James B. Guckert

Determination of monosaturated fatty acid double-bond position and geometry for microbial monocultures and complex consortia by capillary GC-MS of their dimethyl disulphide adducts

Abstract Monounsaturated fatty acid double-bond position and geometry have been determined for microbial monocultures and complex microbial consortia by capillary GC-MS of their dimethyl disulphide (DMDS) adducts. The technique has permitted ( i ) chromatographic separation and positive identification of adducts derived from cis/trans isomers, ( ii ) characterization of long-chain monounsaturated components (up to 26:1), and ( iii ) the identification of a wide range of monounsaturated components derived from methanotrophic soil material. The methanotrophic soil sample contained a high relative proportion of the novel phospholipid ester-linked fatty acid 18:1Δ 10c. The DMDS procedure offers a simple and rapid approach that can be routinely applied to microbial fatty acids derived from environmental samples and monocultures.

Fourier transform-infrared spectroscopic methods for microbial ecology: analysis of bacteria, bacteria-polymer mixtures and biofilms

Fourier transform-infrared (FT-IR) spectroscopy has been used to rapidly and nondestructively analyze bacteria, bacteria-polymer mixtures, digester samples and microbial biofilms. Diffuse reflectance FT-IR (DRIFT) analysis of freeze-dried, powdered samples offered a means of obtaining structural information. The bacteria examined were divided into two groups. The first group was characterized by a dominant amide I band and the second group of organisms displayed an additional strong carbonyl stretch at approximately 1740 cm-1. The differences illustrated by the subtraction spectra obtained for microbes of the two groups suggest that FT-IR spectroscopy can be utilized to recognize differences in microbial community structure. Calculation of specific band ratios has enabled the composition of bacteria and extracellular or intracellular storage product polymer mixtures to be determined for bacteria-gum arabic (amide I/carbohydrate C-O approximately 1150 cm-1) and bacteria-poly-beta-hydroxybutyrate (amide I/carbonyl approximately 1740 cm-1). The key band ratios correlate with the compositions of the material and provide useful information for the application of FT-IR spectroscopy to environmental biofilm samples and for distinguishing bacteria grown under differing nutrient conditions. DRIFT spectra have been obtained for biofilms produced by Vibrio natriegens on stainless steel disks. Between 48 and 144 h, an increase in bands at approximately 1440 and 1090 cm-1 was seen in FT-IR spectra of the V. natriegens biofilm. DRIFT spectra of mixed culture effluents of anaerobic digesters show differences induced by shifts in input feedstocks. The use of flow-through attenuated total reflectance has permitted in situ real-time changes in biofilm formation to be monitored and provides a powerful tool for understanding the interactions within adherent microbial consortia.

Membrane fatty acids as phenotypic markers in the polyphasic taxonomy of methylotrophs within the proteobacteria

A polyphasic approach to bacterial taxonomy attempts to integrate phylogenetic relationships with phenotypic marker analysis. This study describes the application of membrane fatty acids as a phenotypic marker for methylotrophs. Detailed phospholipid, ester-linked fatty acid (PLFA) profiles are reported for 17 methylotrophic eubacterial strains. These profiles included verification of double bond positions and geometries, both critical features for this analysis. Multivariate cluster analysis was used to indicate groupings of these strains along with literature values of both methylotrophs and non-methylotrophs based on the PLFA phenotype. Like many phenotypic characteristics, PLFA profiles were influenced by environmental conditions. The instabilities displayed, however, were predictable from physiological studies including increased trans/cis and cyclopropyl/cis ratios. Cluster analysis of PLFA profiles generated by separate investigators with different culture conditions indicated reproducibility by strain and species. The PLFA phenotype relationships compare favourably with phylogenetic associations based on 16S rRNA data for methylotrophs and will continue to be a valuable phenotypic marker for Proteobacteria taxonomy.

