Fungal biosurfactants, from nature to biotechnological product: bioprospection, production and potential applications

Biosurfactants are in demand by the global market as natural commodities that can be added to commercial products or use in environmental applications. These biomolecules reduce the surface/interfacial tension between fluid phases and exhibit superior stability to chemical surfactants under different physico-chemical conditions. Biotechnological production of biosurfactants is still emerging. Fungi are promising producers of these molecules with unique chemical structures, such as sophorolipids, mannosylerythritol lipids, cellobiose lipids, xylolipids, polyol lipids and hydrophobins. In this review, we aimed to contextualize concepts related to fungal biosurfactant production and its application in industry and the environment. Concepts related to the thermodynamic and physico-chemical properties of biosurfactants are presented, which allows detailed analysis of their structural and application. Promising niches for isolating biosurfactant-producing fungi are presented, as well as screening methodologies are discussed. Finally, strategies related to process parameters and variables, simultaneous production, process optimization through statistical and genetic tools, downstream processing and some aspects of commercial products formulations are presented

Potassium biphthalate buffer for pH control to optimize glycosyl hydrolase production in shake flasks using filamentous fungi

The optimization of culture medium with statistical methods is widely used in filamentous fungi glycosyl hydrolase production. The implementation of such methodology in bioreactors is very expensive as it requires several pH-controlled systems operating in parallel in order to test a large number of culture media components. The objective of this study was to evaluate potassium biphthalate buffer for pH control, which allows the optimization studies to be performed in shake flasks.The results have shown that buffering the culture medium with 0.1 M potassium biphthalate allowed pH control, resulting in a decrease of the standard deviation of triplicates for pH and activities of glycosyl hydrolase measurements. The use of this buffer allowed shake flask culture media optimization of enzyme production by Trichoderma harzianum, increasing the cellulase activity by more than 2 times compared to standard unbuffered culture medium. The same buffer can be used for culture media optimization of other fungi, such as Penicillium echinulatum342439450CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESPsem informaçã

Cellulase and Xylanase Enzymes from Trichoderma reesei RUT-C30 Using Pretreated Sugarcane Bagasse in a Biorefinery Environment

Abstract Biofuels and other bioproducts derived from lignocellulosic feedstocks represent environmentally sustainable pathways that have the potential to compete favorably with conventional petrochemical counterparts. Realizing their economic viability necessitates the development of highly efficient and cost-effective enzymatic preparations for the hydrolysis of lignocellulosic substrates. Within the context of this investigation, it was harnessed the capabilities of T. reesei RUT-C30, a well-recognized hypercellulolytic fungus. Employing delignified pretreated sugarcane bagasse as the main substrate, and amelioration of the culture medium composition enhanced the production of cellulase and xylanase enzymes. In a controlled bench scale bioreactor system, it was achieved noteworthy enzymatic activities, peaking at 3.8 FPU/mL for cellulase and 231 U/mL for xylanase and 26 FPU/L h productivity. Calculated hydrolysis conversion of delignified steam-explosion sugarcane bagasse (DSB) at 3% DSB concentration without the addition of an external source of β-glucosidase was 51.8% for the enzyme preparation DSB(C) and only n this same condition, maximum conversion for commercial enzymes (T reesei, Sigma) was only 34 %. This observation accentuates the competence of the enzymes derived from T. reesei RUT-C30 in efficiently degrading pretreated sugarcane bagasse. While it was demonstrated the potential of T. reesei RUT-C30 for enzyme production in the context of second-generation ethanol generation, our data portrays the enzyme's cost contribution to ethanol production of US$ 0.115 per liter of ethanol produced

Inoculum Influenced Glycohydrolases Production in the endophytic fungus Annulohypoxylon stygium DR47

Abstract Fungal enzymes have potential applicability in plant biomass deconstruction, such as in sugarcane bagasse hydrolysis for biorefinery application. Due to biomass complexity it is necessary to use a set of enzymes in addition to cellulolytic enzymes extracts to hydrolyze the hemicellulose and cellulose efficiently, such as β-glucosidase and pectinases. In this way, this work aimed to study the influence of inoculum in pectinase and β-glucosidase production of Annulohypoxylon stygium DR47 and evaluated culture media components in enzymatic production. Initially, mycelia fragmentation and the carbon source were tested in enzymatic production. The best production occurred with a pre-inoculum, incubated for 48 h, with blender fragmented mycelia cultivated in fructose (20 g∙L-1), as carbon source. Subsequently, tests containing agro-industrial residues (20 g∙L-1) were done to determine the best source for β-glucosidase and pectinases production in the enzyme production medium. Citrus bagasse (CB) was the substrate that induces higher titration of both enzymes (2.36 Upectinase∙mL-1; 1.82 Uβ-glucosidase∙mL-1). In this way, Plackett & Burman experimental design was performed with CB (20 g∙L) as the only carbon source to determine the influence of media components in enzyme production. The pectinase production was negatively affected by ZnSO4 and positively affect by urea, (NH4)2SO4, and MnSO4. Besides significant differences in β-glucosidase production were observed, with a positive effect on urea and peptone addition. In this way, further studies considering C/N ratios and consequently different nitrogen concentrations should be done to better understand the fungus physiology

Cultivation of Podospora anserina on soybean hulls results in an efficient enzyme cocktail for plant biomass hydrolysis

