Riboflavin synthesis from gaseous nitrogen and carbon dioxide by a hybrid inorganic-biological system.

Microbes can provide a more sustainable and energy-efficient method of food and nutrient production compared to plant and animal sources, but energy-intensive carbon (e.g., sugars) and nitrogen (e.g., ammonia) inputs are required. Gas-fixing microorganisms that can grow on H2 from renewable water splitting and gaseous CO2 and N2 offer a renewable path to overcoming these limitations but confront challenges owing to the scarcity of genetic engineering in such organisms. Here, we demonstrate that the hydrogen-oxidizing carbon- and nitrogen-fixing microorganism Xanthobacter autotrophicus grown on a CO2/N2/H2 gas mixture can overproduce the vitamin riboflavin (vitamin B2). We identify plasmids and promoters for use in this bacterium and employ a constitutive promoter to overexpress riboflavin pathway enzymes. Riboflavin production is quantified at 15 times that of the wild-type organism. We demonstrate that riboflavin overproduction is maintained when the bacterium is grown under hybrid inorganic-biological conditions, in which H2 from water splitting, along with CO2 and N2, is fed to the bacterium, establishing the viability of the approach to sustainably produce food and nutrients.

Performance of an air membrane bioreactor for methanol removal under steady and transient state conditions.

The main aim of this study was to evaluate the performance of an air membrane bioreactor (aMBR) for the treatment of gas-phase methanol. A laboratory-scale hollow fiber aMBR was operated for 150 days, at inlet methanol concentrations varying between 2 and 30 g m-3 and at empty bed residence times (EBRT) of 30, 10 and 5 s. Under steady-state conditions, a maximum methanol removal efficiency (RE) of 98% was obtained at an EBRT of 30 s and a decrease in RE of methanol was observed at lower EBRTs. On increasing the inlet loading rate, some portion of gas-phase MeOH was stripped into the liquid phase due to its solubility in water. Under transient conditions, the MeOH removal efficiency dropped from an average value of 95%-90% after 5 h of 10-fold shock load and dropped from an average value of 95%-88% under 5-fold increase in shock load. During transient-state tests, the aMBR performed well under different upset loading conditions and a drop in RE of ∼ 5-10% was observed. However, the aMBR performance was restored within 1-2 days when pre-shock conditions were restored. The results from microbial structure analysis revealed a big shift of the dominant methanol degrader, from Candida boidinii strain TBRC 217 to Xanthobacter sp. and Fusicolla sp., respectively.

Xanthobacter-dominated biofilm as a novel source for high-value rhamnose.

Rhamnose is a high-value carbohydrate used in flavorings, aromatics, and pharmaceuticals. Current demand for rhamnose is filled through plant-based sources; however, microbially originated rhamnolipids have been proposed as an alternative source. A mixed microbial biofilm, cultured from a wastewater sludge, was found to comprise > 8 dry weight% rhamnose when provided volatile fatty acids as carbon source, and 24 dry weight% when given glucose. The latter rhamnose concentration is a fourfold higher production mass than the current plant-based origin and is competitive with yields from pure microbial cultures. The biofilm was characterized based on total carbohydrate production at varying nutrient levels, individual carbohydrate monomer production from varying organic acid substrates, and microbial community composition-based on 16s rRNA. Biofilm carbohydrate production was maximized at a C:N ratio of 28 (mol:mol). The production of rhamnose varied significantly based on carbon substrate; glucose had the greatest yield of rhamnose, followed by propionic acid, lactic acid, acetic acid, valeric acid, and butyric acid. Microbial community analysis indicated an abundance of organisms within the Xanthobacter genus, which is known to produce rhamnose as zeaxanthin rhamnoside. Rhamnose production was heavily correlated with ribose production (R2 = 0.96). Results suggest that mixed microbial biofilms could be a competitive source of monomeric rhamnose that may be produced from mixed organic waste streams of variable composition via volatile fatty acids and glucose.

Biodiversity of aerobic methylobacteria associated with the phyllosphere of the southern Moscow Oblast.

During the summer period (15­25°C), 34 strains of methylotrophic bacteria associated with different species of herbs, shrub, and trees in Pushchino (Moscow oblast, Russia) were isolated on the medium with methanol. Predominance of pink-colored Methylobacterium strains in the phyllosphere of many plants was confirmed by microscopy, enumeration of the colonies from grass leaves, and sequencing of the 16S rRNA genes. Colorless and yellow-pigmented methylotrophs belonged to the genera Methylophilus, Methylobacillus, Hansschlegelia, Methylopila, Xanthobacter, and Paracoccus. All isolates were able to synthesize plant hormones auxins from L-tryptophan (5−50 µg/mL) and are probably plant symbionts.

