Utilization of lasso peptides for biodegradation of polycyclic aromatic hydrocarbons.

Many microbial genes involved in degrading recalcitrant environmental contaminants such as polycyclic aromatic hydrocarbons (PAHs) have been identified and characterized. However, all molecular mechanisms required for PAH utilization have not yet been elucidated. In this work, we demonstrate the proposed involvement of lasso peptides in the utilization of the PAH phenanthrene in Sphingomonas BPH. Transpositional mutagenesis of Sphingomonas BPH with the miniTn5 transposon yielded 3 phenanthrene utilization deficient mutants, #257, #1778, and #1782. In mutant #1782, Tn5 had inserted into the large subunit of the naph/bph dioxygenase gene. In mutant #1778, Tn5 had inserted into the B2 protease gene of a lasso peptide cluster. This finding is the first report on the role of lasso peptides in PAH utilization. Our studies also demonstrate that interruption of the lasso peptide cluster resulted in a significant increase in the amount of biosurfactant produced in the presence of glucose when compared to the wild-type strain. Collectively, these results suggest that the mechanisms Sphingomonas BPH utilizes to degrade phenanthrene are far more complex than previously understood and that the #1778 mutant may be a good candidate for bioremediation when glucose is applied as an amendment due to its higher biosurfactant production.

Foamed glass ceramics-an upcycled scaffold for microbial biofilm development.

Glass, a near infinitely recyclable material, can be upcycled to create new products such as foamed glass ceramics, which are essentially a synthetic pumice-like material. This material has been demonstrated to sustain preserved biofilms which have application in various fields based on the deployability of the product and the preserved microbes. Foamed glass ceramics have increased surface area compared to typical soda-lime glass cullet. This material has been explored for variety of applications including the growth, storage and transport of biofilms and microbial colonies which can be preserved and deployed later. Here, we demonstrate the ability for microbial cultures including BioTiger™, Escherichia coli K-12, Bacillus thuringiensis, and two environmental eukaryotic cells to colonize the upcycled glass products, undergo preservation, and regrow after 84 days of storage. The growth of preserved samples is correlated to the time spent incubating prior to preservation. These results demonstrate the applicability of this novel glass-biofilm combination in which various preserved microorganisms are able to be rapidly grown after storage on an upcycled glass product.

Bioremediation of heavy oily sludge: a microcosms study.

Oily sludge is a residue from the petroleum industry composed of a mixture of sand, water, metals, and high content of hydrocarbons (HCs). The heavy oily sludge used in this study originated from Colombian crude oil with high density and low American Petroleum Institute (API) gravity. The residual waste from heavy oil processing was subject to thermal and centrifugal extraction, resulting in heavy oily sludge with very high density and viscosity. Biodegradation of the total petroleum hydrocarbons (TPH) was tested in microcosms using several bioremediation approaches, including: biostimulation with bulking agents and nutrients, the surfactant Tween 80, and bioaugmentation. Select HC degrading bacteria were isolated based on their ability to grow and produce clear zones on different HCs. Degradation of TPH in the microcosms was monitored gravimetrically and with gas chromatography (GC). The TPH removal in all treatments ranged between 2 and 67%, regardless of the addition of microbial consortiums, amendments, or surfactants within the tested parameters. The results of this study demonstrated that bioremediation of heavy oily sludge presents greater challenges to achieve regulatory requirements. Additional physicochemical treatments analysis to remediate this recalcitrant material may be required to achieve a desirable degradation rate.

Bioremediation of copper in sediments from a constructed wetland ex situ with the novel bacterium Cupriavidus basilensis SRS.

The H-02 constructed wetland was designed to remove metals (primarily copper and zinc) to treat building process water and storm water runoff from multiple sources associated with the Tritium Facility at the DOE-Savannah River Site, Aiken, SC. The concentration of Cu and Zn in the sediments has increased over the lifetime of the wetland and is a concern. A bioremediation option was investigated at the laboratory scale utilizing a newly isolated bacterium of the copper metabolizing genus Cupriavidus isolated from Tim's Branch Creek, a second-order stream that eventually serves as a tributary to the Savannah River, contaminated with uranium and other metals including copper, nickel, and mercury. Cupriavidus basilensis SRS is a rod-shaped, gram-negative bacterium which has been shown to have predatory tendencies. The isolate displayed resistance to the antibiotics ofloxacin, tetracycline, ciprofloxacin, select fungi, as well as Cu2+ and Zn2+. Subsequent ribosomal sequencing demonstrated a 100% confidence for placement in the genus Cupriavidus and a 99.014% match to the C. basilensis type strain. When H-02 wetland samples were inoculated with Cupriavidus basilensis SRS samples showed significant (p < 0.05) decrease in Cu2+ concentrations and variability in Zn2+ concentrations. Over the 72-h incubation there were no significant changes in the inoculate densities (106-108 cells/ML) indicating Cupriavidus basilensis SRS resiliency in this environment. This research expands our understanding of the Cupriavidus genus and demonstrates the potential for Cupriavidus basilensis SRS to bioremediate sites impacted with heavy metals, most notably copper.