The coprophilic ascomycete fungus Podospora anserina was cultivated on three different plant biomasses, i.e. cotton seed hulls (CSH), soybean hulls (SBH) and acid-pretreated wheat straw (WS) for four days, and the potential of the produced enzyme mixtures was compared in the enzymatic saccharification of the corresponding lignocellulose feedstocks. The enzyme cocktail P. anserina produced after three days of growth on SBH showed superior capacity to release reducing sugars from all tested plant biomass feedstocks compared to the enzyme mixtures from CSH and WS cultures. Detailed proteomics analysis of the culture supernatants revealed that SBH contained the most diverse set of enzymes targeted on plant cell wall polymers and was particularly abundant in xylan, mannan and pectin acting enzymes. The importance of lytic polysaccharide monooxygenases (LPMOs) in plant biomass deconstruction was supported by identification of 20 out of 33 AA9 LPMOs in the SBH cultures. The results highlight the suitability of P. anserina as a source of plant cell wall degrading enzymes for biotechnological applications and the importance of selecting the most optimal substrate for the production of enzyme mixtures. (C) 2017 Elsevier B.V. All rights reserved.Peer reviewe

POTENCIAL ENZIMÁTICO DOS FUNGOS ENDOFÍTICOS NA PRODUÇÃO DE BIOCOMBUSTÍVEIS

The growing demand for renewable energy has stimulated the production of biomass-derived biofuels, with bioethanol and biodiesel forming eco-sustainable energy matrixes. Although the composition is highly origin-dependent, when subjected to appropriate pretreatment, biomass can provide a chemical basis for enzymes production. Endophytic fungi are commonly applied to biomasses as biological pre-treatments. Their ability to colonize environments undergoing constant metabolic interaction makes them favorable candidates for the production of extracellular enzymes with potential application in biofuels production. This review contextualizes biomass sources and provides a bibliographic survey of studies of endophytic fungal strains and molecular and bioprocess techniques for enzyme screening and production. The current study summarizes current knowledge of digestion of lignocellulosic biomass with endophytic fungi-derived enzymes and their capacity for catalyzing the esterification reaction, for ethanol and biodiesel production, respectively.A crescente demanda por energias renováveis promovem a produção de biocombustíveis por meio da utilização de biomassas, o bioetanol e biodiesel são matrizes energéticas eco-sustentáveis. Embora sua composição seja variada de acordo com a sua origem, as biomassas quando submetidas a pré-tratamentos adequados são blocos químicos para produção de enzimas. A capacidade dos fungos endofíticos colonizarem ambientes suscetíveis a constantes interações metabólicas os tornam candidatos favoráveis na produção de enzimas extracelulares com potencial aplicação na produção de biocombustíveis, uma vez que são aplicados como pré-tratamento biológico de biomassas. Neste contexto, esta revisão contextualiza as fontes de biomassas e recolhe dados sobre estirpes fúngicas endofíticas e técnicas moleculares e de bioprocessos para o rastreio e produção de enzimas. Nós objetivamos evidenciar o atual cenário dos fungos endofíticos e suas enzimas para desconstrução da biomassa lignocelulósica e para catalise da reação de esterificação, bem como as etapas do processo produtivo de etanol e biodiesel, respectivamente

Hemicelulases e proteínas acessórias de fungos filamentosos e de actinomicetos para desconstrução de biomassa lignocelulósica.

Endophytic microorganisms were screened for hemicellulases production using plate assays and liquid cultivations. Two strains were selected and used in further studies. Aspergillus niger DR02 strain and Annulohypoxylon stigyum DR47. In A.niger fed-batch submerged cultivation approaches were developed using liquor from hydrothermal sugar cane pretreatment, and maximum xylanase activities obtained were 458.1 U/mL for constant fed-batch mode. For A. stygium DR47 media optimization and bioreactor cultivation using citrus bagasse and soybean bran were explored and revealed a maximum production of 6.26 U/mL of pectinase and 10.13 U/mL of β-glucosidase. Improved hemicellulase production was also done by genetic modifications at carbon catabolic repression levels in A. niger. Mass spectrometric studies were done in enzymatic extracts produced and contributed to understand the enzymatic supplementation of Celluclast 1.5L.In addition the extracts produced were tested for an enzyme formulation and that was able to increase sugar cane bagasse hydrolysis.Microrganismos endofíticos foram selecionados para a produção de hemicelulases através de testes em placa e de cultivo líquido. Duas linhagens foram selecionadas para posteriores cultivos, Aspergillus niger DR02 e Annulohypoxylon stigyum DR47. Para o fungo A.niger batelada alimentada em cultivo submerso com licor do tratamento hidrotérmico do bagaço de cana de açúcar permitiram obtenção de máxima atividade de xilanase de 458,1 U/mL. Para a linhagem A. stygium DR47 otimização do cultivo utilizando bagaço cítrico e farelo de soja foram explorados e revelaram produção de 6,26 U/mL de pectinase e 10,13 U/mL de β-glucosidase. Melhoramento da produção de hemicelulase foi realizado com modificações genéticas ao nível de repressão catabólica de carbono em A. niger. Estudos de espectrometria de massas foram realizados nos extratos enzimáticos produzidos e contribuíram para entender a suplementação da Celluclast 1.5L. Além disso, os extratos produzidos foram testados para uma formulação que foi capaz de aumentar a hidrólise do bagaço de cana de açúcar