C and Cl isotope fractionation of 1,2-dichloroethane displays unique δ¹³C/δ³7Cl patterns for pathway identification and reveals surprising C-Cl bond involvement in microbial oxidation.

This study investigates dual element isotope fractionation during aerobic biodegradation of 1,2-dichloroethane (1,2-DCA) via oxidative cleavage of a C-H bond (Pseudomonas sp. strain DCA1) versus C-Cl bond cleavage by S(N)2 reaction (Xanthobacter autotrophicus GJ10 and Ancylobacter aquaticus AD20). Compound-specific chlorine isotope analysis of 1,2-DCA was performed for the first time, and isotope fractionation (ε(bulk)(Cl)) was determined by measurements of the same samples in three different laboratories using two gas chromatography-isotope ratio mass spectrometry systems and one gas chromatography-quadrupole mass spectrometry system. Strongly pathway-dependent slopes (Δδ13C/Δδ37Cl), 0.78 ± 0.03 (oxidation) and 7.7 ± 0.2 (S(N)2), delineate the potential of the dual isotope approach to identify 1,2-DCA degradation pathways in the field. In contrast to different ε(bulk)(C) values [-3.5 ± 0.1‰ (oxidation) and -31.9 ± 0.7 and -32.0 ± 0.9‰ (S(N)2)], the obtained ε(bulk)(Cl) values were surprisingly similar for the two pathways: -3.8 ± 0.2‰ (oxidation) and -4.2 ± 0.1 and -4.4 ± 0.2‰ (S(N)2). Apparent kinetic isotope effects (AKIEs) of 1.0070 ± 0.0002 (13C-AKIE, oxidation), 1.068 ± 0.001 (13C-AKIE, S(N)2), and 1.0087 ± 0.0002 (37Cl-AKIE, S(N)2) fell within expected ranges. In contrast, an unexpectedly large secondary 37Cl-AKIE of 1.0038 ± 0.0002 reveals a hitherto unrecognized involvement of C-Cl bonds in microbial C-H bond oxidation. Our two-dimensional isotope fractionation patterns allow for the first time reliable 1,2-DCA degradation pathway identification in the field, which unlocks the full potential of isotope applications for this important groundwater contaminant.

Shotgun proteomics of Xanthobacter autotrophicus Py2 reveals proteins specific to growth on propylene.

Coenzyme M (CoM, 2-mercaptoethanesulfonate), once thought to be exclusively produced by methanogens, is now known to be the central cofactor in the metabolism of short-chain alkenes by a variety of aerobic bacteria. There is little evidence to suggest how, and under what conditions, CoM is biosynthesized by these organisms. A shotgun proteomics approach was used to investigate CoM-dependent propylene metabolism in the Gram-negative bacterium Xanthobacter autotrophicus Py2. Cells were grown on either glucose or propylene, and the soluble proteomes were analyzed. An average of 395 proteins was identified from glucose-grown replicates, with an average of 419 identified from propylene-grown replicates. A number of linear megaplasmid (pXAUT01)-encoded proteins were found to be specifically produced by growth on propylene. These included all known to be crucial to propylene metabolism, in addition to an aldehyde dehydrogenase, a DNA-binding protein, and five putative CoM biosynthetic enzymes. This work has provided fresh insight into bacterial alkene metabolism and has generated new targets for future studies in X. autotrophicus Py2 and related CoM-dependent alkene-oxidizing bacteria.

Effect of molecule size on carbon isotope fractionation during biodegradation of chlorinated alkanes by Xanthobacter autotrophicus GJ10.

The effect of the number of carbon and chlorine atoms on carbon isotope fractionation during dechlorination of chlorinated alkanes by Xanthobacter autotrophicus GJ10 was studied using pure culture and cell-free extract experiments. The magnitude of carbon isotope fractionation decreased with increasing carbon number. The decrease can be explained by an increasing probability that the heavy isotope is located at a non-reacting position for increasing molecule size. The isotope data were corrected for the number of carbons as well as the number of reactive sites to obtain reacting-site-specific values denoted as apparent kinetic isotope effect (AKIE). Even after the correction, the obtained AKIE values varied (on average 1.0608, 1.0477, 1.0616, and 1.0555 for 1,2-dichloroethane, chloropentane, 1,3-dichloropentane and chlorobutane, respectively). Cell-free extract experiments were carried out to evaluate the effect of transport across the cell membrane on the observed variability in the AKIE values, which revealed that variability still persisted. The study demonstrates that even after differences related to the carbon number and structure of the molecule are taken into account, there still remain differences in AKIE values even for compounds that are degraded by the same pure culture and an identical reaction mechanism.

