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The B12-producing strains Pseudomonas nitroreducens DSM 1650 and Pseudomonas sp. CCUG 2519 (both formerly Pseudomonas denitrificans), with the most distributed pathway among bacteria for exogenous choline/betaine utilization, are promising recombinant hosts for the endogenous production of B12 precursor betaine by direct methylation of bioavailable glycine or non-proteinogenic β-alanine. Two plasmid-based de novo betaine pathways, distinguished by their enzymes, have provided an expression of the genes encoding for N-methyltransferases of the halotolerant cyanobacterium Aphanothece halophytica or plant Limonium latifolium to synthesize the internal glycine betaine or β-alanine betaine, respectively. These betaines equally allowed the recombinant pseudomonads to grow effectively and to synthesize a high level of cobalamin, as well as to increase their protective properties against abiotic stresses to a degree comparable with the supplementation of an exogenous betaine. Both de novo betaine pathways significantly enforced the protection of bacterial cells against lowering temperature to 15 °C and increasing salinity to 400 mM of NaCl. However, the expression of the single plant-derived gene for the β-alanine-specific N-methyltransferase additionally increased the effectiveness of exogenous glycine betaine almost twofold on cobalamin biosynthesis, probably due to the Pseudomonas' ability to use two independent pathways, their own choline/betaine pathway and the plant β-alanine betaine biosynthetic pathway. [ABSTRACT FROM AUTHOR]

Vitamin B12 is a widely used compound in the feed and food, healthcare and medical industries that can only be produced by fermentation because of the complexity of its chemical synthesis. For this reason, finding better producer strains and optimizing their bioprocesses have been the main focus of industrial producers over the last few decades. In this review, we initially provide a historical overview of vitamin B12 research and the main biosynthetic characteristics of the two microorganism families typically used for its industrial production: several strains of Propionibacterium freudenreichii and strains related to Pseudomonas denitrificans. Later, a complete summary of the current state of vitamin B12 industrial production as well as the main advances and challenges for improving it is detailed, with a special focus on bioprocess optimization, which aims not only to increase production but also sustainability. In addition, a comprehensive list of the most important and relevant patents for the present industrial strains is provided. Finally, the potential applications of vitamin B12 in different markets are discussed. [ABSTRACT FROM AUTHOR]

La batata (Ipomoea batatas L.) se cultiva en todo el mundo como fuente de carbohidratos, y su producción comercial requiere un alto aporte de fertilizantes químicos, lo cual eleva los costos de producción. Los inoculantes microbianos, se emplean como una fuente alternativa de nutrición vegetal. El objetivo de esta investigación fue evaluar el efecto de Pseudomonas denitrificans IBVS2 y Azotobacter vinelandii IBVS13 con diferentes niveles fertilización química nitrogenada en el cultivo de batata en la microrregión del Valle del Sinú en el Caribe Colombiano. Para los montajes de los experimentos se utilizó un diseño completamente aleatorizado, ocho tratamientos y tres repeticiones usando como material vegetal plántulas obtenidas in vitro endurecidas en invernadero. Los resultados demostraron que la cepa Azotobacter vinelandii IBVS13 con un 75% de fertilización nitrogenada (FN) mejoró la capacidad de acumulación de materia seca en los tubérculos de batata, generando incrementos de 6,65 t/ha respecto al testigo químico y 3,18 t/ha en relación con el testigo absoluto, garantizando un incremento del rendimiento. Así mismo, el contenido de proteína bruta aumentó 13,93% al realizar la inoculación de las plantas con esta cepa. En el mismo sentido, la cepa Pseudomonas denitrificans IBVS2+ fertilización nitrogenada 50% presentó aumentos en la variable de fibra cruda 31,75% respecto al testigo absoluto, contribuyendo de manera eficaz como bioestimulante microbiano en la agricultura.

