Aquincola Tertiaricarbonis L108 Research Articles

Actinobacterial Degradation of 2-Hydroxyisobutyric Acid Proceeds via Acetone and Formyl-CoA by Employing a Thiamine-Dependent Lyase Reaction.

The tertiary branched short-chain 2-hydroxyisobutyric acid (2-HIBA) has been associated with several metabolic diseases and lysine 2-hydroxyisobutyrylation seems to be a common eukaryotic as well as prokaryotic post-translational modification in proteins. In contrast, the underlying 2-HIBA metabolism has thus far only been detected in a few microorganisms, such as the betaproteobacterium Aquincola tertiaricarbonis L108 and the Bacillus group bacterium Kyrpidia tusciae DSM 2912. In these strains, 2-HIBA can be specifically activated to the corresponding CoA thioester by the 2-HIBA-CoA ligase (HCL) and is then isomerized to 3-hydroxybutyryl-CoA in a reversible and B12-dependent mutase reaction. Here, we demonstrate that the actinobacterial strain Actinomycetospora chiangmaiensis DSM 45062 degrades 2-HIBA and also its precursor 2-methylpropane-1,2-diol via acetone and formic acid by employing a thiamine pyrophosphate-dependent lyase. The corresponding gene is located directly upstream of hcl, which has previously been found only in operonic association with the 2-hydroxyisobutyryl-CoA mutase genes in other bacteria. Heterologous expression of the lyase gene from DSM 45062 in E. coli established a 2-hydroxyisobutyryl-CoA lyase activity in the latter. In line with this, analysis of the DSM 45062 proteome reveals a strong induction of the lyase-HCL gene cluster on 2-HIBA. Acetone is likely degraded via hydroxylation to acetol catalyzed by a MimABCD-related binuclear iron monooxygenase and formic acid appears to be oxidized to CO2 by selenium-dependent dehydrogenases. The presence of the lyase-HCL gene cluster in isoprene-degrading Rhodococcus strains and Pseudonocardia associated with tropical leafcutter ant species points to a role in degradation of biogenic short-chain ketones and highly branched organic compounds.

Structures of 2-Hydroxyisobutyric Acid-CoA Ligase Reveal Determinants of Substrate Specificity and Describe a Multi-Conformational Catalytic Cycle

2-Hydroxyisobutyric acid (2-HIBA) is a biomarker of adiposity and associated metabolic diseases such as diabetes mellitus. It is also formed in the bacterial degradation pathway of the fuel oxygenate methyl tert-butyl ether (MTBE), requiring thioesterification with CoA prior to isomerization to 3-hydroxybutyryl-CoA by B12-dependent acyl-CoA mutases. Here, we identify the adenylating enzymes superfamily member 2-HIBA-CoA ligase (HCL) in the MTBE-degrading bacterium Aquincola tertiaricarbonis L108 by knockout experiments. To characterize this central enzyme of 2-HIBA metabolism, ligase activity kinetics of purified HCL and its X-ray crystal structures were studied. We analyzed the enzyme in three states, which differ in the orientation of the two enzyme domains. A 154° rotation of the C-terminal domain accompanies the switch from the adenylate- into the thioester-forming state. Furthermore, a third conformation was obtained, which differs by 50° and 130° from the adenylation and thioesterification states, respectively. Phylogenetic and structural analysis reveals that HCL defines a new subgroup within phenylacetate-CoA ligases (PCLs) thus far described to exclusively accept aromatic acyl substrates. In contrast, kinetic characterization clearly demonstrated that HCL catalyzes CoA activation of several aliphatic short-chain carboxylic acids, preferentially 2-HIBA. Compared to the classical PCL representatives PaaK1 and PaaK2 of Burkholderia cenocepacia J2315, the acyl binding pocket of HCL is significantly smaller and more polar, due to unique active-site residues Y164 and S239 forming H-bonds with the OH-group of the acyl substrate moiety. Furthermore, HCL and PaaK topologies illustrate the evolutionary steps leading from a homodimeric to the fused monomeric core fold found in other ligases.

Exploiting mixtures of H2, CO2, and O2 for improved production of methacrylate precursor 2-hydroxyisobutyric acid by engineered Cupriavidus necator strains.

