Wool Waste for Keratinase Production Using Glucose Fed-Batch Strategy by Stenotrophomonas

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Wool waste for keratinase production using glucose fed-batch strategy by Stenotrophomonas maltophilia BBE11-1

Zhen Fanga,d, Juan Zhang a,d,*, Baihong Liu a,d, Guocheng Du b,d, Jian Chen c,d

aState Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi 214122, China

bThe Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122, China

cNational Engineering Laboratory for Cereal Fermentation Technology, Jiangnan University, Wuxi 214122, China

dSchool of Biotechnology, Jiangnan University, Wuxi 214122, China

*Corresponding author

J. Zhang, School of Biotechnology, Jiangnan University, 1800 Lihu Road, Wuxi 214122, China.

E-mail: (J. Zhang);

Telephone: +86-510-85918307

Fax: +86-510-85918309

Stenotrophomonas maltophilia BBE11-1(Fang et al, 2013) has the ability to degrade wool waste. In this report, we used it for keratinase production in scale-up fermentation.

The initial medium before optimization was modified from Cao et al. (2009): NaCl 1 g/L, K2HPO41g/L, KH2PO41g/L, wool 10 g/L, pH 7.2,which were incubated in 250 ml shake flask for 48 h. And all medium optimizations including screening signal factor, Plackett-Burman and response surface methodology were based on the initial medium.

Medium optimization in shake-flask

Selection of medium composition by single-factor-at-one-time method

The isolate could grow at wide initial pH range from 5.0 to 11.0, but pH 9.0 was the optimum initial pH for keratinase production reaching to 211 U/ml (Fig. A1). As depicted as in Fig. A1, 20-30℃is benefit for keratinase production, and 25℃is the optimum temperature. However, a slight increase to 35°C, especially reaching 40℃, resulted in a sharp decrease of keratinase production. Cell concentration rose from 15℃to 35℃, whereas obviously dropped at 40℃. Acidic medium (pH 6-7) also inhibited cell growth.

FigureA1.Effects of temperature and pH on cell growth and keratinase production.

As shown inTable A1.a, the strain produced maximum keratinase with the addition of soy peptone (314.6 U/mL). On the opposite side, urea inhibited keratinase production and achieved the lowest value (109.2 U/mL). All carbon sources could enhance keratinase production and glucose was the most excellent (287.2 U/ml).

In this study as shown in Table A1, not only Mg2+ salt but other salts (Ca2+, Zn2+, Mn2+) had a negative impact. Four amino acids were studied the effect on keratinase production and they all did an enhancement, especially the aspartic (Asp) was first reported to improve keratinase production significantly (307.8 U/mL). Furthermore, surfactants were tested, showing positive effect on degradation of wool waste (Table A1.b).

Table A1. Effect of different single factor on keratinase production.

(a)Effect of different nutritional parameters

Nitrogen sources
(5 g/L) / Keratinase production (U/ml) / Carbon sources
(5 g/L) / Keratinase production (U/ml)
Yeast extract / 295.5 / Control / 145.2
Beef extract / 189.8 / Glucose / 287.2
Soy peptone / 314.6 / Sucrose / 241.9
Casein / 155.4 / Maltose / 266.5
Urea / 109.2 / Starch / 196.4
NH4NO3 / 162.2 / Lactose / 247.2
NH4Cl / 148.8 / Dextrin / 192.8

(b)Effect of different chemicals parameters

Inorganic salts 1 g/L / Keratinase production (U/ml) / Amino acids 0.1 g/L / Keratinase production (U/ml) / Surfactants
100 μ/L / Keratinase production (U/ml)
Control / 145.2 / Control / 145.2 / Control / 145.2
MgCl2 / 138.5 / Histidine / 252.5 / Triton X-100 / 185.8
CaCl2 / 72.5 / Aspartic / 307.8 / Tween 20 / 205.5
ZnCl2 / 119.0 / Lysine / 182.6 / Tween 60 / 194.6
MnCl2 / 125.5 / Methionine / 157.3 / Tween 80 / 190.2

Plackett-Burman design for screening important medium factors

In this experiment, the medium was constituted by single factor from single-factor-at-one-time method: NaCl 0.1 g/L, K2HPO41g/L, KH2PO41g/L, soy peptone 5 g/L, glucose 5 g/L, Asp 0.1g/L,wool 10 g/L, Tween 20 100 μ/L, pH 9.0. By employing ‘Design-Expert 8.0.5’ software, a total of 8 factors and 12 experiment groups were analyzed for screening significant factors according to Plackett-Burman design in Factorial tab (Table A2.a). As shown in Table 2a, the lowest keratinase activity achieved in Plackett-Burman experiment was 370.7 U/ml while the highest was 766.6 U/ml. Based on statistical analysis as shown in Table A2.b, soy peptone was negative signal since a high concentration could inhibit keratinase production. On the contrary, the other factors glucose, Asp, wool, K2HPO4, KH2PO4 and Tween-20 did the positive impact. Compared with other data (Table A2.b), the significant factors, the most tremendous effects (Prob F less than 0.05) on keratinase production, were soy peptone, glucose, Asp.

