Utilization of acid hydrolysate of recovered bacterial cell as a novel organic nitrogen source for L-tryptophan fermentation

Figures & data

Figure 1. Preparation and utilization of WBCH. WBC refers to waste bacterial cell recovered by centrifugation. WBCH refers to waste bacterial hydrolysate produced by acid hydrolysis.

Table 1. Experimental design for screening of variables using the Plackett-Burman method.

Trial	A	B	C	D	E	F	G	L-tryptophan yield (g/L)
1	1.00	1.00	1.00	−1.00	−1.00	−1.00	1.00	7.36 ± 0.06
2	−1.00	1.00	1.00	−1.00	1.00	1.00	1.00	7.95 ± 0.08
3	1.00	1.00	−1.00	−1.00	−1.00	1.00	−1.00	8.36 ± 0.06
4	−1.00	−1.00	1.00	−1.00	1.00	1.00	−1.00	9.13 ± 0.08
5	−1.00	1.00	1.00	1.00	−1.00	−1.00	−1.00	8.72 ± 0.07
6	−1.00	1.00	−1.00	1.00	1.00	−1.00	1.00	6.82 ± 0.06
7	−1.00	−1.00	−1.00	1.00	−1.00	1.00	1.00	9.16 ± 0.07
8	1.00	−1.00	−1.00	−1.00	1.00	−1.00	1.00	8.24 ± 0.05
9	1.00	−1.00	1.00	1.00	1.00	−1.00	−1.00	8.93 ± 0.06
10	1.00	−1.00	1.00	1.00	−1.00	1.00	1.00	9.25 ± 0.06
11	−1.00	−1.00	−1.00	−1.00	−1.00	−1.00	−1.00	9.01 ± 0.08
12	1.00	1.00	−1.00	1.00	1.00	1.00	−1.00	8.18 ± 0.06

± represents standard errors of the means.

Table 2. Experimental results of the path of steepest ascent.

Trial	WBCH (g/L)	KH2PO4 (g/L)	VH (mg/L)	L-tryptophan yield (g/L)
1	20	2	1	8.85 ± 0.05
2	22	2.2	1.2	9.32 ± 0.07
3	24	2.4	1.4	9.11 ± 0.06
4	26	2.6	1.6	9.04 ± 0.06
5	28	2.8	1.8	8.76 ± 0.05

± represents standard errors of the means.

Table 3. Optimization of the medium components for improving the L-tryptophan yield using Box-Benhnken design.

Trial	X1 WBCH (g/L)	X2 KH2PO4 (g/L)	X3 VH (mg/L)	L-tryptophan yield (g/L)
1	18	2.6	1.2	10.22 ± 0.05
2	22	2.6	1.6	11.57 ± 0.05
3	22	1.8	0.8	9.73 ± 0.05
4 (c)	22	2.2	1.2	12.50 ± 0.06
5	22	2.6	0.8	10.06 ± 0.05
6	26	1.8	1.2	10.86 ± 0.05
7	22	1.8	1.6	10.92 ± 0.05
8	18	1.8	1.2	9.18 ± 0.05
9	26	2.2	0.8	11.30 ± 0.05
10 (c)	22	2.2	1.2	12.32 ± 0.06
11	26	2.6	1.2	11.18 ± 0.05
12	18	2.2	1.6	10.80 ± 0.05
13	18	2.2	0.8	9.13 ± 0.05
14 (c)	22	2.2	1.2	12.27 ± 0.06
15	26	2.2	1.6	10.65 ± 0.05

± represents standard errors of the means.

Figure 2. Effect of sulfuric acid concentration (a), temperature (b), and reaction duration (c) on WBC hydrolysis. The total amino acid yield is the mass ratio of extracted total amino acid to dry WBC.

Table 4. Comparison of amino acids in YE, WBC extract, and WBCH.

