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PubMedDate not supplied

Kinetics of biotransformation of 2,4-dichlorophenol using UASB-reactor.

Chlorophenol compounds are environmental pollutants that are both anthropogenic and xenobiotics. Some of these chemicals are carcinogens and are both toxic to a number biochemical processes. Biotransformation of 2,4-dichlorophenol (2,4-DCP) was studied in the presence of glucose on an upflow anaerobic sludge blanket reactor (UASB) using mixed culture. A continuously operated UASB reactor was employed using mixed synthetic wastewater. Results obtained from the 1.8 L volume capacity UASB reactor were subjected to kinetic evaluation constants. Results indicate that the degradation of 2,4-DCP in the presence of glucose was strongly influenced by the concentration of the compound. High degradation levels were observed when the concentration of 2,4-DCP was in the range of 50-150 mg L(-1). Concentrations of 2,4-DCP above 160 mg L(-1) were toxic to microbes even in the presence of glucose. The maximum degradation of 2,4-DCP was found to be 70.4% when initial concentration of 2,4-DCP was 124 mg L(-1) and glucose concentration of 500 mg L(-1) at hydraulic retention time of 13.2 hr. The biodegradation followed first order reaction kinetics with a rate constant (K) of 0.67, Vmax of 0.244 kg m(-3) day(-1), Ks of 0.117 kg m(-3) day(-1) and correlation coefficient of 0.766.

Anaerobiosis↗
PubMed1983

Epidemiological study of socioeconomic factors and clinical findings in Hodgkin's disease, and reanalysis of previous data regarding chemical exposure.

An association between malignant lymphoma (both Hodgkin's disease (HD) and non-Hodgkin lymphoma) and exposure to organic solvents, phenoxy acids, or chlorophenols was previously reported. A reanalysis of this investigation regarding the cases with HD and exposure to various chemicals was performed and resulted in comparable findings to the whole group of malignant lymphoma. There was an overrepresentation of cases with primary involvement of the gastrointestinal tract which was associated with exposure to these chemicals. The influence of previous diseases and socioeconomic factors was analysed through a supplementary questionnaire to the cases with HD and their matched controls. No differences were found in cases and controls for such variables except for tonsillectomy which was overrepresented among the cases as well as a history of previous duodenal or ventricular ulceration. These findings were, however, insignificant.

Adult↗
PubMed1986

Case-control study of multiple myeloma and farming.

A previous case-control study which utilised the occupational information available on the New Zealand Cancer Registry found an increased risk of multiple myeloma in agricultural workers consistent with previous findings in the United States. The findings are now presented for the second phase of the study which involved interviewing 76 cases of multiple myeloma (who had been included in the previous study) together with 315 controls with other types of cancer. The previous finding on an excess of farmers in the case group was confirmed by the interview data (odds ratio = 1.7, 95% confidence limits 1.0-2.9, P = 0.04). There were no significant differences between cases and controls regarding potential exposure to phenoxy herbicides or chlorophenols. There were also no significant differences regarding activities involving potential exposure to other agricultural chemicals, although the odds ratio for fencing work, which may involve exposure to arsenic and sodium pentachlorophenate, was 1.6 (95% confidence limits 0.9-2.7, P = 0.11). The odds ratios were significantly elevated for sheep farming (odds ratio = 1.9, 95% confidence limits 1.0-3.6, P = 0.04) and exposure to beef cattle (odds ratio = 1.7, 95% confidence limits 1.0-2.9, P = 0.05). The odds ratio was also elevated for persons reporting a history of hay fever (odds ratio = 1.9, 95% confidence limits 1.0-3.5, P = 0.05). Overall, these findings suggest that the search for the causes of elevated mortality in farmers from multiple myeloma should be directed to potential causes other than pesticide exposure.

Aged↗
PubMed1982

Changes in hepatic microsomal enzyme function in workers exposed to mixtures of chemicals.

