Orientadores: Francisco Maugeri Filho, Gonçalo Amarante Guimarães Pereira / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-20T20:25:34Z (GMT). No. of bitstreams: 1 Goldbeck_Rosana_D.pdf: 2685408 bytes, checksum: 40606034d66dbd81493dcd1bbec17eaa (MD5) Previous issue date: 2012 / Resumo: O etanol vem novamente despertando de modo crescente a atenção de pesquisadores, empresas e governo, devido ao aumento do preço do petróleo e perspectivas de esgotamento das fontes não-renováveis de combustíveis fósseis, bem como, preocupações de natureza ambiental, relacionadas à emissão de substâncias que comprometem o meio ambiente. O estabelecimento de metas extremamente ambiciosas para aumento do consumo do etanol nos próximos anos requer um aumento substancial da produção de etanol e, nesse sentido, estimula a pesquisa e o desenvolvimento de tecnologias que permitem o uso de novas matérias-primas na produção de etanol, como a biomassa lignocelulósica. No entanto a ampliação desta tecnologia é limitante devido ao alto custo das enzimas, indicando desta forma a importância da busca por novas fontes de enzimas capazes de contribuir para este processo. Em face disto, este trabalho teve como objetivo estudar as propriedades lignocelulolíticas de micro-organismos silvestres isolados de diversas regiões brasileiras, bem como realizar um estudo genético visando à produção de bioetanol. Inicialmente foi realizada uma seleção das cepas que apresentaram capacidade de produção de celulases, a partir de micro-organismos silvestres isolados de diversas regiões do país. Após a seleção, a cepa nomeada AAJ6 foi selecionada como potencial produtor de celulases e identificada molecularmente como Acremonium strictum. Posteriormente, foram realizados estudos de recuperação, purificação e caracterização enzimática das enzimas produzidas pelo microorganismo em estudo. A precipitação com acetona 60% foi o método que resultou em melhores porcentagens de recuperação, registrando 80,67% de recuperação para as endoglucanases (CMCase), 65% para a atividade de papel de filtro (FPase) e 25% para celobiase. Em relação à purificação, a resina Q-Sepharose foi selecionada como mais eficiente para a purificação das enzimas do complexo celulase produzidas por Acremonium strictum. Quanto à caracterização enzimática, as faixas de temperatura e pH estudadas não tiveram diferença significativa (p<0,05) em relação à atividade de endoglucanase (CMCase). Já para a atividade de FPase e celobiase, a faixa temperatura ótima foi de 54 a 57 °C e o pH ótimo foi de 4,7. Para a b-glicosidase, apenas a temperatura foi significativa, favorecendo temperaturas mais elevadas (54 a 57 °C) para a atividade enzimática. Paralelamente conduziram-se fermentações para produção de celulases empregando diferentes substratos, tanto substratos comerciais (carboximetilcelulose, SERVACEL® e AVICEL®) como bagaços de cana-de-açúcar pré¿tratados com diferentes intensidades. O bagaço de cana submetido a um pré-tratamento leve (12 kgf/cm²; 188,5°C) foi o que melhor induziu o micro-organismo em estudo a produzir as maiores atividades celulolíticas em comparação aos demais substratos estudados, registrando valores máximos de CMCase de 134,42 U/L em 240 horas de fermentação, 10,82 U/L de FPase em 192 horas, 27,72 U/L de celobiase em 96 horas e 3,48 U/L de b-glicosidase em 240 horas. Com o avanço da biotecnologia e da biologia molecular, a identificação de genes presentes num determinado micro-organismo já se tornou essencial. Em face disto, foi realizado o sequenciamento 454 do genoma do Acremonium strictum e dois genes de celulases foram identificados, sendo um gene de endoglucanase da família 74a e um gene de b-glicosidase. Estas enzimas foram isoladas, sequenciadas e clonadas em E.coli através do vetor pGEM-T Easy de forma que futuros trabalhos possam abordar os produtos de expressão destas enzimas em Saccharomyces cerevisiae visando à produção de bioetanol de segunda geração / Abstract: Ethanol has increasingly attracted the attention of researchers, companies and governments due to the increase in oil prices and prospects of depletion of nonrenewable fossil fuels, as well as environmental concerns related to emissions of substances that compromise the environment. Excessively ambitious goals for the increased