Harry W. Read

Mammalian mitochondria as in vitro monitors of water quality.

To measure acute toxicity effectively, a system must provide a simple, sensitive, and rapid measurement of physiological parameters which are indicative of overall organism (bacteria) or organelle (mitochondria) viability. To detect a broad spectrum of toxicants, the parameters should be associated with a major metabolic process controlled by interdependent enzyme systems. In the bioassay reported here, phosphorylating submitochondrial particles (SMP), prepared by sonic disruption of the heavy fraction of intact bovine heart mitochondria serve as in vitro monitors of aquatic toxicity. The bioassay is based on the phenomenon of energy-coupled reverse electron transfer (RET), discovered in plant mitochondria, and later explored using SMP preparations from mammalian sources. RET responses permit rapid, simple, and sensitive measurement of acute toxicity by spectrophotometric recording of the rate of NAD reduction. The criterion of toxicity in this test is inhibition of NAD reduction in the presence of toxic substances. The RET reaction was chosen over other methods because responses are easily quantifiable and the energy-coupled RET reaction is competent in reconstituted freeze-dried submitochondrial particles. Thus, SMP - a stable biological preparation- can be distributed to other laboratories. This feature is important in selecting a routine bioassay.

Identification of Acyl-Homoserine Lactone Signal Molecules Produced by Nitrosomonas europaea Strain Schmidt

ABSTRACT Nitrosomonas europaea strain Schmidt produces at least three acyl homoserine lactone (AHL) signal molecules: C6-homoserine lactone (HSL), C8-HSL, and C10-HSL. These compounds were identified in extracts of chemostat culture effluent by three independent methods. The concentrations of AHL in effluent were low (0.4 to 2.2 nM) but within the range known to induce AHL-responsive systems. The absence of LuxI and LuxM homologs from the genome of N. europaea strain Schmidt suggested that AHL synthesis occurs by an alternate pathway, possibly mediated by an HdtS homolog. To the best of our knowledge, the present report is the first to document the types and levels of AHLs produced by N. europaea.

Assessment of chemical toxicity using mammalian mitochondrial electron transport particles

New spectrophotometric bioassay procedures have been developed for evaluating chemical toxicity, using electron transport particles isolated from bovine heart mitochondria, based on the ability of many toxic chemicals to interfere with the integrated function of electron transport enzymes. The sensitivity of the mitochondrial assays is compared to published sensitivities of otherin vivo andin vitro toxicity testing methods. Regression analysis of logarithmically transformed toxicity values for 42 chemicals, including 8 pesticides, 5 drugs, 6 metals, 8 alcohols, 5 respiratory inhibitors, 4 phenols, and 2 phthalates, indicates excellent correlation between the sensitivity of the new assays and the sensitivity of mammalian cytotoxicity studies (r2 =0.86). Data from aquatic exposure toxicity tests conducted in fish are also highly correlated with the mitochondrial assay results (r2=0.79). However, correlation of data from these methods with median lethal dose studies conducted in rats is not as good because of the inability ofin vitro and aquatic exposure analyses to account for the gastrointestinal absorption, hepatic metabolism, and excretion processes which modify toxic responses following oral administration.

A jacketed annular membrane photocatalytic reactor for wastewater treatment : degradation of formic acid and atrazine

Abstract An annular photoreactor has been used to study the photocatalytic degradation of aqueous solutions of formic acid and atrazine over a titanium dioxide ceramic membrane. Because of the small degradation rates achieved, a recycle operation was used and, in consequence, the mathematical model of the experimental installation was discussed. Experimental results shows a very low quantum efficiency for atrazine photodegradation.

GC-based detection of aldononitrile acetate derivatized glucosamine and muramic acid for microbial residue determination in soil.

Quantitative approaches to characterizing microorganisms are crucial for a broader understanding of the microbial status and function within ecosystems. Current strategies for microbial analysis include both traditional laboratory culture-dependent techniques and those based on direct extraction and determination of certain biomarkers. Few among the diversity of microbial species inhabiting soil can be cultured, so culture-dependent methods introduce significant biases, a limitation absent in biomarker analysis. The glucosamine, mannosamine, galactosamine and muramic acid have been well served as measures of both the living and dead microbial mass, of these the glucosamine (most abundant) and muramic acid (uniquely from bacterial cell) are most important constituents in the soil systems. However, the lack of knowledge on the analysis restricts the wide popularization among scientific peers. Among all existing analytical methods, derivatization to aldononitrile acetates followed by GC-based analysis has emerged as a good option with respect to optimally balancing precision, sensitivity, simplicity, good chromatographic separation, and stability upon sample storage. Here, we present a detailed protocol for a reliable and relatively simple analysis of glucosamine and muramic acid from soil after their conversion to aldononitrile acetates. The protocol mainly comprises four steps: acid digestion, sample purification, derivatization and GC determination. The step-by-step procedure is modified according to former publications. In addition, we present a strategy to structurally validate the molecular ion of the derivative and its ion fragments formed upon electron ionization. We applied GC-EI-MS-SIM, LC-ESI-TOF-MS and isotopically labeled reagents to determine the molecular weight of aldononitrile acetate derivatized glucosamine and muramic acid; we used the mass shift of isotope-labeled derivatives in the ion spectrum to investigate ion fragments of each derivatives. In addition to the theoretical elucidation, the validation of molecular ion of the derivative and its ion fragments will be useful to researchers using δ(13)C or ion fragments of these biomarkers in biogeochemical studies.

