Media development and process parameter optimization using statistical experimental designs for the production of nonribosomal peptides in Escherichia coli

BACKGROUND: Nonribosomal peptide synthases (NRPS) can synthesize functionally diverse bioactive peptides by incorporating nonproteinogenic amino acids, offering a rich source of new drug leads. The bacterium Escherichia coli is a well-characterized production host and a promising candidate for the synthesis of nonribosomal peptides, but only limited bioprocess engineering has been reported for such molecules. We therefore developed a medium and optimized process parameters using the design of experiments (DoE) approach. RESULTS: We found that glycerol is not suitable as a carbon source for rhabdopeptide production, at least for the NRPS used for this study. Alternative carbon sources from the tricarboxylic acid cycle achieved much higher yields. DoE was used to optimize the pH and temperature in a stirred-tank reactor, revealing that optimal growth and optimal production required substantially different conditions. CONCLUSIONS: We developed a chemically defined adapted M9 medium matching the performance of complex medium (lysogeny broth) in terms of product concentration. The maximum yield in the reactor under optimized conditions was 126 mg L-1, representing a 31-fold increase compared to the first shaking-flask experiments with M9 medium and glycerol as the carbon source. Conditions that promoted cell growth tended to inhibit NRPS productivity. The challenge was therefore to find a compromise between these factors as the basis for further process development.

Biomass of unconventional plants from Brazilian semiarid as substrate for hydrolytic enzymes production by Aspergillus niger under solid and submerged fermentation

Aspergillus niger KIJH was grown in solid and submerged fermentation using leaves and roots (with and without bark) of plants typically from Brazilian semiarid as substrate to produce a multienzymatic extract, which was characterised for its potential biotechnological applications. Solid-state fermentation (SSF) was applied to select the most promising plants biomass as induction substrates for the production of hydrolytic enzymes by fungus. The best biomasses were used as substrate in submerged fermentation (SmF) assays at two scales. Samples of up scale fermented culture were partially purified by ultrafiltration and activity and pH and temperature stability of CMCase and xylanase were evaluated. A. niger KIJH produced hydrolytic enzymes under SSF containing unconventional plants biomass from Brazilian semiarid. In SmF conditions, maximum CMCase (0.264 U mL-1) and xylanase (1.163 U mL-1) activities were induced by Jacaratia corumbensis. Scaling up the SmF to 500 mL of medium was able to maintain constant the production of CMCase (0.346 U mL-1) and xylanase (1.273 U mL-1) on the fermented culture. Ultrafiltered and concentrated extract presented CMCase activities practically constant in all temperature ranges (30-80°C) and pH (3.0-9.0), while xylanase optimum activity temperature was 50°C and pH in the range of 3.0 to 5.0. CMCase activity remained stable for 24 hours at 50°C

Biomass of unconventional plants from Brazilian semiarid as substrate for hydrolytic enzymes production by Aspergillus niger under solid and submerged fermentation

Aspergillus niger KIJH was grown in solid and submerged fermentation using leaves and roots (with and without bark) of plants typically from Brazilian semiarid as substrate to produce a multienzymatic extract, which was characterised for its potential biotechnological applications. Solid-state fermentation (SSF) was applied to select the most promising plants biomass as induction substrates for the production of hydrolytic enzymes by fungus. The best biomasses were used as substrate in submerged fermentation (SmF) assays at two scales. Samples of up scale fermented culture were partially purified by ultrafiltration and activity and pH and temperature stability of CMCase and xylanase were evaluated. A. niger KIJH produced hydrolytic enzymes under SSF containing unconventional plants biomass from Brazilian semiarid. In SmF conditions, maximum CMCase (0.264 U mL-1) and xylanase (1.163 U mL-1) activities were induced by Jacaratia corumbensis. Scaling up the SmF to 500 mL of medium was able to maintain constant the production of CMCase (0.346 U mL-1) and xylanase (1.273 U mL-1) on the fermented culture. Ultrafiltered and concentrated extract presented CMCase activities practically constant in all temperature ranges (30-80°C) and pH (3.0-9.0), while xylanase optimum activity temperature was 50°C and pH in the range of 3.0 to 5.0. CMCase activity remained stable for 24 hours at 50°C a

