Degradation of hydroxyhydroquinone by the strictly anaerobic fermenting bacterium Pelobacter massiliensis sp. nov.

A new rod-shaped, gram-negative, non-sporeforming, strictly anaerobic bacterium (strain HHQ7) was enriched and isolated from marine mud samples with hydroxyhydroquinone (1,2,4-trihydroxybenzene) as sole substrate. Strain HHQ7 fermented hydroxyhydroquinone, pyrogallol (1,2,3-trihydroxybenzene), phloroglucinol (1,3,5-trihydroxybenzene) and gallic acid (3,4,5-trihydroxybenzoate) to 3 mol acetate (plus 1 mol CO₂ in the case of gallic acid) per mol of substrate. Resorcinol accumulated intermediately during growth on hydroxy-hydroquinone. No other aliphatic or aromatic substrates were utilized. Sulfate, sulfite, sulfur, nitrate, and fumarate were not reduced with hydroxyhydroquinone as electron donor. The strain grew in sulfide-reduced mineral medium supplemented with 7 vitamins. The DNA base ratio was 59% G+C. Strain HHQ7 is classified as a new species of the genus Pelobacter, P. massiliensis. Experiments with dense cell suspensions of hydroxyhydroquinone-and pyrogallol-grown cells showed different kinetics of hydroxyhydroquinone and pyrogallol degradation, as well as different patterns of resorcinol accumulation, indicating that these substrates are metabolized by different transhydroxylation reactions.     

Anaerobic degradation of protocatechuate (3,4-dihydroxybenzoate) by Thauera aromatica strain AR-1

The denitrifying bacterium Thauera aromatica strain AR-1 grows anaerobically with protocatechuate (3,4-dihydroxybenzoate (DHB)) as sole energy and carbon source. This bacterium harbors two distinct pathways for degradation of aromatic compounds, the benzoyl-coenzyme A (CoA) pathway for benzoate degradation and the hydroxyhydroquinone (HHQ) pathway for degradation of 3,5-DHB. In order to elucidate whether protocatechuate is degraded via the benzoyl-CoA or the HHQ pathway, induction experiments were carried out. Dense suspensions of cells grown on protocatechuate or benzoate readily degraded benzoate and protocatechuate but not 3,5-DHB. Dense suspensions of 3,5-DHB-grown cells degraded 3,4- and 3,5-DHB at similar rates, but benzoate was not degraded. 3,5-DHB hydroxylating activity was found only in cells grown with this substrate. HHQ dehydrogenase activity was found in extracts of cells grown with 3,5-DHB and at a low rate also in protocatechuate-grown cells, but not in extracts of cells grown with benzoate. Activities of protocatechuyl-CoA synthetase and protocatechuyl-CoA reductase leading to 3-hydroxybenzoyl-CoA were found in extracts of cells grown with protocatechuate. There was no repression of the HHQ pathway by the presence of protocatechuate, unlike by degradation of benzoate. We conclude that protocatechuate is not degraded via the HHQ pathway because there was no evidence of a hydroxylation reaction involved in this process. Instead, our results strongly suggest that protocatechuate is degraded via a pathway which connects to the benzoyl-CoA route of degradation.     

Anaerobic degradation of α-resorcylate by Thauera aromatica strain AR-1 proceeds via oxidation and decarboxylation to hydroxyhydroquinone

Anaerobic degradation of α-resorcylate (3,5-dihydroxybenzoate) was studied with the denitrifying strain AR-1, which was assigned to the described species Thauera aromatica. α-Resorcylate degradation does not proceed via the benzoyl-CoA, the resorcinol, or the phloroglucinol pathway. Instead, α-resorcylate is converted to hydroxyhydroquinone (1,2,4-trihydroxybenzene) by dehydrogenative oxidation and decarboxylation. Nitrate, K₃[Fe(CN)₆], dichlorophenol indophenol, and the NAD⁺ analogue 3-acetylpyridine adeninedinucleotide were suitable electron acceptors for the oxidation reaction; NAD⁺ did not function as an electron acceptor. The oxidation reaction was strongly accelerated by the additional presence of the redox carrier phenazine methosulfate, which could also be used as sole electron acceptor. Oxidation of α-resorcylate with molecular oxygen in cell suspensions or in cell-free extracts of α-resorcylate- and nitrate-grown cells was also detected although this bacterium did not grow with α-resorcylate under an air atmosphere. α-Resorcylate degradation to hydroxyhydroquinone proceeded in two steps. The α-resorcylate-oxidizing enzyme activity was membrane-associated and exhibited maximal activity at pH 8.0. The primary oxidation product was not hydroxyhydroquinone. Rather, formation of hydroxyhydroquinone by decarboxylation of the unknown intermediate in addition required the cytoplasmic fraction and needed lower pH values since hydroxyhydroquinone was not stable at alkaline pH. Received: 8 July 1997 / Accepted: 20 October 1997     

