Bioabsorbable orthopedic fixation devices are conceptually more attractive than metallic devices in repairing damaged tissues or in fastening implants. Our study focuses on investigating bioabsorbable composites for potential use as materials for orthopedic appliances. The study focuses on Poly(l-lactic acid) (PLLA), Polyglycolic acid (PGA), Poly-e-caprolactone (PCL), matrices with Carbon fibers (AS4), Nylon fibers and PLLA fibers. Fiber coating effects have also been investigated, with compliant polymers (1%, 50% and 100% of matrix properties) and with hydroxyapatite (HA). Unidirectional, continuous fiber plies, and multi-directional, random and quasi-random short-fiber composites were considered in our study. NDSANDS a concentric cylinder model computer software, was used to evaluate the stiffness and strength of the bioabsorbable composites with unidirectional fiber orientation. To achieve a better physical understanding, the NDSANDS predictions were also compared with those given by a simple, mechanics of materials approach. The theory for multidirectional short fiber composites, recently developed by Giurgiutiu and Reifsnider was employed with three fiber-orientation distribution functions and three failure mechanisms. Stiffness and strength of bioabsorbable composites were predicted over a range of fiber volume fraction. It was found that AS4/PLLA with 16% fiber volume fraction can have properties close to the bone when used in short fiber composite. Similar results are obtained using AS4/PLLA with hydroxyapatite coating. PLLA/PGA and PLLA/PLLA also demonstrated properties close to those of the bone in the range of 25% and 64%. / Master of Science

Aquifers are an important storage location and source of fresh groundwater. They may become polluted by a number of contaminants including mobile divalent radionuclides such as strontium-90 which is a byproduct of uranium fission. A method for remediating such divalent radionuclides is sequestration through co-precipitation into calcium carbonate. Calcium carbonate precipitation occurs naturally but can be enhanced by the use of ureolytic microorganisms living within the aquifer. The microbial enzyme urease cleaves ammonia from urea (added as a stimulant to the aquifer) increasing the pH and subsequently pushing the bicarbonate equilibrium towards precipitation. Laboratory experimentation is necessary to better predict field scale outcomes of remediation that is driven by ureolytic calcium carbonate co-precipitation. To aid in such laboratory experiments, I constructed two ureolytic organisms which contain green fluorescent protein (GFP) so that the location of the microbes in relation to media flow paths and precipitation can be viewed by microscopy in a 2- dimensional porous medium flow cell reactor. The reactor was operated with a parallel flow regime where the two influent media would not promote microbially induced calcium carbonate precipitation until they were mixed in the flow cell. A demonstration study compared the results of parallel flow and mixing in the reactor operated with and without one of the GFP-containing ureolytic organisms. The growth and precipitation of calcium carbonate within the reactor pore space altered flow paths to promote a wider mixing zone and a more widely distributed overall calcium carbonate precipitation pattern. This study will allow optimization of remediation efforts of contaminants such as strontium-90 in aquifers. / Graduation date: 2012

