Optimization of fungal pigments production through different types of fermentation

Universidade do Minho Escola de Engenharia Bruna Isabel Teixeira Basto Optimization of fungal pigments production through different types of fermentation outubro de 2019 UMinho | 2019 Bruna Basto Optimization of fungal pigments production through different types of fermentation Bruna Isabel Teixeira Basto Optimization of fungal pigments production through different types of fermentation Dissertação de Mestrado Mestrado em Biotecnologia Trabalho efetuado sob a orientação do Professor Doutor José Texeira e da Doutora Sara Silvério Universidade do Minho Escola de Engenharia outubro de 2019 ii DIREITOS DE AUTOR E CONDIÇÕES DE UTILIZAÇÃO DO TRABALHO POR TERCEIROS Este é um trabalho académico que pode ser utilizado por terceiros desde que respeitadas as regras e boas práticas internacionalmente aceites, no que concerne aos direitos de autor e direitos conexos. Assim, o presente trabalho pode ser utilizado nos termos previstos na licença abaixo indicada. Caso o utilizador necessite de permissão para poder fazer um uso do trabalho em condições não previstas no licenciamento indicado, deverá contactar o autor, através do RepositóriUM da Universidade do Minho. Atribuição-NãoComercial-SemDerivações CC BY-NC-ND https://creativecommons.org/licenses/by-nc-nd/4.0/ iii AGRADECIMENTOS Durante estes anos várias pessoas acompanharam e contribuíram para o meu desenvolvimento pessoal e profissional. Ao Professor José Teixeira agradeço todo o apoio e orientação prestado ao longo de todo ano. Agradeço também a oportunidade de integrar um grupo fantástico. Em especial, agradeço à Sara pela orientação exemplar, a simpatia, o companheirismo e por toda a confiança que depositou em mim ao longo de todo o ano. Certamente que se daqui a uns anos me questionarem sobre quem mais contribuiu para o meu desenvolvimento científico e mais me fez enriquecer a nível de conhecimento, não terei dúvida nenhuma em responder que foi a Sara. Obrigada pelo teu espírito crítico, a vontade de inovar, a perseverança e por todas as conversas que permitiram criar uma relação fantástica e desenvolver um trabalho que tanto me deu a todos os níveis. Obrigada por me fazeres ver que a investigação pode ser um caminho a escolher! Ao Nuno agradeço todo o conhecimento, apoio e companheirismo que me deu ao longo de todo este projeto. Posso-me orgulhar de dizer que tive as melhores pessoas a guiarem-me nesta etapa e que ganhei amigos para a vida! A todas as pessoas integrantes do LF agradeço a ótima receção, o ambiente incrível, os momentos de descontração, todas as piadas e todo o apoio. Tudo isto tornou esta experiência especial! Aos meus amigos e em especial à Eduarda, à Marta, à Marlene, à Mika, ao Toni, ao Gonçalo, ao TiGi, à Meme, à Sara, ao Macedo e ao Mário agradeço por fazerem destes anos os melhores anos da minha vida e por termos as melhores histórias e os melhores momentos que vou recordar sempre com tanto carinho. Ao meu Maninho um agradecimento especial por mantermos esta amizade há tantos anos e, por muito tempo que passe sem nos vermos, quando nos reencontramos nada muda, somos irmãos! Ao André um agradecimento do tamanho do mundo foste o meu maior apoio. Obrigada por estares sempre presente, nos bons e nos maus momentos. À minha família agradeço todo o apoio e amor. Ao meu irmão um obrigada e espero que tenhas tanto orgulho em mim como eu tenho em ti, apesar das brincadeiras serás sempre o meu pequenino! Em especial, à minha mãe e ao meu pai por todos os valores, ensinamentos e amor que me deram e continuam a dar e por me apoiarem e permitirem que eu voe mais alto! Espero que se orgulhem no que me tornei como pessoa! Eu sei que toda a gente diz isto, mas os meus são os melhores! iv STATEMENT OF INTEGRITY I hereby declare having conducted this academic work with integrity. I confirm that I have not used plagiarism or any form of undue use of information or falsification of results along the process leading to its elaboration. I further declare that I have fully acknowledged the Code of Ethical Conduct of the University of Minho. v ABSTRACT OPTIMIZATION OF FUNGAL PIGMENTS PRODUCTION THROUGH DIFFERENT TYPES OF FERMENTATION Pigments are organic or inorganic compounds with the ability to provide color, and therefore, are widely applied in several industrial products. Once public awareness concerning environment preservation and safety, sustainability and health issues have been increasing, industries began to call for different pigments from natural sources, obtained by cleaner processes and with less ecological impact. The production of natural pigments through microbial fermentation is considered a very promising alternative. Fungi are known to naturally synthesize and secrete several classes of pigments. Recently, some Penicillium species have been reported as effective pigment producers. The use of fungi cultures to obtain products of interest is generally performed under two types of fermentation: submerged fermentation (SmF) and/or solid-state fermentation (SSF), with SmF being the most industrially used. For fermentation processes to become economically viable, the extractive fermentation (FE) has been proposed as an emerging option which allows the integration of fermentation and selective separation of the product of interest. In this work, the pigments production using alternative low-cost media containing agroindustrial residues was evaluated under SmF (with and without biomass immobilization), SSF and EF conditions. For that purpose, 6 different culture media composed of cheese whey (CW) and/or corn steep liquor (CSL) in different ratios (culture media B-G) were prepared and compared with a reference synthetic medium (A). Regarding the production under SmF conditions, the results revealed no significant differences between the alternative media D, F and G and the reference synthetic medium A. The immobilization of the fungus on a natural support (corn cob, SmFn) under SmF conditions led to higher amount of pigments obtained than with an inert support (nylon sponge, SmFi). Furthermore, most of the culture media tested under SmFn proved to be a suitable alternative to the reference medium A. On the other hand, better pigments yields were achieved using the inert support under SSF conditions. Overall, the best conditions found for pigments production were media D and G both under SmFn conditions. For all types of fermentation processes considered in this work, pigments mixtures were obtained with yellow, orange and red pigments present. The pigments production was also attempted under EF conditions using a PEG8000-NaCit aqueous two-phase system (ATPS) and their recovery and concentration in top phase was observed. In conclusion, the use of alternative low-cost culture media composed of agroindustrial residues to produce pigments by P. brevicompactum was successfully demonstrated. Keywords: agroindustrial residues; fungal pigments; immobilization supports; Penicillium brevicompactum vi RESUMO OTIMIZAÇÃO DA PRODUÇÃO DE PIGMENTOS FÚNGICOS ATRAVÉS DE DIFERENTES TIPOS DE FERMENTAÇÃO Os pigmentos são compostos orgânicos ou inorgânicos com a capacidade de conferir cor sendo amplamente usados em vários produtos industriais. Com o aumento da consciencialização do público sobre a preservação e segurança do meio ambiente, a sustentabilidade e a saúde, as indústrias começaram a exigir pigmentos de fontes naturais, mais limpos e com menos impacto ecológico. A produção de pigmentos naturais por fermentação microbiana é considerada uma alternativa promissora. Sabe-se que os fungos sintetizam e secretam naturalmente várias classes de pigmentos. Recentemente, algumas espécies de Penicillium foram reportadas como produtoras de pigmentos. A utilização de culturas de fungos para obtenção de produtos de interesse é geralmente realizada recorrendo a dois tipos de fermentação: fermentação submersa (SmF) e/ou fermentação sólida (SSF), sendo a SmF a mais utilizada industrialmente. Para tornar os processos de fermentação economicamente viáveis, a fermentação extrativa (EF) tem sido proposta como uma opção emergente que permite a integração da fermentação e a separação seletiva do produto de interesse. Neste trabalho, a produção de pigmentos utilizando meios alternativos de baixo custo contendo resíduos agroindustriais foi avaliada em condições de SmF (com e sem imobilização de biomassa), SSF e EF. Para isso, foram preparados 6 meios de cultura diferentes, compostos por soro de queijo (CW) e/ou milhocina (CSL) em diferentes proporções (meios de cultura B-G) e comparados com um meio sintético de referência (A). Usando condições de SmF, os resultados não revelaram diferenças significativas entre os meios alternativos D, F e G e o meio de referência A. A imobilização do fungo em suporte natural (espiga de milho, SmFn) em SmF permitiu obter uma maior quantidade de pigmentos do que com o suporte inerte (esponja de nylon, SmFi). Além disso, a maioria dos meios de cultura testados em SmFn provou ser uma alternativa adequada ao meio A. Por outro lado, foram obtidos melhores rendimentos de produção de pigmentos usando o suporte inerte em SSF. No geral, as melhores condições encontradas para a produção de pigmentos foram os meios D e G, ambos em SmFn. Todos os tipos de fermentação estudados neste trabalho originaram misturas de pigmentos, constituídos por pigmentos amarelos, laranjas e vermelhos. A produção de pigmentos foi também testada em EF usando um sistema de duas fases aquosas (ATPS) PEG8000-NaCit e verificou-se a recuperação e concentração dos pigmentos na fase superior. Em conclusão, foi demonstrado com sucesso o uso de meios de cultura alternativos de baixo custo, compostos por resíduos agroindustriais, para produção de pigmentos por P. brevicompactum . Palavras-chave: resíduos agroindustriais; pigmentos fúngicos; imobilização em suportes; Penicillium brevicompactum vii TABLE OF CONTENTS Direitos de autor e condições de utilização do trabalho por terceiros ........................................... ii Statement of integrity ................................................................................................................ iv Agradecimentos ........................................................................................................................ iii Abstract..................................................................................................................................... v Resumo.................................................................................................................................... vi Table of Contents .................................................................................................................... vii Table of Figures ......................................................................................................................... x Table of Tables ........................................................................................................................ xiii Abbreviation List ...................................................................................................................... xiv 1. State of art ......................................................................................................................... 