Metabolism of phenol and cresols by Bacillus stearothermophilus.

An obligate thermophilic strain of Bacillus stearothermophilus, strain PH24, isolated from industrial sediment by elective culture, grew readily at 55 C on phenol or on one of the isomers of cresol as the major carbon source. Intact cells grown in the presence of phenol, o-cresol, m-cresol, or p-cresol were induced to oxidize, without lag, these substrates together with catechol, 3-methylcatechol, and 4-methylcatechol. Cell extracts prepared from B. stearothermophilus PH24 after growth in the presence of phenol converted phenol to catechol with a concomitant uptake of 1 mol of oxygen per mol of substrate in reaction mixtures supplemented with reduced nicotinamide adenine dinucleotide. These preparations also catalyzed the oxidation of o-cresol to 3-methylcatechol and of m-cresol and p-cresol to 4-methylcatechol. Enzyme activity was inhibited by 1 mM p-chloromercuribenzoate and by 0.1 mM 0-phenanthroline. Catechol and the corresponding methylcatechol intermediates were further dissimilated by cell extracts of phenol-grown cells via the meta-cleavage route to yield 2-hydroxymuconic semialdehyde and the respective methylated derivatives.     

Anaerobic degradation of indolic compounds by sulfate-reducing enrichment cultures,and description of Desulfobacterium indolicum gen. nov., sp. nov.

Indole (1.5 mmol/l) added to suflate-rich marine mud or sulfate-free sewage digestor sludge was anaerobically degraded within one week. Enrichments from sludge samples in defined indole-containing media with or without sulfate were selective for sulfate-reducing bacteria or mixed methanogenic associations, respectively. Other enrichments of sulfate-reducing bacteria were obtained with skatole, indoleacetate, indolepropionate, quinoline, and pyridine. From a marine enrichment with indole as sole electron donor and carbon source, an oval to rod-shaped, Gram-negative, nonsporing sulfate-reducing bacterium (strain In04) was isolated. Growth occurred in defined bicarbonate-buffered, sulfide-reduced media supplemented with vitamin B₁₂. Furthen aromatic compounds utilized as electron donors and carbon sources were anthranilic acid and quinoline. Nonaromatic compounds used as substrates were formate, acetate, propionate, ethanol, propanol, butanol, pyruvate, malate, fumarate, and succinate. However, growth with substrates other than indole was rather slow. Thiosulfate served as an alternative electron acceptor. Complete oxidation of indole to CO₂ was shown by stoichiometric measurements in batch culture with sulfate as electron acceptor. An average growth yield of 31.3 g cell dry weight was obtained per mol of indole oxidized. Pigment analysis revealed that cytochromes and menaquinone MK-7 (H₂) were present. Desulfoviridin could not be detected. Strain In04 is described as new species of the new genus Desulfobacterium indolicum.     

Anaerobic degradation of phenol by pure cultures of newly isolated denitrifying pseudomonads

From various oxic or anoxic habitats several strains of bacteria were isolated which in the absence of molecular oxygen oxidized phenol to CO₂ with nitrate as the terminal electron acceptor. All strains grew in defined mineral salts medium; two of them were further characterized. The bacteria were facultatively anaerobic Gramnegative rods; metabolism was strictly oxidative with molecular oxygen, nitrate, or nitrite as electron acceptor. The isolates were tentatively identified as pseudomonads. Besides phenol many other benzene derivatives like cresols or aromatic acids were anaerobically oxidized in the presence of nitrate. While benzoate or 4-hydroxybenzoate was degraded both anaerobically and aerobically, phenol was oxidized under anaerobic conditions only. Reduced alicyclic compounds were not degraded. Preliminary evidence is presented that the first reaction in anaerobic phenol oxidation is phenol carboxylation to 4-hydroxybenzoate.     

Anaerobic degradation of o-phenylphenol by mixed and pure cultures

Summary An anaerobic enrichment culture that degraded 0.4 mmol/l per day of o-phenylphenol was selected from sediment of a waste water pond of a sugar factory. From the consortium an o-phenylphenol-degrading bacterium, strain B10, was isolated. Strain B10 could not degrade other aromatic substances, including phenylacetic acid, benzoate, o-hydroxybenzoate, p-hydroxybenzoate and phenol. Best growth was observed with glucose, pyruvate, lactate, methanol and H₂/CO₂ as substrates. o-Phenylphenol was slowly degraded if supplied as the only carbon source and was cometabolized in the presence of >5 mmol/l glucose. Strain B10 has not yet been assigned to a known species or family.     