by Chan Sze Sze. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 193-218). / Abstracts in English and Chinese. / Acknowledgements --- p.i / Abstracts --- p.ii / 摘要 --- p.v / Contents --- p.viii / List of figures --- p.xv / List of tables --- p.xix / Abbreviations --- p.xxii / Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- The North Tsing Yi Abandoned Shipyard area --- p.1 / Chapter 1.2 --- Polycyclic aromatic hydrocarbons (PAHs) in the site --- p.3 / Chapter 1.2.1 --- Characteristics of PAHs --- p.3 / Chapter 1.2.2 --- Sources of PAHs --- p.8 / Chapter 1.2.3 --- Environmental fates of PAHs --- p.9 / Chapter 1.2.4 --- Biodegradation of PAHs --- p.10 / Chapter 1.2.5 --- Toxicity of PAHs --- p.13 / Chapter 1.2.6 --- PAHs contamination in Hong Kong --- p.14 / Chapter 1.2.7 --- Soil decontamination assessment in Hong Kong --- p.16 / Chapter 1.2.8 --- Environmental standards of PAHs --- p.18 / Chapter 1.2.9 --- Remediation technology of PAHs --- p.21 / Chapter 1.2.9.1 --- Bioremediation --- p.22 / Chapter 1.3 --- Heavy metals in the site --- p.28 / Chapter 1.3.1 --- "Characteristics of copper, lead and zinc" --- p.29 / Chapter 1.3.2 --- "Sources of copper, lead and zinc" --- p.32 / Chapter 1.3.3 --- "Environmental fates of copper, lead and zinc" --- p.34 / Chapter 1.3.4 --- "Toxicities of copper, lead and zinc" --- p.36 / Chapter 1.3.5 --- "Copper, lead and zinc contamination in Hong Kong" --- p.39 / Chapter 1.3.6 --- "Environmental standards of copper, lead and zinc" --- p.40 / Chapter 1.3.7 --- Remediation technology of heavy metal --- p.42 / Chapter 1.3.7.1 --- Chemical method --- p.42 / Chapter 1.3.7.2 --- Biological method --- p.43 / Chapter 1.3.7.3 --- Stabilization and Solidification --- p.45 / Chapter 1.4 --- Aim of study --- p.47 / Chapter 1.5 --- Objectives --- p.47 / Chapter 1.6 --- Research Strategy --- p.47 / Chapter 1.7 --- Significance of study --- p.48 / Chapter 2 --- Materials and Methods --- p.49 / Chapter 2.1 --- Soil Collection --- p.49 / Chapter 2.2 --- Characterization of soil --- p.49 / Chapter 2.2.1 --- Sample preparation --- p.49 / Chapter 2.2.2 --- "Soil pH, electrical conductivity & salinity" --- p.50 / Chapter 2.2.3 --- Total organic carbon contents --- p.51 / Chapter 2.2.4 --- Soil texture --- p.51 / Chapter 2.2.5 --- Moisture --- p.53 / Chapter 2.2.6 --- Total nitrogen and total phosphorus --- p.53 / Chapter 2.2.7 --- Available nitrogen --- p.53 / Chapter 2.2.8 --- Available phosphorus --- p.54 / Chapter 2.2.9 --- Soil bacterial and fungal population --- p.54 / Chapter 2.2.10 --- Extraction of PAHs and organic pollutants --- p.55 / Chapter 2.2.10.1 --- Extraction procedure --- p.55 / Chapter 2.2.10.2 --- GC-MS condition --- p.56 / Chapter 2.2.10.3 --- Preparation of mixed PAHs stock solution --- p.56 / Chapter 2.2.11 --- Oil and grease content --- p.57 / Chapter 2.2.12 --- Total Petroleum Hydrocarbons (TPH) --- p.57 / Chapter 2.2.13 --- Total heavy metal analysis --- p.58 / Chapter 2.2.14 --- Toxicity characteristic leaching procedure (TCLP) --- p.59 / Chapter 2.2.15 --- Extraction efficiency --- p.59 / Chapter 2.3 --- Production of mushroom compost --- p.60 / Chapter 2.4 --- Characterization of mushroom compost --- p.62 / Chapter 2.4.1 --- Enzyme assay --- p.62 / Chapter 2.4.1.1 --- Laccase assay --- p.62 / Chapter 2.4.1.2 --- Manganese peroxidase assay --- p.62 / Chapter 2.5 --- Addition of mushroom to soil on site --- p.63 / Chapter 2.5.1 --- Transportation of mushroom compost to Tsing Yi --- p.63 / Chapter 2.5.2 --- Mixing of mushroom compost and soil --- p.64 / Chapter 2.6 --- Soil Monitoring --- p.64 / Chapter 2.6.1 --- On site air and soil measurements --- p.64 / Chapter 2.6.1.1 --- Air temperature and moisture --- p.64 / Chapter 2.6.1.2 --- Light intensity --- p.64 / Chapter 2.6.1.3 --- UV intensity --- p.65 / Chapter 2.6.1.4 --- Rainfall --- p.65 / Chapter 2.6.1.5 --- Soil temperature --- p.65 / Chapter 2.6.2 --- Soil chemical characteristic --- p.65 / Chapter 2.6.3 --- Relative residue pollutant (%) --- p.65 / Chapter 2.7 --- Toxicity of treated soil --- p.66 / Chapter 2.7.1 --- Seed germination test --- p.66 / Chapter 2.7.2 --- Indigenous bacterial toxicity test --- p.67 / Chapter 2.7.3 --- Fungal toxicity test --- p.68 / Chapter 