Enantioselective hydroxylation of 4-alkylphenols by vanillyl alcohol oxidase

Vanillyl alcohol oxidase (VAO) from Penicillium simplicissimum catalyzes the enantioselective hydroxylation of 4-ethylphenol, 4-propylphenol, and 2-methoxy-4-propylphenol into 1-(4'-hydroxyphenyl)ethanol, 1-(4'-hydroxyphenyl)propanol, and 1-(4'-hydroxy-3'-methoxyphenyl)propanol, respectively, with an ee of 94% for the R enantiomer. The stereochemical outcome of the reactions was established by comparing the chiral GC retention times of the products to those of chiral alcohols obtained by the action of the lipases from Candida antarctica and Pseudomonas cepacia. Isotope labeling experiments revealed that the oxygen atom incorporated into the alcoholic products is derived from water. During the VAO-mediated conversion of 4-ethylphenol/4-propylphenol, 4-vinylphenol/4-propenylphenol are formed as side products. With 2-methoxy-4-propylphenol as a substrate, this competing side reaction is nearly abolished, resulting in less than 1% of the vinylic product, isoeugenol. The VAO-mediated conversion of 4-alkylphenols also results in small amounts of phenolic ketones indicative for a consecutive oxidation step. Copyright 1998 John Wiley & Sons, Inc.     

Alkylphenol biotransformations catalyzed by 4-ethylphenol methylenehydroxylase

4-ethylphenol methylenehydroxylase from Pseudomonas putida JD1 acts by dehydrogenation of its substrate to give a quinone methide, which is then hydrated to an alcohol. It was shown to be active with a range of 4-alkylphenols as substrates. 4-n-propylphenol, 4-n-butylphenol, chavicol, and 4-hydroxydiphenylmethane were hydroxylated on the methylene group next to the benzene ring and produced the corresponding chiral alcohol as the major product. The alcohols 1-(4'-hydroxyphenyl)propanol and 1-(4'-hydroxyphenyl)-2-propen-1-ol, produced by the biotransformation of 4-n-propylphenol and chavicol, respectively, were shown to be R(+) enantiomers. 5-Indanol, 6-hydroxytetralin, 4-isopropylphenol, and cyclohexylphenol, with cyclic or branched alkyl groups, gave the corresponding vinyl compounds as their major products.     

Regulation of the biosynthesis of insulin-secretory-granule proteins. Co-ordinate translational control is exerted on some, but not all, granule matrix constituents.

The enzyme 4-ethylphenol methylenehydroxylase was purified from Pseudomonas putida JD1 grown on 4-ethylphenol. It is a flavocytochrome c for which the Mr was found to be 120,000 by ultracentrifuging and 126,000 by gel filtration. The enzyme consists of two flavoprotein subunits each of Mr 50,000 and two cytochrome c subunits each of Mr 10,000. The redox potential of the cytochrome is 240 mV. Hydroxylation proceeds by dehydrogenation and hydration to give 1-(4'-hydroxyphenyl)ethanol, which is also dehydrogenated by the same enzyme to 4-hydroxyacetophenone. The enzyme will hydroxylate p-cresol but is more active with alkylphenols with longer-chain alkyl groups. It is located in the periplasm of the bacterium.     

Anaerobic co-metabolic oxidation of 4-alkylphenols with medium-length or long alkyl chains by <Emphasis Type="Italic">Thauera</Emphasis> sp., strain R5