An alternative to reduce the effect of chemical fertilization on soils and favor the growth of sweet potato crops is the incorporation of microorganisms that promote plant growth in fertilization. In the Colombian Caribbean, sweet potato is currently one of the emerging crops due to its high nutritional properties in the human diet. The objective of this study was to evaluate the effect of two bacterial strains, Pseudomonas denitrificans IBVS2 and Azotobacter vinelandii IBVS13, on the yield of sweet potato clone Tainung 66 in field conditions, in the Valle del Sinú, Córdoba, Colombia. The research was carried out during second semester of 2017, at the Turipaná-Agrosavia Research Center, using apical cuttings from the Tainung 66 clone. A randomized complete block design with a 3x2+2 factorial arrangement was implemented, physiological component, foliar and edaphic chemical analysis, yield and bromatological composition variables were evaluated. The results showed that yield and dry root mass showed significant differences with respect to the chemical control when fertilization consisted of IBVS2 strain plus 50% of chemical fertilizer, obtaining a yield of 11.32 t.ha-1 and 5,78 t.ha-1 respectively. These results showed that inoculation of PGPRs constitutes an alternative for sweet potatoes cultivation with positive effects when compared to complete chemical fertilization.

Excessive nitrate in aquaculture systems has attracted wide attention. To isolate novel aerobic denitrifying strain and characterize its nitrogen removal processes, a facultative anaerobic denitrification bacterium, identified as Pseudomonas denitrificans G1, was isolated from marine sediments. Strain G1 could grow and remove 90–98% of nitrate and 97–99% of nitrite under an aerobic or anaerobic condition in 24 h, with the total nitrogen removal rate of 33–38% (87–100 mg/L). The highest denitrification rate could reach 15.1 mg/(L·h). The suitable condition for the denitrification of G1 is C/N ratio 5–22, dissolved oxygen 0–4.68 mg/L, salinity 0–30 g NaCl/L, pH 7–9.5. Under the aerobic condition, G1 grew fast; however, the mass spectrographic analysis showed that the gas product was N 2 O. Under the anaerobic conditions, G1 grow relatively slowly, but could also achieve effective denitrification and the final product was N 2. In denitrification of aquaculture wastewater, strain G1 can remove 60.57% of nitrate and 36.36% of total nitrogen; meanwhile, there was a slight accumulation of ammonia nitrogen. P. denitrificans strain G1 has potential in denitrification processes for the treatment of aquaculture wastewater. However, the regulation of reaction conditions and gas products needs to be further studied. [ABSTRACT FROM AUTHOR]

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Sulfur, most abundantly found in the environment as sulfate (SO42-), is an essential element in metabolites required by all living cells, including amino acids, co-factors and vitamins. However, current understanding of the cellular delivery of SO42- at the molecular level is limited. CysZ has been described as a SO42- permease, but its sequence family is without known structural precedent. Based on crystallographic structure information, SO42- binding and flux experiments, we provide insight into the molecular mechanism of CysZ-mediated translocation of SO42- across membranes. CysZ structures from three different bacterial species display a hitherto unknown fold and have subunits organized with inverted transmembrane topology. CysZ from Pseudomonas denitrificans assembles as a trimer of antiparallel dimers and the CysZ structures from two other species recapitulate dimers from this assembly. Mutational studies highlight the functional relevance of conserved CysZ residues.

In this study, whole-cell biosensors that detect and indicate the concentration of 3-hydroxypropionic acid (3-HP) by producing a green fluorescent signal were developed using a transcription factor (TF)-responsive 3-HP inducible promoter identified in the Pseudomonas denitrificans 3-HP degradation pathway. Upon forming a complex with 3-HP, the MmsR TF protein, a LysR-type transcriptional regulator (LTTR), binds to the PmmsA promoter and controls the expression of the PmmsA-regulated gfp reporter gene in response to the concentration of 3-HP. Furthermore, by enhancing the expression of MmsR and through mutagenesis of the PmmsA promoter region, three well-performing biosensors were developed that covered a wide dynamic range of 3-HP (0.01-100 mM when added externally) with ~100-fold signal change upon induction in P. denitrificans. The 3-HP biosensor machinery, composed of MmsR, the PmmsA promoter and gfp could also function well in E. coli and P. putida. The developed 3-HP biosensors should be useful for engineering 3-HP-producing strains and the enzymes associated with its production. [ABSTRACT FROM AUTHOR]