Antipryine clearance was measured during occupational exposure and after an exposure-free interval of 3 wk in 26 spray painters, 44 workers in a herbicide-producing (phenoxyacids) plant, and 10 pesticide-exposed greenhouse gardeners. A control group of 39 workers were investigated simultaneously. A noninvasive simplified antipyrine clearance was used. Antipyrine clearance was greater in pesticide-exposed persons, but it was 15% less in workers exposed to spray paints compared to 3 wk after discontinuation of exposure. Clearance was unchanged in the control group examined at the same interval. Thus, the combined action of different spray paints, grinding dust, and cleaners may inhibit, and phenoxyacids, chlorophenols, and various pesticides may induce, the microsomal enzyme function. The workers had been exposed to the chemicals for years, but the change in antipyrine clearance apparently was reversible.

Adolescent↗
PubMed1994

Trace enrichment of chlorinated phenols from drinking water on chemically bonded sorbents for high-performance liquid chromatography.

A method for the determination of seven chlorinated phenols, including those in the US Environmental Protection Agency list of priority pollutants, in drinking water is described. Solid-phase extraction on various alkylsilica bonded phases such as phenyl-, octyl- and octadecylsilica was used for sample preconcentration. The dependences of recovery on the eluent volume, sample pH and sample volume were investigated for all the sorbents in order to find the optimum sorbent. Octadecylsilica was found to be the most efficient sorbent. The sample pH was adjusted to 5.0 and, after concentration, the analytes were eluted with 1.2 cm3 of acetonitrile-0.012 mol dm-3 phosphoric acid (9 + 1). The extract was submitted to reversed-phase high-performance liquid chromatography with acetonitrile-0.007 mol dm-3 phosphoric acid (51 + 49) as the eluent and ultraviolet detection at 225 nm. Recoveries after 28 d of storage in a refrigerator at 3 degrees C were also studied. The results were found to agree with those before storage. A comparison between Bakerbond C18 and Separcol C18 is presented. The results obtained were comparable for both types of octadecylsilica. The recoveries of seven chlorinated phenols were higher than 88%. Extracting a 1 dm3 water sample, a detection limit of 2.6 micrograms dm-3 was determined for 2-chlorophenol and detection limits between 0.12 and 0.38 microgram dm-3 were obtained for the other six chlorinated phenols.

Absorption↗
PubMed2003

Reduction in excess sludge production by addition of chemical uncouplers in activated sludge batch cultures.

AIMS: To investigate the possibility of reducing excess sludge production in activated sludge processes by the addition of chemical uncouplers to greatly dissociate anabolism from catabolism. METHODS AND RESULTS: Ortho-chlorophenol (oCP), 2,4-dichlorophenol (DCP), 3,3',4',5-tetrachlorosalicylanilide (TCS), para-dinitrophenol (pNP) and 2,4-dinitrophenol (DNP) were chosen for short-term tests for their ability to reduce sludge yield by shaking bottle test. The most effective chemicals, DNP and pNP, together with TCS were tested for various uncoupler concentrations and biomass concentrations. TCS was tested in a lab-scale completely mixed activated sludge batch culture. The model (demonstrated by Liu) was verified with experimental data in completely mixed activated sludge batch test, but was inconsistent with the results from the shaking bottle batch test. The observed growth yield (Yobs) decreased with increasing of the ratio of initial uncoupler concentration to initial biomass concentration (Cu/X0). CONCLUSIONS: We suggest that the uncouplers oCP, DCP, TCS, pNP and DNP can cause a significant decrease in sludge production, the metabolism of which can explain the decline in sludge yield. SIGNIFICANCE AND IMPACT OF THE STUDY: The real strength of chemical uncoupler imposing on biomass should be Cu/X0, not initial uncoupler concentration (Cu) alone. Chemical uncouplers can be used to develop the activated sludge processes for minimizing excess sludge production.

Biomass↗
PubMed2000

Monitoring of Desulfitobacterium frappieri PCP-1 in pentachlorophenol-degrading anaerobic soil slurry reactors.

