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1.
World J Microbiol Biotechnol ; 36(3): 46, 2020 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-32140791

RESUMO

Azotobacter vinelandii is a microorganism with biotechnological potential because its ability to produce alginate and polyhydroxybutyrate. Large-scale biotechnological processes are oriented to sustainable production by using biomass hydrolysates that are mainly composed by glucose and xylose. In the present study, it was observed that A. vinelandii was unable to consume xylose as the sole carbon source and that glucose assimilation in the presence of xylose was negatively affected. Adaptive Laboratory Evolution (ALE) was used as a metabolic engineering tool in A. vinelandii, to improve both carbohydrate assimilation. As a result of ALE process, the CT387 strain was obtained. The evolved strain (CT387) grown in shaken flask cultivations with xylose (8 g L-1) and glucose (2 g L-1), showed an increase of its specific growth rate (µ), as well as of its glucose and xylose uptake rates of 2, 6.45 and 3.57-fold, respectively, as compared with the parental strain. At bioreactor level, the µ, the glucose consumption rate and the relative expression of gluP that codes for the glucose permease in the evolved strain were also higher than in the native strain (1.53, 1.29 and 18-fold, respectively). Therefore, in the present study, we demonstrated the potential of ALE as a metabolic engineering tool for improving glucose and xylose consumption in A. vinelandii.


Assuntos
Azotobacter vinelandii/metabolismo , Glucose/metabolismo , Engenharia Metabólica/métodos , Xilose/metabolismo , Azotobacter vinelandii/genética , Azotobacter vinelandii/crescimento & desenvolvimento , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biomassa , Reatores Biológicos , Meios de Cultura/química , Fermentação , Regulação Bacteriana da Expressão Gênica , RNA Bacteriano/genética , RNA Bacteriano/isolamento & purificação
2.
PLoS One ; 13(12): e0209020, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30566461

RESUMO

The influence of nanomaterials on the ecological environment is becoming an increasingly hot research field, and many researchers are exploring the mechanisms of nanomaterial toxicity on microorganisms. Herein, we studied the effect of two different sizes of nanosilver (10 nm and 50 nm) on the soil nitrogen fixation by the model bacteria Azotobacter vinelandii. Smaller size AgNPs correlated with higher toxicity, which was evident from reduced cell numbers. Flow cytometry analysis further confirmed this finding, which was carried out with the same concentration of 10 mg/L for 12 h, the apoptotic rates were20.23% and 3.14% for 10 nm and 50 nm AgNPs, respectively. Structural damage to cells were obvious under scanning electron microscopy. Nitrogenase activity and gene expression assays revealed that AgNPs could inhibit the nitrogen fixation of A. vinelandii. The presence of AgNPs caused intracellular reactive oxygen species (ROS) production and electron spin resonance further demonstrated that AgNPs generated hydroxyl radicals, and that AgNPs could cause oxidative damage to bacteria. A combination of Ag content distribution assays and transmission electron microscopy indicated that AgNPs were internalized in A. vinelandii cells. Overall, this study suggested that the toxicity of AgNPs was size and concentration dependent, and the mechanism of antibacterial effects was determined to involve damage to cell membranes and production of reactive oxygen species leading to enzyme inactivation, gene down-regulation and death by apoptosis.


Assuntos
Apoptose/efeitos dos fármacos , Azotobacter vinelandii/efeitos dos fármacos , Nanopartículas Metálicas/toxicidade , Estresse Oxidativo/efeitos dos fármacos , Compostos de Prata/toxicidade , Azotobacter vinelandii/crescimento & desenvolvimento , Azotobacter vinelandii/metabolismo , Azotobacter vinelandii/ultraestrutura , Proteínas de Bactérias/metabolismo , Poluentes Ambientais , Expressão Gênica/efeitos dos fármacos , Radical Hidroxila/metabolismo , Nanopartículas Metálicas/química , Fixação de Nitrogênio/efeitos dos fármacos , Tamanho da Partícula , Espécies Reativas de Oxigênio/metabolismo , Compostos de Prata/química
3.
PLoS One ; 13(12): e0208975, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30543677

RESUMO

Azotobacter vinelandii is a nitrogen-fixing bacterium of the Pseudomonadaceae family that prefers the use of organic acids rather than carbohydrates. Thus, in a mixture of acetate-glucose, glucose is consumed only after acetate is exhausted. In a previous work, we investigated the molecular basis of this carbon catabolite repression (CCR) process under diazotrophic conditions. In the presence of acetate, Crc-Hfq inhibited translation of the gluP mRNA, encoding the glucose transporter in A. vinelandii. Herein, we investigated the regulation in the expression of the small non-coding RNAs (sRNAs) crcZ and crcY, which are known to antagonize the repressing activity of Hfq-Crc. Our results indicated higher expression levels of the sRNAs crcZ and crcY under low CCR conditions (i.e. glucose), in relation to the strong one (acetate one). In addition, we also explored the process of CCR in the presence of ammonium. Our results revealed that CCR also occurs under non-diazotrophic conditions as we detected a hierarchy in the utilization of the supplied carbon sources, which was consistent with the higher expression level of the crcZ/Y sRNAs during glucose catabolism. Analysis of the promoters driving transcription of crcZ and crcY confirmed that they were RpoN-dependent but we also detected a processed form of CrcZ (CrcZ*) in the RpoN-deficient strain derived from a cbrB-crcZ co-transcript. CrcZ* was functional and sufficient to allow the assimilation of acetate.


