Dissertations / Theses on the topic 'Bioremediation'

Traditional remediation techniques in removing toxic metal contaminants using physical and chemical methods are expensive and may cause other forms of damage to the environment, comparing with these techniques bioremediation can serve as an inexpensive, effective and environmental friendly remediation method. This thesis mainly discusses different bioremediation techniques and identifies possible areas in Hong Kong for bioremediation and suggests bioremediation methods for each potential area. Bioremediation of toxic metals is the use of microorganisms, plants, or even larger sized organisms to decontaminate sites with toxic metals. Bioremediation includes phytoremediation, microremediation and vermiremediation which use plants, microorganisms and earthworms to remediate contaminated environments respectively. The 4 most common mechanisms in phytoremediation of toxic metals are phytoextraction, phytofiltration, phytovolatilization and phytostabilization. Phytoremediation are used frequently for remediation around the world and its development includes using well-understood technology and genetic engineering to increase its effectiveness. Microremediation is another promising technology in bioremediation of toxic metals and consists of 6 major mechanisms which are biosorption, bioaccumulation, biotransformation, bioleaching, biomineralization and microbially-enhanced chemisorption of metals. Microremediation is mainly in research phase and its development includes identifying new species, combining with phytoremediation and genetic engineering. Vermiremediation is another rapidly developing technique in bioremediation of toxic metals, assisting other bioremediation by burrowing actions of earthworms and its excretion, and accumulating toxic metals inside their bodies. Vermiremediation is also in research phase but it is rapidly developing. Generally, bioremediation is around 60% cheaper than traditional remediation methods and no pollutants are emitted during the process. However the remediation process is slow and generally takes longer than a year. Sources of toxic metals in contaminated areas in Hong Kong are mainly due to historic industrial discharge although present activities also contribute. Potential areas include sites for electronic waste activities, sediments of Kwun Tong typhoon shelter and sediments of Tolo Harbour.
published_or_final_version
Environmental Management
Master
Master of Science in Environmental Management

Cyanide-containing waste is an increasingly prevalent problem in today's society. There are many applications that utilize cyanide, such as gold mining and electroplating, and these processes produce cyanide waste with varying conditions. Remediation of this waste is necessary to prevent contamination of soils and water. While there are a variety of processes being used, bioremediation is potentially a more cost effective alternative. A variety of fungal species are known to degrade cyanide through the action of cyanide hydratases, a specialized subset of nitrilases which hydrolyze cyanide to formamide. Here I report on previously unknown and uncharacterized nitrilases from Neurospora crassa, Gibberella zeae, and Aspergillus nidulans. Recombinant forms of four cyanide hydratases from N. crassa, A. nidulans, G. zeae, and Gloeocercospora sorghi were prepared after their genes were cloned with N-terminal hexahistidine purification tags, expressed in Escherichia coli and purified using immobilized metal affinity chromatography. These enzymes were compared according to their relative specific activity, pH activity profiles, thermal stability, and ability to degrade cyanide in the presence of high concentrations of copper and silver. Although all four were relatively similar, the N. crassa cyanide hydratase (CHT) has the greatest thermal stability and widest pH range where activity remained above 50%. N. crassa also demonstrated the highest rate of cyanide degradation in the presence of both metals tested. The CHT of A. nidulans and N. crassa have the highest reaction rate of the four fungal nitrilases evaluated in this work. These data help determine optimization conditions for the possible use of these enzymes in the bioremediation of cyanide-containing waste. Similar to known plant pathogenic fungi, in vivo expression of CHT in both N. crassa and A. nidulans were induced by growth in the presence of KCN (potassium cyanide).

Thesis submitted in fulfilment of the requirements for the degree Magister Technologiae: Chemical Engineering in the Faculty of Engineering at the Cape Peninsula University of Technology 2013
There are significant quantities of free cyanide (F-CN) and heavy metal contaminated effluent being discharged from electroplating operations globally. However, there is an overwhelming tendency in the industry to use physical and/or chemical treatment methods for cyanides (CNs) and heavy metals in effluent. Although these methods may be effective for certain CNs and heavy metals, they produce toxic by-products and also involve high operational and capital investment costs when compared to bioremediation methods. In this study, the design of a two-stage membrane bioreactor (MBR) system was conceptualised for the bioremediation of CNs and heavy metals in the effluent which was collected from an electroplating facility located in the Western Cape, South Africa. The design included a primary inactive bioremediation stage, to reduce the impact of contaminate concentration fluctuations, and a secondary active bioremediation stage, to remove the residual contaminants, in the effluent under alkaline pH conditions which typify most industrial effluent containing these contaminants. An analysis of the electroplating effluent revealed that the effluent contained an average of 149.11 (± 9.31) mg/L, 5.25 (± 0.64) mg/L, 8.12 (± 4.78) mg/L, 9.05 (± 5.26) mg/L and 45.19 (± 25.89) mg/L of total cyanide (T-CN), F-CN, weak acid dissociable cyanides (WAD-CNs), nickel (Ni), zinc (Zn) and copper (Cu), respectively. An Aspergillus sp., which displayed the characteristic black conidiophores of the Aspergillus section Nigri, was isolated from the electroplating facilities’ effluent discharge using a selective pectin agar (PA) and subcultured on 2% (v/v) antibiotic (10,000 units/L penicillin and 10 mg streptomycin/mL) potato dextrose agar (PDA). The isolate was tolerant to F-CN up to 430 mg F-CN/L on F-CN PDA plates which were incubated at 37 ˚C for 5 days. However, a significant decline in microbial growth was observed after 200 mg F-CN/L, thus indicating that the isolate was suitable for the bioremediation of the electroplating effluent. The identification of the isolate as Aspergillus awamori (A. awamori) was definitively determined using a multi-gene phylogenetic analysis, utilising ITS (internal transcribed spacer), -tubulin and calmodulin gene regions. Although an anomaly in the morphology of the conidia of the isolate was observed during the morphological analysis, indicating a possible morphological mutation in the isolate. A comparative study between “sweet orange” (Citrus sinensis (C. sinensis)) pomace, “apple” (Malus domestica (M. domestica)) pomace, “sweetcorn” (Zea mays (Z. mays)) cob and “potato” (Solanum tuberosum (S. tuberosum)) peel, i.e. waste materials considered to be agricultural residues, was conducted in order to assess their potential and as a sole carbon source supplement for A. awamori biomass development for the bioremediation of CNs and heavy metals. The suitability of these agricultural residues for these activities were as follows: C. sinensis pomace ˃ M. domestica pomace ˃ Z. mays cob ˃ S. tuberosum peel. For purpose of the sensitivity analysis, a temperature range of 20 to 50 ˚C and an alkaline pH range of 7 to 12 showed that: (1) optimal conditions for the uptake of Ni, Zn and Cu occurred at pH 12 and a temperature of 37.91 and 39.78 ˚C using active and inactive A. awamori biomass and unhydrolysed and hydrolysed C. sinensis pomace, respectively; (2) F-CN conversion increased linearly with an increase in pH and temperature using unhydrolysed and hydrolysed C. sinensis pomace; and (3) optimal conditions for the F-CN conversion and the respective by-products and sugar metabolism using active A. awamori biomass occurred at 37.02 ˚C and pH 8.75 and at conditions inversely proportional to F-CN conversion, respectively. The heavy metal affinity was Ni > Zn > Cu for all the biomaterials used and with the heavy metal uptake capacity being inactive A. awamori biomass > active A. awamori biomass > hydrolysed C. sinensis pomace > unhydrolysed C. sinensis pomace, respectively. Hydrolysed C. sinensis pomace had a 3.86 fold higher conversion of F-CN compared to the unhydrolysed C. sinensis pomace. The use of C. sinensis pomace extract as a nutrient media, derived from the acid hydrolysis of C. sinensis pomace, showed potential as a rich carbon-based supplement and also that low concentrations, < 0.1% (v/v), were required for the bioremediation of CNs and heavy metals. The two-stage MBR system was operated at 40 ˚C since this temperature was conducive to the bioremediation of CN and heavy metals. The primary bioremediation stage contained hydrolysed C. sinensis pomace while the secondary bioremediation stage contained active A. awamori biomass, supplemented by the C. sinensis pomace extract. After the primary and secondary bioremediation stages, 76.37%, 95.37%, 93.26% and 94.76% (primary bioremediation stage) and 99.55%, 99.91%, 99.92% and 99.92% (secondary bioremediation stage) average bioremediation efficiencies for T-CN, Ni, Zn and Cu were achieved. Furthermore, the secondary bioremediation stage metabolised the CN conversion by-products with an efficiency of 99.81% and 99.75% for formate (CHOO-) and ammonium (NH4+), respectively. After the first, second and third acid regeneration cycles of the hydrolysed C. sinensis pomace, 99.13%, 99.12% and 99.04% (first regeneration cycle), 98.94%, 98.92% and 98.41% (second regeneration cycle) and 98.46%, 98.44% and 97.91% (third regeneration cycle) recovery efficiencies for Ni, Zn and Cu were achieved. However, the design only managed to treat the effluent for safe discharge and the use of a post-treatment stage, such as reverse osmosis, is recommended to remove the remainder of the trace contaminants and colour from the effluent to ensure that the effluent met the potable water standards for reuse. There was a relatively insignificant standard deviation (≤ 3.22%) detected in all the parameters measured in the continuous operation and this indicates the reproducibility of the bioremediation efficiency in this continuous system.

