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BACKGROUND Previous research proved that fermentation via dissolved oxygen (DO) step-wise reduction was favorable for vitamin B12 productivity by Pseudomonas denitrificans, but the high-production mechanism is still unclear. Based on this fact, P. denitrificans metabolic characteristics under different DO supply models were investigated in a 5 L fermenter by assaying the activities of the key enzymes involved in central carbon metabolism and the vitamin B12 biosynthetic pathway. RESULTS Compared with fermentation processes under the DO-stat strategy and DO step-wise increase, it was found that DO step-wise reduction for P. denitrificans fermentation could significantly strengthen the Embden–Meyerhof–Parnas flux and weaken the tricarboxylic acid cycle and hexose monophosphate pathway, but also facilitate the activities of δ-aminolevulinic acid synthase and porphobilinogen synthase. CONCLUSIONS When P. denitrificans fermentation was implemented using DO step-wise reduction, the metabolic characteristics indicated that more precursors for vitamin B12 biosynthesis could be generated. As a result, a significant improvement in vitamin B12 productivity was achieved (69.09 ± 4.48 mg L−1). © 2013 Society of Chemical Industry

3-Hydroxypropionic acid (3-HP) can be produced from glycerol through two sequential enzymatic reactions that are catalyzed by a coenzyme B12 -dependent glycerol dehydratase and an NAD(P)(+) -dependent aldehyde dehydrogenase (ALDH), respectively. Pseudomonas denitrificans synthesizes coenzyme B12 under aerobic conditions, where NAD(P)(+) is regenerated efficiently. Hence, it is considered an ideal host for the production of 3-HP from glycerol under aerobic conditions. In this study, recombinant strains of P. denitrificans were developed and their potential for the production of 3-HP from glycerol was evaluated. When the enzymes, glycerol dehydratase (DhaB) and glycerol dehydratase reactivase (GdrAB), of Klebsiella pneumoniae were expressed heterologously, P. denitrificans could produce 3-HP at 37.7 mmol/L with 62% (mol/mol) yield on glycerol. Glucose was required as the carbon and energy sources for cell growth. The overexpression of heterologous ALDH was not essential; however, the titer and yield of 3-HP were improved to 54.7 mmol/L and 67% (mol/mol), respectively, when an ALDH gene (puuC) from K. pneumoniae was overexpressed. One serious drawback hindering the use of P. denitrificans as a recombinant host for 3-HP production is that it oxidizes 3-HP to malonate and utilizes 3-HP as a carbon source for growth. This is the first report on the development and use of recombinant P. denitrificans for 3-HP production from glycerol.

BACKGROUND Fermentation of the industrial vitamin B12 by Pseudomonas denitrificans usually utilizes sucrose or maltose as the sole carbon source, which results in increased medium costs. In order to decrease the fermentation cost, it is crucial and essential to employ a low-cost and convenient raw material as an alternative medium substrate for industrial vitamin B12 production. RESULTS The results obtained in chemically defined medium showed that glutamate and sucrose were favorable for cell growth and vitamin B12 biosynthesis of P. denitrificans. Due to containing a mass of ingredients such as sucrose, glutamate and betaine, beet molasses was consequently chosen as the main medium substrate for industrial P. denitrificans fermentation in a 120 000 L fermenter. Vitamin B12 production reached 181.75 mg L–1. CONCLUSIONS Beet molasses is a by-product of the sugar industry and is thus very inexpensive, and it was proved that beet molasses was an efficient and economical medium substrate for industiral vitamin B12 fermentation by P. denitrificans. © 2013 Society of Chemical Industry

BACKGROUND: There are two different routes for vitamin B12 biosynthesis, which results in discrepancies and uncertainties of the dissolved oxygen (DO) concentration for vitamin B12 fermentation. In this paper, the DO control strategy was explored for industrial vitamin B12 fermentation by Pesudomonas denitrificans in 120000-L fermenter. RESULTS: A DO-stat strategy was first successfully scaled up from a 9000 L fermenter to a 120 000 L fermenter. Then a multi-stage DO control strategy was further established in the 120 000 L fermenter, in which the DO level was shifted from 8–10% (20–48 h) to 2–5% (49–106 h) and below 2% (107–168 h) by gradually reducing the rate of aeration and agitation. As a result, 198.80 mg L−1 of vitamin B12 was obtained, which was significantly higher than those obtained under the fermentations with one-stage DO control. CONCLUSIONS: The comparatively low DO level was favorable for vitamin B12 biosynthesis, but it would have an extremely negative effect on cell growth. Compared with the low DO level maintained at all times of the fermentation process, a multi-stage DO control strategy could not only increase the biomass but also improve vitamin B12 biosynthesis. Copyright © 2012 Society of Chemical Industry