consumption of ethanol in the for the years ahead requires a substantial increase in ethanol production and, accordingly, encourages research and development of technologies that allow the use of new raw materials for ethanol production, such as lignocellulosic biomass. However the expansion of this technology is limited due to the high cost of enzymes, thus indicating the importance of searching for new sources of enzymes able to contribute to this process. In the face of this, the present work intended to study the lignocellulolytic properties of wild microorganisms isolated from various regions of Brazil as well as conducting a genetic study aimed at producing bioethanol. Initially a selection of strains that were capable of producing cellulases was carried out. Than, the the selected strain, named AAJ6 and molecularly identified as Acremonium strictum, was shown to be a potential producer of cellulases. Subsequently, studies were performed for recovery, purification and characterization of the enzymes produced by this microorganism. Precipitation with 60% acetone was the method that led to improved recovery percentages, about 80%, for the endoglucanases (CMCase), 65% for filter paper activity (FPase) and 25% for cellobiase. With regard to purification, choromatographic column with Q-Sepharose resin was selected as the most efficient for the purification of the cellulase enzyme complex produced by Acremonium strictum. As enzymatic characterization, the temperature and pH ranges studied did not differ significantly (p<0.05) compared to the activity of endoglucanase (CMCase). As for the cellobiase and FPase activity, the optimum temperature range was 54 to 57 °C and optimum pH was 4.7. For the b-glucosidase, only temperature was significant, favoring higher temperatures (54 to 57 °C) for enzyme activity. Parallel fermentations were conducted for cellulase production using different cellulosic substrates (carboxymethylcellulose, SERVACEL® and AVICEL ®) and sugarcane bagasse pretreated with different intensities. Bagasse that underwent t mild pretreatment (12 kgf/cm², 188.5 °C) was the best inducer for microorganism under study, and led to the highest cellulolytic activities, being the maximum values 134.42 U/L U/L for CMCase after 240 hours of fermentation, 10.82 U/L for FPase after 192 hours, 27.72 U/L for cellobiase after 96 hours and 3.48 U/L for b-glucosidase after 240 hours. At the current stage of biotechnology and molecular biology, the identification of genes present in a given micro-organism has become essential. In view of this, the 454 sequencing of the genome of Acremonium strictum was carried out and two cellulase genes were identified, being an endoglucanase of the family 74a gene and b-glucosidase gene. These enzymes were isolated, sequenced and cloned into E. coli using the pGEM-T Easy vector so that future work can address the expression products of these enzymes in Saccharomyces cerevisiae in order to produce second generation bioethanol / Doutorado / Engenharia de Alimentos / Doutora em Engenharia de Alimentos

This project explored the feasibility of using fungi in a constructed wetland for the treatment of pulp mill effluent. The effluent is high in dissolved lignins (some of which are chlorinated), which have proven very difficult to degrade biologically. Mindful of work done with the (terrestrial) white rot fungi, especially Phanerochaete chtysosporium, the question is asked, Is there a fungus which can tolerate submerged conditions while degrading a significant amount of dissolved lignins? Two fungal species with lignin-degrading capability were isolated from submerged films in a log pond. These fungi have been evaluated for decolorization potential under different environmental conditions. Results of laboratory experiments show that one of these fungi, identified as Acremonium sp., was capable of 44% decolorization of pulp mill effluent under sterile, submerged, room temperature conditions. The fungal decolorization was evaluated both in floating cultures and as a film inoculated on wood chips. In addition, bench-scale examination of the potential of this fungus to decolorize pulp mill effluent in non-sterile conditions was completed. / Graduation date: 1994

Diss.--Universität Bochum. / Bibliogr. p. 100-118.