Radionuclide extraction, processing and storage have resulted in a legacy of radionuclide-contaminated groundwater aquifers worldwide. An emerging remediation technology for such sites is the in situ immobilisation of radionuclides via biostimulation of dissimilatory metal reducing bacteria. While this approach has been successfully demonstrated in experimental studies, advances in understanding and optimization of the technique are needed. Mass transfer processes in heterogeneous and structured porous media may significantly affect the geochemical and microbial processes taking place in contaminated sites, impacting remediation efficiency significantly. The objective of this work was to understand better how heterogeneous porous media may affect immobilisation efficiency through interactions with the dominant geochemical, microbial and transport processes. A biogeochemical reactive transport model was developed for uranium immobilisation by DMRB. Physical heterogeneity is conceptually represented by a two-region model. Simulations investigate the parameter sensitivities of the system over wide ranging geochemical, microbial and groundwater transport conditions. The simulations highlight the conditions under which optimal remediation occurs. The relative significance of regional microbial residence patterns, U(VI)-surface complexation, geochemical conditions such as mineralogy, and porous media characteristics such as porosity and regional mass transfer are identified. Additionally, low level radioactive waste disposal sites typically contain significant quantities of cellulose, whose hydrolysis can have a significant impact on the geochemical conditions in these sites. Those geochemical conditions, in turn, can affect radionuclide mobility and bioimmobilisation. To investigate the potentially critical role of cellulose, process-based predictive model was developed, which includes a novel approach to biomass transfer between a cellulose-bound biofilm and biomass in the bulk liquid. A sensitivity analysis of the system parameters revealed the significance of bacterial colonisation of cellulose particles by attachment through contact in solution. The thesis concludes that the processes involved in uranium bioimmobilisation are sensitive to regional residence characteristics, media porosity, surface complexation, microbial efficiency, and mass transfer under varying conditions. Careful characterisation of potential sites and use of a model that includes these processes in sufficient detail is therefore deemed necessary before the remediation effectiveness can be reliably predicted.

Contamination of ecosystems by xenobiotic substances has led to significant negative impacts on the ecologies and on the health and economic livelihood of the human populations in affected environments. Bioremediation, particularly microbial bioremediation, has proven to be a safe, low-cost and environmentally friendly method for remediation of such areas. However, a lack of complete understanding of the metabolic, enzymatic and cellular processes involved has made it difficult to model and predict outcomes of field processes. The ability of researchers to make critical decisions capable of influencing the direction and outcomes of these processes is also hampered. This study outlines the results of a survey and describes the electronic Microbial BioRemediation (eMBR) web portal, designed to improve collaboration in the bioremediation research community. It describes the structure, algorithms and output of three bioinformatics resources developed and deployed via the portal. eMBRLitMine addresses the problem of identifying which microorganisms would be suitable for remediating sites contaminated by named compounds. It combines named-entity recognition algorithms, a mySQL database, graph rendering technologies and Perl scripts to create, from the vast information available within published literature, a statistical co-occurrence matrix which it uses to infer possible associations between microorganisms and particular contaminants. This provides valuable insights into possible bacteria/contaminant relationships and highlights bacterial species that could be used in remediation of specified contaminants. eMBRCatalogue is a moderated and searchable database cataloguing bioremediation case studies. Implemented as an eXtensible Markup Language (XML) database employing a user-generated-content framework, it provides background knowledge necessary for planning and execution of bioremediation activities. Developed following the construction of a comprehensive metabolic biodegradation network, eMBRHelper enables the delineation of possible biodegradation pathways for named contaminants. By integrating relevant chemical, enzymatic and genomics information, it attempts to model the interplay between contaminants, enzymes, microorganisms and degradation pathway, enabling researchers to make informed decisions for improved outcomes, particularly for remediation exercises involving bioaugmentation. The study also analysed usage of the portal and resources, made recommendations for future developments and highlights avenues for further informatics support for the bioremediation research sector.

Introduction 1.1 Occurrence of polycyclic aromatic hydrocarbons (PAH) in the environment Worldwide industrial and agricultural developments have released a large number of natural and synthetic hazardous compounds into the environment due to careless waste disposal, illegal waste dumping and accidental spills. As a result, there are numerous sites in the world that require cleanup of soils and groundwater. Polycyclic aromatic hydrocarbons (PAHs) are one of the major groups of these contaminants (Da Silva et al., 2003). PAHs constitute a diverse class of organic compounds consisting of two or more aromatic rings with various structural configurations (Prabhu and Phale, 2003). Being a derivative of benzene, PAHs are thermodynamically stable. In addition, these chemicals tend to adhere to particle surfaces, such as soils, because of their low water solubility and strong hydrophobicity, and this results in greater persistence under natural conditions. This persistence coupled with their potential carcinogenicity makes PAHs problematic environmental contaminants (Cerniglia, 1992; Sutherland, 1992). PAHs are widely found in high concentrations at many industrial sites, particularly those associated with petroleum, gas production and wood preserving industries (Wilson and Jones, 1993). 1.2 Remediation technologies Conventional techniques used for the remediation of soil polluted with organic contaminants include excavation of the contaminated soil and disposal to a landfill or capping - containment - of the contaminated areas of a site. These methods have some drawbacks. The first method simply moves the contamination elsewhere and may create significant risks in the excavation, handling and transport of hazardous material. Additionally, it is very difficult and increasingly expensive to find new landfill sites for the final disposal of the material. The cap and containment method is only an interim solution since the contamination remains on site, requiring monitoring and maintenance of the isolation barriers long into the future, with all the associated costs and potential liability. A better approach than these traditional methods is to completely destroy the pollutants, if possible, or transform them into harmless substances. Some technologies that have been used are high-temperature incineration and various types of chemical decomposition (for example, base-catalyzed dechlorination, UV oxidation). However, these methods have significant disadvantages, principally their technological complexity, high cost , and the lack of public acceptance. Bioremediation, on the contrast, is a promising option for the complete removal and destruction of contaminants. 1.3 Bioremediation of PAH contaminated soil & groundwater Bioremediation is the use of living organisms, primarily microorganisms, to degrade or detoxify hazardous wastes into harmless substances such as carbon dioxide, water and cell biomass Most PAHs are biodegradable unter natural conditions (Da Silva et al., 2003; Meysami and Baheri, 2003) and bioremediation for cleanup of PAH wastes has been extensively studied at both laboratory and commercial levels- It has been implemented at a number of contaminated sites, including the cleanup of the Exxon Valdez oil spill in Prince William Sound, Alaska in 1989, the Mega Borg spill off the Texas coast in 1990 and the Burgan Oil Field, Kuwait in 1994 (Purwaningsih, 2002). Different strategies for PAH bioremediation, such as in situ , ex situ or on site bioremediation were developed in recent years. In situ bioremediation is a technique that is applied to soil and groundwater at the site without removing the contaminated soil or groundwater, based on the provision of optimum conditions for microbiological contaminant breakdown.. Ex situ bioremediation of PAHs, on the other hand, is a technique applied to soil and groundwater which has been removed from the site via excavation (soil) or pumping (water). Hazardous contaminants are converted in controlled bioreactors into harmless compounds in an efficient manner. 1.4 Bioavailability of PAH in the subsurface Frequently, PAH contamination in the environment is occurs as contaminants that are sorbed onto soilparticles rather than in phase (NAPL, non aqueous phase liquids). It is known that the biodegradation rate of most PAHs sorbed onto soil is far lower than rates measured in solution cultures of microorganisms with pure solid pollutants (Alexander and Scow, 1989; Hamaker, 1972). It is generally believed that only that fraction of PAHs dissolved in the solution can be metabolized by microorganisms in soil. The amount of contaminant that can be readily taken up and degraded by microorganisms is defined as bioavailability (Bosma et al., 1997; Maier, 2000). Two phenomena have been suggested to cause the low bioavailability of PAHs in soil (Danielsson, 2000). The first one is strong adsorption of the contaminants to the soil constituents which then leads to very slow release rates of contaminants to the aqueous phase. Sorption is often well correlated with soil organic matter content (Means, 1980) and significantly reduces biodegradation (Manilal and Alexander, 1991). The second phenomenon is slow mass transfer of pollutants, such as pore diffusion in the soil aggregates or diffusion in the organic matter in the soil. The complex set of these physical, chemical and biological processes is schematically illustrated in Figure 1. As shown in Figure 1, biodegradation processes are taking place in the soil solution while diffusion processes occur in the narrow pores in and between soil aggregates (Danielsson, 2000). Seemingly contradictory studies can be found in the literature that indicate the rate and final extent of metabolism may be either lower or higher for sorbed PAHs by soil than those for pure PAHs (Van Loosdrecht et al., 1990). These contrasting results demonstrate that the bioavailability of organic contaminants sorbed onto soil is far from being well understood. Besides bioavailability, there are several other factors influencing the rate and extent of biodegradation of PAHs in soil including microbial population characteristics, physical and chemical properties of PAHs and environmental factors (temperature, moisture, pH, degree of contamination). Figure 1: Schematic diagram showing possible rate-limiting processes during bioremediation of hydrophobic organic contaminants in a contaminated soil-water system (not to scale) (Danielsson, 2000). 1.5 Increasing the bioavailability of PAH in soil Attempts to improve the biodegradation of PAHs in soil by increasing their bioavailability include the use of surfactants , solvents or solubility enhancers.. However, introduction of synthetic surfactant may result in the addition of one more pollutant. (Wang and Brusseau, 1993).A study conducted by Mulder et al. showed that the introduction of hydropropyl-ß-cyclodextrin (HPCD), a well-known PAH solubility enhancer, significantly increased the solubilization of PAHs although it did not improve the biodegradation rate of PAHs (Mulder et al., 1998), indicating that further research is required in order to develop a feasible and efficient remediation method. Enhancing the extent of PAHs mass transfer from the soil phase to the liquid might prove an efficient and environmentally low-risk alternative way of addressing the problem of slow PAH biodegradation in soil.