Field trials of a TiO2 pellet‐based photocatalytic reactor for off‐gas treatment at a soil vapor extraction well

Abstract A field trial of a pilot‐scale TiO2 photocatalytic reactor for treatment of off‐gases from a soil vapor extraction (SVE) well at a chlorinated solvent spill site at the Savannah River Site near Aiken, SC, is described. Trichloroethylene (TCE), perchloroethylene (PCE), 1,1‐dichloroethylene (1,1 ‐DCE), and 1,1,1 ‐trichloroethane (1,1,1 ‐TCA) were treated at flow rates up to 6 1/min and space times of 5.1 x 107 to 1.2 x 109 g/mol. The TiO2 used was in the form of porous pellets with a surface area of 150 m2/g. Operation of the reactor with the undiluted waste stream (5000 ppmv) at 80, 100, and 110°C yielded many undesirable byproducts, such as phosgene, chloroform, carbon tetrachloride, and penta‐ and hexachloroethane, even though the conversion of PCE and TCE approached 100%. After diluting the waste stream with ambient air to below 1000 ppmv and maintaining space times around 5 x 108 g/mol, a conversion >99.5% was achieved with the production of only small amounts (<10 ppmv) of hexachloroethane. T...

Reliability of Muramic Acid as a Bacterial Biomarker is Influenced by Methodological Artifacts from Streptomycin

The muramic acid (MurA) assay is a powerful tool for the detection and quantification of bacteria with no need to enrich samples by culturing. However, the analysis of MurA in mixed biological and environmental matrices is potentially more complex than analysis in isolated bacterial cells. In this study, we employed one commonly used procedure for extraction of MurA from environmental samples and found that the presence of streptomycin interfered with the determination of MurA by creating chemical species that coeluted with the aldononitrile derivative of MurA prepared in this method. On a molar basis, streptomycin yields a signal that is approximately 0.67 times that of MurA. Mass spectrometry analysis confirmed that the interference from hydrolyzed streptomycin is not actually by MurA, but rather is likely to be N-methyl glucosamine. Because streptomycin is widely applied for selective growth of eukaryotes both in situ and in vitro, our findings may have implications for the significance of results from MurA assays. We conclude that MurA remains an effectual bacterial biomarker due to its unique bacterial origin, but care must be applied in interpreting results from the assay when performed in the presence of streptomycin.

Unique Honey Bee (Apis mellifera) Hive Component-Based Communities as Detected by a Hybrid of Phospholipid Fatty-Acid and Fatty-Acid Methyl Ester Analyses

Microbial communities (microbiomes) are associated with almost all metazoans, including the honey bee Apis mellifera. Honey bees are social insects, maintaining complex hive systems composed of a variety of integral components including bees, comb, propolis, honey, and stored pollen. Given that the different components within hives can be physically separated and are nutritionally variable, we hypothesize that unique microbial communities may occur within the different microenvironments of honey bee colonies. To explore this hypothesis and to provide further insights into the microbiome of honey bees, we use a hybrid of fatty acid methyl ester (FAME) and phospholipid-derived fatty acid (PLFA) analysis to produce broad, lipid-based microbial community profiles of stored pollen, adults, pupae, honey, empty comb, and propolis for 11 honey bee hives. Averaging component lipid profiles by hive, we show that, in decreasing order, lipid markers representing fungi, Gram-negative bacteria, and Gram-positive bacteria have the highest relative abundances within honey bee colonies. Our lipid profiles reveal the presence of viable microbial communities in each of the six hive components sampled, with overall microbial community richness varying from lowest to highest in honey, comb, pupae, pollen, adults and propolis, respectively. Finally, microbial community lipid profiles were more similar when compared by component than by hive, location, or sampling year. Specifically, we found that individual hive components typically exhibited several dominant lipids and that these dominant lipids differ between components. Principal component and two-way clustering analyses both support significant grouping of lipids by hive component. Our findings indicate that in addition to the microbial communities present in individual workers, honey bee hives have resident microbial communities associated with different colony components.

A Lipid Extraction and Analysis Method for Characterizing Soil Microbes in Experiments with Many Samples

Microbial communities are important drivers and regulators of ecosystem processes. To understand how management of ecosystems may affect microbial communities, a relatively precise but effort-intensive technique to assay microbial community composition is phospholipid fatty acid (PLFA) analysis. PLFA was developed to analyze phospholipid biomarkers, which can be used as indicators of microbial biomass and the composition of broad functional groups of fungi and bacteria. It has commonly been used to compare soils under alternative plant communities, ecology, and management regimes. The PLFA method has been shown to be sensitive to detecting shifts in microbial community composition. An alternative method, fatty acid methyl ester extraction and analysis (MIDI-FA) was developed for rapid extraction of total lipids, without separation of the phospholipid fraction, from pure cultures as a microbial identification technique. This method is rapid but is less suited for soil samples because it lacks an initial step separating soil particles and begins instead with a saponification reaction that likely produces artifacts from the background organic matter in the soil. This article describes a method that increases throughput while balancing effort and accuracy for extraction of lipids from the cell membranes of microorganisms for use in characterizing both total lipids and the relative abundance of indicator lipids to determine soil microbial community structure in studies with many samples. The method combines the accuracy achieved through PLFA profiling by extracting and concentrating soil lipids as a first step, and a reduction in effort by saponifying the organic material extracted and processing with the MIDI-FA method as a second step.