基于菌糟固定化假单胞菌MJ的正二十烷绿色处理新工艺的开发

Development of a novel green treatment process for n-eicosane based on the spent mushroom substrate-immobilized Pseudomonas sp. MJ

摘要:

生物强化是处理石油污染的常用技术，然而外源菌株常因环境适应性差而难以在污染场地中有效定殖，限制了其修复效率。为解决外源菌株难以长期维持活性以及农业废弃物菌糟亟待绿色处理的问题，本研究以农业废弃物菌糟为新型固定化载体，制备高效烷烃降解菌剂，并使用正交法对制备工艺进行优化。结果表明，菌糟的纤维素结晶度高达73.63%，同时含有羟基和羧基等官能团及高含量可溶性营养物质，适合作为微生物载体。此外，筛选出一株烷烃降解菌假单胞菌 (Pseudomonas sp.) MJ，将其固定在菌糟上，制备成固定化菌剂，最佳制备条件为：固定化时间72 h、菌糟粒径0.25-0.30 mm、菌糟:菌液=1:4 (质量体积比)。该条件下，菌糟固定化菌剂具有良好的菌株保留能力和长期稳定性，4 ℃保存30 d后的载菌量仍可保持1.05×10¹⁰ CFU/g，同时其具有良好的烷烃降解能力，72 h内对500 mg/L正二十烷的降解率达到95.80%，是游离菌株的11.24倍。本研究为烷烃污染的生物修复提供了一种高效的固定化菌剂，并为农业废弃物菌糟的绿色处理提供了新的途径。

Abstract:

Bioaugmentation is a widely employed strategy for addressing petroleum contamination. However, exogenous strains often struggle to effectively colonize contaminated sites due to their poor environmental adaptability, which constrains the efficacy of remediation efforts. To address the challenges of sustaining long-term activity of exogenous microbial strains and the pressing need for green treatment of the spent mushroom substrate, that is agriculture waste. In this study, we explored the utilization of agricultural waste, specifically spent mushroom substrate, as an innovative immobilization carrier for the development of efficient alkane-degrading microbial agents. The preparation process was optimized by the orthogonal method. Experimental findings indicated that the mushroom substrate had the cellulose crystallinity reaching 73.63%, functional groups such as hydroxyl and carboxyl, and high concentrations of soluble nutrients, serving as an appropriate microbial carrier. Furthermore, an alkane-degrading bacterial strain, Pseudomonas sp. MJ, was isolated and immobilized on the spent mushroom substrate to formulate an immobilized microbial agent. The optimal preparation conditions were determined as follows: immobilization duration of 72 h, a mushroom substrate particle size of 0.25-0.30 mm, and a carrier-to-strain ratio of 1:4 (M/V). Under these conditions, the mushroom substrate-immobilized microbial agent demonstrated excellent strain retention capability and long-term stability. After storage at 4 °C for 30 d, the bacterial load remained at 1.05×10¹⁰ CFU/g. Moreover, the agent demonstrated excellent alkane degradation proficiency, with the degradation rate of 500 mg/L N-eicosane within 72 h reaching 95.80%, which is 11.24 times that of the free strain. This study presents an efficient immobilized microbial agent for the bioremediation of alkane pollution and introduces a novel approach for the sustainable treatment of agricultural waste, specifically spent mushroom substrate.