Groundwater contaminated with radioactive elements is a pressing environmental issue at current and former nuclear sites. Bioremediation, that is the stimulation of sediment microbial communities to remove radionuclides from solution, is a promising technology that may be used to treat groundwater contaminated with uranium and technetium. The application of two different bioremediation techniques has been investigated via a series of sediment microcosm and pure bacterial culture experiments including: the use of an electron donor to stimulate microbial-reduction of soluble and mobile uranium(VI) and technetium(VII) to insoluble U(IV) and Tc(IV) minerals; and the use of glycerol phosphate to stimulate the precipitation of biogenic uranium-phosphate minerals. Sediment samples were collected from the subsurface underlying the Sellafield nuclear site; to our knowledge this is the first time that such samples have been used in biogeochemical experiments. Microbial U(VI) reduction was stimulated in a variety of different lithology Sellafield sediments via the addition of an acetate/lactate electron donor mix. In the majority of samples U(VI) was successfully removed from solution as U(IV), highlighting the potential for biostimulation to be used to remediate groundwater contaminated with uranium at UK nuclear sites. However, this did not occur in two sediments, and investigations suggested that this may have been due to a paucity of bioavailable Fe(III) and hence low numbers of Fe(III)-reducing bacteria. Questions have been raised concerning whether microbially-reduced U(IV) would be suitable for maintaining low concentrations of uranium in groundwater over long time periods, particular if groundwater conditions become oxidising. Therefore a series of experiments were performed with biogenic U(IV) that had been aged for up to 18 months, to assess whether oxidative remobilisation may occur under strongly oxidising, worst case conditions. Microbially-reduced U(IV) was fully reoxidised via exposure to air, and partially reoxidised by nitrate. Evidence for an increase in the crystallinity of microbially-reduced U(IV) was observed during ageing, but despite this it did not become more recalcitrant to oxidative remobilisation. Microbial Tc(VII) reduction was also stimulated in Sellafield sediment using a range of slow-release proprietary electron donors. Further characterisation work, such as column studies and field trials would be required prior to this stimulated microbial reduction technology being implemented at a UK nuclear site. Biomineralisation of uranium phosphates was stimulated by the addition of glycerol phosphate to a Serratia environmental isolate and to Sellafield sediment for comparative studies. The Serratia species was able to remove U(VI) from solution by multiple metabolic pathways, including via the formation of uranyl(VI) phosphates of the autunite group. Uranium was precipitated in sediment systems as a crystalline U(IV) phosphate mineral similar to ningyoite. This was not susceptible to oxidative remobilisation by nitrate, and was only partially reoxidised via exposure to air under strongly oxidising end member conditions. Therefore this bioremediation technique may be more suitable for achieving long-term removal of uranium from groundwater, although again further characterisation work would be required prior to implementation.

Eight isolates (7 species) of white rot fungi were grown on soil extract agar amended with 0, 5 10 and 20 mg l- simazine, trifluralin and dieldrin, individually and as a mixture, under two different water regimes (-0.7 and -2.8 MPa water potential). The best isolates were T.versicolor (R26 and R101) and P.ostreatus, exhibiting good tolerance to the pesticides and water stress and the ability to degrade lignin and produce laccase in the presence of these pesticides. As a result, the activity of those three isolates plus Phanerochaete chrysosporium (well described for its bioremediation potential) was examined in soil extract broth in relation to differential degradation of the pesticide mixture at different concentrations (0-30 mg l-1) under different osmotic stress levels (-0.7 and -2.8 MPa). Enzyme production, relevant to P and N release (phosphomonoesterase, protease), carbon cycling (β-glucosidase, cellulase) and laccase, involved in lignin degradation was quantified. The results suggested that the test isolates have the ability to degrade different groups of pesticides, supported by the capacity for expression of a range of extracellular enzymes at both -0.7 and -2.8 MPa water potential. P.chrysosporium and T.versicolor R101, were able to degrade this mixture of pesticides independently of laccase activity, whereas P.ostreatus and T.versicolor R26 showed higher production of this enzyme. Complete degradation of dieldrin and trifluralin was observed, while about 80% of the simazine was degraded regardless of osmotic stress treatment in a nutritionally poor soil extract broth. The results with toxicity test (Toxalert®10), suggested the pesticides were metabolised. Therefore the capacity for the degradation of high concentrations of mixtures of pesticides and the production of a range of enzymes, even under osmotic stress, suggested potential applications in soil. Subsequently, microcosm studies of soil artificially contaminated with a mixture of pesticides (simazine, trifluralin and dieldrin, 5 and 10 mg kg soil-1) inoculated with P.ostreatus, T.versicolor R26 and P.chrysosporium, grown on wood chips and spent mushroom compost (SMC) were examined for biodegradation capacity at 15ºC. The three test isolates successfully grew and produced extracellular enzymes in soil. Respiratory activity was enhanced in soil inoculated with the test isolates, and was generally higher in the presence of the pesticide mixture, which suggested increased ii mineralization. Cellulase and dehydrogenase was also higher in inoculated soil than in the control especially after 12 weeks incubation. Laccase was produced at very high levels, only when T.versicolor R26 and P.ostreatus were present. Greatest degradation for the three pesticides was achieved by T.versicolor R26, after 6 weeks with degradation rates for simazine, trifluralin and dieldrin 46, 57, and 51% higher than in natural soil. And by P.chrysosporium, after 12 weeks, with degradation rates 58, 74, and 70% higher than the control. The amendment of soil with SMC also improved pesticide degradation (17, 49 and 76% increase in degradation of simazine, trifluralin and dieldrin compared with the control).

New Zealand has a large number (approx. 8000) of sites contaminated by persistent chemicals, of which approximately 10% arecontaminated with pentachlorophenol (PCP) aa a legacy of former timber treatment sites. The fungicide PCP was used extensively by the forestry industry from the late 1940s to prevent sapstaining of wood. New Zealand was a heavy user of industrial grade PCP because of the predominance of radiata pine (Pinus radiata) which is a soft timber and more susceptible than most tree species to sapstain fungi. International research has shown that soils contaminated by such xenobiotics may be ameliorated using white-rot fungi. To avoid the uncertainties associated with the release of foreign organisms into the New Zealand environment, as legislated by the Hazard Substances and New Organisms Act (HSNO) and governed by the Environmental Risk Management Authority (ERMA), a national research initiative was undertaken in 1996 to study the potential of New Zealand native white-rot fungi for bioremediation. Native white-rot isolates were (1) collected (bioprospecting), (2) selected for their ability to degrade xenobiotics - in the initial phase using PCP as the model compound and (3) studied for their mechanisms and pathways of degradation. Organic waste materials were also evaluated for their suitability to serve as a carrier for fungal augmentation to polluted soil. This PhD study formed part of this larger national research programme, with very close interaction between the different researchers and research activities. The aim of this thesis was to optimise white-rot bioremediation of New Zealand isolates. The work described here was led by me, and the principal results are mine. Selected organisms were evaluated for PCP loss and breakdown in soil. Soil limiting factors (such as soil type, moisture, temperature, pollutant concentration) affecting colonisation of augmented isolates were identified. These laboratory results then were transferred into the field and PCP degradation studied using proto-type biopiles.

