Abstract To investigate the simultaneous influence of different non-nutritional factors in production and physical-chemical characteristics of chitosan obtained by Syncephalastrum racemosum we used individually agroindustrial wastes as the only nutritional sources for fungus growth. The growth conditions were evaluated according to Factorial Design, 24 with three central points in order to determine the mainly factors for maximum production of microbiological chitosan in submerged culture. Syncephalastrum racemosum grown in corn steep liquor and yield up to 7.8 g chitosan/kg of substrate in the best condition by factorial design. The microbiological chitosan obtained has deacetilation degree 88.14%, crystallinity rate of 55.96%, mass decomposition process at 304.43 ºC, and low molecular weight. To fast production we performed a kinetic study and confirmed that at 36 h the chitosan production is higher and the physical-chemical characteristics were maintained. This research describes, for the first time, the factorial study of chitosan production by Syncephalastrum racemosum in agroindustrial wastes and its economic potential for commercialization.

RESUMO: Neste estudo, a quitina e a quitosana foram extraídas de resíduos de Litopenaeus vannamei utilizando métodos químicos e do micro-ondas. Os resíduos de camarão foram limpos, secos e peneirados a 16, 32 e 60 mesh, e as amostras foram despigmentadas, desmineralizadas e desproteinizadas. Posteriormente, a quitina foi submetida a processo de desacetilação por solução de NaOH a 45% sob irradiação de micro-ondas a 600w, durante 15 min intermitentes ou utilizando 6 pulsos de 5 min. O estudo mostrou eficácia nas partículas com tamanho de 32 mesh e 6 pulsos de 5 minutos, com 92% grau de desacetilação e rendimento de quitosana (52,2%). A atividade antimicrobiana foi para Staphylococcus aureus, Escherichia coli e Salmonella enterica contra a levedura Candida sp., respectivamente. Os resultados indicaram a viabilidade da radiação de micro-ondas como um método atraente para recuperação de quitina e quitosana a partir de resíduos de camarão.

ABSTRACT: In this study, chitin and chitosan were extracted from Litopenaeus vannamei waste using chemical and microwave methods. Shrimp waste was cleaned, dried and ground sieved to 16, 32 and 60 mesh, and the samples were depigmented, demineralized, and deproteinized. Then, the chitin was submitted to a deacetylation process by 45% NaOH solution under microwave irradiation at 600w, for intermittent 15 min or using 5 pulses of 5 minutes. The study showed that the effectiveness of the particle size of 32 mesh and 6 pulses of 5 min to deacetylation with 92% of degree and chitosan yield (52.2%). The polymer chitosan showed higher antimicrobial activity against to Staphylococcus aureus, Escherichia coli, Salmonella enterica and the yeast Candida sp., respectively. The results indicated the feasibility of the microwave radiation as an attractive method to recover chitin and chitosan from shrimp wastes.

RESUMO: Uma grande variedade de espécies bacterianas é bem mais explorada que os fungos como agentes biossurfactantes (BS) ou bioemulsificantes (BE), usando fontes renováveis. Os BS são considerados ecologicamente seguros e oferecem vantagens sobre os surfactantes sintéticos. Entretanto o rendimento de BS depende grandemente das vias metabólicas dos micro-organismos e do meio nutricional. A produção de BS ou BE utiliza várias condições culturais, em que uma pequena alteração nas fontes de carbono e nitrogênio afeta a produção de BS. O tipo e a quantidade de BS ou BE microbianos produzidos dependem principalmente do organismo produtor e de fatores como fontes de carbono e nitrogênio, oligoelementos, temperatura e aeração. A diversidade de BS ou BE torna-os interessantes para aplicação nos campos farmacêutico, cosmético, da agricultura, da saúde pública, em processos alimentares, detergentes, no tratamento de resíduos oleosos, no controle de poluição ambiental e na biorremediação. Assim, a presente revisão aborda o potencial biotecnológico de leveduras e fungos filamentosos para produção, caracterização e aplicações de BS ou BE .