A 4-alkylphenol-degrading facultative anaerobic bacterium, strain R5, was isolated from paddy soil after enrichment with 4-n-propylphenol, 4-n-butylphenol and 4-hydroxybenzoate (4-HBA) under nitrate-reducing conditions. Strain R5 is a Gram-negative rod bacillus grown on phenolic compounds with short alkyl chains (≤C2), organic acids and ethanol. The sequence of the 16S ribosomal RNA gene revealed that the strain is affiliated with Thauera sp. In the presence of 4-HBA as a carbon source, the strain transformed 4-n-alkylphenols with a medium or long-length alkyl chain (C3–C8) to the corresponding oxidised products as follows: 1-(4-hydroxyphenyl)-1-alkenes, -(4-hydroxyphenyl)-1-alkanones and/or 1-(4-hydroxyphenyl)-1-alcohols. The strain also transformed 4-i-propylphenol and 4-sec-butylphenol to (4-hydroxyphenyl)-i-propene and (4-hydroxyphenyl)-sec-butene but not 4-alkylphenols with tertiary alkyl chains (4-t-butylphenol or 4-t-octylphenol). The biotransformation did not proceed without another carbon source and was coupled with nitrate reduction. Biotransformation activity was high in the presence of p-cresol, 4-ethylphenol, 4′-hydroxyacetophenone and 4-HBA as carbon sources and low in the presence of organic acids and ethanol. We suggest that strain R5 co-metabolically transforms alkylphenols to the corresponding metabolites with oxidised alpha carbon in the alkyl chain during coupling with nitrate reduction.     

Functional relationship between cyclic AMP-dependent protein phosphorylation and platelet inhibition.

The O2-independent hydroxylase 4-ethylphenol methylenehydroxylase (4EPMH) from Pseudomonas putida JD1 catalysed the complete conversion of 4-ethylphenol into 1-(4-hydroxyphenyl)ethanol together with a small amount of 4-hydroxyacetophenone, but with no formation of the side product 4-vinylphenol reported to be formed when the similar enzyme p-cresol methylhydroxylase (PCMH) catalyses this reaction. The enantiomer of 1-(4-hydroxyphenyl)ethanol produced by 4EPMH was R(+) when horse heart cytochrome c or azurin was used as electron acceptor for the enzyme. PCMHs from various bacterial strains produced the S(-)-alcohol. Both enantiomers of 1-(4-hydroxyphenyl)ethanol were substrates for conversion into 4-hydroxyacetophenone by 4EPMH, but the S(-)-isomer was preferred. The Km and kcat. were 1.2 mM and 41 s-1 respectively for the S(-)-alcohol and 4.7 mM and 22 s-1 for the R(+)-alcohol. In addition to the 1-(4-hydroxyphenyl)ethanol dehydrogenase activity of 4-EPMH, NAD(+)-linked dehydrogenase activity for both enantiomers of the alcohol was found in extracts of Ps. putida JD1.     

Alkylphenol Biotransformations Catalyzed by 4-Ethylphenol Methylenehydroxylase

4-Ethylphenol methylenehydroxylase from Pseudomonas putida JD1 acts by dehydrogenation of its substrate to give a quinone methide, which is then hydrated to an alcohol. It was shown to be active with a range of 4-alkylphenols as substrates. 4-n-Propylphenol, 4-n-butylphenol, chavicol, and 4-hydroxydiphenylmethane were hydroxylated on the methylene group next to the benzene ring and produced the corresponding chiral alcohol as the major product. The alcohols 1-(4′-hydroxyphenyl)propanol and 1-(4′-hydroxyphenyl)-2-propen-1-ol, produced by the biotransformation of 4-n-propylphenol and chavicol, respectively, were shown to be R(+) enantiomers. 5-Indanol, 6-hydroxytetralin, 4-isopropylphenol, and cyclohexylphenol, with cyclic or branched alkyl groups, gave the corresponding vinyl compounds as their major products.     

4-Ethylphenol metabolism by Aspergillus fumigatus.

