Isolation and Capability of Dark Septate Endophyte Against Mancozeb Fungicide

Mancozeb is a group member of dithiocarbamat fungicides that most widely applied in the field. The use of mancozeb causes residues that have negative effects on agricultural production system and on agricultural environment. This research aimed to obtain dark septate endophyte (DSE) fungal isolates that are tolerant to mancozeb fungicide. The study was consisted of three steps, i.e., the isolation of DSE fungi from chili roots, the effect of DSE fungal isolates on chili seeds, and the growth ability of DSE isolates on medium supplemented with mancozeb. Thirteen DSE fungal isolates were isolated from chilli pepper roots and grown in the field assumed to be contaminated by pesticides. The isolates of DSE fungi caused the growth of chili seeds were normal and abnormal. Seven DSE isolates were able to grow well at PDA medium suplemented with mancozeb at concentrations of 100, 200, 400, and 800 ppm with different levels of relative growth inhibition. Mancozeb on PDA medium influenced the mycelial growth and colony color of DSE fungal isolates. The biomass of DSE fungal isolates were reduced when they were grown at malt extract broth (MEB) medium suplemented various levels of mancozeb. Keywords: dithiocarbamat, relative growth inhibition, toleran

Computational Studies on Cellular Metabolism:From Biochemical Pathways to Complex Metabolic Networks

Biotechnology promises the biologically and ecologically sustainable production of commodity chemicals, biofuels, pharmaceuticals and other high-value products using industrial platform microorganisms. Metabolic engineering plays a key role in this process, providing the tools for targeted modifications of microbial metabolism to create efficient microbial cell factories that convert low value substrates to value-added chemicals. Engineering microbes for the bioproduction of chemicals has been practiced through three different approaches: (i) optimization of native pathways of a host organism; (ii) incorporation of heterologous pathways in an amenable organism; and finally (iii) design and introduction of synthetic pathways in an organism. So far, the progress that has been made in the biosynthesis of chemicals was mostly achieved using the first two approaches. Nevertheless, many novel biosynthetic pathways for the production of native and non-native compounds that have potential to provide near-theoretical yields and high specific production rates of chemicals remain yet to be discovered. Therefore, the third approach is crucial for the advancement of bio-based production of value-added chemicals. We need to fully comprehend and analyze the existing knowledge of metabolism in order to generate new hypotheses and design de novo pathways. In this thesis, through development and application of efficient computational methods, we took the research path to expand our understanding of cell metabolism with the aim to discover novel knowledge about metabolic networks. We analyze different aspects of metabolism through five distinct studies. In the first study, we begin with a holistic view of the enzymatic reactions across all the species, and we propose a computational approach for identifying all the theoretically possible enzymatic reactions based on the known biochemistry. We organize our results in a web-based database called âAtlas of biochemistryâ. In the second study, we focus on one of the most structurally diverse and ubiquitous constituents of metabolism, the lipid metabolism. Here we propose a computational framework for integrating lipid species with unknown metabolic/catabolic pathways into metabolic networks. In our next study, we investigate the full metabolic capacity of E. coli. We explore computationally all enzymatic potentials of this organism, and we introduce the âSuper E. coliâ, a new and advanced chassis for metabolic engineering studies. Our next contribution concentrates on the development of a new method for the atom-level description of metabolic networks. We demonstrate the significance of our approach through the reconstruction of atom-level map of the E. coli central metabolism. In the last study, we turn our focus on studying the thermodynamics of metabolism and we present our original approach for estimating the thermodynamic properties of an important class of metabolites. So far, the available thermodynamic properties either from experiments or the computational methods are estimated with respect to the standard conditions, which are different from typical biological conditions. Our workflow paves the way for reliable computing of thermochemical properties of biomolecules at biological conditions of temperature and pressure. Finally, in the conclusion chapter, we discuss the outlook of this work and the potential further applications of the computational methods that were developed in this thesis