Se estudiaron los géneros Scedosporium, Acremonium, Phialemonium, Lecythophora y Paecilomyces. En Scedosporium se seleccionó la mejor herramienta para la tipificación de S. aurantiacum y S. prolificans. En los otros géneros se estudiaron la identificación morfológica y molecular para establecer el espectro de especies y sus relaciones filogenéticas. Los resultados indican que el sistema basado en la secuenciación multilocus es la mejor metodología para la tipificación de Scedosporium. Se ha establecido el espectro de las especies asociadas a muestras clínicas de los Estados Unidos, siendo Acremonium kiliense, el complejo Acremonium sclerotigenum-Acremonium egyptiacum, Acremonium implicatum, Phialemonium obovatum, Phialemonium curvatum, Lecythophora hoffmannii, Cephalotheca foveolata y Lecythophora mutabilis las especies más encontradas. Se propone el nuevo género Phialemoniopsis. Se transfiere Sarcopodium oculorum y Phialemonium curvatum a Phialemoniopsis, y Acremonium atrogriseum y Taifanglania inflata a Phialemonium. Se proponen las nuevas especies: Phialemoniopsis cornearis, Phialemoniopsis pluriloculosa, Phialemonium globosum, Lecythophora luteo-rubra, Lecythophora cateniformis y Paecilomyces lavendulus. / Scedosporium, Acremonium, Phialemonium, Lecythophora and Paecilomyces were studied. In Scedosporium, the study has focussed on choosing the best tool for the characterization of S. aurantiacum and S. prolificans. As for the other genera, studies have focussed on morphological and molecular identification. The results showed that the system based on multilocus sequencing is the best methodology for the characterization of Scedosporium. By correlating the results obtained by morphological and molecular techniques, we have been able to establish the species associated with clinical samples from the United States, Acremonium kiliense, complex Acremonium sclerotigenum-Acremonium egyptiacum, Acremonium implicatum, Phialemonium obovatum, Phialemonium curvatum, Lecythophora hoffmannii, Cephalotheca foveolata and Lecythophora mutabilis being the most found species. We proposed Phialemoniopsis as a new genus. We transferred Sarcopodium oculorum and Phialemonium curvatum to Phialemoniopsis, and Acremonium atrogriseum and Taifanglania inflata to Phialemonium. We proposed the new species: Phialemoniopsis cornearis, Phialemoniopsis pluriloculosa, Phialemonium globosum, Lecythophora luteo-rubra, Lecythophora cateniformis and Paecilomyces lavendulus.

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Thesis (MSc)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: Humanity is currently dependant on fossil fuels as an energy source. Increasing economic development and industrialization is, however, raising the demand for this unsustainable energy source. This increased pressure on dwindling reserves and growing concern over detrimental environmental effects associated with the use of these fuels have sparked great interest in the development of alternative sources. Bioethanol has surfaced as a good alternative to fossil fuels, as it can be produced from cheap, abundant, renewable, non-food sources. Bioethanol is also carbonneutral, i.e. utilisation thereof leaves the net level of carbon dioxide in the atmosphere unperturbed. Lignocellulose, more specifically its cellulose fraction, has been identified as a possible feedstock for the production of bioethanol. The use of lignocellulose as feedstock will allow for a more sustainable supply and much needed energy security. Lignocellulosic feedstocks can be divided into two main categories, i.e. wastes from processes other than fuel production and crops grown specifically for fuel production. Cereal crops such as triticale have been identified as good industrial crops for the production of energy. Triticale’s higher biomass yield, moderate water and nutrient requirements, steadily increasing area of