A petroleum-contaminated soil remediation study was conducted in a greenhouse. The system consisted of 36 pots, 12 were vegetated with squash, 12 were vegetated with fescue grass and the last 12 units served as unvegetated controls. For each group, three treatments were applied, 1) the addition of single dose of nutrients, 2) the addition of double dose of nutrients and 3) the addition of double dose of nutrients and acclimated bacteria to the irrigation water. The two plants were selected to represent extremely different species in terms of transpiration potential and root density in order to better understand the mechanisms involved in phytoremediation. Clay sandy soil (3: 1, by weight) was spiked with Fuel oil No.2 and allowed to weather for 1 week before it was placed in the pots. Under all study treatments, units vegetated with fescue grass had significantly less TPH concentration than the unvegetated controls after 10 weeks. Units vegetated with squash had significantly less TPH concentration than the un vegetated controls after 10 weeks only under treatment 3. Squash significantly accumulated TPH in the shoot under all treatments while grass shoot accumulated TPH significantly only under treatment 1. The mechanisms most important in phytoremediation seemed to include plant uptake of TPH, desorption and enhanced bioavailability by transpiration-induced water movement in the rhizosphere and root stimulation of microbial degradation.
Ph. D.

This work focused on using a microbubble dispersion to deliver hydrogen and carbon dioxide to anaerobic consortia to stimulate their ability to reductively dehalogenate tetrachloroethylene all the way to ethene and ethane. A continuous flow system, consisting of six anaerobic soil column bioreactors, inoculated with sediments from Virginia Tech's Duck Pond, was used for this study. Two columns received microbubbles containing hydrogen and carbon dioxide, two received sodium propionate, and two were not fed a substrate. A 30 micromolar PCE solution was delivered to the consortia at 3 ml/min. Microbubbles containing a mixture of 90% hydrogen and 10% carbon dioxide were effectively produced in a closed spinning disk generator, and were acceptable for delivering the gases to the columns. After the biodegradation study was completed, the microbubbles were found to have a pH of 4.4, due to the carbon dioxide. Microbubbles amended with NaOH to 0.01 molar yielded pH neutral microbubbles with improved stability. Methane was measured in all six columns throughout the experiment, verifying that methanogens were present. Methane levels were highest in the propionate columns, showing the the methanogens there were more active. Methane levels in the microbubble columns were similar to those in the control columns. Propionate and acetate were not detected in the columns where propionate was fed, showing that proton reducers and acetoclastic methanogens were both active. Recovery of PCE and the degradation products was almost 90% in the microbubble and control columns where most of the PCE was recovered in the effluent. The predominant product in both systems was TCE, although some ethene was detected in all four columns. The control consortia produced TCE averaging about five micromolar while the microbubble columns averaged about two micromolar TCE. One of the components of the microbubbles probably caused the lowered amounts of PCE reduction. That some ethene was seen in the microbubble columns suggests different conditions can be found to stimulate the further reduction of PCE with hydrogen and carbon dioxide microbubbles. The product recovery in the propionate columns was about 64%. Over half of the injected PCE was dechlorinated to ethene and ethane.
Ph. D.

The area of secondarily salinized lands is increasing at a faster rate over time. Many irrigation districts around the world are shrinking as a result of secondarily salinized soils. This is resulting in crop yield losses. Irrigation practices with low drainage are intensifying this problem. Bioremediation of salinized soils with halophytes is one of the means of reversing this process. In these studies, we tested the growth and performance of four salt tolerant halophytes to varying levels of salinity. We analyzed the salt content of the plant tissues at different salinities, in order to determine how the plants' tissues reflect the increases in salinity. It was discovered that Allenrolfea occidentalis tolerates and grows well at higher salinities than the other plants tested. Furthermore, the concentration of salt in the aerial plant tissue was high and increased further in response to the external salt concentration. Halophytes such as A. occidentalis can be used to remediate abandoned salt affected lands and their biomass can have an added economic value. On the other hand, domestication of wild halophytes for agronomic purposes represents another opportunity to address the increasingly salinized soils and shortages of freshwater around the world. In these studies, we assessed the potential for improvement of an oilseed halophyte, Salicornia bigelovii, through selective breeding. We compared plant characteristics of S. bigelovii cultivars produced in breeding programs with wild germplasm in a green house common garden experiment. We concluded that S. bigelovii has sufficient genetic diversity among wild accessions and cultivars to support a crop improvement program to introduce desirable agronomic characteristics into this wild halophyte.

Uranium contamination of groundwater from mining and milling operations is an environmental concern. Reductive precipitation of soluble and mobile hexavalent uranium (U(VI)) contamination to insoluble and immobile tetravalent uranium (U(IV)) constitutes the most promising remediation approach for uranium in groundwater. Previous research has shown that many microorganisms are able to catalyze this reaction in the presence of suitable electron-donors. The purpose of this work is to explore lowcost, effective alternatives for biologically catalyzed reductive precipitation of U(VI). Methanogenic granular sludge from anaerobic reactors treating industrial wastewaters was tested for its ability to support U(VI)-reduction. Due to their high microbial diversity, methanogenic granules displayed intrinsic activity towards U(VI)-reduction. Endogenous substrates from the slow decomposition of sludge biomass provided electron-equivalents to support efficient U(VI)-reduction without external electrondonors. Continuous columns with methanogenic granules also demonstrated sustained reduction for one year at high uranium loading rates. One column fed with ethanol, only enabled a short-term enhancement in the uranium removal efficiency, and no enhancement over the long term compared to the endogenous column. Nitrate, a common co-contaminant of uranium, remobilized previously deposited biogenic U(IV). U(VI) also caused inhibition to denitrification. An enrichment culture (EC) was developed from a zero-valent iron (Fe⁰)/sand packed-bed bioreactor. During 28 months, the EC enhanced U(VI)-reduction rates by Fe⁰ compared with abiotic Fe⁰ controls. Additional experiments indicated that the EC prevented the passivation of Fe⁰ surfaces through the use of cathodic H₂ for the reduction of Fe(III) in passivating corrosion mineral phases (e.g. magnetite) to Fe²⁺. This contributed to the formation of secondary minerals more enriched with Fe(II), which are known to be chemically reactive with U(VI). To determine the toxicity of U(VI) to different populations present in uranium contaminated sites, including methanogens, denitrifiers and uranium-reducers, experiments were carried out with anaerobic mixed cultures at increasing U(VI) concentrations. Significant inhibition to the presence of U(VI) was observed for methanogens and denitrifiers. On the other hand uranium-reducing microorganisms were tolerant to high U(VI) concentrations. The results of this dissertation indicate that direct microbial reduction of U(VI) and microbially enhanced reduction of U(VI) by Fe⁰ are promising approaches for uranium bioremediation.

Polaromonas sp. strain JS666 is the only isolated bacterium capable of aerobic growth using the groundwater pollutant cis-1,2-dichloroethene (cDCE) as a sole carbon source. Its genome has a wealth of evidence of recent gene acquisition through horizontal gene transfer, and contains gene clusters predicted to encode enzymes allowing the metabolism of a wide variety of xenobiotic compounds. Culture growth using each of these hypothesized substrates was tested experimentally, and many were confirmed as sole carbon sources for strain JS666. In addition to pollutant degradation, many of these metabolic pathways have applicability in the field of biocatalysis, as does the genome-assisted pathway prediction approach to biocatalyst discovery. During (or immediately following) growth on cDCE, cultures of Polaromonas sp. strain JS666 oxidize ethene to epoxyethane at an increased rate, and also cometabolically oxidize several other chlorinated ethenes. Given the involvement of a monooxygenase in other species' 1-chloroethene (vinyl chloride) oxidation, it was hypothesized that alkene oxidation in strain JS666 was due to the activity of a monooxygenase that also was responsible for the first step in cDCE oxidation. The alkene oxidation activity of strain JS666 was investigated using gene expression analysis, proteomics, and whole-cell kinetic assays. Results of these experiments pointed to the upregulation of a cyclohexanone monooxygenase (CHMO) during growth on cDCE and during oxidation of ethene. To determine the activity of this cyclohexanone monooxygenase, its gene was cloned and heterologously expressed in an E. coli host. Our CHMO expression system exhibited activity on cyclohexanone, but not cDCE or ethene, disproving our hypothesis about its involvement in alkene oxidation. The heterologously expressed monooxygenase was also investigated for enantioselective oxidation of racemic cyclic ketones to chiral lactones, and was discovered to have very high enantioselectivity with the tested compounds. Chiral lactones and other single-enantiomer oxidation products are valuable for fine chemical synthesis, and their biocatalytic production is more environmentally sustainable and often less expensive than traditional techniques. The research described in the following chapters illustrates the many opportunities that arise when the fields of bioremediation and biocatalysis converge. The shared research goals and methods of these two areas lend themselves to interdisciplinary research, and increased communication and crossover between them should provide benefits for both environmental remediation and sustainable chemical synthesis.

