ETHANOL PRECIPITATION AS A DOWNSTREAM PROCESSING STEP FOR CONCENTRATION OF XYLANASES PRODUCED BY SUBMERGED AND SOLID-STATE FERMENTATION

Costa, Max A. L.; Farinas, Cristiane S.; Miranda, Everson A.

doi:10.1590/0104-6632.20180352s20160502

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Bioprocess Engineering • Braz. J. Chem. Eng. 35 (2) • Apr-Jun 2018 • https://doi.org/10.1590/0104-6632.20180352s20160502 copy

ETHANOL PRECIPITATION AS A DOWNSTREAM PROCESSING STEP FOR CONCENTRATION OF XYLANASES PRODUCED BY SUBMERGED AND SOLID-STATE FERMENTATION

Authorship SCIMAGO INSTITUTIONS RANKINGS

Abstract

Xylanases have applications in different industries, being produced by microbial cultivations using submerged (SmF) or solid-state fermentation (SSF). Precipitation stands out as a potential method for the concentration of xylanases, especially with the use as ethanol as the precipitant due to its compatibility with the biorefinery concept. This paper presents a comparative laboratory scale study of ethanol precipitation of xylanases produced by Aspergillus niger cultivated under SSF and SmF. Precipitation conditions were selected according to a central composite design. Statistical analysis showed a significant effect of pH on the recoveries of total protein and xylanase activity. The kinetic profiles showed that a relatively short period of time (up to 15 min) was sufficient to recover most of the xylanase activity precipitated under the selected conditions. Xylanase recoveries of 65 and 79% were achieved for the SSF and SmF enzymatic complexes, respectively.

Keywords:
Aspergillus niger; downstream processing; ethanol precipitation; xylanase

	Temperature (°C)	Xylanase activity(IU/mL)			Protein concentration (mg/mL)
	Temperature (°C)	Activ\|₀	a_activ(10 ^-3)	R²	Prot\|₀	a_prot(10 ^-5)	R²
	15	6.50	2.6	0.970	0.23	1.0	0.983
SSF	30	6.84	2.1	0.975	0.22	20.0	0.880
	45	6.90	-1.1	0.984	0.22	2.0	0.964
	15	6.22	-1.2	0.980	0.07	-4.0	0.957
SmF	30	6.34	-1.1	0.983	0.05	8.0	0.931
	45	6.39	1.3	0.977	0.05	10.0	0.906

	pH	Xylanase activity (IU/mL)			Protein concentration (mg/mL)
	pH	Activ\|₀	a_activ(10 ^-3)	R²	Prot\|₀	a_prot(10 ^-5)	R²
	3.0^a a Buffer: 0.2 mol/L sodium citrate	8.78	3.6	0.911	0.16	9.0	0.790
	4.0^a a Buffer: 0.2 mol/L sodium citrate	9.09	-4.2	0.821	0.21	-2.0	0.964
	5.0^a a Buffer: 0.2 mol/L sodium citrate	7.61	4.6	0.917	0.24	-20.0	0.931
SSF	6.0^a a Buffer: 0.2 mol/L sodium citrate	8.59	5.8	0.950	0.24	-10.0	0.918
	6.0^b b Buffer: 0.2 mol/L sodium phosphate	9.65	0.8	0.972	0.25	-5.0	0.988
	7.0^b b Buffer: 0.2 mol/L sodium phosphate	9.32	-3.3	0.967	0.25	-10.0	0.945
	8.0^b b Buffer: 0.2 mol/L sodium phosphate	8.94	-1.0	0.909	0.26	-4.0	0.930
	3.0^a a Buffer: 0.2 mol/L sodium citrate	7.67	-0.6	0.861	0.06	0.7	0.931
	4.0^a a Buffer: 0.2 mol/L sodium citrate	8.96	-4.6	0.841	0.07	3.0	0.890
	5.0^a a Buffer: 0.2 mol/L sodium citrate	6.93	8.3	0.971	0.08	-9.0	0.897
SmF	6.0^a a Buffer: 0.2 mol/L sodium citrate	8.72	6.6	0.898	0.08	-6.0	0.949
	6.0^b b Buffer: 0.2 mol/L sodium phosphate	8.58	0.8	0.895	0.08	1.0	0.970
	7.0^b b Buffer: 0.2 mol/L sodium phosphate	8.67	-1.7	0.967	0.09	-3.0	0.937
	8.0^b b Buffer: 0.2 mol/L sodium phosphate	8.17	-2.7	0.926	0.10	-5.0	0.929