Application of the recently developed wholecell 3-HP biosensor, which detects 3-hydroxypropionic acid (3-HP) and expresses fluorescence as an output signal in response to the 3-HP concentration, was studied in three areas of enzyme and metabolic engineering. First, a sensor was applied to identify active 3-hydroxyaldehyde dehydrogenase (ALDH), a key enzyme in the 3-HP production pathway. Second, with the aid of helper enzymes that catalyze the conversion of 1,3-propanediol (1,3-PDO) to 3-HP, a 3-HP biosensor was converted into a 1,3-PDO biosensor. Third, a 3-HP biosensor, with proper modifications in expression module of the output signal and the use of pH-tolerant red fluorescent protein (RFP), was shown to monitor the rate of 3-HP production under process conditions in which one or more interfering compounds are present in the culture medium and/or the medium pH decreases. This study demonstrates that 3-HP biosensors can be widely used in enzyme and metabolic engineering applications for 3-HP production. [ABSTRACT FROM AUTHOR]

The assessment of the impact of the bacteria Pseudomonas denitrificans , Pseudomonas fluorescens, Bacillus subtilis and yeast Yarrowia lipolytica on commercial poly(ether urethanes) Tecoflex ® and Tecothane ® is presented. The polyurethane samples were incubated with pure cultures of the microorganisms at 30 °C for five months. The changes in the chemical structure of the polymers were evaluated using loss of weight and contact angle measurements, infrared spectroscopy (ATR-FTIR), mass spectrometry (Py-MS), differential scanning calorimetry (DSC) and the thermogravimetric analysis (TG). In addition, scanning electron microscopy (SEM) and atomic force microscopy (AFM) were applied for imaging changes in surface morphology of the poly(ether urethanes). Comparative analysis of these polyurethane features before and after incubation with the microorganism cultures showed that Tecoflex ® was less stable than Tecothane ® . This can be explained by the presence of aromatic rings within the diphenylmethane diisocyanate group in the chemical structure of the latter. Bacterial strains of Bacillus subtilis and Pseudomonas fluorescens showed a much more prominent destructive effect compared to the strain of yeast Y. lipolytica . [ABSTRACT FROM AUTHOR]

The relationship between the morphological character of Pseudomonas denitrificans and vitamin B synthesis based on real-time capacitance measurement and online specific oxygen consumption rate ( Q ) control was established for enhancing vitamin B production. Results demonstrated that the threshold Q value lower than 2.0 mmol/gDCW/l would greatly stimulate the state transfer from the cell number growth phase to the cell elongation phase and promote rapid vitamin B biosynthesis, while the vitamin B biosynthesis rate could also be inhibited when the rate of cell's length-to-width ratio (ratio-LW) was higher than 10:1. Furthermore, the optimal morphology controlling strategy was achieved based on online Q control, which increases the appropriate active cell numbers at the former phase, and then control the elongation of ratio-LW no more than 10:1 at the vitamin B biosynthesis phase. The maximal vitamin B production reached 239.7 mg/l at 168 h, which was improved by 14.7 % compared with the control (208 mg/l). This online controlling strategy would be effectively applied for improving industrial vitamin B fermentation. [ABSTRACT FROM AUTHOR]

The aerobic Pseudomonas denitrificans is widely used for industrial and commercial vitamin B fermentation, due to its higher productivity compared to the anaerobic vitamin B-producing microorganisms. This paper aimed to develop a cost-effective fermentation medium for industrial vitamin B production by P. denitrificans in 120,000-l fermenter. It was found that maltose syrup (a low-cost syrup from corn starch by means of enzymatic or acid hydrolysis) and corn steep liquor (CSL, a by-product of starch industry) were greatly applicable to vitamin B production by P. denitrificans. Under the optimal fermentation medium performed by response surface methodology, 198.27 ± 4.60 mg/l of vitamin B yield was obtained in 120,000-l fermenter, which was close to the fermentation with the refined sucrose (198.80 mg/l) and was obviously higher than that obtained under beet molasses utilization (181.75 mg/l). Therefore, maltose syrups and CSL were the efficient and economical substrates for industrial vitamin B fermentation by P. denitrificans. [ABSTRACT FROM AUTHOR]