Anaerobic biodegradation of pentachlorophenol (PCP) was studied in rotative bioreactors containing 200 g of PCP-contaminated soil and 250 ml of liquid medium. Reactors were bioaugmented with cells of Desulfitobacterium frappieri strain PCP-1, a bacterium able to dehalogenate PCP to 3-chlorophenol. Cells of strain PCP-1 were detected by quantitative PCR for at least 21 days in reactors containing 500 mg of PCP per kg of soil but disappeared after 21 days in reactors with 750 mg of PCP per kg of soil. Generally, PCP was completely removed in less than 9 days in soils contaminated with 189 mg of PCP per kg of soil. Sorption of PCP to soil organic matter reduced its toxicity and enhanced the survival of strain PCP-1. In some non-inoculated reactors, the indigenous microorganisms of some soils were also able to degrade PCP. These results suggest that anaerobic dechlorination of PCP in soils by indigenous PCP-degrading bacteria, or after augmentation with D. frappieri PCP-1, should be possible in situ and ex situ when the conditions are favourable for the survival of the degrading microorganisms.

Anaerobiosis↗
PubMed2003

Poly-beta-hydroxyalkanoates consumption during degradation of 2,4,6-trichlorophenol by Sphingopyxis chilensis S37.

AIMS: To analyse the possible effect of poly-beta-hydroxyalkanoate (PHA) consumption on 2,4,6-trichlorophenol (2,4,6-TCP) degradation during starvation by Sphingopyxis chilensis S37 strain, which stores PHAs and degrades 2,4,6-TCP. METHODS AND RESULTS: The strain was inoculated in saline solution supplemented with 2,4,6-TCP (25-400 microm). Chlorophenol degradation was followed both spectrophotometrically and by chlorine released; viable bacterial counts were also determined. Cells starved for 24, 48 or 72 h were incubated with 25 microm of 2,4,6-TCP and PHA in cells investigated by spectrofluorimetric and flow cytometry. Results demonstrated that starvation decreased the ability to degrade 2,4,6-TCP. After 72 h of starvation, degradation of 2,4,6-TCP decreased to less than 10% and the relative PHA content diminished to ca 50% during the first 24 h. CONCLUSION: Utilization of PHA may be an important factor for the degradation of toxic compounds, such as 2,4,6-TCP, in bacterial strains unable to use this toxic compound as carbon and energy source. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study describing a relationship between intracellular PHA consumption and 2,4,6-TCP degradation. Therefore, PHAs provides an endogenous carbon and energy source under starvation and can play a significant role in the degradation of toxic compounds.

Alphaproteobacteria↗
PubMed2003

Practical applications of the Fenton reaction to the removal of chlorinated aromatic pollutants. Oxidative degradation of 2,4-dichlorophenol.

