Batch and Fed-Batch Production of β-carotene by Rhodotorula Toruloides KP324973 Using Corn Steep Liquor As Sole Carbon Source

doi:10.21203/rs.3.rs-751445/v1

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Batch and Fed-Batch Production of β-carotene by Rhodotorula Toruloides KP324973 Using Corn Steep Liquor As Sole Carbon Source

https://doi.org/10.21203/rs.3.rs-751445/v1

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Application of agro-industrial waste in microbial fermentation is interesting in economic and environmental aspects. Carotenoids production by Rhodotorula toruloides KP324973 was investigated using corn steep liquor (CSL) as sole carbon source. Haldane model with constants µ_max= 0.056 h^-1, K_S= 1.54 vv^-1%, and K_I= 58.58 vv^-1% showed best describe of cell growth kinetics on CLS. A same maximum carotenoid production rate (R_p) about 2.23 μg g_cell^-1h^-1was obtained at initial CSL concentration of 5 v/v% after 72 h and 21 h in batch cultivation in shaken flasks and bubble column reactor (BCR), respectively. Further improvement of carotenogenesis was followed by fed-batch cultivation in BCR where the optimal setting of factors at feed flow rate of 5 mL h^-1, pH of 5.66, and temperature 14 ˚C gained a highest R_p= 8.686 μg g_cell^-1h^-1. Chromatographic analysis showed more than 94% of produced carotenes was β-carotene.

Biotechnology and Bioengineering

Carotenogenesis

Fed-batch operation

Optimization

Bubble column reactor

Table 1 Design of experiments for evaluation of fed-batch carotenoids production in the BCR. The experimental results of R_P and predicted values by Eq. (9) are also presented.

Experiment run no.	Operational factors X₁: Feed flow rate (ml h^-1) X₂: Temperature (˚C) X₃: Initial pH			R_P (μg g_cell^-1h^-1) Experimental Predicted
1	10 (0)	9.91 (-α)	6.5 (0)	3.700	3.508
2	10 (0)	20 (0)	6.5 (0)	2.806	2.550
3	10 (0)	20 (0)	7.34 (+α)	1.103	1.347
4	10 (0)	20 (0)	6.5 (0)	2.562	2.550
5	15 (+1)	14 (-1)	7 (+1)	0.536	0.190
6	5 (-1)	26 (+1)	6 (-1)	3.205	3.390
7	18.41 (+α)	20 (0)	6.5 (0)	1.820	2.168
8	15 (+1)	26 (+1)	6 (-1)	1.755	1.450
9	10 (0)	20 (0)	6.5 (0)	2.337	2.550
10	1.59 (-α)	20 (0)	6.5 (0)	8.325	8.182
11	10 (0)	30.09 (+α)	6.5 (0)	1.625	1.592
12	15 (+1)	14 -1)	6 (-1)	1.339	1.370
13	5 (-1)	26 (+1)	7 (+1)	3.264	3.050
14	10 (0)	20 (0)	5.66 (-α)	2.633	2.624
15	5 (-1)	14 (-1)	6 (-1)	8.475	8.430
16	10 (0)	20 (0)	6.5 (0)	2.518	2.550
17	10 (0)	20 (0)	6.5 (0)	2.126	2.550
18	5 (-1)	14 (-1)	7 (+1)	5.268	5.410
19	15 (+1)	26 (+1)	7 (+1)	3.060	2.950

Table 2 ANOVA of the quadratic model (Eq. 9) developed in this study

Source	Sum of square	Degree of freedom	Mean square	F-value	P-value
Model	82.17	8	10.27	119.92	<0.0001
X₁	43.82	1	43.82	511.61	<0.0001
X₂	4.48	1	4.48	52.30	<0.0001
X₃	1.99	1	1.99	23.27	0.0007
X₁X₂	13.04	1	13.04	152.25	<0.0001
X₁X₃	1.66	1	1.66	19.43	0.0013
X₂X₃	3.61	1	3.61	42.15	<0.0001
X₁²	12.10	1	12.10	141.29	<0.0001
X₃²	0.57	1	0.57	6.61	0.0279
Residual	0.86	10	0.086	-	-
Lack of fit	0.60	6	0.099	1.53	0.3543
Pure error	0.26	4	0.065	-	-
Cor total	83.02	18	-	-	-
SD	0.29	R²	0.9897	Mean	3.08
C.V%	9.51	Adjusted R²	0.9814	PRESS	4.50

Table 3 Comparison of this work with previous fed-batch β-carotene production from CSL as carbone source

Microorganism	CSL concentration (g L⁻¹)	Operational conditions	R_P(μg g_cell^-1 h^-1)	Reference
Phaffia rhodozyma	90	Initial pH 5, temperature 20 °C, constant dissolved oxygen 50%, agitation speed of 120 rpm for 120 h after 48 h of initial batch fermentation in stirred tank reactor	16.0	[6]
Sporidiobolus salmonicolor	40	Initial pH 4, temperature 25 °C, air flow rate of 1.5 vv^-1min^-1, 180 rpm for 84 h after 24 h of initial batch fermentation in stirred tank reactor	8.51	[7]
Phaffia rhodozyma	10	Initial pH 5, temperature 20 °C, air flow rate of 1 vv^-1min^-1 for 95 h after 3 h of initial batch fermentation in stirred tank reactor	3.747	[10]
Sporidiobolus pararoseus	20	Initial pH 6, temperature 28 °C, air flow rate of 0.14 vv^-1min^-1at agitation speed of 90 rpm for 36 h after 24 h of initial batch fermentation in stirred tank reactor	9.265	[25]
Rhodotrula toruloides KP324973	6.7	Initial pH 5.66, temperature 14 °C, aeration rate 0.4 vv^-1min^-1, feed flow rate of 5 mL h^-1 for 30 h after 12 h of initial batch fermentation in bubble column reactor	8.686	This study

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Batch and Fed-Batch Production of β-carotene by Rhodotorula Toruloides KP324973 Using Corn Steep Liquor As Sole Carbon Source

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