Pseudomonas Denitrificans Research Articles

Fermentative production of vitamin B12 by Propionibacterium shermanii and Pseudomonas denitrificans and its promising health benefits: A review

AbstractCobalamin, generally known as vitamin B12, is a crucial component required for humans in several physiological processes. It has been produced from sources that are derived from animals, making it difficult for vegetarians and vegans to consume the recommended amount each day. The importance of vitamin B12 in red blood cell production, DNA synthesis, and brain processes has been highlighted. Recent studies have looked at different methods of producing vitamin B12, such as microbial fermentation. Propionibacterium shermanii and Pseudomonas denitrificans have demonstrated remarkable potential as fermented sources of vitamin B12. Compared to conventional sources, the bioavailability of vitamin B12 produced by P. denitrificans and P. shermanii is more effective in meeting dietary needs. Vitamin B12 can be produced naturally by P. denitrificans. It is equipped with the enzymes and metabolic pathways required to produce this vital vitamin. The fermentation of several dietary substrates by P. shermanii can improve nutrient bioavailability. P. shermanii generates enzymes during fermentation that aid in the breakdown of complex nutrients, facilitating easier absorption and utilization by the body. The motive of the following critical evaluation is to assess the advantages of vitamin B12 for health and the capacity of P. denitrificans and P. shermanii to produce it through fermentation.

Phenol Biodegradation by Three Bacterial Strains Stimulated by Constant Electric Field

Objective: The aim of the present study is to compare phenol biodegradation activity of bacteria from the strains Xanthobacter autotrophicus GJ 10, Pseudomonas denitrificans and Pseudomonas putida enhanced by constant electric field in a potentiostatic mode by free cultures and immobilized cells on granulated activated carbon. Theoretical Framework: The work is based on the concept of biodegradation in constant electric field due to the enhanced removal of intermediate inhibitors produced from phenol. These intermediate may affect slightly microbial growth but the biodegradation capacity is enhanced. Method: The methodology adopted for this research comprises bioelectrochemical oxidative microbial biodegradation of phenol at constant anode potential. Results and Discussion: The obtained results revealed the positive effect of the electric field on the phenol biodegradation rate for all of the considered strains. There is certain anode potential at which the biodegradation rate is strongly affected for each strain. Research Implications: The research have both practical and theoretical implications. The practical importance consists in the enhanced biodegradation of severe pollutant as phenol is. The theoretical importance is within the specific impact of constant electric field on the enzyme activities in the studied strains. Originality/Value: This study contributes to the literature by the innovative approach and the practical aspects. The relevance and value of this research are demosntated by enhancement of phenol biodegradation in wastewater and water ponds.

Differential Deep RNA Sequencing for Diagnostic Detection of Microbial Infections in Inflammatory Cardiomyopathy.

Inflammatory heart disease can be triggered by a variety of causes, both infectious and noninfectious in nature. We hypothesized that inflammatory cardiomyopathy is potentially related to microbial infection. In this retrospective study, we used deep RNA sequencing on formalin-fixed paraffin-embedded heart tissue specimens to detect pathogenic agents. We first investigated 4 single-sample cases to test the feasibility of this diagnostic protocol and further 3 control-sample paired cases to improve the protocol with differential metatranscriptomics next-generation sequencing (mtNGS) analysis. We demonstrate that differential mtNGS allows identification of various microbials as potentially pathogenic, for example, Cutibacterium acnes, Corynebacterium aurimucosum, and Pseudomonas denitrificans, which are usually commensal in healthy individuals. Differential mtNGS also allows characterization of human host response in each individual by profiling alterations of gene expression, networked pathways, and inferred immune cell compositions, information of which is beneficial for us to understand different etiologies and immunity roles in each case. Additionally, differential mtNGS allows the identification of genetic variants in patients that may contribute to their susceptibility to particular microbial infections. The demonstrated power of differential mtNGS in simultaneous capture of both the infectious microbial(s) and the status of human host immune response could help us better understand the pathogenesis of complex inflammatory cardiomyopathy, if conducted on a larger scale of the population. The developed differential mtNGS method could also shed light on its translation and adoption of such a laboratory test in clinic practice, allowing for a more effective diagnosis to guide therapeutic treatment of the disease.

Study of bacterial population dynamics in seed culture developed for ammonia reduction from synthetic wastewater.

