Optimization of succinic acid production from xylose mother liquor (XML) by Actinobacillus succinogenes using response surface methodology

Figures & data

Table 1. Factor levels of PBD for screening of significant variables affecting succinic acid production and corresponding results.

Run	X1^a	X2	X3	X4	X5	X6	X7	X8	X9	Y^b
1	1	−1	1	1	1	−1	−1	−1	1	32.83 ± 0.32^c
2	1	1	−1	−1	−1	1	−1	1	1	38.84 ± 0.41
3	−1	−1	1	−1	1	1	−1	1	1	31.06 ± 0.37
4	−1	1	1	1	−1	−1	−1	1	−1	29.64 ± 0.22
5	1	−1	−1	−1	1	−1	1	1	−1	32.91 ± 0.39
6	1	−1	1	1	−1	1	1	1	−1	28.43 ± 0.44
7	1	1	1	−1	−1	−1	1	−1	1	31.36 ± 0.25
8	−1	−1	−1	1	−1	1	1	−1	1	27.82 ± 0.40
9	−1	−1	−1	−1	−1	−1	−1	−1	−1	26.58 ± 0.29
10	1	1	−1	1	1	1	−1	−1	−1	41.73 ± 0.37
11	−1	1	−1	1	1	−1	1	1	1	31.67 ± 0.46
12	−1	1	1	−1	1	1	1	−1	−1	32.69 ± 0.35

a Each variable was tested at a low (-1) and at a high level (+1) level. The two levels of each variable were: X₁ (xylose mother liquor, XML), 80 and 100 g/L; X₂ (Corn steep liquor powder, CSLP), 14 and 16 g/L; X₃ (NaHCO₃), 2 and 4 g/L; X₄ (MnCl₂), 0.6 and 0.8 g/L; X₅ (MgCO₃), 40 and 50 g/L; X₆ (KH₂PO₄), 1.5 and 2.0 g/L; X₇ (K₂HPO₄), 1.0 and 1.5 g/L; X₈ (MgCl₂), 1.0 and 1.5 g/L; X₉ (CaCl₂), 0.3 and 0.6 g/L;

b Y is the succinic acid content (g/L); ^c Each value is an average of three parallel replicates and is represented as mean ± standard deviation.

Figure 1. The results of single-factor experiment. XML concentration (a), Nitrogen sources (b), CSLP concentration (c), MgCO₃ concentration (d). XML: xylose mother liquor; CSLP: corn steep liquor powder; PM: peanut meal; SM: soybean meal, CM: cotton meal.

Table 2. The Box-Behnken design and the experimental responses of dependent variable Y (succinic acid concentration, g/L).

Run	Coded values and real values			Y (succinic acid g/L)
	X1^a	X2^b	X3^c	Experimental	Predicted
1	110 (0)	16 (-1)	60 (-1)	40.01 ± 0.46	40.81
2	110 (0)	20 (1)	60 (-1)	46.60 ± 0.37	40.83
3	115 (1)	20 (1)	65 (0)	51.71 ± 0.51	51.48
4	110 (0)	20 (1)	70 (1)	56.19 ± 0.29	53.96
5	105 (-1)	18 (0)	70 (1)	46.39 ± 0.53	51.10
6	115 (1)	16 (-1)	65 (0)	45.86 ± 0.47	46.25
7	115 (1)	20 (0)	65 (1)	56.81 ± 0.35	56.84
8	105 (-1)	20 (0)	60 (-1)	43.49 ± 0.53	43.46
9	105 (-1)	16 (-1)	65 (0)	43.26 ± 0.37	43.67
10	115 (1)	18 (0)	60 (-1)	44.25 ± 0.42	44.26
11	105 (-1)	20 (1)	65 (0)	48.98 ± 0.65	39.90
12	110 (0)	16 (-1)	70 (1)	46.46 ± 0.54	46.23
13	110 (0)	18 (0)	65 (0)	55.28 ± 0.66	55.28

a X₁: XML, xylose mother liquor (g/L);

b X₂: CSLP, corn steep liquor powder (g/L);

c X₃: MgCO₃ (g/L).

Table 3. Analysis of variance (ANOVA) and coefficient estimated for succinic acid.

Term	Coefficient	SS^a	DF	MS	F value	Pr>F
model	55.28	392.03	9	43.56	349.23	0.0002*
X1	1.45	16.72	1	16.72	134.04	0.0014*
X2	2.54	51.82	1	51.82	415.44	0.0003*
X3	5.25	220.24	1	220.24	1765.76	<0.0001*
X1*X1	−2.33	12.39	1	12.39	0.32	0.6108
X1*X2	−0.10	0.04	1	0.04	35.11	0.0096*
X1*X3	1.05	4.38	1	4.38	206.09	0.0007*
X2*X2	−5.37	65.84	1	65.84	99.33	0.0021*
X2*X3	2.53	25.70	1	25.70	527.84	0.0002*
X3*X3	−3.85	33.85	1	33.85	271.37	0.0005*
Residual		0.37	3	0.12
Cor Total		392.40	12

a SS: sum of squares; DF: Degrees of freedom; MS: mean square; Pr: probability;

* Significant at p < 0.05, R² = 0.9990, Adjusted R²= 0.9962.

Figure 2. Response surface curves of succinic acid production by A. succinogenes GXAS137 showing interaction between XML and CSLP (a), CSLP and MgCO₃ (b), XML and MgCO₃ (c). XML: xylose mother liquor; CSLP: corn steep liquor powder.

Figure 3. Time course of production of organic acids in batch fermentations with xylose mother liquor (XML). Cells were grown in a 1.3 L stirred bioreactor with a XML content of 110 g/L, corn steep liquor power (CSLP) of 18.86 g/L, and MgCO₃ of 69.12 g/L. Symbols are arabinose (■), glucose (▲), mannose (▼), acetic acid (◆) xylose (△) and succinic acid (○).

Data availability statement

The data that support the findings of this study are available from the corresponding author, [HYZ], upon reasonable request.