I sedimenti marini costituiscono un serbatoio per i contaminanti organici ed inorganici, provenienti da attività industriali, inquinamento atmosferico, fiumi, fuoriuscite accidentali, fognature ed altre fonti. I sedimenti di dragaggio sono spesso caratterizzati dalla presenza di elevate concentrazioni di contaminanti organici e inorganici, influenzando il ri-uso dei sedimenti e/o il loro smaltimento. Diversi tipi di trattamenti possono essere utilizzati per ridurre il livello di contaminazione, e tra le tecnologie disponibili, strategie eco-compatibili di biorisanamento possono essere scelte, come la biostimolazione di comunità microbiche autoctone o la bioaugmentazione, introducendo specifici ceppi microbici. La stimolazione microbica mediante aggiunta di nutrienti è un approccio comune, in grado di migliorare i tassi di biodegradazione di idrocarburi nei sedimenti marini. I metalli, che non possono essere degradati, possono passare da uno stato redox ad un altro, influenzando la loro solubilità. Il biorimedio di contaminanti inorganici può essere indirizzato al cambiamento di speciazione e ripartizione dei metalli, aumentando la loro solubilità in acqua o la loro stabilità nel sedimento. È stato inoltre osservato che l'applicazione di strategie per la bonifica dei sedimenti è in grado di determinare cambiamenti nella composizione della comunità procariote, con la selezione di alcuni ceppi piuttosto che altri. Questa tesi si occupa di biorisanamento di sedimenti contaminati, e le principali domande poste sono: 1- I trattamenti diretti alla degradazione di idrocarburi possono influenzare la ripartizione dei metalli? 2- È possibile valutare la performance di degradazione degli idrocarburi utilizzando semplici modelli cinetici basati sui tassi di crescita dei procarioti? 3- Esiste una relazione tra la degradazione degli idrocarburi e la biodiversità batterica? 4- Quali sono i potenziali ruoli dei filotipi procariotici dominanti nella degradazione di idrocarburi e nella ripartizione dei metalli? Esperimenti di microcosmo sono stati allestiti per rispondere a queste domande. Nel primo esperimento, l'attenzione è stata indirizzata a sedimenti contaminati da idrocarburi del petrolio e metalli pesanti. L'ipotesi era che in esperimenti di microcosmo in condizioni anaerobiche sottoposti a strategie di biostimolazione e bioagumentazione, una maggiore biodegradazione di idrocarburi modificasse la ripartizione dei metalli pesanti, con potenziali ripercussioni sulla la loro mobilità e biodisponibilità. L'approccio di bioremedio utilizzato in questi esperimenti ha determinato una diminuzione significativa della concentrazione di idrocarburi, ma anche cambiamenti della ripartizione dei metalli pesanti. È stata affettuata un'altra serie di esperimenti, e un modello cinetico semi-empirico è stato applicato con successo ai dati sperimentali delle variazioni temporali delle concentrazioni residue di idrocarburi e alle abbondanze microbiche, in grado di prevedere le prestazioni del biorisanamento. Tale modello, eventualmente adattato alle caratteristiche biogeochimiche sito-specifiche del sito, può essere uno strumento utile nella progettazione di tecnologie eco-compatibili per la bonifica di sedimenti contaminati. Cercando di capire meglio gli effetti dovuti alla manipolazione dei sedimenti in prove di laboratorio, sono stati studiati i cambiamenti nella comunità batterica in esperimenti di biorisanamento con sedimenti marini contaminati, sia in condizioni aerobiche sia anaerobiche. Una correlazione positiva è stata trovata tra la biodiversità batterica e l’efficacia di biodegradazione degli idrocarburi, sia in condizioni aerobiche sia anaerobiche. L’efficacia del biorimedio potrebbe essere stata aumentata da possibili interazioni facilitative tra microrganismi. Considerando l'importanza della biodiversità procariotica ai fini del biorimedio, è stato effettuato uno studio per determinare la composizione dei principali procarioti coinvolti nel biorisanamento di sedimenti contaminati in condizioni anaerobiche. Dai dati del sequenziamento dei dominanti geni che codificano per il 16S rRNA, si è riscontrato che la maggior parte degli archaea che componevano la comunità microbica era affiliata al phylum degli Euryarchaeota, in particolare agli ordini del Methanomicrobiales, Methanosarcinales e Thermoplasmatales. Le sequenze geniche batteriche appartenevano agli Alpha-, Gamma- e Deltaproteobacteria, Firmicutes, Chloroflexi, Actinobacteria, Bacteroidetes e Verrucomicrobia. I risultati di questo lavoro suggeriscono che nei sedimenti anossici specifici taxa batterici influenzano l’efficacia di degradazione degli idrocarburi mentre gli archaea potrebbero essere coinvolti nei cambiamenti di ripartizione dei metalli. Questa attività di ricerca ha permesso non solo di avere una migliore comprensione del ruolo delle comunità microbiche in relazione al destino dei contaminanti per i campioni di sedimento specifiche, ma anche di acquisire conoscenze e competenze utili nel campo del biorimedio di sedimenti contaminati. Infatti, si possono incontrare molte difficoltà legate all’eterogeneità del campione, concentrazione in traccia dei contaminanti, differenze sitospecifiche. Quindi risulta fondamentale l’utilizzo di un approccio metodologico interdisciplinare, basato su strumenti di ecologia microbica, biogeochimica e analisi di processo.
Marine sediments represent a sink for organic and inorganic contaminants, coming from industrial activity, air pollution, rivers, spills, sewage and other sources. Dredged sediments are often characterized by the presence of high organic and inorganic concentrations, influencing sediment re-use and/or disposal. Several kinds of treatments can be used to reduce the level of contamination, and among the available technologies the use of eco-compatible bioremediation strategies can be chosen, such as biostimulation of autochthonous microbial communities or bioaugmentation, introducing specific microbial strains. Microbial stimulation by means of nutrient addition is a common approach, able to enhance hydrocarbon biodegradation rate in marine sediments. Metals, which cannot be degraded, can only pass from one redox state to another, which influences their solubility. Bioremediation of inorganic contaminants can be aimed at a change in speciation and partitioning of metals, either increasing their solubility in water or increasing their stability in the sediment. It has also been observed that the application of strategies for sediment remediation can determine shifts in the composition of the prokaryotic community, with the selection of certain strains rather than others. This thesis deals with bioremediation of contaminated sediments, and the main questions addressed are: 1- Can treatments aimed at hydrocarbon degradation influence metal partitioning? 2- Is it possible to assess hydrocarbon degradation extent using simple kinetic models based on prokaryotic growth rates? 3- Is there a relationship between hydrocarbon degradation and bacterial biodiversity? 4- Which are the potential roles of the dominant prokaryotic phylotypes in hydrocarbon degradation and metal partitioning? Microcosm experiments were set up to answer these questions. In the first experiment, the focus was addressed to sediments contaminated with petroleum hydrocarbons and heavy metals. The hypothesis was that in microcosm experiments under anaerobic conditions submitted to biostimulation and bioagumentation strategies, an enhanced biodegradation of hydrocarbons modified the partitioning of heavy metals with potential consequences on their mobility and bioavailability. The bioremediation approach used in these experiments determined a significant decrease in hydrocarbon concentration, but also changes of the heavy metal partitioning. Another set of experiments was designed, and a semi-empirical kinetic model was successfully fitted to experimental temporal changes of hydrocarbon residual concentrations and microbial abundances, able to predict bioremediation performances. Such model, eventually adapted to biogeochemical characteristics that are site-specific, may be a useful tool when designing eco-compatible technologies for contaminated sediment remediation. Trying to better understand the effects due to sediment manipulation in laboratory trials, the changes in the bacterial community were studied in bioremediation experiments with contaminated marine sediments both under aerobic and anaerobic conditions. A positive correlation was found between bacterial biodiversity and hydrocarbon biodegradation performance both under aerobic and anaerobic conditions. Bioremediation extent could be enhanced by possible mutually facilitative interactions among microorganisms. Considering the importance of prokaryotic biodiversity for bioremediation purpose, a study was carried out to characterize the composition of the main prokaryotes involved in bioremediation of contaminated sediments under anaerobic conditions. From sequencing data of the dominant 16S rRNA genes on the sediment samples, it was found that most of the archaea that comprised the microbial community were affiliated to the phylum Euryarchaeota, in particular to the orders of Methanomicrobiales, Methanosarcinales, and Thermoplasmatales. Bacterial gene sequences belonged to the Alpha-, Gamma- and Deltaproteobacteria, Firmicutes, Chloroflexi, Actinobacteria, Bacteroidetes, and Verrucomicrobia. The results of this work suggested that in anoxic sediments specific bacterial taxa influence the extent of hydrocarbon degradation whereas archaea could be involved in changes of metal partitioning. This research activity has allowed not only to have a better understanding of the role of microbial communities in relation to contaminant fate for specific sediment samples, but also to develop a know-how useful when facing with sediment bioremediation. In fact, many difficulties might be encountered related to sample heterogeneity, trace concentration of contaminants and site-specificity. Therefore it is fundamental to use an interdisciplinary methodological approach, based on tools of microbial ecology, biogeochemistry and process analysis.