The performances of betaine on the metabolic processes of vitamin B-producing Pseudomonas denitrificans were investigated in this paper. The results showed that betaine was an indispensable methyl-group donor for vitamin B biosynthesis, but large amounts of the extracellular glycine accompanied by betaine metabolism would impose a severe restriction on the cell growth of P. denitrificans. By further using a comparative metabolomics approach coupled with intracellular free amino acids analysis for the fermentation processes with betaine addition (10 g/l) or not, it was found that betaine could highly strengthen the formation of some key precursors and intermediates facilitating vitamin B biosynthesis, such as δ-aminolevulinic acid (ALA, the first precursor of vitamin B), glutamate (an intermediate of ALA via C pathway), glycine (an intermediate of ALA via C pathway), and methionine (directly participating in the methylation reaction involved in vitamin B biosynthetic pathway). Therefore, the performances of betaine on P. denitrificans metabolic processes were not only serving as a decisive methyl-group donor for vitamin B biosynthesis, but also playing a powerfully promoting role in the generation of vitamin B precursors and intermediates. [ABSTRACT FROM AUTHOR]

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 supplymodels were investigated in a 5 L fermenter by assaying the activities of the key enzymes involved in central carbonmetabolism 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:WhenP.denitrificansfermentationwasimplementedusingDOstep-wise reduction, themetaboliccharacteristics 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±4.48mgL-1). [ABSTRACT FROM AUTHOR]

Our previous research had revealed that the dissolved oxygen limitation was more favorable for vitamin B fermentation, due to its inducement to the increased glycolytic flux in Pseudomonas denitrificans. In this paper, a novel strategy was implemented to further investigate the metabolic characteristics of P. denitrificans under different oxygen supply levels, by exogenously adding rotenone (a respiratory chain inhibitor interfering with the oxygen consumption) to the fermentation broths. Compared to the fermentation process without rotenone treatment, it was observed that 5 mg/L rotenone treatment could significantly strengthen the glycolytic flux of P. denitrificans via activating the key glycolytic enzymes (phosphofructokinase and pyruvate kinase), resulting in the accelerated generations of anterior precursors (glutamate and 5-aminolevulinic acid) for vitamin B biosynthesis. Although 5 mg/L rotenone treatment had a negative effect on cell growth of P. denitrificans, the vitamin B yield was increased from 48.28 ± 0.62 mg/L to 54.70 ± 0.45 mg/L, which further proved that an increased glycolytic flux in P. denitrificans was a consequence of higher vitamin B production. [ABSTRACT FROM AUTHOR]

Pseudomonas denitrificans is a gram-negative bacterium that can produce vitamin B under aerobic conditions. Recently, recombinant strains of P. denitrificans overexpressing a vitamin B-dependent glycerol dehydratase (DhaB) were developed to produce 3-hydroxypropionic acid (3-HP) from glycerol. The recombinant P. denitrificans could produce 3-HP successfully under aerobic conditions without an exogenous supply of vitamin B, but the 3-HP produced disappeared during extended cultivation due to the 3-HP degradation activity in this strain. This study developed mutant strains of P. denitrificans that do not degrade 3-HP. The following eight candidate enzymes, which might be responsible for 3-HP degradation, were selected, cloned, and studied for their activity in Escherichia coli: four (putative) 3-hydroxyisobutyrate dehydrogenases (3HIBDH), a putative 3-HP dehydrogenase (3HPDH), an alcohol dehydrogenase (ADH), and two choline dehydrogenases (CHDH). Among them, 3HIBDHI, 3HIBDHIV, and 3HPDH exhibited 3-HP degrading activity when expressed heterologously in E. coli. When 3hpdh alone or along with 3hibdhIV were disrupted from P. denitrificans, the mutant P. denitrificans exhibited greatly reduced 3-HP degradation activity that could not grow on 3-HP as the sole carbon and energy source. When the double mutant P. denitrificans Δ 3hpdhΔ 3hibdhIV was transformed with DhaB, an improved 3-HP yield (0.78 mol/mol) compared to that of the wild-type counterpart (0.45 mol/mol) was obtained from a 24-h flask culture. This study indicates that 3hpdh and 3hibdhIV (to a lesser extent) are mainly responsible for 3-HP degradation in P. denitrificans and their deletion can prevent 3-HP degradation during its production by recombinant P. denitrificans. [ABSTRACT FROM AUTHOR]