The waterbodies have been polluted by various natural and anthropogenic activities. The aquatic waste includes ammonia as one of the most toxic pollutants. Several biological treatment systems involving anoxic and semi anoxic bacteria have been proposed for reducing nitrogen loads from wastewater and increasing the efficiency and cost effectiveness. These bacteria play a vital role in the processes involved in the nitrogen cycle in nature. However, the enrichment, sustainability and identification of bacterial communities for wastewater treatment is an important aspect. Most of the chemolithotrophs are unculturable hence their identification and measurement of abundance remains a challenging task. In this study the different bacteria involved in total nitrogen removal from the wastewater are enriched for 700days under anoxic condition. The synthetic wastewater containing 0.382g/L of ammonium chloride was used. Molecular identification of the bacteria involved in various steps of the nitrogen cycle was carried out based on amplification of functional genes and 16S rRNA gene Polymerase chain reaction followed by DNA sequencing. Change in the abundance of chemolithotrophs was studied using qPCR. The mutual growth of various nitrifiers along with anaerobic bacteria were identified by molecular characterisation of DNA at various time intervals with the different genes involved in the nitrogen cycle. Nitrosomonas species like Nitrosomonas europaea were identified throughout the batch scale studies possessing the genes associated with ammonia oxidizing bacteria and nitrite oxidizing bacteria which act as a complete ammonia oxidizer. The uncultured species of Nitrospira and anammox bacteria were also observed which predicts the coexistence of the anammox and comammox bacteria in a batch scale study. The coexistence of the semi anoxic and anoxic bacteria helped in the growth of these bacteria for a longer duration of time. The nitrite produced by the comammox during nitrification can be utilized by anammox as an electron carrier. The other species of denitrifiers like Pseudomonas denitrificans and Aminobacter aminovorans were also observed. It is concluded that the enrichment of semi anoxic and anoxic bacteria was faster with the increase in growth of the bacteria involved in nitrification, comammox, anammox and partial denitrification process. The bacterial growth is enhanced and the efficiency is increased which can be further used in the development of small pilot scale bioreactor for total nitrogen removal.

Research on the targeted improvement of the yield of a new VB12-producing strain, Ensifer adhaerens S305, based on genomic and transcriptomic analysis

BackgroundVitamin B12 (VB12) has a wide range of applications and high economic value. In this study, a new strain with high VB12 production potential, Ensifer adhaerens S305, was identified in sewage. Because E. adhaerens strains have become the main strains for VB12 production via fermentation in recent years, the directional modification of the S305 strain to obtain a strain suitable for the industrial production of VB12 has great potential and commercial value.Results16S rRNA and genome-wide phylogenetic tree analysis combined with average nucleotide identity (ANI) analysis showed that the high-yielding VB12 strain was a E. adhaerens strain and that its VB12 synthesis pathway genes were highly similar to related genes of strains of this and other species, including E. adhaerens Casida A, Pseudomonas denitrificans SC 510, and E. adhaerens Corn53. High-pressure liquid chromatography (HPLC) results indicated that the VB12 yields of the S305 strain were more than double those of the Casida A strain under different medium components. Multiple genes with significantly upregulated and downregulated transcription were identified by comparing the transcription intensity of different genes through transcriptome sequencing. KEGG enrichment analysis of the porphyrin metabolism pathway identified 9 significantly upregulated and downregulated differentially expressed genes (DEGs) in the VB12 synthesis pathway, including 7 transcriptionally upregulated genes (cobA, cobT, hemA, cobJ, cobN, cobR, and cobP) that were episomally overexpressed in the Casida A strain. The results showed that the VB12 yield of the overexpressed strain was higher than that of the wild-type strain. Notably, the strains overexpressing the cobA and cobT genes exhibited the most significant increases in VB12 yield, i.e., 31.4% and 24.8%, respectively. The VB12 yield of the S305 strain in shake-flask culture was improved from 176.6 ± 8.21 mg/L to 245.6 ± 4.36 mg/L by integrating the cobA and cobT genes into the strain.ConclusionPhylogenetic tree and ANI analysis showed that the Ensifer and Sinorhizobium strains were quite different at the genome level; the overexpression and integrated expression of significantly upregulated genes in the VB12 synthesis pathway could increase the yield of VB12, further improving the VB12 yield of the E. adhaerens S305 strain.