Current manufacturing of most bulk chemicals through petrochemical routes considerably contributes to common concerns over the depletion of fossil carbon sources and greenhouse gas emissions. Sustainable future production of commodities thus requires the shift to renewable feedstocks in combination with established or newly developed synthesis routes. In this study, the potential of Cupriavidus necator H16 for autotrophic synthesis of the building block chemical 2-hydroxyisobutyric acid (2-HIBA) is evaluated. A novel biosynthetic pathway was implemented by heterologous expression of the 2-hydroxyisobutyryl-coenzyme A (2-HIB-CoA) mutase from Aquincola tertiaricarbonis L108, relying on a main intermediate of strain H16's C4 overflow metabolism, 3-hydroxybutyryl-CoA. The intention was to direct the latter to 2-HIBA instead or in addition to poly-3-hydroxybutyrate (PHB). Autotrophic growth and 2-HIBA (respectively, PHB) synthesis of wild-type and PHB-negative mutant strains were investigated producing maximum 2-HIBA titers of 3.2gL(-1) and maximum specific 2-HIBA synthesis rates (q 2-HIBA) of about 16 and 175μmolg(-1)h(-1), respectively. The obtained specific productivity was the highest reported to date for mutase-dependent 2-HIBA synthesis from heterotrophic and autotrophic substrates. Furthermore, expression of a G protein chaperone (MeaH) in addition to the 2-HIB-CoA mutase subunits yielded improved productivity. Analyzing the inhibition of growth and product synthesis due to substrate availability and product accumulation revealed a strong influence of 2-HIBA, when cells were cultivated at high titers. Nevertheless, the presented results imply that at the time the autotrophic synthesis route is superior to thus far established heterotrophic routes for production of 2-HIBA with C. necator.

Synthesis of the building block 2-hydroxyisobutyrate from fructose and butyrate by Cupriavidus necator H16

2-Hydroxyisobutyryl-coenzyme A mutase, originally discovered in the context of methyl tert-butyl ether degradation in Aquincola tertiaricarbonis L108, catalyzes the isomerization of 3-hydroxybutyryl-coenzyme A (3-HB-CoA) to 2-hydroxyisobutyryl-CoA. It thus constitutes the basis for a biotechnological route from practically any renewable carbon to 2-hydroxyisobutyrate (2-HIB) via the common metabolite 3-hydroxybutyrate. At first sight, recombinant Cupriavidus necator H16 expressing the mutase seems to be well suited for such a synthesis process, as a strong overflow metabolism via (R)-3-HB-CoA is easily induced in this bacterium possessing the poly-3-hydroxybutyrate metabolism. However, the recently established stereospecificity of the mutase, dominantly preferring the (S)-enantiomer of 3-HB-CoA, calls for a closer investigation of C. necator as potential 2-HIB production strain and raised the question about the strain's potential to yield 2-HIB from substrates directly providing (S)-3-HB-CoA. We compared two mutase-expressing C. necator H16 strains for their capability to synthesize 2-HIB from fructose and butyrate, delivering either (R)- or (S)-3-HB-CoA. Our results indicate that due to the enantiospecificity of the mutase, fructose is a weaker substrate for 2-HIB synthesis than butyrate. Production rates achieved with the PHB-negative strain H16 PHB(-)4 on butyrate were higher than on fructose. Using the wild-type did not significantly improve the production rates as the latter showed a 34-fold and a 5-fold lower 2-HIB synthesis rate compared to H16 PHB(-)4 on fructose and butyrate, respectively. Moreover, both strains showed concomitant excretion of undesired side products, such as pyruvate and 3-hydroxybutyrate, significantly decreasing the 2-HIB yield.

Cultivation of Aquincola tertiaricarbonis L108 on the fuel oxygenate intermediate tert-butyl alcohol induces aerobic anoxygenic photosynthesis at extremely low feeding rates