Computational Framework for the Identification of Bioprivileged Molecules

Bioprivileged molecules are biology-derived chemical intermediates that can be efficiently converted to a diversity of chemical products including both novel molecules and drop-in replacements. Bridging chemical and biological catalysis by bioprivileged molecules provides a useful and flexible new paradigm for producing biobased chemicals. However, the discovery of bioprivileged molecules has been demonstrated to require extensive experimental effort over a long period of time. In this work, we developed a computational framework for identification of all possible C6HxOy molecules (29252) that can be honed down to a manageable number of candidate bioprivileged molecules based on analysis of structural features, reactive moieties, and reactivity of species, and the evaluation of the reaction network and resulting products based on automated network generation. Required input is the structure data file (SDF) of the starting molecules and the reaction rules. On-the-fly estimation of thermodynamics by a group contribution method is introduced as a screening criterion to identify the feasibility of reactions and pathways. Generated species are dynamically linked to the PubChem database for identification of novel products and evaluation of the known products as attractive candidates. Application of the proposed computational framework in screening 29252 C6 species and identifying a list of 100 C6HxOy bioprivileged molecule candidates is presented. Each of the 100 candidate molecules falls into one of nine broad compound classes and is typically composed of carbon atoms with a different chemical environment and, as a result, distinct reactivity patterns. Sensitivity analysis of the parameters used in the filtering steps leading to the candidate molecules that were identified is discussed, and analysis of favorable structural features, reactive moieties, and functionalities of C6HxOy candidate bioprivileged molecules is performed

Bioremediation of a pesticide polluted soil : case DDT

RESUMEN: El 1,1,1-tricloro-2,2’bis(p-clorofenil)etano (DDT) Nha sido usado desde la segunda guerra mundial para controlar enfermedades transmitidas por insectos en humanos y animales domésticos. El uso de estos insecticidas organoclorados se ha prohibido en la mayoría de los países, debido a su persistencia en el ambiente, susceptibilidad de biomagnificación y potencial toxicidad a animales superiores. La biorremediación involucra el uso de microorganismos para degradar contaminantes orgánicos presentes en el ambiente, transformándolos en compuestos más simples y de menor peligrosidad, inclusive inocuos. Esta estrategia de descontaminación tiene bajos costos, una amplia aceptación pública y puede llevarse a cabo en el sitio. Comparado con otros métodos, la biorremediación es una forma más promisoria y menos costosa de eliminar los contaminantes presentes en suelos y agua. En suelo los compuestos bifenilos clorados como el DDT, pueden ser parcialmente biodegradados por un grupo de bacterias aerobias que cometabolizan el contaminante. La biodisponibilidad de los contaminantes puede ser mejorada, tratando los suelos en presencia de agentes movilizadores del contaminante como los surfactantes. En esta revisión se discuten las diferentes estrategias de biorremediación de suelo contaminado con DDT, incluyendo mecanismos y rutas de degradación. Se describe la aplicación de estas técnicas en suelo contaminado y se discute cuál es la mejor estrategia para remediación de DDT.ABSTRACT: 1,1,1-trichloro-2, 2’bis (p-chlorophenyl) ethane (DDT) has been used since the Second World War to control insect-borne diseases in humans and domestic animals. The use of these organochlorine insecticides has been banned in most countries because of its persistence in the environment, biomagnification and potential susceptibility to toxicity to higher animals. Bioremediation involves the use of microorganisms to degrade organic contaminants in the environment, transforming them into simpler and less dangerous, even harmless compounds. This decontamination strategy has low costs, and wide public acceptance, also it can take place on the site. Compared to other methods, bioremediation is a more promising and less expensive to eliminate contaminants in soil and water. In soil, compounds such as DDT, chlorinated biphenyls can be partially biodegraded by a group of aerobic bacteria that cometabolize the contaminant. The bioavailability of pollutants may be enhanced by treating the soil in the presence of contaminant mobilizing agents such as surfactants. In this review we discuss the different strategies for bioremediation of soil contaminated with DDT, including mechanisms and degradation pathways. The application of these techniques in contaminated soil is also described. This review also discusses which is the best strategy for bioremediation of DDT

Environmental Hazard Assessment of Heterocyclic Polyaromatic Hydrocarbons in Aquatic Compartment: Biodegradation, Bioaccumulation, Acute Toxicity and Chronic Toxicity

