Metalworking fluids (MWFs) are coolants and lubricants, which are widely employed in metal cutting works. They are designed to be a long lasting product. Manufacturers have designed MWFs with lack of awareness of end-of-life disposal by including biocides, which make biological treatment challenging. Here, Syntilo 9913 was used as a case study to develop a cradle-to-grave product that was biologically stable in use but amenable to sustainable hybrid biological treatment at end-of-life. The product was reverse engineered employing factorial design approach based on a priori knowledge of the product components. From the combinatorial work, it was observed that chemical interactions can results in synergistic and antagonistic effects in terms of the toxicity and biodegradability. One of the major components of most MWFs are amines such as Triethanolamine (TEA). TEA does not biodeteriorate in single compound screening, but in combination with many other components TEA was found to cause "softening" of MWF formulations. Octylamine was found to be best for "bio-hardening" but it was not economically sustainable. Hence, the modified biocide-free synthetic MWF, Syntilo 1601, was reformulated with TEA, isononanoic acid, neodecnoic acid, Cobratec TT50S, and pluronic 17R40, which were resistant to biological treatment. Although, no change in the overall oxidation state of the MWF, metabolic activity did occur as breakdown products were observed. This suggested that both raw materials and metabolic breakdown products were recalcitrant. Thus, immobilisation agents were applied to aid further biodegradation by removing toxic bottleneck compounds. It was found that hybrid nano-iron and kaffir lime leaf performed similarly in removing chemical oxygen demand and ammonium from the system. Work in this Thesis demonstrated that the combined use of biological treatment and immobilisation agents effectively overcome the limitations of biological treatment alone by removing bottleneck compounds, which allowed greater COD reduction. This laboratory scale is a proof of principle, which needs to be tested at full scale.

Este projeto teve como objetivo a construção de biosensores para a detecção de compostos monoaromáticos do grupo BTEX a partir dos componentes da via de degradação de compostos monoaromáticos codificada no plasmídeo TOL de Pseudomonas putida, mais precisamente: o promotor Pu e a proteína reguladora XylR, que ativa o promotor Pu após a ligação ao efetor monoaromático. Um objetivo secundário foi a verificação da existência de sequências reguladoras desconhecidas a montante do promotor Pu, construindo três variantes com fragmentos de Pu que se estendem por diferentes comprimentos a montante do promotor (Pu202 pb, Pu396 pb e Pu802 pb), cuja existência foi sugerida em trabalho anterior do laboratório. O promotor Pu foi ligado ao gene indicador para fosfatase alcalina isolado de E. coli. Todos os componentes das três variantes de biossensores foram clonados com sucesso. A construção de um dos plasmídeos de biossensoramento com a variante mais curta de Pu (Pu202) foi concluída. / Monoaromatic compounds are mainly responsible for the contamination of areas, this is because they are components of gas and the fuel stations most of accidents sites. Such compounds are highly toxic and, in between non-polar compounds, present high solubility and vapor pressure which assists them in dispersion at groundwaters and soil. This work aim to develop a biosensor based on alkaline phosphatase indicator gene under the regulation of the Pu promoter and its regulatory protein, XylR, that is activated by monoaromatic. Moreover, this work will continue a previous project of the research group that indicated a possible regulatory region not described for Pu, that hypothesis will be tested by producing different plasmids biosensors with varying sizes of Pu (202 bp, 396 bp and 802 bp). All biosensor fragments were purified and cloned on pGem T Easy and biosensor with Pu 202 pb was produced. Next goals are finishing others biosensors assemble and perform induction tests.

Este projeto teve como objetivo a construção de biosensores para a detecção de compostos monoaromáticos do grupo BTEX a partir dos componentes da via de degradação de compostos monoaromáticos codificada no plasmídeo TOL de Pseudomonas putida, mais precisamente: o promotor Pu e a proteína reguladora XylR, que ativa o promotor Pu após a ligação ao efetor monoaromático. Um objetivo secundário foi a verificação da existência de sequências reguladoras desconhecidas a montante do promotor Pu, construindo três variantes com fragmentos de Pu que se estendem por diferentes comprimentos a montante do promotor (Pu202 pb, Pu396 pb e Pu802 pb), cuja existência foi sugerida em trabalho anterior do laboratório. O promotor Pu foi ligado ao gene indicador para fosfatase alcalina isolado de E. coli. Todos os componentes das três variantes de biossensores foram clonados com sucesso. A construção de um dos plasmídeos de biossensoramento com a variante mais curta de Pu (Pu202) foi concluída. / Monoaromatic compounds are mainly responsible for the contamination of areas, this is because they are components of gas and the fuel stations most of accidents sites. Such compounds are highly toxic and, in between non-polar compounds, present high solubility and vapor pressure which assists them in dispersion at groundwaters and soil. This work aim to develop a biosensor based on alkaline phosphatase indicator gene under the regulation of the Pu promoter and its regulatory protein, XylR, that is activated by monoaromatic. Moreover, this work will continue a previous project of the research group that indicated a possible regulatory region not described for Pu, that hypothesis will be tested by producing different plasmids biosensors with varying sizes of Pu (202 bp, 396 bp and 802 bp). All biosensor fragments were purified and cloned on pGem T Easy and biosensor with Pu 202 pb was produced. Next goals are finishing others biosensors assemble and perform induction tests.