Isolation of a Xanthobacter sp. degrading dichloromethane and characterization of the gene involved in the degradation.

A bacterial strain able to degrade dichloromethane (DCM) as the sole carbon source was isolated from a wastewater treatment plant receiving domestic and pharmaceutical effluent. 16S rDNA studies revealed the strain to be a Xanthobacter sp. (strain TM1). The new isolated strain when grown aerobically on DCM showed Luong type growth kinetics, with 1(max) of 0.094 h(-1) and S (m) of 1,435 mg l(-1). Strain TM1 was able to degrade other aromatic and aliphatic halogenated compounds, such as halobenzoates, 2-chloroethanol and dichloroethane. The gene for DCM dehalogenase, which is the key enzyme in DCM degradation, was amplified through PCR reactions. Strain TM1 contains type A DCM dehalogenase (dcmAa), while no product could be obtained for type B dehalogense (dcmAb). The sequence was compared against 12 dcmAa from other DCM degrading strains and 98% or 99% similarity was observed with all other previously isolated DCM dehalogenase genes. This is the first time a Xanthobacter sp. is reported to degrade DCM.

Getting a handle on the role of coenzyme M in alkene metabolism.

Coenzyme M (2-mercaptoethanesulfonate; CoM) is one of several atypical cofactors discovered in methanogenic archaea which participate in the biological reduction of CO(2) to methane. Elegantly simple, CoM, so named for its role as a methyl carrier in all methanogenic archaea, is the smallest known organic cofactor. It was thought that this cofactor was used exclusively in methanogenesis until it was recently discovered that CoM is a key cofactor in the pathway of propylene metabolism in the gram-negative soil microorganism Xanthobacter autotrophicus Py2. A four-step pathway requiring CoM converts propylene and CO(2) to acetoacetate, which feeds into central metabolism. In this process, CoM is used to activate and convert highly electrophilic epoxypropane, formed from propylene epoxidation, into a nucleophilic species that undergoes carboxylation. The unique properties of CoM provide a chemical handle for orienting compounds for site-specific redox chemistry and stereospecific catalysis. The three-dimensional structures of several of the enzymes in the pathway of propylene metabolism in defined states have been determined, providing significant insights into both the enzyme mechanisms and the role of CoM in this pathway. These studies provide the structural basis for understanding the efficacy of CoM as a handle to direct organic substrate transformations at the active sites of enzymes.

Metabolism of mono- and dihalogenated C1 and C2 compounds by Xanthobacter autotrophicus growing on 1,2-dichloroethane.

The conversion of and toxic effects exerted by several mono- and dihalogenated C1 and C2 compounds on cultures of Xanthobacter autotrophicus GJ10 growing on 1,2-dichloroethane were investigated. Bromochloromethane, dibromomethane and 1-bromo-2-chloroethane were utilized by strain GJ10 in batch culture as a cosubstrate and sole carbon source. The rate of degradation of dihalomethanes by whole cells was lower than that of 1,2-dichloroethane, but a significant increase of the rate of dihalomethane biodegradation was observed when methanol or ethanol were added as a cosubstrate. Products of the degradation of several tested compounds by haloalkane dehalogenase were analyzed and a new metabolic pathway based on hydrolytic conversion to formaldehyde was proposed for the dihalomethanes. Strain GJ10 growing on 1,2-dichloroethane converted 2-fluoroethanol and 1-chloro-2-fluoroethane to 2-fluoroacetate, which was tolerated up to a concentration of 2.5 mM. On the basis of the results from batch cultures an inert (dichloromethane), a growth-supporting (dibromomethane) and a toxic (1,2-dibromoethane) compound were selected for testing their effects on a continuous culture of strain GJ10 growing on 1,2-dichloroethane. The compounds were added as pulses to a steady-state chemostat and the response of the culture was followed. The effects varied from a temporary decrease in cell density for dibromomethane to severe toxicity and culture washout with 1,2-dibromoethane. Our results extend the spectrum of halogenated C1 and C2 compounds that are known to be degraded by strain GJ10 and provide information on toxic effects and transformation of compounds not serving as a carbon source for this bacterium.

