高效利用纤维素原料产聚羟基脂肪酸酯菌种的选育及其发酵特性

Selection and fermentation characterization of strains for efficient utilization of cellulose-based feedstocks to produce polyhydroxyalkanoates (PHA)

摘要:

背景 直接利用纤维素原料作微生物碳源来合成聚羟基脂肪酸酯(polyhydroxyalknoates, PHA)，能有效降低其生产成本，但目前仍缺乏合适的微生物菌种。目的 获得能够直接利用纤维素原料作碳源且高效合成PHA的微生物菌种，对分离菌种进行鉴定并对其发酵特性进行研究。方法 利用羧甲基纤维素(sodium carboxymethyl cellulose, CMC)-Na为唯一碳源，结合苏丹黑B染色和尼罗红染色法从自然环境中筛选目的菌株，研究分离菌株的滤纸崩解及纤维素酶活情况，通过形态学观察、生理生化反应及16S rRNA基因序列同源性比较对菌株进行分类鉴定，对菌株利用纤维素发酵产PHA的相关发酵条件如氮源种类、温度、pH、接种量等进行优化，最后分别利用傅里叶红外光谱(Fourier transform infrared spectroscopy, FT-IR)和核磁共振氢谱(nuclear magnetic resonance hydrogen spectroscopy, ¹H-NMR)对发酵产物进行分析。结果 筛选到1株高效利用纤维素产PHA的菌株FX-9，该菌能够让纤维素滤纸在短时间内崩解明显，并表现出较高的纤维素酶活性。经鉴定，该菌株为产碱杆菌属(Cupriavidus)。优化结果表明，当氮源为酵母粉，温度为30 ℃，pH 7.0，接种量为10%时，该分离菌株经84 h发酵后PHA浓度达到最大值为0.49 g/L，此时细胞内PHA含量为42.44%。经FT-IR、¹H-NMR鉴定该菌株利用纤维素原料发酵所提取的产物为聚羟基丁酸酯(polyhydroxybutyrate, PHB)。结论 分离菌株Cupriavidus sp. FX-9能直接利用纤维素原料作为碳源且高效合成PHA，这将为实现直接利用木质纤维素原料发酵生产PHA提供菌种资源及经验借鉴。

Abstract:

Background Utilizing cellulose-based feedstocks directly as carbon sources for microbial production of polyhydroxyalkanoates (PHAs) can significantly lower costs, while suitable microbial strains remain scarce.Objective To isolate and identify the microbial strains capable of directly utilizing cellulose-based feedstocks as a carbon source for efficient PHA production and then investigate their fermentation characteristics.Methods With sodium carboxymethyl cellulose as the sole carbon source, Sudan black B staining and Nile red staining were employed to screen the target strains from the natural environment. The filter paper disintegration and cellulase activity of the isolated strains were studied. The strains were identified through morphological observation, physiological and biochemical tests, and 16S rRNA sequence comparison. The fermentation conditions such as nitrogen source, temperature, pH value, and inoculation amount for the strains to produce PHAs by fermentation with cellulose were optimized. Finally, the fermentation products were analyzed by Fourier transform infrared spectroscopy (FT-IR) and nuclear magnetic resonance hydrogen spectroscopy (¹H-NMR).Results The strain FX-9 capable of efficiently utilizing cellulose to produce PHAs was screened out. This strain caused obvious disintegration of filter paper in a short period of time and showed high cellulase activity. The strain was identified as Cupriavidus sp. The optimization results showed that under the conditions of yeast powder as the nitrogen source, 30 ℃, pH 7.0, and the inoculation amount of 10%, the PHA concentration reached a maximum of 0.49 g/L after 84 h of fermentation with this strain, and the intracellular PHA content was 42.44%. FT-IR and ¹H-NMR identified that the product from fermentation of the strain using cellulose as the raw material was polyhydroxybutyrate.Conclusion The isolated strain Cupriavidus sp. FX-9 can directly utilize cellulose-based feedstocks as a carbon source for efficient PHA production. The findings provide microbial resources and practical insights for the direct fermentation of lignocellulosic biomass into PHAs.