Table A2.Plackett–Burman design with 8 factors in 2 levels for screening significant factors.

(a)Plackett–Burman design
Run / Soy peptone (g/L) / pH / Glucose (g/L) / Wool (g/L) / Asp (g/L) / K2HPO4 (g/L) / KH2PO4 (g/L) / Tween 20 (μL) / Keratinase activity (U/ml)
1 / 5 / 6 / 2 / 15 / 0.1 / 0.2 / 1 / 50 / 555.4
2 / 5 / 10 / 0.5 / 15 / 0.1 / 0.2 / 0.2 / 150 / 402.5
3 / 1 / 10 / 2 / 2 / 1 / 0.2 / 0.2 / 50 / 699.3
4 / 5 / 6 / 2 / 15 / 1 / 1 / 0.2 / 50 / 706.5
5 / 1 / 10 / 0.5 / 15 / 1 / 1 / 1 / 50 / 686.3
6 / 1 / 6 / 2 / 2 / 0.1 / 1 / 1 / 150 / 691.7
7 / 1 / 6 / 0.5 / 15 / 1 / 0.2 / 1 / 150 / 698.2
8 / 5 / 6 / 0.5 / 2 / 1 / 1 / 0.2 / 150 / 565.1
9 / 5 / 10 / 0.5 / 2 / 0.1 / 1 / 1 / 50 / 370.7
10 / 5 / 10 / 2 / 2 / 1 / 0.2 / 1 / 150 / 673.1
11 / 1 / 10 / 2 / 15 / 0.1 / 1 / 0.2 / 150 / 766.6
12 / 1 / 6 / 0.5 / 2 / 0.1 / 0.2 / 0.2 / 50 / 517.2
(b)Screening significant factors
Factors / Coefficient Estimate / Prob F / Remarks
Soy peptone / -65.4917 / 0.0104 / Negative
pH / -11.3117 / 0.3934 / Negative
Glucose / 71.05833 / 0.0083 / Positive
Wool / 24.86833 / 0.1167 / Positive
Asp / 60.37333 / 0.0131 / Positive
K2HPO4 / 20.11 / 0.1753 / Positive
KH2PO4 / 1.526667 / 0.9018 / Positive
Tween 20 / 21.815 / 0.1510 / Positive

Optimization by response surface methodology (RSM)

Three selected significant factors for the maximum production of keratinase namely soy peptone, glucose and Asp were optimized using central composite design (CCD) as shown in Table A.3a. Medium of RSM experiments was based on Plackett–Burman design (soy peptone 1.4 g/L, glucose 4 g/L, Asp 2 g/L,NaCl 0.1 g/L, K2HPO41 g/L, KH2PO41 g/L, wool 10 g/L, Tween 20 100 μ/L, pH 9.0) and incubated at 23℃for 48 h. The highest keratinase production was 782.6 U/mL.

As shown in Table A3.b, adequate precision representing the signal to noise ratio was 8.794, greater than common ratio (Ade. Pre=4). The p-values (ProbF) of model and ‘Lack of fit’ were less than 0.05, implying they are all significant.The value of R2 (multiple correlation coefﬁcient) was 0.8958 and adjusted determination coefﬁcient (Adj-R2) was 0.8021 indicating that regression equation has a better fit to navigate the design space. A low coefﬁcient of variation (6.72%) indicated that experiment was highly reliable. Consequently, the experimental data could perfectly fit with the model.

Fitting the experimental data with polynomial regression analysis, a quadratic regression equation was established as following:

Y=770.84 + 11.88A + 7.36B－42.86C－1.80AB + 1.38AC + 3.10BC－90.15A2－30.26B2－44.41C2 (2)

A is soy peptone; B is glucose; C is Asp, they are all in coded levels.

Table A3.Central composite design with three factors (g/L) for keratinase production and ANOVA analysis.

