Production of natural colorants by liquid fermentation with Chlorociboria aeruginascens and Laetiporus sulphureus and prospective applications.

The replacement of potentially hazardous synthetic dyes with natural dyes and pigments are of great interest for a sustainable economy. In order to obtain cost-efficient, environmentally friendly and competitive products, improvements in the cultivation and extraction of pigment-producing organisms and in dyeing processes are necessary. In our study, we were able to scale up the production of xylindein by Chlorociboria aeruginascens from 3 to 70 L bioreactor cultivations. We have identified important bioprocess parameters like low shear stress (150 rpm, tip speed <0.5 m/s) for optimal pigment yield (4.8 mg/L/d). Additionally, we have demonstrated the potential of laetiporic acid production by Laetiporus sulphureus in various cultivation systems and media, achieving dried biomass concentrations of almost 10 g/L with a 7 L bioreactor cultivation after 17 days. Extractions performed at 70°C and 15 min incubation time showed optimal results. To the best of our knowledge, we have described for the first time the use of this pigment in silk dyeing, which results in a brilliant hue that cannot easily be produced by other natural pigments.

Influence of Environmental Growth Factors on the Biomass and Pigment Production of Chlorociboria aeruginascens.

The soft rot fungus Chlorociboria aeruginascens produces a blue-green pigment xylindein, which is of considerable interest for various applications such as in the veneer industry or in organic semiconductors. To understand the fungal growth as well as pigment production of C. aeruginascens, several studies were performed, the results of which are presented here. These studies investigated various growth conditions such as temperature, pH value, oxygen level and light intensity. It was observed that the formation of xylindein by C. aeruginascens decoupled from growth. In the primary metabolismus, the uncolored biomass is formed. Pigment production took place within the secondary metabolism, while biomass growth as well as pigment production depended on various growth conditions. It was also found that certain conditions encourage the switch in metabolism, leading to pigment production.

Influence of the Nutrients on the Biomass and Pigment Production of Chlorociboria aeruginascens.

The blue-green pigment xylindein, produced by the soft rot fungus Chlorociboria aeruginascens, is of considerable interest for various applications such as the veneer industry or organic semiconductors. The studies presented were performed in order to understand the fungal growth as well as the pigment production of C. aeruginascens. Therefore, various nutrient compositions were investigated. As a result, observations of the formation of xylindein through C. aeruginascens decoupling from growth were made. In the primary metabolism the uncolored biomass is formed. Various carbohydrates were determined as nutrients for the fungus and as a nitrogen source it was observed that the fungus prefers the complex organic nitrogen source, that being yeast extract. Furthermore, it was discovered that the ratio between carbohydrate and nitrogen sources encourages the switch of the metabolism and therewith the production of the blue-green pigment xylindein.

It Is the Mix that Matters: Substrate-Specific Enzyme Production from Filamentous Fungi and Bacteria Through Solid-State Fermentation.

Fungi have a diverse spectrum of extracellular enzymes. In nature, extracellular enzymes primarily serve to procure nutrients for the survival and growth of the fungi. Complex polymers such as lignocellulose and starch as well as proteins and fats are broken down into their basic building blocks by extracellular enzymes such as amylases, proteases, lipases, xylanases, laccases, and many more.The abilities of these enzymes are made use of in diverse areas of industry, including food technology, textiles, and pharmaceuticals, and they have become indispensable for today's technology. Enzyme production is usually carried out using submerged fermentation (SmF). However, as part of the search for more sustainable uses of raw materials, solid-state fermentation (SSF) has become the focus of research.The rate of enzyme formation depends on different factors, for example, microorganism, temperature, or oxygen supply. However, one of the most important factors in enzyme production is the choice of substrate, which varies depending on the desired target enzyme. Substrates with proven effectiveness include wheat bran and straw, but unusual agricultural residues such as forage cactus pears and orange peels have surprisingly positive effects on enzyme formation as well.This review gives an overview of various technically relevant enzymes produced by filamentous fungi and suitable substrates for the production of the enzymes by SSF. Graphical Abstract.

"Fungal elicitors combined with a sucrose feed significantly enhance triterpene production of a Salvia fruticosa cell suspension".