Amino acid	Yeast extract/(mg/g)^a	WBC extract/(mg/g)^b	WBCH/(mg/g)^c
Asparagine	16.10 ± 0.08	5.52 ± 0.08	0.86 ± 0.02
Tyrosine	14.20 ± 0.07	3.06 ± 0.07	0.48 ± 0.03
Histidine	5.50 ± 0.06	28.38 ± 0.15	4.43 ± 0.05
Proline	8.90 ± 0.05	2.22 ± 0.06	0.35 ± 0.02
Glycine	11.10 ± 0.10	11.58 ± 0.08	1.81 ± 0.02
Methionine	12.00 ± 0.09	6.54 ± 0.05	1.02 ± 0.02
Phenylalanine	12.20 ± 0.11	5.94 ± 0.06	0.93 ± 0.02
Serine	14.60 ± 0.13	2.28 ± 0.08	0.36 ± 0.03
Isoleucine	14.80 ± 0.12	3.78 ± 0.03	0.59 ± 0.01
Lysine	15.20 ± 0.11	8.94 ± 0.06	1.40 ± 0.03
Threonine	15.09 ± 0.14	10.02 ± 0.08	1.57 ± 0.02
Arginine	21.00 ± 0.16	3.18 ± 0.05	0.50 ± 0.03
Valine	22.10 ± 0.16	10.62 ± 0.09	1.66 ± 0.04
Leucine	30.60 ± 0.18	9.72 ± 0.06	1.52 ± 0.03
Alanine	41.10 ± 0.12	14.1 ± 0.06	2.20 ± 0.05
Glutamic Total	58.20 ± 0.15 312.69	19.44 ± 0.12 145.32	3.04 ± 0.06 22.72

^aMass ratio of extracted total amino acids to dry yeast cell.

^bMass ratio of extracted total amino acids to dry WBC.

^cMass ratio of extracted total amino acids to WBCH.

± represents standard errors of the means.

Table 5. F-test and significance levels calculated from Plackett-Burman experiments.

Trial	Factors (g/L)	Level		F value	Pr > F	Significance
		Low (−1) High (+1)
A	(NH4)2SO4	4	5	0.17	0.7052	NS
B	WBCH	20	25	29.98	0.0054	*
C	Citric acid	2	2.5	1.84	0.2460	NS
D	KH2PO4	2	2.5	6.51	0.0432	*
E	MgSO4·7H2O	2	2.5	5.10	0.0869	S
F	FeSO4·7H2O	0.15	0.188	0.76	0.4316	NS
G	VH (mg/L)	1	1.25	9.43	0.0373	*

Table 6. Variance analysis of regression equation of Box-Benhnken design.

Factor	Sum of squares	Degree of freedom	Mean square	F value	Pr > F
Model	16.29	9	1.81	18.76	0.0024*
X1-WBCH	2.71	1	2.71	28.15	0.0032*
X2-KH2PO4	0.68	1	0.68	7.10	0.0447*
X3-VH	1.73	1	1.73	17.94	0.0082*
X1X2	0.13	1	0.13	1.34	0.2987
X1X3	1.35	1	1.35	13.95	0.0135*
X2X3	0.026	1	0.026	0.27	0.6284
X1^2	4.48	1	4.48	46.47	0.0010*
X2^2	3.70	1	3.70	38.42	0.0016*
X3^2	2.94	1	2.94	30.44	0.0027*
Residual	0.48	5	0.096
Lack of fit	0.42	3	0.14	4.41	0.1902 (NS)
Pure error	0.063	2	0.032
Total variation	16.77	14
R^2 = 0.9712			Adjustment R^2 = 0.9595

Figure 3. Combined effect of (a) WBCH and KH₂PO₄ concentration; (b) WBCH and V_H concentration; (c) KH₂PO₄ and V_H concentration on L-tryptophan production.

Figure 4. Time courses of (a) biomass formation; (b) specific growth rate; (c) L-tryptophan titer; and (d) L-tryptophan production rate by E. coli using WBCH medium and YE medium.

Table 7. Calculation of production cost of WBCH.

Materials	Consumption	Unit price (RMB)	Cost(RMB/kg)
WBC	0.16 (kg)	2.0	0.320
Sulfuric acid	0.23 (kg)	0.6	0.138
Ammonium hydroxide	0.06 (kg)	2.0	0.12
Steam (0.8 MPa)	0.07 (kg)	0.08	0.056
Electricity	0.04 (KW·h)	0.8	0.032
Total			0.666