This study examined the role and physiological relevance of 3-hydroxyisobutyrate dehydrogenase-I (3HIBDHI) of Pseudomonas denitrificans ATCC 13867 in the degradation of 3-hydroxypropionic acid (3-HP) during 3-HP production. The gene encoding 3HIBDH-I of P. denitrificans ATCC 13867 was cloned and expressed in Escherichia coli BL21 (DE3). The recombinant 3HIBDH-I was then purified on a Ni-NTA-HP column and characterized for its choice of substrates, cofactors, metals, reductants, and the optimal temperature and pH. The recombinant 3HIBDH-I showed a high catalytic constant ( k/ K) of 604.1 ± 71.1 mM/S on ( S)-3-hydroxyisobutyrate, but no detectable activity on ( R)-3-hydroxyisobutyrate. 3HIBDH-I preferred NAD over NADP as a cofactor for its catalytic activity. The k/ K determined for 3-HP was 15.40 ± 1.43 mM/S in the presence of NAD at 37°C and pH 9.0. In addition to ( S)-3-hydroxyisobutyrate and 3-HP, 3HIBDH-I utilized l-serine, methyl- d, l-serine, and methyl-( S)-(+)-3-hydroxy-2-methylpropionate; on the other hand, the k/ K values determined for these substrates were less than 5.0mM/S. Ethylenediaminetetraacetic acid, 2-mercaptoethanol, dithiothreitol and Mn increased the activity of 3HIBDHI significantly, whereas the presence of Fe, Hg and Ag in the reaction mixture at 1.0 mM completely inhibited its activity. This study revealed the characteristics of 3HIBDH-I and its significance in 3-HP degradation. [ABSTRACT FROM AUTHOR]

ABSTRACT 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. Biotechnol. Bioeng. 2013;110: 3177-3187. © 2013 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

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 [ABSTRACT FROM AUTHOR]

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 [ABSTRACT FROM AUTHOR]

Abstract: δ-aminolevulinate (ALA) is an important intermediate involved in tetrapyrrole synthesis (precursor for vitamin B12, chlorophyll and heme) in vivo. It has been widely applied in agriculture and medicine. On account of many disadvantages of its chemical synthesis, microbial production of ALA has been received much attention as an alternative because of less expensive raw materials, low pollution, and high productivity. Vitamin B12, one of ALA derivatives, which plays a vital role in prevention of anaemia has also attracted intensive works. In this review, recent advances on the production of ALA and vitamin B12 with novel approaches such as whole-cell enzyme-transformation and metabolic engineering are described. Furthermore, the direction for future research and perspective are also summarized. [Copyright &y& Elsevier]

Abstract: The network topology and metabolic fluxes of central carbon metabolism in the industrial vitamin B12 producing strain Pseudomonas denitrificans were characterized under oxygen limiting levels. Cultivations were carried out with 100% [1-13C] or 20% [U-13C] glucose as substrates under different oxygen supply conditions. The labeling patterns of the proteinogenic amino acids of exponentially growing cells were used to accurately estimate the fluxes in the central carbon metabolism of P. denitrificans. Metabolic flux analysis showed that glucose was mostly catabolized by the Entner–Doudoroff and pentose phosphate pathways. Up to 33% of glucose was consumed via the PP pathway under high specific oxygen uptake rate (SOUR) conditions. This amount was 77.9% higher than that under low oxygen uptake conditions. Quantitative evidence was also found for reversible serine hydroxymethyl transferase and threonine aldolase activities. Metabolic flux and cofactor analyses further showed that higher SOUR accelerated the supply of precursors and methyl groups. SOUR also provided more NADPH for higher vitamin B12 production under the same glucose consumption. [Copyright &y& Elsevier]

Abstract: Effects of different oxygen transfer rates (OTR) on the cell growth and vitamin B12 biosynthesis of Pseudomonas denitrificans were first investigated under dissolved oxygen limiting conditions. The results demonstrated that high OTR accelerated cell growth and initial vitamin B12 biosynthesis rate, while lower OTR was critical for higher productivity in the late fermentation process. The oxygen uptake rates (OUR) corresponded well with OTR. Based on the metabolic intermediate analysis, a step-wise OUR control strategy was proposed. The strategy was successfully implemented in scale-up to an industrial fermenter (120,000l). A stable maximum vitamin B12 production of 208±2.5mg/l was achieved, which was increased by 17.3% compared with the control. Furthermore, the glucose consumption coefficient to vitamin B12 was 34.4% lower than that of the control. An efficient and economical fermentation process based on OUR criterion was established for industrial vitamin B12 fermentation by P. denitrificans. [Copyright &y& Elsevier]

