Characterisation of 2,5-diketo-D-gluconic acid reductase from Corynebacterium sp.

Summary 2,5-diketo-D-gluconic acid reductase, that converts 2,5-diketo-D-gluconic acid into 2-keto-L-gulonic acid (the direct precursor of vitamin C) was extracted and purified from Corynebacterium sp.. The enzyme was characterised in terms of kinetic parameters, molecular weight and isoelectric point. Enzyme stability at different operating temperatures was investigated, as well.     

Kinetic model for the bioconversion of glucose to 2,5-diketo-D-gluconic acid

Summary A simple unstructured mathematical model for the oxidation of glucose to 2,5-diketo-D-gluconic acid with Erwinia citreus was developed. The kinetic parameters of the model were estimated by the oxygen partial pressure method and directly from the temporal response obtained from the experimental data collected in the batch fermentation. The simple developed model based on the kinetic measurements was able to simulate quite well the dynamic behaviour of the batch fermentation.     

Structural alteration of cofactor specificity in Corynebacterium 2,5-diketo-D-gluconic acid reductase

Corynebacterium 2,5-Diketo-D-gluconic acid reductase (2,5-DKGR) catalyzes the reduction of 2,5-diketo-D-gluconic acid (2,5-DKG) to 2-Keto-L-gulonic acid (2-KLG). 2-KLG is an immediate precursor to L-ascorbic acid (vitamin C), and 2,5-DKGR is, therefore, an important enzyme in a novel industrial method for the production of vitamin C. 2,5-DKGR, as with most other members of the aldo-keto reductase (AKR) superfamily, exhibits a preference for NADPH compared to NADH as a cofactor in the stereo-specific reduction of substrate. The application of 2,5-DKGR in the industrial production of vitamin C would be greatly enhanced if NADH could be efficiently utilized as a cofactor. A mutant form of 2,5-DKGR has previously been identified that exhibits two orders of magnitude higher activity with NADH in comparison to the wild-type enzyme, while retaining a high level of activity with NADPH. We report here an X-ray crystal structure of the holo form of this mutant in complex with NADH cofactor, as well as thermodynamic stability data. By comparing the results to our previously reported X-ray structure of the holo form of wild-type 2,5-DKGR in complex with NADPH, the structural basis of the differential NAD(P)H selectivity of wild-type and mutant 2,5-DKGR enzymes has been identified.     

DNA from uncultured organisms as a source of 2,5-diketo-D-gluconic acid reductases

Total DNA of a population of uncultured organisms was extracted from soil samples, and by using PCR methods, the genes encoding two different 2,5-diketo-D-gluconic acid reductases (DKGRs) were recovered. Degenerate PCR primers based on published sequence information gave internal gene fragments homologous to known DKGRs. Nested primers specific for the internal fragments were combined with random primers to amplify flanking gene fragments from the environmental DNA, and two hypothetical full-length genes were predicted from the combined sequences. Based on these predictions, specific primers were used to amplify the two complete genes in single PCRs. These genes were cloned and expressed in Escherichia coli. The purified gene products catalyzed the reduction of 2,5-diketo-D-gluconic acid to 2-keto-L-gulonic acid. Compared to previously described DKGRs isolated from Corynebacterium spp., these environmental reductases possessed some valuable properties. Both exhibited greater than 20-fold-higher kcat/Km values than those previously determined, primarily as a result of better binding of substrate. The Km values for the two new reductases were 57 and 67 microM, versus 2 and 13 mM for the Corynebacterium enzymes. Both environmental DKGRs accepted NADH as well as NADPH as a cosubstrate; other DKGRs and most related aldo-keto reductases use only NADPH. In addition, one of the new reductases was more thermostable than known DKGRs.     

Synthesis of 2-keto-L-gulonic acid from gluconic acid by co-immobilized Gluconobacter oxydans and Corynebacterium sp.