Oleanolic (OA) and ursolic acid (UA) are plant secondary metabolites with diverse pharmacological properties. To reach reasonable productivities with plant cell suspension cultures, elicitation is a widely used strategy. Within the presented work, the effects of different elicitors on growth and production of OA and UA in a Salvia fruticosa cell suspension culture were examined. Beside commonly used elicitors like jasmonic acid (JA) and yeast extract, the influence of medium filtrates of the endophytic fungi Aspergillus niger and Trichoderma virens was investigated. The best eliciting effects were achieved with JA and fungal medium filtrates. Both increased the triterpene content by approximately 70 %. Since JA showed significant growth inhibition, the volumetric triterpene yield did not increase. But, adding fungal filtrates increased the volumetric triterpene yield by approximately 70 % to 32.6 mgOA l(-1) and 65.9 mgUA l(-1) for T. virens compared to the control with 19.4 mgOA l(-1) and 33.3 mgUA l(-1). An elicitation strategy combining fungal medium filtrate of T. virens with sucrose feeding significantly enhanced cell dry weight concentration to 22.2 g l(-1) as well as triterpene content by approximately 140 %. In total, this led to an approximately 500 % increase of volumetric triterpene yield referring to the control with final values of 112.9 mgOA l(-1) and 210.4 mgUA l(-1). Despite the doubled cultivation duration, productivities of 6.7 mgOA l(-1) day(-1) and 12.4 mgUA l(-1) day(-1) were reached. These results demonstrate methods by which increased productivities of triterpenes can be achieved to attain yields competing with intact plants.

Better One-Eyed than Blind--Challenges and Opportunities of Biomass Measurement During Solid-State Fermentation of Basidiomycetes.

Filamentous fungi, especially basidiomycetes, produce a wide range of metabolites, many of which have potential biotechnological and industrial applications. Solid-state fermentation (SSF) is very suitable for the cultivation of basidiomycetes since it mimics the natural habitat of these fungi. Some of the major advantages of SSF are the robustness of the process, the use of low-cost residual materials as substrates, and the reduced usage of water. However, monitoring key variables is difficult, which makes process control a challenge. Specifically, it is very difficult to determine the biomass during SSF process involving basidiomycetes. This is problematic, as the biomass is normally a key variable in mass and energy balance equations. Further, the success of fungal SSF processes is often evaluated, in part, based on the growth of the fungus. Direct determination of the dry weight of biomass is impossible and indirect quantification techniques must be used. Over the years, various determination techniques have been developed for the quantification of fungal biomass in SSF processes. The current review gives an overview of various direct and indirect biomass determination methods, discussing their advantages and disadvantages.

Biomass estimation during macro-scale solid-state fermentation of basidiomycetes using established and novel approaches.

Solid-state fermentation (SSF) has been utilised in food production for millennia and is well suited for the cultivation of basidiomycetes, due to the robustness of the process and the possibility of using lignocellulose as the substrate. Basidiomycetes produce diverse enzymes and various primary and secondary metabolites, many of which have biotechnological potential. The quantification of the fungal biomass present is essential for the characterisation of growth kinetics in processes such as SSF. In SSF, fungi grow into the substrate and use it as a nutrient source. Therefore, direct biomass determination is not possible and indirect methods have to be employed. In the presented study, we compared 11 methods for quantifying fungal biomass during SSF of the basidiomycete Trametes hirsuta in a newly developed laboratory reactor (working volume 10 L). The methods were based on measuring the levels of six cell-specific components (ergosterol, glucosamine, nucleic acids, number of fungal nuclei, protein and genomic DNA) and estimations of biological activity (respiration, activities of lignolytic and cellulolytic enzymes, and the glucose and protein contents of the liquid). The methods were evaluated with regards to reproducibility and plausibility of the results, time and resource requirements, possible influential factors, and matrix effects. The most reliable biomass estimates were obtained from measurements of ergosterol content, number of nuclei, and respiration. Thus, these three methods were deemed most suitable for process control and modelling.

Biomass measurement by flow cytometry during solid-state fermentation of basidiomycetes.

Solid-state fermentation (SSF) is a robust process that is well suited to the on-site cultivation of basidiomycetes that produce enzymes for the treatment of lignocellulosics. Reliable methods for biomass quantification are essential for the analysis of fungal growth kinetics. However, direct biomass determination is not possible during SSF because the fungi grow into the substrate and use it as a nutrient source. This necessitates the use of indirect methods that are either very laborious and time consuming or can only provide biomass measurements during certain growth periods. Here, we describe the development and optimization of a new rapid method for fungal biomass determination during SSF that is based on counting fungal nuclei by flow cytometry. Fungal biomass was grown on an organic substrate and its concentration was measured by isolating the nuclei from the fungal hyphae after cell disruption, staining them with SYTOX(®) Green, and then counting them using a flow cytometer. A calibration curve relating the dry biomass of the samples to their concentrations of nuclei was established. Multiple buffers and disruption methods were tested. The results obtained were compared with values determined using the method of ergosterol determination, a classical technique for fungal biomass measurement during SSF. Our new approach can be used to measure fungal biomass on a range of different scales, from 15 mL cultures to a laboratory reactor with a working volume of 10 L (developed by the Research Center for Medical Technology and Biotechnology (fzmb GmbH)). © 2014 International Society for Advancement of Cytometry.