Resorcinol degradation by a <Emphasis Type="Italic">Penicillium chrysogenum</Emphasis> strain under osmotic stress: mono and binary substrate matrices with phenol

A phenol-degrading Penicillium chrysogenum strain previously isolated from a salt mine was able to grow at 1,000 mg l⁻¹ of resorcinol on solid medium. The aerobic degradation of resorcinol by P. chrysogenum CLONA2 was studied in batch cultures in minimal mineral medium with 58.5 g l⁻¹ of sodium chloride using resorcinol as the sole carbon source. The fungal strain showed the ability to degrade up to 250 mg l⁻¹ of resorcinol. Resorcinol and phenol efficiency degradation by P. chrysogenum CLONA2 was compared. This strain removes phenol faster than resorcinol. When phenol and resorcinol were in binary substrate matrices, phenol enhanced resorcinol degradation, and organic load decreased with respect to the mono substrate matrices. The acute toxicity of phenol and resorcinol, individually and in combination, to Artemia franciscana larvae has been verified before and after the bioremediation process with P. chrysogenum CLONA2. The remediation process was effective in mono and binary substrate systems.     

Catabolism of benzene compounds by ascomycetous and basidiomycetous yeasts and yeastlike fungi

A literature review is given on growth of yeasts on benzene compounds and on the catabolic pathways involved. Additionally, a yeast collection was screened for assimilation of phenol and 3-hydroxybenzoic acid. Fifteen ascomycetous and thirteen basidiomycetous yeast species were selected and were tested for growth on 84 benzene compounds. It appeared that 63 of these compounds supported growth of one or more yeast species. The black yeastExophiala jeanselmei assimilated 54 of these compounds.The catechol branch of the 3-oxoadipate pathway and its hydroxyhydroquinone variant were involved in phenol and resorcinol catabolism of ascomycetes as well as of basidiomycetes. However, these two groups of yeasts showed characteristic differences in hydroxybenzoate catabolism. In the yeastlike fungusE. jeanselmei and in basidiomycetes of the generaCryptococcus, Leucosporidium andRhodotorula, the protocatechuate branch of the 3-oxoadipate pathway was induced by growth on 3- and 4-hydroxybenzoic acids. In threeTrichosporon species and in all ascomycetous yeasts tested, 4-hydroxybenzoic acid was catabolyzed via protocatechuate and hydroxyhydroquinone. These yeasts were unable to cleave protocatechuate. 3-Hydroxybenzoic and 3-hydroxycinnamic acids were catabolized in ascomycetous yeasts via the gentisate pathway, but in basidiomycetes via protocatechuate.Incomplete oxidation of phenol, some chlorophenols, cresols and xylenols was observed in cultures ofCandida parapsilosis growing on hydroquinone. Most compounds transformed by the growing culture were also converted by the phenol monooxygenase present in cell-free extracts of this yeast. They did not support growth.The relationship between the ability of ascomycetous yeasts to assimilate n-alkanes, amines and benzene compounds, and the presence of Coenzyme Q₉ is discussed.     

Phenolic compounds inhibit the aldose reductase enzyme from the sheep kidney

Aldose reductase (AR) is the key enzyme for the polyol pathway and responsible for sorbitol accumulation during the hyperglycemia. The present article focuses on the role of phenol, pyrogallol, hydroquinone, resorcinol, catechol, and phloroglucinol in in vitro inhibition of AR. For this purpose, AR was purified from the sheep kidney with 5.33 EU mg^?1 specific activity and 0.64% yield using several chromatographic methods. Various concentrations of the compounds were tested on in vitro AR activity. IC₅₀ values were found for phenol, pyrogallol, hydroquinone, resorcinol, catechol, and phloroglucinol as 6.5, 1.13, 5.45, 2.21, 1.8, and 2.09 mM, respectively, and their K_i constant was calculated as 3.45 ± 0.92, 0.96 ± 0.28, 3.07 ± 0.46, 1.59 ± 0.43, 2.5 ± 0.35, and 2.54 ± 0.45 mM, respectively. Pyrogallol showed better inhibitory effect compared to the other compounds. The inhibition mechanisms of all compounds were noncompetitive. In the presents study, in vitro AR inhibition was examined by the phenolic compounds.     