1 1.1. Pigments and their importance in different industries ........................................................... 2 1.2. Organic natural pigments and their production ..................................................................... 3 1.3. Pigments produced by filamentous fungi .............................................................................. 5 1.3.1. Penicillium spp. as potential pigment producers ........................................................... 8 1.3.1.1. Penicillium brevicompactum .................................................................................... 9 1.4. Production of fungal pigments by fermentation..................................................................... 9 1.4.1. Submerged Fermentation .......................................................................................... 10 1.4.2. Solid-State Fermentation ............................................................................................ 12 1.4.3. Agroindustrial residues as alternative culture media in fermentation processes ........... 14 1.5. Extractive fermentation using Aqueous two-phase systems ................................................. 15 1.5.1. Polymer-Polymer Aqueous Two-Phase Systems .......................................................... 16 1.5.2. Polymer-Salt Aqueous Two-Phase Systems ................................................................. 17 2. Objectives of the thesis ................................................................................................... 17 3. Materials and Methods .................................................................................................... 19 3.1. Microorganism .................................................................................................................. 20 xiv ABBREVIATION LIST ATPSAqueous Two-Phase Systems CSLCorn Steep Liquor CWCheese whey/Cheese whey powder EEEthanolic extract EFExtractive fermentation MMedium MCMedium change MEAMalt extract agar MPAMycophenolic Acid P-YEPeptone and yeast extract mixture SmFSubmerged fermentation SmFiSubmerged fermentation with immobilization on inert support SmFnSubmerged fermentation with immobilization on natural support SSFSolid-state fermentation SSFiSolid-state fermentation using inert support SSFnSolid-state fermentation using natural support 1 1. STATE OF ART 2 1.1. PIGMENTS AND THEIR IMPORTANCE IN DIFFERENT INDUSTRIES Color has always attracted humans and plays an important role in the choice of various products of commercial value, since consumers tend to evaluate the quality of a product or choose it, by its visual appearance. Thus, the use of pigments as color-conferring molecules has been widely explored by various industries1. Pigments can be classified as organic or inorganic, both classes being subdivided into natural or synthetic pigments. Natural pigments can be obtained from living organisms such as plants, animals, fungi and other microorganisms and by ores, while the synthetic ones are obtained by chemical synthesis in the laboratory (Figure 1)2,3. Figure 1. Representative scheme of the classification of pigments and their sources of production. Pigments are compounds with important characteristics to several industries, such as food, cosmetics, textile, pharmaceutical and tanneries. Among these characteristics stands out the wide variety of existing colors, which led many pigments to begin to be produced, isolated and characterized4. Currently, approximately 43 pigments are allowed as food additives in the European Union, while in the United States approximately 30 are approved. Of these 43, a large number belongs to the class of organic natural pigments and are essentially obtained from plants5. In the food industry, pigments are used as color additives6. Color additives are mainly applied as: antioxidants, compensating the loss of color due to the exposure to light, air, temperature and storage Pigments Organic Natural Plants Animals Microorganisms Synthetic Laboratory Inorganic Natural Minerals Synthetic Laboratory 3 conditions; color enhancers, to turn food more attractive; suppliers of color to colorless foods7. Carotenoids, which are responsible for the attractive color of most of the fruits and vegetables and present diverse functions and biological properties, are a typical example of pigments used as color aditives8. Some studies have shown the potential of application of some pigmented secondary metabolites in the pharmaceutical industry, especially in the treatment of several diseases, since some pigments also present antibiotic, anticancer and immunosuppressive properties9–11. An example of such compounds are the anthocyanins, which present a wide range of biological properties such as potential to reduce cancer and inflammatory risks, and modulate the inflammatory response12. The textile industry is another industrial sector with great interest in pigments. Data found in the literature suggest that in 2013 approximately 1.3 million tons of dyes, pigments and color precursors are used in this sector per year, valued at around 19 billion euros, being almost all produced synthetically13. Currently, synthetic pigments derived from non-renewable resources, such as fossil fuels, are mainly used in this industry due to their simple application, reproducibility and possibility to obtain quality at a reasonable price to consumers. However, the biosynthesis of natural pigments for use in this industry has increased considerably in the recent years, mostly due to the issues associated with the synthetic dyes14. Among these issues, the production dependency on non-renewable petroleum resources, their environmental toxicity, sustainability matters and, particularly, the increasing concerns regarding their danger to human health can be highlighted. Due to all these factors, in the recent years there has been an increase in the preference for organic natural pigments obtained from sustainable processes15. 1.2. ORGANIC NATURAL PIGMENTS AND THEIR PRODUCTION Public consciousness of ecological preservation and safety, sustainability and health issues has been increasing. Thus, the development of biological, non-toxic and biocompatible pigments has become necessary. All these aspects have contributed to put pressure on the industries to take the steps needed to invest in the research and development of new types of pigments and to explore new sources for their production. In this way, various pigments derived from natural sources, cleaner and more ecological began to be reported and applied16. In 2010, natural pigments represented about 31% of the world market of pigments, and its application is growing and may even exceed the synthetic pigments in the near future17. Organic natural pigments are obtained and/or extracted from natural sources, mainly from plants. In most cases, they are mixtures of varied compositions, which depend on the climatic conditions 4 and form of cultivation. Therefore, they are not easy to obtain or characterize. Also, a great variation in the stability and functionality of different classes of existing organic natural pigments can be observed, being the pigments often affected by light, pH and temperature. Once certain products require the use of stable pigments, this lack of stability can limit their range of use18. Despite the disadvantages mentioned above, there are also undoubted advantages that justify the preference for these pigments. In terms of human metabolism, substances of natural origin are more easily metabolized by the body, since there are many metabolic pathways in common with other organisms. In addition, there are several organic natural pigments that have antioxidant activity, a biological property of extreme importance and that accompanies the trend of development of food with health benefits19. Thus, replacing artificial pigments by natural pigments, two desired effects are obtained, on the one hand, color matching (an important feature for the food industry) and on the other its biological properties, such as protection against free radicals, having a two in one effect20. Traditionally, organic natural pigments are extracted from plants with flowers, such as the blue color pigment indigo obtained from Indigofera tinctoria, or from insect tissues, such as red cochineal obtained from Dactylopius coccus 4,21. However, most of the pigments obtained from these sources are produced in insufficient amounts for industrial use, being dependent on the supply of raw materials (due to seasonality) and being also affected by ethical reasons, particularly when considering an animal source22. Organic natural pigments obtained through microbial fermentation are considered a very promising alternative, namely to overcome the associated availability and ethical issues of the raw materials. Many bacteria, fungi and yeasts are known to produce a wide variety of natural pigments23–26. Pigments obtained from microorganisms are of industrial interest because these organisms are generally more accessible, can grow rapidly and still have the potential to be genetically modified, which consequently can provide better yields and productivities. Efforts have been made to reduce the costs associated with the production of pigments by microbial fermentation, to efficiently compete with the production of synthetics and derivatives from other natural sources, such as plants and animals22,27. Among the different microorganisms, fungi have been reported as the major pigment producers due to their versatility in producing pigments of different colors and easy large-scale cultivation. So far, 5 there are several fungi species well studied to produce pigments, and some examples will be presented and briefly discussed below27–29. 