Changes in periphyton fatty acid composition in chlorine-polluted streams

A manipulative experiment was conducted to evaluate a biochemical method for using periphyton in water quality assessment. Periphyton assemblages that developed in situ on ceramic tiles placed in a reference site in one stream were transferred into a reference site and a polluted site in each of three streams in eastern Tennessee. Samples of periphyton from each site were analyzed for chlorophyll a content, rate of photosynthesis (by 14C uptake), and fatty acid composition, 3, 17, and 35 days after being transferred to the sites. Chlorophyll a and photosynthetic rates per unit of chlorophyll at polluted sites were generally lower than at corresponding reference sites. More than 50 different fatty acids were separated and identified in the periphyton phospholipids; 12 of the 50 accounted for more than 90% of the total fatty acids. Periphyton fatty-acid biomarkers revealed differences in the periphyton taxonomic composition between reference and polluted sites. Fatty acid profiles indicated a shift from diatom to green alga-dominated assemblages in reference and chlorine contaminated sites respectively. Individual fatty-acid biomarkers specific for green algae (18:3ω3 or α-linolenic acid) and diatoms (20:5ω3 or eicosapentaenoic acid) proved particularly useful in quantifying the periphyton response to chlorine. The use of algal signature fatty acids may evolve into a quantitative automated method for measuring chlorine effect on stream periphyton.

Determination of microbial fatty acid profiles at femtomolar levels in human urine and the initial marine microfouling community by capillary gas chromatography-chemical ionization mass spectrometry with negative ion detection

Room temperature esterification with the electron capturing pentafluorobenzyl bromide in glass capillaries, with analysis by capillary gas-liquid chromatography coupled with chemical ionization mass spectrometry and negative ion detection in the selected ion mode, allowed detection and identification of fatty acids from microbial biofilms at the femtomolar level. This sensitivity was achieved without loss of specificity of the mass spectrometric analysis. The detection of all the fatty acids commonly associated with bacteria was quantitative and linearly related to their concentration at a sensitivity permitting detection of about 600 bacteria the size of Escherichia coli. With this technique it was possible to detect the characteristic 3-hydroxy fatty acid of the lipopolysaccharide lipid A of E. coli infecting human urine at concentrations representing 10 4 bacteria and define the community structure of the initial marine microfouling community attached to a teflon surface at concentrations below the detectability by gas chromatography with flame ionization detection.

Radiotracer Determination of Ingestion and Assimilation of Periphytic Algae, Bacteria, and Adsorbed Amino Acids by Snails

We examined the use of radiotracers to determine if ³H-amino acids (AA) and ¹⁴C-bicarbonate could differentiate grazing by Physella virgata, a pulmonate gastropod, on the bacterial and algal components in periphyton. Large amounts of ³H-AA became associated with periphyton as a result of adsorption to periphyton, rather than microbial uptake. As a consequence of this abiotic adsorption of ³H-AA by periphyton, uptake of ³H by grazers could be due to ingestion of either ³H-labeled bacteria or detritivory on material to which ³H-AA were adsorbed. Physella was able to ingest and assimilate adsorbed ³H-AA directly in the absence of any bacterial uptake of amino acids. In contrast to ³H-AA, relatively little ¹⁴C-bicarbonate adsorbed to periphyton, and essentially all of the uptake could be attributed to photosynthetic activity. ¹⁴C-bicarbonate is, therefore, a reliable tracer for determining grazing activity on the algal component of periphyton assemblages. Calculated assimilation efficiencies for snails grazing on periphyton to which ³H-AA was adsorbed (78%) were greater than those of snails grazing on periphyton in which bacteria had incorporated ³H-AA (50%) or periphyton algae labeled with ¹⁴C-bicarbonate (32%) suggesting that adsorbed organic carbon and detritivory may be important to this snails nutrition.

Evaluation of a hexane/isopropanol lipid solvent system for analysis of bacterial analysis of bacterial phospholipids and application to chloroform-soluble Nuclepore (polycarbonate) membranes with retained bacteria

Abstract The recovery of phospholipid, ester-linked fatty acids and phospholipid phosphate from the eubacterium Pseudomonas atlantica was shown to be equivalent for the modified Bligh ad Dyer (chloroform/methanol/water) procedure and hexane/isopropanol (HIP) lipid extraction. In addition to its previously suggested lower toxicity, the HIP solvent system allows lipid analyses to be routinely done on chloroform-soluble materials such as Nuclepore polycarbonates, useful for well-defined size fractionation studies of bacterioplankton communities.