Evaluation of Dunaliella salina growth and corresponding ß-carotene production in tubular photobioreactor / Avaliação do crescimento da Dunaliella salina e correspondente produção de ß-caroteno em fotobiorreator tubula

Microalgae, photosynthetic microorganisms, are rich in lipids, polyunsaturated fatty acids, carbohydrates, proteins, vitamins, as well as carotenoids, which are antioxidants that may protect human body from various diseases including obesity, cardiovascular disease, vision-related diseases such as macular degeneration and certain types of cancer. These natural pigments have applications in the pharmaceutical (nutraceutical), food (coloring, functional food, and supplements), and cosmetics industries (e.g. sunscreen), as well as in aquaculture (animal feed). The Dunaliella salina microalga can synthesize 10% of dry weight in ß-carotene (orange pigment, pro-vitamin A activity) under high light intensity and nitrogen and phosphorus limitation, among other stress conditions. The first chapter of this thesis presents a review focused on microalgae carotenoids: culture systems, mode of operation, and applications. In this bibliographic survey, the advantages of microalgae cultivation in relation to traditional sources (higher plants) were discussed, as well as a discussion of the main cultivation systems and their importance in cell growth. This review presented a critical analysis of the different operational regimes like batch, fed-batch, semi-continuous and continuous. Relevant information on the most important world producers of microalgae carotenoids were presented. Chapter II presents the development of a modified method of dispersive liquid-liquid microextraction (DLLME) for rapid extraction of ß-carotene from Dunaliella salina cultivated in tubular photobioreactor, with subsequent development of a rapid chromatographic screening method using a C4 column for separation of geometric isomer of ß-carotene. The use of benzene as extraction solvent and water with 50% acetone as dispersant provided the best condition for the extraction of this carotenoid. In HPLC (High Performance Liquid Chromatography), employing mobile phase composed of methanol and water (95:5, v/v), it was possible to detect/quantify ß-carotene at 14 min (retention time). Besides the short analysis time (<20 min), by the miniaturized extraction (< 10 mL organic waste) this method abide by green chemistry analytical principles. It is known that nitrogen, phosphorus, as well as carbon and vitamins are vital elements for the growth of microalgae, also determining the biochemical composition of biomass. In this sense, Chapter III presents the study of the influence of different amounts of sodium nitrate (1N = 75 mg L-1; 1.5N = 112.5 mg L-1, and 3N = 225 mg L-1) and phosphate monobasic dehydrate (1P = 5.65 mg L-1, 1.5P = 8.47 mg L-1, and 3P = 16.95 mg L-1) in seawater-based f/2 medium on the growth of Dunaliella salina and ß-carotene biosynthesis, by continuous process with different replenishment proportions (R = 20% and 80%). Best results of cell productivity were obtained by semicontinuous process (mean values of Px up to 6.7 x 104 cells mL-1 d-1 with medium 1N:1P; R =20%) in comparison with batch process cultivation. Maximum cell density (Xm) obtained in this work was not dependent of R, but the best results were obtained when using medium 1.5N:1.5P (mean values up to 5.6 x 105 cells mL-1 with R =80%) instead of 1N:1P. The content of ß-carotene in the cells, in general, was higher in cells grown in medium 1N:1P (mean yield values up to 57.5 mg g-1 with R =80%) in comparison with medium 1.5N:1.5P. The cultivation of D. salina with media 3N:3P led to a long lag phase, followed by decrease in cell density and cell lysis. The use of a tubular photobioreactor contributed to successfully cultivate this microalga without contamination by protozoa. The cultivation of Dunaliella salina in tubular photobioreactor with the use of 12:12 photoperiod was appropriate, as well as to induce carotenogenesis, in the second stage, by increasing the light intensity and absence of pH control

