Exopolysaccharide production from <i>Bacillus velezensis</i> KY471306 using statistical experimental design

Moghannem, Saad A.M.; Farag, Mohamed M.S.; Shehab, Amr M.; Azab, Mohamed S.

doi:10.1016/j.bjm.2017.05.012

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Brazilian Journal of Microbiology

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Environmental Microbiology • Braz. J. Microbiol. 49 (3) • Jul-Sep 2018 • https://doi.org/10.1016/j.bjm.2017.05.012 copy

Exopolysaccharide production from Bacillus velezensis KY471306 using statistical experimental design

Authorship SCIMAGO INSTITUTIONS RANKINGS

Abstract

Exopolysaccharide (EPS) biopolymers produced by microorganisms play a crucial role in the environment such as health and bio-nanotechnology sectors, gelling agents in food and cosmetic industries in addition to bio-flocculants in the environmental sector as they are degradable, nontoxic. This study focuses on the improvement of EPS production through manipulation of different culture and environmental conditions using response surface methodology (RSM). Plackett-Burman design indicated that; molasses, yeast extract and incubation temperature are the most effective parameters. Box-Behnken RSM indicated that; the optimum concentration for each parameter was 12% (w/v) for molasses, 6 g/L yeast extract and 30 °C for incubation temperature. The most potent bacterial isolate was identified as Bacillus velezensis KY498625. After production, EPS was extracted, purified using DEAE-cellulose, identified using Fourier transform infrared (FTIR), gel permeation chromatography (GPC) and gas chromatography-mass spectroscopy (GC-MS). The result indicated that; it has molecular weight 1.14 × 10⁵ D consisting of glucose, mannose and galactose.

Keywords:
Bacterial exopolysaccharide; Plackett-Burman; Box-Behnken; DEAE-cellulose

RUN	X ₁	X ₂	X ₃	X ₄	X ₅	X ₆	X ₇	X ₈	X ₉	X ₁₀	X ₁₁	EPS g/L observed	Predicted
1	-1.00	-1.00	-1.00	1.00	-1.00	1.00	1.00	-1.00	1.00	1.00	1.00	5.8 ± 0.21	5.8
2	1.00	-1.00	-1.00	1.00	1.00	1.00	-1.00	-1.00	-1.00	1.00	-1.00	5.42 ± 0.16	5.55
3	1.00	1.00	1.00	-1.00	-1.00	-1.00	1.00	-1.00	1.00	1.00	-1.00	6.48 ± 0.22	6.61
4	-1.00	-1.00	-1.00	1.00	1.00	-1.00	1.00	1.00	1.00	-1.00	-1.00	3.01 ± 0.31	3.14
5	1.00	-1.00	-1.00	-1.00	-1.00	1.00	-1.00	1.00	1.00	-1.00	1.00	7.05 ± 0.28	7.18
6	1.00	1.00	1.00	1.00	-1.00	1.00	1.00	1.00	-1.00	-1.00	-1.00	6.76 ± 0.44	6.76
7	-1.00	1.00	1.00	-1.00	1.00	1.00	1.00	-1.00	-1.00	-1.00	1.00	2.92 ± 0.13	3.05
8	-1.00	-1.00	-1.00	-1.00	-1.00	-1.00	-1.00	-1.00	-1.00	-1.00	-1.00	2.15 ± 0.35	2.15
9	-1.00	1.00	1.00	-1.00	1.00	1.00	-1.00	1.00	1.00	1.00	-1.00	3.69 ± 0.37	3.69
10	-1.00	1.00	1.00	1.00	-1.00	-1.00	-1.00	1.00	-1.00	1.00	1.00	2.44 ± 0.21	2.57
11	1.00	-1.00	-1.00	-1.00	1.00	-1.00	1.00	1.00	-1.00	1.00	1.00	1.96 ± 0.14	1.96
12	1.00	1.00	1.00	1.00	1.00	-1.00	-1.00	-1.00	1.00	-1.00	1.00	4.65 ± 0.65	4.65
13	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	4.84 ± 0.29	4.69
14	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	5.13 ± 0.22	4.69
15	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	4.94 ± 0.51	4.69