Promotion or suppression of glucose isomerization in subcritical aqueous straight- and branched-chain alcohols

The influence of water-miscible alcohols (methanol, 1-propanol, 2-propanol, and t-butyl alcohol) on the isomerization of glucose to fructose and mannose was investigated under subcritical aqueous conditions (180–200 °C). Primary and secondary alcohols promoted the conversion and isomerization of glucose to afford fructose and mannose with high and low selectivity, respectively. On the other hand, the decomposition (side-reaction) of glucose was suppressed in the presence of the primary and secondary alcohols compared with that in subcritical water. The yield of fructose increased with increasing concentration of the primary and secondary alcohols, and the species of the primary and secondary alcohols tested had little effect on the isomerization behavior of glucose. In contrast, the isomerization of glucose was suppressed in subcritical aqueous t-butyl alcohol. Both the conversion of glucose and the yield of fructose decreased with increasing concentration of t-butyl alcohol. In addition, mannose was not detected in reactions using subcritical aqueous t-butyl alcohol.     

Enhancement of specific nitrogenase activity inAzospirillum brasilense andKlebsiella pneumoniae,inhibition inRhizobium japonicum under air by phenol

Specific nitrogenase activity inAzospirillum brasilense ATCC 29145 in surface cultures under air is enhanced from about 50 nmol C₂H₄·mg protein^-1·h^-1 to 400 nmol C₂H₄ by the addition of 1 mM phenol. 0.5 and 2 mM phenol added increase the rate 5-fold and 4-fold. This enhancement effect is observed only between 2 and 3 days after inoculation, with only a small reduction of the growth of the cells by the phenol added. In surface cultures under 1% O₂, nitrogenase activity is slightly reduced by the addition of 1–0.01 mM phenol. Utilization of succinate is enhanced during the period of maximum enhancement of nitrogenase activity by 60% by addition of 1 mM phenol. The cells did not produce¹⁴CO₂ from [U-¹⁴C] phenol, neither in surface cultures nor in liquid cultures and less than 0.1% of the phenol was incorporated into the cells. A smaller but significant enhancement of nitrogenase activity by about 100% in surface cultures under air was found withKlebsiella pneumoniae K 11 after addition of 1 mM phenol. However, inRhizobium japonicum 61-A-101 all phenol concentrations above 0.01 mM reduced nitrogenase activity. With 1 mM phenol added activity was reduced to less than 10% with no effect on the growth in the same cultivation system. With thisRhizobium japonicum strain significant quantities of phenol (25 mol in 24 h by 2·10¹² cells) were metabolized to¹⁴CO₂, with phenol as sole carbon source. WithAzospirillum brasilense in liquid culture under 1% and 2% O₂ in the gas phase, no enhancement of nitrogenase activity by phenol was noticed.     

Peroxidase Oxidation of Phenols

Partially purified preparations of horseradish peroxidase were able to catalyze the effective transformation of such phenol compounds as phenol, o-chlorophenol, 2,4,6-trichlorophenol, pentachlorophenol (giving rise to the formation of polymer products insoluble in water), resorcinol, and thymol (giving rise to the formation of low-molecular-weight products). The following conditions were found to be optimal for peroxidase oxidation and provide the maximum extent of elimination of phenol compounds: temperature, 15–25 and 25–30°C for phenol and chlorophenol compounds, respectively; molar ratio H₂O₂/phenol, 1 : 1; and transformation time, 1–3 h. Although effective transformation was observed within a broad range of pH, the efficiency of the process slightly increased at a pH from 6.0 to 7.5. It was suggested to carry out multiple peroxidase oxidations of phenols using partially purified peroxidase enclosed in a dialysis membrane bag placed into a solution of a phenol compound containing hydrogen peroxide.     