(a)Central composite design of RSM
Run / Soy peptone / Glucose / Asp / Keratinase production (U/mL)
Actual / Predicted
1 / 0.8 (-1) / 2 (-1) / 1 (-1) / 658.2 / 632.32
2 / 2 (+1) / 2 (-1) / 1 (-1) / 660.8 / 656.93
3 / 0.8 (-1) / 6 (+1) / 1 (-1) / 656.2 / 644.44
4 / 2 (+1) / 6 (+1) / 1 (-1) / 642.2 / 661.85
5 / 0.8 (-1) / 2 (-1) / 3 (+1) / 599.6 / 537.64
6 / 2 (+1) / 2 (-1) / 3 (+1) / 598.3 / 567.76
7 / 0.8 (-1) / 6 (+1) / 3 (+1) / 600.6 / 562.16
8 / 2 (+1) / 6 (+1) / 3 (+1) / 601.5 / 585.07
9 / 0.391 (-α) / 4 (0) / 2 (0) / 434.2 / 495.88
10 / 2.409 (+α) / 4 (0) / 2 (0) / 537.7 / 535.85
11 / 1.4 (0) / 0.636 (-α) / 2 (0) / 620.6 / 672.89
12 / 1.4 (0) / 7.364 (+α) / 2 (0) / 690.1 / 697.64
13 / 1.4 (0) / 4 (0) / 0.318 (-α) / 724.7 / 717.30
14 / 1.4 (0) / 4 (0) / 3.682 (+α) / 505.9 / 573.13
15 / 1.4 (0) / 4 (0) / 2 (0) / 752.6 / 770.84
16 / 1.4 (0) / 4 (0) / 2 (0) / 782.6 / 770.84
17 / 1.4 (0) / 4 (0) / 2 (0) / 780.4 / 770.84
18 / 1.4 (0) / 4 (0) / 2 (0) / 771.4 / 770.84
19 / 1.4 (0) / 4 (0) / 2 (0) / 779.8 / 770.84
20 / 1.4 (0) / 4 (0) / 2 (0) / 768.5 / 770.84
(b)ANOVA for response surface quadratic model
Source / F-value / ProbF / Value
Model / 9.55 / 0.0008
Lack of fit / 29.99 / 0.0010
Mean / 658.29
Coefﬁcient of variance / 6.72%
R2 / 0.8958
Adj-R2 / 0.8021
Adequate precision / 8.794

The factors havingsignificant influence on the response (keratinase production) were C, squared terms A, B and C. As shown in Fig. A.2a-c, circular contour plots are the equivalent value of keratinase production, corresponding to X-Y axis when the third variable is fixed. Design Expert software showed the optimal levels of three factors were 1.5 g/L Asp, 1.45 g/L soy peptone and 4.25 g/L glucose. In order to verify the predicted values, the optimal medium parameters were used for keratinase production. A total activity of 782.7 U/mL was obtained, close to the predicted value and more than 5-fold increase of keratinase production compared to the non-optimization medium. It was interesting that medium optimization with response surface methodology (RSM) was a powerful implement to promote keratinase production. The optimal media were 1.5 g/L Asp, 1.45 g/L soy peptone, 4.25 g/L glucose, 10 g/L wool, 1 g/L K2HPO4, 1 g/L KH2PO4, 1 g/L NaCl, 100 μL Tween-20 and initial pH 9.0, incubated at 23℃and 200 rpm for 48 h.

Figure A2.Response surface plot of two factors on keratinase production. (a) glucose and soy peptone as factors at a ﬁxed concentration of Asp; (b) Asp and soy peptone as factors at a ﬁxed concentration of glucose; (c) glucose and Asp as factors at a ﬁxed concentration of soy peptone.

Figure A3. Scanning electron micrographs (SEM) and Fourier transform infrared spectroscopy (FTIR) of wool structure. (a) Native wool; (b) wool after 2 days biodegradation by S. maltophilia BBE11-1.

Figure A4. Effects of pH and temperature on crude keratinase activity.

References

Cao, Z.J., Zhang, Q., Wei, D.K., Chen, L., Wang, J., Zhang, X.Q., Zhou, M.H., 2009. Characterization of a novel Stenotrophomonas isolate with high keratinase activity and purification of the enzyme. J. Ind. Microbiol. Biotechnol. 36,181-188.
Fang, Z., Zhang, J., Liu, B.H., Du G.C., Chen, J, 2013. Biochemical characterization of three keratinolytic enzymes from Stenotrophomonas maltophilia BBE11-1 for biodegrading keratin wastes. Int. Biodeter. Biodegr. 82, 166-172.