.

Genotypic differences in growth and yield of two common bean (Phaseolus vulgaris L) cultivars to Rhizobium inoculation and management were investigated. In 2003-04, the two bean cultivars (Scylla and T-49) were combined with three inoculant treatments (strains CC 511 and RCR 3644, and a control of no inoculation), two fertiliser levels (0 and 150 kg N ha⁻¹) and two irrigation treatments (irrigated and rainfed). There was no nodulation on either cultivar. To further investigate the symbiotic relationship, 16 rhizobial isolates, including the two used in the first field experiment, were combined with the cultivar Scylla and evaluated in a greenhouse. Subsequently, five Rhizobium isolates were chosen for further field evaluation, based on signs of early nodulation in the greenhouse trial. The second field experiment in 2004-05 combined the five inoculant strains (RCR 3644, UK 2, H 20, PRF 81, PhP 17 and a control) with two bean cultivars (Scylla and T-49). In the greenhouse, nodule number varied from 7 (UK 2) to 347 (H 441) nodules plant⁻¹ at 51 DAS and from 13 (UK 1) to 335 (CIAT 899) nodules plant⁻¹ at 85 DAS. In 2004-05, in the field, nodulation was also variable, ranging between 1 and approximately 70 nodules plant⁻¹, with higher nodules numbers plant⁻¹ being found on cultivar T-49. Of the isolates used in the field, strains H 20, PRF 81 and PhP 17 produced 70, 25 and 12 nodules plant⁻¹ at 70, 40 and 54 DAS respectively. Nodules formed were of various sizes and more than 80 % were pink to dark red in colour denoting the presence of leghaemoglobin and active N fixation. The remaining nodules were either green or white. The importance of selecting an appropriate cultivar for the growing conditions was highlighted in these experiments. Leaf area index, leaf area duration intercepted radiation and final utilisation efficiency were significantly affected by cultivar. In both seasons cv. T-49 reached maturity (dry seed) before Scylla, while unirrigated plants reached green pod maturity seven days before irrigated plants. Plants of cv. Scylla gave a final TDM of 730 g m⁻²; compared to the 530 g m⁻² produced by T-49. The average growth rate was 7.0 and 5.2 g m⁻² day⁻¹ for Scylla and T-49 respectively (2003-04). Plants receiving 150 kg N ha⁻¹ produced 665 g m⁻² TDM which was 12 % more than was produced by unfertilised plants. The application of 150 kg N ha⁻¹ gave an average growth rate of 6.4 g m⁻² day⁻¹ compared to 5.7 g m⁻² day⁻¹ from plants with no N. Inoculation in the field had no significant effect on TDM in both seasons. Temperature affected growth and DM accumulation. Accumulated DM was highly dependent on cumulative intercepted PAR. Air temperatures below the base temperature (10 °C) affected growth in 2004-05, resulting in plants accumulating just 0.24 g DM MJ⁻¹ PAR during early growth. This increased to 2.26 g DM MJ⁻¹ PAR when the temperature was increased above the base temperature. There was a strong relationship between LAI and intercepted PAR. A LAI of 4.0-4.5 was required to intercept 90-95 % of incident solar radiation. Cultivar significantly (p < 0.001) affected radiation use efficiency (RUE). Scylla had a RUE of 1.02 g DM MJ⁻¹ PAR compared to T-49 at 1.18 g DM MJ⁻¹ PAR. Seed yield was significantly (p < 0.001) affected by cultivar and fertiliser application. Cultivar Scylla produced 467 g m⁻² which was 76 % more than T-49, while a 12 % increase in seed yield was observed in N fertilised plants over unfertilised plants. Only cultivar significantly affected HI, while the yield components that had the greatest effect on seed yield were hundred seed weight and pods plant⁻¹. Inoculation significantly (p< 0.05) affected 100 seed weight (2004-05). Plants inoculated with strain H 20 had the highest 100 seed weight at 25.2 g with cv. Scylla producing larger seeds than T-49. The belief that local environmental conditions play a major role on field survival of bacteria, led to the use of PCR methods to identify field nodulating organisms. Amplification of genomic DNA from parent isolates using primers fC and rD generated a single band for each isolate. Isolates were identified to the species level as either Rhizobium or Agrobacterium, using the highly conserved internally transcribed spacer (ITS) region and are known to nodulate common bean. The DNA extracted from the isolates recovered from nodules of field grown beans gave multiple bands with primers fC and rD. Five distinct banding patterns were observed. All of these were different from those of parent isolates. Sequencing of the 16S rRNA demonstrated that nodules of field grown beans in Canterbury were inhabited by Pseudomonads either alone or in association with other root nodulating organisms. The inability to identify the inoculant strains in nodules of field grown beans does not rule out their infection and nodulating function in the cultivars used. The results suggest the possibility of both Rhizobium and Pseudomonads cohabiting in the nodules of field grown beans. The aggressive nature of Pseudomonads on artificial media, possibly out competing the inoculant rhizobia is proposed, leading to the inability to identify the inoculant strain from the nodules of the field grown beans by PCR methods. The need to identify the nodule forming or nodule inhabiting bacteria in the nodules is necessary to classify the importance of these organisms and their economic benefit to agricultural production. This study also underlines the importance of using PCR methods to gain valuable insights into the ecological behaviour of Rhizobium inoculants and nodule inhabiting organisms.