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pH-adjusted bleach was one of the agents used to disinfect contaminated public buildings in the USA following the 2001 bioterrorist attack with Bacillus anthracis spores. A USEPA fact sheet describes the preparation of pH-adjusted bleach by combining diluted sodium hypochlorite (NaOCl) with a controlled amount of 5 % acetic acid. This paper reports a modification of this procedure to qualify the use of pH-adjusted bleach for routine disinfection of cleanroom surfaces in pharmaceutical manufacturing facilities whenever a short contact time is desirable or there is a need for enhanced germicidal or sporicidal activity. Adjustment of pH was obtained reproducibly with either acetic acid or HCl, confirming the feasibility of developing standard procedures for the controlled addition of acid to diluted NaOCl solutions without compromising operator safety and convenience. Efficacy testing using spores from an in-house isolate of Bacillus pumilus confirmed that NaOCl solutions in the pH 5-8 range have much greater sporicidal activity on surfaces than do unadjusted alkaline solutions (pH > 11). With a contact time of 0.5 min, the log10 reduction in spore viable counts was >5.4 for the five representative surfaces tested relative to untreated controls. Solutions of pH-adjusted NaOCl are known to be less stable than unadjusted alkaline solutions. Stability studies were performed by monitoring sporicidal efficacy, level of free available chlorine (FAC), and pH. Testing included several NaOCl concentrations and adjustment to different starting pHs. The efficacy of pH-adjusted solutions persisted in open containers for at least 12 h even though some FAC degradation occurred. In addition, solutions of 0.29 or 0.50 % NaOCl stored at room temperature protected from light retained efficacy for at least 4 weeks, indicating that short-term storage of solutions is possible following pH adjustment. The inorganic chemical degradation of pH-adjusted NaOCl solutions generates chlorate ion, an undesirable by-product. A comparison of chemical stability for 0.12, 0.25, and 0.50 % NaOCl solutions adjusted to different initial pHs indicated that the least chlorate formation occurred with 0.12 % NaOCl.

Anaerobic O demethylation by acetogenic bacteria often is the first step in the mineralization of methoxylated aromatic compounds in anoxic environments. In this reaction, an ether bond is cleaved and the resulting methyl group is metabolized via the acetyl coenzyme A pathway (acetogenesis). Anaerobic O demethylation was used to assess acetogen populations. Environmental samples were diluted in anaerobic medium containing a methoxylated aromatic substrate (vanillate) and titanium(III), and acetogen titers were estimated by the most-probable-number (MPN) method. Complex formation between Ti(III) and vicinal hydroxyl groups of the aromatic products of anaerobic O demethylation results in the development of a yellow color in the medium, which can be detected by eye and monitored spectrophotometrically. High-performance liquid chromatography analysis of the yellow MPN tubes showed that they contained the product of anaerobic O demethylation of vanillate (protocatechuate). This assay was used to enumerate O-demethylating acetogen populations in environmental samples.

The denitrifying strain T1 utilizes toluene anaerobically. We now report that anaerobic toluene degradation is inducible in strain T1. Fluoracetate treatment of cell suspensions inhibited both the rate of toluene metabolism and the formation of the toluene dead-end products benzylsuccinate and benzylfumarate, which is consistent with the pathway proposed by Evans et al. (Appl. Environ. Microbiol. 58:496-501, 1992). In addition, when either nitrate was limiting or fluoroacetate was added, benzoate was detected during toluene metabolism.

DL-Tetrahydrofolate (THF) and ATP were necessary for the anaerobic O-demethylation of phenylmethylethers in cell extracts of the type strain (ATCC 29683) of the homoacetogen Acetobacterium woodii. The reactants for this enzymatic activity have not been previously demonstrated in any system, nor has the mediating enzyme been studied. An assay using reaction mixtures containing 1 mM THF, 2 mM ATP, and 2 mM hydroferulate (i.e., 4-hydroxy,3-methoxyphenylpropionate) was developed and was performed under stringent anaerobic conditions. Pyridine nucleotides and several other possible cofactors were tested but had no effect on the activity. After centrifugation of disrupted cells at 27,000 x g, the activity was found primarily in the supernatant, which had a specific activity of 14.2 +/- 0.5 nmol/min/mg of protein. At saturating levels of each of the other two substrates, apparent Km values for the variable substrate were 0.65 mM hydroferulate, 0.27 mM ATP, and 0.17 mM THF. Activity was significantly decreased when extract was preincubated at 60 degrees C and was completely lost after preincubation in air for 30 min. Thus, the soluble anaerobic O-demethylating enzyme system of A. woodii is oxygen sensitive. The THF- and ATP-dependent activity measurable in the soluble fraction of cell extracts constituted about 34% of the activity seen with intact cells.

Aryl-O-methyl ethers are abundant in aerobic and anaerobic environments. In particular, lignin is composed of units of this type. Lignin monomers specifically radiolabeled in methoxy, side chain, and ring carbons have been synthesized by chemical procedures and are important in studies of lignin synthesis and degradation, humus formation, and microbial O-demethylation. In this paper attention is drawn to an enzymatic procedure for preparing O-methyl-C-labeled aromatic lignin monomers which has not previously been exploited in microbial ecology and physiology studies and which has several advantages compared with chemical synthesis procedures. O-[methyl-C]vanillic and O-[methyl-C]ferulic acids were prepared with S-[methyl-C]adenosyl-l-methionine as the methyl donor, using commercially obtained porcine liver catechol-O-methyltransferase (EC 2.1.1.6). The specific activity of the methylated products was the same as that of the methyl donor, a maximum of about 58 muCi/mumol, and the yields were 42% (vanillate) and 35% (ferulate). Thus lignin monomers are readily prepared as O-methylated products of the catechol-O-methyltransferase reaction and, with this enzyme method of preparation, would be more widely available than labeled compounds which require chemical synthesis.

The O-methyl substituents of aromatic compounds constitute a C(1) growth substrate for a number of taxonomically diverse anaerobic acetogens. In this study, strain TH-001, an O-demethylating obligate anaerobe, was chosen to represent this physiological group, and the carbon flow when cells were grown on O-methyl substituents as a C(1) substrate was determined by C radiotracer techniques. O-[methyl-C]vanillate (4-hydroxy-3-methoxy-benzoate) was used as the labeled C(1) substrate. The data showed that for every O-methyl carbon converted to [C]acetate, two were oxidized to CO(2). Quantitation of the carbon recovered in the two products, acetate and CO(2), indicated that acetate was formed in part by the fixation of unlabeled CO(2). The specific activity of C in acetate was 70% of that in the O-methyl substrate, suggesting that only one carbon of acetate was derived from the O-methyl group. Thus, it is postulated that the carboxyl carbon of the product acetate is derived from CO(2) and the methyl carbon is derived from the O-methyl substituent of vanillate. The metabolism of O-[methyl-C]vanillate by strain TH-001 can be described as follows: 3CH(3)OC(7)H(5)O(3) + CO(2) + 4H(2)O --> CH(3)COOH + 2CO(2) + 10H + 10e + 3HOC(7)H(5)O(3).

A novel strain of gram-negative anaerobic rods which utilized O-methyl substituents of monoaromatic acids as a sole organic source of carbon was isolated from municipal sewage sludge. Energy for growth seemed to be generated by an acetate formation pathway. The growth yield in defined medium was 7.9 g (dry weight) of cells per mol of ferulate utilized. This isolate and other O-demethylating anaerobes may play a role in the turnover of acetate and the metabolism of highly methoxylated lignaceous materials in anaerobic environments.