Run	pH	Ethanol (%)	SSF				SmF
Run	pH	Ethanol (%)	X_r (%)	P_r (%)	A_e (IU/mg)	P_F	X_r (%)	P_r (%)	A_e (IU/mg)	P_F
1	-1(4.0)	-1(80)	42.6	59.4	14.9	0.83	54.2	54.6	27.3	0.46
2	-1(4.0)	1(90)	45.1	63.0	14.8	0.83	54.6	68.3	53.4	0.90
3	1(7.0)	-1(80)	56.1	83.0	14.0	0.78	65.4	61.0	51.1	0.86
4	1(7.0)	1(90)	55.1	76.4	15.0	0.84	55.9	76.5	48.9	0.83
5	-1.41(3.4)	0(85)	48.6	45.1	22.4	1.25	58.3	46.3	40.7	0.69
6	1.41(7.6)	0(85)	46.6	69.8	13.8	0.77	51.9	51.1	60.0	1.02
7	0(5.5)	-1.41(78)	60.0	78.9	15.8	0.88	57.8	63.3	60.3	1.02
8	0(5.5)	1.41(92)	59.8	85.6	14.5	0.81	73.5	85.3	46.0	0.78
9	0(5.5)	0(85)	62.1	81.4	15.8	0.88	65.8	72.1	48.7	0.82
10	0(5.5)	0(85)	64.4	86.2	14.9	0.83	68.7	76.9	47.7	0.80
11	0(5.5)	0(85)	61.8	82.7	15.6	0.87	66.4	73.4	48.3	0.82

	SSF				SmF
	Total protein concentration		Xylanase activity		Total protein concentration		Xylanase activity
	Coefficient	p-value	Coefficient	p-value	Coefficient	p-value	Coefficient	p-value
Mean	83.43^* * Significant at p<0.05.	0.000	66.30^* * Significant at p<0.05.	0.000	74.13^* * Significant at p<0.05.	0.000	66.97^* * Significant at p<0.05.	0.000
pH	8.99^* * Significant at p<0.05.	0.009	2.58^* * Significant at p<0.05.	0.036	2.67	0.093	0.43	0.509
pH²	-12.84^* * Significant at p<0.05.	0.007	-8.59^* * Significant at p<0.05.	0.005	-11.82^* * Significant at p<0.05.	0.008	-6.65^* * Significant at p<0.05.	0.009
EtOH	0.81	0.454	0.15	0.791	7.54^* * Significant at p<0.05.	0.013	1.64	0.094
EtOH²	-0.44	0.714	-2.44	0.055	0.98	0.446	-1.37	0.167
pHxEtOH	-2.55	0.176	-0.88	0.344	0.45	0.752	-2.48	0.084
R	0.98		0.79		0.98		0.60
F-value	50.77		3.83		30.72		1.48

Cultivation System	Microorganism	Precipitant (v/v)	Time (min)	X_R (%)	PF	Reference
SSF	A. niger	85% ethanol	15	65.0	0.74	This work
	A. niger	80% ethanol	180	23.0	-	(Farinas et al., 2011Farinas, C., Scarpelini, L., Miranda, E. and Neto, V., Evaluation of operational parameters on the precipitation of endoglucanase and xylanase produced by solid state fermentation of Aspergillus niger. Brazilian Journal of Chemical Engineering, 28(1) 17-26 (2011).)
		80% ammonium sulfate		27.0	-
	T. harzianum	80% ethanol	30	94.0 97.0	-	(Fadel, 2001Fadel, M., High-level xylanase production from sorghum flour by a newly isolate of Trichoderma harzianum cultivated under solid state fermentation. Annals of Microbiology, 51(1) 61-78 (2001).)
SmF	A. niger	85% ethanol	15	79.0	1.03	This work
	T. harzianum	90% ethanol	0	100.0	-	(Marino et al., 2015Marino, M.A., Freitas, S. and Miranda, E.A., Ethanol precipitation of glycosyl hydrolases produced by Trichoderma harzianum P49P11. Brazilian Journal of Chemical Engineering, 32(2) 325-333 (2015).)
	Chainia sp.	66% ethanol	overnight	78.5	13.70	(Varma et al., 1999Varma, R.J., Nene, S., Baliga, B.A. and Elias, C., Studies on commercial aspects of xylanase from Chainia species. Journal of Scientific & Industrial Research, 58(11) 878-882 (1999).)
		80% ethanol		99.0	15.97

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[1] *Corresponding author: Everson Alves Miranda, Phone: 55 19 3521 3918, E-mail: everson@feq.unicamp.br