cultivation and main use as an animal feed and not a human food source, makes it attractive as feedstock for the production of bioethanol. The combined activity of endoglucanases, exoglucanases and β-glucosidases is needed to hydrolyse crystalline cellulose to fermentable sugars. The high cost of these enzymes is, however, the most significant barrier to the economical production of bioethanol from cellulosic biomass. A promising strategy for a reduction in costs is the production of these cellulolytic enzymes, hydrolysis of biomass and fermentation of the resulting sugars to bioethanol in a single process step via a cellulolytic microorganism. The development of such a consolidated bioprocessing (CBP) organism can be achieved by the introduction of cellulolytic activity into a noncellulolytic microorganism that is able to ferment glucose to ethanol. Saccharomyces cerevisiae is a good host candidate for CBP as this yeast’s high tolerance towards ethanol and its use in industrial applications has been established. The enzymatic activities of endoglucanases and exoglucanases are, however, inhibited by the build-up of cellobiose during the hydrolysis of cellulose. This effect may be alleviated with the introduction of a better functioning β-glucosidase into the system. β-Glucosidases hydrolyse cellobiose to glucose, alleviating the inhibition on the enzymatic activities of endoglucanases and exoglucanases. Despite advances in enzyme production systems and engineering enzymes currently in use for higher stability and activity, there is still a demand to expand the current collection of enzymes. Bioprospecting for novel cellulolytic enzymes focuses on specific environment, with high turnover rates of cellulosic material or extreme conditions, such as the composting process. These enzymes are becoming more attractive compared to their mesophillic counterparts due to their potential industrial applications and the fact that they represent the lower natural limits of protein stability. / AFRIKAANSE OPSOMMING: Die mensdom is hoofsaaklik van fossielbrandstowwe as 'n energiebron afhanklik. Toenemende ekonomiese ontwikkeling en industrialisasie verhoog egter die aanvraag na hierdie onvolhoubare energiebron. Druk op kwynende reserwes en groeiende kommer oor die nadelige gevolge vir die omgewing wat met die gebruik van hierdie brandstowwe gepaard gaan, het tot groot belangstelling in die ontwikkeling van alternatiewe bronne gelei. Bio-etanol is 'n goeie alternatief vir fossielbrandstowwe, want dit kan van goedkoop, vollop, hernubare nievoedselbronne geproduseer word. Bio-etanol is ook koolstof-neutraal; die gebruik daarvan laat die netto vlak van koolstofdioksied in die atmosfeer onverstoord. Lignosellulose, en meer spesifiek die sellulose fraksie, is as moontlike grondstof vir die vervaardiging van bio-etanol geïdentifiseer. Die gebruik van lignosellulose as grondstof sal meer volhoubare voorsiening en broodnodige energie-sekuriteit verseker. Sellulose grondstowwe kan in twee hoof kategorieë verdeel word, nl. Newe produkteafval van prosesse anders as brandstofproduksie en gewasse wat spesifiek vir brandstofproduksie gekweek word. Graangewasse soos korog is geïdentifiseer as 'n goeie industriële gewas vir die produksie van energie. Korog se hoër biomassa opbrengs, matige water en voedingstofvereistes, groeiende bewerkingsgebied en die gebruik as 'n veevoergewas eerder as 'n menslike voedselbron, maak dit aantreklik as 'n grondstof vir die vervaardiging van bio-etanol. Die gesamentlike aktiwiteit van endoglukanases, eksoglukanases en β-glukosidases is nodig om kristallyne sellulose tot fermenteerbare suikers te hidroliseer. Die hoë koste van hierdie ensieme is egter die grootste hindernis vir die ekonomiese produksie van bio-etanol vanaf sellulosiese biomassa. 