Análise Técnico-Econômica para a produção de biofármacos de L-asparaginase / Technical-Economic analysis for the production of L-asparaginase biopharmaceuticals

A enzima L-asparaginase é comumente utilizada como biofármaco para o tratamento da Leucemia Linfoblástica Aguda e possui altas taxas de cura com o medicamento disponível no mercado. Atualmente a aquisição deste biofármaco é fruto integral de importação, não sendo realizada produção nacional, muito embora existam grupos de pesquisas nacionais que trabalham em pesquisas e no desenvolvimento de biofármacos alternativos da L-asparaginase. Assim, a presente dissertação tem como objetivo realizar análises técnico-econômicas para avaliar a viabilidade de implementação industrial de bioprocessos para a produção da L-asparaginase do tipo Erwinase PEGuilada e não PEGuilada, que foram previamente desenvolvidos na FCF-USP. As análises técnico-econômicas foram conduzidas por meio do software SuperPro Design® (Intelligen, Inc.) e permitiram adaptar o processo laboratorial para um processo piloto e possibilitaram estimar os valores de custo de produção unitário (Unity Cost of Production - UPC) de US$ 12,37/mg e US$ 3,46/mg para a L-asparaginase monoPEGuilada e nativa obtida por processo similar, respectivamente. O custo unitário de produção para a enzima peguilada foi, portanto, estimado em cerca de 4 vezes o mesmo custo para a produção da enzima peguilada, sendo tal aumento de custo devido às operações de peguilação, já que ambas as plantas foram mantidas nas mesmas dimensões. Ainda, foram obtidos indicadores econômicos, que indicam a atratividade do processo desenvolvido, muito embora tenham sido identificados diversos gargalos de processo e fatores a serem otimizados e melhorados de forma a tornar o processo mais atrativo sob os pontos de vista técnico e econômico. Em uma análise de sensibilidade preliminar um aumento factível da densidade celular já mostra que é possível reduzir em mais de 30% o UPC. De toda forma, ainda que não otimizado, o processo apresentou valores e dados compatíveis com os biofármacos de L-asparaginase já disponíveis no mercado

The enzyme L-asparaginase is commonly used as a biopharmaceutical in the treatment of Acute Lymphoblastic Leukemia, presenting high cure rates with the formulations available on the market. Nowadays, the acquisition of this biopharmaceutical is only from importation, given that there is no national production being carried out, although there are national research groups working on research and development of alternative L-asparaginase biopharmaceuticals. Thus, this project aims at carrying out technical-economic analyzes to evaluate the viability of industrial implementation of bioprocesses for the production of L-asparaginase of the PEGylated and non-PEGylated Erwinase type previously developed at FCF-USP. The technical-economic analyzes, conducted by means of the software SuperPro Design® (Intelligen, Inc.), allowed to adapt the laboratory process to a pilot process and made it possible to estimate the unit cost of production (UPC) values of US $ 12.37 / mg and US $ 3.56 / mg for monoPEGylated L-asparaginase and bare obtained by similar process, respectively. The unit cost of production for the pegylated enzyme was, therefore, estimated at about 4 times the same cost for the production of the pegylated enzyme, such an increase in cost due to pegylation operations, since both plants were maintained in the same dimensions. Moreover, economic indicators were obtained, which indicate the attractiveness of the developed process. However, several process bottlenecks and factors to be optimized and improved were identified to make the process more attractive from the technical and economic point of view. In a preliminary sensitivity analysis, a feasible increase in cell density already shows that it is possible to reduce UPC by more than 30%. Accordingly, although not optimized, the process presented values and data compatible with the L-asparaginase biopharmaceuticals already available on the market