ABSTRACT: A wide variety of bacteria is far more exploited than fungi as biosurfactants (BS) or bioemulsifiers (BE), using renewable sources. BS are considered to be environmentally safe and offer advantages over synthetic surfactants. However, the BS yield depends largely on the metabolic pathways of the microorganisms and the nutritional medium. The production of BS or BE uses several cultural conditions, in which a small change in carbon and nitrogen sources affects the quantity of BS or BE produced. The type and quantity of microbial BS or BE produced depend mainly on the producer organism, and factors such as carbon and nitrogen sources, trace elements, temperature and aeration. The diversity of BS or BE makes it interesting to apply them in the pharmaceutical and cosmetics industries, agriculture, public health, food processes, detergents, when treating oily residues, environmental pollution control and bioremediation. Thus, this paper reviews and addresses the biotechnological potential of yeasts and filamentous fungi for producing, characterizing and applying BS or BE.

Background The effects of exposure to copper, during growth, on the production of biomass, total protein, catalase, glutathione-S transferase, glutathione peroxidase, peroxidase, polyphosphate, acid and alkaline phosphatases, ultrastructure and the ability to remove this metal from Aspergillus niger, obtained from caatinga soil, were evaluated. Results All parameters tested were influenced by the concentration of metal in the culture medium. The presence of metal induced high levels of antioxidant enzymes, including lipid peroxidation, thereby revealing the appearance of an oxidative stress response. The variation in polyphosphate levels indicates the participation of the polymer in response to stress induced by copper. The activities of the phosphatases were positively influenced by growing them in the presence of copper. Ultrastructure changes in the cell surface, electron density, thickness, and septation were visualized by exposing cells to increasingly larger concentrations of metal. The isolate was able to remove the agent from the growth medium, while maintaining its physiological functions. The metal removed from the cultures exposed to 0.5 mM, 1 mM and 2 mM copper exhibited percentages of removal equivalent to 75.78%, 66.04% and 33.51%. Conclusions The results indicate that the isolate was able to grow in high concentrations of copper, activates mechanisms for adaptation and tolerance in the presence of metal, and is highly efficient at removing the agent. Such data are fundamental if a better understanding is to be reached of the cellular and molecular abilities of native isolates, which can be used to develop bioprocesses in environmental and industrial areas.

Background: Biotechnological processes are costly, especially for the production of biosurfactants. The successful production of a biosurfactant is dependent on the development of processes using low cost raw materials. Considering the importance of the characteristics of a biosurfactant to facilitate its industrial application, the properties of the biosurfactant produced by Candida lipolytica through previously optimized medium have been established. Results: The yeast was grown for 72 h to determine the kinetics of growth and production. The surface tension of the cell-free broth was reduced from 55 to 25 mN/m. The yield of biosurfactant was 8.0 g/l with a CMC of 0.03%. The biosurfactant was characterized as an anionic lipopeptide composed of 50% protein, 20% lipids, and 8% of carbohydrates. Conclusions: The isolated biosurfactant showed no toxicity against different vegetable seeds: Brassica oleracea, Solanum gilo and Lactuca sativa L. and the micro-crustacean Artemia salina. The properties of the biosurfactant produced suggest its potential application in industries that require the use of effective compounds at low cost.

O interesse em surfactantes de origem microbiana tem aumentado consideravelmente nos últimos anos. A preocupação ambiental entre os consumidores, combinada às novas legislações de controle do meio ambiente, aumentou a procura por surfactantes naturais em substituição aos derivados petroquímicos. Os surfactantes microbianos têm sido testados em muitas aplicações ambientais e industriais, como na biorremediação, na dispersão de manchas oleosas e na recuperação de petróleo, substituindo os surfactantes químicos. Além disso, também podem ser utilizados nas indústrias de alimentos, cosméticos, detergentes e na agricultura. Neste trabalho foi produzido biossurfactante por Chromobacterium violaceum ATCC 12472, utilizando nutrientes de baixo valor agregado. A tensão superficial do biossurfactante produzido por C. violaceum ATCC 12472 teve valores entre 29 e 40 mN/m. O melhor resultado foi observado no ensaio 8 (milhocina 8,0%, lactose 1,0% e óleo de milho pós-fritura 7,5%) do planejamento fatorial, com 72 horas de fermentação, reduzindo a tensão superficial da água de 71 para 29 mN/m. Os melhores resultados do índice (E24) e da atividade de emulsificação (UEA) foram observados no ponto central, sendo utilizados para ambos os métodos o óleo de milho, que resultou no E24 de 2% e atividade com valores acima de 6 UAE. Os resultados obtidos demonstram o elevado potencial de C. violaceum na produção de um promissor biossurfactante.