Complete oxidation of benzoate and 4-hydroxybenzoate by a new sulfate-reducing bacterium resembling Desulfoarculus

A new sulfate-reducer strain SAX was isolated from an anaerobic marine sediment [Saxild, Denmark]. The isolate was a gram-negative, motile and non-spore-forming rod which sometimes appeared as a curved rod. Strain SAX differed from all described Desulfovibrio-, Desulfobotulus- and Desulfoarculus-species by the ability to degrade aromatic compounds such as benzoate, 4-hydroxybenzoate and phenol completely to CO₂. Electron donors used included lactate, pyruvate, malate, fumarate, crotonate and butyrate, while pyruvate was fermented in the absence of an external electron acceptor. Sulfate, thiosulfate or sulfite served as electron acceptors with benzoate as the donor, while nitrate and nitrite did not. The sulfate-reducing bacterium required vitamins and NaCl-concentrations of about 20 g/l. The optimum temperature for growth of strain SAX was 30°C and the optimum pH value was 7.3. The DNA base composition was 62.4 mol% G+C. The strain possessed cytochrome c₃, but no desulfoviridin. On the basis of these characteristics and because strain SAX could not be ascribed to any of the existing species therefore assignment as a new species to the genus Desulfoarculus was suggested.Abbreviations G+C Guanine plus Cytosine     

Studies on dissimilatory sulfate-reducing bacteria that decompose fatty acids II. Incomplete oxidation of propionate by Desulfobulbus propionicus gen. nov., sp. nov.

A new type of sulfate-reducing bacteria with ellipsoidal to lemon-shaped cells was regularly enriched from anaerobic freshwater and marine mud samples when mineral media with propionate and sulfate were used. Three strains (1pr3, 2pr4, 3pr10) were isolated in pure culture. Propionate, lactate and alcohols were used as electron donors and carbon sources. Growth on H₂ required acetate as a carbon source in the presence of CO₂. Stoichiometric measurements revealed that oxidation of propionate was incomplete and led to acetate as an endproduct. Instead of sulfate, strain 1pr3 was shown to reduce sulfite and thiosulfate to H₂S; nitrate also served as electron acceptor and was reduced to ammonia. With lactate or pyruvate, all three strains were able to grow without external electron acceptor and formed propionate and acetate as fermentation products. None of the strains contained desulfoviridin. In strain 1pr3 cytochromes of the b- and c-type were identified. Strain 1pr3 is described as type strain of the new species and genus, Desulfobulbus propionicus.     

Biodegradation of phenol and cresol isomer mixtures by Arthrobacter

The Arthrobacter species can degrade phenol, o-cresol and p-cresol much faster (as reflected in high specific growth rates) than other microbes which are reported to degrade toxic compounds. In mixtures, phenol and p-cresol mutually inhibited each other; the inhibition constants show that phenol degradation is strongly inhibited in the presence of p-cresol rather than reverse. o-Cresol enhanced phenol degradation marginally but o-cresol degradation was not affected by the presence of phenol.     

Syntrophorhabdus aromaticivorans gen. nov., sp. nov., the first cultured anaerobe capable of degrading phenol to acetate in obligate syntrophic associations with a hydrogenotrophic methanogen

Phenol degradation under methanogenic conditions has long been studied, but the anaerobes responsible for the degradation reaction are still largely unknown. An anaerobe, designated strain UI(T), was isolated in a pure syntrophic culture. This isolate is the first tangible, obligately anaerobic, syntrophic substrate-degrading organism capable of oxidizing phenol in association with an H(2)-scavenging methanogen partner. Besides phenol, it could metabolize p-cresol, 4-hydroxybenzoate, isophthalate, and benzoate. During the degradation of phenol, a small amount of 4-hydroxybenzoate (a maximum of 4 microM) and benzoate (a maximum of 11 microM) were formed as transient intermediates. When 4-hydroxybenzoate was used as the substrate, phenol (maximum, 20 microM) and benzoate (maximum, 92 microM) were detected as intermediates, which were then further degraded to acetate and methane by the coculture. No substrates were found to support the fermentative growth of strain UI(T) in pure culture, although 88 different substrates were tested for growth. 16S rRNA gene sequence analysis indicated that strain UI(T) belongs to an uncultured clone cluster (group TA) at the family (or order) level in the class Deltaproteobacteria. Syntrophorhabdus aromaticivorans gen. nov., sp. nov., is proposed for strain UI(T), and the novel family Syntrophorhabdaceae fam. nov. is described. Peripheral 16S rRNA gene sequences in the databases indicated that the proposed new family Syntrophorhabdaceae is largely represented by abundant bacteria within anaerobic ecosystems mainly decomposing aromatic compounds.     

