Este trabalho tem como finalidade estudar as melhores condições do cultivo da microalga Chlamydomonas reindhartii geneticamente modificada para a produção da proteína fluorescente mCherry a partir do estudo dos macronutrientes contidos no meio de cultivo. A proteína mCherry possui a vantagem de ser facilmente detectada no meio de cultivo por espectofotometria convencional, convertendo-se, desta forma, em uma molécula interessante para o estudo como modelo de expressão. Inicialmente, foram estudadas três diferentes fontes de nitrogênio para avaliar a expressão da proteína recombinante. Os resultados indicaram que a melhor fonte de nitrogênio para a produção da mCherry foi o NH4NO3. Em seguida, para avaliar os efeitos gerados pelos macronutrientes (acetato, cloreto de cálcio, sultato de magnésio, nitrato de amônio e fostato total) contidos no meio de cultivo TAP, foi realizado um planejamento composto central 25, em cultivos em microplacas, sendo os resultados avaliados por regressão multivariada. Além disso, a análise realizada por regressão multivariada indicou que, dos níveis avaliados das variáveis, as condições que melhor atendem à otimização da produção de mCherry e crescimento celular são: acetato, 33,35 mM; cloreto de cálcio, 0,45 mM; sulfato de magnésio, 0,83 mM; nitrato de amônio, 10,31 mM; fosfato total, 1,96 mM. Essas condições foram escolhidas para cultivo em fotobiorreator tubular, onde foi obtido título de fluorescência de mCherry a 608 nm de 59209 UF, correspondendo a um aumento de 118,5% maior que o título de fluorescência obtido com uso de meio TAP padrão. Com a finalidade de seguir com os processos de produção foi disenhado um biorreator tipo coluna e foi reaizado um estudio de produção em sistema semicontinuo. Os resultados obtidos demostraram que o sistema semicontinuo aumento 2,6 veces a produtividade da biomassa. / This work aims to study the best conditions of the cultivation of the microalgae Chlamydomonas reindhartii genetically modified for the production of the fluorescent protein mCherry from the study of the macronutrients contained in the culture medium. The mCherry protein has the advantage of being easily detected in the culture medium by conventional spectrophotometry, thus becoming an interesting molecule for the study as an expression model. Initially, three different nitrogen sources were studied to evaluate the expression of the recombinant protein. The results indicated that the best source of nitrogen for the production of mCherry was NH4NO3. Then, to evaluate the effects generated by macronutrients (acetate, calcium chloride, magnesium sulphate, ammonium nitrate and total phosphate) contained in the TAP culture medium, a central composite 25 was carried out in cultures on microplates, Results evaluated by multivariate regression. In addition, multivariate regression analysis indicated that, from the evaluated levels of the variables, the conditions that best serve the optimization of mCherry production and cell growth are: acetate, 33.35 mM; Calcium chloride, 0.45 mM; Magnesium sulfate, 0.83 mM; 10.31 mM ammonium nitrate; Total phosphate, 1.96 mM. These conditions were chosen for cultivation in tubular photobioreactor where fluorescence titre of mCherry at 608 nm of 59209 UF was obtained, corresponding to an increase of 118.5% greater than the titer of fluorescence obtained using standard TAP medium. In order to follow the production processes, a column type bioreactor was designed and a production study was carried out in a semicontinuous system. The results showed that the semicontinuous system increased 2.6 times the productivity of the biomass.