Nitrogen-, sulfur-, and oxygen-substituted heterocyclic polyaromatic hydrocarbons (heterocyclic PAHs) are ubiquitous in all environmental compartments, often co-occurring with their unsubstituted carbocyclic analogues. However, they have received little attention in the hazard assessment of contaminated sites and little is known about their fate and impacts. Due to their higher polarity resulting from the presence of electronegative atoms possessing lone electron pairs, most heterocycles are expected to be less toxic and bioaccumulative than their homocyclic counterparts, which is perhaps why research on their hazard and fate is much more limited. If this assumption is not true (e.g., because factors other than polarity/hydrophobicity drive their hazard), this may lead to an underestimation of the risks posed by contaminated sites or emissions. Therefore, the main objectives of this thesis are (1) to improve the understanding of the physicochemical properties, biodegradability, ecotoxicity, bioaccumulation, and mobility of these compounds in environmental contexts, (2) to evaluate the effectiveness of different modeling approaches in accurately predicting their properties, and (3) to tackle experimental barriers by designing robust techniques that can produce reliable and reproducible data output in the long run. From the large family of heterocycles, structurally comparable representative compounds with log KOW values (as a measure of hydrophobicity) ranging over four orders of magnitude were selected as model structures. First, the most environmentally relevant physicochemical properties of heterocyclic PAHs were investigated, including water solubility (SW), n-octanol-water partition coefficient (KOW), and organic carbon-water partition coefficient (KOC). The effect of molecular size, type and number of heteroatoms on the solubility and partition coefficients was investigated. These properties were then used to test the performance of in silico models, including Quantitative Structure-Property Relationship (QSPR), Polyparameter Linear Free Energy Relationship (pp-LFER), and Conductor-like Screening Model for Realistic Solvation (COSMO-RS), that predict these properties. A screening-level environmental hazard assessment was performed by integrating the data and evidence from multiple sources. The screening results revealed (i) high hazard for 4- and 5-ring compounds mainly due to high persistence and bioaccumulation potential, (ii) limited availability of experimental data, and (iii) notable uncertainty due to the limited applicability domain of the models, stressing the urgent need for a detailed hazard assessment for this group of compounds. The persistence of heterocyclic PAHs in the environment was assessed experimentally through a series of biodegradation tests under aerobic conditions. These included ultimate biodegradation, primary biodegradation and inoculum toxicity tests at different exposure concentrations. None of the test substances (≥3 rings) were found to be readily biodegradable. Therefore, to stimulate the biodegradation process, the microbial community from a wastewater treatment plant was adapted to a mixture of heterocyclic PAHs and biodegradation tests were performed with either adapted or non-adapted bacteria. Surprisingly, there were trade-offs in bacterial adaptation: a positive outcome (degradation of carbazole) is accompanied by a less desirable outcome (increased sensitivity to toxic effects of benzo[c]carbazole). Four of the compounds were identified as primarily biodegradable, while five compounds showed no evidence of biodegradation in any of the tests, indicating a high potential for environmental persistence. Predictions from mathematical models were also compared with measured results and it was found that the models were only partially successful in predicting the degradation timeframe of the heterocycles tested. Furthermore, the short-term acute aquatic toxicity of heterocyclic PAHs was investigated using three surrogate species from different trophic levels of the aquatic food web, including bacteria (Aliivibrio fischeri), unicellular algae (Raphidocelis subcapitata), and water fleas (Daphnia magna). In order to maintain a stable exposure and to avoid loss of concentration caused mainly due to sorption, the passive dosing method was adapted to routine test protocols. Most of the compounds were identified as highly toxic to the tested organisms, and non-polar narcosis (baseline toxicity) was found to be the most likely mode of toxic action for the tested chemicals. Membrane-water partition coefficients (KMW) of heterocyclic PAHs were also determined by solid-supported lipid membrane