1.3. PIGMENTS PRODUCED BY FILAMENTOUS FUNGI Fungal secondary metabolites, such as pigments, can be divided into four different classes depending on their structural properties: terpenes, polyketides, non-ribosomal peptides, and amino acidderived compounds30. These metabolites, also known as exometabolites, are characterized as bioactive small molecules that are not essential for the growth of the host organism but instead aid survival in harsh environments, resisting desiccation and UV stress and improving competition with other microbes31,32. Exometabolites are produced during morphological and chemical differentiation and then are secreted, deposited or accumulated in the cell wall32. Most of the exometabolites produced by ascomycetous filamentous fungi are polyketides. Polyketides are typically synthesized by multifunctional polyketide synthases (PKA) from acetylcoenzyme A (CoA) and malonyl-CoA33,34. Polyketides represent an array of structurally complex natural products such as anthraquinones, hydroxyanthraquinone, naphthoquinone and flavonoid pigments. Some species of ascomycetous filamentous fungi belonging to the families Monascaceae , Trichocomaceae , Pleosporaceae and Nectriaceae are responsible for the production of polyketide-based pigments with different colors (red, orange, yellow and/or brown)27,35. Certain pigments produced by filamentous fungi also belong to the terpene class. Terpene synthases and terpene cyclases generate terpenes from activated isoprene units. Carotene and antheraxanthin are examples of secondary metabolites belonging to this class36. This two class of secondary metabolites are derived from central metabolic pathways and primary metabolite pools, with acyl-CoAs being the critical initial building block (Figure 2). 6 Figure 2. Acyl-CoAs as critical initial building block for polyketides and terpenes. Adapted from Keller36. Fungi, particularly filamentous fungi, are known for their ability to produce a wide range of pigments that are generally more soluble than plant-derived pigments27,37. Some pigments produced by filamentous fungi, and their respective color and class, are shown in Table 1. 7 Table 1. Pigments produced by filamentous fungi and their respective color and class. Adapted from Caro et al .30, Mapari et al. 38 and Rao et al. 39 . Filamentous Fungi Pigment Colour Class Monascus spp. Ankaflavin Rubropunctatin Monascorubramin Yellow Orange Red Polyketides Aspergillus versicolor Asperversin Yellow Terpene Fusarium sp. JN158 Benzoquinon Yellow Polyketide Fusarium oxysporum Anthraquinone Yellow Polyketide Stemphylium lycopersici Anthraquinone Yellow Polyketide Ashbya gossip Riboflavin Yellow Polyketide Cordyceps unilateralis Naphthoquinone Deep blood red Polyketide Penicillium oxalicum Anthraquinone Red Polyketide Blakeslea trispora Lycopene Red Terpene Blakeslea trispora ß-carotene Yellow–orange Terpene Mucor circinelloides ß-carotene Yellow–orange Terpene Penicillium freii Viomellein Xanthomegnin Reddish-brown Orange Polyketide Penicillium purpurogenum Mitorubrin Mitorubrinol Purpurogenone Yellow Orange-red Yellow-orange Polyketide Penicillium rugulosum Rugulosin Yellow Polyketide The first fungal pigment production known is ang-kak (red rice) using Monascus purpureus , which consists in fermenting rice with this fungus getting the rice a bright purplish red color. Currently, more than 50 pigments produced by Monascus species have been identified and studied. Also, more than 50 patents have been issued worldwide for the use of Monascus pigments in 8 foods40,41. In addition to the variety of available colors, pigments produced by this genus of fungus have been shown to have antimicrobial42,43, anticancer44,45, antimutagenic44 and antiobesity properties46,47. Although there are several fungal pigments reported in the literature, their practical use is conditioned by regulatory approval criteria concerning their toxicity and stability. In addition to these criteria, capital investment should also be considered, which represents the main bottleneck for transposing Petri dish products to the market48. The best example of these approval issues is the pigments obtained from Monascus which have been used in Asia for centuries as food pigments, but banned in Europe and in the United States due to the presence of mycotoxins in the final formulations40. Several methods have been developed to avoid obtaining mycotoxin-contaminated pigments such as: (a) selection of non-pathogenic strains (several fungal strains have been tested for toxicity and it was found that toxin production is only present in some lineages); (b) control of metabolites biosynthesis (the study of metabolic pathways has shown that toxin production can be controlled through the biosynthesis process); (c) selection of the culture media (it has been observed that the addition or removal of metal ions, carbon sources and nitrogen sources may affect toxin production)40,49. In addition to the absence of toxins during production and commercialization, microbial pigments must be stable under extreme pH and temperature conditions so that they can be used at industrial level. It is currently known that there are several strains of non-pathogenic filamentous fungi that can be used as potential producers of non-toxic and non-pathogenic pigments for humans, such as Talaromyces , Cordyceps unilateralis , Herpotrichia rhodosticta , Curvularia lunata , several species of Drechslera and some species of Penicillium 5,50,51 . 1.3.1. PENICILLIUM SPP. AS POTENTIAL PIGMENT PRODUCERS As mentioned previously, pigments obtained from fungi are produced as secondary metabolites of known or unknown function. Although Monascus pigments are the most commercially available, especially in the East as food pigments, it is known that several strains of Monascus produce, among other potentially toxic metabolites, the mycotoxin citrinin18. This mycotoxin is known to be nephrotoxic, hepatotoxic and possibly carcinogenic in humans and animals52. Therefore, the search for alternative microorganisms to produce safe pigments has become an important and demanding field53. Recently, Penicillium species have been identified as potential pigment producers. The pigments produced by these microorganisms are homologous to the those produced by Monascus , with similar 9 chromophores4. Also, these fungal pigments can be more easily accepted in the food industry since they come from microorganisms that are known to not produce the mycotoxin citrinin. However, the presence of other mycotoxins should be carefully evaluated prior their practical application at industrial level. Several studies have demonstrated the production of pigments by Penicillium species by fermentation, thus evidencing the potential of using this genus as an industrial pigment producer4,54,55. 1.3.1.1. PENICILLIUM BREVICOMPACTUM P. brevicompactum is a mold usually found inside the production chambers of the food industry56. This specie of Penicillium is able to grow in environments with low water activity57. This fungus has the following lineage: Eukaryota , Fungi , Dikarya , Ascomycota , Pezizomycotina , Eurotiomycetes , Eurotiomycetidae , Eurotiales , Aspergillaceae , Penicillium . According to the literature, this specie is able to produce mycophenolic acid (MPA), an immunosuppressive compound that is increasingly associated with cases of allergic and pulmonary fibrosis58. This compound is reported as a mycotoxin, which is synthesized during the secondary metabolism of the fungus while contaminating foods and feeds56. However, when found in contaminated foods, the levels of MPA are lower in comparison to the therapeutic doses commonly administered to humans, so its presence should not be a health concern. Some authors reported that MPA should not be classified as a mycotoxin since the risk and the severity of MPA effects after consuming potential contaminated foods are both reduced59. Until today, there are no reports in the literature about the ability of P. brevicompactum to produce pigments, but this fungus showed a high potential to produce this type of compounds from a yellow to a red color range, depending on the growth conditions. Recently, this strain was isolated from the air of a cheese curing chamber in Ponte de Lima, Portugal. Previous studies of our research group showed that P. brevicompactum has great potential for pigment production60,61. 1.4. PRODUCTION OF FUNGAL PIGMENTS BY FERMENTATION Fermentation is an important biological technology that allows to produce several chemical or pharmaceutical compounds by the cultivation of microorganisms such as bacteria and fungi in the presence of complex substrates. In the course of the metabolic decomposition of the substrates, various secondary compounds (e.g. pigments) are released in addition to the usual fermentation products, such as carbon dioxide or alcohol. 16 should not be used in concentrations that are toxic or inhibit cell growth; (b) the physicochemical nature of the two-phase system, such as polymer integrity and stability, should not be affected by the fermentation process; (c) the cells and the substrates should be fully or predominantly split into one of the phases (preferably the opposite phase of the product accumulation); (d) feed constituents such as mineral salts or substrates should not drastically affect phase behavior; (e) the system should be cost effective in terms of the recycling and reuse of its constituents (polymer and/or salt)87. In Table 2, some selected examples of extractive fermentation using ATPSs for the recovery of different products of interest, including pigments, are presented. Table 2 . A list of some selected examples of extractive fermentation using ATPSs. ATPS Product Reference PEG(1)8000/NaCit(2) Protease Silva et al. 88 Thermo-separating EOPO(3) Polyhydroxyalkanoates Leong et al. 89 PEG3350/Dextran 66900 β-carotene Chavez-Santoscoy et al .90 PEG8000/Dextran 66900 Lutein Chavez-Santoscoy et al. 90 PEG8000/Phosphate salts Clavulanic acid Viana Marques et al. 91 (1)PEG - polyethylene glycol; (2)NaCit - sodium citrate; (3)EOPO - copolymer of ethylene oxide-propylene oxide. 