BACKGROUND: Chlorophenols (CPs) constitute a group of organic pollutants that are introduced into the environment as a result of several man-made activities, such as uncontrolled use of pesticides and herbicides, and as byproducts in the paper pulp bleaching. Promising removal technologies of chlorinated aromatics consist in the application of advanced oxidation processes (AOPs) that can provide an almost total degradation of a variety of contaminants. Among these, wide application find Fenton systems based on generation of reactive species having a high oxidizing power, such as hydroxyl radical HO*. Our objective was that of determining the overall degradation efficiency of the model compound 2,4-dichlorophenol (DCP) by thermal Fenton-type oxidation systems with a view toward defining in more details relevant process parameters, the effect of reaction temperature and of co-catalyst Cu2+. METHODS: Reaction conditions were similar to those generally adopted as optimal in many practical applications, i.e. pollutant/Fe2+ (as FeSO4) ratio ca. 20, Fe2+/Cu2+ (co-catalyst) 2:1, pH adjusted and controlled at pH 3, and H2O2 in excess (up to four-fold over the stoichiometric amount required for complete mineralization). RESULTS AND DISCUSSION: The results demonstrate that it is advantageous to carry out the reaction at a temperature markedly higher (70 degrees C) than ambient. The stepwise addition of H2O2 in aliquots yields an efficient transformation, while allowing a convenient control of the reaction exothermicity. Under these conditions, the essentially complete removal of the initial DCP is accomplished using just one equiv of H2O2 during 15 min; excess H2O2 (5 equivalents) yields extensive substrate mineralization. Also relevant, at 70 degrees C dechlorination of the initial DCP (and of derived reaction intermediates) is remarkably extensive (3-5% residual TOX), already with the addition of 1 equiv of H2O2. At the end of the reaction, IC and IC-MS analyses of the solution reveal that only low-molecular weight carboxylic acid (acetic, formic, oxalic, malonic, tartaric, etc.) contribute to the residual TOC. CONCLUSIONS: The whole of the results herein point to the advantage of performing the process at temperatures substantially higher than ambient (70 degrees C). Under the conditions adopted, almost complete degradation of the initial toxic compound can be achieved using hydrogen peroxide in fair excess (e.g., 3.5 equiv H2O2). RECOMMENDATIONS AND OUTLOOK: In applying practical Fenton-type degradation systems to heavily polluted wastes, either for the pre-treatment of waters with a high COD value prior to biodegradation or for complete mineralization of pollutants, the set up of appropriate reaction conditions appears to be a key factor. Also, it is desirable to keep the concentration of iron salts within the lower limits in order to minimize the production and disposal of iron oxide sludges.

Chlorophenols↗
PubMed1997

Microperoxidase/H2O2-mediated alkoxylating dehalogenation of halophenol derivatives in alcoholic media.

The results of this study report the H2O2-driven microperoxidase-8 (MP8)-catalyzed dehalogenation of halophenols such as 4-fluorophenol, 4-chlorophenol, 4-bromophenol, and 2-fluorophenol in alcoholic solvents. In methanol, the conversion of the para-halophenols and 2-fluorophenol to, respectively, 4-methoxyphenol and 2-methoxyphenol, as the major dehalogenated products is observed. In ethanol, 4-ethoxyphenol is the principal dehalogenated product formed from 4-fluorophenol. Two mechanisms are suggested for this MP8-dependent alkoxylating dehalogenation reaction. In one of these mechanisms the oxene resonant form of compound I of MP8 is suggested to react with methanol forming a cofactor-peroxide-alkyl intermediate. This intermediate reacts with the reactive pi-electrons of the substrate, leading to the formation of the alkoxyphenols and the release of the fluorine substituent as fluoride anion.

Chlorophenols↗
PubMed2004

Regulation of anaerobic dehalorespiration by the transcriptional activator CprK.

Desulfomonile, Desulfitobacterium, and Dehalobacter are anaerobic microbes that can derive energy from the reductive dehalogenation of chlorinated organic compounds, many of which are environmental pollutants. There is very little information about how anaerobic dehalorespiration is regulated. An open reading frame within the Desulfitobacterium dehalogenans chlorophenol reductase (cpr) gene cluster (cprK) was proposed to be a transcriptional regulatory protein (Smidt, H., van Leest, M., van der Oost, J., and deVos, W. M. (2000) J. Bacteriol. 182, 5683-5691). We have cloned, actively overexpressed in Escherichia coli, and purified to homogeneity the D. dehalogenans CprK. The results of electrophoretic mobility shift assays, DNA footprinting studies, and promoter-lac fusion experiments indicate that CprK is a transcriptional activator of the cpr gene cluster. CprK binds 3-chloro-4-hydroxyphenylacetate (CHPA) with high affinity (K(d) = 3.5 mum, determined by isothermal titration calorimetry), which promotes its specific interaction with a DNA sequence (TTAAT-N4-ACTAA) located upstream of the -35 and -10 promoter regions of several cpr genes and activates transcription of these genes. Binding to the upstream "box" sequence increases the affinity of CprK for CHPA by approximately 10-fold (K(d) = 0.4 mum, determined by electrophoretic mobility shift assays). Chlorophenylacetate, which lacks the ortho-hydroxy group, and hydroxyphenylacetate, lacking the chlorine group, do not activate transcription or promote DNA binding, even at millimolar concentrations, at least 1000-fold higher than the K(d) value for CHPA. Lacking metals, CprK is oxygen-sensitive. Oxidation by diamide, which converts thiols to the disulfide, inactivates CprK, and reduction of the oxidized protein by dithiothreitol fully restores DNA binding, indicating that CprK is redox-regulated and is active only when reduced. This is the first reported characterization of a transcriptional regulator of anaerobic dehalorespiration.