Advances in Microalgal Research in Brazil

Abstract Microalgae research has attracted interest worldwide and in order to advance algal biotechnology in Brazil, government has been funding several projects. In the last 10 years, two main funds were provided by the National Council of Scientific and Technological Development (CNPq) agency to researchers in Brazil, who study the potential uses of microalgae for biomass, bioproducts and biofuels production. These funded projects addressed aspects of algal strain identification, development of algal cultivation techniques, designing photobioreactors and raceway ponds, modeling harvesting and dewatering process, maximizing biomass and oil productivities, characterizing chemical composition with different extractions systems and determining physiochemical properties of biodiesel. This review presents the state of art of algal research conducted by Brazilian institutions. Special attention is given to the recent progress on microalgal cultivation, high-value products extracted from microalgae and potential biofuels production. This review may serve as a policy instrument for planning next steps for algal research in Brazil as well as for attracting attention from international researchers who work with microalgae and would like to pursue a future partnership on algal research with Brazilian research institutions.

Amino Acid Supplementation Improves the Production of Extracellular Peptidases by Aspergillus Section Flavi and their Ionic Immobilization

Abstract Bioprocess studies have been highlighted due to the importance of physiological processes and industrial applications of enzymes. The potential of peptidase production from Aspergillus section Flavi using different amino acids as a supplemental nitrogen source was investigated. A production profile revealed that amino acids had positive effects on peptidase production when compared to the control without amino acids. Optimal production (100 U/mL) was obtained with Arginine amino acid in 96 h of fermentation. Extracellular peptidase from Aspergillus section Flavi was identified in submerged bioprocesses by in situ activity. Biochemical studies revealed that the maximum activities of the enzyme extract were obtained at pH 6.5 and a temperature of 55°C. The inhibition by EDTA and PMSF suggests the presence of more than one peptidase while the Ni2+ and Cu2+ had a negative influence on the enzyme activity. When the crude extract was reversibly immobilized on ionic supports, DEAE-Agarose and MANAE-Agarose the derivative showed different profiles of thermal and pH stabilities. Hence, this study revealed the basic properties and biochemical characteristics that allowed the production improvement of this class of enzyme. Moreover, with known properties stabilization and immobilization process is required to further explore its biotechnological capacities.

Produção de biossurfactantes: manoproteínas intracelulares e soforolipídios extracelulares por Saccharomyces cerevisiae / Biosurfactant production: intracellular manoproteins and extracellular sophorolipids by Saccharomyces cerevisiae

RESUMO Os biossurfactantes apresentam inúmeras aplicações ambientais e são produzidos por diversos microrganismos. Os provenientes da levedura Saccharomyces cerevisiae são pouco estudados para fins ambientais, sendo atóxicos. Objetivou-se o estudo da produção de biossurfactantes intra e extracelular por essa levedura, desenvolvida em meio de cultivo contendo 0,5% de extrato de levedura e 1% de peptona, além de concentrações variadas de sacarose e indutores oleosos - glicerol e óleos de soja e diesel. Os experimentos foram realizados durante 96 horas, e a produção de biossurfactantes foi avaliada diariamente, por meio da redução da tensão superficial e de estabilização de emulsões. O biossurfactante extracelular foi extraído da biomassa obtida, com posterior precipitação e caracterização química por intermédio de espectrometria de massa. As maiores produtividades de emulsificantes extracelulares foram obtidas com glicerol (0,20 UE.h-1) e óleo de soja (0,21 UE.h-1), em 48 horas de cultivo. Em ensaios posteriores, realizados com aumento da concentração de indutor, foi verificado um aumento das produtividades extracelulares para 0,45 UE.h-1 para o glicerol e 0,30 UE.h-1 para o óleo de soja. A maior redução da tensão superficial foi de 9,89%, em 72 horas, para o indutor óleo diesel. A diminuição dessa tensão, aliada ao aumento das atividades emulsificantes, é um importante indicativo da utilização do substrato hidrofóbico pelo microrganismo. O estudo comprova aumento na produção de biossurfactantes extracelulares quando realizada otimização de cultivo. Para a produção dos intracelulares, a necessidade de processo de rompimento celular aumenta os custos do bioprocesso.