2.7.3.1 --- Preparation of ergosterol standard solution --- p.70 / Chapter 2.8 --- Soil Washing --- p.70 / Chapter 2.8.1 --- Optimization of soil washing --- p.70 / Chapter 2.8.1.1 --- Effect of hydrochloric acid concentration --- p.70 / Chapter 2.8.1.2 --- Effect of incubation time --- p.71 / Chapter 2.9 --- Phytoremediation --- p.71 / Chapter 2.10 --- Mycoextraction --- p.72 / Chapter 2.11 --- Integrated bioextraction --- p.72 / Chapter 2.12 --- Cementation --- p.73 / Chapter 2.13 --- Glass encapsulation --- p.73 / Chapter 2.14 --- Statistical analysis --- p.74 / Chapter 3 --- Results --- p.75 / Chapter 3.1 --- Characterization of soil --- p.75 / Chapter 3.2 --- Characterization of mushroom compost --- p.78 / Chapter 3.2.1 --- Enzyme activity --- p.78 / Chapter 3.2.2 --- Total nitrogen and total phosphorus contents --- p.78 / Chapter 3.3 --- Soil monitoring --- p.79 / Chapter 3.3.1 --- Initial pollutant content in biopile and fungal treatment soils --- p.79 / Chapter 3.3.2 --- On site air and soil physical characteristics --- p.81 / Chapter 3.3.2.1 --- Soil temperature and air temperature --- p.81 / Chapter 3.3.3 --- Soil chemical characteristic --- p.84 / Chapter 3.3.3.1 --- Effect of type of treatment on total petroleum hydrocarbon content --- p.85 / Chapter 3.3.3.2 --- Effect of type of treatment on oil and grease content --- p.87 / Chapter 3.3.3.3 --- Soil pH --- p.89 / Chapter 3.3.3.4 --- Moisture --- p.91 / Chapter 3.3.3.5 --- Electrical conductivity --- p.92 / Chapter 3.3.3.6 --- Salinity --- p.93 / Chapter 3.3.3.7 --- Microbial population --- p.95 / Chapter 3.3.3.8 --- Removal of organopollutant PAHs in biopile and fungal treatment --- p.98 / Chapter 3.3.3.9 --- Effect of type of treatment on residual PAHs at Day 4 --- p.104 / Chapter 3.3.3.10 --- Effect of type of treatment on residual PAHs at peak levels --- p.107 / Chapter 3.3.3.11 --- Effect of type of treatment on residual organopollutants at the end of treatments --- p.109 / Chapter 3.3.3.12 --- Effect of type of treatment on total nitrogen and phosphorus contents --- p.111 / Chapter 3.3.3.13 --- Effect of type of treatment on physical and chemical properties of soil --- p.113 / Chapter 3.4 --- Toxicity of treated soil --- p.116 / Chapter 3.4.1 --- Seed germination test --- p.116 / Chapter 3.4.2 --- Indigenous bacterial toxicity test --- p.120 / Chapter 3.4.3 --- Fungal toxicity test --- p.125 / Chapter 3.5 --- Soil washing --- p.129 / Chapter 3.5.1 --- Optimisation of soil washing --- p.129 / Chapter 3.5.1.1 --- The effect of hydrochloric acid concentration --- p.129 / Chapter 3.5.1.2 --- The effect of incubation time --- p.134 / Chapter 3.6 --- Mycoextraction --- p.139 / Chapter 3.7 --- Phytoextraction and integrated bioextraction --- p.146 / Chapter 3.8 --- Cementation --- p.153 / Chapter 3.9 --- Glass encapsulation --- p.158 / Chapter 4 --- Discussion --- p.160 / Chapter 4.1 --- Characterization of soil --- p.160 / Chapter 4.2 --- Characterization of mushroom compost --- p.162 / Chapter 4.2.1 --- Enzyme activity --- p.162 / Chapter 4.2.2 --- Total nitrogen and total phosphorus contents --- p.163 / Chapter 4.3 --- Soil monitoring --- p.163 / Chapter 4.3.1 --- Initial pollutant content in biopile and fungal treatment soil --- p.163 / Chapter 4.3.2 --- On site air and soil physical characteristics --- p.164 / Chapter 4.3.3 --- Soil chemical characteristic --- p.164 / Chapter 4.3.3.1 --- Soil pH --- p.164 / Chapter 4.3.3.2 --- Moisture --- p.165 / Chapter 4.3.3.3 --- Electrical conductivity --- p.165 / Chapter 4.3.3.4 --- Salinity --- p.166 / Chapter 4.3.3.5 --- Microbial population in biopile and fungal treatments --- p.166 / Chapter 4.3.3.6 --- Removal of organopollutant PAHs in biopile and fungal treatments --- p.168 / Chapter 4.3.3.7 --- Effect of type of treatment on residual PAHs at peak levels --- p.170 / Chapter 4.3.3.8 --- Effect of type of treatment on residual oil and grease and TPH contents --- p.171 / Chapter 4.3.3.9 --- Effect of type of treatment on total nitrogen and phosphorus contents --- p.172 / Chapter 4.3.3.10 --- Effect of type of treatment on physical and chemical properties of the soil --- p.173 / Chapter 4.4 --- Toxicity of treated soil --- p.174 / Chapter 4.5 --- Summary of Pleurotus pulmonarius mushroom compost on organopollutant remediation --- p.177 / Chapter 4.6 --- Soil washing --- p.178 / Chapter 4.7 --- Mycoextraction --- p.180 / Chapter 4.8 --- Phytoextraction and integrated bioextraction --- p.182 / Chapter 4.9 --- Cementation --- p.184 / Chapter 4.10 --- Glass encapsulation --- p.185 / Chapter 4.11 --- "Summary of physical, chemical and biological heavy metal removal treatments" --- p.186 / Chapter 4.12 --- Future studies --- p.187 / Chapter 5 --- Conclusion --- p.190 / Chapter 6 --- References --- p.193

<p>Soil bottles and soil slurry experiments were conducted to investigate the effect of some additives on the aerobic and anaerobic biodegradation of chlorinated aliphatic hydrocarbons; tetrachloroethylene (PCE), trichloroethylene (TCE) and dichloroethylene (DCE) in a contaminated soil from Startvätten AB Linköping Sweden. For the aerobic degradation study the soil sample was divided into two groups, one was fertilised. The two groups of soil in the experimental bottles were treated to varying amount of methane in pairs. DCE and TCE were added to all samples while PCE was found in the contaminated soil. Both aerobic and anaerobic experiments were conducted. For aerobic study air was added to all bottles to serve as electron acceptor (oxygen). It was observed that all the samples showed a very small amount of methane consumption while the fertilised soil samples showed more oxygen consumption. For the chlorinated compounds the expected degradation could not be ascertained since the control and experimental set up were more or less the same.</p><p>For the anaerobic biodegradation study soil slurry was made with different media i.e. basic mineral medium (BM), BM and an organic compound (lactate), water and sulphide, phosphate buffer and sulphide and phosphate buffer, sulphide and ammonia. To assure anaerobic conditions, the headspace in the experimental bottles was changed to N2/CO2. As for the aerobic study all the samples were added DCE and TCE while PCE was found in the contaminated soil. The sample without the soil i.e. the control was also given PCE. It was observed that there was no clear decrease in the GC peak area of the pollutants in the different media. The decrease in GC peak area of the pollutants could not be seen, this may be so because more susceptible microorganisms are required, stringent addition of nutrients and to lower the risk of the high concentration of PCE and petroleum products in the soil from Startvätten AB.</p>

Using ����C cross polarization magic angle spinning nuclear magnetic resonance techniques on 5 species of dead trees from the northwest (western hemlock, Douglas fir, Sitka spruce, lodgepole pine and ponderosa pine) I tracked the lignin and cellulose content over a 22 to 36 year period in order to determine the effects of decay fungi, if any, that is attacking certain species of tree. I had samples from the wood of the roots, the bark on the roots and, in some cases, the resin core of the roots. The Department of Forest Science at Oregon State University has studied this problem by using wet chemical analysis, and direct visual observation. Mark Harmon and Hua Chen of the Department of Forest Science believe that white rot occurred most frequently in the lodgepole pine and ponderosa pine and brown rot was more frequent in the Douglas-fir and Sitka spruce. Western hemlock seemed to have both brown and white rots active. The Douglas fir bark sample showed definite