In situmeasurement of microbial biomass, community structure and nutritional status

Abstract

In sediments and soils the extant microbiota that can be counted by direct microscopy have proved exceedingly difficult to isolate and culture. Classical tests are time consuming and provide little indication of the interactions within the community, the community nutritional status or metabolic activity. Thein situmethod is based on the extraction of ‘signature’ lipid biomarkers (SLB) from the cell membranes and walls of microorganisms. Lipids are cellular components that are recoverable by extraction with organic solvents. Lipids are an essential component of the membrane of all cells and play a role as storage materials. Extraction of the lipid components of the microbiota from soils and sediments provides both purification and concentration together with anin situquantitative analysis of the microbial biomass, community structure, and nutritional status. The determination of the total phospholipid ester-linked fatty acids (PLFA) provides a quantitative measure of the viable biomass. Viable microbes have an intact membrane which contains phospholipids (and PLFA). With cell death enzymes hydrolyze the phosphate group within minutes to hours. The lipid core remains as diglyceride (DG). The resulting DG has the same signature fatty acids as the phospholipids (until it degrades) so a comparison of the ratio of PLFA to DG provides an indication of the viable and nonviable microbes. Analysis by SLB technique provides a quantitative definition of the microbial community structure as specific groups of microbes contain characteristic PLFA patterns. The analysis of other lipids such as the sterols (for the microeukaryotes -nematodes, algae, protozoa), glycolipids (for the phototrophs, gram-positive bacteria), or the hydroxy fatty acids in the lipopolysaccharide of the lipid A (gramnegative bacteria) can provide more detailed community structure analysis. The formation of poly (3-hydroxyalkanoic acid (PHA) in bacteria or triglyceride (TG) in the microeukaryotes relative to the PLFA provides a measure of the nutritional status. Bacteria grown with adequate carbon and terminal electron acceptors form PHA when they cannot divide, because some essential component is missing. Rates of incorporation of14C-acetate into PHA relative to PLFA is a sensitive indicator of disturbance artifacts in estimates of metabolic activity in sediments with redox gradients. Exposure to toxic environments can lead to minicell formation and increases in specific PLFAS. Respiratory quinone structure indicates the proportions of aerobic/anaerobic activities in the community. The SLB technology provides quantitativein situinformation that define the microbial ecology in sedimentary geochemical processes.