The interest in microbial surfactants has been increased considerably in the recent years. Environmental concern among consumers, combined with new laws control of the environment, the demand for natural surfactants, to replace petrochemicals. The microbial surfactants have been tested in many industrial and environmental applications, such as in bioremediation, oil dispersion and oil recovery, replacing chemical surfactants. Furthermore, they can also be used in the food, cosmetic, detergent and agriculture. However, increased environmental concern among consumers to new legislation combined control of the environment has led to demand for natural surfactants as alternatives to existing products. This work aimed to produce biosurfactant by Chromobacterium violaceum ATCC 12472, using low nutrient value. The surface tension of the biosurfactant produced by C. violaceum ATCC 12472 had values between 29 and 40 mN/m. The best result was observed on the assay 8 (8.0% of corn steep liquor, 1.0% of lactose and corn oil post-frying 7.5%) factorial design, within 72 hours of fermentation, lowering the surface tension of water from 71 to 29 mN/m. The best results of the index (E24) and emulsification activity (UEA) were observed in the central point, and using the both methods and corn post-frying oil resulted in 72% of emulsification, and the emulsification activity value of 6 UAE. The results demonstrate the high potential of C. violaceum promising in producing a biosurfactant.

A produção de um novo biossurfactante por Candida glabrata UCP1002 foi inicialmente investigada com a finalidade de avaliar a influência da concentração dos substratos óleo de algodão, glicose e extrato de levedura. As cinéticas de crescimento e de produção do surfactante foram demonstradas para todas as condições de cultivo testadas. A melhor emulsificação do nhexadecano, quantificada através do índice de emulsificação foi observada na condição de cultivo contendo 7,5% de óleo de algodão, 5% de glicose e 0,3% de extrato de levedura. O biossurfactante produzido apresentou uma concentração micelar crítica de 2,5%, sendo a tensão superficial nesse ponto de 31mN/m. O potencial de aplicação do biossurfactante na recuperação de óleo de areia contaminada, em condições ácidas e alcalinas, bem como sob exposição a altas salinidades e diferentes temperaturas, foram demonstrados com base no percentual de remoção do óleo e na estabilidade da tensão superficial, respectivamente.

The production of a new biosurfactant by Candida glabrata UCP1002 was studied to evaluate the influence of the concentration of the cotton seed oil, glucose and yeast extract. The dynamics of the growth and surfactant production were showed for all the cultivation conditions studied. The best emulsification of the n-hexadecane, quantified by the emulsifying index was observed in the medium containing 7.5% cotton seed oil, 5% glucose and 0.3% yeast extract. The isolated biosurfactant showed a CMC of 2.5% and the surface tension at that point showed to be 31mN/m. The potential application of the biosurfactant in oil recovery from the sand, in acid and alkaline environments and over exposure to high salinity and different temperatures was demonstrated by the percentage of oil removal and by the stability of the surface tension, respectively.

O mutante fisiológico de Rhizopus arrhizus foi obtido pelo teste do gradiente de resistência ao pireno. Estudos comparativos conduzidos sobre o comportamento do processo de germinação e o crescimento radial foram realizados entre as amostras selvagem e mutante de R. arrhizus UCP 402. Os meios Sabouraud Sacarose e Caldo de Levedura e Malte contendo pireno (10 mg/L) induziram ao processo de germinação de esporangiosporos das amostras selvagem e mutante de R. arrhizus. O crescimento radial das amostras foi inversamente proporcional à concentração de pireno no meio de cultura. Os resultados demonstraram uma excelente adaptação da amostra mutante de R. arrhizus UCP 402x na concentração de pireno (50 mg/L), sugerindo uma alta habilidade e possibilidade de aplicação na remoção de pireno em áreas contaminadas.