Aerobic anoxygenic photosynthesis (AAP) is found in an increasing number of proteobacterial strains thriving in ecosystems ranging from extremely oligotrophic to eutrophic. Here, we have investigated whether the fuel oxygenate-degrading betaproteobacterium Aquincola tertiaricarbonis L108 can use AAP to compensate kinetic limitations at low heterotrophic substrate fluxes. In a fermenter experiment with complete biomass retention and also during chemostat cultivation, strain L108 was challenged with extremely low substrate feeding rates of tert-butyl alcohol (TBA), an intermediate of methyl tert-butyl ether (MTBE). Interestingly, formation of photosynthetic pigments, identified as bacteriochlorophyll a and spirilloxanthin, was only induced in growing cells at TBA feeding rates less than or equal to maintenance requirements observed under energy excess conditions. Growth continued at rates between 0.001 and 0.002 h(-1) even when the TBA feed was decreased to values close to 30 % of this maintenance rate. Partial sequencing of genomic DNA of strain L108 revealed a bacteriochlorophyll synthesis gene cluster (bchFNBHL) and photosynthesis regulator genes (ppsR and ppaA) typically found in AAP and other photosynthetic proteobacteria. The usage of light as auxiliary energy source enabling evolution of efficient degradation pathways for kinetically limited heterotrophic substrates and for lowering the threshold substrate concentration Smin at which growth becomes zero is discussed.

Constitutive Expression of the Cytochrome P450 EthABCD Monooxygenase System Enables Degradation of Synthetic Dialkyl Ethers in Aquincola tertiaricarbonis L108

In Rhodococcus ruber IFP 2001, Rhodococcus zopfii IFP 2005, and Gordonia sp. strain IFP 2009, the cytochrome P450 monooxygenase EthABCD catalyzes hydroxylation of methoxy and ethoxy residues in the fuel oxygenates methyl tert-butyl ether (MTBE), ethyl tert-butyl ether (ETBE), and tert-amyl methyl ether (TAME). The expression of the IS3-type transposase-flanked eth genes is ETBE dependent and controlled by the regulator EthR (C. Malandain et al., FEMS Microbiol. Ecol. 72:289-296, 2010). In contrast, we demonstrated by reverse transcription-quantitative PCR (RT-qPCR) that the betaproteobacterium Aquincola tertiaricarbonis L108, which possesses the ethABCD genes but lacks ethR, constitutively expresses the P450 system at high levels even when growing on nonether substrates, such as glucose. The mutant strain A. tertiaricarbonis L10, which is unable to degrade dialkyl ethers, resulted from a transposition event mediated by a rolling-circle IS91-type element flanking the eth gene cluster in the wild-type strain L108. The constitutive expression of Eth monooxygenase is likely initiated by the housekeeping sigma factor σ(70), as indicated by the presence in strain L108 of characteristic -10 and -35 binding sites upstream of ethA which are lacking in strain IFP 2001. This enables efficient degradation of diethyl ether, diisopropyl ether, MTBE, ETBE, TAME, and tert-amyl ethyl ether (TAEE) without any lag phase in strain L108. However, ethers with larger residues, n-hexyl methyl ether, tetrahydrofuran, and alkyl aryl ethers, were not attacked by the Eth system at significant rates in resting-cell experiments, indicating that the residue in the ether molecule which is not hydroxylated also contributes to the determination of substrate specificity.

Third-generation feed stocks for the clean and sustainable biotechnological production of bulk chemicals: synthesis of 2-hydroxyisobutyric acid

Abstract Background The synthesis of 2-hydroxyisobutyric acid (2-HIB), a promising building block for, e.g., Plexiglas® production, is described as an example for a clean and sustainable bioproduction. Methods A derivative strain of Cupriavidus necator H16, impaired in the poly-ß-hydroxybutyrate synthesis pathway and equipped with xenogenic 2-hydroxyisobutyryl-coenzyme A mutase from Aquincola tertiaricarbonis L108, was applied. Batch cultivation was performed in the presence of vitamin B12 by supplying a gas mixture comprising hydrogen, oxygen, and carbon dioxide. Results Exploiting the chemo-litho-autotrophic potential of this so-called knallgas bacterium, 2-HIB was synthesized and excreted into the cultivation broth under aerobic conditions when inorganic nitrogen-limited conditions allowed an overflow metabolism of carbon metabolites. 2-HIB synthesis proceeded at a rate of 8.58 mg/[(g bacterial dry mass)·h]. Approximately 400 mg/L in total was obtained. The results were subsequently compared to calculated model data to evaluate the efficiency of the conversion of the substrates into the product. To achieve overall yield data regarding the substrate conversion, the model describes an integral process which includes both 2-HIB synthesis and biomass formation. Conclusions This study has confirmed the feasibility of the microbial synthesis of the bulk chemical 2-HIB from hydrogen and carbon dioxide by exploiting the chemo-litho-autotrophic metabolism of C. necator H16 PHB−4, additionally expressing the foreign 2-HIB-coenzyme A mutase. The product synthesis was satisfying as a proof of principle but does not yet approach the maximum value as derived from the model data. Furthermore, the biosynthesis potential of an optimized process is discussed in view of its technical application.