2-Keto-L-gulonic acid was produced from gluconic acid using co-immobilized cells of Gluconobacter oxydans and Corynebacterium sp. with 2,5-diketo-D-gluconic acid. Gluconobacter oxydans and Corynebacterium sp. were entrapped together with polyvinylalcohol and alginate. 50 g/l glucose, 50 g/l gluconic acid, and the mixture of equal volume of 50 g/l glucose and 50 g/l gluconic acid were used as substrates. When the ratio of two cells was 1 to 1 with 100 mg cells/ml, the conversion of 2-KLG from gluconic acid was 38% (g/g). © Rapid Science Ltd. 1998     

Mathematical modeling of in vitro enzymatic production of 2-Keto-L-gulonic acid using NAD(H) or NADP(H) as cofactors

A 2-Keto-L-gulonic acid (2-KLG) production process using stationary Pantoea citrea cells and a Corynebacterium 2,5-diketo-D-gluconic acid (2,5-DKG) reductase enzyme has been developed which may represent an improved method of vitamin C biosynthesis. Experimental data was collected using the F22Y/A272G 2,5-DKG reductase mutant and NADP(H) as a cofactor. An extensive kinetic analysis was performed and a kinetic rate equation model for this process was developed. A recent protein engineering effort has resulted in several 2,5-DKG reductase mutants exhibiting improved activity with NADH as a cofactor. The use of NAD(H) in the bioreactor may be preferable due to its increased stability and lower cost. The kinetic parameters in the rate equation model have been replaced in order to predict 2-KLG production with NAD(H) as a cofactor. The model was also extended to predict 2-KLG production in the presence of a range of combined cofactor concentrations. This analysis suggests that the use of the F22Y/K232G/R238H/A272G 2,5-DKG reductase mutant with NAD(H) combined with a small amount of NADP(H) could provide a significant cost benefit for in vitro enzymatic 2-KLG production.     

Alteration of the specificity of the cofactor-binding pocket of Corynebacterium 2,5-diketo-D-gluconic acid reductase A

The NADPH-dependent 2,5-diketo-D-gluconic acid (2,5-DKG) reductase enzyme is a required component in some novel biosynthetic vitamin C production processes. This enzyme catalyzes the conversion of 2,5-DKG to 2-keto-L-gulonic acid, which is an immediate precursor to L-ascorbic acid. Forty unique site-directed mutations were made at five residues in the cofactor-binding pocket of 2,5-DKG reductase A in an attempt to improve its ability to use NADH as a cofactor. NADH is more stable, less expensive and more prevalent in the cell than is NADPH. To the best of our knowledge, this is the first focused attempt to alter the cofactor specificity of a member of the aldo-keto reductase superfamily by engineering improved activity with NADH into the enzyme. Activity of the mutants with NADH or NADPH was assayed using activity-stained native polyacrylamide gels. Eight of the mutants at three different sites were identified as having improved activity with NADH. These mutants were purified and subjected to a kinetic characterization with NADH as a cofactor. The best mutant obtained, R238H, produced an almost 7-fold improvement in catalysis with NADH compared with the wild-type enzyme. Surprisingly, most of this catalytic improvement appeared to be due to an improvement in the apparent kcat for the reaction rather than a large improvement in the affinity of the enzyme for NADH.     

Verification of a novel NADH-binding motif: combinatorial mutagenesis of three amino acids in the cofactor-binding pocket of Corynebacterium 2,5-diketo-D-gluconic acid reductase

A screening method has been developed to support randomized mutagenesis of amino acids in the cofactor-binding pocket of the NADPH-dependent 2,5-diketo-D-gluconic acid (2,5-DKG) reductase. Such an approach could enable the isolation of an enzyme that can better catalyze the reduction of 2,5-DKG to 2-keto-L-gulonic acid (2-KLG) using NADH as a cofactor. 2-KLG is a valuable precursor to ascorbic acid, or vitamin C, and an enzyme with increased activity with NADH may be able to improve two potential vitamin C production processes. Previously we have identified three amino acid residues that can be mutated to improve activity with NADH as a cofactor. As a pilot study to show feasibility, a library was made with these three amino acids randomized, and 300 random colonies were screened for increased NADH activity. The activities of seven mutants with apparent improvements were verified using activity-stained native gels, and sequencing showed that the amino acids obtained were similar to some of those already discovered using rational design. The four most active mutants were purified and kinetically characterized. All of the new mutations resulted in apparent kcat values that were equal to or higher than that of the best mutant obtained through rational design. At saturating levels of cofactor, the best mutant obtained was almost twice as active with NADH as a cofactor as the wild-type enzyme is with NADPH. This screen is a valuable tool for improving 2,5-DKG reductase, and it could easily be modified for improving other aspects of this protein or similar enzymes.     