The physiological mutant of Rhizopus arrhizus was obtained in the pyrene resistance gradient test. Comparative studies were carried out about the behavior of the germination process and the radial growth of the mutant and wild strains of R. arrhizus UCP 402. Sabouraud Sucrose and Yeast Malt Broth cultures containing pyrene (10 mg/L) induced the germination process of the sporangiospores of the wild and mutant strains of R. arrhizus. The radial growth of the strains was inversely proportional to the pyrene concentration in the culture medium. The results showed an adaptation of R. arrhizus UCP 402x (mutant) in the pyrene (50mg/L) and suggested a higher ability of application in the removal of pyrene from the contaminated areas.

Microbiological processes were used for chitin and chitosan productions by Cunninghamella elegans (UCP 542) grown in a new economic culture medium. The assay was carried out to evaluate the growth of C. elegans using yam bean (Pachyrhizus erosus L. Urban) medium, in different times of growth (24, 48, 72 and 96 hrs), incubated at 28ºC in an orbital shaker at 150 rpm. The lyophilized biomass was determined by gravimetry. The polysaccharides were extracted by alkali-acid treatment, and characterized by infrared spectroscopy, titration and viscosity. C. elegans grown in the yam bean medium and produced higher yields of biomass (24.3 g/ mL) in 96 hrs. The high level was chitosan (66 mg/g), and chitin (440 mg/g) were produced at 48 and 72 hrs of growth, respectively. The polysaccharides showed degree of deacetilation and viscosimetric molecular weight as: 6.2% and 3.25 x10(4) g/mol for chitin, and 85% and 2.72 x 10(4) g/mol for chitosan, respectively. The results obtained suggest high biotechnological potential of yam bean as an economic source to produce chitin and chitosan by C. elegans. In addition, the new medium using yam bean for production of the chitin and chitosan may be used for many purposes to reduce the cost price of fermentation processes.

Evaluation of both tenso-active and emulsifying activities indicated that a biosurfactant was produced by the newly isolated and promising strain Candida glabrata isolated from mangrove sediments. The extracellular water-soluble emulsifying agent was isolated and identified as a heteropolymer. The maximum of bioemulsifier production was observed when the strain was grown on soluble and insoluble substrates cotton seed oil plus glucose, reaching values of 10.0 g/l after 144 hrs at 200 rpm. The cell-free culture broth containing the examined agent lowered the surface tension of the medium to 31 mN/m. Stable and compact emulsions with emulsifying activity of 75% of cotton seed oil were detected. The emulsification capacity remained practically unaltered within a wide pH (2-12), temperature (4-80ºC) ranges and under NaCl concentrations up to 10%.

Quitina e quitosana foram extraídas a partir da massa micelial de Cunninghamella elegans (IFM 46109) e avaliou-se a aplicação destes polissacarídeos na remoção dos metais pesados cobre, chumbo e ferro preparados em solução aquosa. O crescimento de C. elegans foi acompanhado através da determinação de biomassa, pH, consumo de glicose e de nitrogênio. A extração de quitina e quitosana realizou-se através de tratamento álcali-ácido e a produção dos polissacarídeos foi de 23,8 e 7,8 %, respectivamente. A avaliação do processo de remoção dos metais pesados foi realizada utilizando-se os polissacarídeos em solução a 1% (p/v). Os níveis de biossorção de metais foram dependentes da concentração e do pH das soluções. Os melhores resultados foram obtidos em pH 4,0. A quitosana mostrou maior índice de biossorção para o íon cobre e a quitina para o ferro. Os resultados sugerem que C.elegans pode ser considerada uma fonte atrativa para a produção alternativa de quitina e quitosana, e que demonstra grande potencial de biossorção de metais pesados em ambientes poluídos.