The impact of tritium phytoremediation on plant health as measured by fluorescence.

Phytoremediation, using plants for soil, sediment, or water contaminant clean-up, is an established technology dependent on plant health. Tritium (3H), a radioactive isotope of hydrogen that is generally found in the environment as tritiated water (HTO), is a low-level beta emitter with a half-life of 12.32 years. Chlorophyll fluorescence (CF) for monitoring risk assessment of tritium to plant health was conducted at the Tritium Irrigation Facility (TIF) located on the US Department of Energy's Savannah River Site (SRS) near Aiken, SC. Two fluorometers were evaluated in conjunction with phytoremediation at the 25 -acre TIF where tritiated groundwater is being spray-irrigated on a mixed coniferous/deciduous forested watershed as a means of reducing tritium release to a nearby stream that serves as a tributary to the Savannah River. Tritium activity in irrigated water averaged 104 + 42 pCi mL-1 during the 2003 project. Fluorescence parameters measured by the two fluorometers were well correlated with each other (p < 0.0001). Tritium in water respired from oak leaves ranged up to 1845.13 pCi ml-1 and 2138.22 pCi ml-1 in pine needles. Trees in both the test and control sites were approximately 15 years old. Here we demonstrated that fluorescence parameters provide an effective way to estimate the impact of HTO on plant health in a noninvasive, extremely rapid, and cost-effective manner. In the current study applying fluorometry, plants within the TIF phytoremediation site exposed to the site tritiated water were not significantly impacted by the tritium phytoremediation based on CF parameters as compared to the control, a nascent non-irrigated site.

Draft Genome Sequence of Cupriavidus basilensis SRS, a Bacterium Isolated from Stream Sediments.

Cupriavidus basilensis SRS was isolated from stream sediments from the Savannah River Site in South Carolina. Here, we report the draft genome sequence and annotation of Cupriavidus basilensis SRS. The genome contains 8,918,236 bp and 7,916 predicted protein-coding genes, with a total G+C content of 65.2%.

Infectious Diseases Impact on Biomedical Devices and Materials.

Infectious diseases and nosocomial infections may play a significant role in healthcare issues associated with biomedical materials and devices. Many current polymer materials employed are inadequate for resisting microbial growth. The increase in microbial antibiotic resistance is also a factor in problematic biomedical implants. In this work, the difficulty in diagnosing biomedical device-related infections is reviewed and how this leads to an increase in microbial antibiotic resistance. A conceptualization of device-related infection pathogenesis and current and future treatments is made. Within this conceptualization, we focus specifically on biofilm formation and the role of host immune and antimicrobial therapies. Using this framework, we describe how current and developing preventative strategies target infectious disease. In light of the significant increase in antimicrobial resistance, we also emphasize the need for parallel development of improved treatment strategies. We also review potential production methods for manufacturing specific nanostructured materials with antimicrobial functionality for implantable devices. Specific examples of both preventative and novel treatments and how they align with the improved care with biomedical devices are described.

The Impact of Storms on Legionella pneumophila in Cooling Tower Water, Implications for Human Health.