Molecular modeling of substrate binding in wild-type and mutant Corynebacteria 2,5-diketo-D-gluconate reductases

2,5-diketo-D-gluconic acid reductase (2,5-DKGR; E.C. 1.1.1.-) catalyzes the Nicotinamide adenine dinucleotide phosphate (NADPH)-dependent stereo-specific reduction of 2, 5-diketo-D-gluconate (2,5-DKG) to 2-keto-L-gulonate (2-KLG), a precursor in the industrial production of vitamin C (L-ascorbate). Microorganisms that naturally ferment D-glucose to 2,5-DKG can be genetically modified to express the gene for 2,5-DKGR, and thus directly produce vitamin C from D-glucose. Two naturally occurring variants of DKGR (DKGR A and DKGR B) have been reported. DKGR B exhibits higher specific activity toward 2,5-DKG than DKGR A; however, DKGR A exhibits a greater selectivity for this substrate and significantly higher thermal stability. Thus, a modified form of DKGR, combining desirable properties from both enzymes, would be of substantial commercial interest. In the present study we use a molecular dynamics-based approach to understand the conformational changes in DKGR A as the active site is mutated to include two active site residue changes that occur in the B form. The results indicate that the enhanced kinetic properties of the B form are due, in part, to residue substitutions in the binding pocket. These substitutions augment interactions with the substrate or alter the alignment with respect to the putative proton donor group. Proteins 2000;39:68-75.     

Reduction of wobble-position GC bases in Corynebacteria genes and enhancement of PCR and heterologous expression

Corynebacteria codon usage exhibits an overall GC content of 67%, and a wobble-position GC content of 88%. Escherichia coli, on the other hand has an overall GC content of 51%, and a wobble-position GC content of 55%. The high GC content of Corynebacteria genes results in an unfavorable codon preference for heterologous expression, and can present difficulties for polymerase-based manipulations due to secondary-structure effects. Since these characteristics are due primarily to base composition at the wobble-position, synthetic genes can, in principle, be designed to eliminate these problems and retain the wild-type amino acid sequence. Such genes would obviate the need for special additives or bases during in vitro polymerase-based manipulation and mutant host strains containing uncommon tRNA's for heterologous expression. We have evaluated synthetic genes with reduced wobble-position G/C content using two variants of the enzyme 2,5-diketo-D-gluconic acid reductase (2,5-DKGR A and B) from Corynebacterium. The wild-type genes are refractory to polymerase-based manipulations and exhibit poor heterologous expression in enteric bacteria. The results indicate that a subset of codons for five amino acids (alanine, arginine, glutamate, glycine and valine) contribute the greatest contribution to reduction in G/C content at the wobble-position. Furthermore, changes in codons for two amino acids (leucine and proline) enhance bias for expression in enteric bacteria without affecting the overall G/C content. The synthetic genes are readily amplified using polymerase-based methodologies, and exhibit high levels of heterologous expression in E. coli.     

Microbial aldo-keto reductases

The aldo-keto reductases (AKR) are a superfamily of enzymes with diverse functions in the reduction of aldehydes and ketones. AKR enzymes are found in a wide range of microorganisms, and many open reading frames encoding related putative enzymes have been identified through genome sequencing projects. Established microbial members of the superfamily include the xylose reductases, 2,5-diketo-D-gluconic acid reductases and beta-keto ester reductases. The AKR enzymes share a common (alpha/beta)(8) structure, and conserved catalytic mechanism, although there is considerable variation in the substrate-binding pocket. The physiological function of many of these enzymes is unknown, but a variety of methods including gene disruptions, heterologous expression systems and expression profiling are being employed to deduce the roles of these enzymes in cell metabolism. Several microbial AKR are already being exploited in biotransformation reactions and there is potential for other novel members of this important superfamily to be identified, studied and utilized in this way.     

