Optimization of Degradation Conditions of Paraffin⁃Degrading Mixed Bacteria Based on Orthogonal Experiment and Response Surface Methodology

doi:10.12422/j.issn.1672-6952.2024.05.004

Abstract

Abstract:

Microbial dewaxing of crude oil is an efficient and simple method. In order to further improve the paraffin degradation rate of microorganisms, two strains L and K were used to form paraffin degradation mixed bacteria, and their degradation conditions were optimized. The factors affecting the degradation of mixed bacteria (culture temperature, initial pH value of culture medium, salt concentration, and V（L strain ）/V（K strain）) were determined by orthogonal experiment and single factor experiment. Box-Behnken method was used to design four?factor three?level experiment and response surface optimization, and a mathematical model was established to explore the best conditions for the degradation of paraffin by mixed bacteria. The experimental results showed that the influence of four single factors is as follows: V（L strain）/V（K strain）>culture temperature>salt concentration>initial pH value of culture medium. Their primary term, secondary term and individual interaction term had significant influence on the paraffin degradation rate. The optimal experimental operating conditions for the mixed bacteria to degrade paraffin were determined as follows: culture temperature 37.2 ℃, initial pH value of culture medium 7.3, salt concentration of culture medium 1.2%, ratio of mixed bacteria V（L strain）/V（K strain）=1.0∶1.6, and paraffin degradation rate 58.67%. The mixed bacteria were used to act on crude oil under the optimized conditions, and the biodegradation rate was 41.38%,have good application prospects.

Key words: Orthogonal experiment, Response surface optimization, Paraffin degradation rate, Mixed bacteria, Influence factor

摘要：

微生物作用于原油除蜡是一种高效简约的方法。为进一步提高微生物的石蜡降解率，混合两种菌株L和K配制了石蜡降解混合菌；通过正交实验和单因素实验确定了影响混合菌降解的因素（培养温度、培养基初始pH、盐质量分数、V（L菌）/V（K菌））；采用Box-Behnken方法设计四因素三水平实验及响应面优化，并建立数学模型，探究了混合菌降解石蜡的最佳条件。结果表明，4种单因素的影响大小顺序为V（L菌）/V（K菌）>培养温度>盐质量分数>培养基初始pH，其中一次项、二次项以及个别交互项对石蜡降解率影响显著；混合菌降解石蜡的最优条件：培养温度为37.2 ℃、培养基初始pH=7.3，培养基盐质量分数为1.2%，V（L菌）/V（K菌）=1.0∶1.6，石蜡降解率可达58.67%；混合菌在优化条件下作用于原油，生物降解率为41.38%，有良好的应用前景。

关键词: 正交实验, 响应面优化, 石蜡降解率, 混合菌, 影响因素

CLC Number:

TE832

Kuo LI, Hui WANG, Shengnan DU, Weiqiang WANG. Optimization of Degradation Conditions of Paraffin⁃Degrading Mixed Bacteria Based on Orthogonal Experiment and Response Surface Methodology[J]. Journal of Liaoning Petrochemical University, 2024, 44(5): 22-30.

李阔, 王慧, 杜胜男, 王卫强. 基于正交实验和响应面法优化石蜡降解混合菌的降解条件研究[J]. 辽宁石油化工大学学报, 2024, 44(5): 22-30.

Figures/Tables 10

Table 1 Horizontal factor design of orthogonal experiment

水平	因素
水平	温度（A）/℃	初始pH（B）	w（盐）（C）/%	培养时间（D）/h	碳源（E）	摇床转速（F）/(r $⋅$ min^-1)
1	31	5.0	0.1	24	淀粉	0
2	35	6.0	0.5	48	蔗糖	90
3	39	7.0	1.0	72	液体石蜡	120
4	43	8.0	2.0	96	固体石蜡	150
5	47	9.0	3.0	120	C16	180

