Timeline of Advancements in the Field of Biomarkers

1964 Worked with Richard E. Shope at Rockefeller to characterize what was then called by us Haemophilus pleuropneumonia a serious infection in pigs now a widely studied porcine pathogen known as Actinobacillus pleuropheumonia and the focus of our current DARPA program to test if breath condensate lipids and eicosanoids are presymptomatic harbingers of infection

1963-65 Whilst at Rockefeller and at Kentucky worked with Marvin P. Bryant (a member of the NAS from Illinois and his student Dan Caldwell recently retired from University of Wyoming to first demonstrate cytochrome based electron transport and heme synthesis in a strict anaerobe.

1964-65 Worked on cytochrome systems using spectrophotometry to study respiratory pigments in bacterial cellular membranes in Lucile Smith Lab at Dartmouth Medical school, Hanover NH whilst graduate student Rockefeller.

1963-1972 Learned lipid extraction, deacylation and separation of subsequent glycerol phosphoryl esters by liquid chromatography and classical assessment from R. E. Lester (retired from University of Kentucky) and John E. Dittmer (deceased) who had one of the first gas chromatographs for separation of fatty acid methyl esters with University of Kentucky colleagues in the Department of biochemistry.

1963–1972 Described demethyl vitamin k2 isoprenologues and showed its essential role in electron transport by removing it and then replacing it whilst monitoring the oxygen utilization capacity.

1966 Showed a functional electron transport system could be formed in spheroplasts with Elizabeth A. Wright a medical student at UKMC

1967 Showed heme in a bacterial cytochrome oxidase is formed from protoheme

1968 Showed that the genes for heme biosynthesis in Staphylococcus aureus were linear in the order of the metabolic pathway with a graduate student from microbiology.

1969 Found new and then unique branched sphingophospholipids and demonstrated electron transport in the oral pathogen and halitosis generating Bacteroides melaninogenicus with Anne Tucker and medical student J. P. LaBlock and Masters student Victor Rizza

1963–1972 Described demethyl vitamin k2 isoprenologues and showed its essential role in electron transport by removing it and then replacing it whilst monitoring the oxygen utilization capacity.

1966 Showed a functional electron transport system could be formed in spheroplasts with Elizabeth A. Wright a medical student at UKMC

1967 Showed heme in a bacterial cytochrome oxidase is formed from protoheme

1968 Showed that the genes for heme biosynthesis in Staphylococcus aureus were linear in the order of the metabolic pathway with a graduate student from microbiology.

1972 After becoming increasingly frustrated at what I though was a far to great emphasis on microbial metabolism in the teaching of medical students and and constraints of lab space (I was moved to the parking lot in “surge space” of the medical school building I took the opportunity to move my family to the new Program in Medical Sciences at Florida State University. I needed to retake the Medical Licensure Examination to renew a valid MD in Florida and set up the clinical training program for our 35 first year medical students. This teachng took a great deal of time and my research clearly needed a change from the intensely competitive of electron transport membrane organization. I went from 5 lectures to medical and graduate students per year in biochemistry to 5 lectures in various basic medical sciences per week.

I took the opportunity to join “Skip” Livingston in his study of the microbial ecology Apalachicola estuary.

1977 The new laboratory direction was functioning with grants, technicians, and graduate students. We showed polar lipids were nifty quantitative measures of viable microbial biomass , and had a GC/assay for muramic acid in bacterial cell walls. The quest for
biomarkers had begun.

1978 had found poly beta-hydroxybutyrate as a measure of unbalanced growth in bacterial activity.

1979 We had the opportunity to apply the new quantitative lipid based measures of microbial ecology to biofouling in the ocean thermal energy program. (OTEC).

1980 We applied the phospholipid ester-liked fatty acids (PLFA) as measures of biomass and community composition and did the funguss heaven (formed ergosterol) and hell experiments in microcosms to show that rational manipulations of biomarkers actually worked. Bob Findlay used the methods to document what and dollars actually ate (mostly protozoa) as they sifted through the sand.

1980. We developed a sensitive HPLC fluorometric method for determining adenosine energy charge and showed the reactions to maintain the energy charge ratio at the expense of ADP by the formation of adenine was the most sensitive measure of bacterial stress..

1981. We were able to apply the new methods to show effects of substratum degradability, sedimentary grain size, substratum composition, and light on the biofilm community. Susan Morrison was also able to show different amphipods with different mouthparts selectively grazed on the detrital community.