Degradation of phenol via carboxylation to benzoate by a defined,obligate syntrophic consortium of anaerobic bacteria

Summary An obligate syntrophic culture was selected in mineral medium with phenol as the only carbon and energy source. The consortium consisted of a short and a long rod-shaped bacterium and of low numbers of Desulfovibrio cells, and grew only in syntrophy with methanogens, e. g. Methanospirillum hungatei. Under N₂/CO₂, phenol was degraded via benzoate to acetate, CH₄ and CO₂, while in the presence of H₂/CO₂ benzoate was formed, but not further degraded. When 4-hydroxybenzoate was fed to the mixed culture, it was decarboxylated to phenol prior to benzoate formation and subsequent ring cleavage. Isolation of pure cultures of the two rod-shaped bacteria failed. Microscopic observations during feeding of either 4-hydroxybenzoate, phenol or benzoate implied an obligate syntrophic interdependence of the two different rod-shaped bacteria and of the methanogen. The non-motile rods formed phenol from 4-hydroxybenzoate and benzoate from phenol, requiring an as yet unknown co-substrate or co-factor, probably cross-fed by the short, motile rod. The short, motile rodshaped bacterium grew only in syntrophy with methanogens and degraded benzoate to acetate, CO₂ and methane. Desulfovibrio sp., present in low numbers, apparently could not contribute to the degradation of phenol or 4-hydroxybenzoate.     

Anaerobic degradation of phenol via carboxylation to 4-hydroxybenzoate: in vitro study of isotope exchange between 14CO2 and 4-hydroxybenzoate

Extracts of denitrifying bacteria grown anaerobically with phenol and nitrate catalyzed an isotope exchange between ¹⁴CO₂ and the carboxyl group of 4-hydroxybenzoate. This exchange reaction is ascribed to a novel enzyme, phenol carboxylase, initiating the anaerobic degradation of phenol by para-carboxylation to 4-hydroxybenzoate. Some properties of this enzyme were determined by studying the isotope exchange reaction. Phenol carboxylase was rapidly inactivated by oxygen; strictly anoxic conditions were essential for preserving enzyme activity. The exchange reaction specifically was catalyzed with 4-hydroxybenzoate but not with other aromatic acids. Only the carboxyl group was exchanged; [U-¹⁴C]phenol was not exchanged with the aromatic ring of 4-hydroxybenzoate. Exchange activity depended on Mn²⁺ and inorganic phosphate and was not inhibited by avidin. Ortho-phosphate could not be substituted by organic phosphates nor by inorganic anions; arsenate had no effect. The pH optimum was between pH 6.5–7.0. The specific activity was 100 nmol ¹⁴CO₂ exchange · min^-1 · mg^-1 protein. Phenol grown cells contained 4-hydroxybenzoyl CoA synthetase activity (40 nmol · min^-1 · mg^-1 protein). The possible role of phenol carboxylase and 4-hydroxybenzoyl CoA synthetase in anaerobic phenol metabolism is discussed.     

Anaerobic degradation of aniline and dihydroxybenzenes by newly isolated sulfate-reducing bacteria and description of Desulfobacterium anilini