ABSTRACT Biosurfactants implicate many environmental applications, being produced by a wide range of microorganisms. Those from the Saccharomyces cerevisiae yeast are still poorly studied for environmental purposes and are non-toxic. The aim of the study was the production of intra- and extracellular biosurfactants by the Saccharomyces cerevisiae yeast. The yeast was grown in cultured medium containing 0.5% yeast extract, 1% peptone and variable concentrations of sucrose and oily inducers. Inducers used were glycerol, soybean oil and diesel oil. Experiments were conducted for 96 h, and the daily production of biosurfactants was evaluated by reducing surface tension and stabilizing emulsions. Extracellular biosurfactant was extracted from the obtained biomass, with subsequent precipitation and chemical characterization by mass spectrometry. The highest extracellular emulsifier yields were achieved with glycerol inductor (0.20 UE h-1) and soybean oil (0.21 UE h-1) in 48h of cultivation. In later tests performed with increasing concentration of inducer, an increase in extracellular yields was noticed in these experiments (0.45 UE h-1 for glycerol and 0.30 UE h-1 for the soybean oil). The greatest reduction in surface tension was 9.89% in 72 h for diesel oil inducer. The reduction of surface tension combines with the increase of emulsifying activities in an important indicator of the use of hydrophobic substrate by the microorganism. The study confirms an increase in the production of extracellular biosurfactants when optimizing cultivation. The production of intracellular biosurfactants has also been verified, however the process of cellular disruption increases the cost of the bioprocess.

An efficient method of Yarrowia lipolytica immobilization using oil- and emulsion-modified bacterial cellulose carriers

Background: Yarrowia lipolytica is a nonconventional, dimorphic yeast with multiple biotechnological applications. Considering the size of Y. lipolytica cells and a plethora of its morphological forms (spherical cells or hyphae and pseudohyphae), it is highly difficult to select a suitable carrier for this useful microorganism. Bacterial cellulose (BC) is currently considered one of the most promising immobilization carriers. In the current study, the usefulness of oil- and emulsion-modified BCs as a carrier for Y. lipolytica immobilization was investigated. Static and agitated cultures were conducted in media supplemented with oil or emulsion to improve carrier porosity. Results: It was found that the application of oil- and emulsion-modified BCs correlated with significantly higher efficiency of Y. lipolytica immobilization and hence higher yield than the yield achieved with an unmodified carrier. Increased efficiency of immobilization correlated with BC porosity-related parameters, which, in turn, depended on the size of oil droplets introduced into the culture medium. Moreover, changes in porosity-related parameters caused by the addition of oil or emulsion to the medium were observed when the cultures were conducted only under static conditions and not under agitated conditions. Conclusion: The application of oil- and emulsion-modified BCs as carriers significantly increased the efficiency of Y. lipolytica immobilization as compared to unmodified BC. The addition of oil or emulsion to the culture medium can be a simple but effective method to modify the porosity of BC-based carriers.

Co-production of Proteases and Bioactive Protein Hydrolysates from Bioprocessing of Feather Meal

Abstract: Feather meal conversion through submerged cultivations with Bacillus strains (CL33A, CL14) yielded proteases and protein hydrolysates. After 4-day (CL33A) and 10-day (CL14) cultivations, protease activities reached 461 U/mL; hydrolysates presented antioxidant (radicals-scavenging, 57-77%; Fe2+-chelation, 14-28%; Fe3+-reduction) and antidiabetic (dipeptidyl peptidase-IV inhibition, 49-52%) potentials. The obtained bioproducts present prospective commercial/industrial applications.

Progress in intelligent control of industrial bioprocess / 生物工程学报

Industrial bioprocess is a complex systematic process and bio-manufacturing can be realized on the basis of understanding the metabolism process of living cells. In this article, the multi-scale optimization principle and practice of industrial fermentation process are reviewed, including multi-scale optimizing theory and equipment, on-line sensing technology for cellular macroscopic metabolism, and correlated analysis of physiological parameters. Furthermore, intelligent control of industrial bioprocess is further addressed, in terms of new sensing technology for intracellular physiological metabolism, big database establishment and data depth calculation, intelligent decision.