Characterization of 2-bromoethanesulfonate as a selective inhibitor of the coenzyme m-dependent pathway and enzymes of bacterial aliphatic epoxide metabolism.

Bacterial growth with short-chain aliphatic alkenes requires coenzyme M (CoM) (2-mercaptoethanesulfonic acid), which serves as the nucleophile for activation and conversion of epoxide products formed from alkene oxidation to central metabolites. In the present work the CoM analog 2-bromoethanesulfonate (BES) was shown to be a specific inhibitor of propylene-dependent growth of and epoxypropane metabolism by Xanthobacter autotrophicus strain Py2. BES (at low [millimolar] concentrations) completely prevented growth with propylene but had no effect on growth with acetone or n-propanol. Propylene consumption by cells was largely unaffected by the presence of BES, but epoxypropane accumulated in the medium in a time-dependent fashion with BES present. The addition of BES to cells resulted in time-dependent loss of epoxypropane degradation activity that was restored upon removal of BES and addition of CoM. Exposure of cells to BES resulted in a loss of epoxypropane-dependent CO(2) fixation activity that was restored only upon synthesis of new protein. Addition of BES to cell extracts resulted in an irreversible loss of epoxide carboxylase activity that was restored by addition of purified 2-ketopropyl-CoM carboxylase/oxidoreductase (2-KPCC), the terminal enzyme of epoxide carboxylation, but not by addition of epoxyalkane:CoM transferase or 2-hydroxypropyl-CoM dehydrogenase, the enzymes which catalyze the first two reactions of epoxide carboxylation. Comparative studies of the propylene-oxidizing actinomycete Rhodococcus rhodochrous strain B276 showed that BES is an inhibitor of propylene-dependent growth in this organism as well but is not an inhibitor of CoM-independent growth with propane. These results suggest that BES inhibits propylene-dependent growth and epoxide metabolism via irreversible inactivation of the key CO(2)-fixing enzyme 2-KPCC.

Growth and denitrifying activity of Xanthobacter autotrophicus CECT 7064 in the presence of selected pesticides.

The effects of the application of nine pesticides used commonly in agriculture (aldrin, lindane, dimetoate, methylparathion, methidation, atrazine, simazine, captan and diflubenzuron) on growth, CO2 production, denitrifying activity [as nitrous oxide (N2O) released] and nitrite accumulation in the culture medium by Xanthobacter autotrophicus strain CECT 7064 (Spanish Type Culture Collection) (a micro-organism isolated from a submerged fixed-film) were studied. The herbicide atrazine and the insecticide dimetoate totally inhibited growth and biological activity of X. autotrophicus at 10 mg l(-1), while the rest of the tested pesticides delayed the growth of strain CECT 7064 but did not drastically affect the bacterial growth after 96 h of culture. The denitrifying activity of X. autotrophicus was negatively affected by the pesticides application with the exception of fungicide captan. The release of N2O was strongly inhibited by several pesticides (aldrin, lindane, methylparathion, methidation and diflubenzuron), while dimetoate, atrazine and simazine inhibited totally the denitrifying activity of the strain. The effects of the pesticides on denitrifying submerged fixed-film reactor are discussed.

Strain stability in biological systems treating recalcitrant organic compounds.

The availability of molecular probing technology in recent years has facilitated investigation of microbial community composition during bio-treatment of organic wastes. Particularly, it has allowed the study of microbial culture stability and correlation between stability and treatment performance. However, most studies to date have only addressed mixed cultures and there is limited information regarding single strain stability. Here we have investigated the microbial community dynamics in two bioreactors, each inoculated with a pure bacterial strain capable of degrading a recalcitrant substrate, namely Xanthobacter aut. GJ10 degrading 1,2-dichloroethane (DCE) and Burkholderia sp. JS150 degrading monochlorobenzene (MCB). Universal and strain specific 16S rRNA oligonucleotide probes were designed and used to follow strain stability. The bioreactor fed with DCE was functionally stable and the percentage of GJ10 cells in the community remained high (around 95% of total cells) throughout, even after introduction of foreign microorganisms. The bioreactor fed with MCB was also functionally stable, but in contrast to the DCE bioreactor, probing results revealed the disappearance of strain JS150 from the bioreactor within a week. The difference in behavior between the two systems is attributed to the specific pathway required to degrade DCE.