Production of Methyl Ketones from Secondary Alcohols by Cell Suspensions of C2 to C4n-Alkane-Grown Bacteria

Nineteen new C₂ to C₄n-alkane-grown cultures were isolated from lake water from Warinanco Park, Linden, N.J., and from lake and soil samples from Bayway Refinery, Linden, N.J. Fifteen known liquid alkane-utilizing cultures were also found to be able to grow on C₂ to C₄n-alkanes. Cell suspensions of these C₂ to C₄n-alkane-grown bacteria oxidized 2-alcohols (2-propanol, 2-butanol, 2-pentanol, and 2-hexanol) to their corresponding methyl ketones. The product methyl ketones accumulated extracellularly. Cells grown on 1-propanol or 2-propanol oxidized both primary and secondary alcohols. In addition, the activity for production of methyl ketones from secondary alcohols was found in cells grown on either alkanes, alcohols, or alkylamines, indicating that the enzyme(s) responsible for this reaction is constitutive. The optimum conditions for in vivo methyl ketone formation from secondary alcohols were compared among selected strains: Brevibacterium sp. strain CRL56, Nocardia paraffinica ATCC 21198, and Pseudomonas fluorescens NRRL B-1244. The rates for the oxidation of secondary alcohols were linear for the first 3 h of incubation. Among secondary alcohols, 2-propanol and 2-butanol were oxidized at the highest rate. A pH around 8.0 to 9.0 was found to be the optimum for acetone or 2-butanone formation from 2-alcohols. The temperature optimum for the production of acetone or 2-butanone from 2-propanol or 2-butanol was rather high at 60°C, indicating that the enzyme involved in the reaction is relatively thermally stable. Metal-chelating agents inhibit the production of methyl ketones, suggesting the involvement of a metal(s) in the oxidation of secondary alcohols. Secondary alcohol dehydrogenase activity was found in the cell-free soluble fraction; this activity requires a cofactor, specifically NAD. Propane monooxygenase activity was also found in the cell-free soluble fraction. It is a nonspecific enzyme catalyzing both terminal and subterminal oxidation of n-alkanes.     

Classification of secondary alcohol-utilizing methanogens including a new thermophilic isolate

A thermophilic coccoid methanogenic bacterium, strain TCI, that grew optimally around 55° C was isolated with 2-propanol as hydrogen donor for methanogenesis from CO₂. H₂, formate or 2-butanol were used in addition. Each secondary alcohol was oxidized to its ketone. Growth occurred in defined freshwater as well as salt (2% NaCl, w/v) medium. Acetate was required as carbon source, and 4-aminobenzoate and biotin as growth factors. A need for molybdate or alternatively tungstate was shown.Strain TCI was further characterized together with two formerly isolated mesophilic secondary alcohol-utilizing methanogens, the coccoid strain CV and the spirilloid strain SK. The guanine plus cytosine content of the DNA of the three strains was 55,47, and 39 mol%, respectively. Determination of the molecular weights of the methylreductase subunits and sequencing of ribosomal 16S RNA of strains TCI and CV revealed close relationships to the genus Methanogenium. The new isolate TCI is classified as a strain of the existing species, Methanogenium thermophilum (thermophilicum). For strain CV, that uses ethanol or 1-propanol in addition, a classification as new species, Methanogenium organophilum, is proposed. Strain SK is affiliated with the existing species, Methanospirillum hungatei. The ability to use secondary alcohols was also tested with described species of methanogens. Growth with secondary alcohols was observed with Methanogenium marisnigri, Methanospirillum hungatei strain GP1 and Methanobacterium bryantii, but not with Methanospirillum strains JF1 and M1h, Methanosarcina barkeri, Methanococcus species or thermophilic strains or species other than the new isolate TCI.     

Occurrence of phenolsulfotransferase in primary glial culture cells of rat

Phenolsulfotransferase (PST) activity towards phenol and monoamines was determined in rat brain and in primary cultures of rat astrocytes. The pH requirement.K _m values and the proportion of PST activity with respect to phenol and dopamine as substrates were similar between PST from the glial cells and the rat cortex. The enzyme activity increased with age in the brain of older animals, and also with increasing incubation time in the primary culture of astroglia. The specific PST activity of the astroglia appeared to be higher than that of the brain enzyme. In glial cultures treated with 0.25 mM dibutyryl cyclic AMP in the same culture conditions, PST activity is suppressed to about 25% of its untreated counterpart, even though dibutyryl cyclic AMP at concentrations of 1 mM only slightly inhibited PST activity in vitro.     

