Pigment producing microorganisms have gained significant attention due to the existing genetic diversity [28] and various industrial application. Whereas filamentous fungi are more attractive due to their potency to produce wide range of pigments [11]. The researches have focused their attention on process optimization for the production of pigments by Talaromyces spp [16]. Hence developing a new bioprocess requires several steps that includes designing and feasible optimizing process conditions to achieve high yields, using low cost fermentative substrate like wheat bran and its hydrolysate (WBH).
3.1 Solid-state fermentation
It was observed after 10 days of solid-state fermentation on WB at 30ºC, T. purpureogenus CFRM02 culture with deep green spores. The production of red colour pigment was negligible under various concentration (w/v) of moisture condition (Fig S1). Similarly, the pigment production by Penicillium sp was less in solid-state fermentation compared to submerged fermentation [3]. Kantifedaki et al [10] have also reported lower level of pigment production by P. purpurogenum CBS 113139 during solid-state fermentation using orange peel waste. Moreover, the medium used for fermentation process significantly affects the product yield in terms of biomass and pigment. It was confirmed that solid-state fermentation is not appropriate to produce T. purpureogenus CFRM02 pigment by utilizing WB.
3.2 Submerged fermentation
Consequently, submerged fermentation conditions were adopted to produce T. purpureogenus CFRM02 pigment in higher quantities. It was observed that the pH plays an essential role for the production of secondary metabolites and biomass yield [30]. The fungus grown in the PDB medium at different pH condition (Fig S2) has produced more red pigment (0.362 ± 0.00 OD units/ml) at pH 6 and 8 (0.346 ± 0.03 OD units/ml). Even though the OD value (0.552 ± 0.05 OD unit/ml) of the culture broth was higher at pH 10, the pigment was not produced (Fig S2). The higher OD values of culture broth of pH 10 was due to the interference of the other metabolites produced during the growth. This was evidenced by the analysis of T. purpureogenus CFRM02 culture broth containing pigment through UV-visible spectra and CIE Lab colour values. The prominent peak at 494 nm (λ max) was observed in the spectrum of the culture broth of pH 6 and 8 (Fig S2). Further the CIE Lab colour values for pH 6 and 8 in terms of redness (a* value) were 16.32 ± 1.40 and 11.65 ± 1.82 respectively. The biomass production was ranging between (0.263 ± 0.02) to (0.377 ± 0.06). Even though the higher biomass yield was observed at pH 10, the pigment production was not increased significantly. Similarly, the temperature also affect the production of secondary metabolites. The higher pigment production was observed in the culture medium grown at 30 ºC (Fig S3). Similar studies with Monascus spp., Penicillium spp., Talaromyces spp. the optimal pH was found to be 5.5–6.5 and optimal temperature was 24–30ºC for pigment production [30]. Accordingly for further cultivation of fungus on WBH medium and other nutrient supplementation, the pH and temperature was adjusted to 6 and 30ºC respectively.
3.3 Cost effective medium
The production of pigments through microbial fermentation process using the commercially available media is expensive process. Hence, the development of a cost-effective bioprocess is essential for the production of T. purpureogenus CFRM02 pigment by using wheat bran as waste raw material. The main carbohydrate in WBH comprises starch, hemicellulose and cellulose [26]. It is also that, the pigment production in Penicillium sp was influenced by the presence of nitrogen in the medium [17]. Accordingly, the study was designed in four tasks. Initially, the WBH was evaluated with addition of individual carbon and nitrogen source after acid hydrolysis (Tables 1 and 2). The conditions of xylose levels and combination of C/N were evaluated to obtain a liquor to be used as a fermentation medium.
Table 1
Effect of carbon and xylose concentration supplementation to WBH medium on T. purpureogenus CFRM02 pigment characteristics and the CIELAB value.