Base Sequence↗
PubMed2006

Sorption of pesticides on kaolinite and montmorillonite as a function of hydrophilicity.

Pesticides and other organic species are adsorbed by soil via different mechanisms, with bond strengths that depend on the properties of both the soil and the pesticide. Since the clay fraction in soil is a preferential sorbent for organic matter, reference kaolinite and montmorillonite are useful models for studying the mechanism and the strength of sorption. This paper presents the results of batch experiments to investigate the interactions of kaolinite KGa-1 and montmorillonite SWy-1 with the following pesticides and organic species resulting from the natural degradation of pesticides in the environment: atrazine (1-chloro-3-ethylamino-5-isopropylamino-2,4,6-triazine), simazine (1-chloro-3,5-bisethylamino-2,4,6-triazine), diuron [1,1-dimethyl-3-(3,4-dichlorophenyl)urea], aniline, 4-chlorophenol, 2,4-dichlorophenol, and 2,4,6-trichlorophenol. Each of these chemicals has different hydrophilicity. Systems containing 2.0 g of clay were put in contact with 100.0 mL of solutions of the pesticides at known concentration ranging from 1.0 to 5.0 mg/L, and the amount of solute adsorbed was evaluated through RP-HPLC analysis of the pesticide still present in the aqueous suspension. To test for electrostatic interactions between the clay surface and the pesticides, potentiometric titration was used to determine the permanent surface charge of clays. Experiments were performed at different pH values. The results indicate that, for the chemicals studied, neutral molecules are preferentially retained relative to ionized ones, and that montmorillonite is a more effective sorbent than kaolinite.

Adsorption↗
PubMed1978

The in vitro metabolism of lindane by an enzyme preparation from chicken liver.

The soluble fraction (105,000 x g, 30 min) of chicken liver homogenates contained an enzyme(s), probably a dehydrochlorinase(s), which metabolized lindane in vitro. The reaction was glutathione dependent and took place anaerobically. The enzyme(s) also metabolized the alpha- and delta-isomers but not the beta-isomer. About 66% of the in vitro metabolites were soluble in petroleum ether. From the ether-soluble fraction, o-, p- amd m-dichlorobenzene, 1,2,3- and 1,2,4-trichlorobenzene, gamma-2,3,4,5,6-pentachlorocyclohex-1-ene (gamma-PCCH), 2,3- and 2,4-dichlorophenol and 2,4,6-trichlorophenol were identified by comparison of mass spectra and gas chromatographic data with those of reference compounds. Seven additional metabolites were tentatively identified from their mass spectra data. These metabolites were: chlorobenzene, 2 isomers of trichlorocyclohexene, dichlorocyclohexadienetriol, chlorophenol, trichlorocyclohexenol and trichlorocyclohexanediol. The petroleum ether-extracted aqueous phase contained a number of unidentified conjugated metabolites. It was concluded that the metabolic pathway for lindane in the chicken is quite similar to that in the pheasant.

Animals↗
PubMed1999

Microbial dechlorination of 2,4,6-trichlorophenol in anaerobic sewage sludge.