Bacterial Degradation of tert-Amyl Alcohol Proceeds via Hemiterpene 2-Methyl-3-Buten-2-ol by Employing the Tertiary Alcohol Desaturase Function of the Rieske Nonheme Mononuclear Iron Oxygenase MdpJ

Tertiary alcohols, such as tert-butyl alcohol (TBA) and tert-amyl alcohol (TAA) and higher homologues, are only slowly degraded microbially. The conversion of TBA seems to proceed via hydroxylation to 2-methylpropan-1,2-diol, which is further oxidized to 2-hydroxyisobutyric acid. By analogy, a branched pathway is expected for the degradation of TAA, as this molecule possesses several potential hydroxylation sites. In Aquincola tertiaricarbonis L108 and Methylibium petroleiphilum PM1, a likely candidate catalyst for hydroxylations is the putative tertiary alcohol monooxygenase MdpJ. However, by comparing metabolite accumulations in wild-type strains of L108 and PM1 and in two mdpJ knockout mutants of strain L108, we could clearly show that MdpJ is not hydroxylating TAA to diols but functions as a desaturase, resulting in the formation of the hemiterpene 2-methyl-3-buten-2-ol. The latter is further processed via the hemiterpenes prenol, prenal, and 3-methylcrotonic acid. Likewise, 3-methyl-3-pentanol is degraded via 3-methyl-1-penten-3-ol. Wild-type strain L108 and mdpJ knockout mutants formed isoamylene and isoprene from TAA and 2-methyl-3-buten-2-ol, respectively. It is likely that this dehydratase activity is catalyzed by a not-yet-characterized enzyme postulated for the isomerization of 2-methyl-3-buten-2-ol and prenol. The vitamin requirements of strain L108 growing on TAA and the occurrence of 3-methylcrotonic acid as a metabolite indicate that TAA and hemiterpene degradation are linked with the catabolic route of the amino acid leucine, including an involvement of the biotin-dependent 3-methylcrotonyl coenzyme A (3-methylcrotonyl-CoA) carboxylase LiuBD. Evolutionary aspects of favored desaturase versus hydroxylation pathways for TAA conversion and the possible role of MdpJ in the degradation of higher tertiary alcohols are discussed.

Formation of Alkenes via Degradation of tert -Alkyl Ethers and Alcohols by Aquincola tertiaricarbonis L108 and Methylibium spp

Bacterial degradation pathways of fuel oxygenates such as methyl tert-butyl and tert-amyl methyl ether (MTBE and TAME, respectively) have already been studied in some detail. However, many of the involved enzymes are still unknown, and possible side reactions have not yet been considered. In Aquincola tertiaricarbonis L108, Methylibium petroleiphilum PM1, and Methylibium sp. strain R8, we have now detected volatile hydrocarbons as by-products of the degradation of the tert-alkyl ether metabolites tert-butyl and tert-amyl alcohol (TBA and TAA, respectively). The alkene isobutene was formed only during TBA catabolism, while the beta and gamma isomers of isoamylene were produced only during TAA conversion. Both tert-alkyl alcohol degradation and alkene production were strictly oxygen dependent. However, the relative contribution of the dehydration reaction to total alcohol conversion increased with decreasing oxygen concentrations. In resting-cell experiments where the headspace oxygen content was adjusted to less than 2%, more than 50% of the TAA was converted to isoamylene. Isobutene formation from TBA was about 20-fold lower, reaching up to 4% alcohol turnover at low oxygen concentrations. It is likely that the putative tert-alkyl alcohol monooxygenase MdpJ, belonging to the Rieske nonheme mononuclear iron enzymes and found in all three strains tested, or an associated enzymatic step catalyzed the unusual elimination reaction. This was also supported by the detection of mdpJK genes in MTBE-degrading and isobutene-emitting enrichment cultures obtained from two treatment ponds operating at Leuna, Germany. The possible use of alkene formation as an easy-to-measure indicator of aerobic fuel oxygenate biodegradation in contaminated aquifers is discussed.