Media | L lightness | a value | b value | h hue angle | c chroma |
| WBH | 78.77 ± 2.21a | 7.38 ± 1.38 b | 27.22 ± 1.11 ab | 74.87 ± 2.35 d | 28.22 ± 1.33 a |
Carbon Source (1%) | Glu | 84.59 ± 3.44 bc | 11.56 ± 0.69 e | 26.26 ± 1.05 a | 66.26 ± 0.43 a | 28.69 ± 1.24 a |
Fru | 86.13 ± 3.32 bc | 8.25 ± 0.64 bc | 28.48 ± 0.82 b | 73.84 ± 1.38 cd | 29.66 ± 0.75 a |
Lac | 81.57 ± 2.06 ab | 10.08 ± 0.37 de | 28.03 ± 1.03 ab | 70.21 ± 0.39 b | 29.79 ± 1.08 a |
Mal | 85.57 ± 1.69 bc | 9.33 ± 0.35 cd | 27.47 ± 0.62 ab | 71.24 ± 0.30 bc | 29.01 ± 0.70 a |
Suc | 88.11 ± 1.53 d | 5.30 ± 1.13 a | 27.26 ± 1.58 ab | 78.92 ± 2.69 e | 27.79 ± 1.43 a |
Xylose concentration (%) | Xyl 1 | 57.58 ± 2.34 a | 49.55 ± 1.62 d | 52.98 ± 4.00c | 46.86 ± 3.09 a | 72.61 ± 1.81 c |
Xyl 2 | 76.98 ± 2.45 b | 20.00 ± 3.15 c | 35.49 ± 3.48b | 60.71 ± 1.48 b | 40.74 ± 4.57 b |
Xyl 3 | 88.32 ± 1.48 c | 5.08 ± 1.66 a | 30.56 ± 0.91 a | 80.52 ± 3.30 c | 31.01 ± 0.63 a |
Xyl 4 | 88.34 ± 1.34 c | 3.83 ± 0.55 a | 31.22 ± 0.30 a | 83.01 ± 1.07 c | 31.46 ± 0.23 a |
WBH wheat bran hydrolysate, Glu glucose, Fru fructose, Lac lactose, Mal maltose, Suc sucrose, Xyl xylose. |
The concentration of carbon sources were 1% and xylose was 1–4% in the WBH medium. |
The mean values within the column with different superscript were significantly (P < 0.05) different |
3.4 Supplementation of Carbon source to WBH
Carbon source type had a significant effect on pH, biomass and pigment yield as shown in Fig. 1. The pH of the fermented broth was in the range of 4.5 to 6.5. The higher biomass observed in the fructose (1.55 ± 0.13 gm) containing media compared to other sugars. Whereas the significant increase in the pigment (0.945 ± 0.14 OD units/ml) production was observed in the xylose medium (Fig. 1A and B). While there was no significant difference in the pigment production in other carbon containing medium (Fig. 1A). Similarly, the highest redness a٭ value was found in basal medium supplemented with 1% xylose (49.55 ± 1.62), whereas the least was in sucrose (5.30 ± 1.13). The production of pigments by T. amestolkiae DPUA 1275 was explained utilizing glucose as carbon source and careful selection of nitrogen source [32]. Carbon source addition (glucose, fructose, sucrose, xylose, maltose, and lactose) to the WBH basal medium produced a significant increase in both pigment and biomass yield (Table 1). While the biomass yield in the basal medium containing the carbon sources, except xylose (1.090 ± 0.14) was significantly higher than the basal medium (1.044 ± 0.05) alone, but with higher pigment yields. It is due to the fact that simple sugars have induced more of the growth and inhibited the secondary metabolite production [17]. In all the fermented broth containing different carbon source, the L*, a*, and b* values were positive, indicating yellowness and redness hue (Table 1 and Fig S4). The lightness values were in the range of 70–78. The hue angle of the pigment produced was in the range of 66–78° indicating the minor shade changes in the red colour characteristics of the pigment (Table 1). But the significant difference was observed in the chroma value of the pigment produced in the medium containing xylose (72.61 ± 1.81). This revealed the saturation intensity of the pigment produced in the 1% xylose medium (Table 1) and correlated with the UV-visible spectrum colour appearance in the culture flask (Fig S4).
3.5 Supplementation of Xylose to WBH
Since 1% xylose has significantly induced the pigment production, further to observe the effect of higher concentration of xylose, the concentration was gradually increased to 4%. The results indicated that the increased concentration of xylose was inversely proportional to the pigment production (Fig. 1B). One gram of xylose has shown significant increase in red pigment production in the medium (49.55 ± 2.29) and least in 4g xylose composition (3.83 ± 0.78). Similar results were reported that higher concentration of xylose did not induce the pigment production in T. atroroseus GH2 utilising the corn cob hydrolysate [16]. However, the higher concentration of xylose has positively affected the growth of fungus (Fig. 1B). The biomass yield was highest in 4g xylose composition (1.450 ± 0.16), whereas the least biomass yield was in 1g xylose composition (0.915 ± 0.03). The medium containing 1g of xylose has shown maximum absorbance (1.565 ± 0.07) at 494nm indicated the higher pigment yield while less pigment yield was observed in the medium containing 4g xylose (Fig S5).