Evidence of Two Oxidative Reaction Steps Initiating Anaerobic Degradation of Resorcinol (1,3-Dihydroxybenzene) by the Denitrifying Bacterium Azoarcus anaerobius

The denitrifying bacterium Azoarcus anaerobius LuFRes1 grows anaerobically with resorcinol (1,3-dihydroxybenzene) as the sole source of carbon and energy. The anaerobic degradation of this compound was investigated in cell extracts. Resorcinol reductase, the key enzyme for resorcinol catabolism in fermenting bacteria, was not present in this organism. Instead, resorcinol was hydroxylated to hydroxyhydroquinone (HHQ; 1,2,4-trihydroxybenzene) with nitrate or K₃Fe(CN)₆ as the electron acceptor. HHQ was further oxidized with nitrate to 2-hydroxy-1,4-benzoquinone as identified by high-pressure liquid chromatography, UV/visible light spectroscopy, and mass spectroscopy. Average specific activities were 60 mU mg of protein⁻¹ for resorcinol hydroxylation and 150 mU mg of protein⁻¹ for HHQ dehydrogenation. Both activities were found nearly exclusively in the membrane fraction and were only barely detectable in extracts of cells grown with benzoate, indicating that both reactions were specific for resorcinol degradation. These findings suggest a new strategy of anaerobic degradation of aromatic compounds involving oxidative steps for destabilization of the aromatic ring, different from the reductive dearomatization mechanisms described so far.     

Hydroxyhydroquinone reductase, the initial enzyme involved in the degradation of hydroxyhydroquinone (1,2,4-trihydroxybenzene) by Desulfovibrio inopinatus

The recently isolated sulfate reducer Desulfovibrio inopinatus oxidizes hydroxyhydroquinone (1,2,4trihydroxybenzene; HHQ) to 2 mol acetate and 2 mol CO2 (mol substrate)-1, with stoichiometric reduction of sulfate to sulfide. None of the key enzymes of fermentative HHQ degradation, i.e. HHQ-1,2,3,5-tetrahydroxybenzene transhydroxylase or phloroglucinol reductase, were detected in cell-free extracts of D. inopinatus, indicating that this bacterium uses a different pathway for anaerobic HHQ degradation. HHQ was reduced with NADH in cell-free extracts to a nonaromatic compound, which was identified as dihydrohydroxyhydroquinone by its retention time in HPLC separation and by HPLC-mass spectrometry. The compound was identical with the product of chemical reduction of HHQ with sodium borohydride. Dihydrohydroxyhydroquinone was converted stoichiometrically to acetate and to an unknown coproduct. HHQ reduction was an enzymatic activity which was present in the cell-free extract at 0.25-0.30 U (mg protein)-1, with a pH optimum at 7.5. The enzyme was sensitive to sodium chloride, potassium chloride, EDTA, and o-phenanthroline, and exhibited little sensitivity towards sulfhydryl group reagents, such as copper chloride or p-chloromercuribenzoate.     

Heterologous expression and identification of the genes involved in anaerobic degradation of 1,3-dihydroxybenzene (resorcinol) in Azoarcus anaerobius