Fruit Peel from Astrocaryum aculeatum G. Mey. AS Substrate for Lipases Production by Aspergillus niger A2B1

The fruit of Astrocaryum aculeatum G. Mey. are consumed by population in the Brazil Northern resulting fruit peels. These peels are rich in lignocellulose and fat. The present study investigated peels from the Astrocaryum aculeatum G. Mey. as a substrate for lipases production by solid state bioprocess. To reach this objective: 1) we isolated fungi from peels from the fruit of Astrocaryum aculeatum G. Mey; 2) we screened the isolates for lipase production (screening in petri dish and screening in submerged bioprocess), 3) we investigated the production of lipases by using peels from Astrocaryum aculeatum G. Mey as substrate. The isolates belonged to the genera Aspergillus (16), Penicillium (3) and Fusarium (1). These strains were submitted to petri dishes and submerged fermentation for lipases production, these experiments resulted in the selection of five strains belonging to the Aspergillus genera. The lipases produced by these five strains performed enzymatic transesterification; however, the lipases from the strain Aspergillus niger A2B1 produced the highest ester content. The utilisation of fruit peel from Astrocaryum aculeatum G. Mey. as the main substrate, fruit peel from Astrocaryum aculeatum G. Mey. oil (13%), moisture (70%) and 75 h of incubation were the optimal conditions identified for the production of lipases by Aspergillus niger A2B1(17.42 U/g) in solid state bioprocess (SSB).

Cost Effective Pilot-scale Ajmalicine Production by Catharanthus roseus Cell Suspension Cultures in a 100 Lit Bioreactor

Aims: The cosmic production of biomass and bioactive compounds at pilot scale with minimum production costs is an important task to achieve feasible production process of corresponding secondary metabolites at a commercial level. Materials and Methods: The cell suspension cultures of Catharanthus roseus in MS medium supplemented with 2, 4-dichlorophenoxyacetic acid (9.05 µM), kinetin (4.52 µM) were scaled up in a pilot plant bioreactor (100 lit). The cost of production was reduced by addition of substitute carbon source in a basal medium which hardly costs 30% in the medium. Preliminary studies were performed in the 7-lit bioreactor. A 100 lit stainless steel bioreactor equipped with helical impeller top mounted was used for scale-up of C. roseus suspension cultures and ajmalicine production. Results: The culture medium reduced the cost by 36% by addition of commercial grade sugar whereas medium consist of tissue culture grade sucrose costs 53 USD per 100 lit. The suspension cultures were cultivated in a 100 lit bioreactor containing MS medium fortified with cost-effective carbon source produced ajmalicine 73.18 mg/l DW and achieved 36 kg of fresh biomass on day 20. Conclusion: The results of the present finding demonstrated the feasible and cost-effective production process of ajmalicine at pilot scale.

Filamentous fungi producing enzymes under fermentation in cassava liquid waste / Fungos filamentosos produtores de enzimas sob fermentação em manipueira

The conversion of agroindustrial residues by microorganisms has been explored from fermentative processes to obtain several bioactive molecules. The objective of this work was to isolate and select filamentous fungi present in cassava liquid waste for the production of amylase, carboxymethylcellulose (CMCase), pectinase and xylanase using the same residue as induction substrate in fermentative processes. A total of 65 filamentous fungi were isolated and qualitative tests indicated that approximately 86% of these strains were able to produce at least one of the enzymes and 32% capable of producing the four enzymes. Fermentation assays in cassava liquid residue-containing medium showed 6 fungal lines as potential enzyme producers. The maximum activities of pectinase, xylanase, amylase and CMCase were respectively observed at 96 hours of fermentation by the strain by the strain Aspergillus sp. B5C; at 120 hours (163.6 ± 0.13 nKat mL-1), by Aspergillus sp. B4I; at 144 hours (99.8 ± 0.24 nKat mL-1), by Penicillium sp. B3A; and at 48 hours (55.5 ± 0.21 nKat mL-1), by Aspergillus sp. B4O. These results suggest that cassava liquid waste was source of filamentous fungi producing amylase, CMCase, pectinase and xylanase, as well as a promising alternative substrate for bioprocesses aiming the production of enzymes.