It was also observed that the colour characteristics of the pigments was significantly affected by varying xylose (1–4%) concentration. Even though the L*, a*, and b* values were positive, indicating yellowness and redness (Table 1). The a٭ value (redness), 50 to 4 and chroma intensity, 73 to 32 was significantly decreased and inversely proportional to the xylose concentration (Table 1). The increase in b٭ value (yellowness), 31 to 53 and hue angle 47° to 83° towards 90° indicted that, higher concentration of xylose induced yellow pigment production.
Considering the utilization of carbon source, the cultivation of Corydopsis militaris strain TBRC6039 demonstrated that although xylose was less favourable than glucose or sucrose in terms of biomass productivity, this particular strain could efficiently metabolize xylose. Further analyses of key metabolites showed that a high production yield of cordycepin was obtained when it was grown in xylose rather than in favourable carbon sources. Similarly, it was also observed in T. purpureogenus CFRM02 that contain alternative gene(s)/pathway(s) for metabolizing xylose. Therefore, most of the assimilated xylose in this strain might be channeled towards the biosynthetic pathway of pigment rather than towards central carbon metabolism [24].
3.6 Supplementation of nitrogen to WBH
It was reported that the red pigment produced by Talaromyces are similar to Monascus purpureus pigments. The yield and characteristics of the pigments were affected by the nitrogen source in the growth medium [4, 14]. Accordingly, the effect of nitrogen source was evaluated. The pH of the fermented medium was in the range of 5–7. The individual nitrogen source added to the basal medium significantly affected the yield of biomass. No significant changes in the pigment production was observed (Fig. 1C). The significant increase in the biomass was observed in the medium containing NaNO3 (2.591 ± 0.59g/100ml) compared other nitrogen sources. The biomass yield was less in the medium containing peptone and NH4NO3. While the yeast extract positively influenced the biomass yield (2.078 ± 0.20gm/100ml). Even though the biomass yield was significantly affected by type of nitrogen sources, the pigment production in all the nitrogen containing medium (inorganic or organic), remained almost same (Fig. 1C). Similar type of behaviour was observed by Penicillium sp for pigment production utilising waste stream cellulose as culture medium [27]. The nitrogen sources added individually to the medium did not affect the colour characteristics (Table 2). In all the fermented broth containing different nitrogen source, the L*, a*, and b* values were positive, indicating yellowness and redness (Table 2). The lightness values were 85 to 88. The hue angle of the pigment produced in the medium was in the range of 74 to 84° indicating the minor shade changes in the red colour characteristics of the pigment (Table 2). Whereas the chroma value was ranging from 28 to 36.
Table 2
Effect of nitrogen concentration supplementation to WBH medium on T. purpureogenus CFRM02 pigment characteristics and the CIELAB value.
|
|
L lightness
|
a value
|
b value
|
h hue angle
|
c chroma
|
|
WBH
|
85.24 ± 2.33 b
|
6.82 ± 2.03 c
|
28.16 ± 1.67 ab
|
76.27 ± 4.64 a
|
29.03 ± 1.17 ab
|
Nitrogen
source
|
KNO3
|
84.91 ± 0.92 b
|
4.30 ± 0.88 ab
|
30.04 ± 1.43 ab
|
81.81 ± 1.94 b
|
30.36 ± 1.32 ab
|
NH4NO3
|
85.26 ± 1.46 b
|
3.60 ± 0.47 a
|
27.36 ± 1.43 a
|
82.48 ± 1.17 b
|
27.60 ± 1.39 a
|
NaNO3
|
87.82 ± 1.71 bc
|
3.49 ± 0.43 a
|
30.69 ± 1.82 b
|
83.49 ± 0.96 b
|
30.89 ± 1.79 b
|
PEP
|
88.87 ± 1.81 c
|
4.44 ± 2.66 ab
|
35.60 ± 1.91 c
|
82.93 ± 4.19 b
|
35.94 ± 1.97 c
|
YE
|
81.69 ± 2.06 a
|
7.99 ± 0.61 c
|
27.11 ± 1.50 a
|
73.53 ± 1.72 a
|
28.27 ± 1.37 a
|
Nitrogen concentration
|
N1
|
68.28 ± 1.55 b
|
13.10 ± 1.25 a
|
71.87 ± 2.06 a
|
79.65 ± 1.25 c
|
73.06 ± 1.80 a
|
N2
|
66.17 ± 2.80 b
|
18.90 ± 2.04 b
|
78.25 ± 0.41 b
|
76.43 ± 1.34 bc
|
80.51 ± 0.87 b
|
N3
|
59.16 ± 0.69 a