1982. I was invited to present our work using biomarkers to quantitatively define sedimentary and estuarine detrital microbial communities at the Society for General microbiology in Wawrick University in England. It was the first exposure of the quantitative methods for microbial ecology biomarker methods to an international audience. WE also devised a quantitative assay for the hydroxy fatty acids of the lipopoysaccharides of Gram-negative bacteria. We found that in the environment that poly-beta hydroxy alkonates containe a complex mixture of poly beta hydorxy-fatty acids in the polymers.

1983. I was asked to found and develop the Journal of Microbiological methods for Elsevier in Amsterdam. It had been difficult to publish methods prior to this in journals such as Applied and Environmental Microbiology. I had determined when I worked in the Johnson Foundation for Medical Physics in 1958 while I was waiting to a Medical Residency at the Hospital for the University Pennsylvania in July watching the marvels of rapid spectrometry of electron transport in a darkened room with oscilloscopes that I would heed the advice of Otto Warburg the honorary Aryan Nobel Lauriat that if you can measure something no one else can measure you will be useful in science. The success of the Journal of Microbiological Methods prompted major microbiology journals including AEM to now have sections for Methods.

1984. We added methods fro teichoic acids of Gram-positive bacteria that unfortunately required use of strong HF which is toxic and phosphoipid glycerol. We began our studies of deep subsurface microbial communities with biomarkers, sulfur oxidizing bacteria in concrete corrosion, drilling fluids on coral and sedimentary communities, and microbial influenced corrosion (MIC).

1985. We applied the biomarker methods to seagrass beds with Dave Moriarty in Australia and in deep sea sediments in areas of disturbance. We defined huge disturbance artifacts in the classical determinations of activities in marine sediments. We documented low biomass of viable bacteria by PLFA in the abyssal plains of the Venezula Basin and Puerto Rico Trench sediments. The position of double bonds in unsaturated PLFA by DMDS (so good it has been reprinted 3 times without attribution to Nichols et al), the utilization of 15-N enrichment n cell wall D-alanine, the effects and shfts in rhizosphere microbiota, and the use or Fourier transform infrared spectroscopy to non-destructively define chemical structures in biofilms was reported.

1986. We found a unique microbial community in the sponge spicule mats with a huge uncultured diatom that showed no evidence of photoinhibition asand a shade adapted community in Antarctic sea ice. Sufate-reducing bacteria and methane oxidizing bacteria were definable by their unique patterns of PLFA, respiratory quinones in communities were correlated to the in-situ redox level and the ether lipids of the archae, archaeol and caldarcheol were quantitated. Biomarkers of starving (viable but not culturable) Vibrio were identified and the toxicity markers of ratios of cis to trans monoenoic PLFA. Our first proposal to utilize these methods in detection of bacterial warfare agents was presented. .

1987. The community composition of an aerobic consortia utilizing methane or propane id degrading pentachoroethane acid ground water pollution was defined. And the chemical methods for monitoring methane-producing digesters was initiated.

1988. The utility of monitoring effects of predation on microbial communities with biomarkers was made. Equivalence of microbial biomass measures based on membrane lipid and cell wall components, adenosine triphosphate, and direct counts in subsurface sediments was demonstrated. Effects of weldments in metal substrata on MIC were determined

1989. Microbial community structure and function as quantitative indicators of environmental health was first proposed. Characterization of Bacteria that suppress Rhizoctania Damping-Off in Bark Compost Media showed the utility of the biomarkers in studies of biocontrol agents. Biomarkers of filamentous bacteria colonizing subtidal hydrothermal vents were characterized.

1990. Differential adhesion, activity, and carbohydrate:protein ratios of Pseudomonas atlantica monocultures and the use of The use of current density mapping in the study of microbial influenced corrosion were reported

1991. Anaerobic hydrogen mediated acetogenesis in natural gas transmission lines, pitting corrosion by bacteria on carbon steel, determined by the scanning vibrating electrode technique, disturbance, starvation, and overfeeding stress influenced detected by microbial lipid biomarkers in high-solids high-yield methanogenic reactors, effect of electrochemical impedance spectroscopy on microbial cell numbers, viability, and activity.and detection of microbial biofilms using on-line monitoring techniques.were reported.