Aspergillus fumigatus ATCC 28282 was found to be capable of growth on 4-ethylphenol as its sole carbon and energy source. A pathway for the metabolism of this compound has been proposed. The initial step involves hydroxylation of the methylene group of 4-ethylphenol to form 1-(4'-hydroxyphenyl)ethanol, followed by oxidation to 4-hydroxyacetophenone. The hydroxylase was NADPH and oxygen dependent, which is a characteristic of a monooxygenase type of enzyme. The 1-(4'-hydroxyphenyl)ethanol isolated from growth medium was a racemic mixture of R-(+) and S-(-) enantiomers. 4-Hydroxyacetophenone undergoes an NADPH-dependent Baeyer-Villiger type of oxygenation to give 4-hydroxyphenyl acetate, which is hydrolyzed to form hydroquinone (1,4-dihydroxybenzene). Hydroxylation of hydroquinone by an NADPH-dependent enzyme produces 1,2,4-trihydroxybenzene, the ring fission substrate, which is cleaved by ortho fission to form maleylacetate. The pathway was elucidated by various kinds of investigations. Analysis of culture medium sampled during growth on 4-ethylphenol revealed the transient appearance of 1-(4'-hydroxyphenyl)ethanol, 4-hydroxyacetophenone, and hydroquinone. Cells grown on 4-ethylphenol were able to oxidize all of these compounds immediately, whereas oxidation by succinate-grown cells showed a lag period. Extracts prepared from cells grown on 4-ethylphenol contained enzyme activities for all of the proposed steps. Apart from a low level of esterase activity towards 4-hydroxyphenyl acetate, extracts prepared from cells grown on succinate did not contain any of these enzyme activities.     

Studies on Cow’s Urine

An oil obtained from cow’s urine was examined by means of gas chromatography. Ethylbenzene, phenol, m-cresol, p-cresol, and p-ethylphenol were identified as the major components of the oil, while there were at least four components still remaining unknown.

A hypothesis concerning the degradation of equol,¹⁾ 7-hydroxy-3-(4’-hydroxy) chroman, to p-cresol and p-ethylphenol in the urine was proposed.     

Effect of environmental pollutants and their metabolites on a soil mycobacterium

The relative toxicity of seven major ground-water pollutants (benzene, chlorobenzene, propylbenzene, ethylbenzene, trichloroethylene, toluene, and styrene) and their metabolites to a soil mycobacterium (Mycobacterium vaccae strain JOB-5) that can catabolize all of these pollutants was determined. The metabolites of chlorobenzene, styrene and trichloroethylene degradation (4-chlorophenol, styrene oxide, and 2,2,2-trichloroethanol, respectively) were less toxic to M. vaccae than was their parent compound. The pollutants propylbenzene, ethylbenzene and benzene were less toxic than their metabolites (4-propylphenol, 4-ethylphenol, and phenol). Metabolites were also examined for their ability to interfere with the biodegradation of selected groundwater pollutants. The metabolites of ethylbenzene, propylbenzene and chlorobenzene biotransformation by M. vaccae were found to adversely affect biodegradation by M. vaccae. Toluene degradation by M. vaccae was inhibited by 4-chlorophenol, 4-ethylphenol and 4-propylphenol at 0.2 mm, 0.4 mm, and 0.4 mm, respectively.Correspondence to: J. J. Perry     

Anaerobic biodegradability of alkylphenols and fuel oxygenates in the presence of alternative electron acceptors

Alkylphenols and fuel oxygenates are important environmental pollutants produced by the petrochemical industry. A batch biodegradability test was conducted with selected ortho-substituted alkylphenols (2-cresol, 2,6-dimethylphenol and 2-ethylphenol), fuel oxygenates (methyl tert-butyl ether, ethyl tert-butyl ether and tert-amylmethyl ether) and tert-butyl alcohol (TBA) as model compounds. The ortho-substituted alkylphenols were not biodegraded after 100 days of incubation under methanogenic, sulfate-, or nitrate-reducing conditions. However, biodegradation of 2-cresol and 2-ethylphenol (150 mg l⁻¹) was observed in the presence of Mn (IV) as electron acceptor. The biodegradation of these two compounds took place in less than 15 days and more than 90% removal was observed for both compounds. Mineralization was indicated since no UV-absorbing metabolites accumulated after 23 days of incubation. These alkylphenols were also slowly chemically oxidized by Mn (IV). No biodegradation of fuel oxygenates or TBA (1 g l⁻¹) was observed after 80 or more days of incubation under methanogenic, Fe (III)-, or Mn (IV)-reducing conditions, suggesting that these compounds are recalcitrant under anaerobic conditions. The fuel oxygenates caused no toxicity towards acetoclastic methanogens activity in anaerobic granular sludge. Received: 8 February 2000 / Received revision: 15 May 2000 / Accepted: 19 May 2000     