The work undertaken during the PhD was aimed at evaluating alternative bioremediation methods, trying to focus on biological and low environmental impact techniques. At first, the main purpose was to create a robust and replicable method, capable to break-down priority pollutants for the environment, using natural microorganisms or their consortia. Was decided to study the degradation of a particular class of priority micro-pollutants, the Polycyclic Aromatic Hydrocarbons (PAHs), and evaluate the bioremediation capabilities of a white-rot fungus, the Pleurotus sajor caju, and a selected bacteria consortium (BULAB 5738). PAHs are organic compounds, composed of multiple aromatic rings, that persist in the environment because they are very stable due to their molecular structure and therefore are difficult to be attacked by microorganisms present in nature. The study of PAHs and their bio-degradation is also motivated by the difficulties encountered in the scientific literature, as appears from the current state of the art. As far as this study is concerned, the use of 'white rot' fungi and selected bacterial consortia is a valid and real opportunity for the degradation of many toxic molecules, including PAHs. Another part of the PhD work was dedicated to the study of microalgae’s degradation capabilities, with particular regard to the potential of these microorganisms to remove nutrients from waste water. The microalgae are often studied for energy purposes and for high added-value molecules production; in this work the focus was on their capabilities on nitrogen and phosphorus removal. These compounds are present on wastewater and on aquacolture processes that can be treated by microalgae. This alternative was considered feasible in the short term, in terms of applicability and practical results. Furthermore, the capacity of Nannochloropsis oculata to use high loads of nitrogen and phosphorus as a source of nourishment, has been explored, in order to remove these pollutants from the wastewater. The Nannochloropsis oculata was chosen for its ability to survive in adverse conditions and for its relatively fast growth velocity, if compared to other photosynthetic microorganisms. Design of Experimental approach was used for all the experimental campaigns, to maximize the obtained information and minimize the number of experiments. Thanks to the statistical analysis it was possible to obtain significant information about single and multiple effect, that influence the studied process. Both experiments with the Pleurotus s.c. and the bacterial consortium showed how they tolerate and break-down the selected PAHs. The main differences lay on the dynamics of growth and on the timing of the two different kind of microorganism, as well as the different pathways of degradation process. In particular, the fungi growth is slower than the one of bacterial consortium, but both are able to degrade the selected pollutants. As far as the selected microalgal species are concerned, the Nannochloropsis o. showed a good removal capacity for nitrogen, evaluated as nitrates and urea, obtaining the complete degradation, even on high loads up to concentrations of 1000 mg/l of nitrogen, as a nitrate.
The work undertaken during the PhD was aimed at evaluating alternative bioremediation methods, trying to focus on biological and low environmental impact techniques. At first, the main purpose was to create a robust and replicable method, capable to break-down priority pollutants for the environment, using natural microorganisms or their consortia. Was decided to study the degradation of a particular class of priority micro-pollutants, the Polycyclic Aromatic Hydrocarbons (PAHs), and evaluate the bioremediation capabilities of a white-rot fungus, the Pleurotus sajor caju, and a selected bacteria consortium (BULAB 5738). PAHs are organic compounds, composed of multiple aromatic rings, that persist in the environment because they are very stable due to their molecular structure and therefore are difficult to be attacked by microorganisms present in nature. The study of PAHs and their bio-degradation is also motivated by the difficulties encountered in the scientific literature, as appears from the current state of the art. As far as this study is concerned, the use of 'white rot' fungi and selected bacterial consortia is a valid and real opportunity for the degradation of many toxic molecules, including PAHs. Another part of the PhD work was dedicated to the study of microalgae’s degradation capabilities, with particular regard to the potential of these microorganisms to remove nutrients from waste water. The microalgae are often studied for energy purposes and for high added-value molecules production; in this work the focus was on their capabilities on nitrogen and phosphorus removal. These compounds are present on wastewater and on aquacolture processes that can be treated by microalgae. This alternative was considered feasible in the short term, in terms of applicability and practical results. Furthermore, the capacity of Nannochloropsis oculata to use high loads of nitrogen and phosphorus as a source of nourishment, has been explored, in order to remove these pollutants from the wastewater. The Nannochloropsis oculata was chosen for its ability to survive in adverse conditions and for its relatively fast growth velocity, if compared to other photosynthetic microorganisms. Design of Experimental approach was used for all the experimental campaigns, to maximize the obtained information and minimize the number of experiments. Thanks to the statistical analysis it was possible to obtain significant information about single and multiple effect, that influence the studied process. Both experiments with the Pleurotus s.c. and the bacterial consortium showed how they tolerate and break-down the selected PAHs. The main differences lay on the dynamics of growth and on the timing of the two different kind of microorganism, as well as the different pathways of degradation process. In particular, the fungi growth is slower than the one of bacterial consortium, but both are able to degrade the selected pollutants. As far as the selected microalgal species are concerned, the Nannochloropsis o. showed a good removal capacity for nitrogen, evaluated as nitrates and urea, obtaining the complete degradation, even on high loads up to concentrations of 1000 mg/l of nitrogen, as a nitrate.

Seven aerobic field trichloroethene (TCE) bioremediation projects were evaluated to determine key parameters leading to in situ TCE bioremediation effectiveness. Key parameters identified were: 1) presence of other contaminants, 2) efficacy of the cometabolic inducer, 3) technology design, and 4) site soils and hydraulics. These four parameters were then used to evaluate the pilot bioremediation operation at Air Force Plant #44 in Tucson, Arizona. The pilot operation was poorly designed. Site characterization appeared insufficient; laboratory studies were not representative of site conditions; 1, 1-dichloroethene appeared to inhibit TCE degradation; the purpose of the injected methanol ( cometabolic inducer) was unclear. Well design, specifically screen interval location, also contributed to technology deficiency. Soil type appeared to be the most limiting component; hydraulic conductivity (K) representative of the contaminated clay at the APP #44 site was estimated at 1.5 x 10-5 cm/sec. Over the course of the trial, spatially averaged TCE concentrations decreased by 41 %. Well chloride data calculations indicated that a 27% reduction may be attributable to dilution, thereby suggesting that only a 14% decrease in concentrations may be attributable to biological degradation.

The presence of nitrates (NO3-) in groundwater is a worldwide concern. The high energy demand and environmental impact of available technologies requires investigating new technologies. This thesis was focused on investigating the usage of bioelectrochemical systems (BES) for treating nitrate-polluted groundwater. BES uses microorganisms able to catalyze oxidation/reduction processes by delivering/obtaining electrons from an electrode. In this thesis, microorganisms able to use the electrode as an electron donor (biocathode) to reduce nitrates into dinitrogen gas (inert) were investigated. As a result, a process could be patented, in which BES are able to treat nitrates at high denitrification rates (up to 700 gN•m-3NCC•d-1), with a competitive energy demand (0.68•10-2 – 1.27•10-2 kWh•gN-1treated), without sludge generation nor chemical dosing. Moreover, the microorganisms were electrochemically characterized, and the key subcommunities of the process were elucidated. In summary, bioelectrochemical systems have the potential for becoming a competitive alternative for the treatment of nitrate-polluted groundwater
La presència de nitrats (NO3-) en aigües subterrànies és una preocupació global. L’alt cost energètic i ambiental de les tecnologies actuals requereixen la investigació de noves estratègies. Aquesta tesi ha investigat la utilització de sistemes bioelectroquímics (BES) pel tractament d’aigües subterrànies contaminades per nitrats. Les BES es basen en microorganismes capaços de realitzar oxidacions/reduccions tot alliberant/captant electrons d’un elèctrode. Aquesta tesi ha investigat l’ús de bactèries capaçes d’utilitzar l’elèctrode com a donador d’electrons (biocàtode) per reduir el nitrat a dinitrogen gas (compost inert). Com a resultat, s’ha patentat un procés que permet desnitrificar a altes velocitats (700 gN•m-3NCC•d-1), a un cost energètic competitiu (0.68•10-2 – 1.27•10-2 kWh•gN-1tractat), sense generar fangs ni addicionar substàncies químiques. També s’ha caracteritzat electroquímicament els microorganismes i s’ha elucidat les subcomunitats microbianes responsables de la desnitrificació. En definitiva, aquesta tesi demostra que els sistemes bioelectroquímics poden esdevenir una alternativa competitiva pel tractament d’aigües subterrànies contaminades per nitrats

The poorly controlled disposal of chromium ore processing residue (COPR) is a globally widespread problem due to its potential to form chromium contaminated hyperalkaline (pH > 12) leachates. These highly oxidising leachates typically contain chromium in the Cr(VI) oxidation state as its chromate anion (CrO42-). This anion is highly mobile, toxic, carcinogenic, and exhibits a high degree of bioavailability. Under reducing conditions chromium exists in the non-toxic and poorly soluble Cr(III) oxidation state. Thus, the reduction of Cr(VI) to Cr(III) is often the goal of remediative strategies. In anaerobic subsurface environments where reducing conditions are established by the indigenous microbial population, chromium reduction can occur naturally. The microbial transformation of Cr(VI) to Cr(III) can be both a result of its direct use in microbial metabolism, or through its indirect reaction with microbially produced reduced species, e.g. Fe(II). This study has used a multidisciplinary approach to investigate the biogeochemical influences on the fate and stability of Cr(VI) leaching from a site of COPR in the north of England. Reducing sediments encountered directly beneath the COPR waste were found contain elevated concentrations of chromium. These sediments were shown to be able to remove aqueous Cr(VI) from solution when incubated with contaminated site groundwater in microcosm incubation experiments. This removal is likely a result of the abiotic reduction by soil associated microbially produced Fe(II), followed by precipitation as insoluble Cr(III) hydroxides. X-ray absorption spectroscopy (XAS) and electron microscopy confirms the association of chromium as Cr(III) with iron in these soils, hosted as a mixed Cr(III)-Fe(III) oxyhydroxide phase. Upon air oxidation, only minor amounts of chromium was remobilised from these sediments as Cr(VI). A diverse population of alkaliphilic microorganisms are indigenous to this horizon, capable of successful metabolism despite elevated pH values. This population was found to contain a consortium of microorganisms capable of iron reduction when incubated at pH 9 to 9.5. Microbial community analysis found taxonomic similarity to several known metal reducing alkaliphiles from the phylum Firmicutes. These results suggest that the novel action of iron reducing alkaliphiles indigenous to reducing sediments beneath COPR sites may provide zones of natural chromium attenuation via microbially mediated mechanisms of Cr(VI) transformation.