At the U.S. Department of Energy's Savannah River Site (SRS) in Aiken, SC, cooling tower water is routinely monitored for Legionella pneumophila concentrations using a direct fluorescent antibody (DFA) technique. Historically, 25-30 operating SRS cooling towers have varying concentrations of Legionella in all seasons of the year, with patterns that are unpredictable. Legionellosis, or Legionnaires' disease (LD), is a pneumonia caused by Legionella bacteria that thrive both in man-made water distribution systems and natural surface waters including lakes, streams, and wet soil. Legionnaires' disease is typically contracted by inhaling L. pneumophila, most often in aerosolized mists that contain the bacteria. At the SRS, L. pneumophila is typically found in cooling towers ranging from non-detectable up to 108 cells/L in cooling tower water systems. Extreme weather conditions contributed to elevations in L. pneumophila to 107-108 cells/L in SRS cooling tower water systems in July-August 2017. L. pneumophila concentrations in Cooling Tower 785-A/2A located in SRS A-Area, stayed in the 108 cells/L range despite biocide addition. During this time, other SRS cooling towers did not demonstrate this L. pneumophila increase. No significant difference was observed in the mean L. pneumophila mean concentrations for the towers (p < 0.05). There was a significant variance observed in the 285-2A/A Tower L. pneumophila results (p < 0.05). Looking to see if we could find "effects" led to model development by analyzing 13 months of water chemistry and microbial data for the main factors influencing the L. pneumophila concentrations in five cooling towers for this year. It indicated chlorine and dissolved oxygen had a significant impact (p < 0.0002) on cooling tower 785A/2A. Thus, while the variation in the log count data for the A-area tower is statistically greater than that of the other four towers, the average of the log count data for the A-Area tower was in line with that of the other towers. It was also observed that the location of 785A/2A and basin resulted in more debris entering the system during storm events. Our results suggest that future analyses should evaluate the impact of environmental conditions and cooling tower design on L. pneumophila water concentrations and human health.

Bioremediation of Hexanoic Acid and Phenanthrene in Oil Sands Tailings by the Microbial Consortium BioTiger™.

Polycyclic aromatic hydrocarbons (PAHs) and naphthenic acids (NAs) are toxic contaminants of environmental concern found in process water and mature fine tailings, or tailings, from the oil sands industry. BioTiger™, a patented microbial consortium of twelve natural environmental isolates, was found to cometabolically biodegrade the NA hexanoic acid and the PAH phenanthrene in the presence of tailings. Hexanoamide was found to be produced and consumed during cometabolism of hexanoic acid. Mechanistic analysis demonstrated three of the BioTiger™ strains generated biosurfactants with the bacterial adhesion to hydrocarbons assay, seven with the methylene blue active substances assay, and nine with a hemolysis assay. Serial transfers of the BioTiger™ consortium demonstrated the stability of hexanoic acid degradation over several generations. The results demonstrate that BioTiger™ cometabolically biodegrades combinations of phenanthrene and hexanoic acid in tailings. This work reveals the potential for in situ bioremediation of tailings with this natural microbial consortium.

Influence of triethyl phosphate on phosphatase activity in shooting range soil: Isolation of a zinc-resistant bacterium with an acid phosphatase.

Phosphatase-mediated hydrolysis of organic phosphate may be a viable means of stabilizing heavy metals via precipitation as a metal phosphate in bioremediation applications. We investigated the effect of triethyl phosphate (TEP) on soil microbial-phosphatase activity in a heavy-metal contaminated soil. Gaseous TEP has been used at subsurface sites for bioremediation of organic contaminants but not applied in heavy-metal contaminated areas. Little is known about how TEP affects microbial activity in soils and it is postulated that TEP can serve as a phosphate source in nutrient-poor groundwater and soil/sediments. Over a 3-week period, TEP amendment to microcosms containing heavy-metal contaminated soil resulted in increased activity of soil acid-phosphatase and repression of alkaline phosphatase, indicating a stimulatory effect on the microbial population. A soil-free enrichment of microorganisms adapted to heavy-metal and acidic conditions was derived from the TEP-amended soil microcosms using TEP as the sole phosphate source and the selected microbial consortium maintained a high acid-phosphatase activity with repression of alkaline phosphatase. Addition of 5mM zinc to soil-free microcosms had little effect on acid phosphatase but inhibited alkaline phosphatase. One bacterial member from the consortium, identified as Burkholderia cepacia sp., expressed an acid-phosphatase activity uninhibited by high concentrations of zinc and produced a soluble, indigo pigment under phosphate limitation. The pigment was produced in a phosphate-free medium and was not produced in the presence of TEP or phosphate ion, indicative of purple acid-phosphatase types that are pressed by bioavailable phosphate. These results demonstrate that TEP amendment was bioavailable and increased overall phosphatase activity in both soil and soil-free microcosms supporting the possibility of positive outcomes in bioremediation applications.

Kinetics of 1,2-dichloroethane and 1,2-dibromoethane biodegradation in anaerobic enrichment cultures.

