Highly efficient screening and optimal combination of functional isolates for bioremediation of hydrocarbon-polluted soil.

The key to enhancing the efficacy of bioremediation of hydrocarbon-contaminated soil is the precise and highly efficient screening of functional isolates. Low screening effectiveness, narrow screening range and an unstable structure of the constructed microflora during bioremediation are the shortcomings of the traditional shaking culture (TSC) method. To improve the secondary screening of isolates and microflora implemented for alkane degradation, this work evaluated the characterization relationship between bacterial function and enzyme activity and devised an enzyme activity assay (EAA) method. The results indicated a substantial positive correlation (r = 0.97) between 24 candidate isolates and their whole enzymes, proving that whole enzyme activity properly reflects the metabolic functions of microorganisms. The functional analysis of the isolates demonstrated that the EAA method in conjunction with microbial abundance and metabolite determination could broaden the screening range of functional isolates, including aliphatic acid-metabolizing isolates (isolates H4 and H7) and aliphatic acid-sensitive isolates (isolate H2) with n-hexadecane degradation ability. The EAA method also guided the construction of functional microflora and optimized the mode of application using combinations of alkane-degrading bacteria and aliphatic acid-degrading bacteria successively (e.g., F1+H7+H7). The combinations maintained a high abundance of functional isolates and stable α diversity and community composition throughout the experiment, which contributed to more advanced alkane degradation and mineralization ability (p < 0.01). Assuming a workload of 100 tests, the screening efficiency of the EAA method is more than 16 times that of the TSC method, and the greater the quantity of isolates, the higher the screening efficiency, enabling high-throughput screening. In conclusion, the EAA method has a broad-spectrum, accurate and highly efficient screening ability for functional isolates and microflora, which can provide intensive technical support for the development of bioremediation materials and the application of bioremediation technology.