Fatty acid composition of lipids and polyhydroxyalkanoate determination in the air-air biofilm using a two-stage sample extraction and gas chromatography with flame ionization detector

Modifications in the fatty acyl chain composition of cell lipids significantly influence bacterial biofilm development, and biofilm-forming bacteria synthesise polyhydroxyalkanoates as carbon and energy reserves under challenging environmental conditions. Despite the close relationship between fatty acid composition in lipids and polyhydroxyalkanoates, comprehensive research examining both constituents within biofilms remains limited, and no analytical methodologies support their concurrent analysis. In this study, a two-stage extraction procedure combined with GC-FID analysis was developed to determine both the lipid fatty acid profiles and the polyhydroxyalkanoate content in microbial biofilms. Air-air interfaced bubble biofilms obtained from a mine tunnel environment were analysed to investigate microbial adaptation to low-light and metal-enriched conditions. Nine bubble biofilm samples collected from Ytterby mine, Sweden, demonstrated a consistent fatty acid composition dominated by four main components: mono-unsaturated C16:1 (n-7) and C18:1 (n-7), along with saturated C14:0 and C16:0. Mono-unsaturated and saturated fatty acids accounted for over 90 % of total extracts, while bacterial fatty acids, polyhydroxyalkanoates, and polyunsaturated fatty acids represented approximately 5 %, 2 %, and 1 %, respectively. Two polyhydroxyalkanoate monomers were found in the samples: 3OHC14:0 and 3OHC16:0. The observed fatty acid and polyhydroxyalkanoate profiles closely aligned with those of Nevskia ramosa strains, confirming its predominance within these biofilm communities. The developed analytical methodology enables efficient and cost-effective simultaneous determination of fatty acids of lipids and polyhydroxyalkanoates in microbial biofilms, offering a useful tool for future studies in this field.