1,2-Dichloroethane (1,2-DCA) and 1,2-dibromoethane (ethylene dibromide [EDB]) contaminate groundwater at many hazardous waste sites. The objectives of this study were to measure yields, maximum specific growth rates (µ), and half-saturation coefficients (K(S)) in enrichment cultures that use 1,2-DCA and EDB as terminal electron acceptors and lactate as the electron donor and to evaluate if the presence of EDB has an effect on the kinetics of 1,2-DCA dehalogenation and vice versa. Biodegradation was evaluated at the high concentrations found at some industrial sites (>10 mg/liter) and at lower concentrations found at former leaded-gasoline sites (1.9 to 3.7 mg/liter). At higher concentrations, the Dehalococcoides yield was 1 order of magnitude higher when bacteria were grown with 1,2-DCA than when they were grown with EDB, while µ's were similar for the two compounds, ranging from 0.19 to 0.52 day(-1) with 1,2-DCA to 0.28 to 0.36 day(-1) for EDB. K(S) was larger for 1,2-DCA (15 to 25 mg/liter) than for EDB (1.8 to 3.7 mg/liter). In treatments that received both compounds, EDB was always consumed first and adversely impacted the kinetics of 1,2-DCA utilization. Furthermore, 1,2-DCA dechlorination was interrupted by the addition of EDB at a concentration 100 times lower than that of the remaining 1,2-DCA; use of 1,2-DCA did not resume until the EDB level decreased close to its maximum contaminant level (MCL). In lower-concentration experiments, the preferential consumption of EDB over 1,2-DCA was confirmed; both compounds were eventually dehalogenated to their respective MCLs (5 µg/liter for 1,2-DCA, 0.05 µg/liter for EDB). The enrichment culture grown with 1,2-DCA has the advantage of a more rapid transition to 1,2-DCA after EDB is consumed.

Reduction of petroleum hydrocarbons and toxicity in refinery wastewater by bioremediation.

The aim of the study was to investigate petroleum waste remediation and toxicity reduction by five bacterial strains: Ralstonia picketti SRS (BP-20), Alcaligenes piechaudii SRS (CZOR L-1B), Bacillus subtilis (I'-1a), Bacillus sp. (T-1), and Bacillus sp. (T'-1), previously isolated from petroleum-contaminated soils. Petroleum hydrocarbons were significantly degraded (91%) by the mixed bacterial cultures in 30 days (reaching up to 29% in the first 72 h). Similarly, the toxicity of the biodegraded petroleum waste decreased 3-fold after 30 days. This work shows the influence of bacteria on hydrocarbon degradation and associated toxicity, and its dependence on the specific microorganisms present. The ability of these mixed cultures to degrade hydrocarbons and reduce toxicity makes them candidates for environmental restoration applications at other hydrocarbon-contaminated environments.

Phytoremediation of chlorinated ethenes in seepline sediments: tree selection.

Phytoremediation of chlorinated ethene (CE)-contaminated water was investigated at the Savannah River Site in Aiken, SC, USA. Perchloroethylene (PCE) and trichloroethylene (TCE) are present where CE-contaminated groundwater currently outcrops in seepline soils. Results of constructed and planted test cells, filled with soil from a noncontaminated seepline area and supplied with CE-contaminated groundwater (48 ppb) in the field for one season are presented. These test cells were planted with loblolly pines, hybrid poplars, coyote willow, and sweet gum. Cis-dichloroethylene (cDCE), a byproduct from rhizosphere microbial activity, was detected in the soils as well as some tree tissues. All trees tested were found to uptake both PCE and TCE (5-50 pbb/gm dry wt).

Assessment of genotoxic activity of petroleum hydrocarbon-bioremediated soil.

The relationship between toxicity and soil contamination must be understood to develop reliable indicators of environmental restoration for bioremediation. Two bacterial rapid bioassays, SOS chromotest and the umu test with and without metabolic activation (S-9 mixture), were used to evaluate the genotoxicity of petroleum hydrocarbon-contaminated soil following bioremediation treatment. The soil was taken from an engineered biopile at the Czechowice-Dziedzice Polish oil refinery (CZOR). The bioremediation process in the biopile lasted 4 years, and the toxicity measurements were done after this treatment. Carcinogens detected in the soil, polyaromatic hydrocarbons (PAHs), were reduced to low concentrations (2mg/kg dry wt) by the bioremediation process. Genotoxicity was not observed for soils tested with and without metabolic activation by a liver homogenate (S-9 mixture). However, the umu test was more sensitive than the SOS chromotest in the analysis of petroleum hydrocarbon-bioremediated soil. Analytical results of soil used in the bioassays confirmed that the bioremediation process reduced 81% of the petroleum hydrocarbons including PAHs. We conclude that the combined test systems employed in this study are useful tools for the genotoxic examination of remediated petroleum hydrocarbon-contaminated soil.