Chitin and chitosan were extracted from mycelial biomass of Cunninghamella elegans and the performance for copper, lead and iron biosorption in aqueous solution was evaluated. The growth curve of C. elegans was accomplished by determination of biomass, pH, glucose and nitrogen consumption. Chitin and chitosan were extracted by alkali-acid treatment and the yields were 23.8 and 7.8%, respectively. For the adsorption analysis, the process of heavy uptake metal sorption was evaluated using polysaccharides solutions (1% w/v). The rate of metallic biosorption was dependent upon the concentration and pH of metal solutions, and the best results were observed with pH 4.0. Chitosan showed the highest affinity for copper and chitin for iron adsorption. The results suggest that C. elegans (IFM 46109) is an attractive source of production of chitin and chitosan, with a great potential of heavy metals bioremediation in polluted environments.

Uma coluna de discos perfurados rotativos foi utilizada na extração da enzima ascorbato oxidorredutase (E.C.1.10.3.3), obtida do extrato bruto de Curcubita maxima, através da utilização do sistema bifásico aquoso Polietilenoglicol-sais de fosfato. Os efeitos da velocidade da fase dispersa nos coeficientes de transferência de massa e na eficiência de separação para valores de 1, 2 e 3 mL/min foram estudados. Observou-se um aumento da transferência de massa com a velocidade da fase dispersa, enquanto que a eficiência de separação demonstrou uma ligeira redução com o aumento deste parâmetro. Os resultados experimentais obtidos durante a extração contínua demonstraram que a atividade da ascorbato oxidorredutase se concentrou preferencialmente na fase rica em sal para todas as condições estudadas. A maior recuperação da atividade enzimática foi de 236%, com um fator de purificação de 34 para o valor de 1 mL/min para a fase dispersa.

A continuous perforated rotating disc contactor was used to extract the enzyme ascorbic oxidoreductase (E.C.1.10.3.3) from crude extract of Curcubita maxima with an aqueous two-phase system of poly (ethylene glycol) and phosphate salts. The effect of dispersed phase velocity on either protein mass transfer coefficients or separation efficiency at 1, 2 and 3 mL/min was studied. An increase of the mass transfer coefficients was observed with the dispersed phase velocity, while the separation efficiency showed a small decrease with the increase of this parameter. The experimental results obtained during continuous extraction showed that the ascorbic oxidoreductase activity was partitioned preferentially into the salt-rich phase in all conditions studied. The best recovery of enzyme activity was 236%, with a purification factor of 34 in flow rates of 1 mL/min for dispersed phase.

A partição de duas proteínas, albumina de soro bovino (BSA) e tripsina foi estudada no sistema bifásico aquoso Polietileno glicol(PEG) - Goma do cajueiro. O diagrama de fases foi estabelecido para a Goma do Cajueiro e para PEG de peso molecular 1500 em duas diferentes temperaturas. A influência de vários parâmetros na partição destas proteínas, incluindo concentração dos polímeros, pH, adição de sal e temperatura foi investigada. Os resultados desta pesquisa demonstraram a importância das características da proteína na partição em sistemas bifásicos aquosos.

The partitioning of two proteins, bovine serum albumin (BSA) and trypsin was studied in an aqueous poly(ethylene glycol)(PEG)- Cashew-nut tree gum system. The phase diagram was provided for Cashew-nut tree gum and PEG molecular weight of 1500 at two different temperatures. The influence of several parameters including concentrations of polymers, pH, salt addition and temperature on the partitioning of these proteins were investigated.. The results of this research demonstrated the importance of the protein characteristics for partitioning in aqueous biphasic system.