Azoarcus anaerobius, a strictly anaerobic, gram-negative bacterium, utilizes resorcinol as a sole carbon and energy source with nitrate as an electron acceptor. Previously, we showed that resorcinol degradation by this bacterium is initiated by two oxidative steps, both catalyzed by membrane-associated enzymes that lead to the formation of hydroxyhydroquinone (HHQ; 1,2,4-benzenetriol) and 2-hydroxy-1,4-benzoquinone (HBQ). This study presents evidence for the further degradation of HBQ in cell extracts to form acetic and malic acids. To identify the A. anaerobius genes required for anaerobic resorcinol catabolism, a cosmid library with genomic DNA was constructed and transformed into the phylogenetically related species Thauera aromatica, which cannot grow with resorcinol. By heterologous complementation, a transconjugant was identified that gained the ability to metabolize resorcinol. Its cosmid, designated R(+), carries a 29.88-kb chromosomal DNA fragment containing 22 putative genes. In cell extracts of T. aromatica transconjugants, resorcinol was degraded to HHQ, HBQ, and acetate, suggesting that cosmid R(+) carried all of the genes necessary for resorcinol degradation. On the basis of the physiological characterization of T. aromatica transconjugants carrying transposon insertions in different genes of cosmid R(+), eight open reading frames were found to be essential for resorcinol mineralization. Resorcinol hydroxylase-encoding genes were assigned on the basis of sequence analysis and enzyme assays with two mutants. Putative genes for hydroxyhydroquinone dehydrogenase and enzymes involved in ring fission have also been proposed. This work provides the first example of the identification of genes involved in the anaerobic degradation of aromatic compounds by heterologous expression of a cosmid library in a phylogenetically related organism.     

Degradation of some phenols and hydroxybenzoates by the imperfect ascomycetous yeastsCandida parapsilosis andArxula adeninivorans: evidence for an operative gentisate pathway

The imperfect ascomycetous yeastsCandida parapsilosis andArxula adeninivorans degraded 3-hydroxybenzoic acid via gentisate which was the cleavage substrate. 4-Hydroxybenzoic acid was metabolized via protocatechuate. No cleavage enzyme for the latter was detected. In stead of this NADH- and NADPH-dependent monooxygenases were present. In cells grown at the expense of hydroquinone and 4-hydroxygenzoic acid, enzymes of the hydroxyhydroquinone variant of the 3-oxoadipate pathway were demonstrated, which also took part in the degradation of 2,4-dihydroxybenzoic acid byC. parapsilosis.Abbreviations HHQ Hydroxyhydroquinone (1,2,4-trihydroxybenzene) - GSH reduced Glutathione     

Degradation of prochloraz and 2,4,6-trichlorophenol by environmental bacterial strains

 Eight bacterial isolates from enrichment with 2,4,6-trichlorophenol (TCP) as sole carbon source were tested for their potential to degrade prochloraz. None of them could grow on prochloraz. Strain C964, identified as Aureobacterium sp., effectively reduced the fungitoxic activity of prochloraz in a bioassay and degradation was confirmed by HPLC. Two other isolates, strain C611 and C961, using TCP as a carbon source, belong to the β subclass of the proteobacteria and presumely degrade TCP via 2,4-dichlorohydroquinone and hydroxyhydroquinone as indicated by oxygen-consumption tests. Received: 3 July 1995/Received revision: 27 July 1995/Accepted: 31 July 1995     

Isolation and characterization of Thermus thermophilus Gy1211 from a deep-sea hydrothermal vent

We examined a single, non-spore-forming, aerobic, thermophilic strain that was isolated from a deep-sea hydrothermal vent in the Guaymas Basin at a depth of 2000 m and initially placed in a phenetic group with Thermus scotoductus (X-1). We identified this deep-sea isolate as a new strain belonging to Thermus thermophilus using several parameters. DNA–DNA hybridization under stringent conditions showed 74% similarity between the deep-sea isolate and T. thermophilus HB-8^T (T = type strain). Phenotypic characteristics, such as the utilization of carbon sources, hydrolysis of different compounds, and antibiotic sensitivity were identical in the two strains. The polar lipids composition showed that strain Gy1211 belonged to the genus Thermus. The fatty acids composition indicated that this strain was related to the marine T. thermophilus strain isolated from the Azores. The new isolate T. thermophilus strain Gy1211 grew optimally at 75°C, pH 8.0, and 2% NaCl. A hydrostatic pressure of 20 MPa, similar to the in situ hydrostatic pressure of the deep-sea vent from which the strain was isolated, had no effect on growth. Strain HB-8^T, however, showed slower growth under these conditions. Received: November 26, 1997 / Accepted: May 20, 1999     

Fermentation of phenoxyethanol to phenol and acetate by a homoacetogenic bacterium