Surface active properties of bacterial strains isolated from petroleum hydrocarbon-bioremediated soil.

Two bacterial strains identified as Ralstonia picketti (BP 20) and Alcaligenes piechaudii (CZOR L-1B) were isolated from petroleum hydrocarbon-contaminated soil following bioremediation treatment. The surface active properties, e.g. surface tension, emulsification and foamability of their culture filtrates were evaluated. Bacterial cell-surface hydrophobicity (BAH) as measured by analyzing cell affinity towards aliphatic and aromatic compounds was also determinated. The bacteria grew in liquid cultures containing 1% (v/v) of crude oil as carbon and energy source at 30 degrees C under aerobic conditions. The surface tensions were reduced to 61 mN/m and 55 mN/m by Ralstonia picketti and Alcaligenes piechaudii, respectively. The emulsification index (EI24) was almost 100% for all tested compounds except diesel oil. The stability of the emulsions was deteminated at 4 degrees C, 45 degrees C and 65 degrees C. The emulsions were stable at 4 degrees C. Ralstonia picketti was better foam inducer (FV = 50 ml) compared to Alcaligenes piechaudii (FV = 10 ml). The BAH measurements revealed higher adhesion of Alcaligenes piechaudii cells towards different hydrocarbons compared to Ralstonia picketti cells. The strains were found to have a surface hydrophobicity in the following order: aliphatic hydrocarbons, BTEX, and PAHs. The ability to adhere to bulk hydrocarbon is mostly a characteristic of hydrocarbon-degrading bacteria. The strains were found to be better emulsifiers than surface tension reducers. They produce water-soluble extracellular bioemulsifiers. Both bacterial isolates have good properties to use them, mainly in the petroleum industry, e.g. in enhanced oil recovery and in bioremediation processes-primarily due to their emulsification property, i.e. emulsion forming and stabilizing capacity.

Lipid biomarkers and carbon isotope signatures of a microbial (Beggiatoa) mat associated with gas hydrates in the gulf of Mexico.

White and orange mats are ubiquitous on surface sediments associated with gas hydrates and cold seeps in the Gulf of Mexico. The goal of this study was to determine the predominant pathways for carbon cycling within an orange mat in Green Canyon (GC) block GC 234 in the Gulf of Mexico. Our approach incorporated laser-scanning confocal microscopy, lipid biomarkers, stable carbon isotopes, and 16S rRNA gene sequencing. Confocal microscopy showed the predominance of filamentous microorganisms (4 to 5 mum in diameter) in the mat sample, which are characteristic of Beggiatoa. The phospholipid fatty acids extracted from the mat sample were dominated by 16:1omega7c/t (67%), 18:1omega7c (17%), and 16:0 (8%), which are consistent with lipid profiles of known sulfur-oxidizing bacteria, including Beggiatoa. These results are supported by the 16S rRNA gene analysis of the mat material, which yielded sequences that are all related to the vacuolated sulfur-oxidizing bacteria, including Beggiatoa, Thioploca, and Thiomargarita. The delta13C value of total biomass was -28.6 per thousand; those of individual fatty acids were -29.4 to -33.7 per thousand. These values suggested heterotrophic growth of Beggiatoa on organic substrates that may have delta13C values characteristic of crude oil or on their by-products from microbial degradation. This study demonstrated that integrating lipid biomarkers, stable isotopes, and molecular DNA could enhance our understanding of the metabolic functions of Beggiatoa mats in sulfide-rich marine sediments associated with gas hydrates in the Gulf of Mexico and other locations.

The application of bioassays as indicators of petroleum-contaminated soil remediation.