Anaerobic 2-Propanol Degradation in Anoxic Paddy Soil and the Possible Role of Methanogens in Its Degradation

The anaerobic degradation of 2-propanol in anoxic paddy soil was studied with soil cultures and a 2-propanol-utilizing methanogen. Acetone was the first and the major intermediate involved in the methanogenic degradation of 2-propanol. Analyses with a methanogenesis inhibitor, bacteria antibiotics, and the addition of H₂ to the gas phase revealed that 2-propanol oxidation to acetone directly occurred using 2-propanol-utilizing methanogens, but not with H₂-producing syntrophic bacteria, for which the removal of acetone is required for complete 2-propanol oxidation. The 2-propanol-utilizing strain IIE1, which is phylogenetically closely related to Methanoculleus palmolei, was isolated from paddy soil, and the potential role of the strain in 2-propanol degradation was investigated. 2-Propanol is one of the representative fermentation intermediates in anaerobic environments. This is the first report on the anaerobic 2-propanol degradation process.     

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.     

Kinetic study of phenol hydroxylase and catechol 1,2-dioxygenase biosynthesis by <Emphasis Type="Italic">Candida tropicalis</Emphasis> cells grown on different phenolic substrates

When Candida tropicalis was grown on phenol, catechol or resorcinol, the highest levels of specific activity of phenol hydroxylase (EC. 1.14.13.7) and catechol 1,2-dioxygenase (EC. 1.13.11.1) were attained with phenol. With the three aromatic compounds tested, the yeast cells exhibited sharp peaks of specific activity of both enzymes at particular incubation times. Phenol-induced cells containing high levels of both enzymes were capable of degrading rapidly and without delay 4-chlorophenol and 2,6-dichlorophenol, and to a lesser extend pentachlorophenol. However, the yeast could not grow on chlorophenols as major carbon and energy source.     

Utilization of formate as an additional energy source by glucose-limited chemostat cultures ofCandida utilis CBS 621 andSaccharomyces cerevisiae CBS 8066

The oxidation of benzene to phenol by whole cells of Nitrosomonas europaea is catalysed by ammonia monooxygenase, and therefore requires a source of reducing power. Endogenous substrates, hydrazine, hydroxylamine and ammonium ions were compared as reductants. The highest rates of benzene oxidation were obtained with 4 mM benzene and hydrazine as reductant, and equalled 6 mol· h^-1·mg protein^-1. The specificity of ammonia monooxygenase for benzene as a substrate was determined by measuring k _cat/K _m for benzene relative to k _cat/K _m for uncharged ammonia, a value of 0.4 being obtained. Phenol was found to be further hydroxylated to yield hydroquinone. This reaction, like benzene oxidation, was sensitive to the ammonia monooxygenase inhibitor allylthiourea. Catechol and resorcinol were not detected as products of phenol oxidation, implying that at least 88% of the hydroxylation is para-directed.     

Formation of higher alcohols and phenol by strains of zymomonas sp.

Strains of the bacteria Zymomonas sp. were studied for their ability to form higher alcohols. In a complex growth medium, six strains were shown to produce significant amounts of 1-propanol, 1-butanol, 2-methyl-1-butanol, 3-methyl-1-butanol, 2-methyl-2-butanol, pentanols, secondary hexyl-alcohols, and trace amounts of n-hexanol. When resting cells of these organisms were placed into a fermentation medium containing glucose and Tris-buffer, Z. mobilis 8938 produced increased levels of 1-butanol, and secondary hexyl-alcohols at concentrations of 13.5 mg/liter and 5.8 mg/liter, respectively. Another strain, Z. mobilis subsp. mobilis B 806, stimulated the formation of 1-propanol and 1-butanol at concentrations of 14.9 mg/liter and 23.52 mg/liter, respectively. Amino acids or amino acid precursors were then added to the fermentation medium. The presence of threonine and α-ketobutyric acid stimulated Z. mobilis 8938 to produce 82.6 mg/liter secondary hexyl-alcohols and 8.0 mg/liter n-hexanol, respectively. Isoleucine and valine increased the production of 2-methyl-1-butanol (394.0 mg/liter) and 3-methyl-1-butanol (113.4 mg/liter), respectively, by Z. mobilis subsp. mobilis B 806. Glutamine enhanced the formation of 2-methyl-2-butanol production to concentrations 38.8 mg/liter in Zymomonas strain B 806. Additional experiments suggested that higher alcohol production could also be accomplished in the absence of glucose when cells were allowed to metabolize the precursors only. The effect of aromatic amino acids on phenol production was determined using resting cells of Zymomonas sp. The maximum yield of phenol (111.6 mg/liter) was found by Zymomonas strain 8938 in the presence of tyrosine. The addition of phenylalanine also stimulated this strain to form 71.4 mg/liter of phenol.     