A strictly anaerobic gram-positive, rod-shaped bacterium, strain LuPhet1, was isolated from sewage sludge with phenoxyethanol as sole carbon and energy source, and was assigned to the genus Acetobacterium. The new isolate fermented the alkylaryl ether compound phenoxyethanol stoichiometrically to phenol and acetate, whereas phenoxyacetic acid was not degraded. In cell-free extracts of strain LuPhet1, cleavage of the ether linkage was shown, and acetaldehyde was detected as reaction product. Coenzyme A-dependent acetaldehyde: acceptor oxidoreductase, phosphate acetyltransferase, acetate kinase, and carbon monoxide dehydrogenase were measured in cell-free extracts of this strain. Our results indicate that the ether linkage of phenoxyethanol is cleaved by a shift of the hydroxyl group to the subterminal carbon atom, analogous to a corrinoid-dependent diol dehydratase reaction, to form an unstable hemiacetal that releases phenol and acetaldehyde. Obviously, phenoxyethanol is degraded by the same strategy as in anaerobic degradation of the alkyl ether polyethylene glycol.     

Reduction in hydroxyhydroquinone from coffee increases postprandial fat utilization in healthy humans: a randomized double-blind,cross-over trial

The present study aimed to clarify the effect of reduction in hydroxyhydroquinone (HHQ) from roasted coffee on energy utilization in humans. Indirect calorimetry showed that one-week ingestion of HHQ-reduced coffee led to significantly higher postprandial fat utilization than that of HHQ-containing coffee. This finding indicates that reduction in HHQ from coffee increases postprandial fat utilization.     

Conversion of the Pseudomonas aeruginosa Quinolone Signal and Related Alkylhydroxyquinolines by Rhodococcus sp. Strain BG43

A bacterial strain, which based on the sequences of its 16S rRNA, gyrB, catA, and qsdA genes, was identified as a Rhodococcus sp. closely related to Rhodococcus erythropolis, was isolated from soil by enrichment on the Pseudomonas quinolone signal [PQS; 2-heptyl-3-hydroxy-4(1H)-quinolone], a quorum sensing signal employed by the opportunistic pathogen Pseudomonas aeruginosa. The isolate, termed Rhodococcus sp. strain BG43, cometabolically degraded PQS and its biosynthetic precursor 2-heptyl-4(1H)-quinolone (HHQ) to anthranilic acid. HHQ degradation was accompanied by transient formation of PQS, and HHQ hydroxylation by cell extracts required NADH, indicating that strain BG43 has a HHQ monooxygenase isofunctional to the biosynthetic enzyme PqsH of P. aeruginosa. The enzymes catalyzing HHQ hydroxylation and PQS degradation were inducible by PQS, suggesting a specific pathway. Remarkably, Rhodococcus sp. BG43 is also capable of transforming 2-heptyl-4-hydroxyquinoline-N-oxide to PQS. It thus converts an antibacterial secondary metabolite of P. aeruginosa to a quorum sensing signal molecule.     

Degradation of polyvinyl alcohol by <Emphasis Type="Italic">Sphingomonas</Emphasis> sp. SA3 and its symbiote

A total of 800 samples was taken from Taegu province, Korea, where many textile factories provide a source of polyvinyl alcohol (PVA) waste. These samples were screened for PVA-degrading bacteria. A new strain, SA3, was discovered which formed yellow colonies and used PVA as the sole carbon and energy source. Strain SA3 was identified as a Sphingomonas sp., based on the partial nucleotide sequence analysis of 16S ribosomal RNA, the presence of 2-hydroxymyristic acid (14:O 2-OH) and sphingolipids with d-17:0, d-18:0, d-19:1, and d-20:1 as the main dihydrosphingosines. This genus has not previously been reported as a PVA-degrading bacterium. Sphingomonas sp. SA3 needs a symbiote strain, SA2, for PVA degradation as a growth factor producer. In mixed cultures of these strains, the optimum temperature for PVA biodegradation ranged from 30 °C to 35 °C. The optimum pH was 8.0 and the most effective nitrogen source was NH₄ ⁺. Electronic Publication     