Methylotrophic bacteria with cobalamin-dependent mutases in primary metabolism as potential strains for vitamin B12 production

Several bacterial species are known for their ability to synthesize vitamin B12 but biotechnological vitamin B12 production today is restricted to Pseudomonas denitrificans and Propionibacterium freudenreichii. Nevertheless, the rising popularity of veganism leads to a growing demand for vitamin B12 and thereby interest in alternative strains which can be used as efficient vitamin B12 sources. In this work, we demonstrate that methylotrophic microorganisms which utilize the ethylmalonyl-CoA pathway containing B12-dependent enzymes are capable of active vitamin B12 production. Several bacteria with an essential function of the pathway were tested for vitamin B12 synthesis. Among the identified strains, Hyphomicrobium sp. DSM3646 demonstrated the highest vitamin B12 levels reaching up to 17.9 ± 5.05 µg per g dry cell weight. These relatively high vitamin B12 concentrations achieved in simple cultivation experiments were performed in a mineral methanol medium, which makes Hyphomicrobium sp. DSM3646 a new promising cobalamin-producing strain.

Shift of Choline/Betaine Pathway in Recombinant Pseudomonas for Cobalamin Biosynthesis and Abiotic Stress Protection.

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.

Bioprocess Strategies for Vitamin B12 Production by Microbial Fermentation and Its Market Applications.

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.

Zng1 is a GTP-dependent zinc transferase needed for activation of methionine aminopeptidase.

The evolution of zinc (Zn) as a protein cofactor altered the functional landscape of biology, but dependency on Zn also created an Achilles' heel, necessitating adaptive mechanisms to ensure Zn availability to proteins. A debated strategy is whether metallochaperones exist to prioritize essential Zn-dependent proteins. Here, we present evidence for a conserved family of putative metal transferases in human and fungi, which interact with Zn-dependent methionine aminopeptidase type I (MetAP1/Map1p/Fma1). Deletion of the putative metal transferase in Saccharomyces cerevisiae (ZNG1; formerly YNR029c) leads to defective Map1p function and a Zn-deficiency growth defect. Invitro, Zng1p can transfer Zn2+ or Co2+ to apo-Map1p, but unlike characterized copper chaperones, transfer is dependent on GTP hydrolysis. Proteomics reveal mis-regulation of the Zap1p transcription factor regulon because of loss of ZNG1 and Map1p activity, suggesting that Zng1p is required to avoid a compounding effect of Map1p dysfunction on survival during Zn limitation.

Improvement of 3-hydroxypropionic acid tolerance in Klebsiella pneumoniae by novel transporter YohJK

3-Hydroxypropionic acid (3-HP) is a platform chemical which has potential applications in cosmetic and polymer industries. Microbial production of 3-HP is hampered by its toxic effect when its concentration is high (>300 mM). In this study, the effect of yohJK overexpression (via yieP deletion or episomal overexpression) on 3-HP tolerance was investigated in Klebsiella pneumoniae, Pseudomonas denitrificans and P. asiatica. The deletion of yieP homolog could improve 3-HP tolerance in K. pneumoniae. Transcriptional analysis suggested that, among the two yohJK homologs of K. pneumoniae, expression of yohJK1, not yohJK2, was under the negative control of YieP. Furthermore, deletion of yieP significantly reduced cytoplasmic 3-HP concentration when determined by 3-HP biosensor and enhanced 3-HP tolerance and 3-HP production. This study demonstrates that the YohJK1 functions as 3-HP transporter in K. pneumoniae and their overexpression by the yieP deletion is a good strategy to enhance 3-HP tolerance and its production.

In vivo Characterization of the Inducible Promoter System of 3-hydroxypropionic Dehydrogenase in Pseudomonas denitrificans

In vivo Characterization of the Inducible Promoter System of 3-hydroxypropionic Dehydrogenase in Pseudomonas denitrificans

English

In this study we assayed on antimicrobial activity of peel, pulp, waxy coating of Benincasa hispida. Various parts of the plant are reported as antibacterial agents worldwide. Leaves, flower, root, seed are the most studied elements as antibacterial agent. Some fruits are also proved as antibacterial agent and used as herbal medicine as well as nutritional supplements during disease. Different parts of mature and immature winter melon fruits were extracted with different organic solvents viz., methanol, ethyl acetate and chloroform. These extracts were subjected to test against selected pathogenic bacterial (Bacillus subtilis IFO 3026, Sarcina lutea IFO 3232, Xanthomonas campestris IAM 1671, Escherichia coli iw 3310 IFO 3007, Pseudomonas denitrificans KACC 32026, and fungal (Fusarium oxysporum, Aspergillus niger KTCC 1700, Collectotrichum melongenae) strains using the well diffusion method. The waxy coating and peel of mature fruit of B. hispida extracts has strong antibacterial activities than immature fruit. In these cases, the minimum inhibitory concentration was 128 μg/ml. Ethyl acetate and chloroform extracts of waxy coating of B. hispida showed inhibition rate against fungal infection 43% and 33%, respectively against F. oxysporum but there is no inhibition against A. niger, C. melongenae. These results suggested that. So, the B. hispida would be a potential source that may help to develop natural antimicrobial agents. Key words: Antibacterial, a fruit, winter melon, infectious, extracts, minimum inhibitory concentration (MIC).