Field studies on efficacy of host odour baits for the biting midge Culicoides impunctatus in Scotland

The efficacy of some putative attractants for the biting midge Culicoides impunctatus (Goetghebuer) (Diptera: Ceratopogonidae) was assessed using odour-baited 'delta traps' and suction traps. 1-octen-3-ol was confirmed as a potent olfactory attractant for C. impunctatus when released at 0.06mg/h. Acetone (23mg/h) and a mix of six phenolic compounds (phenol, 3-ethylphenol, 4-ethylphenol, 3-methylphenol, 4-methylphenol and 4-propylphenol), at undetermined release rate, also significantly increased delta trap catches compared to unbaited controls. When tested in combination, there was evidence of synergism between CO2 (0.2L/min) and acetone, 1-octen-3-ol or cow urine, trap catches being, respectively, 4.7, 6.2 and 9.3-fold greater than for CO2 alone. Highest catches were obtained with triple bait combinations comprising cow urine + acetone + CO2 or cow urine + 1-octen-3-ol+CO2, which increased trap catches by X 22 and X 24, respectively, compared to CO2 alone. Culicoides impunctatus was found to be extremely sensitive to CO2 and responses, gauged over two field seasons, showed a significant dose-dependent increase in catch across the entire range of release rates (0.2-2.5 L/min). Responses to these release rates, ranging from small to large mammal equivalents, emphasized the important role of CO2 in host location by C. impunctatus. Uses of olfactory attractants for monitoring and control of Culicoides are reviewed on the basis of these results.     

The effect of sugar concentration and temperature on growth and volatile phenol production by Dekkera bruxellensis in wine

The wine spoilage yeast Dekkera bruxellensis was evaluated for the production of 4-ethylphenol under low concentrations (0.02-20 g L(-1)) of glucose and fructose in synthetic media. Measurable amounts of 4-ethylphenol were produced over 0.2 g L(-1) of each sugar. The yeast growth rate and amount of biomass formed increased from 0.2 to 20 g L(-1) of glucose or fructose, being accompanied by increasing production of 4-ethylphenol. In red wines, the production of 4-ethylphenol was only observed in the presence of growing populations of indigenous or inoculated strains of D. bruxellensis. The production rate of 4-ethylphenol varied between 22 and 93 mug day(-1) either with inoculated strains or wild populations in bottled wines. The production rate of 4-ethylphenol as a function of the increase in the number of cells varied from 349 to 1882 mug L(-1) per one log CFU mL(-1). The effect of temperature on cellular viability and 4-ethylphenol production was tested in red wines with indigenous or inoculated strains of D. bruxellensis. Incubation temperatures of 15, 20 and 25 degrees C allowed cellular growth and volatile phenol production. Increasing incubation temperatures to 36 degrees C induced full viability loss of 10 strains of D. bruxellensis within <12 h.     

Morpho-functional identification of abdominal olfactory receptors in the midge Culicoides imicola

The aim of the present study was to examine the presence and the possible role of abdominal olfactory sensilla in Culicoides imicola mediating the search for potential hosts and oviposition sites, by means of a morphological, electrophysiological and behavioural approach. The results reported here show that in the midge C. imicola the whole abdomen, comprising the ovipositor, are endowed with three morphotypes of multiporous sensilla that display olfactory sensitivity towards kairomones related to the host-animal skin such as l-(+)-lactic acid and 1-octen-3-ol, to the host-animal urine such as 3-ethylphenol and 4-propylphenol, and to the potent attractant sesame seed oil. Electrophysiological and behavioural data for the first time suggest in the midge the involvement of abdominal olfactory structures in the choice of the oviposition sites and allow in discussing their possible role in the host-animal localisation. Field experiments showed that light traps baited with the aforementioned compounds elicited a stronger degree of attractiveness on midges with respect to the unbaited traps (control), although to a different extent. Our results, while implying a number of considerations concerning the role of molecules tested as kairomones, also suggest their use in the control of the midge C. imicola population.     