'n Belowende koste verminderingstrategie is die produksie van hierdie sellulolitiese ensieme, die hidrolise van biomassa, en die fermentasie van die suikers na bio-etanol in 'n enkelstap-proses via 'n sellulolitiese mikro-organisme. Die ontwikkeling van so 'n gekonsolideerde bioprosesserings (CBP) organisme kan deur die uitdrukking van sellulolitiese aktiwiteite in 'n nie-sellulolitiese mikro-organisme wat wel in staat is om glukose na etanol om te fermenteer, gerealiseer word. Saccharomyces cerevisiae is 'n goeie kandidaat gasheer vir CBP, omdat hierdie gis ‘n hoë verdraagsaamheid teenoor etanol toon en sy gebruik in industriële toepassings gevestig is. Die ensiematiese aktiwiteite van endoglukanases en eksoglukanases word egter deur die ophoop van sellobiose gedurende die hidrolise van sellulose geïnhibeer. Hierdie effek kan met die byvoeging van meer effektiewe β-glukosidases verlig word. β-Glukosidases hidroliseer sellobiose na glukose en verlig dus die inhibisie op die endoglukanase en eksoglukanase ensiematiese aktiwiteite. Ten spyte van vooruitgang in ensiemproduksie stelsels en ensiemmodifiserings strategieë wat tans vir hoër stabiliteit en aktiwiteit in gebruik is, bestaan daar steeds 'n behoefte om die bestaande versameling van ensieme uit te brei. Bioprospektering vir nuwe sellulolitiese ensieme fokus op spesifieke omgewings, met hoë omsetkoerse van sellulose materiaal of omgewings met uiterste toestande, soos die komposterings-proses. Hierdie ensieme is besig om meer aantreklik in vergelyking met hul mesofieliese eweknieë te raak as gevolg van hul potensiele industriële toepassings en die feit dat hulle die laer natuurlike grense van proteïen-stabiliteit verteenwoordig. / Stellenbosch University and the Technology Innovation Agency for financial support

Some aspects of the presence of systemic endophytic fungi in agriculturally important New Zealand grasses were studied in relation to plant breeding. Seedling resistance to adult Argentine stem weevil feeding in perennial ryegrass, Italian ryegrass and tall fescue was found to be related to the presence of their respective Acremonium endophytes in the seed rather than to plant genetic resistance. In addition a study of perennial ryegrass revealed that this resistance was independent of endophyte viability. The seedling resistance conferred by the endophyte of Italian ryegrass was found to be beneficial for field establishment. This endophyte differs from that in perennial ryegrass and tall fescue in that it does not confer resistance to Argentine stem weevil on mature plants, but only on seedlings. The extent of plant genetic seedling tolerance to adult Argentine stem weevil feeding was limited to broad inter-specific differences, with tall fescue more tolerant than perennial ryegrass and both of these more tolerant than Italian ryegrass. This ranking corresponds with previous observations on feeding preference on mature plants. A study of factors affecting the concentration of endophyte mycelia in infected seed of perennial ryegrass revealed that plant genetic factors had little effect. The major factors studied were: 1) the endophyte concentration in the maternal parent plant directly influenced the endophyte concentration in the seed. 2) nitrogen fertilizer applications to a seed crop reduced the concentration of mycelia in the seed, with earlier applications having a greater effect. 3) application of the fungicide propiconazole (Tilt) to a seed crop reduced the endophyte concentration in the seed. 4) the endophyte concentration in the seed was found to directly influence the endophyte concentration in seedlings, six month old plants and that of seed harvested from a first year seed crop. As there have been no previous reports of tetraploid perennial ryegrass cultivars with endophyte an experiment was conducted to determine if these could be developed by the standard procedure of colchicine treatment. The results revealed that endophyte was retained following colchicine treatment.