Revealing the Promoting Effect of Betaine on Vitamin B12 Biosynthetic Pathway of Pseudomonas denitrificans by Using a Proteomics Analysis.

Our previous comparative metabolomics research revealed that betaine (N,N,Ntrimethylglycine, a typically essential methyl-group donor for vitamin B12 biosynthesis) had powerful promoting effect on the generation of vitamin B12 precursors and intermediates in vitamin B12-producing Pseudomonas denitrificans. However, the integral effect of betaine on the vitamin B12 biosynthetic pathway is still unclear. Considering the vitamin B12 biosynthetic pathway of P. denitrificans as a whole, this work aimed to reveal the biological function of betaine on the vitamin B12 biosynthetic pathway in P. denitrificans, which would sharpen and expand understanding of betaine as the methyl-group donor for vitamin B12 biosynthesis. By using a proteomics method based on the iTRAQ technique, the present study compared and analyzed the differential expression of proteins involved in vitamin B12 biosynthetic pathway under 10 g/L betaine in addition to P. denitrificans fermentation medium. The results showed that betaine could significantly up-regulate the expression of proteins related to the vitamin B12 biosynthetic pathway, which was mainly reflected in the following three aspects: 1) the δ-aminolevulinic acid (ALA) synthase and porphobilinogen synthase that were responsible for the formation of the committed precursors for tetrapyrrole-derived macrocycle in vitamin B12 molecule; 2) the C-methylation-related enzymes (such as precorrin-4 C(11)-methyltransferase, precorrin-2 C(20)- methyltransferase, precorrin-8X methylmutase, and precorrin-6Y C5,15-methyltransferase) and methionine synthase that were crucial to the C-methylation reactions for vitamin B12 biosynthesis; 3) the latestage key enzymes (Cobaltochelatase, and Cob(I)yrinic acid a,c-diamide adenosyltransferase) that were related to cobalt chelation of vitamin B12 molecule. The present study demonstrated clearly that betaine could significantly promote the expression of the integral enzymes involved in the vitamin B12 biosynthetic pathway of P. denitrificans, thus promoting vitamin B12 biosynthesis.

Microbial and Genetic Resources for Cobalamin (Vitamin B12) Biosynthesis: From Ecosystems to Industrial Biotechnology.

Many microbial producers of coenzyme B12 family cofactors together with their metabolically interdependent pathways are comprehensively studied and successfully used both in natural ecosystems dominated by auxotrophs, including bacteria and mammals, and in the safe industrial production of vitamin B12. Metabolic reconstruction for genomic and metagenomic data and functional genomics continue to mine the microbial and genetic resources for biosynthesis of the vital vitamin B12. Availability of metabolic engineering techniques and usage of affordable and renewable sources allowed improving bioprocess of vitamins, providing a positive impact on both economics and environment. The commercial production of vitamin B12 is mainly achieved through the use of the two major industrial strains, Propionobacterium shermanii and Pseudomonas denitrificans, that involves about 30 enzymatic steps in the biosynthesis of cobalamin and completely replaces chemical synthesis. However, there are still unresolved issues in cobalamin biosynthesis that need to be elucidated for future bioprocess improvements. In the present work, we review the current state of development and challenges for cobalamin (vitamin B12) biosynthesis, describing the major and novel prospective strains, and the studies of environmental factors and genetic tools effecting on the fermentation process are reported.