A conversão de resíduos agroindustriais por micro-organismos tem sido explorada a partir de processos fermentativos para obtenção de diversas moléculas bioativas. O objetivo deste trabalho foi isolar e selecionar fungos filamentosos presentes em manipueira para produção de amilase, carboximetilcelulase (CMCase), pectinase e xilanase utilizando o próprio resíduo como substrato indutor. Um total de 65 fungos filamentosos foi isolado e testes qualitativos indicaram que, aproximadamente, 86% dessas linhagens foram hábeis em produzir pelo menos uma das enzimas e 32% capazes de produzir as quatro enzimas. Ensaios fermentativos em meio contendo manipueira apontaram 6 linhagens fúngicas como potenciais produtoras de enzimas. As atividades máximas de pectinase, xilanase, amilase e CMCase foram observadas, respectivamente, às 96 horas de fermentação (67.4 ± 0,6 nKat mL-1), pela linhagem Aspergillus sp. B5C; às 120 horas (163.6 ± 0,13 nKat mL-1), por Aspergillus sp. B4I; às 144 horas (99.8 ± 0,24 nKat mL-1), por Penicillium sp. B3A; e às 48 horas (55.5 ± 0,21 nKat mL-1), por Aspergillus sp. B4O. Estes resultados sugerem a manipueira como fonte de fungos filamentosos produtores de amilase, CMCase, pectinase e xilanase, além de um promissor substrato alternativo para bioprocessos visando a produção dessas enzimas.

Produção de proteínas heterólogas em microalga / Production of heterologous protein in microalga

O objetivo desta tese foi explorar o sistema de produção de proteínas heterólogas em microalga com ênfase em Chlamydomonas reinhardtii por meio de: (1) desenvolvimento de um fotobiorreator tubular fechado de escala laboratorial, utilizando técnicas de manufatura digital; (2) avaliação de 7 diferentes proteínas fluorescentes (mTagBFP, Cerulean, Emerald, crGFP, cOFP, tdTomato e mCherry), como sistema reporter de secreção de proteínas em microalga; (3) avaliação do fotobiorreator desenvolvido utilizando cultivo de cepas recombinantes; (4) desenvolvimento de novos peptídeos sinais para secreção de proteínas em C. reinhardtii; (5) avaliação da produção de um biofármaco (hialuronidase) em microalgas, por meio da expressão de duas isoenzimas codificadas pelos genes HYA1 e SPAM1 em C. reinhardtii. O fotobiorreator tubular foi avaliado quanto a sua capacidade de resistir ao processo de esterilização por autoclavação e seu desempenho por meio do cultivo de cepa recombinante secretando mCherry. A fluorescência das proteínas fluorescentes foi medida por leitor de placas de fluorescência e visualizada intracelularmente por microscopia confocal de fluorescência. A atividade de hialuronidase foi determinada através de um ensaio enzimático turbidimétrico. O desenvolvimento do fotobiorreator resultou em um sistema fechado resistente a autoclavação, com capacidade de cultivo de cepas recombinantes de C. reinhardtii. Esse fotobiorreator proporcionou uma produtividade máxima de 10 mg/L.d de mCherry da cepa recombinante em sistema fechado, com velocidade específica de crescimento máxima de 1,27 d-1 para a cepa recombinante testada. Todas as proteínas fluorescentes avaliadas apresentaram capacidade de secreção por C. reinhardtii, com diferentes níveis de interferências em sua medição, permitindo a escolha da mCherry como proteína reporter. Entre os peptídeos sinais avaliados (quatro descritos na literatura - BiP, ARS1, CAH1 e IBP1 - e seis preditos), o peptídeo predito "SP5" foi o que apresentou maior capacidade de secreção, determinado por níveis de fluorescência no sobrenadante. A avaliação dos peptídeos sinais constatou a necessidade de explorar o desenvolvimento de sistemas de expressão (e.g. vetores de expressão) aliados a análises computacionais, como o SignalP 4.0. Por último, os dados desse estudo mostram que C. reinhardtii transformadas com o vetor de expressão foi capaz de produzir as duas isoformas de hialuronidase em sua forma ativa, evidenciando a capacidade desse sistema para a produção de biofármacos. Portanto, nesta tese o sistema de expressão de proteínas heterólogas baseado em microalgas foi explorado, atingindo os objetivos propostos. O fotobiorreator desenvolvido tem a capacidade de esterilização em escala laboratorial (1) e em cultivo com cepa recombinante propiciou elevada produtividade (3). As proteínas vermelhas fluorescentes apresentaram-se como as proteínas com menores interferências para estudos de secreção em C. reinhardtii (2). Além disso, o peptídeo predito SP5 apresentou o melhor desempenho na secreção de proteínas (4) e o vetor de expressão empregado permitiu a identificação de cepas produtoras de biofármaco hialuronidase (5). Portanto, o sistema de produção de proteínas heterólogas por microalgas é um sistema promissor e poderá permitir, utilizando sistemas de secreção, obter proteínas de alto valor comercial a baixos custos, empregando a secreção e técnicas de cultivo como a fermentação extrativa