1.5.1. POLYMER-POLYMER AQUEOUS TWO-PHASE SYSTEMS When mixing two hydrophilic polymers in water a partially immiscible aqueous two-phase system (ATPS polymer-polymer) is formed above certain critical conditions87. The composition of the two phases depends, among others, on the amount of polymer added, their molecular weights and the equilibrium temperature. The concentration of each polymer in the equilibrium phases of a given system can be determined by a phase diagram, which is specific for the temperature used92. Originally, these systems were based on aqueous mixtures of polymers such as polyethylene glycol (PEG), dextran and/or maltodextrin. The combination of dextran and PEG represents one of the most studied systems, where dextran, the most dense and hydrophilic polymer, predominantly accumulates in the bottom phase and PEG, more hydrophobic and less dense, forms the top phase93. However, the major limitations of this biphasic system are the high cost and viscosity of dextran, which economically and technically impairs its application at industrial scale. Therefore, new and cheaper polymers have emerged as an alternative to dextran such as crude dextran, waxy starch, hydroxypropyl starch or polyvinyl alcohol68,94. However, polymer-polymer ATPS performance is not as promising for the separation of compounds of interest because they usually have low selectivity95. 17 Additionally, thermoseparating copolymers such as Ucon (an ethylene oxide and propylene oxide copolymer, EOPO) were proposed as phase forming components for cheaper and reusable systems. An aqueous solution containing 10-40 (% w/w) of this copolymer, forms a biphasic system with the Ucon predominantly concentrated in the bottom phase, when heated above 47 °C. The advantage of these copolymers is that at the end of the process, about 95 % of the initial copolymer can be recovered by increasing the temperature, thus allowing its recycling and potential reuse96–98. The PEG-polyacrylic acid ATPSs were also reported as an interesting alternative to the PEGdextran systems, since they present a lower cost, constituting a suitable biphasic system for the industrial separation of biomolecules99. 1.5.2. POLYMER-SALT AQUEOUS TWO-PHASE SYSTEMS ATPSs formed by polymers and electrolytes (salt) have a vast range of applications due to the significantly different chemical environments achieved in top and bottom phases, which consequently results in different physicochemical properties. Therefore, separation processes are generally more efficient in such ATPSs with the additional advantage that they are relatively cheaper and thus more suitable for large scale operations100. Different salts, such as phosphates, citrates, tartrates, succinates and oxalates may form an ATPS when combined with polymers, namely PEG101. Polymer/salts systems generally present higher selectivity in the partitioning process, with high yields in first extraction step87. PEG-potassium phosphate ATPSs are ones of the most common biphasic systems due to several advantages such as extensive physicochemical characterization, low cost and wide range of applications. However, the use of phosphates represents an environmental threat, particularly their waste disposal, because they are responsible for the eutrophication of several watercourses87,102. Thus, alternative ATPSs such as PEG-citrate and PEG-formate may be used, representing a lower environmental risk due to their higher biodegradability103,104. 2. OBJECTIVES OF THE THESIS Previously results obtained by our research group demonstrated the potential of P. brevicompactum to produce pigments under submerged fermentation using a synthetic culture medium. Thus, the present work aimed to improve pigment production using alternative low-cost media containing agroindustrial residues. Culture media composed of residues offer recognized economic advantages and therefore can 18 result in a more attractive approach for pigment production at industrial level. Additionally, other alternative strategies for pigment production, such as solid and extractive fermentations, were explored and compared in terms of production effectiveness. In order to successfully achieve the proposed main goals, the following secondary objectives were established: • pigment production under submerged fermentation conditions (with and without immobilization support) using fermentation media composed of CW and CSL; • pigment production under solid fermentation conditions using fermentation media composed of CW and CSL; • scale-up of pigment production under submerged fermentation conditions using synthetic medium in a 3.7 L bioreactor; • pigment production under extractive fermentation conditions using a PEG-salt ATPS. 19 3. MATERIALS AND METHODS 20 3.1. MICROORGANISM P. brevicompactum (MUM 02.07) was obtained from the Mycology collection of University of Minho (MUM). Stock cultures were maintained at room temperature (around 25 °C) in spore suspensions of semi-solid agar medium (2 g/L). For inoculum preparation, stock cultures were then sub-cultured on Petri dishes containing the so-called malt extract agar (MEA) medium (g/L): malt extract (20), glucose (20), peptone (1) and agar (20), and grown at 25 °C for 7 days. 3.2. AGRO-INDUSTRY RESIDUES Cheese whey powder (CW) was kindly provided by Lactogal Produtos Alimentares S.A. and corn steep liquor (CSL) by COPAM (Companhia Portuguesa de Amidos, S.A.), Portugal. The composition (% w/w) of CW was 58.5 % lactose, 12.6 % protein, less than 0.2 % fat and 1.2 % moisture. The composition of CSL was 75 g/L sugars and 5 g/L proteins105 . 3.3. SUPPORTS 3.3.1. INERT SUPPORT Cubes (0.125 cm3) of commercial nylon sponge (Vileda Ultra Fresh®, Freudenberg) were used as inert supports. Prior to use, the nylon sponge cubes were pre-treated by boiling for 15 min and washing with distilled water. After that, the cubes were dried at 60 °C. Prior to use, the support was autoclaved at 121 °C for 15 min. Figure 5 shows the inert support before and after cutting into cubs. Figure 5. Inert support before (left side) and after cutting into cubs (right side). 3.3.2. NATURAL SUPPORT Corn cobs were obtained from local harvest (Lousada, Portugal). Corn cobs were cut into small pieces with approximately 0.1 cm3. The prepared material was soaked in deionized water at 80 °C for 6 h to wash and increase porosity81. Then, the material was dried at 60 °C. Prior to use, the support was autoclaved at 121 °C for 15 min. Figure 6 shows the natural support before and after cutting into pieces. 21 Figure 6. Natural support before (left side) and after cutting into pieces (right side). 3.4. INOCULUM PREPARATION Inoculum for fermentations was prepared by adding 1 - 2 mL of a sterile saline solution (8.5 g/L NaCl) containing 0.1 g/L Tween 80 to fully sporulated agar plate culture of P. brevicompactum 75. The spores were then scraped from the agar plates under aseptic conditions and the conidia suspension was used as the inoculum. Conidia density was adjusted to 106 conidia/mL using a Neubauer chamber. 3.5. PIGMENTS PRODUCTION The experiments were performed in triplicate using 250 mL cotton-plugged Erlenmeyer flasks containing an initial density of 106 conidia/mL. Several fermentation media were prepared as indicated in Table 3. Prior to use, all the media and material were autoclaved at 121 °C during 15 min. 22 Table 3. Different culture media studied and their composition. Culture media Composition (g/L) Synthetic (A) Peptone (8), yeast extract (8), KH2PO4 (2), Na2HPO4.12H2O (8), MgSO4.6H2O (0.25) and lactose (20) Cheese whey (CW) (B) CW (34.6) CW synthetic supplementation 0.5 (C) CW (34.6), yeast extract (0.5) and peptone (0.5) CW synthetic supplementation 4 (D) CW (34.6), yeast extract (4) and peptone (4) Corn steep liquor (CSL) (E) CSL (12.55) CW CSL supplementation 1 (F) CW (34.6) and CSL (1) CW CSL supplementation 8 (G) CW (34.6) and CSL (8) The different culture media (A, B, C, D, E, F and G) were studied under different types of fermentation. Initial tests were performed in submerged fermentation (SmF) using a volume of 50 mL for each type of medium presented in Table 3. Submerged fermentation tests were also carried out with fungus immobilization on an inert support (nylon sponge; SmFi) and a natural support (corn cob; SmFn). SmFi and SmFn were performed with 50 mL of each medium and approximately 20 pieces of support (corresponding to 0.8 g of nylon sponge and 2 g of corn cob, respectively). Solid-state fermentation using inert (SSFi) and natural support (SSFn) was also tested. Initial moisture content was previously evaluated using 3 g of nylon sponge cubes (75 cubes) and synthetic medium (A). The moisture % (w/w) contents tested were 75, 80, 85 and 90 % since the maximum volume that this support can absorb without resulting in visible free medium, corresponds to 90 % moisture. The 23 amount of support used was selected according to the volume occupied by the cubes in the flask. The moisture content (wet basis) was calculated according Ahmad and Munaim106 and is shown in Eq. (1). Moisture content = Mass of liquid component Total mass (liquid + support) ×100 % (1) All the media present in Table 3 were studied under SSFi conditions using 3 g of nylon sponge cubes and 85 % moisture content. For SSFn, the amount of support used was the same used in SSFi, 75 pieces of corn cobs, corresponding to 7 g. For this support the moisture content used was 65 %, being this the maximum that it can absorb. All the fermentations were performed in an orbital shaker at 23 °C and 150 rpm for 12 days. These experimental conditions were determined