Biodegradation of monochloroacetic acid used as a sole carbon and energy source by Xanthobacter autotrophicus GJ10 strain in batch and continuous culture.

Batch and continuous mode degradation of monochloroacetic acid used as a sole carbon and energy source in the concentration range of 0.9-48.4 mM by pure culture of Xanthobacter autotrophicus GJ10 was investigated. The substrate was completely degraded in each flask in batch system. Partial substrate inhibition occurred at the concentrations exceeding 25.4 mM. Temporary accumulation of glycolic acid in the medium indicated that dehalogenation was undergoing faster than further utilization of glycolate. Three different carbon substrates were used for inoculum preparation--1,2-dichloroethane, tri-sodium citrate and a nutrient broth. The fastest growth on monochloroacetate occurred for 1,2-dichloroethane-grown inoculum. The assays of haloacid dehalogenase in crude extract indicated that the bacteria grown on 1,2-dichloroethane possessed higher level of the enzyme. The response of the GJ10 culture towards spikes of 20 mM monochloroacetate was tested in 2.5-1 continuously stirred tank fermentor. The substrate was readily utilized within 7-8 h. Continuous degradation of monochloroacetate in the fermentor was demonstrated for monochloroacetate concentration of 20 mM and dilution rate 0.016 h(-1). Quantitative agreement between the amount of monochloroacetate introduced and chloride released was found. The results demonstrated that the strain X. autotrophicus GJ10 might be suitable for biodegradation of monochloroacetate contaminated media.

Denitrifying activity of Xanthobacter autotrophicus strains isolated from a submerged fixed-film reactor.

Xanthobacter autotrophicus strains with the ability to reduce nitrate and nitrite to either nitrous oxide or molecular nitrogen were isolated from submerged fixed-film reactors. Isolated strains were Gram-negative rods able to grow on methanol, ethanol and sucrose. The yellow cellular pigmentation, pleomorphic appearance, and the presence of poly-beta-hydroxybutyrate granules suggest that the organisms might belong to the genus Xanthobacter. Comparison of 16S rDNA gene sequences demonstrated the affiliation of the strains to X. autotrophicus species. The results show that X. autotrophicus may play a role in inorganic nitrogen removal from a denitrifying submerged filter used for the treatment of contaminated groundwater. To our knowledge, no data on denitrifying activity in X. autotrophicus strains have been reported previously.

Cloning of Baeyer-Villiger monooxygenases from Comamonas, Xanthobacter and Rhodococcus using polymerase chain reaction with highly degenerate primers.

To clone novel type 1 Baeyer-Villiger monooxygenase (BVMO) genes, we isolated or collected 25 bacterial strains able to grow on alicyclic compounds. Twelve of the bacterial strains yielded polymerase chain reaction (PCR) fragments with highly degenerate primers based on the sequences of known and putative BVMOs. All these fragments were found to encode peptides homologous to published BVMO sequences. The complete BVMO genes and flanking DNA were cloned from a Comamonas, a Xanthobacter and a Rhodococcus strain using the PCR fragments as probes. BVMO genes cloned from the first two strains could be expressed to high levels in Escherichia coli using standard expression vectors, and the recombinants converted cyclopentanone and cyclohexanone to the corresponding lactones. The Rhodococcus BVMO, a putative steroid monooxygenase, could be expressed after modification of the N-terminal sequence. However, recombinants expressing this protein did not show activity towards progesterone. An esterase homologue located directly upstream of the Xanthobacter BVMO gene and a dehydrogenase homologue encoded directly downstream of the Comamonas sp. NCIMB 9872 BVMO gene were also expressed in E. coli and shown to specify lactone hydrolase and cyclohexanol dehydrogenase activity respectively.

Is the contribution of bacteria to terrestrial carbon budget greatly underestimated?

Some commonly found species of soil bacteria use low molecular weight organic acids as their sole source of carbon and energy. This study shows that acids such as citrate and oxalate (produced in large amounts by fungi and plants) can rapidly be consumed by these bacteria. Two strains, Ralstonia eutropha and Xanthobacter autotrophicus, were cultured on acetate- and citrate-rich media. The resulting CO2 and/or HCO3- reacted with calcium ions to precipitate two polymorphs of calcium carbonate (CaCO3), calcite and vaterite, depending on the quantity of slime produced by the strains. This production of primary calcium carbonate crystals by oxalate- and citrate-degrading bacteria from soil organic carbon sources highlights the existence of an important and underestimated potential carbon sink.