Synthesis of Poly-3-Hydroxybutyrate Reduces Maintenance Demand In Bacteria Growing Slowly on Methyl Tert-Butyl Ether

Availability of usable energy for biosynthesis and maintenance is a primary condition of life. In the case of microbial degradation of so-called recalcitrant compounds, growth is often prevented due to low energy production rates not exceeding maintenance requirements. However, any process that reduces biomass maintenance under these threshold conditions will increase the chance that productive degradation can occur. We have developed a growth model to explain the surprising observation that the growth of Aquincola tertiaricarbonis L108 and other bacterial strains on the groundwater pollutant methyl tert-butyl ether (MTBE) and its degradation product tert-butyl alcohol is accompanied by the accumulation of poly-3-hydroxybutyrate (PHB). The growth-coupled production of this storage polymer is remarkable since slow growth of all MTBE degraders indicates the difficulty to utilize this substrate. The modified model distinguished an active biomass fraction for which maintenance energy is required and a passive PHB fraction without maintenance requirements. Consequently, the presence of a PHB fraction saved specific maintenance costs and due to PHB accumulation, calculated overall growth yields were increased. More important, the structured model predicted an increased specific growth rate and a decreased minimum substrate concentration for growth Smin. Using experimentally determined parameters, it could be demonstrated that at low maximal growth rates PHB synthesis allowed for growth, whereas in its absence Smin became infinite. Additional reduction of maintenance in dependence on the rate of PHB formation was considered as the stoichiometry of PHB synthesis from MTBE resulted in a gain of energy equivalents. Our model thus showed that coupling of growth on slowly metabolized substrates with PHB synthesis is energetically advantageous. Hence, we propose a not yet considered role of PHB in delivering energy to compensate high maintenance costs while growing on recalcitrant xenobiotics.

Characterization and expression of the genes encoding the 2-hydroxyisobutyryl-coa mutase, a key enzyme in methyl Tert-Butylether degradation by Aquincola tertiaricarbonis L108

Characterization and expression of the genes encoding the 2-hydroxyisobutyryl-coa mutase, a key enzyme in methyl Tert-Butylether degradation by Aquincola tertiaricarbonis L108

Biodegradation of fuel oxygenates by sol–gel immobilized bacteria Aquincola tertiaricarbonis L108

The development of long-term storable biofilters containing bacteria which are capable to degrade recalcitrant environmental compounds like the fuel oxygenates methyl tert-butyl ether (MTBE) and ethyl tert-butyl ether (ETBE) is of special interest for the treatment of contaminated water. Fuel oxygenates have been proven to be more persistent to biodegradation than other gasoline components. Only a few microorganisms, e.g. Aquincola tertiaricarbonis L108, are capable to biodegrade these substances.In order to develop highly efficient and long-term stable biocomposite materials for bioremediation, the applicability of sol–gel matrices for the immobilization of A. tertiaricarbonis was tested. Two different sol–gel immobilization techniques were used: (A) the immobilization within ceramic-like shapes by using the freeze-gelation technique and (B) sol–gel coatings on an inert, porous material. For comparison, the widely used alginate-bead immobilization technique has been applied.The embedding of A. tertiaricarbonis within freeze-gelation composites (method A) proved to be not applicable because this strain was too sensitive regarding the applied freezing and drying conditions even though cell-protecting additives were added. In contrast, A. tertiaricarbonis immobilized on porous expanded clay pellets (method B) could be stored under humid atmosphere at least for a time of 8 months without a significant reduction of their metabolic activity.

Degradation of fuel oxygenates and their main intermediates by Aquincola tertiaricarbonis L108

