The worldwide demand for natural food and textile colorants is rapidly increasing. Natural colorants are about to overpass synthetic colorants in market value in the future due to the continued consumer demands for natural food ingredients. Currently, natural pigments are derived from sources like plants, insects and microorganisms. The production, extraction and the existing genetic diversity in microorganisms and sophistication of technology have made their choice more feasible. Within the different groups of microorganisms like bacteria, yeast, algae, fungi and actinomycetes, filamentous fungi seem to be a more efficient, attractive source of bio-colorants and some fungal species are rich in stable colorants. Pigments produced by Penicillium purpurogenum are excreted out of the cell; however most of the pigments remain inside of the cell. This could lead to a high intracellular product concentration inhibition. Recently, export of intracellular product by microbial fermentation in a water-organic solvent two-phase system, which is known as ‘milking processing’, has attracted the attention of bioprocessing engineers. Permeability and biocompatibility are critical issues in selection of an organic solvent for ‘milking processing’. Modification of cell membrane structure by addition of surfactant has been proved to be effective for the secretion of intracellular metabolites. The present work studied the effect of various surfactants on secretion of intracellular pigments into its fermentation broth. Czapex-dox modified medium was used for pigment production (pH 5-6). A mycelial suspension of Penicillium purpurogenum GH2 was used as inoculum. The inoculated flasks were incubated at 30±2°C on an orbital shaker (Inova 94, New Brunswick Scientific, USA) at 200 rpm for 8 days. A total of 4 surfactants were used to evaluate their effect on the pigment production and growth in submerged fermentation of Penicillium purpurogenum GH2. The surfactants analyzed were Tween 80, Span 20, Triton X-100 and polyethylene glycerol polymer PEG 8000 at 3 different concentrations (5, 20, 35 gL-1), surfactants were added at the beginning of the fermentation. For pigment extraction, each sample was centrifuged at 8000 rpm for 20 min at 4oC (Sorball, Primo R Biofuge Centrifugation Thermo, USA). The supernatant was then filtered through a 0.45 μm cellulose filter (Millipore, USA). The concentration of red pigments was quantified indirectly measuring the optical density at 500 nm using a spectrophotometer (Cary 50, UV-Visible Varian, USA). After second centrifugation, mycelia was collected and soaked in 70% (V/V) ethanol aqueous solution for 1 h. Corresponding ethanol aqueous solution was subjected to the intracellular pigment concentration analysis. Biomass concentration was determined using the gravimetric method. The highest production of pigments (18 OD500nm) was reached using Triton X-100 (35 gL-1) which is 70% higher than the control (no surfactant added). Furthermore the effect of addition time (0-144) of Triton X-100 (35 gL-1) on secretion of intracellular pigments into the fermentation broth was studied. The highest extracellular pigment concentration (22 OD500nm) was achieved when surfactant was added at 120 h of fermentation. To further investigate the effect of Triton X-100 on pigments production, kinetics of extracellular and intracellular pigments production were examined both in control and in Triton X-100 aqueous solution (Surfactant added at 120 h). In the control, intracellular pigments were accumulated to reach the maximum production at 120 h then a light release of pigments is excreted into the broth, therefore no marked increase of extracellular pigment concentration was observed. In contrast it can be seen that 24 h after Triton X-100 is added into the fermentation the extracellular pigment concentration was increased by 57% in comparison with the control. Also, it was observed a considerable decrease in cell biomass weight after Triton X-100 was added, which is due to the releasing of intracellular product. Total pigment (extracellular pigment and intracellular pigment) at the end of the fermentation was 27 % higher when Triton X-100 was used comparing with control, confirming that Triton X-100 had at least partially alleviated the product inhibition. A remarkable increase of extracellular pigment was achieved by the addition of Triton X-100. Use of surfactants represents a potential for replacement of organic solvent for perstractive fermentation of intracellular product.
Keywords: natural pigments, surfactant, perstraction, kinetics.