The dechlorination of 2,4,6-trichlorophenol (TCP) in municipal sewage sludge with a chlorophenol (CP)-adapted consortium was investigated. Results show that dechlorination rates differed according to the source of the sludge samples used in the batch experiments. No significant differences in 2,4,6-TCP dechlorination were observed following treatment with inoculum at densities ranging from 10% to 50% (V/V), but a significant delay was noted at 5% (V/V) density. Overall, results show that the higher the 2,4,6-TCP concentration, the slower the dechlorination rate. The addition of acetate, lactate, pyruvate, vitamin B12 or manganese dioxide did not results in a significant change in 2,4,6-TCP dechlorination. Data collected from a bioreactor experiment revealed that pH 7.0 and a total solid concentration of 10 g/L were optimal for dechlorination. Dechlorination rates decreased significantly at higher agitation speeds. 2,4,6-TCP dechlorination was enhanced under methanogenic conditions, but it was inhibited under denitrifying and sulfate-reducing conditions.

Anaerobiosis↗
PubMed2004

Investigation on the usage of clay modified electrode for the electrochemical determination of some pollutants.

Redox behavior of three pollutants, namely endosulfan (EN), o-chlorophenol (OCP) and direct orange 8 (DO8) were investigated electrochemically using sodium montmorillonite clay modified glassy carbon electrode. Influence of pH, scan rate and concentration were studied on the voltammetric response. Suitable medium for the electrochemical studies of EN was pH 1.0 and for other two pollutants pH 13.0. EN exhibited one well-defined reduction peak accounting for irreversible 2e(-) transfer and leading to the removal of one chlorine atom. OCP underwent one electron oxidation to the formation of phenoxy radical resulting in an oxidation peak. DO8 showed two peaks for reductions and two peaks for oxidation as a result of the reduction of azo group and oxidation of phenoxide group. These electrochemical reactions of the three pollutants reveals the suitability of clay modified electrode for the electroanalysis. A differential pulse stripping voltammetric procedure for the determination of these pollutants was developed. The limits of determination for EN, OCP and DO8 are 5-300, 10-800 and 50-800 ppb, respectively.

Aluminum Silicates↗
PubMed1996

Rhodococcus percolatus sp. nov., a bacterium degrading 2,4,6-trichlorophenol.

A bacterial strain that was able to mineralize 2,4,6-trichlorophenol was isolated from a chlorophenol-fed percolator and was identified as a member of the genus Rhodococcus on the basis of chemotaxonomic characteristics and 16S RNA phylogenetic inference data. This organism (strain MBS1T [T = type strain]) exhibited a typical irregular rod-coccus cycle, and the cells had fimbria-like structures on their surfaces. The diagnostic cell wall amino acid was meso-diaminopimelic acid, and the sugars were arabinose and galactose; the mycolic acids contained 46 to 54 carbon atoms. The main menaquinone was MK-8(H2), and MK-9(H2) was a minor component. The cellular phospholipids were phosphatidylethanolamine, phosphatidylinositol, phosphatidylinositolmannoside, phosphatidylglycerol, and diphosphatidylglycerol. Tuberculostearic acid was present. The whole-cell fatty acids were straight-chain acids with 14 to 18 C atoms. The G+C content of the DNA was 67.4 mol%. This organism grew on sucrose, pyruvate, and 2,4,6-trichlorophenol, and it oxidized a large number of carbon compounds, including catechol, 3-hydroxyphenylacetic acid, and phenol. It also exhibited beta-galactosidase, urease, and 2-acetyl-lactate decarboxylase activities. On a phylogenetic tree that was based on 16S ribosomal DNA gene sequences strain MBS1T was found among the rhodococci on an independent branch. On the basis of the chemotaxonomic and phenotypic characteristics of strain MBS1T and its phylogenetic position we suggest that this bacterium should be placed in a new species, Rhodococcus percolatus; the specific epithet was chosen because the organism was isolated by using an enriched percolator. The type strain is strain MBS1.

Base Sequence↗
PubMed2005

Novosphingobium lentum sp. nov., a psychrotolerant bacterium from a polychlorophenol bioremediation process.