Isolation and characterization of a novel 2-<Emphasis Type="Italic">sec</Emphasis>-butylphenol-degrading bacterium <Emphasis Type="Italic">Pseudomonas</Emphasis> sp. strain MS-1

A novel bacterium capable of utilizing 2-sec-butylphenol as the sole carbon and energy source, Pseudomonas sp. strain MS-1, was isolated from freshwater sediment. Within 30 h, strain MS-1 completely degraded 1.5 mM 2-sec-butylphenol in basal salt medium, with concomitant cell growth. A pathway for the metabolism of 2-sec-butylphenol by strain MS-1 was proposed on the basis of the identification of 3 internal metabolites—3-sec-butylcatechol, 2-hydroxy-6-oxo-7-methylnona-2,4-dienoic acid, and 2-methylbutyric acid—by gas chromatography-mass spectrometry analysis. Strain MS-1 degraded 2-sec-butylphenol through 3-sec-butylcatechol along a meta-cleavage pathway. Degradation experiments with various alkylphenols showed that the degradability of alkylphenols by strain MS-1 depended strongly on the position (ortho ≫ meta = para) of the alkyl substitute, and that strain MS-1 could degrade 2-alkylphenols with various sized and branched alkyl chain (o-cresol, 2-ethylphenol, 2-n-propylphenol, 2-isopropylphenol, 2-sec-butylphenol, and 2-tert-butylphenol), as well as a dialkylphenol (namely, 6-tert-butyl-m-cresol).     

Altering toluene 4-monooxygenase by active-site engineering for the synthesis of 3-methoxycatechol, methoxyhydroquinone, and methylhydroquinone

Wild-type toluene 4-monooxygenase (T4MO) of Pseudomonas mendocina KR1 oxidizes toluene to p-cresol (96%) and oxidizes benzene sequentially to phenol, to catechol, and to 1,2,3-trihydroxybenzene. In this study T4MO was found to oxidize o-cresol to 3-methylcatechol (91%) and methylhydroquinone (9%), to oxidize m-cresol and p-cresol to 4-methylcatechol (100%), and to oxidize o-methoxyphenol to 4-methoxyresorcinol (87%), 3-methoxycatechol (11%), and methoxyhydroquinone (2%). Apparent V_max values of 6.6 ± 0.9 to 10.7 ± 0.1 nmol/min/ mg of protein were obtained for o-, m-, and p-cresol oxidation by wild-type T4MO, which are comparable to the toluene oxidation rate (15.1 ± 0.8 nmol/min/mg of protein). After these new reactions were discovered, saturation mutagenesis was performed near the diiron catalytic center at positions I100, G103, and A107 of the alpha subunit of the hydroxylase (TmoA) based on directed evolution of the related toluene o-monooxygenase of Burkholderia cepacia G4 (K. A. Canada, S. Iwashita, H. Shim, and T. K. Wood, J. Bacteriol. 184:344-349, 2002) and a previously reported T4MO G103L regiospecific mutant (K. H. Mitchell, J. M. Studts, and B. G. Fox, Biochemistry 41:3176-3188, 2002). By using o-cresol and o-methoxyphenol as model substrates, regiospecific mutants of T4MO were created; for example, TmoA variant G103A/A107S produced 3-methylcatechol (98%) from o-cresol twofold faster and produced 3-methoxycatechol (82%) from 1 mM o-methoxyphenol seven times faster than the wild-type T4MO (1.5 ± 0.2 versus 0.21 ± 0.01 nmol/min/mg of protein). Variant I100L produced 3-methoxycatechol from o-methoxyphenol four times faster than wild-type T4MO, and G103S/A107T produced methylhydroquinone (92%) from o-cresol fourfold faster than wild-type T4MO and there was 10 times more in terms of the percentage of the product. Variant G103S produced 40-fold more methoxyhydroquinone from o-methoxyphenol than the wild-type enzyme produced (80 versus 2%) and produced methylhydroquinone (80%) from o-cresol. Hence, the regiospecific oxidation of o-methoxyphenol and o-cresol was changed for significant synthesis of 3-methoxycatechol, methoxyhydroquinone, 3-methylcatechol, and methylhydroquinone. The enzyme variants also demonstrated altered monohydroxylation regiospecificity for toluene; for example, G103S/A107G formed 82% o-cresol, so saturation mutagenesis converted T4MO into an ortho-hydroxylating enzyme. Furthermore, G103S/A107T formed 100% p-cresol from toluene; hence, a better para-hydroxylating enzyme than wild-type T4MO was formed. Structure homology modeling suggested that hydrogen bonding interactions of the hydroxyl groups of altered residues S103, S107, and T107 influence the regiospecificity of the oxygenase reaction.     