Groundwater Nitrate Removal Performance of Selected Pseudomonas Strains Carrying nosZ Gene in Aerobic Granular Sequential Batch Reactors

Four granular sequencing batch reactors (GSBRs) were inoculated with four denitrifying Pseudomonas strains carrying nosZ to study the process of granule formation, the operational conditions of the bioreactors, and the carbon concentration needed for nitrate removal. The selected Pseudomonas strains were P. stutzeri I1, P. fluorescens 376, P. denitrificans Z1, and P. fluorescens PSC26, previously reported as denitrifying microorganisms carrying the nosZ gene. Pseudomonas denitrificans Z1 produced fluffy, low-density granules, with a decantation speed below 10 m h−1. However, P. fluorescens PSC26, P. stutzeri I1, and P. fluorescens 376 formed stable granules, with mean size from 7 to 15 mm, related to the strain and carbon concentration. P. stutzeri I1 and P. fluorescens 376 removed nitrate efficiently with a ratio in the range of 96%, depending on the source and concentration of organic matter. Therefore, the findings suggest that the inoculation of GSBR systems with denitrifying strains of Pseudomonas spp. containing the nosZ gene enables the formation of stable granules, the efficient removal of nitrate, and the transformation of nitrate into nitrogen gas, a result of considerable environmental interest to avoid the generation of nitrous oxide.

Use of acetate for the production of 3-hydroxypropionic acid by metabolically-engineered Pseudomonas denitrificans

The use of acetate as carbon feedstock can enhance sustainability and economics of the current bio-productions. This study explored the potential of acetate for the production of 3-hydroxypropionic acid by engineered Pseudomonas denitrificans. Heterologous mcr (encoding malonyl-CoA reductase) from Chloroflexus aurantiacus and endogenous accABCD (encoding acetyl-CoA carboxylase) were overexpressed in P. denitrificans. Carbon flux to 3-HP synthesis at the malonyl-CoA node was promoted by suppressing fatty acid synthesis through addition of cerulenin or deletion of fabF gene. In addition, stimulation of glyoxylate shunt and/or TCA cycle were attempted. Recombinant P. denitrificans overexpressing mcr and accABCD produced 19.3 mM 3-HP with cerulenin addition, and 14.2 mM with fabF deletion, respectively. Furthermore, the non-growing cells devoid of fabF could continuously produce 3-HP up to 40.4 mM without losing its production activity for 22 h. This study demonstrates that acetate is a good substrate for 3-HP production by recombinant P. denitrificans.

Denitrification characteristics and pathways of a facultative anaerobic denitrifying strain, Pseudomonas denitrificans G1

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 24h, with the total nitrogen removal rate of 33-38% (87-100mg/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 0-4.68mg/L, salinity 0-30g NaCl/L, pH 7-9.5. Under the aerobic condition, G1 grew fast; however, the mass spectrographic analysis showed that the gas product was N2O. Under the anaerobic conditions, G1 grow relatively slowly, but could also achieve effective denitrification and the final product was N2. 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.

Betaine biosynthesis in a heterologous expression system based on the B12 producer Pseudomonas denitrificans

Glycine betaine is an important donor of methyl groups in various metabolic processes of the cell and acts as an osmoprotector when exposed to various abiotic stresses in pro- and eukaryotic organisms. Moreover, exogenous application of betaine activates the production of vitamin B12 in industrial strains-producers. In this work, we have developed a new technology for microbiological betaine synthesis that can be used in biotechnology to activate B12 biosynthesis during large-scale fermentation of Pseudomonas denitrificans.

Author Correction: Improving Substrate Consumption and Decrease of Growth Yield in Aerobic Cultures of Pseudomonas denitrificans By Applying Low Voltages in Bioelectric Systems.

The original version of this article unfortunately contained a mistake in Equation 1.

Improving Substrate Consumption and Decrease of Growth Yield in Aerobic Cultures of Pseudomonas denitrificans By Applying Low Voltages in Bioelectric Systems.

It is well known that activated sludge treatment systems generate a lot of surplus sludge having environmental and economic impacts. Although several approaches have been proposed for the treatment/reuse of the excess of sludge, there are few studies focused on decreasing the biomass yield without affecting the metabolic activity. This work reports the effect of low magnitude electrical fields (0.07 to 0.2 V/cm) on the growth yield of a pure strain of Pseudomonas denitrificans (used as model microorganism). Cell potentials between 0.2 and 0.57 V were measured during 24 h to the aerobic culture; biomass production and substrate consumption were evaluated at regular intervals. Results indicated that the substrate (lactate) consumption efficiency increased with the applied potential, up to 100%, while the yield diminished 31% (0.34 g biomass/g lactate consumed) at 0.7 V vs. NHE. Bioenergetics showed that the fraction of electron equivalents toward biomass synthesis decreased from 0.68 (when no potential was applied) to 0.47 at 0.57 V, pointing out the redirection of the energy flow toward maintenance to cope with the stress caused by the imposed voltage. Therefore, the electrical stimulus could be used as control of biomass growth in aerobic wastewater treatment lines.