Analytical techniques applicable to the assay and remediation of cutting/mud matrices have been developed, utilising soxhlet extraction with dichloromethane and a drying agent followed by analysis using Gas Chromatography (FID). Calibration curves of oil content were produced for Novatec and Versaplus coated cuttings that were also sized by wet and dry sieving techniques, demonstrating their variable nature. The oil in each size fraction was assessed and showed that the finer fractions preferentially adsorbed the oil. Bacteria were isolated from the cuttings, muds and the pure oils to see if any indigenous species could, with optimum conditions, remediate the oil they contained. The resulting isolates were batch-tested in the laboratory in a minimal medium, with the drill cuttings providing the sole carbon source. Each isolate was scored for remediation performance, with reduction in oil varying from 50% to 6% within one week. Subsequently three bacteria (A,D & J) were identified using 16SrRNA sequencing; they were Bacillus Thuringiensls (A&D) and a novel species related to Bacillus oleronius. These were then tested slurry-phase in a rotating drum bioreactor designed and fabricated for the research against a known remediator, Rhodococcus 9737, and a non-inoculated control for four weeks. All the reactors remediated, but Rhodococcus 9737 reduced the oil to 35% of the original, A, D and other isolates as a consortium to 83% and J, 90%. Further tests in the bioreactors, after a modification to improve the air supply gave reductions of around 50% after four weeks. The high clay content of the cuttings was detrimental to significant levels of bioremediation in a slurry-phase bioreactor. Manures were added to the drill cuttings and tested in the bioreactors as a solid-phase system. These degraded the cuttings oil to 2% (v/v), a 96% reduction. Composting was thus more applicable for a high clay content drilling waste bioremediation system.

Arsenic is a toxic metalloid existing everywhere in the nature. It is toxic to most organisms and considered as human carcinogen. Arsenic contamination leads to severe health problems with diseases like damage of skin, lung, bladder, liver and kidney as well as central nervous system. As arsenic can be found everywhere in nature it may come in contact with food chain very easily through either water or cultivated crops. My thesis works include studies of bioremediation of arsenic by microorganisms. In this experiment the test organisms were collected from the Hazaribagh tanning industrial area of Dhaka, Bangladesh. The whole laboratory works were performed with two types of bacterial strains. Genomic DNA isolation and restriction digestion of genomic DNA, plasmid DNA isolation, Growth response to different concentrations of Arsenic, minimum inhibitory concentration (MIC), plasmid degradation procedures were carried out during this experiment. The MIC value for amoxicillin of these test organisms was 300 μg/ml and they are able to degrade 5 mM arsenite (AsIII) and 40 mM arsenate (AsV). Though the experiment was carried out with two bacterial strains but by observing all experimental data such as restriction digestion, growth response to the arsenic before and after treated with ethidium bromide and minimum inhibitory concentration it can be concluded that these two strains were not different. These bacteria are able to survive in high concentration of antibiotics and arsenic (AsV and AsIII). Loss of plasmid resulted no growth on media containing arsenic. These results support that plasmid contains important genes that are responsible for surviving bacteria in stress conditions.

Thesis submitted in fulfilment of the requirements for the degree Magister Technologiae: Chemical Engineering in the Faculty of Engineering at the Cape Peninsula University of Technology 2014
The legislative requirements for handling cyanide containing wastewater have become stringent internationally. Cyanide properties make it indispensable in the mining industry especially for gold recovery. The resultant wastewater generated is discarded to tailing ponds. Any leakages or total collapse of tailing ponds can result in the contamination of surface water bodies; endangering aquatic organisms’ and humans’ alike. The over reliance on physical and/or chemical treatment methods for cyanide wastewater treatment is not sustainable due to high input costs and the generation of by-products. A feasible alternative treatment method for cyanide contaminated wastewater is the biodegradation method, as a wide range of microorganisms can degrade cyanide. In this study, the cyanide biodegradation ability of Fusarium oxysporum was assessed in two stages. Firstly, optimal operating conditions for maximum cyanide biodegradation were determined using a central composite design (CCD) at an elevated cyanide concentration, i.e. 500 mg F-CN/L. Thereafter, using the optimum conditions obtained, (i.e temperature 22°C and pH 11), cyanide biodegradation kinetics and microbial growth kinetics in the cultures at lower cyanide concentrations of 100, 200 and 300 mg F-CN/L were assessed. This was followed by the assessment of cyanide biodegradation at a temperature of 5°C, which was used to simulate winter conditions. In general, lower cyanide concentrations are used in the extraction of gold, therefore, the resultant wastewater will contain free cyanide concentration less than 300 mg F-CN/L. For the first stage of experiments, an isolate, Fusarium oxysporum from cyanide containing pesticides was cultured on potato dextrose agar (PDA) plates, followed by incubation at 25°C for 5 days. A response surface methodology (RSM) was used to evaluate design parameters for the biodegradation of cyanide by this fungus. The temperature evaluated at this stage ranged from 9°C to 30°C and pH range of 6 to 11 in cultures solely supplemented with agrowaste, i.e Beta vulgaris waste. Beta vulgaris is commonly known as Beetroot. The Fusarium oxysporum inoculum (2% v/v) was grown on a Beta vulgaris waste solution (20% v/v), as the sole carbon source in a synthetic gold mine wastewater (39% v/v) containing heavy metals; arsenic (7.1 mg/L), iron (4.5 mg/L), copper (8 mg/L), lead (0.2 mg/L) and zinc (0.2 mg/L), for 48 hours using a rotary shaker at 70 rpm. Thereafter, free cyanide as a potassium cyanide solution (39% v/v), was added to the cultures to make a final cyanide concentration of 500 mg F-CN/L in the culture medium which was incubated for a further 72 hours at 70 rpm. Optimal operating conditions for the biodegradation of cyanide were then determined using a numerical option in the Design-Expert® software version 6.0.8 (Stat-Ease Inc., USA). Subsequently, using the optimal pH obtained (pH =11) and a preselected temperature of 5°C (to represent winter conditions), cyanide biodegradation rates and microbial growth kinetic studies were carried out using Beta vulgaris waste containing a Fusarium oxysporum (0.7% v/v; grown overnight) inoculum in wastewater (32.7% v/v) and potassium cyanide in phosphate buffer (53.7% v/v). The cultures contained 100, 200 and 300 mg F-CN/L. The cultures were incubated in an orbital shaker at 70 rpm for 144 hours and samples taking every 24 hours. An Ordinary Differential Equation (ODE) solver (Polymath) was used for modelling cyanide degradation kinetics while the Monod’s growth kinetic model was used to monitor the microbial growth parameters of the cultures. For the first stage, the optimum operating conditions were determined as a temperature of 22°C and a pH of 11 for maximum cyanide biodegradation of 277 mg F-CN/L from an initial cyanide concentration of 500 mg F-CN/L over a 72 hour period, with residual ammonium-nitrogen and nitrate-nitrogen of 150 mg NH4+-N/L and 37 mg NO3--N/L, respectively. Although, the residual ammonium-nitrogen inhibited cyanide biodegradation, it was consumed as a nitrogen source for microbial growth. The Beta vulgaris waste was determined to be a suitable substrate for cyanide degradation. From the biodegradation response quadratic model, temperature was determined to influence cyanide biodegradation. For the cyanide degradation kinetics, at an optimum temperature of 22°C, the biodegradation efficiency was 77%, 58% and 62% with the corresponding maximum microbial population of 1.56 x 107, 1.55 x 107 and 1.57 x 107 CFU/mL for 100, 200 and 300 mg F-CN/L, being achieved. An indication that the F. oxysporum cultures were efficient at lower cyanide concentration. Furthermore, at a temperature of 5°C, the biodegradation efficiency, although slightly lower, was 51%, 43% and 44% with the corresponding maximum microbial population of 1.21 x107, 1.11 x 107 and 1.12 x 107 CFU/mL for 100, 200 and 300 mg F-CN/L cultures, respectively, with minimal differences observed for cultures with 200 and 300 mg F-CN/L. The cyanide biodegradation rates increased with temperature increases and varied with different cyanide concentrations below 500 mg F-CN/L. The estimated energy of activation for cyanide degradation for a change in temperature from 5°C to 22°C using the Arrhenius model was 19.6, 12.7 and 14.9 kJ/mol for 100, 200 and 300 mg F-CN/L, respectively. The means and standard deviations for rate of degradation of cyanide at 5°C and 22°C for the ODE models was 0.0052 (± 0.0011) h-1 and 0.0084 (± 0.0027) h-1, respectively. The inhibitory effect of the cyanide was quantitatively pronounced under cold temperature as the heavy metals, residual ammonium-nitrogen and nitrate-nitrogen hindered the cyanide degradation. Similarly, microbial growth rates increased with a temperature rise (from 5°C to 22°C), resulting with a reduction in the microbial populations’ doubling time. When compared with the simulated winter conditions, the specific population growth rate increased 4-fold, 5-fold and 6-fold in 100, 200 and 300 mg F-CN/L, respectively, for higher temperatures; an indication that the Fusarium oxysporum isolate prefers higher temperature. The estimated energy of activation for cellular respiration was 44.9, 54 and 63.5 kJ/mol for 100, 200 and 300 mg F-CN/L cultures, respectively, for the change in temperature from 5°C to 22°C. The means and standard deviations of microbial growth rate at 5°C and 22°C were: 0.0033 (± 0.0013) h-1 and 0.0151 (± 0.0027) h-1, respectively. The difference in error (standard deviation) of the cyanide biodegradation rate and microbial growth rate was insignificant (0.02% at 5°C) especially at temperature 22°C where there were minimal differences, indicating the reliability and reproducibility of this biodegradation system in batch operated bioreactors.