In this thesis, the heterologous protein production in microalgae with emphasis on Chlamydomonas reinhardtii was explored through: (1) development of a laboratory scale closed tubular photobioreactor using digital manufacturing techniques; (2) evaluation of different fluorescent proteins (mTagBFP, Cerulean, Emerald, crGFP, cOFP, tdTomato and mCherry) as a reporter system for protein secretion in microalgae (3) evaluation of photobioreactor developed using recombinant strains culture; (4) development of new signals peptides for protein secretion in C. reinhardtii (5) expression evaluation of a biopharmaceutical (Hyaluronidase) in microalgae, through the expression of two isoenzymes encoded by the HYA1 and SPAM1 genes in C. reinhardtii. The tubular photobioreactor was evaluated for its ability to resist sterilization process by autoclaving and its performance by culturing recombinant strain secreting mCherry. Fluorescence of fluorescent proteins was measured by fluorescence plate reader and observed intracellularly by confocal fluorescence microscopy. The hyaluronidase activity was determined by a turbidimetric enzymatic assay. The development of the photobioreactor resulted in a closed system resistant to autoclaving, capable of culturing recombinant strains of C. reinhardtii. This recombinant strain achieved a maximum productivity of 10 mg/L.day of mcherry in the closed system, with a maximum growth rate of 1.27 d-1 for the recombinant strain tested. All the fluorescent proteins evaluated had C. reinhardtii secretion capacity, with different interference levels in their measurement, allowing the selection of mCherry as a reporter protein. Among the evaluated peptides (four described in the literature - BiP, ARS1, CAH1 and IBP1 - and six predicted), the predicted peptide "SP5" was the one that presented greater capacity of secretion, determined by levels of fluorescence in the supernatant. The results of this study point out the need to explore the development of biological systems (i.e., expression vectors) allied to computational analysis. Finally, the data from this study showed that C. reinhardtii could produce the two isoforms of hyaluronidase in its active form, evidencing the capacity of this system to produce biopharmaceuticals. Therefore, in this thesis the heterologous protein expression system based on microalgae was explored, reaching the proposed objectives. The developed photobioreator has sterilization capabilityin laboratorial scale (1) and in culture with recombinant strain had high productivity (3). The red fluorescent proteins was found as the most suitable proteins for studies of secretion in C. reinhardtii with lower interference levels(2). In addition, the predicted SP5 peptide showed the best performance in protein secretion (4) and the expression vector employed allowed the identification of strains producing biopharmaceutical hyaluronidase (5). Therefore, the system of heterologous proteins production by microalgae is promising and will allow, using secretion systems, to obtain proteins of high commercial value at low costs, using secretion and cultivation techniques such as extractive fermentation