decomposition consistent with white rot during the first 10 years. The ponderosa pine sap showed decomposition consistent with white rot in the 10 to 22 year period. Sitka Spruce showed some decomposition consistent with white rot in the bark from 7 to 33 years, and the western hemlock showed some decomposition consistent with white rot in the sap in the first 10 years. The decompositions consistent with brown rot were much easier to see in this study. Virtually all the sap and bark samples showed decomposition consistent with brown rot at some point. The Douglas fir was the only species, other than lodgepole pine, not to show any decomposition consistent with brown rot in the bark of the tree, only decomposition consistent with white rot. The Douglas fir did show a decay consistent with brown rot in the sap for the first ten years. Ponderosa pine showed evidence of decay that brown rot would cause for the first 10 years in the sap and the bark. The Sitka spruce species analysis showed brown rot type decay in the bark for the first 7 years and in the sap for the entire time studied of 33 years. The lodgepole pine was the only species to not show any brown rot type decay in the sap or bark for the entire 22 year period studied. The western hemlock was distinct by not showing any definitive brown rot type decay for the first 10 years, but showed massive decay consistent with brown rot in both sap and bark during the following 26 years studied. I used an 8 Tesla magnet and the MAS frequency was at 5 kHz. The recycle time was 1.5 seconds and the contact time was 1 ms. I generally took about 10,000 acquisitions per sample, which added up to about 4 hours total acquisition time per sample. Presence of these rots shows that certain species are more susceptible than others, and also shows that local environmental conditions can contribute to rot susceptibility. / Graduation date: 2003

Soil bottles and soil slurry experiments were conducted to investigate the effect of some additives on the aerobic and anaerobic biodegradation of chlorinated aliphatic hydrocarbons; tetrachloroethylene (PCE), trichloroethylene (TCE) and dichloroethylene (DCE) in a contaminated soil from Startvätten AB Linköping Sweden. For the aerobic degradation study the soil sample was divided into two groups, one was fertilised. The two groups of soil in the experimental bottles were treated to varying amount of methane in pairs. DCE and TCE were added to all samples while PCE was found in the contaminated soil. Both aerobic and anaerobic experiments were conducted. For aerobic study air was added to all bottles to serve as electron acceptor (oxygen). It was observed that all the samples showed a very small amount of methane consumption while the fertilised soil samples showed more oxygen consumption. For the chlorinated compounds the expected degradation could not be ascertained since the control and experimental set up were more or less the same. For the anaerobic biodegradation study soil slurry was made with different media i.e. basic mineral medium (BM), BM and an organic compound (lactate), water and sulphide, phosphate buffer and sulphide and phosphate buffer, sulphide and ammonia. To assure anaerobic conditions, the headspace in the experimental bottles was changed to N2/CO2. As for the aerobic study all the samples were added DCE and TCE while PCE was found in the contaminated soil. The sample without the soil i.e. the control was also given PCE. It was observed that there was no clear decrease in the GC peak area of the pollutants in the different media. The decrease in GC peak area of the pollutants could not be seen, this may be so because more susceptible microorganisms are required, stringent addition of nutrients and to lower the risk of the high concentration of PCE and petroleum products in the soil from Startvätten AB.