Characterization of the 2-[(R)-2-hydroxypropylthio]ethanesulfonate dehydrogenase from Xanthobacter strain Py2: product inhibition, pH dependence of kinetic parameters, site-directed mutagenesis, rapid equilibrium inhibition, and chemical modification.

Although the short-chain dehydrogenase/reductase (SDR) superfamily contains a very large number of members defined in annotated databases and by biochemical and structural studies, very few SDR enzymes have been identified that have a homologous partner catalyzing the same reaction but with an opposite stereospecificity. In the present study we have cloned and expressed one of these enzymes, the 2-[(R)-2-hydroxypropylthio]ethanesulfonate (R-HPC) dehydrogenase, that is part of the coenzyme M-dependent pathway of alkene and epoxide metabolism in Xanthobacter strain Py2. Investigation of the kinetic mechanism using product inhibition suggested that a compulsory-ordered ternary complex mechanism was followed. The pH dependence of k(cat)/K(m) indicated the presence of a single ionizable residue of catalytic importance (pK(a) = 6.9) that was proposed to be Y155 of the catalytic triad. Amino acid substitutions of the putative catalytic triad residues produced inactive enzymes (S142C, Y155F, Y155E, and K159A) or enzyme with a greatly decreased activity (S142A). Inhibitors were investigated as probes of the molecular features of R-HPC that contribute to substrate binding. 2-[(S)-2-Hydroxypropylthio]ethanesulfonate (S-HPC) and 2-(2-methyl-2-hydroxypropylthio)ethanesulfonate were found to be competitive inhibitors of R-HPC with K(ic) values close to the K(m) for R-HPC. The arginine-specific modifiers 2,3-butanedione and phenylglyoxal were found to be inactivators, and inactivation could be protected against by the addition of R-HPC. 2,3-Butanedione was found to reduce enzyme activity with R-HPC as a substrate much more dramatically than with substrates that lacked a sulfonate moiety [e.g., 2-propanol, (R)-2-pentanol, and (R)-2-heptanol]. Amino acid analyses of enzyme modified by 2,3-butanedione in the presence and absence of S-HPC suggested protection of a single arginine residue. On the basis of these results, we propose that one or more active site arginines play a key role in substrate binding via an ionic interaction with the sulfonate moiety of R-HPC.

Evidence of substantial carbon isotope fractionation among substrate, inorganic carbon, and biomass during aerobic mineralization of 1, 2-dichloroethane by Xanthobacter autotrophicus.

Carbon isotope fractionation during aerobic mineralization of 1, 2-dichloroethane (1,2-DCA) by Xanthobacter autotrophicus GJ10 was investigated. A strong enrichment of (13)C in residual 1,2-DCA was observed, with a mean fractionation factor alpha +/- standard deviation of 0.968 +/- 0.0013 to 0.973 +/- 0.0015. In addition, a large carbon isotope fractionation between biomass and inorganic carbon occurred. A mechanistic model that links the fractionation factor alpha to the rate constants of the first catabolic enzyme was developed. Based on the model, it was concluded that the strong enrichment of (13)C in 1,2-DCA arises because the first irreversible step of the initial enzymatic transformation of 1,2-DCA consists of an S(N)2 nucleophilic substitution. S(N)2 reactions are accompanied by a large kinetic isotope effect. The substantial carbon isotope fractionation between biomass and inorganic carbon could be explained by the kinetic isotope effect associated with the initial 1,2-DCA transformation and by the metabolic pathway of 1,2-DCA degradation. Carbon isotope fractionation during 1,2-DCA mineralization leads to 1,2-DCA, inorganic carbon, and biomass with characteristic carbon isotope compositions, which may be used to trace the process in contaminated environments.

Effects of the Calvin cycle on nicotinamide adenine dinucleotide concentrations and redox balances of Xanthobacter flavus.

The levels of reduced and oxidized nicotinamide adenine dinucleotides were determined in Xanthobacter flavus during a transition from heterotrophic to autotrophic growth. Excess reducing equivalents are rapidly dissipated following induction of the Calvin cycle, indicating that the Calvin cycle serves as a sink for excess reducing equivalents. The physiological data support the conclusion previously derived from molecular studies in that expression of the Calvin cycle genes is controlled by the intracellular concentration of NADPH.