Fungi provide a readily available alternative source of natural pigments with potential industrial applications due to the wide range of colors that are able to produce. Despite of their major properties (antioxidant, antibacterial and cytotoxic activities), the industrial application of natural pigments is still uncompetitive to synthetic colorants due to their production costs. Efforts need to be addressed in developing a low cost bioprocess for the production of such natural fungal pigments by using inexpensive raw materials and a process improvement. Lignocellulosic materials have gained extensive attention as a promising alternative source for the industrial production of valuable compounds by fermentation. Selection of a suitable lignocellulosic waste will depend on the availability of the raw materials for any geographical zone and the bioprocess required. It has been reported that corncob hydrolysate contains a high level of xylose due to its high composition of hemicellulose and thus represent an abundant and inexpensive carbon source that can be converted to pigments by microorganisms.
This study aimed to determine the feasibility of using a corncob hydrolysate as an alternative substrate for pigment production by Penicillium purpurogenumGH2 that has been previously reported as a potential pigment producer strain.
To hydrolyse the sugars oligomers to the correspondent monomers a dilute sulphuric acid hydrolysis treatment was used (temperature: 120 oC, pressure: 15 psi, H2SO4: 1%, 90 min, solid-liquid ratio: 10% (w/w)). The hydrolysate was detoxified using activated charcoal. The detoxified liquor without any nutrient supplementation was diluted to obtain a xylose concentration of 15 g/L and used as fermentation medium (HM). A synthetic Czapeck-dox modified medium (CDM) previously optimized for the production of pigments by this strain containing (g/L): D-xylose 15.0, NaNO3 3.0, MgSO4· 7H2O 0.5, FeSO4· 7H2O 0.1, K2HPO4 1.0, KCl 1.0 and ethanol 20.0 was used as control. Submerged fermentation experiments were carried out at 200 rpm, 30 oC for 8 days.
In both media the microorganism utilized two carbon sources (xylose and ethanol in CMD and xylose and glucose in HM) for its growth and production of metabolites. For future process optimization using a hydrolysate-based media, it is important to fully understand how the microorganism is consuming each substrate and how this simultaneous uptake affects cell growth and pigment production. Therefore pigment production, growth and substrate consumption kinetics using CMD and HM were studied. Unstructured fermentation models were applied to obtain valuable information as parameters with clear biological meaning.
The Verlhust logistic model was applied to describe the carbohydrate consumption during fermentation. This model describes substrate consumption as function of the residual substrate (Sres,g/L), the ratio between the initial volumetric rate of substrate consumption and the initial substrate concentration (So', h-1) and the lag time (k, h) before the carbohydrate is consumed. Biomass growth (X, g/L) was expected to be function of the consumption of both carbon sources (Si, g/L) in each medium (xylose and ethanol in CDM; xylose and glucose in HM) taking in account the yield (Yx/si, g/g) of biomass formed by each substrate. Production of pigments was described using the Luedeking-Piret model. This model assumes that the production rate (dP/dt) depends on the instantaneous biomass concentration (X,g/L), the growth rate (dX/dt) and takes into account when the production is limited by the exhaustion of a carbon source defined as limiting substrate (SL, g/L). It also contains 2 parameters with significant biological meaning, α for production related to growth and βfor production related to maintenance.
The models described accurately the experimental kinetic data with correlation coefficients (R2) of 0.97 and 0.99 for CDM and HM substrates respectively. Total pigment production obtained with HM was 6% lower that the pigments produced in the control CDM, however cell growth, production and substrate consumption presented different patterns in both media.
When CMD was used, the microorganism rapidly adapted to the medium showing a lag phase less than 24 h. Initial consumption rates differed from xylose (4.13 g/L.h) and ethanol (1.65 g/L.h) thus even though the obtained lag time for the utilization of ethanol was low (k=24 h), its assimilation started at a low rate. The microorganism used ethanol and xylose for growing and showed a typical diauxic growth due to the utilization of two carbon sources. The biomass yield obtained by ethanol (YX/S=0.11) was higher than with xylose (YX/S=0.08). Pigment production was related to grow with α=4.91 and β=0.
In HM medium, biomass growth presented an extended lag phase, indicating that the microorganism needed more time to adapt to the medium. The microorganism first used the most assailable and simple carbohydrate, glucose. The initial consumption rates varied from 0.38 g/L.h in glucose to 1.12 g/L.h in xylose. This confirmed the poor adaption of the microorganism to the hydrolysate. The time lag k for xylose assimilation was nearly 80 h. The highest yield of biomass per substrate was achieved by glucose (YX/S=0.53) meanwhile the biomass formed with xylose was almost negligible (YX/S=0.02) meaning that the microorganism did not used xylose to continue growing thus it did not show a diauxic type growth. Pigments production started after 80 h that corresponded to the lag time in xylose. Pigments production was associated with maintenance with values of α=0 and β=4.70.
Corncob hydrolysate was proved to be an efficient alternative medium for pigment production by Penicillium purpurogenum GH2. The hydrolysate without any nutrient supplementation showed high production yield of total pigment comparable to that obtained with the synthetic medium. The production of pigments was related to cell maintenance when the hydrolysate was used as fermentation media, thus for future process optimization the first approach would be the reduction of the extended initial lag phase.
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