As microalgas, micro-organismos fotossintetizantes, são ricas em lipídios, ácidos graxos poli-insaturados, carboidratos, proteínas, vitaminas, além de carotenoides que são antioxidantes com potencial de proteger o organismo humano de várias doenças incluindo a obesidade, doenças cardiovasculares, doenças relacionadas à visão como a degeneração macular e certos tipos de câncer, entre outras. Esses pigmentos naturais têm aplicações em indústrias farmacêuticas (nutracêuticos), alimentícias (colorantes, alimentos funcionais e suplementos) e de cosméticos (exemplo: filtro solar) e na aquacultura (ração animal). A microalga Dunaliella salina é capaz de sintetizar, sob alta intensidade luminosa e limitação de nutrientes como fontes de fósforo e nitrogênio, dentre outras condições de estresse, 10 % do peso seco em ß-caroteno (pigmento laranja com atividade pró-vitamina A). Assim, neste trabalho, numa primeira etapa, foi feita uma revisão da literatura abordando a produção de carotenoides por microalgas, bem como sua aplicação. Nesse levantamento bibliográfico abordou-se, dentre outros assuntos, as vantagens do cultivo de microalgas em relação as fontes tradicionais (plantas superiores), assim como uma discussão dos diferentes sistemas de cultivos e sua importância no crescimento celular. Esse review apresentou uma análise crítica dos principais regimes operacionais como batch, fed-batch, semicontínuo e contínuo. Apresentou-se também informações relevantes sobre os mais importantes produtores mundiais de carotenoides de microalgas. Numa segunda etapa, foi desenvolvido um método modificado de microextração líquido-líquido dispersivo modificado (DLLME) para a rápida extração de ß-caroteno de Dunaliella salina cultivada em fotobiorreatores tubulares, com subsequente desenvolvimento de método cromatográfico em uma coluna C4 para a separação do isômero geométrico de ß-caroteno. A extração ótima de ß-caroteno foi obtida com benzeno como solvente extrator e água com 50% de acetona como dispersante. Empregando uma fase móvel composta por metanol e água (95:5, v/v) em HPLC, foi possível a detecção/quantificação de ß-caroteno com 14 minutos de tempo de retenção. Além dos tempos curtos de análises (<20 min), pela extração em volume reduzido (< 10 mL resíduos orgânicos) este método obedece aos princípios da química verde. Sabe-se que nitrogênio, fósforo, assim como carbono e vitaminas são elementos vitais para o crescimento das microalgas e também exercem influência na composição bioquímica da biomassa. Assim, na terceira etapa deste trabalho, estudou-se a influência das quantidades de nitrato de sódio (75 mg L-1, denominado 1N; 112,5 mg L-1, denominado 1,5N; 225 mg L-1, denominado 3N) e de fosfato monobásico dihidratado (5,65 mg L-1, denominado 1P; 8,47 mg L-1, denominado 1,5P; 16,95 mg L-1, denominado 3P) em meio f/2, que tem como base a água do mar, no crescimento e na síntese de ß-caroteno da Dunaliella salina por processo semicontínuo, com uso de frações de corte (R) de 20% e 80%. Foram obtidas produtividades celulares mais elevadas em processos semicontínuos do que em processo descontínuo, com produtividades médias de até 6,7 x 104 células mL-1 d-1 (meio 1N:1P; R =20%). A máxima concentração celular (Xm) obtida neste trabalho não foi dependente de R. Os melhores resultados de Xm foram obtidos quando se usou meio 1,5N:1,5P em vez de meio, com 1N:1P, com valores médios de até 5,6 x 105 células m L-1 (R =80%). O conteúdo de ß-caroteno nas células, de maneira geral, foi maior nas células cultivadas em meio 1N:1P do que no meio 1,5N:1,5P, com valores até 57,5 mg g-1 (R =80%). O cultivo de D. salina com o meio 3N:3P levou a uma longa fase lag, seguida por uma diminuição na concentração celular e sua lise. O cultivo de células em um fotobiorreator tubular contribuiu para um crescimento celular sem contaminação por protozoários. O cultivo de Dunaliella salina em fotobiorreator tubular com o uso de fotoperíodo 12:12 foi apropriado, assim como induzir a carotenogênese, no segundo estágio, por meio do aumento da intensidade luminosa e ausência de controle de pH

Volatile Components and Antibacterial Profile of Essential Oils Extracted from Leaves and Twigs of Pistacia lentiscus L.