in a previous work performed by our research group to optimize pigment production by P. brevicompactum in synthetic fermentation medium60 . Samples were taken on days 0, 3, 5, 7, 9 and 12 days in aseptic conditions for SmF, SmFi and SmFn. 3.6. PIGMENTS RECOVERY At the end of the SmF, the culture media containing the extracellular pigments were recovered through vacuum filtration using a 0.45 µm Whatman filter. The biomass was dried at 60 °C until constant weight (dry weight). For SmFi and SmFn, the culture media with the extracellular pigments were treated as described above for SmF. Additionally, the support containing biomass was taken for intracellular pigment extraction using a solution with 95 % (v/v) ethanol and 5 % (v/v) water (20 mL total volume). The mixture was kept on a rotary shaker at 150 rpm for 24 h at room temperature. The support was squeezed with the aid of a syringe and the liquid was filtered through a 0.45 µm Whatman filter. The ethanol was then evaporated at 60 °C. For SSF, pigments were also extracted from supports using a solution of 95 % (v/v) ethanol and 5 % (v/v) water. For that purpose, all the content removed from the Erlenmeyer was submerged in ethanol. The volume of ethanol solution used corresponded to three times the culture medium volume initially added107. Apart from the volume of ethanol solution, the extraction method was performed similarly to that described above for intracellular pigment extraction in SmFi and SmFn. 24 All the mixtures containing the extracellular pigments collected by vacuum filtration were freezedried. The dry extracts obtained by ethanolic extraction were diluted in 2.5 mL of distilled water and their absorbance was measured (section 3.10) before freeze-drying. 3.7. BIOMASS REUSE UNDER SUBMERGED FERMENTATION CONDITIONS To evaluate if biomass can be reused to increase the pigments production, two types of fermentation were carried out: SmF and SmFi. For each type of fermentation, the experiments were performed in triplicate using 250 mL cottonplugged Erlenmeyer flasks containing 50 mL of culture medium G (Table 3) and an initial density of 106 conidia/mL. This test was divided in 3 cycles of fermentation with 12 days. After 12 and 24 days of fermentation (first and second cycle, respectively), the medium present in the Erlenmeyer was removed with aid of a sterile net and replaced by 50 mL of fresh culture medium G. Simultaneously, for each condition (SmF and SmFi) parallel fermentation studies were performed for 36 days without replacing the fermentation medium. All the material and media were previously autoclaved at 121 °C during 15 min. The fermentations were performed in an orbital shaker at 23 °C and 150 rpm for a total of 36 days. In the three cycles, samples were aseptically taken at 0, 3, 5, 7, 9 and 12 days. In the fermentation without replacing the culture medium, samples were aseptically taken at 0, 3, 5, 7, 10, 12, 14, 24, 26 and 36 days. For each fermentation condition, pigment recovery was performed as described in section 3.6.. 3.8. EXTRACTIVE FERMENTATION The EF was carried out using culture medium A and a polymer-salt ATPS, composed of polyethylene glycol average molecular weight 8,000 (PEG 8000) purchase from Sigma Aldrich (Lot# BCBS0176V) and sodium citrate (NaCit). To find the more suitable composition, different mixtures of PEG 8000 and NaCit were previously prepared by weighting appropriate amounts of each solid component into 2mL tubes. Then adequate amounts of culture medium A were added to obtain the required final systems compositions (PEG 8000-NaCit: 10-10, 10-8, 10-6, 9-9, 8-8, 7-7 % (w/w)) with 1 g total weight. The EF was performed in 250 ml cotton-plugged Erlenmeyer flasks containing a 9 % wt. PEG 8000 and 9 % wt. NaCit ATPS with 50 g total weight. Two strategies to inoculate this fermentation medium 25 with an initial spore density of 106 conidia/mL were investigated: the direct addition of the spore suspension to the medium or the addition of biomass from a 2-day pre-inoculum. Furthermore, other two initial spore densities (102 and 104 conidia/mL) were tested. Prior to use, all the material and media were autoclaved at 121 °C during 15 min. Figure 7 represent a scheme of the extractive fermentation process. Figure 7. Scheme of a typical extractive fermentation process. The fermentations were performed in an orbital shaker at 23 °C and 150 rpm for 12 days. Samples from the top phase were taken at 0, 2, 5, 7, 9 and 12 days in aseptic conditions and centrifuged at 4500 rpm during 5 min before reading the absorbance (section 3.10). 3.9. SUBMERGED FERMENTATION IN BIOREACTOR Pigment production by SmF was performed in a bioreactor Bioengineering RALF 3500 mL containing 2500 mL of culture medium A. A pre-inoculum was prepared with the same medium composition, initial density of 106 conidia/mL and growth for 48 h. The culture medium and the bioreactor were autoclaved at 121 °C during 30 min. The medium used for pre-inoculum was autoclaved at 121 °C during 15 min. The pre-inoculum was added to the bioreactor under aseptic conditions. The fermentation was performed at controlled temperature of 23 °C, agitation of 150 rpm and aeration rate of approximately 2 vvm (volume flow per unit of liquid volume per minute). Additionally, another fermentation trial was performed under the same experimental conditions but without mechanical agitation. 3.10. PIGMENT ANALYSIS The samples taken during fermentations were used to spectrophotometrically monitor the production of pigments. For all the conditions tested, the samples were centrifuged prior the absorbance 32 days) and samples corresponding to the final fermentation time (12 days) were analyzed by HPLC (Appendix B). For media A and F, lactose was completely consumed during the fermentation process. The media C, D and G presented a lactose concentration lower than 1 g/L at the final time. For medium B, the concentration of lactose was higher than 8 g/L. Through this analysis it was also possible to verify that the medium E had no lactose in its composition. However, it is known that CSL has other sugars in its composition which may contribute to the fungal growth105. This is in line with a previous work performed by our research group which concluded that lactose is the best carbon source for P. brevicompactum pigment production under SmF conditions60. Thus, the results suggest that lactose supplementation with an adequate nitrogen source is important to pigment production, since its absence (medium E) yielded in a lower pigment production. On the other hand, its total or almost total consumption led to a higher pigment production. Surprisingly it was observed a possible association with the timing of lactose initial consumption and color appearance in the fermentation broth, which led us to assume that the use of this carbon source by the fungus may be favoring or activating secondary metabolic pathways and consequently promoting pigment production. The work performed by Gmoser et al. 111 aiming the production of carotenoids by Neurospora sp. suggested that the change from glucose to mannose as carbon source could direct the metabolic pathway for to acetyl-CoA pool (the most important precursor for pigments production) rather than cell growth pathways. However, additional studies are needed to unequivocally conclude about this specific effect of the carbon source in pigments production by P. brevicompactum . In order to study if the biomass originated could influence the pigment production, the amount of biomass was gravimetrically determined (dry weight) (Table 4). 33 Table 4. Dry weight of biomass obtained for all the culture media studied under submerged fermentation conditions. Values are the mean ± SD (n=3). Biomass (g) A 0.445 ± 0,043 B 0.442 ± 0,017 C 0.547 ± 0,012 D 0.655 ± 0,099 E 0.080 ± 0,001 F 0.529 ± 0,007 G 0.641 ± 0,010 The results in Table 4 show that for media D and G the maximal cellular growth was achieved, which also correspond to the media where higher production of pigments was obtained. However, with medium A (also with the highest pigment production), the cellular growth was not so high. These results seem to be concordant with Méndez et al .4 who showed that does not exist a direct relationship between maximal growth and pigment production by P. purpurogenum . It is important to mention that more biomass was obtained in media with the maximal supplementation with nitrogen sources (media D and G), probably because the microorganism can easily assimilate the nutrients present in the supplements and use them to grow, since they are more available (higher concentrations). According to Lebeau et al. 112 an increase in the biomass content is attributed to the richness of nutrients in the medium and the pigment production is related to stress conditions, as a way of protection. Pigments mixtures obtained from the several fermentations media studied were qualitatively analyzed by Thin Layer Chromatography (TLC) using a silica plate. Pigments were separated using a (50:50) mixture of water:ethanol as eluent, which was shown in previous studies to be the best eluent for their separation60. The separation obtained by TLC under visible light and exposition to UV light at 254 and 366 nm is presented in Figure 9. 34 Figure 9. Silica gel TLC showing the pigments separation obtained for the extracellular culture medium from submerged fermentation: visible light (A) and exposed to UV light at 254 (B) and 366 nm (C), respectively. The results obtained by TLC suggested that different pigments mixtures were produced according with the fermentation medium used. The migration pattern for CW culture media supplemented with P-YE (C and D) was very similar. For CW culture media supplemented with a different nitrogen source, CSL (F and G), also a similar migration pattern was observed (but distinct from the C and D). This result suggests that different nitrogen supplementation can probably originate different pigments mixtures. 