Paracoccus pantotrophus expresses two nitrate reductases—membrane bound nitrate reductase (Nar) and periplasmic nitrate reductase (Nap). In growth experiments with two denitrifying species (Paracoccus pantotrophus and Alcaligenes eutrophus) that have both Nap and Nar and two species (Pseudomonas denitrificans and Pseudomonas fluorescens) with Nar only, it was found that diauxic lag is shorter for bacteria that express Nap. In P. pantotrophus, napEDABC encodes the periplasmic nitrate reductase. To analyze the effect of Nap on diauxic lag, the nap operon was deleted from P. pantotrophus. The growth experiments with nap− mutant resulted in increased diauxic lag when switched from aerobic to anoxic respiration, suggesting Nap is responsible for shorter lags and helps in adaptation to anoxic metabolism after transition from aerobic conditions. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009

The mechanism of the ring contraction process during vitamin B(12) biosynthesis by the anaerobe Propionibacterium shermanii was investigated under both aerobic and anaerobic conditions by means of feeding experiments with delta-amino[1-(13)C]levulinic acid (a biosynthetic intermediate of tetrapyrrole) and delta-amino[1-(13)C,1,1,4-(18)O(3)]levulinic acid in combination with (13)C-NMR spectroscopy. We showed that the characteristic mechanism of the ring contraction process (the generation of precorrin-3x from formation of the gamma-lactone from the ring A acetate group at C1 and hydroxylation at C20 by molecular oxygen catalyzed by CobG, and the migration of ring D by cleavage of the carbon-oxygen bond at C1 of precorrin-3x) in the aerobe Pseudomonas denitrificans was not seen in P. shermanii under aerobic conditions, and the mechanism of the ring contraction process in P. shermanii was the same irrespective of the presence or absence of oxygen.

We present a model for diauxic growth of denitrifying bacteria in which nitrate reductase synthesis kinetics dominate the overall growth kinetics. The model is based on the assumption of the existence of a nitrate respiration operon, thereby linking the rate of nitrate uptake to the activity of nitrate reductase. We show that this approach can model diauxic growth of Pseudomonas denitrificans by conducting experiments in which nitrate reductase activity was measured during both lag and ensuing exponential growth phases. We consistently observed the pattern of low nitrate reductase enzyme activity during the lag phase, increasing before the onset of growth. By fitting model parameters we were able to successfully match experimental data for growth, nitrate uptake, and enzyme activity level.

Bacteria were isolated from the rhizosphere of cotton, wheat, alfalfa, and tomato grown in field locations within a semi-arid region of Uzbekistan. Strains were identified as Pseudomonas denitrificans, P. rathonis, Bacillus laevolacticus, Bacillus amyloliquefaciens, and Arthrobacter simplex. The isolated strains produced different enzymes, phytohormone auxin and were antagonists against specific plant-pathogenic fungi. Most of the strains are tolerant with respect to salt and temperature. All of the bacterial strains isolated in this study have been found to increase plant growth of wheat and maize in pot experiments.

Denitrification of a synthetic wastewater containing nitrates and methanol as carbon source was carried out in two systems - a fluidized-bed biofilm reactor (FBBR) and a stirred tank reactor (STR) - using Pseudomonas denitrificans over a period of five months. Nitrogen loading was varied during operation of both reactors to assess differences in the response to transient conditions. Experimental data were analyzed to obtain a comparison of denitrification kinetics in biofilm and suspended growth reactors. The comparison showed that the volumetric degradation capacity in the FBBR (5.36 kg N m -3 d -1 ) was higher than in the STR, due to higher biomass concentration (10 kg BM .m -3 vs 1.2 kg BM m -3 ).