1992. Accelerated testing of MIC resistance of materials and weldments using flow-through testing with indigenous microbiota, on-line detection of bacterial adhesion in a shear gradient with bioluminescence by a Pseudomonas fluorescens (lux) strain, Lipid components in anal scent sacs of three mongoose species, Periphyton response along an industrial effluent gradient: Lipid-based physiological stress analysis and pattern recognition of microbial community structure, and differential corrosion of carbon steel by combinations of Bacillus sp., Hafnia alvei, and Desulfovibrio gigas established by phospholipid analysis of electrode biofilm were reported. In a study of flanges recovered from hydrotherjmal vents percolated with fluid at temperature of 350ºC contained archea and traces of bacteria with intact polar lipid membranes

1993. Supercritical fluid extraction of polar analytes using in situ chemical derivatization, FTIRT monitoring microbial adhesion and biofilm formation, and long-term, on-line monitoring of microbial biofilms using a quartz crystal microbalance was reported.

1994. Evaluating trichloroethylene biodegradation by measuring the in situ status and activities of microbial populations, parallel phylogenetic relationships based on phospholipid fatty acid profiles and ribosomal RNA sequence similarities in dissimilatory sulfate-reducing bacteria, detection of the anaerobic declorinator Desulfomonile tiedjei in soil by signature lipopolysaccharide branched-long-chain hydroxy fatty acids, and algD-bioluminescent reporter to monitor alginate production in biofilms were reported.

1995. Aromatic-degrading Sphingomonas were isolated from the deep subsurface and the publication of on-line monitoring of biofilm biomass and activity on antifouling and fouling-release surface using bioluminescence and fluorescence measurements during laminar-flow.

1996. Soil microbial communities beneath Populus grandidentata grown under elevated atmospheric CO2 showed reproducible changes in biomarkers and Combined lipid/DNA extraction method for environmental samples showed lipid extraction facilitated amplifiable DNA recovery.

1997. Post-sampling changes in microbial community composition and activity in a subsurface paleosol, survival and phospholipid fatty aid profiles of surface and subsurface bacteria in natural microcosms, compositional and functional shifts in microbial communities related to soil warming, monitoring biofilm-induced persistence of Mycobacterium in drinking water systems using GFP fluorescence, quantitative lipid biomarker analysis of airborne microorganisms in indoor environments, and quantitative comparison of the in situ microbial communities in different biomes were published.

1998. Physiological considerations of environmental applications of lux reporter fusions, and in situ microbial ecology for quantitative appraisal, monitoring, mapping changes in soil microbial community composition signaling for bioremediation, and risk assessment of pollution remediation in soils, the subsurface and in biofilms were published

1999. Biofilm ecology of microbial biofouling, biocide resistance, and corrosion, fate of a metal-resistant inoculum in contaminated and pristine soils assessed by denaturating gradient gel electrophoresis,. microbial population changes during bioremediation of an experimental oil spill, and comparison of relative photon flux from single cells Vibrio fisheri and Vibrio harveyi using photon-counting microscopy were published.

2000. Quantitative lipid biomarker detection of unculturable microbes and chlorine exposure in water distribution system biofilms, physiological status and community composition of microbial mats of the Ebro Delta, Spain by signature lipid biomarkers, electrospray ionization/mass spectrometry compatible reversed-phase separation of phospholipids: piperidine as a post column modifier for negative ion detection, phylogenetic characterization and description of novel heat-tolerant Bacillus species isolated from spacecraft assembly facility and landscape-level patterns of microbial community composition and substrate use in forest ecosystems was published.

2001. Sensitive Characterization of microbial ubiquinones from biofilms by electrospray/mass spectrometry, soil microbial community response to dairy manure or ammonium nitrate applications, control of bacterial community composition in macrofaunal burrows, lipid and carbon isotopic evidence of methane-oxidizing and sulfate-reducing bacteria in association with gas hydrates from the Gulf of Mexico and diversity and characterization of sulfate-reducing bacteria in groundwater at a uranium mill tailings site were published.

2002. Phylogenetic characterization and description of novel heat-tolerant Bacillus species isolated from spacecraft assembly facility, control of bacterial community composition in macrofaunal burrows, microbial community composition and function beneath temperate trees exposed to elevated atmospheric carbon dioxide and ozone, and diversity, and characterization of sulfate-reducing bacteria in groundwater at a uranium mill tailings site.were published.

2003. Carbon isotope signatures of fatty acids in Geobacter metallireducens and Shewanella putreficiens, and forensic Analysis by Comprehensive Rapid Detection of Pathogens and Contamination Concentrated in Biofilms in Drinking Water Systems for Water Resource Protection and Management