4.1.1. EFFECT OF THE FERMENTATION TIME IN THE ALTERNATIVE MEDIUM In previous studies it was determined that 12 days of fermentation was the suitable time for pigments production using the culture medium A60,61. In order to evaluate if the same fermentation time would be appropriated for media composed only by agroindustrial residues, a 36 days fermentation was performed using medium G. Medium G was chosen preferentially to medium F because it presented higher pigments production (Figure 8) and larger amounts of residues are used in its preparation, which may represent an environmental advantage. Two tests were performed: (i) a continuous 36 days fermentation and (ii) a 3x12 days fermentation with culture medium replacement by a fresh one (with the same initial composition) at the end of each 12 days cycle. The sum of the absorbances obtained for these two assays, with medium change (MC) and without medium change (M) is presented in Figure 10. The bar corresponding to MC includes the absorbance obtained after each 12 days cycle of fermentation. 35 Figure 10. Absorbances obtained for the 36 days submerged fermentations: medium change condition (MC) and condition without medium change (M). In medium change condition it was represented the absorbance measured after culture medium recovery in the 12 days of each cycle: first cycle (black); second cycle (gray); and third cycle (dark gray). Values are the mean ± SD (n=3). Statistical analysis was performed between each media recovery and with the total absorbance for MC by one-way ANOVA. ***p<0.001 Regarding test (ii) (MC bar in Figure 10) no significant differences were found between each of the 3 cycles of biomass reuse. Furthermore, the absorbance values obtained indicate that a fresh medium replacing led to a higher pigment production in comparison with the approach performed in test (i). It is known that secondary metabolites may be produced under stress conditions31,32. However, some authors referred that the secondary metabolites production in fungi depends on some factors, such as nutrient, concentration sources and their adequate ratio in the culture media40,113. The obtained results suggest that the presence of adequate carbon and nitrogen sources in the culture media lead to higher pigments production than in their absence. Thus, these results allowed to establish 12 days of fermentation as the minimum suitable fermentation time for pigment production using media composed by CW and CSL. This conclusion was supported by the lack of statistically differences between the production after the first 12 cycle (MC) and 36 days fermentations (M). However, the substitution of the medium allowed a higher total pigments recovery at the end of 36 days of fermentation. This can be explained by the fact that in MC condition biomass always has nutrients available, producing pigments whenever the fermentation medium is renewed. In contrast, in M condition the biomass probably consumes all the nutrients supplied in the first 12 days. These results 36 also support the assumption that the carbon and the nitrogen sources in the medium are essential to stimulate the metabolic pathways involved in pigments production. Since the best pigments production was observed following the fresh medium replacing and biomass reuse approach, it was decided to immobilize the biomass on an inert support, nylon sponge114, in order to facilitate its handling and transfer. Therefore, the two previous assays were repeated under submerged fermentation conditions with fungus immobilization in nylon sponge. However, it is important to mention that this type of fermentation (SmF with immobilization) will be discussed in detail in section 4.2.1, being presented here only the effect of the fermentation time on pigment production for direct comparison with the SmF with free P. brevicompactum . The sum of absorbances for the two conditions evaluated, medium change (MC) and without medium change (M), is present in Figure 11. The bar corresponding to MC includes the absorbance obtained for each fermentation cycle. Figure 11. Absorbance for medium change condition (MC) and for condition without medium change (M) with immobilization of biomass on inert support. In medium change condition was represented the absorbance from medium recovery (left) in first (black), second (gray) and third 12 days cycle (dark gray) and ethanolic extract obtained in the end of 36 days. In medium change condition was represented the absorbance from medium (left) and from ethanolic extract obtained from immobilized biomass. Values are the mean ± SD (n=3). Statistical analysis was performed between each media recovery and with the total absorbance for MC (letters) by one-way ANOVA. Bar graphs which have the same letter are significantly equal therefore p is not bellow 0.05. On contrary bar graph with different letters are significantly different from each other (p<0.05). Similarly the previous test, the absorbance values obtained indicate that a fresh medium replacement led to a higher pigment production in comparison with the approach performed without medium change. Also, for this type of fermentation, there are no statistical differences between the 37 production after the first 12 cycle (MC) and the 36 days fermentations (M) for medium. Thus, 12 days of fermentation seems to be the minimum suitable fermentation time. Interestingly, the absorbance of the ethanolic extracts was equal for MC and M and it was higher than the absorbance of the medium after 36 days of fermentation (when statistical test was performed with the sum of absorbances of each cycle in MC). Comparing the two tests performed, the SmF with immobilization of biomass (Figure 11) allows to obtain more extracellular pigments than submerged fermentation without immobilization (Figure 10) under MC conditions. Under M conditions the absorbance values for the mixtures of extracellular pigments were similar for both types of fermentation. 4.2. PIGMENTS PRODUCTION BY SUBMERGED FERMENTATION WITH IMMOBILIZATION 4.2.1. THE USE OF AN INERT SUPPORT The immobilization of filamentous fungi is a relatively simple procedure since this kind of microorganism adheres easily to the surfaces of several materials. Fungal immobilization can be advantageous since it facilitates the recovery and reuse of the cells and, consequently, the bioproducts115. In a study conducted by Domínguez-Espinosa and Webb116 the immobilization of Monascus on an inert support (foam) was tested and resulted in an enhanced production of red pigments compared with submerged fermentation using the free fungus. To investigate whether biomass immobilization influenced pigment production, all the media (AG) were tested under submerged fermentation conditions with immobilization of the fungus on an inert support (SmFi), namely nylon sponge. The amount of support used, 20 pieces, was chosen based on the amount of support that could be used and still be completely submerged in the total volume of culture medium. Since the extraction of pigments from biomass was necessary, ethanol was used due to its wellrecognized classification as GRAS solvent105. Moreover, ethanol can be easily recovered and recycled using distillations. This solvent was used in all the extraction of pigments from biomass procedures performed in the present work. The ethanolic extraction of pigments was carried out in these fermentation conditions because the biomass obtained at the end of the fermentation presented a strong coloration, thus indicating a considerable amount of (intracellular) pigments. 38 The results of pigment production were expressed in relative absorbance (%) for the pigments mixtures obtained from extracellular medium (M) and from ethanolic extraction (EE) from biomass. All the alternative media were compared with the medium A, used as reference and corresponding to 100 % absorbance (Figure 12). Figure 12. Relative absorbance (%) obtained for each culture media (A-G) under submerged fermentation conditions with immobilization on an inert support, extracellular medium (M) and ethanolic extracts (EE) from biomass. The composition of media (g/L) was: A) Peptone (8), yeast extract (8), KH2PO4 (2), Na2HPO4.12H2O (8), MgSO4.6H2O (0.25) and lactose (20); B) CW (34.6); C) CW (34.6), yeast extract (0.5) and peptone (0.5); D) CW (34.6), yeast extract (4) and peptone (4); E) CSL (12.55); F) CW (34.6) and CSL (1) and G) CW (34.6) and CSL (8). Values are the mean ± SD (n=3). Statistical analysis was performed for medium samples, ethanolic samples and with each other separately by two-way ANOVA. **p<0.01, ***p<0.001, ****p<0.0001, ###p<0.001 and ####p<0.0001 Analyzing the results corresponding to M samples it can be observed that none of the alternative media tested allowed to reach the same relative absorbance obtained with the reference medium A. On the other hand, examining the results of the EE it is verified that the media C and G do not present statistically significant differences comparing with medium A, which means that with these alternative media the same amount of pigments can be obtained by ethanolic extraction from the immobilized biomass. Comparing the relative absorbances measured for the same culture medium (M and EE) it is possible to establish that there is only a significant difference in media C, E and G. In the media C and G, there is a higher content of pigments in the ethanolic extract (EE) than in the fermentation broth (M). In contrast, in medium E higher amount of pigments was obtained in the fermentation broth (M). 