O crescimento, consumo de fosfato e glicose, bem como o conteúdo de fósforo, a distribuição, estrutura e localização de polifosfato foram avaliados no micélio de Cunninghamella elegans cultivado em meios contendo diferentes concentrações de fosfato. Os resultados permitiram verificar a influência dessas concentrações de fosfato sobre o crescimento do fungo estudado. A maior concentração de fosfato proporcionou maior rendimento da biomassa ao longo do crescimento. Uma relação entre consumo de fosfato e glicose do meio foi observada em relação ao crescimento e a quantidade de polifosfato total nos micélios cultivados nos diferentes meios de cultivo. Distintos métodos de extração permitiram identificar e quantificar as diferentes frações do polifosfato celular de Cunninghamella elegans. A citoquímica ultrastrutural foi utilizada com sucesso para identificar a localização e a distribuição de polifosfato em Cunninghamella elegans. Os resultados revelaram diferenças no padrão de marcação citoquímica nas diferentes fases do crescimento e meios de cultivo. Uma marcação uniforme do polifosfato foi observada sobre a superfície celular, em especial, na parede celular e na membrana citoplasmática. Produtos de reação resultantes da marcação citoquímica foram também visualizados em estruturas trabeculares, vacuolares, vesiculares, sob a forma de corpos eletrondensos e grânulos dispersos no citoplasma. Os resultados demonstraram o potencial de Cunninghamella elegans na acumulação de polifosfato, sugerindo uma possível aplicação em processos biotecnológicos.

The content of inorganic polyphosphate and the polymeric degree of these compounds were evaluated during the growth of Cunninghamella elegans in medium containing varying orthophosphate (Pi) concentrations. For this purpose, a combination of chemical methods for polyphosphate extraction and ultrastructural cytochemistry were used. The orthophosphate and glucose consumption was also determined during the fungal cultivation. At Pi concentrations of 0.5, 2.5 and 0.0 g/L, the maximum amounts of biomass were 3.18, 3.29 and 0.24 g/L, respectively. During growth the cells accumulated Pi from the medium. At three days of growth the biomass consumed up to 100 and 95% of Pi from the media at initial concentrations of 0.5 and 2.5 g/L, respectively. Polyphosphate was observed at different Pi concentrations in medium and at different stages of growth. Polyphosphate was assayed by the content of labile phosphorus in water, acid-insoluble and alkali-soluble fractions. The content of fractions changed according to phosphorus concentration in the media and growth phase. During growth on all three media used, the cytochecmical behavior of polyphosphate changed considerably. The results obtained in this study reveal a potential of Cunninghamella elegans in the polyphosphate accumulation, and suggest a future application in the biotechnological processes.

Candida lipolytica IA 1055 produziu biosurfactantes extracelulares com atividade de emulsificação, através de fermentação utilizando óleo de babaçu e D-glicose como fontes de carbono. A água do mar diluída a 50% suplementada com uréia, sulfato de amônio e fosfato foi usada como meio basal. Os melhores resultados foram atingidos com os meios YSW-B2 (contendo uréia, sulfato de amônio e óleo de babaçu)e YSW-B3 (contendo uréia, sulfato de amônio, fosfato e óleo de babaçu), através de fermentação em batelada alimentada com 5% de óleo de babaçu. A velocidade específica de crescimento foi de 0,02 h-1 e 0,04 h-1; tempo de geração de 34,6 h-1 e 17,3 h-1 e atividade de emulsificação igual a 0,666 e 0,158 unidades, respectivamente. As moléculas dos novos bioemulsificantes demonstraram ser constituídas por carboidratos, proteínas e lipídeos.

Candida lipolytica IA 1055 produced extracellular biosurfactants with emulsification activity by fermentation using babassu oil and D-glucose as carbon sources. Natural seawater diluted at 50% supplemented with urea, ammonium sulfate, and phosphate was used as economic basal medium. The best results were achieved with the YSW-B2 medium, which contained urea, ammonium sulfate, and babassu oil and with YSW-B3 medium, which contained urea, ammonium sulfate, phosphate, and babassu oil, kept under fed batch fermentation for 60 hours with 5% of babassu oil. For the two media, the maximum specific growth rates were 0.02 h-1 and 0.04 h-1; the generation times were 34.6 h-1 and 17.3 h-1, and the emulsification activities were 0.666 and 0.158 units, respectively. The molecules of these new bioemulsifiers were contituted of carbohydrates, proteins and lipids.