|
24.45 ± 0.56 c
|
81.40 ± 1.54 c
|
73.27 ± 0.66 ab
|
84.99 ± 1.31 c
|
N4
|
59.29 ± 1.40 a
|
26.95 ± 2.90 c
|
82.80 ± 1.51 c
|
72.00 ± 1.51 a
|
87.10 ± 2.33 c
|
WBH wheat bran hydrolysate, Pep peptone, YE yeast extract |
The concentration of nitrogen sources were 0.3% and N1 = 0.3%YE, 0.4% KNO3, 0.3% NaNO3, N2 = N1 X 2, N3 = N1 X 3 and N4 = N1 X 4 in the WBH medium. |
The mean values within the column with different superscript were significantly (P < 0.05) different. |
3.7 Supplementation NaNO3 and yeast extract to WBH
Since NaNO3 and yeast extract have positively affected the growth, these two nitrogen source were selected for further process of pigment production along with KNO3 for potassium ion. Interestingly, the pigment production was increased, when these nitrogen sources were added to the medium (Fig. 1D). Further, significant increase in biomass and pigment yield was also observed by increasing the concentration of these nitrogen (0.3–0.12%) sources (Fig. 1D). The nitrogen sources added to the medium influenced colour characteristics of the pigment. In all the fermented broth containing different nitrogen source, the L*, a*, and b* values were positive, indicating yellowness and redness (Table 2). Whereas the b* values (71 to 83) were higher compared to a* values (13 to 27). These results indicted the nitrogen sources influence more yellow pigment production compared carbon sources (Table 2). There was significant changes in the colour characteristics of the pigment as the nitrogen concentration increased from 1X to 4X. The lightness values ranged from 59 to 68. The hue angle was ranging from 72° to 80° indicating production of orangish red colour characteristic (Table 2).
3.8 Combined effect of carbon and nitrogen
It was reported earlier that, the type of pigment or hue produced by ascomycetous filamentous fungi might vary within the same species by growing in different media [9]. Herein also, the culture medium influenced the pigment yield and characteristics of T. purpureogenus CFRM02. Hence, the sixteen different WBH medium were prepared for the cultivation of T. purpureogenus CFRM02 by addition of carbon and nitrogen (Fig S6). Fermentation broths were used for pigment estimation and analysis of colour characteristics. Interestingly it was observed that, the significant increase in the higher pigment production in the medium containing high xylose concentration (Fig. 2). Whereas, the production of pigment was higher in the WBH medium containing 1% xylose only (Fig. 1A and B). Similarly, the biomass yield was more when high concentration of carbon and nitrogen was added to the medium (Fig. 2).
The values of L*, a*, and b* were all positive, indicating yellowness and redness (Table 3). The significant difference was observed in the L*, a*, and b* values. The lightness values were ranging from 52–80. The a* values were ranging from 7 to 33 indicating the light red to middle red intensity. The b* values were ranging from 41 to 75 indicating middle reddish-orange to deep reddish-orange pigment. The a* value was higher (33 ± 3) in the medium containing 1–2% carbon and nitrogen sources as presented in Table 3. While the hue angle was ranging from 65° to 82° corresponding to light red and middle orange. This indicated changes in the hue of the pigment produced from red to orange to yellow in the culture medium of the T. puprureuogenus CFRM02. The chroma values varied in a range from 42 (gray) to 78 (bright) according to the medium used for cultivation. On the basis of the hue angle and chroma values the pigments derived with carbon and nitrogen source had various shades of red. The higher chroma values were observed in the higher concentration of the xylose suggested the brightest colour hue. These results suggested that, higher chroma values can be observed by increasing the concentration of the xylose (Table 3). The colours varied according to the concentration of the carbon and nitrogen source used in the fermentation medium.