Submitted by orjuela@ibilce.unesp.br (orjuela@ibilce.unesp.br) on 2016-02-17T12:16:38Z No. of bitstreams: 1 Isolamento de bactérias degradadoras de hidrocarbonetos aromáticos 1-107.pdf: 3940758 bytes, checksum: c39dfe1dada0d918848470d3f0de5b83 (MD5) / Approved for entry into archive by Juliano Benedito Ferreira (julianoferreira@reitoria.unesp.br) on 2016-02-17T13:16:08Z (GMT) No. of bitstreams: 1 orjuela_gl_dr_rcla.pdf: 3940758 bytes, checksum: c39dfe1dada0d918848470d3f0de5b83 (MD5) / Made available in DSpace on 2016-02-17T13:16:08Z (GMT). No. of bitstreams: 1 orjuela_gl_dr_rcla.pdf: 3940758 bytes, checksum: c39dfe1dada0d918848470d3f0de5b83 (MD5) Previous issue date: 2016-02-11 / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Este documento foi organizado em dois capítulos. O Capítulo I é um artigo de revisão intitulado “Metabolic Pathways for Aromatic Compounds Degradation by Bacteria”, no qual são descritas as fontes naturais e antrópicas dos hidrocarbonetos aromáticos e suas características químicas. Relacionaram-se os fatores ambientais que afetam a degradação aeróbia e anaeróbia desses compostos por bactérias e os principais compostos intermediários produzidos. É descrita passo a passo a sequência de preparação e desaromatização do anel benzênico e os compostos finais dessa degradação. O artigo foi publicado no volume 237 da série Reviews of Environmental Contamination and Toxicology em janeiro de 2016 (Springer http://dx.doi.org/10.1007/978-3-319-23573-8_5). O Capítulo II contém os resultados da pesquisa desenvolvida. O objetivo geral foi isolar cepas bacterianas de amostras de solo e avaliar o potencial degradador de fenol e outros hidrocarbonetos aromáticos. Foram realizadas coletas de amostras de solo de cinco postos de combustíveis, para a seleção das cepas bacterianas e para análises químicas e granulométricas. Alíquotas das amostras de solo foram transferidas para meio de cultura seletivo contendo querosene como única fonte de carbono. Testes morfológicos e bioquímicos indicaram que as cepas isoladas são Gram negativas, móveis, catalase positivas, produtoras de cápsula e de biossurfactantes. O sequenciamento do gene 16S rRNA mostrou 99 a 100% de similaridade com o gênero Pseudomonas sp. Todas as cepas degradaram o fenol em concentração de 120 mg L-1 em menos de 24 horas. Testes da atividade enzimática mostraram que algumas das cepas expressaram a catecol 1,2-dioxigenase que catalisa a orto-clivagem do anel benzênico e outras expressaram a catecol 2,3 dioxigenase da meta-clivagem do anel aromático. Uma cepa não apresentou atividade para nenhuma dessas duas enzimas e uma apresentou atividade de ambas. Todas as cepas foram capazes de crescer em presença de fenantreno, fluoranteno e pireno. / This document is organized in two chapters. The Chapter I is a review paper entitled “Metabolic Pathways for Aromatic Compounds Degradation by Bacteria” in which are described natural and anthropogenic sources of aromatic hydrocarbons and their chemical characteristics. There are listed environmental factors that affect the aerobic and anaerobic degradation by bacteria and the central intermediates yielded. It is described step to step of sequence of preparation and dearomatization of benzene ring and the final metabolites of breakdown. The manuscript was publicated in the volume 237 of Reviews of Environmental Contamination and Toxicology in January of 2016 (Springer http://dx.doi.org/10.1007/978-3- 319-23573-8_5). Chapter II has the results of developed research. The general objective was to isolate bacterial strains from samples of soil and to evaluate the potential of them for breakdown hydrocarbons. Soil samples from five gas stations were collected to isolate the bacterial strains and chemical and granulometric analysis was made. Aliquots of the soil samples were cultured with selective media with kerosene as only carbon and energy sources. Morphological and biochemical tests showed that bacterial strains were Gram negatives, motile, positive catalase, capsule-producers and biosurfactant producers. The sequencing of 16S rRNA gene showed 99 to 100% similarity with Pseudomonas sp genera. All bacterial strains were able to degrade phenol 120 mg L-1 in less than 24 hours. Tests of enzymatic activity showed that some bacteria expressed the catecol 1,2-dioxygenase that catalyze ortoclivage of benzene ring, others showed activity for the catecol 2,3-dioxygenase that catalyze the meta-cleavage of the ring. One strain did not show activity for any of these enzymes and one strain had activity for both. All strains were able to growth with fenantrene, fluoranthene and pyrene. / CNPq: 140704/2012-4