Purification and Properties of Primary and Secondary Alcohol Dehydrogenases from Thermoanaerobacter ethanolicus

Thermoanaerobacter ethanolicus (ATCC 31550) has primary and secondary alcohol dehydrogenases. The two enzymes were purified to homogeneity as judged from sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration. The apparent M_rs of the primary and secondary alcohol dehydrogenases are 184,000 and 172,000, respectively. Both enzymes have high thermostability. They are tetrameric with apparently identical subunits and contain from 3.2 to 5.5 atoms of Zn per subunit. The two dehydrogenases are NADP dependent and reversibly convert ethanol and 1-propanol to the respective aldehydes. The V_m values with ethanol as a substrate are 45.6 μmol/min per mg for the primary alcohol dehydrogenase and 13 μmol/min per mg for the secondary alcohol dehydrogenase at pH 8.9 and 60°C. The primary enzyme oxidizes primary alcohols, including up to heptanol, at rates similar to that of ethanol. It is inactive with secondary alcohols. The secondary enzyme is inactive with 1-pentanol or longer chain alcohols. Its best substrate is 2-propanol, which is oxidized 15 times faster than ethanol. The secondary alcohol dehydrogenase is formed early during the growth cycle. It is stimulated by pyruvate and has a low K_m for acetaldehyde (44.8 mM) in comparison to that of the primary alcohol dehydrogenase (210 mM). The latter enzyme is formed late in the growth cycle. It is postulated that the secondary alcohol dehydrogenase is largely responsible for the formation of ethanol in fermentations of carbohydrates by T. ethanolicus.     

Growth of Methanogenic Bacteria in Pure Culture with 2-Propanol and Other Alcohols as Hydrogen Donors

Two types of mesophilic, methanogenic bacteria were isolated in pure culture from anaerobic freshwater and marine mud with 2-propanol as the hydrogen donor. The freshwater strain (SK) was a Methanospirillum species, the marine, salt-requiring strain (CV), which had irregular coccoid cells, resembled Methanogenium sp. Stoichiometric measurements revealed formation of 1 mol of CH₄ by CO₂ reduction, with 4 mol of 2-propanol being converted to acetone. In addition to 2-propanol, the isolates used 2-butanol, H₂, or formate but not methanol or polyols. Acetate did not serve as an energy substrate but was necessary as a carbon source. Strain CV also oxidized ethanol or 1-propanol to acetate or propionate, respectively; growth on the latter alcohols was slower, but final cell densities were about threefold higher than on 2-propanol. Both strains grew well in defined, bicarbonate-buffered, sulfide-reduced media. For cultivation of strain CV, additions of biotin, vitamin B₁₂, and tungstate were necessary. The newly isolated strains are the first methanogens that were shown to grow in pure culture with alcohols other than methanol. Bioenergetic aspects of secondary and primary alcohol utilization by methanogens are discussed.     

Anaerobic degradation of phenol in batch and continuous cultures by a denitrifying bacterial consortium

Summary The anaerobic degradation of phenol under denitrifying conditions by a bacterial consortium was studied both in batch and continuous cultures. Anaerobic degradation was dependent on NO_f3 ^p– and concentrations up to 4 mm phenol were degraded within 2–5 days. During continuous growth in a fermenter, steady states could be maintained at eight dilution rates (D) corresponding to residence times between 12.5 and 50 h. Culture wash-out occurred at D=0.084 h^–1. The kinetic parameters obtained for anaerobic degradation of phenol under denitrifying conditions by the consortium were: maximam specific growth rate = 0.091 h^–1; saturation constant = 4.91 mg phenol/l; true growth yield = 0.57 mg dry wt/mg phenol; maintenance coefficient = 0.013 mg phenol/mg dry wt per hour. The Haldane model inhibition constant was estimated from batch culture data giving a value of 101 mg/l. The requirement of CO₂ for the anaerobic degradation of phenol with NO_f3 ^p– indicates that phenol carboxylation to 4-hydroxybenzoate was the first step of phenol degradation by this culture. 4-Hydroxybenzoate, proposed as an intermediate of phenol carboxylation under these conditions, was detected only in continuous cultures at very low growth rates (D=0.02 h^–1), but was never detected as a free intermediary metabolite either in batch or in continuous cultures. Correspondence to: N. Khoury     