A new, rod-shaped, Gram-negative, non-sporing sulfate reducer (strain Ani1) was enriched and isolated from marine sediment with aniline as sole electron donor and carbon source. The strain degraded aniline completely to CO2 and NH3 with stoichiometric reduction of sulfate to sulfide. Strain Ani1 also degraded aminobenzoates and further aromatic and aliphatic compounds. The strain grew in sulfide-reduced mineral medium supplemented only with vitamin B12 and thiamine. Cells contained cytochromes, carbon monoxide dehydrogenase, and sulfite reductase P582, but no desulfoviridin. Strain Ani1 is described as a new species of the genus Desulfobacterium D. anilini. Marine enrichments with the three dihydroxybenzene isomers led to three different strains of sulfate-reducing bacteria; each of them could grow only with the isomer used for enrichment. Two strains isolated with catechol (strain Cat2) or resorcinol (strain Re10) were studied in detail. Both strains oxidized their substrates completely to CO2, and contained cytochromes, carbon monoxide dehydrogenase, and sulfite reductase P 582. Desulfoviridin was not present. Whereas the rod-shaped catechol oxidizer (strain Cat2) was able to grow on 18 aromatic compounds and several aliphatic substrates, the coccoid resorcinol-degrading bacterium (strain Re10) utilized only resorcinol, 2,4-dihydroxybenzoate and 1,3-cyclohexanedion. These strains could not be affiliated with existing species of sulfate-reducing bacteria. A further coccoid sulfate-reducing bacterium (strain Hy5) was isolated with hydroquinone and identified as a subspecies of Desulfococcus multivorans. Most-probable-number enumerations with catechol, phenol, and resorcinol showed relatively large numbers (10(4)-10(6) per ml) of aryl compound-degrading sulfate reducers in marine sediment samples.     

Studies on dissimilatory sulfate-reducing bacteria that decompose fatty acids

Three strains (2ac9, 3ac10 and 4ac11) of oval to rodshaped, Gram negative, nonsporing sulfate-reducing bacteria were isolated from brackish water and marine mud samples with acetate as sole electron donor. All three strains grew in simple defined media supplemented with biotin and 4-aminobenzoic acid as growth factors. Acetate was the only electron donor utilized by strain 2ac9, while the other two strains used in addition ethanol and/or lactate. Sulfate served as electron acceptor and was reduced to H₂S. Complete oxidation of acetate to CO₂ was shown by stoichiometric measurements with strain 2ac9 in batch cultures using sulfate, sulfite or thiosulfate as electron acceptors. With sulfate an average growth yield of 4.8 g cell dry weight was obtained per mol of acetate oxidized; with sulfite or thiosulfate the growth yield on acetate was about twice as high. None of the strains contained desulfoviridin. In strain 2ac9 cytochromes of the b- and c-type were detected. Strain 2ac9 is described as type strain of the new species and genus, Desulfobacter postgatei.     

Ability of intestinal lactic bacteria to bind or/and metabolise phenol and p-cresol

Intestinal microflora can contribute to colon cancer by the production of substances playing a role in carcinogenesis. Metabolites of protein fermentation in the colon, such as ammonia, H₂S, indole, phenol, skatole are toxic. Lactic bacteria existing in the colon may exert an anti-carcinogenic action, but the mechanism is poorly understood. In the present study the ability of intestin|al lactobacilli to bind or metabolise phenol and p-cresolin vitro was determined.Lactobacillus strains were cultivated in MRS and in a modified MRS broth with reduced concentrations of carbon source. Phenol and p-cresol content in the media were from 2 to 10 μg/ml. In MRS medium lactobacilli could decrease the concentration of phenol and p-cresol and it was 0.2-5.8 μg/ml for phenol and 0.2-1.4 μg/ml for p-cresol. After cultivation in a modified MRS broth, the decrease was 0.5-2.0 μg/ml for phenol and 0.5-2.4 μg/ml for p-cresol. The binding capacity of bacterial cells was rather low. After incubation of non-growing bacteria the decrease of phenol concentration was 0.1-0.5 μg/ml and p-cresol 0.1-2.8 μg/ml. But the ability of growing lactobacilli to metabolise the compounds cannot be excluded. After interaction of lactobacilli with 10 μg/ml of phenol they displayed a lower genotoxicity, as evaluated by the alkaline comet assay. The phenomenon not always depended on the decrease of phenol concentration, but on the medium, the strain of bacteria and for phenol it ranged from 32 to 48%.Lactobacillus strains tested did not lower the genotoxicity of p-cresol.     

Biodegradation of p-Cresol by Bacillus sp. Strain PHN 1

A bacterium capable of utilizing p-cresol as sole source of carbon and energy was isolated from soil and identified as a Bacillus species. The organism also utilized phenol, o-cresol, m-cresol, 4-hydroxybenzoic acid, and gentisic acid as growth substrates. The organism degraded p-cresol to 4-hydroxybenzoic acid, which was further metabolized by a gentisate pathway, as evidenced by isolation and identification of metabolites and enzyme activities in the cell-free extract. Such a bacterial strain can be used for bioremediation of environments contaminated with phenolic compounds.     