Made available in DSpace on 2017-06-01T18:20:26Z (GMT). No. of bitstreams: 1 dissertacao_fabiana_america.pdf: 1099131 bytes, checksum: 9135e1cca2e3ecf79ae358085f3e7e31 (MD5) Previous issue date: 2009-04-15 / The biodegradation of hydrocarbons by natural population of microorganism represents one of the primary mechanisms by which diesel oil and others hydrocarbons pollutants are eliminated or transformed in the environment. It is generally accepted today that petroleum hydrocarbon, can be degraded by microorganisms as long as a few factors, such as nutrients, organic compound bioavailability, pH and temperture are controlled and optimized. In this study biodegradationof diesel oil by Candida lipolytica in sea water supplemented with nitrogen and phosphorus sources was investigated in skake flask fermentation scale. A set of three full factorial designs was carried out to investigate the effects and interactions of pH and the seawater, diesel oil, urea, ammonium sulfate and potassium dihydrogen orthophosphate concentrations on the C.lipolytica growth, the emulsification activity and the surface tension of the free cell broth. The biodegradation of diesel oil was confirmed through four laboratory experiments using: (1) seawater + diesel oil; (2) distilled water + diesel oil; (3) seawater + corn oil and (4) distilled water + corn oil. The best result for 5% (v/v) diesel degradation was obtained at condition 1, using seawater supplemented with 1,0% (p/v) of ammonium sulfate and 1,0 % (p/v) of potassium dihydrogen orthophosphate. In this condition, after 96 h, the pH, the salinity, the surface tension and the emulsification activities to emulsions with corn oil and with motor oil were equal to 9.47, 44 , 46.63 mN/m, 5.49 e 6.00 UAE, respectively. Whereas C.lipolytica has potential application in biotechnological process, the production medium conditions and bioemulsifiers and biosurfactants produced are candidates to be optimized and used in bioremediation of marine environments contaminated by diesel and other oil products. / A biodegradação de hidrocarbonetos por população natural de microrganismos representa um dos mecanismos primários pelos quais óleo diesel e outros hidrocarbonetos poluentes são eliminados ou transformados no ambiente. Atualmente aceita-se que hidrocarbonetos de petróleo possam ser degradados por microrganismos, desde que alguns fatores, tais como nutrientes, disponibilidade de compostos orgânicos, pH e temperatura sejam controlados e otimizados. Neste estudo, biodegradação de óleo diesel por Candida lipolytica em água do mar suplementada com fontes de nitrogênio e fósforo foi investigada em escala de frascos de fermentação agitados. Um conjunto de três planejamentos fatoriais completos foi realizado para investigar os efeitos e as interações do pH e das concentrações de água do mar, óleo diesel, uréia, sulfato de amônio e fosfato monobásico de potássio sobre o crescimento de C. lipolytica, a atividade emulsificação e a tensão superficial do cultivo livre de células. A biodegradação de óleo diesel foi confirmada através de quatro experimentos na presença de: (1) água do mar + óleo diesel (2); água destilada + óleo diesel; (3) + água do mar + óleo de milho (4) água destilada + óleo de milho. O melhor resultado para degradação de óleo diesel 5% (v/v) foi obtido, utilizando água do mar suplementada com 1,0% (p/v) de sulfato de amônio e 1,0% (p/v) de fosfato monobásico de potássio. Nesta condição, após 96 h, o pH, a salinidade, a tensão superficial e as atividades de emulsificação para emulsões com óleo de milho e com óleo de motor foram iguais a 9,47, 44 , 46,63 mN/m, 5,49 e 6,00 UAE, respectivamente. Considerando que a C. lipolytica tem potencial de aplicação em processos biotecnológicos, as condições dos meios de produção e os bioemulsificantes e biossurfactantes produzidos são candidatos a serem otimizados e utilizados na biorremediação de ambientes marinhos contaminados por óleo diesel e outros derivados de petróleo.