39 The differences observed between M and EE for the same culture medium might indicate that the amount of pigment excreted may vary with the fungal immobilization. It was visually verified that the immobilization did not occur evenly in all media. Plus, in some cases, the biomass immobilization suffered variations among the replicates (all biomass immobilized and/or some suspended biomass). In addition, it was found that in media A, D and E pigment production started on the third day, while in the others it happened on the fifth day of fermentation. Based on the absorbance measurements, it was also observed that in all the media a higher amount of yellow pigments was produced than orange or red (Appendix C). Similarly, to SmF, samples from culture medium corresponding to the initial time (0 days), the time at which color appeared in the culture broth (3/5 days) and the final fermentation time (12 days) were analyzed by HPLC to assess the consumption of lactose over the fermentation (Appendix D). The results showed that the medium which allowed the best pigment production (medium A) presented less lactose concentration in the final fermentation time (less than 5 g/L). All the other media presented a lactose concentration in the final time over 5 g/L. As the nylon sponge is a considerable porous material, the growth of biomass occurs both inside of the support and on its surface, forming a dense coating. This fact can probably explain why lactose was not completely consumed. The biomass inside the support did not have the same access to the medium nutrients as the free biomass or the biomass on the support surface. Pigments mixtures corresponding to the fermentation media (M) and to the ethanolic extracts (EE) obtained from the several fermentations were qualitatively analyzed by TLC using a silica plate. The produced pigments mixtures were separated using water:ethanol (50:50) as eluent, as previously described. The separation obtained by TLC under visible light and exposition to UV light at 254 and 366 nm is presented in Figure 13. 40 Figure 13. Silica gel TLC showing the pigments separation obtained for the extracellular culture medium (M) and ethanolic extracts (EE) from submerged fermentation with immobilization on inert support: visible light (A) and exposed to UV light at 254 (B) and 366 nm (C), respectively. The results obtained by TLC suggested that different pigments mixtures were produced according with the fermentation medium used. Also, it was possible to verify that the migration pattern of M samples was different from that of EE. The migration pattern in M samples suggests that the migration order of pigments was a blue spot (visible at 366 nm) followed by yellow-orange-red. In EE, initially a blue spot (visible at 366 nm) can be detected, which is followed by yellow-orange-red spots and finally a new blue/purple spot. It is important to mention that the yellow-orange-red migration order was observed for all the conditions. 4.2.2. IMMOBILIZATION IN A NATURAL SUPPORT Some studies showed the potential of corn cob hydrolysate to be used both as a substrate in SmF as well as a support/substrate in SSF81,117. In this way, it was decided to test all the culture media defined before under submerged fermentation using the corn cob as a natural immobilization support (SmFn). Similarly, to SmFi, the amount of support used in these experiments was 20 pieces of corn cob. The results were expressed in relative absorbance (%) for culture medium samples (M) and ethanolic extract samples (EE), always comparing the alternative media with the reference medium A, corresponding to 100 % of absorbance (Figure 14). 41 Figure 14. Relative absorbance (%) obtained for each culture media (A-G) under submerged fermentation conditions with immobilization on natural support, extracellular medium (M) and ethanolic extracts (EE) from biomass. The composition of media (g/L) was: A) Peptone (8), yeast extract (8), KH2PO4 (2), Na2HPO4.12H2O (8), MgSO4.6H2O (0.25) and lactose (20); B) CW (34.6); C) CW (34.6), yeast extract (0.5) and peptone (0.5); D) CW (34.6), yeast extract (4) and peptone (4); E) CSL (12.55); F) CW (34.6) and CSL (1) and G) CW (34.6) and CSL (8). Values are the mean ± SD (n=3). Statistical analysis was performed for medium samples and ethanolic samples separately by two-way ANOVA. *p<0.05, ****p<0.0001, ##p<0.01, ###p<0.001, ####p<0.0001 Analyzing the results corresponding to M it can be seen that all the alternative media promoted the production of the same amount of pigments (media C, E, F and G) or even more (medium D) than the medium A, exception for medium B. Regarding EE, the media B, D and F yielded the same amount of pigments than the medium A; and media C and G allowed to produce more pigments than medium A. Medium E is the only one that did not reach the same pigments production obtained with the reference medium A. Comparing M and EE from the same culture medium, significant statistical differences were found between media B, D, E and G. For media B and G more pigments were obtained in EE conditions. In contrast, for media D and E more pigments were obtained in M conditions. A remarkable result was observed in M samples from medium D where the relative absorbance exceeds 136 % the absorbance obtained with the reference medium (A), indicating that this medium is an effective alternative for the synthetic one. The use of corn cob as immobilization support showed more potential than the use of nylon sponge (Figure 13). As the corn cob is less porous than the nylon sponge, the fungus probably adhered mostly on the surface of this support. In this way, all or almost all the biomass was in contact with the 48 Figure 21. Sum of the absorbances (400, 470 and 500 nm) obtained for the best conditions found for pigments production using the respective culture media tested under: submerged fermentation (SmF); submerged fermentation with immobilization on an inert support (SmFi) and on a natural support (SmFn), which include the extracellular pigments present in the fermentation broth (M) and the intracellular pigments extracted from biomass using ethanol (EE); solid state fermentation using an inert support (SSFi); and solid state fermentation using a natural support (SSFn). Values are the mean ± SD (n=3). Statistical analysis was performed by one-way ANOVA. *p<0.05, ****p<0.0001 The data presented in Figure 21 shows that from all the selected conditions the best ones belong to the SmFn, namely with the use of the medium D when pigments amount in the fermentation broth is considered and the media C and G when ethanolic extracts are evaluated. In order to verify whether there were significant differences between these three media, a oneway ANOVA (p <0.05) was performed. The results obtained through this statistical analysis showed that there are differences between media D and C, but not between media D and G. Thus, based on all the studies performed, media D and G can be indicated as the best conditions for pigments production using P. brevicompactum under SmFn. These are considered a promising result because the use of agroresidues as alternative media allows the reuse of wastes generated from the agroindustry sector, for the production of valuable products. In this way, the pigments production is more environmentally friendly and more interesting economically. In a previous work developed by our research group, a fraction containing a red pigment was chromatographically separated from an ethanolic extract of the mycelium grown in agar plate (P). After 49 some structural characterization studies, it was found that this fraction could be an antheraxanthin-type pigment. Thus, the best condition obtained in this work and a P sample were qualitatively analyzed by TLC using a silica plate and the results are presented in Figures 22. Medium D (SmFn) and ethanolic extracts C and G (SmFn) correspond to 1, 2 and 3, respectively. Figure 22. Silica gel TLC showing the pigments separation obtained for the best fermentation conditions (1, 2 and 3) and for P sample: visible light (A) and exposed to UV light at 254 (B) and 366 nm (C), respectively. The samples obtained in the present work show a more complex composition, since they correspond to a mixture of pigments, as already mentioned. Although this TLC analysis showed not to be conclusive, the pigments samples obtained in this work seem to present some similarities, mainly at visible light, with the P sample. Further studies are needed to obtain more isolated fractions in order to determine their chemical structures and then conclude about the presence of antheraxanthin-type pigments in the mixtures produced in this work. 4.5. PIGMENTS PRODUCTION BY EXTRACTIVE FERMENTATION In this study a polymer-salt ATPS was used to perform extractive fermentation. The biodegradable and non-toxic NaCit was the selected salt mainly due to its high biocompatibility and considering its lower environmental impact and toxicity issues120. Regarding the polymer choice, PEG was selected once it is the most well-studied and used polymer engaged in polymer-salt ATPS formation. Besides, this polymer is known to be non-toxic, being frequently used in several bioapplications. Before performing the extractive fermentation, the most suitable composition for PEG 8000/NaCit ATPS was determined. It was verified that a system composed by 9% wt. PEG 8000 and 9% wt. NaCit was the most appropriate due to the stability of both phases and the lower amounts of PEG and NaCit required as well. 50 The best way to inoculate the medium was also tested and the direct method was chosen (instead of using 2 days pre-inoculum) as it allowed a more controlled biomass growth that grant the collection of samples. However, even by adjusting the amount of inoculum (which needs to be optimized in the future) sampling from the bottom phase was impossible since biomass steered to this phase of the biphasic system being completely spread over it. The sum of the absorbances in the top phase at the end of the fermentation for each initial spore density is presented in Table 5. Table 5. Sum of absorbances (400, 470 and 500 nm) for top phase sample in the end of the fermentation for each initial spore density (conidia/mL) tested. The initial absorbance of medium was subtracted to final absorbance. Values are the mean ± SD (n=3). Statistical analysis was performed by one-way ANOVA. Values which have the same letter are significantly equal therefore P is not bellow 0.05. On contrary bar graph with different letters are significantly different from each other (p<0.05). Initial spore density (conidia/mL) Absorbance (Top phase) 106 0.719 ± 0.043a 104 1.284 ± 0.105b 102 1.273 ± 0.130b The results indicate that PEG 8000-NaCit ATPS can be a suitable biphasic system for extractive fermentation that allows the growth of P. brevicompactum and the production of pigments. The reduction of the initial spore density from 106 to 104 conidia/mL resulted in a higher total absorbance (meaning that more pigments were produced). The reduction of the initial spore density from 104 to 102 does not present statistical differences regarding pigments production. Although the sampling of bottom phase was not possible, it was possible to verify that the top phase had color, being the pigments concentrated in that equilibrium phase. The use of EF for pigments production requires an additional optimization but the present results show that this technique is a promising alternative to reduce downstream processes for pigments extraction. However, this result is not conclusive, since the clogging of the bottom phase with biomass was verified. Therefore, this approach should be optimized to avoid clogging of bottom phase. Increasing the volume of the culture medium (i.e. the total volume of the biphasic system) and using a thinner and taller flask may help to overcome the issues faced. In this way, biomass can have room to accumulate at the interface of the ATPS (as frequently reported in the literature) instead of occupying all the bottom phase. 