Anaerobic degradation of cyclohexane-1,2-diol by a new Azoarcus species

A bacterium, strain 22Lin, was isolated on cyclohexane-1,2-diol as sole electron donor and carbon source and nitrate as electron acceptor. Cells are motile rods and are facultatively anaerobic. A phylogenetic comparison based on the total 16S rRNA gene sequence allowed the assignment of the isolate to the genus Azoarcus. Cyclohexanol, cyclohexanone, cyclohexane-1,3-diol, and cyclohexane-1,3-dione, which are oxidized by a different denitrifying strain, did not support denitrifying growth of isolate 22Lin. On the contrary, cyclohexanol (I₅₀ = 37 μM) and cyclohexanone (I₅₀ = 28 μM) inhibited growth on cyclohexane-1,2-diol, but not on acetate. NAD was reduced by crude extracts of strain 22Lin in the presence of cyclohexane-1,2-dione, but not in the presence of cyclohexanone or cyclohexane-1,3-dione. The formation of 6-oxohexanoate from cyclohexane-1,2-dione and of adipate during NAD reduction suggests that strain 22Lin possesses a carbon–carbon hydrolase that transforms cyclohexane-1,2-dione into 6-oxohexanoate. This pathway was once observed in an aerobic pseudomonad that was lost and could not be reisolated. Here, the application of strictly anoxic enrichment conditions enabled the reisolation of another strain (22Lin) that uses this pathway. Received: 3 February 1997 / Accepted: 12 May 1997     

Isolation and characterization of a marine bacterium capable of utilizing 2-methylphenanthrene

A marine bacterium isolated from a coastal hydrocarbon-polluted sediment has been described and attributed on the basis of its phenotypic and genotypic characteristics to the genus Sphingomonas sp. This strain was capable of using an alkylated phenanthrene 2-methylphenanthrene, as sole source of carbon and energy. In experiments, 2-methylphenanthrene (0.2 g/l) was added as crystals to the culture medium. After 5 days of aerobic growth at 30 °C, 70% was degraded and the complete dissipation occurred after 20 days. Furthermore, the strain could degrade various kinds of polyaromatic compounds, but failed to grow on aliphatic hydrocarbons. Received: 27 December 1996 / Received last revision: 10 June 1997 / Accepted: 14 June 1997     

Fermentative degradation of triethanolamine by a homoacetogenic bacterium

With triethanolamine as sole source of energy and organic carbon, a strictly anaerobic, gram-positive, rod-shaped bacterium, strain LuTria 3, was isolated from sewage sludge and was assigned to the genus Acetobacterium on the basis of morphological and physiological properties. The G+C content of the DNA was 34.9±1.0 mol %. The new isolate fermented triethanolamine to acetate and ammonia. In cell-free extracts, a triethanolamine-degrading enzyme activity was detected that formed acetaldehyde as reaction product. Triethanolamine cleavage was stimulated 30-fold by added adenosylcobalamin (co-enzyme B₁₂) and inhibited by cyanocobalamin or hydroxocobalamin. Ethanolamine ammonia lyase, acetaldehyde:acceptor oxidoreductase, phosphate acetyltransferase, acetate kinase, and carbon monoxide dehydrogenase were measured in cell-free extracts of this strain. Our results establish that triethanolamine is degraded by a corrinoid-dependent shifting of the terminal hydroxyl group to the subterminal carbon atom, analogous to a diol dehydratase reaction, to form an unstable intermediate that releases acetaldehyde. No anaerobic degradation of triethylamine was observed in similar enrichment assays.Abbreviation NTA nitrilotriacetate     

Isolation of new 6-methylnicotinic-acid-degrading bacteria, one of which catalyses the regioselective hydroxylation of nicotinic acid at position C2

2-Hydroxynicotinic acid is an important building block for herbicides and pharmaceuticals. Enrichment strategies to increase the chances of finding microorganisms capable of hydroxylating at the C2 position and to avoid the degradation of nicotinic acid via the usual intermediate, 6-hydroxynicotinic acid, were used. Three bacterial strains (Mena 23/3–3c, Mena 25/4–1, and Mena 25/ 4–3) were isolated from enrichment cultures with 6-methylnicotinic acid as the sole source of carbon and energy. Partial characterization of these strains indicated that they represent new bacterial species. All three strains completely degraded 6-methylnicotinic acid, and evidence is presented that the first step in the degradation pathway of strain Mena 23/3–3c is hydroxylation at the C2 position. Resting cells of this strain grown on 6-methylnicotinic acid also hydroxylated nicotinic acid at the C2 position, but did not further degrade the product. Strain Mena 23/ 3–3c showed the highest degree of 16S rRNA sequence similarity to members of the genera Ralstonia and Burkholderia. Received: 4 April 1997 / Accepted: 10 June 1997     

The phylogenetic position of the Thermococcus isolate AN1 based on 16S rRNA gene sequence analysis: a proposal that AN1 represents a new species, Thermococcus zilligii sp. nov.