Biodegradation of VOCs from printing press air by an on-site pilot plant bioscrubber and laboratory scale continuous yeast cultures

The volatile organic compound composition (VOCs) of printing press air wasfound to contain mostly ethanol, but also ethyl acetate, 1-propanol, 2-propanol,1-methoxy-2-propanol and 3-ethoxy-1-propanol. A pilot plant bioscrubber inoculatedwith a mixed microbial population was constructed on-site. The bioscrubber wasable to treat the polluted gas efficiently. It, however, suffered from strong wall growthand blockages in the column. The efficiencies of the pilot plant and a bioreactor iscompared. The yeasts Candida guilliermondii and Saccharomyces cerevisiae known to tolerate ethanol were selected instead of mixed population to avoid the wall growth and blockages in the bioreactor. The removal of the VOCs both individually and as a complex mixture was tested in a microcultivation system and in continuous chemostat cultures with and without cell recycling. The Candida yeast could use all the compounds as a carbon source while growth of S. cerevisiae was markedly slower on the methoxylated and ethoxylated propanols. Best total removal of the VOCs was 99% and achieved by C. guilliermondii. The only compound that was not totally removable in the chemostat experiment with C. guilliermondii was 1-methoxy-2-propanol. In laboratory scale the total and volumetric removal of VOCs by C. guilliermondii was more efficient compared to the pilot plant encouraging to scale up and applying the yeast bioreactor to real field conditions.     

Methanogenic degradation of hydroquinone and catechol via reductive dehydroxylation to phenol

Abstract Fermentative degradation of hydroquinone, catechol, and phenol was demonstrated with nearly-homogeneous mixed methanogenic cultures obtained from freshwater sediments and sewage sludge by enrichment with the respective phenolic substrates. Gram-negative short rods predominated in these cultures, together with hydrogen- and acetate-utilizing methanogens. Acetate and methane were the only degradation products. Bacteria enriched with hydroquinone or catechol also degraded phenol and p -hydroxy-benzoate, but not resorcinol or resorcylic acids. Phenol was formed as an intermediate during catechol and hydroquinone degradation, indicating that reductive dehydroxylation was the primary event in degradation of these substrates. Inhibition experiments with bromoethanesulfonate and acetylene indicated that catechol, hydroquinone, and phenol degradation depended on a syntrophic co-operation of fermenting bacteria and hydrogen-oxidizing methanogens.     

Safrole Analysis by GC-MS of Prototrophic (Ocotea odorifera (Vell.) Rohwer) Cell Cultures

Ocotea odorifera, a tree native to the Atlantic rainforest in south Brazil, has been used as a source of sassafras oil rich in safrole [5-(2-propenyl)-1,3-benzodioxole], an aromatic ether used as a flavoring agent and also in the manufacture of insecticides. The intensive deforestation process of the Brazilian Atlantic rainforest has threatened with extinction some species including O. odorifera. In this context, O. odorifera cell cultures might be an interesting alternative for the production of secondary metabolites of value (i.e., safrole), without risk of damage to the native germplasm. Insights into the secondary metabolites of organosolvent extracts of prototrophic cell cultures were performed by GC-MS and MALDI-TOF MS. GC-MS analysis revealed the occurrence of safrole in concentration of ca. 62.6 μg ml⁻¹ and allowed the identification of other compounds besides safrole in the crude extract of these cultures. Thus, alkyl phenol (C₈) [m/z= 206], C₁₄ myristic acid [m/z= 228], long chain olefin or alcohol and essential oil were detected. However, safrole [m/z= 162] was not detected by MALDI-TOF MS, indicating that it is not easily protonated (M + H)⁺.