THE BIOLOGICAL OXIDATION OF SPENT GAS LIQUOR

SUMMARY: Mixed cultures of bacteria grown in spent gas liquor readily oxidized phenol, o -, m - and p -cresol, catechol, 3-methyl catechol, 4-methyl catechol, resorcinol, 2-methyl resorcinol, and 4-methyl resorcinol. Quinol, pyrogallol and phloroglucinol were more resistant. The optimum temperature was 30° and the best pH range 6·5–7·8. Yeast extract and sterile sewage sludge both increased the rate of growth of organisms in liquor when the inoculum was small. Five phenol oxidizing organisms were isolated in pure culture. Copper in concentrations greater than 1 p/m inhibited both growth and phenol oxidation by one of these.
Mixed cultures grown in an ammonium thiocyanate medium originally inoculated with Thiobacillus thiocyanoxidans oxidized potassium thiocyanate and sodium thiosulphate. Chloride inhibited thiocyanate oxidation in concentrations above 5,000 p/m, although adaptation to 15,000 p/m was possible. Phenol inhibited thiocyanate oxidation in concentrations of 300 p/m or more. Mixed cultures grown on sodium thiosulphate oxidized sodium trithionate and tetrathionate, potassium pentathionate and hexa-thionate, and potassium and ammonium thiocyanate
Manometric determinations of the 5 day biological oxygen demand of effluents after treatment showed good agreement with the values obtained by the conventional method, the manometric values being usually somewhat higher.     

Demonstration of two functionally heterogenous groups within the activities of UDP-glucuronosyltransferase towards a series of 4-alkyl-substituted phenols.

1. A simple colorimetric assay for UDP-glucuronosyltransferase activities towards phenolic substrates, using Folin & Ciocalteu's phenol reagent, is described. The assay is used to measure rat liver transferase activities towards substrates from a series of 4-alkyl-substituted phenols. 2. Activities towards phenol, 4-methylphenol and 4-ethylphenol develop near-adult values before birth, are precociously stimulated by dexa methasone in utero and are stimulated 3--4-fold by 3-methylcholanthrene in adult liver. These are assigned to a "late-foetal" group of transferase activities. 3. Activities towards 4-n-propylphenol, 4-s-butylphenol and 4-t-butylphenol are negligible in late-foetal liver, developing to near-adult values in the first 4 postnatal days, and are not affected by dexamethasone or 3-methylcholanthrene. They are assigned to a "neonatal" group of transferase activities. 4. Although 4-ethylphenol and 4-n-propylphenol differ only by a single --CH2-- moiety, this is sufficient to change the acceptability of these substrates respectively from the late-foetal to the neonatal group of transferase activities. The change is distinct, with no overlapping of substrate acceptability between the two groups of transferase activities. 5. From consideration of the above and other substrates, the two groups of transferase activities do not distinguish substrates on the basis of their molecular weights or lipophilicity. The distinguishing feature appears to be the specific molecular configurations of the substrates.     