Ph. D. (Department of Biotechnology, Faculty of Applied and Computer Sciences), Vaal University of Technology. / Hydrocarbon pollution is becoming a growing environmental concern in South Africa and globally. This inadvertently supports the need to identify enzymes for their targeted degradation. The search for novel biocatalysts such as monooxygenases, alcohol dehydrogenases and aldehyde dehydrogenases, have relied on conventional culture-based techniques but this allows sourcing of the biomolecules from only 1-10 % of the microbial population leaving the majority of the biomolecules unaccounted for in 90-99 % of the microbial community. The implementation of a metagenomics approach, a culture-independent technique, ensures that more or less than 100 % of the microbial community is assessed. This increases the chance of finding novel enzymes with superior physico-chemical and catalytic traits. Hydrocarbon polluted soils present a rich environment with an adapted microbial diversity. It was thus extrapolated that it could be a potential source of novel monooxygenases, alcohol dehydrogenases (ADH) and aldehyde dehydrogenases (ALDH) involved in hydrocarbon degradation pathways. Therefore, the aim of the study was to extract metagenomic DNA from hydrocarbon contaminated soils and construct a metagenomic fosmid library and screen the library for monooxygenases, alcohol dehydrogenases (ADH) and aldehyde dehydrogenases (ALDH). Accordingly, the fosmid library was constructed from metagenome of hydrocarbon-contaminated soil. Then the library was functionally screened using hexadecane, octadecene and cyclohexane as substrates and fifteen positive clones were selected. The fosmid constructs of the positive clones were sequenced using PacBio next generation sequencing platform. The sequences were de novo assembled and analysed using CLC Genomic Workbench. The open reading frames (ORF) of the contigs were identified by blasting the contigs against uniport database. Accordingly, four novel genes namely amo-vut1, aol-vut3, dhy-sc-vut5 and dhy-g-vut7 that showed close similarity with our target enzymes were further analysed in silico and codon-optimized as per Escherichia coli codon preference. The codon adjusted sequences were synthesised and cloned into pET30a(+) expression vector. However, it is worth noting that expression of amo-vut1 was not successful since it was later identified to be a multi-pass member protein, which made it insoluble despite the use of detergent to the effect. There is a need to meticulously genetically engineer amo-vut1 to remove the signal and other membrane-bound peptides while maintaining its activity. Yet the other three constructs were successfully transformed and expressed in E. coli BL21 (DE3). The enzymes were purified and characterized and cocktail for hydrolysis of hexanol was succesfully engineered based on AOL-VUT3, DHY-SC-VUT5 and DHY-G-VUT7. Therefore, novel enzymes were mined from metagenome of fossil-oil contaminated soil and effective hydrocarbon-degrading enzyme cocktails containing their combination were successfully engineered.

Call number: LD2668 .T4 1985 L435 / Master of Science