binding experiments and modeling. The measured KMW values were closely aligned with the KOW values, suggesting that both may be good descriptors for predicting the baseline toxicity and bioaccumulation potential of the tested heterocyclic PAHs. Lastly, the long-term chronic reproductive toxicity and bioaccumulation of large heterocyclic PAHs were investigated using Daphnia magna as an aquatic model organism. The passive dosing method ensured constant exposure even at very low concentration levels (70 ng L-1 to 68 µg L-1) in large and complex experimental systems. Given the enormous time and effort required to conduct long-term toxicity tests with minimal data output, a method was developed to assess chronic toxicity and bioaccumulation potential in a single test. All four substances tested were highly toxic and bioaccumulative in Daphnia magna. However, daphnids’ fecundity recovered rapidly from the toxic effects of the heterocycles during the depuration period in the absence of chemicals. The Predicted No Effect Concentrations (PNECs) of the test compounds were also estimated from the chronic toxicity data and ranged from 1 to 150 ng L-1 in a freshwater environment. Due to the lack of regulation and attention to these compounds, no systematic environmental monitoring data are available for their concentrations in freshwater. However, environmental concentrations of heterocyclic PAHs appear to be higher than the PNECs, particularly at contaminated sites. Thus, the tested heterocycles are of high concern in terms of risks to ecosystems and human health. Overall, the results of this research not only contribute to a comprehensive understanding and progress in the environmental hazard assessment of heterocyclic PAHs, but also present several methodological advances that can be applied to future testing of other challenging substances commonly referred to as 'difficult-to-test'.Stickstoff-, schwefel- und sauerstoffsubstituierte heterocyclische polyaromatische Kohlenwasserstoffe (heterocyclische PAK) sind in allen Umweltkompartimenten gegenwärtig und treten häufig zusammen mit ihren homocyclischen Analoga auf. Bei der Gefährdungsbeurteilung kontaminierter Standorte wurde ihnen bisher verhältnismäßig wenig Aufmerksamkeit geschenkt, zudem ist über ihr Verhalten und ihre Ökotoxizität nur wenig bekannt. Aufgrund ihrer höheren Polarität, die auf das Vorhandensein elektronegativer Atome zurückzuführen ist, ging man bislang davon aus, dass die meisten heterocyclischen PAK mobiler und weniger toxisch sind als ihre homocyclischen Pendants, weshalb sie wahrscheinlich bislang vergleichsweise wenig studiert wurden. Sollte die Annahme einer geringeren Toxizität nicht zutreffen, weil beispielsweise andere Mechanismen als die Polarität/Hydrophobie für ihre Gefährdung verantwortlich sind, könnte dies zu einer Unterschätzung der Risiken, die von kontaminierten Standorten oder anderen Emissionen ausgehen, führen. Die Hauptziele dieser Arbeit bestehen daher darin, (1) ein umfassenderes Verständnis der physikalisch-chemischen Eigenschaften, der biologischen Abbaubarkeit, der Ökotoxizität, der Bioakkumulation und der Mobilität dieser Verbindungen in der Umwelt zu erlangen; (2) eine Bewertung verschiedener Modellierungsansätze zur Vorhersage dieser Eigenschaften durchzuführen und (3) experimentelle Hindernisse zu überwinden, indem robustere Techniken entwickelt werden, die langfristig zuverlässige und reproduzierbare Daten liefern können. Aus der sehr breiten Gruppe der heterocyclischen Verbindungen wurden strukturell vergleichbare repräsentative Verbindungen mit unterschiedlichen log KOW-Werten (als Maß für die Hydrophobie), die vier Größenordnungen abdecken, als Modellstrukturen ausgewählt. Zunächst erfolgte eine Untersuchung umweltrelevanter physikalisch-chemischer Eigenschaften dieser heterocyclischen PAK, wie die Wasserlöslichkeit (SW), der n-Octanol-Wasser-Verteilungskoeffizient (KOW) und der Verteilungskoeffizient bezogen auf den organischen Kohlenstoff und Wasser (KOC). Dabei wurden die Auswirkungen von Molekülgröße, Art und Anzahl der Heteroatome auf die Löslichkeit und die Verteilungskoeffizienten systematisch untersucht. Diese experimentell ermittelten Parameter wurden dann verwendet, um die Leistung von In-silico-Modellen zu testen, einschließlich der quantitativen Struktur-Eigenschafts-Beziehung (QSPR), der linearen Polyparameter-Energie-Beziehung (pp-LFER) und des Conductor-like Screening Model for Realistic Solvation (COSMO-RS), die diese Eigenschaften vorhersagen. Zudem wurde eine Umweltverträglichkeitsprüfung auf Screening-Ebene durchgeführt, bei der Daten aus verschiedenen Quellen berücksichtigt wurden. Den Screening-Ergebnissen