Este trabalho tem como finalidade estudar as melhores condições do cultivo da microalga Chlamydomonas reindhartii geneticamente modificada para a produção da proteína fluorescente mCherry a partir do estudo dos macronutrientes contidos no meio de cultivo. A proteína mCherry possui a vantagem de ser facilmente detectada no meio de cultivo por espectofotometria convencional, convertendo-se, desta forma, em uma molécula interessante para o estudo como modelo de expressão. Inicialmente, foram estudadas três diferentes fontes de nitrogênio para avaliar a expressão da proteína recombinante. Os resultados indicaram que a melhor fonte de nitrogênio para a produção da mCherry foi o NH4NO3. Em seguida, para avaliar os efeitos gerados pelos macronutrientes (acetato, cloreto de cálcio, sultato de magnésio, nitrato de amônio e fostato total) contidos no meio de cultivo TAP, foi realizado um planejamento composto central 25, em cultivos em microplacas, sendo os resultados avaliados por regressão multivariada. Além disso, a análise realizada por regressão multivariada indicou que, dos níveis avaliados das variáveis, as condições que melhor atendem à otimização da produção de mCherry e crescimento celular são: acetato, 33,35 mM; cloreto de cálcio, 0,45 mM; sulfato de magnésio, 0,83 mM; nitrato de amônio, 10,31 mM; fosfato total, 1,96 mM. Essas condições foram escolhidas para cultivo em fotobiorreator tubular, onde foi obtido título de fluorescência de mCherry a 608 nm de 59209 UF, correspondendo a um aumento de 118,5% maior que o título de fluorescência obtido com uso de meio TAP padrão. Com a finalidade de seguir com os processos de produção foi disenhado um biorreator tipo coluna e foi reaizado um estudio de produção em sistema semicontinuo. Os resultados obtidos demostraram que o sistema semicontinuo aumento 2,6 veces a produtividade da biomassa. / This work aims to study the best conditions of the cultivation of the microalgae Chlamydomonas reindhartii genetically modified for the production of the fluorescent protein mCherry from the study of the macronutrients contained in the culture medium. The mCherry protein has the advantage of being easily detected in the culture medium by conventional spectrophotometry, thus becoming an interesting molecule for the study as an expression model. Initially, three different nitrogen sources were studied to evaluate the expression of the recombinant protein. The results indicated that the best source of nitrogen for the production of mCherry was NH4NO3. Then, to evaluate the effects generated by macronutrients (acetate, calcium chloride, magnesium sulphate, ammonium nitrate and total phosphate) contained in the TAP culture medium, a central composite 25 was carried out in cultures on microplates, Results evaluated by multivariate regression. In addition, multivariate regression analysis indicated that, from the evaluated levels of the variables, the conditions that best serve the optimization of mCherry production and cell growth are: acetate, 33.35 mM; Calcium chloride, 0.45 mM; Magnesium sulfate, 0.83 mM; 10.31 mM ammonium nitrate; Total phosphate, 1.96 mM. These conditions were chosen for cultivation in tubular photobioreactor where fluorescence titre of mCherry at 608 nm of 59209 UF was obtained, corresponding to an increase of 118.5% greater than the titer of fluorescence obtained using standard TAP medium. In order to follow the production processes, a column type bioreactor was designed and a production study was carried out in a semicontinuous system. The results showed that the semicontinuous system increased 2.6 times the productivity of the biomass.

The Great Brak temporarily open/closed estuary was subjected to a drought during the spring and summer of 2009/2010 resulting in the mouth remaining closed for a prolonged period. According to the Great Brak Estuary management programme, the mouth of the estuary had to be open for a total of 308 days during spring and summer of 2009/2010, respectively, but was closed for almost the entire two years (693 days). The aim of this study was to assess monthly changes in the abiotic characteristics (salinity, temperature, oxygen, pH and nutrients) and the biotic responses of phytoplankton and macroalgae; identify sources of nutrient input into the estuary and determine the response of the salt marsh to water level and salinity changes. The results indicated that physico-chemical parameters were similar to that previously recorded during the closed mouth condition. However mouth closure combined with elevated nutrient concentrations led to a shift from rooted submerged macrophytes to one where either microalgae or macroalgae were dominant. Soluble reactive phosphorus concentrations were significantly higher in bottom compared with surface waters. There was a significant negative correlation with SRP and dissolved oxygen for the sampling period indicating potential release of phosphorus from the sediment during closed mouth conditions. Microalgal biomass increased in response to remineralised nutrients and freshwater pulses. Flagellates were the dominant microalgal group (21718 ± 3336 cells m l-1, p < 0.05) because of their morphological ability to migrate vertically within the water column. The macroalgal cover was highest during the closed mouth state but only during winter (August 2010) when temperatures were below 20 oC. Five major point sources of nutrient input into the Great Brak Estuary were identified during rainfall periods. Point sources 4 and 5 in the upper reaches of the estuary had the highest DIN input whereas point source 3 in the middle reaches of the estuary had the highest DIP input. As a result of the drought and low water level, the salt marsh was never inundated for longer than 3 months. Die-back of Sarcocornia decumbens (r 2= -0.62, p < 0.05) was related to smothering by dead macroalgae whereas dieback of Sporobolus virginicus was related to decreasing nutrient (r2 = 0.59, p < 0.05) and salinity (r2 = 0.55, p < 0.05) levels. The physico-chemical characteristics alone did not convey the true health status of the Great Brak Estuary for the duration of the sampling (April 2010-April 2011). The study showed that microalgae and macroalgae are valuable indicators of the status of the estuary. Therefore it is suggested that bio-indicators are incorporated into the management/monitoring plan in order to assist in improving the health assessment of the Great Brak Estuary.