51 4.6. PIGMENTS PRODUCTION BY SUBMERGED FERMENTATION IN BIOREACTOR It is well-known that scaling up a bioprocess aiming the production of a desired product is easier when this one is performed under submerged fermentation70. In order to increase the amount of fungal pigments, a scale up of their production by P. brevicompactum under submerged fermentation was carried out in a bioreactor using the synthetic culture medium A. Two experiments were carried out, one using a mechanical agitation of 150 rpm and another one without mechanical agitation (only aeration). It was verified that when the fermentation was performed with mechanical agitation the shear stress caused by the bioreactor blades compromise the biomass integrity, increasing the viscosity of the culture broth. The fermentation with mechanical agitation was carried out for 12 days, the same time used for the production tests in Erlenmeyer flasks, and no pigment production was observed. This result suggested that the morphology of the fungus, the viscosity of the broth after some cell disruption and/or the design/configuration of the bioreactor negatively influenced the pigment production. However, even with cell disruption the growth of fungus was verified. These observations seem to be corroborated by the work carried out by Lu et al. 121 who demonstrated that the viscosity triggered by shear stress affects negatively the production of desirable products. In order to avoid shear stress and the disruption of the biomass a fermentation without mechanical agitation was carried out instead. After 5 days it was observed pigment production. However, the increase of broth viscosity was also verified. At the end of the fermentation, the absorbance was measured and the sum of absorbances was considered. The total absorbance obtained was 0.787 ± 0.052. In this test it was possible to verify that the type of agitation influenced the fungus morphology and that the type of morphology can be related with pigments production. Although some pigment production was observed in the bioreactor fermentation, the absorbance value is around 2.5 times lower than that obtained under submerged fermentation in Erlenmeyer flasks (Appendix A). Thus, to perform an effective scale up for pigments production by P. brevicompactum more studies involving the optimization of some operational parameters such as the amount of the inoculum, temperature, fermentation time, agitation and aeration is needed. 52 5. CONCLUSIONS AND FUTURE PERSPECTIVES Fungi, particularly filamentous fungi, are known for their ability to produce a wide range of pigments. It was demonstrated in this work that P. brevicompactum is a promising pigments producer using alternative media composed of agroindustrial residues. For SmF, no direct relationship between maximal growth and pigment production was established. It was demonstrated that 12 days of fermentation is the minimal suitable fermentation time for pigments production. An increase in pigments production was observed after the replacement of the culture medium after 12 days of fermentation. Under these conditions the immobilization of the biomass (3 cycles of 12 days of fermentation) allowed to obtain more pigments than with free biomass. It was also shown that the immobilization of the fungus on a natural support (corn cob) allowed the production of a larger amount of pigments than obtained with an inert support (nylon sponge). In the future it would be interesting to optimize the SSFn conditions using only corn cob and water, since pigments production in this condition was observed. The most suitable moisture contents were shown to be 85 and 65 % for SSFi and SSFn, respectively. The highest pigments productions were achieved with medium D (CW supplemented with 4 g/L P-YE, extracellular pigments mixture) and medium G (CW supplemented with 4 g/L CSL, ethanolic extract), both under SmFn conditions. It was also concluded that for all the types of fermentation studied, a pigment mixture was produced possibly containing antheraxanthin-type pigments. Under EF conditions, promising results were obtained since the pigments production and their concentration in top phase was noticed. Therefore, process integration using a PEG 8000/NaCit ATPS can be a suitable alterative to produce and separate pigments from the biomass and the remaining contaminants of the fermentation broth. However, the optimization of some parameters such as fermentation volume, fermentation apparatus, fermentation time and type of inoculum are necessary. Optimization of this process may allow to produce interesting amounts of pigments, reducing downstream processes and making pigment production by P. brevicompactum more environmentally friendly and more industrially attractive. 53 Under SmF conditions in bioreactor, the amount of pigments produced was 2.5 lower than using the 250 mL Erlenmeyer. To scale up the pigments production by P. brevicompactum , additional studies involving the optimization of several operational parameters such as the amount of the inoculum, temperature, fermentation time, agitation and aeration are needed. Additionally, it would be interesting to investigate the pigments production using an air-lift bioreactor, where no mechanical agitation is present and thus the biomass morphology (which is probably related with pigments production) would not be negatively affected by the harsh action of the impellers. Overall, this work demonstrated alternative low-cost culture media composed of agroindustrial residues as an efficient substitute of the synthetic ones to produce pigments by P. brevicompactum . These results also address the potential of profuse agroindustrial residues to be used as suitable resources of nutrients for fungal growth and metabolites biosynthesis. 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C., & Teixeira, J. A. Production, extraction and characterization of natural fungal pigments from Penicillium sp. in 12th European Symposium on 64 Appendix B. Lactose concentration determined by HPLC for all the culture media at initial time (It), the time where pigment was visible in the medium (Pt) and final time (Ft) under submerged fermentation conditions. Values are the mean ± SD (n=3). [Lactose] (g/L) It Pt Ft A 19.203 ± 1.382 17.046 ± 3.116 0.000 ± 0.000 B 23.068 ± 1.923 17.294 ± 3,737 8.961 ± 1,049 C 22.815 ± 0.263 17.084 ± 4,943 0.508 ± 0,443 D 22.648 ± 0.730 16.733 ± 0.496 0.966 ± 0.043 E 0.000 ± 0.000 0.000 ± 0.000 0.000 ± 0.000 F 24,091 ± 0,638 19.672 ± 0,344 0.000 ± 0.000 G 25,528 ± 0,564 11.398 ± 0,434 0,184 ± 0,730 2. SUBMERGED FERMENTATION WITH IMMOBILIZATION ON INERT SUPPORT Appendix C contain all the results for absorbances and the sum of the absorbances obtained through the 12 days of fermentation at the three wavelengths measured (400, 470 and 500 nm) under SmFi conditions. 65 Appendix C. Absorbance and the sum of absorbances versus fermentation time for each culture media tested under submerged fermentation conditions with fungus immobilization on inert support. Values are the mean ± SD (n=3). Appendix D shows the lactose concentration determined by HPLC for all the culture media at initial time (It), the time where pigment was visible in the medium (Pt) and final time (Ft) under SmFi conditions. Appendix D. Lactose concentration determined by HPLC for all the culture media in initial time (It), the time where pigment was visible in the medium (Pt) and final time (Ft) under submerged fermentation conditions with fungus immobilization on inert support. Values are the mean ± SD (n=3). [Lactose] (g/L) It Pt Ft A 19.203 ± 1.382 17.789 ± 0.395 4.259 ± 2.901 B 23.068 ± 1.923 18.595 ± 1.033 9.834 ± 1.359 C 22.815 ± 0.263 18.359 ± 0.252 9.399 ± 1.358 D 22.648 ± 0.730 20.157 ± 1.391 7.997 ± 3.257 E 0.000 ± 0.000 0.000 ± 0.000 0.000 ± 0.000 F 24.091 ± 0.638 22.371 ± 0.329 10.685 ± 1.628 G 25.528 ± 0.564 23.978 ± 0.914 12.727 1.394 66 3. SUBMERGED FERMENTATION WITH IMMOBILIZATION ON NATURAL SUPPORT Appendix E contain all the results for absorbances and the sum of the absorbances obtained through the 12 days of fermentation at the three wavelengths measured (400, 470 and 500 nm) under SmFn conditions. Appendix E. Absorbance and the sum of absorbances versus fermentation time for each culture media tested under submerged fermentation conditions with fungus immobilization on natural support. Values are the mean ± SD (n=3). Appendix F shows the lactose concentration determined by HPLC for all the culture media at initial time (It), the time where pigment was visible in the medium (Pt) and final time (Ft) under SmFn conditions. 67 Appendix F. Lactose concentration determined by HPLC for all the culture media in initial time (It), the time where pigment was visible in the medium (Pt) and final time (Ft) under submerged fermentation conditions with fungus immobilization on natural support. Values are the mean ± SD (n=3). [Lactose] (g/L) It Pt Ft A 19.203 ± 1.382 17.565 ± 0.250 0.000 ± 0.000 B 23.068 ± 1.923 19.804 ± 0.885 5.644 ± 0.795 C 22.815 ± 0.263 18.359 ± 0.252 0.760 ± 0.219 D 22.648 ± 0.730 14.319 ± 1.820 0.000 ± 0.000 E 0.000 ± 0.000 0.000 ± 0.000 0.000 ± 0.000 F 24.091 ± 0.638 17.662 ± 1.166 3.739 ± 0.772 G 25.528 ± 0.564 17.698 ± 1.233 1.244 ± 0.241