Growth of Aquincola tertiaricarbonis L108 on the fuel oxygenates methyl tert-butyl ether (MTBE), ethyl tert-butyl ether (ETBE) and tert-amyl methyl ether (TAME), as well as on their main metabolites tert-butyl alcohol (TBA), tert-amyl alcohol (TAA) and 2-hydroxyisobutyrate (2-HIBA) was systematically investigated to characterize the range and rates of oxygenate degradation by this strain. The effective maximum growth rates for MTBE, ETBE and TAME at pH 7 and 30 degrees C were 0.045 h(-1), 0.06 h(-1) and 0.055 h(-1), respectively, whereas TAA, TBA and 2-HIBA permitted growth at rates up to 0.08 h(-1), 0.1 h(-1) and 0.17 h(-1), respectively. The experimental growth yields with all these substrates were high. Yields of 0.55 g dry mass (dm) (g MTBE)(-1), 0.53 g dm (g ETBE)(-1), 0.81 g dm (g TAME)(-1), 0.48 g dm (g TBA)(-1), 0.76 g dm (g TAA)(-1) and 0.54 g dm (g 2-HIBA)(-1) were obtained. Maximum specific degradation rates were 0.92 mmol MTBE h(-1) (g dm)(-1), 1.11 mmol ETBE h(-1) g(-1), 0.66 mmol TAME h(-1) g(-1), 1.19 mmol TAA h(-1) g(-1), 2.82 mmol TBA h(-1) g(-1), and 3.27 mmol 2-HIBA h(-1) g(-1). The relatively high rates with TBA, TAA and 2-HIBA indicate that the transformations of these metabolites did not limit the metabolism of MTBE and the related ether compounds. Despite the fact that these metabolites still carry a tertiary carbon atom that is commonly suspected to confer recalcitrance to the ether oxygenates, the transformation rates were in the same range as those with succinate and fructose. With MTBE, strain L108 grew at pHs between 5.5 and 8.0 at near-maximal rate, whereas no growth was found below pH 5.0 and above pH 9.0. The optimum growth temperature was 30 degrees C, but at 5 degrees C still about 15 % of the maximum rate remained, whereas no growth occurred at 42 degrees C. This indicates that MTBE metabolites are valuable substrates and that A. tertiaricarbonis L108 is a good candidate for bioremediation purposes. The possible origin of its exceptional metabolic capability is discussed in terms of the evolution of enzymic activities involved in the conversion of compounds carrying tertiary butyl groups.

Growth of Aquincola tertiaricarbonis L108 on tert‐Butyl Alcohol Leads to the Induction of a Phthalate Dioxygenase‐related Protein and its Associated Oxidoreductase Subunit

AbstractThe microbial degradation of tert‐butyl alcohol (TBA), an important environmental pollutant and an intermediate in the degradation of methyl tert‐butyl ether (MTBE), was proposed to involve a monooxygenase for the initial oxidation of TBA, but up to now a specific enzyme with that activity has not been described except the well‐known AlkB for the Gram‐positive strain Mycobacterium austroafricanum IFP2012 (Lopez Ferreira et al., Appl. Microbiol. Biotechnol. 2007, 75, 909–919). In the course of our studies of the MTBE pathway, the proteome patterns in one‐ and two‐dimensional gels of Aquincola tertiaricarbonis L108 which was grown on lactate, on hydroxyisobutyrate (2‐HIBA) and TBA, were compared. A protein of about 55 kDa was detected after growth on TBA and 2‐HIBA, which, after mass spectrometric analysis of the tryptic digested peptides, was assigned with a high score to phthalate dioxygenase. Sequence analysis of PCR products obtained with primers derived from the amino acid sequences in the above peptides supported the assignment to the hydroxylase subunit of phthalate dioxygenase‐like proteins by covering 96.7 % of a corresponding gene from Methylibium petroleiphilum PM1. The conserved amino acid motifs ‐R‐x12‐CxHRxxxLxxG‐x8‐CxYHR‐x6‐G‐ for the Rieske [2Fe‐2S] binding domain and (‐D/E)xxxDxxHxxxxH‐ for the mononuclear iron binding domain were found. A second protein of about 38 kDa was detected after growth on TBA with a lower score and attributed to a putative iron‐sulfur oxidoreductase subunit. Primers derived from the peptides resulted in an amplicon, which covered 75.7 % of a corresponding gene from M. petroleiphilum PM1. Conserved motifs ‐RxYSL‐x20‐22‐RGGS‐ for FMN binding and ‐GGIGxTPxxxM‐ for NAD binding were detected, which suggests that this protein is the small subunit of a two‐component phthalate dioxygenase‐like enzyme typically containing FMN. Dioxygenase‐related enzymes are known to catalyze also monooxygenase reactions (see e.g. Zhou et al. J. Bacteriol. 2002, 184, 1547–1555), which makes it likely that the two proteins induced in the presence of TBA are involved in TBA oxidation.