Optimizing an artificial metabolic pathway: engineering the cofactor specificity of Corynebacterium 2,5-diketo-D-gluconic acid reductase for use in vitamin C biosynthesis

The strict cofactor specificity of many enzymes can potentially become a liability when these enzymes are to be employed in an artificial metabolic pathway. The preference for NADPH over NADH exhibited by the Corynebacterium 2,5-diketo-D-gluconic acid (2,5-DKG) reductase may not be ideal for use in industrial scale vitamin C biosynthesis. We have previously reported making a number of site-directed mutations at five sites located in the cofactor-binding pocket that interact with the 2'-phosphate group of NADPH. These mutations conferred greater activity with NADH upon the Corynebacterium 2,5-DKG reductase [Banta, S., Swanson, B. A., Wu, S., Jarnagin, A., and Anderson, S. (2002) Protein Eng. 15, 131-140; (1)]. The best of these mutations have now been combined to see if further improvements can be obtained. In addition, several chimeric mutants have been produced that contain the same residues as are found in other members of the aldo-keto reductase superfamily that are naturally able to use NADH as a cofactor. The most active mutants obtained in this work were also combined with a previously reported substrate-binding pocket double mutant, F22Y/A272G. Mutant activity was assayed using activity-stained native polyacrylamide gels. Superior mutants were purified and subjected to a simplified kinetic analysis. The simplified kinetic analysis was extended for the most active mutants in order to obtain the kinetic parameters in the full-ordered bi bi rate equation in the absence of products, with both NADH and NADPH as cofactors. The best mutant 2,5-DKG reductase produced in this work was the F22Y/K232G/R238H/A272G quadruple mutant, which exhibits activity with NADH that is more than 2 orders of magnitude higher than that of the wild-type enzyme, and it retains a high level of activity with NADPH. This new 2,5-DKG reductase may be a valuable new catalyst for use in vitamin C biosynthesis.     

The developmentally regulated P100/11E gene of Leishmania major shows homology to a superfamily of reductase genes

The life cycle transformation of the protozoan parasite Leishmania from promastigote to amastigote is accompanied by changes in the level of expression of a number of proteins whose function may be necessary for parasite survival in the sandfly vector or mammalian host. To genetically characterize these proteins, we have cloned and characterized cDNA sequences that vary in abundance during the life cycle of Leishmania major. One sequence (P100/11E) encodes a poly(A+) RNA whose abundance is markedly elevated in promastigotes of L. major. The DNA sequence of the P100/11E cDNA predicts an acidic polypeptide of Mr = 32,000 which shows 40-46% similarity to the superfamily of reductase proteins including 2,5-diketo-D-gluconic acid reductase, aldose reductase, aldehyde reductase, and rho-crystallin. The P100/11E sequence of L. major contains the IPKS motif located at the active site of both aldose and aldehyde reductases. The P100/11H sequence was expressed in Escherichia coli, and the purified polypeptide was used to raise rabbit antisera which detect a protein of Mr = 35,000 in promastigotes of L. major. These results provide direct genetic evidence that L. major expresses a sequence homologous to the reductase superfamily as a developmentally regulated gene product in promastigotes.     

Pantoea punctata sp. nov., Pantoea citrea sp. nov., and Pantoea terrea sp. nov. isolated from fruit and soil samples.

A total of 37 bacterial strains with the general characteristics of the family Enterobacteriaceae were isolated from fruit and soil samples in Japan as producers of 2,5-diketo-D-gluconic acid from D-glucose. These organisms were phenotypically most closely related to the genus Pantoea (F. Gavini, J. Mergaert, A. Beji, C. Mielearek, D. Izard, K. Kersters, and J. De Ley, Int. J. Syst. Bacteriol. 39:337-345, 1989) and were divided into three phenotypic groups. We selected nine representative strains from the three groups for an examination of DNA relatedness, as determined by the S1 nuclease method at 60 degrees C. Strain SHS 2003T (T = type strain) exhibited 30 to 41 and 28 to 33% DNA relatedness to the strains belonging to the strain SHS 2006T group (strains SHS 2004, SHS 2005, SHS 2006T, and SHS 2007) and to the strains belonging to the strain SHS 2008T group (strains SHS 2008T, SHS 2009, SHS 2010, and SHS 2011), respectively. Strain SHS 2006T exhibited 38 to 46% DNA relatedness to the strains belonging to the strain SHS 2008T group. The levels of DNA relatedness within the strain SHS 2006T group and within the strain SHS 2008T group were more than 85 and 71%, respectively. Strain SHS 2003T, SHS 2006T, and SHS 2008T DNAs exhibited less than 18% binding to Pantoea dispersa ATCC 14589T and Pantoea agglomerans ATCC 27155T DNAs.(ABSTRACT TRUNCATED AT 250 WORDS)     

