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Research on safety and compliance of imported microbial inoculants using high-throughput sequencing
Lin Dong1, Zilong Zhang2, Biyun Zhu1
1School of Perfume and Aroma Technology, Shanghai Institute of Technology, Shanghai, China.
Abstract:
Microbial inoculants are widely used in wastewater treatment, soil remediation, and biological control. Safety and compliance for active constituents are considered to be the most important measures of imported microbial inoculants. Microbial inoculants composition was commonly identified by phenotypic culture, which is time-consuming and labor intense with occasionally false negative results provided, and can only be tested for specific species. High-throughput sequencing (HTS), known for its non-targeted detection of unknown species composition in samples, is suitable for composition consistency identification and biosafety analysis of imported microbial inoculants. In this study, the application of HTS for microflora distribution and resistance gene was verified in microbial inoculants for environmental protection and then applicated in imported microbial inoculants. Both Illumina- and Nanopore-based HTS methods identified the same dominant bacterial species successfully in the imported microbial inoculants. The main component of bacterial species was Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus licheniformis, and Enterococcus faecium, and further confirmed with traditional methods. The antibiotic resistance genes Bacillus subtilis mprF, bcrA, blt, lmrB, rphB, tet(L), tmrB, vmlR, ykkC, and ykkD were detected in all samples. Our results indicated that HTS processes the application potential to identify the active ingredients of microbial inoculants. Therefore, rapid and accurate identification of the microbial compositions in microbial formulation products is of high importance for port biosafety supervision.
Insights
High-throughput sequencing (HTS) accurately identifies microbial inoculant composition and resistance genes, improving biosafety assessments for imported products. This method offers a rapid and reliable alternative to traditional techniques for environmental protection applications.
Area of Science:
- Environmental microbiology
- Biotechnology
- Molecular biology
Background:
- Microbial inoculants are crucial for wastewater treatment, soil remediation, and biological control.
- Assessing the safety and composition of imported microbial inoculants is vital for regulatory compliance.
- Traditional culture-based methods for microbial identification are time-consuming, labor-intensive, and may yield inaccurate results.
Purpose of the Study:
- To evaluate the application of high-throughput sequencing (HTS) for identifying microbial composition and resistance genes in microbial inoculants.
- To compare HTS methods with traditional techniques for accuracy and efficiency in analyzing imported microbial inoculants.
- To assess the biosafety of imported microbial inoculants for environmental protection applications.
Main Methods:
- Application of both Illumina- and Nanopore-based high-throughput sequencing (HTS) for microflora distribution and resistance gene analysis.
- Verification of HTS findings using traditional culture-based identification methods.
- Analysis of microbial inoculants intended for environmental protection and imported products.
Main Results:
- Both Illumina and Nanopore HTS platforms successfully identified dominant bacterial species, including *Bacillus subtilis*, *Bacillus amyloliquefaciens*, *Bacillus licheniformis*, and *Enterococcus faecium*.
- Identified bacterial species were further confirmed by traditional methods.
- Specific antibiotic resistance genes, such as *mprF*, *bcrA*, *blt*, *lmrB*, *rphB*, *tet(L)*, *tmrB*, *vmlR*, *ykkC*, and *ykkD*, were detected in all analyzed samples.
Conclusions:
- High-throughput sequencing (HTS) demonstrates significant potential for accurately identifying active ingredients in microbial inoculants.
- HTS provides a rapid and reliable method for microbial composition analysis, crucial for biosafety supervision of imported products.
- The findings support the use of HTS for ensuring the quality and safety of microbial formulations in environmental applications.