The strategy of betaine control for vitamin B12 large-scale fermentation by Pseudomonas denitrificans was investigated in this paper. The results obtained in shake-flask experiments demonstrated that betaine could greatly stimulate vitamin B12 biosynthesis but had an inhibition to cell growth. Based on the influence of betaine on the fermentation of P. denitrificans, betaine feeding was a beneficial strategy to solve the inconsistency between cell growth and vitamin B12 production. As a result, an effective and economical strategy of betaine feeding was established for vitamin B12 fermentation in 120-m3 fermenter, in which betaine was continuously fed to maintain betaine concentration of the broth at the range of 5–7g/l during 50–140h of fermentation. [Copyright &y& Elsevier]

Abstract: A simple model has been formulated to simulate diauxic growth of denitrifying bacteria. It is capable of fitting the experimental results of batch growth experiments with Pseudomonas denitrificans under various conditions. It successfully predicts the observed lags when a pure culture of this bacterium switches from oxygen to nitrate as terminal electron acceptor. The model includes the effect of carbon substrate limitation and length of aerobic phase and does not run into problems when switching from anoxic to aerobic conditions, unlike prior models of diauxic growth. [Copyright &y& Elsevier]

The crystallographic structure of the Pseudomonas denitrificans S-adenosyl-l-methionine-dependent uroporphyrinogen III methyltransferase (SUMT), which is encoded by the cobA gene, has been solved by molecular replacement to 2.7Å resolution. SUMT is a branchpoint enzyme that plays a key role in the biosynthesis of modified tetrapyrroles by controlling flux to compounds such as vitamin B12 and sirohaem, and catalysing the transformation of uroporphyrinogen III into precorrin-2. The overall topology of the enzyme is similar to that of the SUMT module of sirohaem synthase (CysG) and the cobalt-precorrin-4 methyltransferase CbiF and, as with the latter structures, SUMT has the product S-adenosyl-l-homocysteine bound in the crystal. The roles of a number of residues within the SUMT structure are discussed with respect to their conservation either across the broader family of cobalamin biosynthetic methyltransferases or within the sub-group of SUMT members. The D47N, L49A, F106A, T130A, Y183A and M184A variants of SUMT were generated by mutagenesis of the cobA gene, and tested for SAM binding and enzymatic activity. Of these variants, only D47N and L49A bound the co-substrate S-adenosyl-l-methionine. Consequently, all the mutants were severely restricted in their capacity to synthesise precorrin-2, although both the D47N and L49A variants produced significant quantities of precorrin-1, the monomethylated derivative of uroporphyrinogen III. The activity of these variants is interpreted with respect to the structure of the enzyme. [Copyright &y& Elsevier]

The kinetics of biological denitrification have been studied and several models, with varying degree of complexity, to be used for design purposes have been presented in the recent years. However, most of these kinetic studies were performed with mixed (and not well defined) microbial systems, such as activated sludge. In the present work, kinetic experiments were carried out in order to study the dynamic characteristics of a defined mixed culture of the denitrifiers Pseudomonas denitrificans and Bacillus subtilis under anoxic and aerobic conditions in a defined synthetic medium involving a mixture of organic substrates, in the presence of nitrates and/or nitrites. 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 behavior of these defined mixed cultures was predicted using a kinetic model based on the kinetic models that have already been developed for each bacterium separately and the predictions were compared with the results from mixed culture experiments. The overall mathematical model that was developed and validated in the present work is capable of describing the behavior of the mixed culture in the above conditions, i.e. the nitrates and nitrites reduction kinetics, the cell growth, and the organic carbon utilization rates. [Copyright &y& Elsevier]

The growth of Pseudomonas denitrificans ATCC 13867 under denitrifying conditions was significantly stimulated by adding an appropriate amount of formate (2.5 mM or above) to the growth medium. The accumulation of nitrite in the culture was markedly depressed so long as formate remained in the culture above a certain level. Cellular activities of enzymes participating in denitrification also changed. The cells grown in the presence of formate exhibited a lower nitrate reductase activity and, in contrast, a higher nitrite reductase activity than the cells grown without added formate. [ABSTRACT FROM AUTHOR]