Gentisic acid and its 3- and 4-methyl-substituted homologues as intermediates in the bacterial degradation of m-cresol, 3,5-xylenol and 2,5-xylenol

1. Intact cells of a non-fluorescent Pseudomonas grown with m-cresol, 2,5-xylenol, 3,5-xylenol, 3-ethyl-5-methylphenol or 2,3,5-trimethylphenol rapidly oxidized all these phenols to completion. 3-Hydroxybenzoate and 2,5-dihydroxybenzoate (gentisate) were also readily oxidized. 2. 3-Hydroxybenzoic acid and 2,5-dihydroxybenzoic acid were isolated as products of m-cresol oxidation by cells inhibited by alphaalpha'-bipyridyl. Alkyl-substituted 3-hydroxybenzoic acids and alkyl-substituted gentisic acids were formed similarly from 2,5-xylenol, 3,5-xylenol, 3-ethyl-5-methylphenol and 2,3,5-trimethylphenol. 3. When supplemented with NADH, not NADPH, extracts of cells grown with 2,5-xylenol catalysed the oxidation of all five phenols and accumulated the corresponding gentisic acids in the presence of alphaalpha'-bipyridyl. 4. Cells of a fluorescent Pseudomonas grown with m-cresol oxidized m-cresol, 3,5-xylenol and 3-ethyl-5-methylphenol to completion and oxidized 2,5-xylenol and 2,3,5-trimethylphenol partially. The oxidation product of 2,5-xylenol was identified as 3-hydroxy-4-methylbenzoic acid. In the presence of alphaalpha'-bipyridyl, 3-hydroxy-5-methylbenzoic acid and 3-methylgentisic acid were formed from 3,5-xylenol.     

The preparation of some bromodeoxy- and dibromodideoxy-pentonolactones

Treatment of ammonium d-xylonate with hydrogen bromide in acetic acid yields 2,5-dibromo-2,5-dideoxy-d-lyxono-1,4-lactone (2a), whereas similar treatment of potassium d-arabinonate gives 5-bromo-5-deoxy-d-arabinono-1,4-lactone (8a) as the main product. Two isomeric 2,5-dibromo-2,5-dideoxy-1,4-lactones are also formed in minor amounts. Selective hydrogenolysis of 2a affords 5-bromo-2,5-dideoxy-d-threo-pentono-1,4-lactone, while prolonged treatment results in the formation of 3-hydroxypentanoic acid. Similarly, hydrogenolysis of 8a produces a 2,3-dihydroxypentanoic acid together with smaller amounts of 5-deoxy-d-arabinono-1,4-lactone; the latter also results from hydrogenolysis of 5-deoxy-5-iodo-d-arabinono-1,4-lactone with Raney nickel.     

Oxygenated monoterpenoids from badea (Passiflora quadrangularis) fruit pulp

The new monoterpenoids (2E)-2,6-dimethyl-2,5-heptadienoic acid, (2E)-2,6-dimethyl-2,5-heptadienoic acid beta-D-glucopyranosyl ester, (5E)-2,6-dimethyl-5,7-octadiene-2,3-diol, and (3E)-3,7-dimethyl-3-octene-1,2,6,7-tetrol were isolated from the fruit pulp of Passiflora quadrangularis along with the known 2,5-dimethyl-4-hydroxy-3(2H)-furanone beta-D-glucopyranoside.     

Degradation of mono- and dichlorobenzoic acid isomers by two natural isolates of Alcaligenes denitrificans

Two strains of Alcaligenes denitrificans, designated BRI 3010 and BRI 6011, were isolated from polychlorinated biphenyl (PCB)-contaminated soil using 2,5-dichlorobenzoic acid (2,5-DCBA) and 2,4-DCBA, respectively, as sole carbon and energy sources. Both strains degraded 2-chlorobenzoic acid (2-CBA), 2,3-DCBA, and 2,5-DCBA, and were unable to degrade 2,6-DCBA. BRI 6011 alone degraded 2,4-DCBA. Growth of BRI 6011 in yeast extract and 2,6-DCBA induced pyrocatechase activity, but 2,6-DCBA was not degraded, suggesting the importance of an unsubstituted carbon six of the aromatic ring. Metabolism of the chlorinated substrates resulted in the stoichiometric release of chloride, and degradation proceeded by intradiol cleavage of the aromatic ring. Growth of both strains on 2,5-DCBA induced pyrocatechase activities with catechol and chlorocatechols as substrates. In contrast to dichlorobenzoic acids, growth on 2-CBA, benzoic acid, mono- and dihydroxybenzoic acids induced a pyrocatechase activity against catechol only. Although 2,4-DCBA was a more potent inducer of both pyrocatechase activities, its utilization by BRI 6011 was inhibited by 2,5-DCBA. Specific uptake rates using resting cells were highest with 2-CBA, except when the resting cells had been previously grown on 2,5-DCBA, in which case 2,5-DCBA was the preferred substrate. The higher rates of 2,5-DCBA uptake obtained by growth on that substrate, suggested the existence of a separately induced uptake system for 2,5-DCBA.     