zufolge ist davon auszugehen, dass kleine Verbindungen mit zwei oder drei Ringen in der Umwelt mobil sind, während größere heterocyclische PAK im Allgemeinen als bioakkumulierbare Stoffe identifiziert wurden, was auf ein erhebliches Gefahrenpotenzial hinweist. Die Persistenz von heterocyclischen PAK wurde experimentell durch eine Reihe von Tests zum biologischen Abbau unter aeroben Bedingungen untersucht. Dazu gehörten Tests zur Mineralisierung, zum biologischen Primärabbau und zur Inokulumtoxizität bei unterschiedlichen Expositionskonzentrationen. Keine der Prüfsubstanzen mit drei oder mehr Kohlenstoffringen erwies sich als biologisch gut/schnell abbaubar. Um den biologischen Abbauprozess zu stimulieren, wurde daher die mikrobielle Gemeinschaft aus einer Kläranlage an das Gemisch heterocyclischer PAK angepasst, wobei die Tests zum biologischen Abbau entweder mit angepassten oder nicht angepassten Bakterien durchgeführt wurden. Überraschenderweise gab es bei der bakteriellen Anpassung Widersprüche: positive Effekte (wie der bessere Abbau von Carbazol) gehen mit weniger wünschenswerten Ergebnissen (erhöhte Toxizität gegenüber Benzo[c]carbazol) einher. Für vier der betrachteten Verbindungen wurde ein biologischer Primärabbau festgestellt, während fünf Substanzen in keinem der Tests Anzeichen für eine mikrobielle Umsetzung zeigten, was auf ein hohes Potenzial für eine Persistenz in der Umwelt hindeutet. Die Vorhersagen softwaregestützter Modelle wurden ebenfalls mit den Messergebnissen verglichen, und es zeigte sich, dass die Modelle bei der Vorhersage der Abbaukinetik der getesteten Heterocyclen nur teilweise übereinstimmende Ergebnisse lieferten. Darüber hinaus wurde die akute Kurzzeittoxizität heterocyclischer PAK anhand von drei Arten aus verschiedenen trophischen Ebenen des aquatischen Nahrungsnetzes untersucht: Bakterien (Aliivibrio fischeri), einzellige Algen (Raphidocelis subcapitata) und Wasserflöhe (Daphnia magna). Um eine stabile Exposition aufrechtzuerhalten und Verluste, zum Beispiel durch Sorption an Gefäßwandungen, zu vermeiden, wurde die Methode des „passive dosing“ an Routinetestprotokolle angepasst. Die meisten Verbindungen wurden in diesen Tests als hochgiftig für die getesteten Organismen identifiziert, wobei festgestellt werden konnte, dass unpolare Narkose (Basistoxizität) die wahrscheinlichste Art der toxischen Wirkung der getesteten Chemikalien ist. Darüber hinaus wurden die Membran-Wasser-Verteilungskoeffizienten (KMW) der heterocyclischen PAK durch Lipidmembran-Bindungsexperimente auf festem Trägermaterial sowie mit Hilfe von Vorausberechnungen (Modellierung) bestimmt. Die gemessenen KMW-Werte stimmten eng mit den KOW-Werten überein, was darauf hindeutet, dass beide Werte gute Deskriptoren für die Vorhersage der Basistoxizität bzw. des Bioakkumulationspotenzials der getesteten heterocyclischen PAK sein können. Schließlich wurden die langfristige chronische Reproduktionstoxizität und die Bioakkumulation großer heterocyclischer PAK mit Daphnia magna als aquatischer Modellorganismus untersucht. Die „passive dosing method“ gewährleistete eine konstante Exposition selbst bei sehr niedrigen Konzentrationen (70 ng L-1 bis 68 µg L-1) in groß angelegten und komplexen Versuchssystemen. Angesichts des enormen Zeit- und Arbeitsaufwands, der für die Durchführung von Langzeit-Toxizitätstests erforderlich ist, wurde eine Methode entwickelt, um die chronische Toxizität sowie das Bioakkumulationspotenzial in einem einzigen Test zu bewerten. Alle vier getesteten Substanzen waren gegenüber Daphnia magna hochtoxisch und stark bioakkumulierend. Unerwarteterweise erholte sich die Reproduktionsfähigkeit der Daphnien jedoch schnell von den toxischen Wirkungen der Heterocyclen, wenn die Chemikalienexposition endete. Die PNECs (Predicted No Effect Concentrations) der Testsubstanzen wurden ebenfalls anhand der Daten zur chronischen Toxizität verglichen. Die Werte lagen in einer Süßwasserumgebung zwischen 1 und 150 ng L-1 (PNEC). Für die meisten Testverbindungen gibt es keine Informationen zu Monitoringdaten in der Umwelt. Die Umweltkonzentrationen heterocyclischer PAK scheinen jedoch höher zu sein als die PNEC-Werte, insbesondere an kontaminierten Standorten, was eine Gefahr für die Ökosysteme und die menschliche Gesundheit darstellt. Insgesamt tragen die Ergebnisse dieser Forschungsarbeit nicht nur zu einem umfassenderen Verständnis und zu Fortschritten bei der Bewertung der von heterocyclischen PAK ausgehenden Umweltgefahren bei, sondern liefern auch mehrere methodische Verbesserungen, die in Zukunft bei der Prüfung anderer Stoffe, die gemeinhin als 'schwierig zu testen' bezeichnet werden, angewendet werden können