Studies have reported that organic nitrogen sources are better than inorganic ones to promote pigment production by Talaromyces/Penicillium [32]. However, in this study, significant changes were not observed in the pigment production and colour characteristics when supplemented with organic and inorganic nitrogen (Table 2). There are not more data on the CIE Lab colour characteristics of T. purpureogenus CFRM02 especially from the prospective of food use. Moreover, our previous studies on the toxicity evaluation of the T. purpureogenus CFRM02 pigment confirmed its nontoxic effect [21, 22]. The color hues of T. purpureogenus CFRM02 pigment was similar to color hues of the red and orange Monascus pigments [14]. This signifies the contribution of the present findings and the reddish-orange hues of T. purpureogenus CFRM02 pigments can be worth to consider in food use.
Table 3
The combined effect of carbon and nitrogen concentration supplementation to WBH medium on T. purpureogenus CFRM02 pigment characteristics and the CIELAB value.
|
L lightness
|
a value
|
b value
|
h hue angle
|
c chroma
|
N1C1
|
80.13 ± 1.39 j
|
8.63 ± 1.11 a
|
42.05 ± 1.61 a
|
78.44 ± 1.02 fgh
|
42.93 ± 1.79 a
|
N1C2
|
78.96 ± 1.02 ij
|
14.56 ± 1.08 b
|
41.11 ± 1.66 a
|
70.51 ± 0.61 b
|
43.61 ± 1.92 a
|
N1C3
|
60.63 ± 7.24 cde
|
30.44 ± 1.81 d
|
67.09 ± 1.76 def
|
65.58 ± 1.85 a
|
73.70 ± 0.85 de
|
N1C4
|
54.80 ± 8.05abc
|
32.52 ± 2.85 d
|
70.25 ± 1.51 fg
|
65.16 ± 2.38 a
|
77.45 ± 0.17 e
|
N2C1
|
72.05 ± 4.20 ghi
|
17.99 ± 1.33 c
|
40.97 ± 0.45 a
|
66.30 ± 1.78 a
|
44.76 ± 0.12 a
|
N2C2
|
80.29 ± 2.23 j
|
9.07 ± 0.33 a
|
52.41 ± 7.33 b
|
80.03 ± 1.73 hi
|
53.20 ± 7.16 b
|
N2C3
|
61.68 ± 6.18 cde
|
21.56 ± 1.80
|
67.88 ± 1.08 def
|
72.37 ± 1.64 bc
|
71.24 ± 0.48 d
|
N2C4
|
49.23 ± 2.50 a
|
32.49 ± 2.75 d
|
70.45 ± 3.87 fg
|
65.18 ± 3.04 a
|
77.65 ± 2.36 e
|
N3C1
|
76.40 ± 2.44 hij
|
12.54 ± 0.99 b
|
52.32 ± 5.52 b
|
76.50 ± 0.35 defg
|
53.80 ± 5.60 b
|
N3C2
|
75.30 ± 1.24 hji
|
13.32 ± 1.31 b
|
55.80 ± 3.96 bc
|
76.59 ± 0.36 defg
|
57.37 ± 4.16 b
|
N3C3
|
69.52 ± 0.69 fgh
|
13.60 ± 1.96 b
|
62.55 ± 5.09 de
|
77.61 ± 2.70 efg
|
64.06 ± 4.55 c
|
N3C4
|
51.63 ± 6.34 ab
|
29.82 ± 4.06 d
|
69.82 ± 1.84 fg
|
66.87 ± 3.35 a
|
76.00 ± 0.12 de
|
N4C1
|
76.45 ± 3.31 hij
|
7.56 ± 1.82 a
|
52.84 ± 4.03 b
|
81.74 ± 2.55 i
|
53.41 ± 3.73 b
|
N4C2
|
67.02 ± 2.82 efg
|
18.95 ± 1.08 c
|
61.61 ± 3.03 cd
|
72.86 ± 1.71 bcd
|
64.47 ± 2.57 c
|
N4C3
|
57.76 ± 1.98 bcd
|
21.57 ± 1.71 c
|
74.88 ± 4.06 g
|
73.87 ± 2.04 bcde
|
77.96 ± 3.43 e
|
N4C4
|
63.80 ± 4.04 def
|
18.39 ± 2.63 c
|
68.23 ± 1.46 ef
|
74.90 ± 2.37 cdef
|
70.71 ± 0.73 d
|
The N and C corresponds to the nitrogen and carbon at different concentration in the WBH medium. |
The N nitrogen sources, N1 = 0.3%YE, 0.4% KNO3, 0.3% NaNO3, N2 = N1 X 2, N3 = N1 X 3 and N4 = N1 X 4. |
The C1-C4 corresponds to 1–4% of xylose concentration. |
The mean values within the column with different superscript were significantly (P < 0.05) different. |