The 16S rRNA gene from the Thermococcus New Zealand isolate AN1 was cloned and sequenced. Analysis of the gene revealed the presence of signature sequences, indicating that strain AN1 represents a new species of the genus Thermococcus. Since the isolate AN1 differed from other thermococci in both its lower optimal NaCl concentration and generally lower optimal temperature for growth, in its unusual lipid membrane composition, and in its sensitivity to antibiotics, we propose that strain AN1 represents a new species of Thermococcus. The proposed name is Thermococcus zilligii, and the type strain is DSM 2770. Received: 13 February 1997 / Accepted: 30 May 1997     

Biodegradation of methyl parathion and <Emphasis Type="Italic">p</Emphasis>-nitrophenol: evidence for the presence of a <Emphasis Type="Italic">p</Emphasis>-nitrophenol 2-hydroxylase in a Gram-negative <Emphasis Type="Italic">Serratia</Emphasis> sp. strain DS001

A soil bacterium capable of utilizing methyl parathion as sole carbon and energy source was isolated by selective enrichment on minimal medium containing methyl parathion. The strain was identified as belonging to the genus Serratia based on a phylogram constructed using the complete sequence of the 16S rRNA. Serratia sp. strain DS001 utilized methyl parathion, p-nitrophenol, 4-nitrocatechol, and 1,2,4-benzenetriol as sole carbon and energy sources but could not grow using hydroquinone as a source of carbon. p-Nitrophenol and dimethylthiophosphoric acid were found to be the major degradation products of methyl parathion. Growth on p-nitrophenol led to release of stoichiometric amounts of nitrite and to the formation of 4-nitrocatechol and benzenetriol. When these catabolic intermediates of p-nitrophenol were added to resting cells of Serratia sp. strain DS001 oxygen consumption was detected whereas no oxygen consumption was apparent when hydroquinone was added to the resting cells suggesting that it is not part of the p-nitrophenol degradation pathway. Key enzymes involved in degradation of methyl parathion and in conversion of p-nitrophenol to 4-nitrocatechol, namely parathion hydrolase and p-nitrophenol hydroxylase component “A” were detected in the proteomes of the methyl parathion and p-nitrophenol grown cultures, respectively. These studies report for the first time the existence of a p-nitrophenol hydroxylase component “A”, typically found in Gram-positive bacteria, in a Gram-negative strain of the genus Serratia. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Degradation of polychlorinated phenols by Streptomyces rochei 303

The strain Streptomyces rochei 303 (VKM Ac-1284D) is capable of utilizing 2-chloro-,2,4-,2,6-dichloro- and 2,4,6-trichlorophenols as the sole source of carbon. Its resting cells completely dechlorinated and degraded 2-, 3-chloro-; 2,4-, 2,6-, 2,3-, 2,5-, 3,4-, 3,5-dichloro-; 2,4-, 2,6-dibromo-; 2,4,6-, 2,4,5-, 2,3,4-, 2,3,5-, 2,3,6-trichlorophenols; 2,3,5,6-tetrachloro- and pentachlorophenol. During chlorophenol degradation, a stoichiometric amount of chloride ions was released and chlorohydroquinols were formed as intermediates. In cell-free extracts of S. rochei, the activity of hydroxyquinol 1,2-dioxygenase was found. The enzyme was induced with chlorophenols. Of all so far described strains degrading polychlorophenols, S. rochei 303 utilized a wider range of chlorinated phenols as the sole sourse of carbon and energy.Abbreviations CP chlorophenol - DCP dichlorophenol - TCP trichlorophenol - TeCP tetrachlorophenol - PCP pentachlorophenol - DBrP dibromophenol - CHQ chlorohydroquinol - DCHQ dichlorohydroquinol - HHQ hydroxyhydroquinol - CHHQ chlorohydroxyhydroquinol - CC chlorocatechol - TLC thin layer chromatography - GC/MC chromato-mass-spectrometry - HPLC high-performance liquid chromatography