Table 1 Horizontal factor design of orthogonal experiment

水平	因素
水平	温度（A）/℃	初始pH（B）	w（盐）（C）/%	培养时间（D）/h	碳源（E）	摇床转速（F）/(r $⋅$ min^-1)
1	31	5.0	0.1	24	淀粉	0
2	35	6.0	0.5	48	蔗糖	90
3	39	7.0	1.0	72	液体石蜡	120
4	43	8.0	2.0	96	固体石蜡	150
5	47	9.0	3.0	120	C16	180

Table 2 Experimental design results of orthogonal experiments for L bacteria

序号	水平						D₆₀₀
序号	A	B	C	D	E	F	D₆₀₀
1	1	1	1	1	1	1	0.045
2	1	2	2	2	2	2	0.311
3	1	3	3	3	3	3	0.392
4	1	4	4	4	4	4	0.320
5	1	5	5	5	5	5	0.114
6	2	1	2	3	4	5	0.324
7	2	2	3	4	5	1	0.428
8	2	3	4	5	1	2	0.339
9	2	4	5	1	2	3	0.192
10	2	5	1	2	3	4	0.332
11	3	1	3	5	2	4	0.220
12	3	2	4	1	3	5	0.135
13	3	3	5	2	4	1	0.383
14	3	4	1	3	5	2	0.333
15	3	5	2	4	1	3	0.250
16	4	1	4	2	5	3	0.226
17	4	2	5	3	1	4	0.309
18	4	3	1	4	2	5	0.250
19	4	4	2	5	3	1	0.146
20	4	5	3	1	4	2	0.096
21	5	1	5	4	3	2	0.135
22	5	2	1	5	4	3	0.088
23	5	3	2	1	5	4	0.113
24	5	4	3	2	1	5	0.222
25	5	5	4	3	2	1	0.215
x₁	0.236	0.190	0.210	0.116	0.233	0.243
x₂	0.323	0.254	0.229	0.295	0.238	0.243
x₃	0.264	0.295	0.272	0.315	0.228	0.230
x₄	0.205	0.243	0.247	0.277	0.242	0.259
x₅	0.155	0.201	0.227	0.181	0.243	0.209
R	0.168	0.105	0.062	0.199	0.015	0.050
最佳值	A2	B3	C3	D3	E5	F4

Table 3 Experimental design results of orthogonal experiments for K bacteria

序号	水平						D₆₀₀
序号	A	B	C	D	E	F	D₆₀₀
1	1	1	1	1	1	1	0.089
2	1	2	2	2	2	2	0.295
3	1	3	3	3	3	3	0.486
4	1	4	4	4	4	4	0.340
5	1	5	5	5	5	5	0.144
6	2	1	2	3	4	5	0.423
7	2	2	3	4	5	1	0.417
8	2	3	4	5	1	2	0.355
9	2	4	5	1	2	3	0.137
10	2	5	1	2	3	4	0.274
11	3	1	3	5	2	4	0.325
12	3	2	4	1	3	5	0.223
13	3	3	5	2	4	1	0.262
14	3	4	1	3	5	2	0.396
15	3	5	2	4	1	3	0.456
16	4	1	4	2	5	3	0.207
17	4	2	5	3	1	4	0.260
18	4	3	1	4	2	5	0.386
19	4	4	2	5	3	1	0.367
20	4	5	3	1	4	2	0.274
21	5	1	5	4	3	2	0.102
22	5	2	1	5	4	3	0.113
23	5	3	2	1	5	4	0.089
24	5	4	3	2	1	5	0.135
25	5	5	4	3	2	1	0.110
x₁	0.271	0.229	0.252	0.162	0.259	0.249
x₂	0.321	0.262	0.326	0.235	0.251	0.284
x₃	0.332	0.316	0.327	0.335	0.290	0.280
x₄	0.299	0.275	0.247	0.340	0.282	0.258
x₅	0.110	0.252	0.181	0.261	0.251	0.262
R	0.222	0.087	0.146	0.178	0.039	0.035
最佳值	A3	B3	C3	D4	E3	F2