Pseudomonas knackmussii B13 was the first strain to be isolated in 1974 that could degrade chlorinated aromatic hydrocarbons. This discovery was the prologue for subsequent characterization of numerous bacterial metabolic pathways, for genetic and biochemical studies, and which spurred ideas for pollutant bioremediation. In this study, we determined the complete genome sequence of B13 using next generation sequencing technologies and optical mapping. Genome annotation indicated that B13 has a variety of metabolic pathways for degrading monoaromatic hydrocarbons including chlorobenzoate, aminophenol, anthranilate and hydroxyquinol, but not polyaromatic compounds. Comparative genome analysis revealed that B13 is closest to Pseudomonas denitrificans and Pseudomonas aeruginosa. The B13 genome contains at least eight genomic islands [prophages and integrative conjugative elements (ICEs)], which were absent in closely related pseudomonads. We confirm that two ICEs are identical copies of the 103 kb self-transmissible element ICEclc that carries the genes for chlorocatechol metabolism. Comparison of ICEclc showed that it is composed of a variable and a 'core' region, which is very conserved among proteobacterial genomes, suggesting a widely distributed family of so far uncharacterized ICE. Resequencing of two spontaneous B13 mutants revealed a number of single nucleotide substitutions, as well as excision of a large 220 kb region and a prophage that drastically change the host metabolic capacity and survivability.

We have shown that Pseudomonas denitrificans undergo a diauxie when switching from dissolved oxygen to nitrate as terminal electron acceptor. The length of time under aeration significantly affected the length of the diauxic lag, whereas the presence or absence of nitrate in the culture under aeration had a marginal effect. Nitrate consumption was very low during the lag period and then increased rapidly, coinciding with exponentially increasing biomass concentrations. Biochemical rate expressions that account for enzyme synthesis and activity in response to culture conditions and enzyme specific levels were developed. The new model successfully predicts the different lengths of diauxic lags observed in the experiments as well as the growth pattern and nitrate uptake.

Kinetic experiments were carried out for a pure culture of Pseudomonas denitrificans to develop a mathematical model that adequately describes denitrification under both carbon and nitrate or nitrite electron acceptor limited conditions. Denitrification was assumed to occur by the consecutive reduction of nitrates to nitrites and then to nitrogen gas without accumulation of intermediate gaseous products. The denitrification kinetics, assuming they are associated with cell growth, could be very well described by using Monod expressions with double substrate limitation (terminal electron acceptor and organic carbon). The kinetic parameters of the model were evaluated exclusively from batch experimental data. The proposed model is able to predict accurately the biomass growth, the nitrate and nitrite reduction rates, as well as the accumulation of nitrite-nitrogen as an intermediate, due to nitrate inhibition on the nitrite reduction rate. Water Environ. Res., 68, 934 (1996).

According to the endosymbiont hypothesis, mitochondria are descended from ancient aerobic bacteria that were engulfed by protoeukaryotic cells. Experiments described here show that a synthetic peptide corresponding to a yeast mitochondrial targeting sequence can be imported into Paracoccusdenitrificans, a soil bacterium thought to be closely related to the protomitochondrion. The import is very similar to that observed with isolated yeast mitochondria. The results suggest that the protomitochondrion may have been inherently able to translocate mitochondrial presequences. This ability may partly explain the development of the protein import process during the evolution of the mitochondrion.

The reduction of nitrate by Pseudomonas denitrificans in a culture medium containing glycerol, yeast extract and 700 mg/1 NO3–N, was antagonized by Aeromonas hydrophila, Escherichia coli and Enterobacter aerogenes. Nitrate reduction by Ps. denitrificans in mixed culture with a fermentative heterotroph was inhibited when 100–150 mg/1 NO2–N had accumulated in the medium. The number of Ps. denitrificans declined concomitantly with the appearance of NO2 in the culture medium, but there was only a slight increase in the numbers of fermentative hetero-trophs. The fermentative heterotrophs did not antagonize nitrate reduction by Ps. denitrificans, when the culture medium contained 140 mg/1 NO3–N. When mixtures of equal parts of Ps. denitrificans and Esch. coli cultures were added to autoclaved river water relatively high concentrations of NO2 were produced from the nitrate present in the water.