贪铜菌利用混合餐厨废油合成聚羟基丁酸酯

doi:10.13560/j.cnki.biotech.bull.1985.2020-1107

摘要/Abstract

摘要：

餐厨废油是餐厨垃圾处理过程中的附加产物,是一种高含碳量的碳氢化合物。利用贪铜菌（Cupriavidus necator）以混合餐厨废油为单一碳源合成可生物降解塑料——聚羟基丁酸酯（PHB）,分析菌株利用餐厨废油合成PHB的最佳条件,并对所合成的PHB进行了性质研究。通过进一步发酵培养,探究对比了不同发酵模式下菌株利用餐厨废油合成PHB的产量,为未来产业化奠定基础。结果表明：在30℃,150 r/min,pH 7.0,3%餐厨废油添加量和接种量条件下,Cupriavidus necator的生长量与 PHB 合成量均达到最大值,分别为9.5 g/L和7.6 g/L。利用5 L发酵罐进行分批培养,PHB的产量在发酵96 h后达到8.25 g/L。本研究利用餐厨废油合成的PHB平均分子量为30 kD,分散性指数（PDI）为1.44。PHB的熔化温度为175.7℃,分解温度为285.5℃,可以满足热塑性材料的需求。

关键词: 餐厨废油, 生物塑料, 聚羟基脂肪酸酯, Cupriavidus necator

Abstract:

Waste cooking oil,a kind of hydrocarbon with high carbon content,is one of the main valuable products from the disposal process of kitchen waste. In this study,Cupriavidus necator was employed to synthesize degradable bio-plastics,i.e.,polyhydroxybutyrate（PHB）,with mixed waste cooking oil as sole carbon source. The operational conditions of the PHB synthesis from the waste cooking oil were optimized,and the properties of the synthesized PHB were studied. To lay a foundation for the industrialization of PHB in the future,the PHB yields under different modes of fermentation were explored and compared. The results showed that the maximum yields of the C. necator cell dry weight and PHB were 9.5 g/L and 7.6 g/L under the conditions of 30℃,150 r/min,and pH = 7.0 with amount of 3% waste oil and inoculation addition. Furthermore,the yield of PHB reached 8.25 g/L after fermentation for 96 h by batch culture in a 5 L bio-reactor. The average molecular mass of the synthesized PHB in this study was 30 kD,and the polydispersity index（PDI）was 1.44. The melting temperature of PHB was 175.7℃ and a decomposition temperature of it was 285.5 ^oC,which may meet the requirements of thermoplastic materials.

Key words: waste cooking oil, bio-plastics, polyhydroxybutyrate, Cupriavidus necator

潘兰佳, 李杰, 林清怀, 汪印. 贪铜菌利用混合餐厨废油合成聚羟基丁酸酯[J]. 生物技术通报, 2021, 37(4): 127-136.

PAN Lan-jia, LI Jie, LIN Qing-huai, WANG Yin. Polyhydroxybutyrate Production from Mixed Waste Cooking Oil by Cupriavidus necator[J]. Biotechnology Bulletin, 2021, 37(4): 127-136.

图/表 12

表1 餐厨废油的脂肪酸含量

Table 1 Fatty acid content of the waste cooking oil

脂肪酸Fatty acid	含量Content/%
十六烷酸Palmitic acid（C16：0）	14.887
十八烷酸Stearic acid（C18：0）	4.097
十八碳烯酸Octadecenoic acid（C18：1）	31.000
亚麻酸Linolenic acid（C18：3）	28.380
亚油酸Linoleic acid（C18：2）	18.086
其他Others	3.550

图1 PHA甲酯化产物气相色谱质谱鉴定图

Fig. 1 Identification of methylated products of PHA by gas chromatography-mass spectrometry

图2 温度对菌株C. necator细胞干重和PHB合成的影响不同大写字母表示各处理组中的菌体干重有显著差异（P<0.05）;不同小写字母表示各处理组中的PHA占比有显著差异（P<0.05）,下同

Fig. 2 Effect of temperature on the cell dry weight of strain C. necator and PHB synthesis Different capital letters represent significant difference (P < 0.05); different lowercase letters represent the percentage of PHB has significant difference (P < 0.05), the same below

图3 pH对菌株C. necator细胞干重和PHB合成的影响

Fig.3 Effect of pH on the cell dry weight of strain C. necator and PHB synthesis

图4 摇床转速对菌株C. necator 细胞干重和PHB合成的影响

Fig.4 Effect of rotation speed on the cell dry weight of strain C. necator and PHB synthesis

图5 餐厨废油添加量对菌株C. necator 细胞干重和PHB合成的影响

Fig.5 Effect of waste cooking oil amount on the cell dry weight of strain C. necator and PHB synthesis

图6 接种量对菌株C. necator细胞干重和PHB合成的影响

Fig.6 Effect of inoculum amount on the cell dry weight of strain C. necator and PHB synthesis

图7 培养时间对菌株C. necator细胞干重和PHB合成的影响

Fig.7 Effect of culture time on the cell dry weight of strain C. necator and PHB synthesis

表2 不同发酵模式下菌体和PHB产量

Table 2 Bacterial cell and PHB production under different fermentation modes

模式 Mode	菌体干重 Cell dry weight/（g·L^-1）	PHB含量 PHB content/wt%	PHB/（g·L^-1）
M1	10.75	76.75	8.25
M2	7.62	86.88	6.62
M3	6.79	82.06	5.57

表3 模式一发酵培养实验结果

Table 3 Experiment results of fermentation model 1

循环 Circulation	菌体干重 Cell dry weight/（g·L^-1）	PHB含量 PHB content /wt%	PHB/（g·L^-1）
1	10.75	76.75	8.25
2	12.38	76.77	9.50
3	11.10	76.22	8.46
4	12.12	77.36	9.38

表4 细菌合成PHB的分子量分布和热力学性质

Table 4 Molecular weight distribution and thermodynamic properties of PHB synthesized by bacteria

菌种Strain	碳源Carbon source	M_n/kD	M_w/kD	PDI	熔化温度 Melting temperature/℃	分解温度Decomposition temperature/℃	参考文献Reference
B. thailandensis	餐厨废油 Waste cooking oil	179	511	2.86	166.4	279.3	[17]
C. necator DSM428	餐厨废油 Waste cooking oil	110	170	1.5	168.6	-	[19]
C. necator DSM428	咖啡渣油 Coffee residue oil	-	234	1.2	172.3	-	[20]
C. necator（CGMCC 1.7092）	混合餐厨废油 Mixed waste cooking oil	20	30	1.44	175.7	285.5	本研究 This study

图8 差示扫描量热分析（A）和热重分析（B）结果图

Fig.8 Differential scanning calorimetry analysis (A) and thermogravimetric analysis (B)

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