Anaerobic degradation of p-ethylphenol by "Aromatoleum aromaticum" strain EbN1: pathway, regulation, and involved proteins

The denitrifying “Aromatoleum aromaticum” strain EbN1 was demonstrated to utilize p-ethylphenol under anoxic conditions and was suggested to employ a degradation pathway which is reminiscent of known anaerobic ethylbenzene degradation in the same bacterium: initial hydroxylation of p-ethylphenol to 1-(4-hydroxyphenyl)-ethanol followed by dehydrogenation to p-hydroxyacetophenone. Possibly, subsequent carboxylation and thiolytic cleavage yield p-hydroxybenzoyl-coenzyme A (CoA), which is channeled into the central benzoyl-CoA pathway. Substrate-specific formation of three of the four proposed intermediates was confirmed by gas chromatographic-mass spectrometric analysis and also by applying deuterated p-ethylphenol. Proteins suggested to be involved in this degradation pathway are encoded in a single large operon-like structure (~15 kb). Among them are a p-cresol methylhydroxylase-like protein (PchCF), two predicted alcohol dehydrogenases (ChnA and EbA309), a biotin-dependent carboxylase (XccABC), and a thiolase (TioL). Proteomic analysis (two-dimensional difference gel electrophoresis) revealed their specific and coordinated upregulation in cells adapted to anaerobic growth with p-ethylphenol and p-hydroxyacetophenone (e.g., PchF up to 29-fold). Coregulated proteins of currently unknown function (e.g., EbA329) are possibly involved in p-ethylphenol- and p-hydroxyacetophenone-specific solvent stress responses and related to other aromatic solvent-induced proteins of strain EbN1.     

Anaerobic production and transformation of aromatic hydrocarbons and substituted phenols by ferulic acid-degrading BESA-inhibited methanogenic consortia

Abstract Sewage sludge-derived methanogenic enrichments degrading ferulic acid as sole carbon and energy source were partially inhibited with 2-bromoethanesulfonic acid. The various intermediates and products formed under inhibition of methanogenesis were studied using gas chromatography/mass spectrometry (GC/MS). In addition to aromatic, alicyclic, and aliphatic acids previously shown to be intermediates of ferulate degradation to CO₂ and CH₄, the following compounds were detected: toluene, ethylbenzene, phenol, p -cresol, 2-ethylphenol, catechol, and 3-hydroxy-4-ethylphenol. The character and the sequence of appearance of the compounds indicate that fermentative bacteria which initiate the anaerobic transformation of ferulic acid, in case of disruption of interspecies hydrogen transfer, dispose of electrons by converting part of the substrate to reduced derivatives. Aromatic hydrocarbons are further partially oxidized through hydroxylation of the ring (and, to a lesser extent, the side-chain), and partially reduced to saturated alicyclic rings. Some of these compounds seem to be gradually degraded to branched or straight- chain aliphatic acids. Some compounds, like catechol and ethylphenol, accumulate transiently or persistently in high concentrations (up to 16 mM carbon out of the initial concentration of 30 mM substrate carbon), indicating that hydroxylation of the aromatic ring might be an important metabolic reaction in these systems.     

Responses of individual antennal olfactory cells of tsetse flies (Glossina m. morsitans) to phenols from cattle urine

Abstract. Action potentials from olfactory cells in antennae (funiculi) of living tsetse flies, Glossina morsitans morsitans Westwood, were recorded using a surface-contact technique. Stimuli were the vapours of the seven alkylphenols identified in cattle urine: phenol, 3-methyl-, 3-ethyl-, 3- n -propyl-, 4-methyl-, 4-ethyl-, and 4- n -propylphenol. In addition, responses to the vapours of 1-octen-3-ol, acetone and dichloromethane were recorded. The phenol-sensitive cells could be grouped into four subclasses. Subclass, 1, 2 and 3 cells responded to the phenols only, cells of subclass 1 and 2 being activated by these substances, those of subclass 3 inhibited. Cells of subclass 4 were activated to a similar degree by all phenols and by one or more of the other chemicals tested. Subclass 1 cells were strongly activated by the 3-alkylphenols, whereas subclass 2 cells were most sensitive to 4-methylphenol. Subclass 3 cells were most strongly inhibited by phenol, and 3- and 4-methylphenol. The results suggest that though individual phenols may be attractive to G. m. morsitans , preference for certain blends of phenols may exist; for example, blends composed of moderate doses of 4-methylphenol and 3-methyl-, 3-ethyl- or 3- n -propylphenol.