Cresol metabolism by the sulfate-reducing bacterium Desulfotomaculum sp. strain Groll.

The metabolism of cresols under sulfate-reducing conditions was investigated in Desulfotomaculum sp. strain Groll. This strain grows on a variety of aromatic compounds, including para- and meta- but not ortho-cresol. Degradation of p-cresol proceeded by oxidation reactions of the methyl group to yield p-hydroxybenzoate, which was then dehydroxylated to benzoate. The aromatic intermediates expected for this pathway, p-hydroxybenzyl alcohol, p-hydroxybenzaldehyde, p-hydroxybenzoate, and benzoate, were readily metabolized by strain Groll. Utilization of these intermediates generally preceded and inhibited the degradation of p-cresol. p-Hydroxybenzoate and benzoate were detected in culture fluid as metabolites of p-cresol. p-Hydroxybenzaldehyde and p-hydroxybenzoate were detected in cultures degrading p-hydroxybenzyl alcohol. Enzyme activities responsible for utilization of p- and m-cresol, induced by growth on the respective cresol, were detected in cell-free extracts of strain Groll. The compounds detected in culture fluids and the enzyme activities detected in cell-free extracts indicate that the pathways for the degradation of p- and m-cresol converge on benzoate, followed by metabolism to benzoyl-coenzyme A (CoA). Strain Groll can utilize both cresol isomers under sulfate-reducing conditions by similar reactions, but the enzyme activities catalyzing these transformations of the two isomers appear distinct.     

Biodegradation of trichloroethylene and involvement of an aromatic biodegradative pathway.

Biodegradation of trichloroethylene (TCE) by bacterial strain G4 resulted in complete dechlorination of the compound, as indicated by the production of inorganic chloride. A component of the water from which strain G4 was isolated that was required for TCE degradation was identified as phenol. Strain G4 degraded TCE in the presence of chloramphenicol only when preinduced with phenol. Toluene, o-cresol. and m-cresol could replace the phenol requirement. Two of the inducers of TCE metabolism, phenol and toluene, apparently induced the same aromatic degradative pathway that cleaved the aromatic ring by meta fission. Cells induced with either phenol or toluene had similar oxidation rates for several aromatic compounds and had similar levels of catechol-2,3-dioxygenase. The results indicate that one or more enzymes of an inducible pathway for aromatic degradation in strain G4 are responsible for the degradation of TCE.     

Chemolithotrophic growth of Desulfovibrio sulfodismutans sp. nov. by disproportionation of inorganic sulfur compounds

A new Desulfovibrio strain ThAc01 was isolated from freshwater mud; the strain conserved energy for growth under strictly anaerobic conditions by disproportionation of thiosulfate or sulfite to sulfate and sulfide according to the following reactions: $$\begin{gathered} S_2 O_3^{2 - } + H_2 O \to SO_4^{2 - } + HS^ - + H^ + \hfill \\ 4SO_3^{2 - } + H^ + {\text{ }} \to 3SO_4^{2 - } + HS^ - \hfill \\ \end{gathered}$$ Strain ThAc01 required acetate as a carbon source, but was unable to utilize acetate as an oxidizable energy source. In a defined medium with acetate and bicarbonate as carbon sources, the growth yields per mol of substrate disproportionated were 2.1 g or 3.2 g dry cell mass on thiosulfate or sulfite, respectively. Strain ThAc01 was also able to grow by dissimilatory sulfate reduction with lactate, ethanol, propanol, or butanol as electron donors and carbon sources which were incompletely oxidized to the corresponding fatty acids. However, growth by sulfate reduction was slower than by disproportionation. Elemental sulfur, nitrate, fumarate, or malate did not serve as electron acceptors. Strain ThAc01 contained desulfoviridin and cytochromes; it required panthothenate and biotin as growth factors and had a DNA base ratio of 64.1 mol% G+C. Disproportionating bacteria similar to strain ThAc01 were enriched with either thiosulfate or sulfite from various freshwater, brackish or marine mud samples. Most probable number enumeration indicated that 2×10⁶ thiosulfate-disproportionating bacteria were present per ml freshwater mud. Of various other sulfate-reducing bacteria tested, only Desulfobacter curvatus (strain AcRM3) was able to disproportionate thiosulfate or sulfite. Desulfovibrio vulgaris (strain Marburg) slowly disproportionated sulfite, but effected only a slight increase in cell density. Strain ThAc01 is proposed as the type strain of a new species, Desulfovibrio sulfodismutans.     