Cette thèse se compose de deux parties. Dans la première partie, nous étudions les stratégies de temps minimum pour le traitement de la pollution dans de grandes ressources en eau, par exemple des lacs ou réservoirs naturels, à l'aide d'un bioréacteur continu qui fonctionne à un état quasi stationnaire. On contrôle le débit d'entrée d'eau au bioréacteur, dont la sortie revient à la ressource avec le même débit. Nous disposons de l'hypothèse d'homogénéité de la concentration de polluant dans la ressource en proposant trois modèles spatialement structurés. Le premier modèle considère deux zones connectées l'une à l'autre par diffusion et seulement une d'entre elles connectée au bioréacteur. Avec l'aide du Principe du Maximum de Pontryagin, nous montrons que le contrôle optimal en boucle fermée dépend seulement des mesures de pollution dans la zone traitée, sans influence des paramètres de volume, diffusion, ou la concentration dans la zone non traitée. Nous montrons que l'effet d'une pompe de recirculation qui aide à homogénéiser les deux zones est avantageux si opérée à vitesse maximale. Nous prouvons que la famille de fonctions de temps minimal en fonction du paramètre de diffusion est décroissante. Le deuxième modèle consiste en deux zones connectées l'une à l'autre par diffusion et les deux connectées au bioréacteur. Ceci est un problème dont l'ensemble des vitesses est non convexe, pour lequel il n'est pas possible de prouver directement l'existence des solutions. Nous surmontons cette difficulté et résolvons entièrement le problème étudié en appliquant le principe de Pontryagin au problème de contrôle relaxé associé, obtenant un contrôle en boucle fermée qui traite la zone la plus polluée jusqu'au l'homogénéisation des deux concentrations. Nous obtenons des limites explicites sur la fonction valeur via des techniques de Hamilton-Jacobi-Bellman. Nous prouvons que la fonction de temps minimal est non monotone par rapport au paramètre de diffusion. Le troisième modèle consiste en deux zones connectées au bioréacteur en série et une pompe de recirculation entre elles. L'ensemble des contrôles dépend de l'état, et nous montrons que la contrainte est active à partir d'un temps jusqu'à la fin du processus. Nous montrons que le contrôle optimal consiste à l'atteinte d'un temps à partir duquel il est optimal de recirculer à vitesse maximale et ensuite ré-polluer la deuxième zone avec la concentration de la première. Ce résultat est non intuitif. Des simulations numériques illustrent les résultats théoriques, et les stratégies optimales obtenues sont testées sur des modèles hydrodynamiques, en montrant qu'elles sont de bonnes approximations de la solution du problème inhomogène. La deuxième partie consiste au développement et l'étude d'un modèle stochastique de réacteur biologique séquentiel. Le modèle est obtenu comme une limite des processus de naissance et de mort. Nous établissons l'existence et l'unicité des solutions de l'équation contrôlée qui ne satisfait pas les hypothèses habituelles. Nous prouvons que pour n'importe quelle loi de contrôle la probabilité d'extinction de la biomasse est positive. Nous étudions le problème de la maximisation de la probabilité d'atteindre un niveau de pollution cible, avec le réacteur à sa capacité maximale, avant l'extinction. Ce problème ne satisfait aucune des suppositions habituelles (la dynamique n'est pas lipschitzienne, diffusion dégénérée localement hölderienne, contraintes d'état, ensembles cible et absorbant s'intersectent), donc le problème doit être étudié dans deux étapes: en premier lieu, nous prouvons la continuité de la fonction de coût non contrôlée pour les conditions initiales avec le volume maximal et ensuite nous développons un principe de programmation dynamique pour une modification du problème original comme un problème de contrôle optimal avec coût final sans contrainte sur l'état
This thesis consists of two parts. In the first part we study minimal time strategies for the treatment of pollution in large water volumes, such as lakes or natural reservoirs, using a single continuous bioreactor that operates in a quasi-steady state. The control consists of feeding the bioreactor from the resource, with clean output returning to the resource with the same flow rate. We drop the hypothesis of homogeneity of the pollutant concentration in the water resource by proposing three spatially structured models. The first model considers two zones connected to each other by diffusion and only one of them treated by the bioreactor. With the help of the Pontryagin Maximum Principle, we show that the optimal state feedback depends only on the measurements of pollution in the treated zone, with no influence of volume, diffusion parameter, or pollutant concentration in the untreated zone. We show that the effect of a recirculation pump that helps to mix the two zones is beneficial if operated at full speed. We prove that the family of minimal time functions depending on the diffusion parameter is decreasing. The second model consists of two zones connected to each other by diffusion and each of them connected to the bioreactor. This is a problem with a non convex velocity set for which it is not possible to directly prove the existence of its solutions. We overcome this difficulty and fully solve the studied problem applying Pontryagin's principle to the associated problem with relaxed controls, obtaining a feedback control that treats the most polluted zone up to the homogenization of the two concentrations. We also obtain explicit bounds on its value function via Hamilton-Jacobi-Bellman techniques. We prove that the minimal time function is nonmonotone as a function of the diffusion parameter. The third model consists of a system of two zones connected to the bioreactor in series, and a recirculation pump between them. The control set depends on the state variable; we show that this constraint is active from some time up to the final time. We show that the optimal control consists of waiting up to a time from which it is optimal the mixing at maximum speed, and then to repollute the second zone with the concentration of the first zone. This is a non intuitive result. Numerical simulations illustrate the theoretical results, and the obtained optimal strategies are tested in hydrodynamic models, showing to be good approximations of the solution of the inhomogeneous problem. The second part consists of the development and study of a stochastic model of sequencing batch reactor. We obtain the model as a limit of birth and death processes. We establish the existence and uniqueness of solutions of the controlled equation that does not satisfy the usual assumptions. We prove that with any control law the probability of extinction is positive, which is a non classical result. We study the problem of the maximization of the probability of attaining a target pollution level, with the reactor at maximum capacity, prior to extinction. This problem does not satisfy any of the usual assumptions (non Lipschitz dynamics, degenerate locally H"older diffusion parameter, restricted state space, intersecting reach and avoid sets), so the problem must be studied in two stages: first, we prove the continuity of the uncontrolled cost function for initial conditions with maximum volume, and then we develop a dynamic programming principle for a modification of the problem as an optimal control problem with final cost and without state constraint

The design and development of a system for disinfecting medical waste at the site of origin is presented. Investigation of the current commercial systems that accomplish this task shows that they all expose the waste to physical conditions that are harmful to all forms of life. Further, most are very expensive to install and to operate. A recently developed biochemical process promises to effectively inactivate harmful pathogenic organisms economically and without the danger of extreme heat or poisonous chemicals. The biochemical process is not yet fully developed. Nonetheless, the development of a marketable system to take advantage of this technology has been initiated. The motivation for developing this technology and the particular system that will employ it is presented. A general overview of the system and components is presented. Previous and suggested future testing strategies are explained. Component interactions and process control are described.
Master of Science