Evaluating T-RFLP protocols to sensitively analyze the genetic diversity and community changes of soil alkane degrading bacteria

• Published in: European journal of soil biology Vol. 65; no. 65; pp. 107 - 113
• Main Authors: Giebler, Julia, Wick, Lukas Y., Harms, Hauke, Chatzinotas, Antonis
• Format: Journal Article
• Language: English
• Published: Paris Elsevier Masson SAS 01-11-2014; Elsevier
• Subjects: 16S rRNA gene T-RFLP; Agronomy. Soil science and plant productions; Alkane degradation; alkB T-RFLP; Biochemistry and biology; Biological and medical sciences; Chemical, physicochemical, biochemical and biological properties; Fundamental and applied biological sciences. Psychology; Physics, chemistry, biochemistry and biology of agricultural and forest soils; Protocol evaluation; Restriction enzyme selection; Soil bacterial communities; Soil science; Alkane degradation; Restriction enzyme selection; alkB T-RFLP; Protocol evaluation; 16S rRNA gene T-RFLP; Soil bacterial communities; Biodegradation; Enzyme; Ribosomal DNA; Genetic diversity; Soils; Restriction fragment length polymorphism; Ribosomal RNA; Alkane; Bacteria; Soil science; Microbial community
• ISSN: 1164-5563
• DOI: 10.1016/j.ejsobi.2014.10.006

Due to the enormous complexity of soil bacterial communities, functional gene markers, like the alkB gene coding for the bacterial alkane monooxygenase, might be more suitable to monitor specific ecosystem functions, i.e. alkane degradation, compared to phylogenetic gene markers which include species not providing the respective service. However, the routine application of such functional fingerprints needs careful method development and evaluation. This study aimed at identifying a reliable, highly resolving fingerprinting method to monitor alkane degrading communities in soil. Therefore, we (i) evaluated various available terminal restriction fragment length polymorphism (T-RFLP) protocols for the alkB gene, (ii) comparatively analyzed the same soil communities targeting either a functional (alkB) or a phylogenetic marker gene (16S rRNA gene) in the T-RFLP, and (iii) judged on the applicability of diversity indices to follow diversity changes in the degrader community during alkane input. In silico restriction of 83 alkB gene sequences from public databases identified HpyCH4V as the enzyme yielding the highest T-RF richness for alkB. Analyzing DNA extracts from microcosms of two different agricultural soils (sandy and silty, amended with either maize or pea litter as alkane source) revealed a higher sensitivity of the alkB T-RFLP compared to 16S rRNA gene T-RFLP for following spatiotemporal changes in the alkane degrader community. As expected, the diversity indices based on either phylogenetic or functional fingerprinting profiles deviated significantly, but both resulted in lower richness estimates compared to an alkane degrading enrichment culture obtained from the same soil samples. The latter indicated that alkane degradation is a very common capability of soil bacteria. However, as the two fingerprinting methods monitor different kinds of diversity, they might provide complementary answers (i.e. phylogenetic and functional) to the same research questions, i.e. which is the robustness of an ecosystem function in a given system.
•We evaluated alkB TRFLP protocols for monitoring soil alkane degrading bacteria.•We challenged the method by following alkane degrader communities in two soils.•Environmental influences were better resolved by alkB- than by 16S TRFLP.•Diversity indices underestimated the alkane degrader richness in soil.•We propose a complementarity of alkB/16S TRFLP when analyzing complex soil samples.