Evidence that phenol phosphorylation to phenylphosphate is the first step in anaerobic phenol metabolism in a denitrifying Pseudomonas sp.

Anaerobic phenol degradation has been shown to proceed via carboxylation of phenol to 4-hydroxybenzoate. However, in vitro the carboxylating enzyme was inactive with phenol; only phenylphosphate (phosphoric acid monophenyl ester) was readily carboxylated. We demonstrate in a denitrifying Pseudomonas strain that phenylphosphate is the first detectable product formed from phenol in whole cells and that subsequent phenylphosphate consumption parallels 4-hydroxybenzoate formation. These kinetics are consistent with phosphorylation being the first step in anaerobic phenol degradation. Various cosubstrates failed so far to act as phosphoryl donor for net phosphorylation of phenol in cell extracts. Yet, cells anaerobically grown with phenol contained an enzyme that catalyzed an isotope exchange between [U-¹⁴C]phenol and phenylphosphate. This transphosphorylation activity was anaerobically induced by phenol but was stable under aerobic conditions and required Mn²⁺ and polyethylene glycol. Activity was optimal at pH 5.5 and half-maximal with 0.6 mM Mn²⁺, 0.2 mM phenylphosphate, and 1 mM phenol. It is proposed that the phenol exchange/transphosphorylation reaction is catalyzed as partial reaction by an inducible phenol phosphorylating enzyme. The isotope exchange demands that a phosphorylated enzyme was formed in the course of the reaction, which might be similar to the phosphotransferase system of sugar transport.     

Biodegradation of trichloroethylene and involvement of an aromatic biodegradative pathway

Biodegradation of trichloroethylene (TCE) by bacterial strain G4 resulted in complete dechlorination of the compound, as indicated by the production of inorganic chloride. A component of the water from which strain G4 was isolated that was required for TCE degradation was identified as phenol. Strain G4 degraded TCE in the presence of chloramphenicol only when preinduced with phenol. Toluene, o-cresol. and m-cresol could replace the phenol requirement. Two of the inducers of TCE metabolism, phenol and toluene, apparently induced the same aromatic degradative pathway that cleaved the aromatic ring by meta fission. Cells induced with either phenol or toluene had similar oxidation rates for several aromatic compounds and had similar levels of catechol-2,3-dioxygenase. The results indicate that one or more enzymes of an inducible pathway for aromatic degradation in strain G4 are responsible for the degradation of TCE.     

Comparison of 4-hydroxynzoate decarboxylase and phenol carboxylase activities in cell-free extracts of a defined, 4-hydroxybenzoate and phenol-degrading anaerobic consortium

Summary Two 4-hydroxybenzoate decarboxylase activities and a phenol carboxylase activity were found in cell-free extracts of a defined, 4-hydroxybenzoate- or phenol-grown consortium. Both decarboxylase activities were loosely membrane-associated and required K⁺ but a different pH and ion strength. Loss of activity of both decarboxylases by EDTA could be compensated by Zn²⁺ ions. The K _m values for 4-hydroxybenzoate and K⁺ of the decarboxylase activities with pH optima at 6.4 or 7.8 were 0.02 and 2.5 or 0.004 and 0.5 mm, respectively. 3,4-Dihydroxybenzoate, 3,4,5-tridydroxybenzoate, 3,5-dimethoxy-4-hydroxybenzoate and 3-chloro-4-hydroxybenzoate were also decarboxylated by both enzyme activities. The phenol carboxylase was a soluble enzyme with its pH optimum at 6.5. It required K⁺, Rb⁺ or NH _inf4 ^sup+ as monovalent, Zn²⁺, Mg²⁺, Mn²⁺ or Ni²⁺ as divalent cations and catalysed the carboxylation of phenol if 2,4-,2,3,4- or 2,4,6-hydroxybezoates were absent. The three enzyme activities were not influenced by Avidin and thus were probably not biotin-dependent enzymes. Offprint requests to: J. Winter