Using chemical structure and inocula characteristics to predictively model biodegradation rate

PhD ThesisPredictive biodegradation models [i.e. Quantitative Structure Biodegradation Relationship (QSBR) models] might be used as an alternative to current regulatory biodegradation tests to predict chemical persistence. Current models are mostly based on the results derived from regulatory Ready Biodegradability Tests (RBTs), which are highly variable and were not designed to provide half-life data and therefore fundamentally undermines efforts to reliably predict chemical persistence. Improvement to existing approaches for developing and verifying predictive models and their reliability, respectively, have been proposed, and the use of functional gene and 16S rRNA amplicon sequencing techniques towards identifying and quantifying the putative chemical degraders have been studied. Several QSBR models for aromatic chemicals were developed according to OECD principles. Models for mono-aromatic chemicals were verified and calibrated with experimentally determined rates (both from pure culture and natural mixed communities). Traditional test methods were combined with functional genes and 16S amplicon sequence analyses to develop a relationship between rate, chemical concentration and competent putative chemical degrader abundance. QSBR models for mono-aromatic chemicals were stable (R2 = 0.8924), robust (Q2LOO = 0.8718) and had good predictive ability (Q2F1 = 0.8829, Q2F2 = 0.8835, and Q2F3 = 0.9178). In these models, biodegradation rates were associated with electronic, lipophilic and steric descriptors, and thus provided information on the mechanisms of different rate-limiting steps associated with the biodegradation process. However, all the variation in biodegradation rates cannot be explained by the structure alone, the prevailing environmental conditions have a significant role in determining the extent of chemical degradation. Biodegradation rates (k) of chemicals in natural mixed communities were significantly correlated with the ratio of abundance of initial putative degrader abundances (X0) and the starting chemical concentration (C0) (Pearson correlation coefficient (r) > 0.9 and p-value < 0.05). Predictive models developed by relating k with X0 and C0 reliably predicted the rate of studied chemicals. Experimentally determined rates further formed the basis towards calibrating the developed QSBR models. The molecular analysis revealed that majority of identified putative chemical degraders were rare taxa, and their enrichment did not necessarily influence the overall biomass count of the microbial community, and therefore biodegradation models that only consider the overall biomass would not account for the kind of relationships found in this study. Application of 16S amplicon sequencing and functional gene analyses techniques in biodegradation studies will help in depth screening of diversity and function of microbial community in an inoculum and enables better understanding of biodegradation outcomes.funded by the Engineering and Physical Sciences Research Counci