Medios de cultivo económicos para la producción de un biocatalizador a células enteras para el tratamiento de aguas residuales que contienen Cr(VI) / Economical fermentation media for the production of a whole cell catalyst for the treatment of Cr(VI)-containing wastewaters

The biotechnology sector is continually seeking sustainable and more economical bioprocesses. Fermentation media produced with cheap components or wastes reduce production costs. Moreover, if wastes are used, they contribute to avoid environmental pollution. In this work, microbial growth media based on molasses or acidified glycerol as carbon sources and fertilizer as nitrogen source were tested for the production of a whole-cell catalyst that could be used in Cr(VI)-containing wastewater treatments. Results showed that the highest biomass production yield was obtained with a medium containing acidified glycerol 5% v/v and fertilizer 0.6% v/v. The biomass produced using this medium was immobilized in calcium alginate beads and used as catalyst in the biotransformation of Cr(VI) into Cr(III). The catalyst could be efficiently used for 5 reduction cycles of 40 mg/l Cr(VI) each. Cr(III) retention assays were performed to determine whether Cr(III) could be retained by the catalyst avoiding its solubilization in the supernatants. The retention capacity of the catalyst at 32 °C and pH 3.0 was 3 mg Cr(III)/g. Both an alternative and economical fermentation medium is here proposed for the optimization of Cr(VI)-containing wastewater treatment.

El sector industrial biotecnológico continuamente busca bioprocesos más económicos y sustentables. El uso de medios de cultivo producidos con componentes de bajo costo o con residuos reduce el presupuesto global del proceso y, particularmente si se utilizan residuos, se contribuye, además, a evitar la contaminación ambiental. En este trabajo se probaron medios de cultivo basados en melaza de caña o glicerol ácido como fuentes de carbono y energía, y fertilizante como fuente de nitrógeno, para la producción de un biocatalizador que podría ser usado para el tratamiento de aguas residuales que contienen Cr(VI). Los resultados mostraron que el mayor rendimiento de producción de biomasa se obtuvo con un medio que contenía 5% v/v de glicerol ácido y 0,6% v/v de fertilizante. Utilizando este medio se produjo la biomasa suficiente para la biotransformación de Cr(VI) a Cr(III), luego de ser inmovilizada en alginato de calcio. El proceso pudo ser aplicado eficientemente durante 5 ciclos de reducción de 40 mg/l de Cr(VI) cada uno. Además, se realizaron ensayos de retención de Cr(III) para determinar si esta especie química podría ser removida de la solución por interacción con el biocatalizador. La capacidad de retención obtenida por el biocatalizador a 32 °C y pH 3 fue de 3 mg de Cr(III)/g. De esta manera, se propone un medio de cultivo alternativo y económico para la efectivización de un tratamiento de aguas residuales que contengan Cr(VI).

Surface response methodology for the optimization of lipase production under submerged fermentation by filamentous fungi

Abstract A Plackett–Burman Factorial Design of 16 experiments was conducted to assess the influence of nine factors on the production of lipases by filamentous fungi. The factors investigated were bran type (used as the main carbon source), nitrogen source, nitrogen source concentration, inducer, inducer concentration, fungal strain (Aspergillus niger or Aspergillus flavus were selected as good lipase producers via submerged fermentation), pH and agitation. The concentration of the yeast extract and soybean oil and the pH had a significant effect (p < 0.05) on lipase production and were consecutively studied through a Full Factorial Design 23, with the concentration of yeast extract and pH being significant (p < 0.05). These variables were optimized using a central composite design, obtaining maximum lipolytic activities with the use of 45 g/L of yeast extract and pH 7.15. The statistical model showed a 94.12% correlation with the experimental data.

Top-quality Course Construction for Bioprocess Equipment / 微生物学通报

Bioprocess equipment is of great importance in application of modern industrial biotechnology.With the rapid development of industrial biotechnology,demands for talents capable of understanding the theory,design and manipulation of modern bioprocess equipment increased.The experiences in aspects such as the building of teachers' contingent,construction of teaching materials,innovation of teaching method from the top-quality course construction of Bioprocess Equipment was discussed in this paper.