Bioremediation has proven successful in numerous applications to petroleum contaminated soils. However, questions remain as to the efficiency of bioremediation in lowering long-term soil toxicity. In the present study, the bioassays Spirotox, Microtox, Ostracodtoxkit F, umu-test with S-9 activation, and plant assays were applied, and compared to evaluate bioremediation processes in heavily petroleum contaminated soils. Six higher plant species (Secale cereale L., Lactuca sativa L., Zea mays L., Lepidium sativum L., Triticum vulgare L., Brassica oleracea L.) were used for bioassay tests based on seed germination and root elongation. The ecotoxicological analyses were made in DMSO/H2O and DCM/DMSO soil extracts. Soils were tested from two biopiles at the Czechowice oil refinery, Poland, that have been subjected to different bioremediation applications. In biopile 1 the active or engineered bioremediation process lasted four years, while biopile 2 was treated passively or non-engineered for eight months. The test species demonstrated varying sensitivity to soils from both biopiles. The effects on test organisms exposed to biopile 2 soils were several times higher compared to those in biopile 1 soils, which correlated with the soil contaminants concentration. Soil hydrocarbon concentrations indeed decreased an average of 81% in biopile 1, whereas in biopile 2 TPH/TPOC concentrations only decreased by 30% after eight months of bioremediation. The bioassays were presented to be sensitive indicators of soil quality and can be used to evaluate the quality of bioremediated soil. The study encourages the need to combine the bioassays with chemical monitoring for evaluation of the bioremediation effectiveness and assessing of the contaminated/remediated soils.

Involvement of linear plasmids in aerobic biodegradation of vinyl chloride.

Pseudomonas putida strain AJ and Ochrobactrum strain TD were isolated from hazardous waste sites based on their ability to use vinyl chloride (VC) as the sole source of carbon and energy under aerobic conditions. Strains AJ and TD also use ethene and ethylene oxide as growth substrates. Strain AJ contained a linear megaplasmid (approximately 260 kb) when grown on VC or ethene, but it contained no circular plasmids. While strain AJ was growing on ethylene oxide, it was observed to contain a 100-kb linear plasmid, and its ability to use VC as a substrate was retained. The linear plasmids in strain AJ were cured, and the ability of strain AJ to consume VC, ethene, and ethylene oxide was lost following growth on a rich substrate (Luria-Bertani broth) through at least three transfers. Strain TD contained three linear plasmids, ranging in size from approximately 90 kb to 320 kb, when growing on VC or ethene. As with strain AJ, the linear plasmids in strain TD were cured following growth on Luria-Bertani broth and its ability to consume VC and ethene was lost. Further analysis of these linear plasmids may help reveal the pathway for VC biodegradation in strains AJ and TD and explain why this process occurs at many but not all sites where groundwater is contaminated with chloroethenes. Metabolism of VC and ethene by strains AJ and TD is initiated by an alkene monooxygenase. Their yields during growth on VC (0.15 to 0.20 mg of total suspended solids per mg of VC) are similar to the yields reported for other isolates (i.e., Mycobacterium sp., Nocardioides sp., and Pseudomonas sp.).

Relationship between soil microbial diversity and bioremediation process at an oil refinery.

Microbial diversity in hydrocarbon-contaminated soil was characterized during a bioremediation project at an oil refinery. The project consisted of isolation and cultivation of microbes on laboratory media and the subsequent characterization of pure isolates. In a lagoon at the Czechowice Oil Refinery, Poland, a biopile with actively and passively aerated sections was constructed and has been operated since 1997. The bioremediation process has been continuously monitored by physical, chemical, and microbiological methods. One hundred and forty nine bacterial and fungal strains were isolated from site soils by standard procedures. Analysis of cultivable microorganisms revealed a diverse microbial population within the cultured isolates. Among isolated strains, Pseudomonas and Chryseomonas genera predominated in the bacterial population while Candida, Fusarium, and Trichophyton dominated the fungal population. This paper describes the application of traditional microbiological methods (plating and microscopic methods) to evaluate cultivable microbial diversity in bioremediated soil.

Adaptation of an automated assay for determination of beta-hydroxybutyrate in dogs using a random access analyzer.

An automated method for measuring beta-hydroxybutyrate was adapted to the Ciba-Corning 550 Express trade mark random access analyzer. The assay was based on a kinetic reaction utilizing hydroxybutyrate-dehydrogenase. Beta-hydroxybutyrate concentration (mmol/L) was calculated ratiometrically using a 1.0 mmol/l standard. Canine serum, plasma, and urine were used without prior deproteinization and only a 30-microliter sample was required. The method demonstrated good linearity between 0 to 2 mmol/l of beta-hydroxybutyrate. Analytical recovery (accuracy) within these concentrations ranged from 85.8 to 113.3%. Both within-run and day-to-day precision were determined, as was specificity of the assay in the presence of a variety of interfering substances. The automated assay was rapid and economical, with reagent stability maintained for at least 2 weeks at 4 degrees C. This assay can readily be applied toward the assessment of ketoacidosis in dogs, and with further validation, other species.