A fragment of the Claviceps purpurea pyr4 gene, encoding the enzyme orotidine-5'-monophosphate decarboxylase (OMPdecarboxylase) was used to screen a genomic library to an isolate (designated Lp1) of an Acremonium sp. which grows as an endophyte in perennial ryegrass (Lolium perenne). Four positive clones, λMC11, λMC12, λMC14 and λMC20 were isolated. Three of these clones, λMC12, λMC14 and λMC20 were overlapping clones from the same locus, while λMC11 was from a different locus. Fragments of these clones which hybridised with C. purpurea pyr4 were sequenced and found to have similarity with pyr4 from other fungi of the Pyrenomycetes and related Deuteromycetes, suggesting that Lp1 has evolved from a sexual Pyrenomycetes species. The pyr4 from λMC12, λMC14 and λMC20 was designated pyr4-1 and that from λMC11 was designated pyr4-2. The predicted ORFs of the two genes were highly conserved and the 5' non-coding nucleotide sequences were the least conserved regions. RT-PCR and northern analysis of total RNA from Lp1 demonstrated that transcripts approximately 1.4 kb in length were produced from the two genes and present at similar levels. Genomic fragments containing pyr4-1 or pyr4-2 were transformed into a strain of Aspergillus nidulans which has a mutation in the pyrG gene (encoding OMPdecarboxylase). Both of the Lp1 pyr4 complemented a pyrG mutation in Aspergillus nidulans, confirming that both pyr4-1 and pyr4-2 encode functional OMPdecarboxylases. Comparisons of pyr4 restriction fragment length polymorphisms (RFLPs) from Lp1 and isolates of Epichloë typhina, E. festucae, A. lolii, A. uncinatum, and three endophyte taxonomic groupings from Festuca arundinacea: FaTG-1 (=A. coenophialum), FaTG-2 and FaTG-3 suggested that pyr4-1 originated from E. typhina, the ryegrass choke pathogen, and pyr4-2 originated from A. lolii, another endophyte from perennial ryegrass. This suggested that Lp1 is an interspecific hybrid, between E. typhina and A. lolii. Comparisons of the variable 5' non-coding nucleotide sequences from pyr4 of Lp1 and other isolates demonstrated that E. typhina, and A. lolii or E. festucae were the most likely ancestors of the two pyr4 found in Lp1. The A. lolii and E. festucae sequences were very similar, suggesting they are closely related. A. lolii has most probably evolved from an E. festucae, and in the process lost the sexual cycle. Analysis of single spore purified isolates of Lp1 revealed that Lp1 was a homokaryon for pyr4. A Southern blot of a CHEF gel of Lp1 and these single spored isolates was hybridised to a pyr4 probe and demonstrated that pyr4-1 and pyr4-2 were present on either two chromosomes of similar size, or one chromosome. The hybridisation that gave rise to Lp1 was concluded to have been a relatively recent event, given the similarity of pyr4-1 and pyr4-2 nucleotide sequences to those of their probable ancestors, and the fact that both genes are expressed and functional. Interspecific hybridisation is probably widespread in the asexual endophytes, and may be an important event in their evolution, and the evolution of other fungal species.

Made available in DSpace on 2016-06-02T19:55:29Z (GMT). No. of bitstreams: 1 DoutAAF.pdf: 3275307 bytes, checksum: f45ed0ce056382979fefde457b9cfe5f (MD5) Previous issue date: 2003-10-10 / The scope of the current work was to investigate the effect of glucose, amonium acetate, DL-methionine, phosphorus, oleic acid and salt solution on the maximum specific growth rate (µmax), as well as the effect of sucrose and DL-methionine on the specific cefalosporin C production rate (µp), by Cephalosporium acremonium ATCC 48272, using factorial design and response surface techniques. The initial medium of fermentation was used according to factorial design and the experiments were carried out in shaker flasks at controlled temperature at 28 °C, initial pH of 7,0+0,1 and agitation speed 250 rpm. For both specific rate the variables involved were correlated by a fitted second order model. The canonic and surface response analysis showed a maximum specific growth rate of 0.22 h-1 obtained when glucose and amonium acetate concentration, were 4.2 , 2.5 g/L, respectively, and specific cefalosporin C production rate of 1.12 mgCPC/gcel.h when sucrose and DL-methionine concentration, were 10.5, 2.5 g/L, respectively, for a celular concentration of 14.0 