Aims: The present work aims to evaluate the antibacterial activity of essential oils of two different parts of Pistacia lentiscus (leaves and twigs) and to determine their chemical composition. Study Design: An experimental study Place and Duration of Study: 1- Laboratory of Microbial Biotechnology, Faculty of Science and Technology Saïss, Sidi Mohamed Ben Abdellah University, Fez, MOROCCO. 2- Laboratory of Aromatic Plants, Medicinal and Natural Substances, National Institute of Medicinal and Aromatic Plants, Sidi Mohamed Ben Abdellah University, Fez. MOROCCO. Between November 2012 and April 2013. Methodology: The study of antibacterial activity was performed on gram-negative bacteria Salmonella sp., Pseudomonas aeruginosa, Mycobacterium smegmatis and Mycobacterium MC² 155 aurum A+ and gram positive bacteria Staphylococcus aureus, Bacillus sp. and Enterococcus faecalis by the method of agar diffusion. The chemical composition of essential oils was identified by gas chromathography. Results: These oils presented an important antibacterial activity against Salmonella sp., Pseudomonas aeruginosa, Staphylococcus aureus, Bacillus sp., Enterococcus faecalis, Mycobacterium aurum, Mycobacterium smegmatis. The use of gas chromatography enabled us to identify a total of 43 volatile components in the essential oil of twigs and 36 in leaves, representing respectively 95.9% and 86.8% of the chemical composition of these essences. These compositions are quantitatively and qualitatively different, which seems to be due to large structure differences of the two plant parts tested. Conclusion: Current study supports the traditional use of aromatic plants as antibacterial agents.

A new approach for quantifying furanodiene and curzerene: a case study on the essential oils of Eugenia uniflora L., Myrtaceae (pitangueira) leaves

The essential oil obtained from the leaves of Eugenia uniflora L., Myrtaceae, which grows in the Brazilian savannah, was studied by gas chromatography mass spectrometry (GC-MS). Furanodiene (1.2 percent) was thermally rearranged to curzerene (85.1 percent) to produce a combined content of 86.3 percent. GC analysis carried out under mild conditions (with a constant temperature of 100 ºC) showed that the furanodiene concentration was three-fold greater than the curzerene concentration, i.e., the essential oil contained 64.7 percent furanodiene and 21.6 percent curzerene. Germacrene B also rearranged to γ-elemene and the concentration of both was 2.3 percent. Special care should be taken when conventional gas chromatography analysis is used for quantifying compounds that can rearrange at high temperatures.

Gas chromatographic analysis of 2,3,4,5,6-Pentafluorobenzyl monofluoroacetate / 中国法医学杂志

Objective To determine 2, 3, 4, 5, 6-pentafluorobenzyl monofluoroacetate (PFB-MFA) as the derivative of monofluoroacetate anion ion by gas chromatography, by which fluoroacetamide, one of the raticides may be analyzed. Methods The calibration curve and limit of detection were obtained by quantification of standardized PFB-MFA using GC/ECD and GC/MS. Results The linear ranges for PFB-MFA were 0.01～0.1ng/?for GC/ECD,1～100ng/?l for GC/MS SCAN and 5?10~(-3)～1 ng/?l for GC/MS SIM. The limit of detections for fluoroacetate anoin were 1.31?10~(-4)ng/?l for GC/ECD, 0.13 ng/?l for GC/MS SCAN, and 1.76?10~(-4) ng/?l for GC/MS SIM. Conclusion The monofluoroacetate anion derivative (MFAPFB) can be deterimined accurately with high sensitivity by GC/ECD and GC/MS SIM, which may be used for analysis of fluoroacetamide in forensic practice.

Pseudonomas sp.W2 Metabolic Pathway of Bisphenol A / 微生物学通报

With GC-MS、LC-UV and gene analysis,we studied Pseudonomas sp.W2 metabolic pathway of bisphenol A(Bpa).It was discovered that 4'-(trimethylsiloxy)-Acetophenone、p-Hydroxy benzaldehyde and p-Hydroxy benzoic acid are medium metabolites and that the bacteria has pcaG.