The interactions between xenobiotics and soil microbial communities

Excessive use and lack of appropriate disposal technology for industrial xenobiotics have resulted in the contamination of ecosystems globally impacting the self-regulating capacity of the biosphere. This often results in irreversible alterations of ecosystem’s structure and function, but the outcomes of these events on soil microbial communities (and their functional capabilities) are poorly understood. Assessing the impact of xenobiotics on soil microbial communities is of paramount importance as they play a vital role in ecosystem services and maintain soil health, which are key requirements for sustainable land use in terms of food security and environmental sustainability. Bacteria are the most abundant and diverse soil micro-flora and play a key role in the biogeochemical cycles of important elements including carbon (C), nitrogen (N), and phosphorus (P) and sulphur (S). The current work aimed to unravel the two-way interactions between xenobiotics and soil microbial communities; i.e., how soil microbial communities modulate xenobiotic persistence through biodegradation and what impacts xenobiotic have on soil microbial community’s structure and functions, with particular focus on widely used pesticides (chlorpyrifos and imidacloprid) and industrial solvents (trichloroethene). In Chapter 2, characterisation of active methanotrophs involved in trichloroethene (TCE) degradation under different methane (CH4) concentrations was evaluated. Methane (CH4) enriched methanotrophic consortia from three Australian soils (Sydney University, Victoria Park and Botany Industrial Park) were examined for their effectiveness in TCE (50μM) degradation at 1%, 10% and 33% CH4 concentration at 20oC. Only the methanotrophic consortium from Sydney University (SU) soil was able to co-metabolically degrade TCE. The (SU) methanotrophic growth and TCE degradation was accelerated under high CH4 concentration degrading up to 30% (within 2 days) and 20% (within 5 days) TCE under 33% and 10% CH4, respectively. No degradation of TCE was observed at 1% CH4 concentration or in the absence of CH4 suggesting the dependence on relatively high CH4 availability for TCE degradation. pmoA-based stable isotope probing (SIP), terminal restriction fragment length polymorphism (T-RFLP), clone library construction and sequencing of TCE degrading SU methanotroph consortium revealed the dominance of novel uncultivable Type I methanotrophs (distantly related to Methylovulum-88%) belonging to TRF-53 in TCE degradation. In Chapter 3, the effects of the pesticides chlorpyrifos (CP) and imidacloprid (IC) on soil microbial processes (e.g. biodegradation and respiration) and community structure were evaluated. Two soil treatments (from five sugarcane farms), one with no history of pesticide application (non-treated; 1H, 2H, 3H, 4H and 5H) and the other with ~20 years pesticide application (pesticide-treated; 1R, 2R, 3R, 4R and 5R), were used in this study. MicroRespTM, q-PCR and T-RFLP analyses were combined to explore the relationship between pesticide degradation and soil microbial communities in soils spiked (3 times) with 10 mg/kg of CP or IC, under lab conditions. The results showed that the half-lives of CP decreased with application frequency and were 23-47, 8-20 and 3-17 days following the first, second and third application, respectively (for soils from five sugarcane farms). In particular, the soils from 4R, 4H and 5R showed enhanced CP degradation even when not exposed to CP for last 13 years due to legacy effect of the pesticide. Parallel analyses of IC degradation (10 mg/kg) showed high persistence of this pesticide in soil where repeated application increased half-lives from 30-60 days for the first treatment to 45-65 days for second treatment. The application of both pesticides (CP and IC) reduced soil respiration (basal and substrate-induced) between 7-76% with the lowest respiration found in 5R and highest in 1R after the pesticides treatment, indicating that application of pesticides had an adverse impact on soil functional activity. The molecular analyses showed that both CP and IC significantly altered the soil bacterial community structure and reduced diversity, evenness and richness. In Chapter 4, sequential soil and liquid culture enrichments enabled the isolation of six bacterial CP degraders with sequence homologies to Xanthomonas sp. (3), Pseudomonas sp. (1), Rhizobium sp. (1) and Lysobacter sp. (1). The efficacy of the isolated strains: Xanthomonas sp. 4R3-M1, Pseudomonas sp. 4H1-M3 and Rhizobium sp. 4H1-M1 were further investigated for biodegradation of CP and its primary metabolic product, TCP (3,5,6-trichloro-2-pyridinol). The results indicated that all three bacterial strains utilised CP (10 mg/l) and TCP (as CP degradation product) in mineral salt media (MSM) as a sole source of C and N. Bacterial strains Xanthomonas sp. 4R3-M1 and Pseudomonas sp. 4H1-M3 could also degrade 10 mg/l TCP as a sole C- and N-source, when provided externally. Thus, these bacterial strains promise to be effective in practical application of bioremediation of both CP and TCP. In Chapter 5, using next-generation sequencing, the structure and potential functions of bacterial communities in pesticide-treated and non-treated reference sites was compared at finer levels. Across all soils, the functional beta diversity was correlated with taxonomic diversity indicating possible linkages between the structure and functioning of soil microbial communities. The pesticide-treated sites had higher relative abundance of Proteobacteria and Bacteroidetes, with Archaea exhibiting the opposite pattern. Metagenomic analysis revealed increases in the relative abundance of genes associated with key specialised functions (iron acquisition and metabolism, motility, cell signalling, stress response) at pesticide-treated sites. The results suggested impacts of long-term pesticide application on soil microbial community composition and potential functions. Despite, a CP legacy effect, no marked difference was observed in abundance of genes related to P-metabolism between pesticide-treated and non-treated sites. Overall, the results supported taxonomic and functional adaptations in the soil microbial communities following pesticide treatment. Overall, this study provides the novel insights into the interaction between xenobiotics and soil microbial communities both at structural (diversity, community structure) and functional (degradation) levels and should be considered in developing new bioremediation technologies and agronomic practices such as number and frequency of pesticide applications