g/L. The experimental design showed to be very efficcient in evaluating the bioprocess, as long as high values of µmax e µp were obtained in comparison with literature data. The feedforward neural network (FNN) was employed for modelling and simulation the effect of glucose, amonium acetate, DL-methionine, phosphorus, oleic acid and salt solution on the maximum specific growth rate. The network topologies were used with 6 neurons in the input layer, 22 hidden layer and 1 output layer. The data base for training the FNN was obtained from results of the factorial designs processed with a backpropagation learning algorithm. The surfaces response obtained by simulations for all range of glucose and amonium acetate concentrations showed inhibitory effect for the two substrates on the maximum specific growth rate. The results of the simulation are in good agreement with the experimental and statistical model results. / Neste trabalho verificou-se o efeito das variáveis concentração de glicose, acetato de amônio, DL-metionina, fósforo, ácido oléico e solução de sais na velocidade específica máxima de crescimento (µmax), assim como sacarose e DL-metionina na velocidade específica de produção do antibiótico cefalosporina C (µp), pelo fungo Cephalosporium acremonium ATCC 48272, usando como ferramenta estatística a técnica de planejamento experimental e a análise por superfície de resposta. Na otimização destas variáveis, os ensaios foram conduzidos de acordo com os planejamentos fatoriais, fracionário, completo e composto central, em fermentações em incubador rotatório, variando as concentrações iniciais dos nutrientes do meio, a 28 oC, pH 7,0+0,1, com agitação de 250 rpm. Para ambos os casos, um modelo ajustado de 2a ordem foi o que melhor correlacionou as variáveis. Os resultados da otimização mostraram que uma velocidade máxima de crescimento de 0,22 h-1 pode ser atingida para uma concentração de glicose e acetato de amônio em torno de 4,20 e 2,5 g/L, respectivamente, e uma velocidade específica de produção de cefalosporina C de 1,12 mgCPC/gcel.h para uma concentração de sacarose de 10,5 g/L e DL-metionina 2,5 g/L com uma massa celular média, para produção, de 14,0 g/L. O planejamento estatístico mostrou-se uma ferramenta bastante eficiente para avaliação do bioprocesso, pois com a realização dos ensaios propostos, foi possível obter valores de µmax e µp superiores aos encontrados na literatura. Desenvolveu-se um modelo baseado na técnica de redes neurais para a simulação de todo o domínio das concentrações de glicose, acetato de amônio, DL-metionina, fósforo, ácido oléico e solução de sais na velocidade específica máxima de crescimento. A rede que melhor representou os valores experimentais foi uma do tipo "feedforward", com três camadas e 22 neurônios na camada oculta, treinada com o algoritmo de retropropagação, por um conjunto de resultados obtidos através de ensaios do planejamento estatístico. As superfícies obtidas pela simulação em amplo domínio de concentrações de glicose e acetato de amônio, mostraram que o crescimento do microrganismo segue um modelo cinético de inibição por este dois nutrientes, prevendo altos valores de µmax para concentrações de glicose e acetato de amônio, comparáveis com os obtidos pelo modelo estatístico.

Effect of an extraction method on the structure of glucan and its immunostimulatory response in Fenneropenaeus indicus was investigated. Here we extracted alkali insoluble glucan (AIG) and alkali soluble glucan (ASG) from a filamentous fungi Acremonium diospyri following alkali-acid hydrolysis and the sodium hypochlorite oxidation and dimethyl sulphoxide extraction method respectively. Structural analysis showed that 85% of glucan in AIG was a (1→3)-β-d-glucan and it increased the prophenoloxidase and reactive oxygen intermediate activity when administered to F. indicus. On the other hand, ASG, which contained 93% (1→3)-α-glucan, did not induce significant immune response in shrimp. Here we report that the difference in immunostimulatory potential between AIG and ASG is due to the difference in the percentage of (1→3)-β-d-glucans present in each preparation, which varies with the method of extraction employed. Also our observations suggest that glucan can be used as a potential immunostimulant to shrimp, provided it contains (1→3)-β-d-glucan as the major fraction.