Fig.1 Wax reduction rate at different V（L strain）/V（K strain）

Table 4 Box?Behnken experimental factor level design

水平	因素
水平	温度/℃	初始pH	w（盐）/%	V（L菌）/ V（K菌）
-1	35	6.0	0	1 $∶$ 1
0	37	7.0	1.0	2 $∶$ 3
1	39	8.0	2.0	1 $∶$ 2

Table 4 Box?Behnken experimental factor level design

水平	因素
水平	温度/℃	初始pH	w（盐）/%	V（L菌）/ V（K菌）
-1	35	6.0	0	1 $∶$ 1
0	37	7.0	1.0	2 $∶$ 3
1	39	8.0	2.0	1 $∶$ 2

Table 5 Experimental design and results of Box?Behnken

序号	温度	初始pH	w（盐）	V（L菌）/V（K菌）	石蜡降解率/%	序号	温度	初始pH	w（盐）	V（L菌）/ V（K菌）	石蜡降解率/%
1	-1	0	-1	0	51.00	7	0	1	1	0	56.12
2	0	0	1	1	54.81	8	0	0	1	-1	50.29
3	0	0	0	0	58.12	9	0	0	0	0	58.60
4	-1	0	0	-1	43.18	10	0	1	0	-1	48.43
5	0	1	0	1	55.99	11	0	0	0	0	57.95
6	0	-1	0	1	52.61	12	1	0	0	1	52.40
13	1	0	1	0	52.89	22	0	0	-1	1	53.96
14	1	1	0	0	54.15	23	1	0	0	-1	45.88
15	1	-1	0	0	52.51	24	0	0	-1	-1	46.40
16	0	0	0	0	59.06	25	0	-1	1	0	56.13
17	0	-1	-1	0	55.15	26	-1	-1	0	0	51.69
18	1	0	-1	0	52.39	27	0	1	-1	0	55.36
19	-1	1	0	0	52.36	28	-1	0	1	0	52.10
20	0	0	0	0	57.30	29	-1	0	0	1	50.07
21	0	-1	0	-1	48.22

Fig.2 The relationship between the measured values and the predicted values of the regression model on the degradation rate of paraffin

Table 6 Analysis results of variance of multiple regression model

来源	平方和	自由度	均方	F	P	显著性
模型	433.02	14	30.93	64.28	<0.000 1	**
A	8.04	1	8.04	17.05	0.001 1	**
B	3.10	1	3.10	6.45	0.023 8	*
C	5.44	1	5.44	11.03	0.004 7	**
D	116.81	1	116.81	242.81	<0.000 1	**
AB	0.235 2	1	0.235 2	0.488 9	0.496 6	——
AC	0.090 0	1	0.090 0	0.254 6	0.672 5	——
AD	0.034 2	1	0.034 2	0.071 1	0.793 9	——
BC	0.012 1	1	0.012 1	0.025 2	0.876 5	——
BD	2.51	1	2.51	5.22	0.038 7	*
CD	2.31	1	2.31	4.80	0.046 1	*
A²	138.01	1	138.01	285.82	<0.000 1	**
B²	7.85	1	7.85	16.44	0.001 2	**
C²	12.09	1	12.09	24.82	0.000 2	**
D²	207.80	1	207.80	432.58	<0.000 1	**
残差	6.76	14	0.482 7
失拟项	4.98	10	0.497 9	1.12	0.497 0	——
纯误差	1.78	4	0.444 6
总和	439.77	28

Fig.3 Response surface and contour plots of each factor affecting the wax reduction rate

Table 7 The quality of crude oil before and after biodegradation by mixed bacteria

组别	初始原油质量/g	剩余原油质量/g
对照组	5.000	4.455
实验组	5.000	2.386

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