Trials	X₁ (molasses)		X₂ (yeast extract)		X₃ (incubation temp.)		EPS (g/L)
	Coded level	Real level (%)	Coded level	Real level (g/L)	Coded level	Real level (ºC)	Observed	Predicted
1	0.00	8	1.00	6	-1.00	30	6.92 ± 0.21	7.05
2	1.00	12	-1.00	2	0.00	35	4.55 ± 0.07	4.53
3	0.00	8	1.00	6	1.00	40	7.78 ± 0.51	7.48
4	1.00	12	0.00	4	-1.00	30	6.90 ± 0.09	6.62
5	-1.00	4	0.00	4	1.00	40	5.31 ± 0.02	4.99
6	1.00	12	0.00	4	1.00	40	5.44 ± 0.11	5.59
7	1.00	12	1.00	6	0.00	35	7.16 ± 0.19	7.29
8	0.00	8	-1.00	2	-1.00	30	5.36 ± 0.44	5.65
9	-1.00	4	0.00	4	-1.00	30	4.78 ± 0.66	4.62
10	-1.00	4	-1.00	2	0.00	35	3.97 ± 0.08	3.83
11	0.00	8	-1.00	2	1.00	40	4.69 ± 0.02	4.55
12	0.00	8	0.00	4	0.00	35	5.88 ± 0.11	5.64
13	-1.00	4	1.00	6	0.00	35	5.39 ± 0.09	5.40
14	-1.00	4	0.00	4	1.00	40	4.41 ± 0.58	4.99
15	0.00	8	0.00	4	0.00	35	5.41 ± 0.06	5.64

EPS production
Variables		Coefficients	Standard error	t Stat	p-Value
Intercept		4.69	0.12	39.01	3.71
X ₁	Yeast extract	1.28	0.14	9.13	0.002
X ₂	Sugarcane molasses	1.57	0.57	2.75	0.070
X ₃	MgSO₄	-1.18	0.48	-2.42	0.093
X ₄	K₂HPO₄	0.58	0.14	4.12	0.025
X ₅	KH₂PO₄	-0.49	0.14	-3.47	0.04
X ₆	Peptone	0.65	0.14	4.61	0.01
X ₇	Incubation time	-0.13	0.14	-0.95	0.41
X ₈	Inoculum size	-0.47	0.14	-3.34	0.044
X ₉	Temperature	1.01	0.14	7.19	0.005
X ₁₀	pH	-0.32	0.14	-2.30	0.10
X ₁₁	Shaking	0.037	0.14	0.26	0.80
Regression statistics
Multiple R		0.995	Adjusted R square	0.954
R square		0.990	Standard error	0.360

Sociedade Brasileira de Microbiologia USP - ICB III - Dep. de Microbiologia, Sociedade Brasileira de Microbiologia, Av. Prof. Lineu Prestes, 2415, Cidade Universitária, 05508-900 São Paulo, SP - Brasil, Ramal USP 7979, Tel. / Fax: (55 11) 3813-9647 ou 3037-7095 - São Paulo - SP - Brazil
E-mail: bjm@sbmicrobiologia.org.br

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[1] *Corresponding author. E-mail: saad_moghannem@yahoo.com (S.A. Moghannem).

ANOVA	df	SS	MS	F	Significance F
Regression	9	16.7	1.85	10.08	0.01
Residual	5	0.91	0.18
Total	14	17.6
	Coefficients		Standard error		t Stat
Intercept	5.64		0.30		18.61
X ₁	0.64		0.14		4.43
X ₂	1.08		0.15		7.15
X ₃	-0.16		0.14		-1.13
X ₁ X ₂	0.29		0.21		1.38
X ₁ X ₃	-0.35		0.19		-1.76
X ₂ X ₃	0.38		0.21		1.78
X ₁ X ₁	-0.55		0.23		-2.33
X ₂ X ₂	0.17		0.23		0.73
X ₃ X ₃	0.36		0.23		1.55