A polychlorophenol-degrading strain, designated MT1(T), and three MT1-like strains, MT101, MT103 and MT104, were isolated from a cold (4-8 degrees C) fluidized-bed process treating chlorophenol-contaminated groundwater in southern Finland. The organisms were Gram-negative, rod-shaped, catalase-positive, non-spore-forming and non-motile. Phylogenetic analysis based on 16S rRNA gene sequences indicated that the strains belonged to the alpha-4 subclass of the Proteobacteria and were members of the genus Novosphingobium. The highest 16S rRNA gene sequence similarity observed for these strains was 96.5 % with the type strains of Novosphingobium hassiacum, Novosphingobium aromaticivorans and Novosphingobium subterraneum. Chemotaxonomic data (major ubiquinone: Q-10; major polyamine: spermidine; major polar lipids: phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, phosphatidylcholine and sphingoglycolipid; major fatty acids: 18 : 1omega7c, 16 : 1omega7c and 2-OH 14 : 0) as well as the ability to reduce nitrate supported the affiliation of the strains to the genus Novosphingobium. Based on the phylogenetic analysis, whole-cell fatty acid composition as well as biochemical and physiological characteristics, the MT1-like strains were highly similar and could be separated from all recognized Novosphingobium species. The novel species Novosphingobium lentum sp. nov. is proposed to accommodate strains MT1(T) (=DSM 13663(T)=CCUG 45847(T)), MT101 (=CCUG 45849), MT103 (=CCUG 45850) and MT104 (=CCUG 45851).

Bacterial Typing Techniques↗
PubMed2007

Analysis of factors influencing the rate of germination of spores of Bacillus subtilis by very high pressure.

AIMS: To elucidate the factors that determine the rate of germination of Bacillus subtilis spores with very high pressure (VHP) and the mechanism of VHP germination. METHODS AND RESULTS: Spores of B. subtilis were germinated rapidly with a VHP of 500 MPa at 50 degrees C. This VHP germination did not require the spore's nutrient-germinant receptors, as found previously, and did not require diacylglycerylation of membrane proteins. However, the spore's pool of dipicolinic acid (DPA) was essential. Either of the two redundant enzymes that degrade the spore's peptidoglycan cortex, and thus allow completion of spore germination, was essential for completion of VHP germination. However, neither of these enzymes was needed for DPA release triggered by VHP treatment. Completion of spore germination as well as DPA release with VHP had an optimum temperature of approx. 60 degrees C, in contrast to an optimum temperature of 40 degrees C for germination with the moderately high pressure of 150 MPa. The rate of spore germination by VHP decreased approx. fourfold when the sporulation temperature increased from 23 degrees C to 44 degrees C, and decreased twofold when 1 mol l(-1) salt was present in sporulation. However, large variations in levels of unsaturated fatty acids in the spore's inner membranes did not affect rates of VHP germination. Complete germination of spores by VHP was not inhibited significantly by killing of spores with several oxidizing agents, and was not inhibited by ethanol, octanol or o-chlorophenol at concentrations that abolish nutrient germination. Completion of spore germination by VHP was also inhibited by Hg(2+), but this ion did not inhibit DPA release caused by VHP. In contrast, dodecylamine, a surfactant that can trigger spore germination, strongly inhibited DPA release caused by VHP treatment. CONCLUSIONS: VHP does not cause spore germination by acting upon the spore's nutrient-germinant receptors, but by directly causing DPA release. This DPA release then leads to subsequent completion of germination. VHP likely acts on the spore's inner membrane to cause DPA release, targeting either a membrane protein or the membrane itself. However, the precise identity of this target is not yet clear. SIGNIFICANCE AND IMPACT OF THE STUDY: There is significant interest in the use of VHP to eliminate or reduce levels of bacterial spores in foods. As at least partial spore germination by pressure is almost certainly essential for subsequent spore killing, knowledge of factors involved and the mechanism of VHP germination are crucial to the understanding of spore killing by VHP. This work provides new insight into factors that can affect the rate of B. subtilis spore germination by VHP, and into the mechanism of VHP germination itself.

Amines↗