Degradation of 2,5-dichlorobenzoic acid byPseudomonas aeruginosa JB2 at low oxygen tensions

From long-term chemostat experiments, variants ofPseudomonas aeruginosa JB2 were obtained which exhibited altered properties with respect to the metabolism of 2,5-dichlorobenzoic acid (2,5-DBA). Thus, unlike the original strain JB2-WT, strain JB2-var1 is able to grow in continuous culture on 2,5-DBA as the sole limiting carbon and energy source. Yet, at a dilution rate of 0.07 h^–1 and a dissolved oxygen concentration of 12 µM, even with this strain no steady states with 2,5-DBA alone could be established in continuous cultures. Yet another strain was obtained after prolonged continuous growth of JB2-var1 in the chemostat. It has improved 2,5-DBA degrading capabilities which become apparent only during growth in continuous culture: a lower apparent K _m for 2,5-DBA and lowered steady-state residual concentrations of 2,5 DBA. Although with this strain steady states were obtained at oxygen concentrations as low as 11 µM, at further lowered concentrations this was no longer possible. In C-limited continuous cultures of JB2-var1 or JB2-var2, addition of benzoic acid (BA) to the feed reduced the amounts of 2,5-DBA degraded, which was most apparent at low oxygen concentrations (< 30 µM). At higher dissolved oxygen concentrations the addition of BA resulted in increasing cell-densities but did not affect the residual steady state concentration of 2,5-DBA. Indeed, whole cell suspensions from chemostat cultures grown on BA plus 2,5-DBA did show a lower apparent affinity for 2,5-DBA than those from cultures grown on 2,5-DBA alone. These results indicate that in environments with low oxygen concentrations and alternative, more easily degradable, substrates the degradation rates of chloroaromatic compounds by aerobic organisms may be negatively affected.Abbreviations BA benzoic acid - 2,5-DBA 2,5-dichlorobenzoic acid - QO ₂ ^max maximum specific respiration rate     

Microbial metabolism of some 2,5-substituted thiophenes

2,5-Dialkylthiophenes are found in bitumens and crude oils, and previous studies showed that bacterial metabolism of some with a methyl substituent lead to the formation of 5-methyl-2-thiophenecarboxylic acid, which persisted in the culture medium (Fedorak PM & Peakman TM 1992 Biodegradation 2: 223–236). The objectives of this investigation were to study the further metabolism of this acid, and of two dialkylthiophenes, 2,5-diundecylthiophene and 2-(3,7-dimethyloctyl)-5-methylthiophene. Undefined, oil-degrading mixed cultures were used. 5-Methyl-2-thiophenecarboxylic acid was oxidized to 2,5-thiophenedicarboxylic acid which was identified by gas chromatography-mass spectrometry (GC-MS). This dicarboxylic acid was degraded and supported the growth of a mixed microbial population, and approximately 50% of the sulfur in this substrate was detected as sulfate in the medium at the end of the 15-day incubation time. Mixed cultures were incubated with 2,5-diundecylthiophene or 2-(3,7-dimethyloctyl)-5-methylthiophene as their sole carbon source, and at various times some of these were freezedried and the residues were treated to form methyl esters of any carboxylic acids produced. GC-MS analyses showed the presence of several dicarboxylic acids, indicating that both alkyl groups were oxidized. A small amount of the dimethyl ester of 2,5-thiophenedicarboxylic acid was detected in the culture grown on 2,5-diundecylthiophene, and 37% of the sulfur from this dialkylthiophene was detected as sulfate in the medium after 35 days of incubation.