Assuntos
Azotobacter vinelandii/genética , Repressão Catabólica/genética , Glucose/metabolismo , Pequeno RNA não Traduzido/genética , Acetatos/metabolismo , Azotobacter vinelandii/crescimento & desenvolvimento , Azotobacter vinelandii/metabolismo , Carbono/química , Carbono/metabolismo , Regulação Bacteriana da Expressão Gênica/genética , Glucose/genética , Fixação de Nitrogênio/genética , Regiões Promotoras Genéticas , RNA Mensageiro/genética
4.
Mol Biotechnol ; 60(9): 670-680, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-29987520

RESUMO

The GacS/A system in Azotobacter vinelandii regulates alginate and alkylresorcinols production through RsmZ1, a small regulatory RNA (sRNA) that releases the translational repression of the algD and arpR mRNAs caused by the RsmA protein. In the Pseudomonadaceae family, the Rsm-sRNAs are grouped into three families: RsmX, RmsY and RsmZ. Besides RsmZ1, A. vinelandii has six other isoforms belonging to the RsmZ family and another one to the RsmY. Environmental signals controlling rsmsRNAs genes in A. vinelandii are unknown. In this work, we present a transcriptional study of the A. vinelandii rsmZ1-7-sRNAs genes, whose transcriptional profiles showed a differential expression pattern, but all of them exhibited their maximal expression at the stationary growth phase. Furthermore, we found that succinate promoted higher expression levels of all the rsmZ1-7 genes compared to glycolytic carbon sources. Single mutants of the rsmZ-sRNAs family were constructed and their impact on alginate production was assessed. We did not observe correlation between the alginate phenotype of each rsmZ-sRNA mutant and the expression level of the corresponding sRNA, which suggests the existence of additional factors affecting their impact on alginate production. Similar results were found in the regulation exerted by the RsmZ-sRNAs on alkylresorcinol synthesis.


Assuntos
Alginatos/metabolismo , Azotobacter vinelandii/genética , Regulação Bacteriana da Expressão Gênica , RNA Bacteriano/genética , Pequeno RNA não Traduzido/genética , Resorcinóis/metabolismo , Azotobacter vinelandii/crescimento & desenvolvimento , Azotobacter vinelandii/metabolismo , Metabolismo dos Carboidratos , Meios de Cultura/química , Perfilação da Expressão Gênica , Mutação , Regiões Promotoras Genéticas/genética , RNA Bacteriano/metabolismo , Pequeno RNA não Traduzido/metabolismo , Resorcinóis/química , Transcrição Genética
5.
Methods Mol Biol ; 1772: 45-60, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29754222

RESUMO

This work is motivated by both (1) the desire to make interesting products without the reliance on fixed carbon or fixed nitrogen using microbial cocultures, and (2) the desire to develop new methods for growing microbial communities. The bioplastic polyhydroxybutyrate (PHB), for example, is considered prohibitively expensive to make from sugar (compared to polypropylene). Utilizing and building on engineered strains of Synechococcus elongatus and Azotobacter vinelandii, we have combined a nitrogen-fixing organism with a carbon-fixing organism to make PHB from air, water, sunlight, and trace minerals. Our observations of coculture growth in batch culture led us to develop an improved system based on manipulating the osmotic pressure within a hydrogel. The methods we used to develop this coculture are described in detail in the following chapter, including notes detailing some of our additional observations or thoughts on this system.


Assuntos
Azotobacter vinelandii/crescimento & desenvolvimento , Carbono/metabolismo , Técnicas de Cocultura/métodos , Nitrogênio/metabolismo , Synechococcus/crescimento & desenvolvimento , Hidrogéis , Hidroxibutiratos/metabolismo , Água/metabolismo
6.
World J Microbiol Biotechnol ; 33(11): 198, 2017 Oct 07.
Artigo em Inglês | MEDLINE | ID: mdl-28988302

RESUMO

Alginate is a linear polysaccharide that can be used for different applications in the food and pharmaceutical industries. These polysaccharides have a chemical structure composed of subunits of (1-4)-ß-D-mannuronic acid (M) and its C-5 epimer α-L-guluronic acid (G). The monomer composition and molecular weight of alginates are known to have effects on their properties. Currently, these polysaccharides are commercially extracted from seaweed but can also be produced by Azotobacter vinelandii and Pseudomonas spp. as an extracellular polymer. One strategy to produce alginates with different molecular weights and with reproducible physicochemical characteristics is through the manipulation of the culture conditions during fermentation. This mini-review provides a comparative analysis of the metabolic pathways and molecular mechanisms involved in alginate polymerization from A. vinelandii and Pseudomonas spp. Different fermentation strategies used to produce alginates at a bioreactor laboratory scale are described.


Assuntos
Alginatos/metabolismo , Azotobacter vinelandii/crescimento & desenvolvimento , Pseudomonas/crescimento & desenvolvimento , Alginatos/química , Azotobacter vinelandii/genética , Azotobacter vinelandii/metabolismo , Reatores Biológicos , Fermentação , Ácido Glucurônico/química , Ácido Glucurônico/metabolismo , Ácidos Hexurônicos/química , Ácidos Hexurônicos/metabolismo , Redes e Vias Metabólicas , Peso Molecular , Pseudomonas/genética , Pseudomonas/metabolismo
7.
Methods Enzymol ; 595: 261-302, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28882203

RESUMO

Nitrogenase is a metalloenzyme system that plays a critical role in biological nitrogen fixation, and the study of how its metallocenters are assembled into functional entities to facilitate the catalytic reduction of dinitrogen to ammonia is an active area of interest. The diazotroph Azotobacter vinelandii is especially amenable to culturing and genetic manipulation, and this organism has provided the basis for many insights into the assembly of nitrogenase proteins and their respective metallocofactors. This chapter will cover the basic procedures necessary for growing A. vinelandii cultures and subsequent recombinant transformation and protein expression techniques. Furthermore, protocols for nitrogenase protein purification and substrate reduction activity assays are described. These methods provide a solid framework for the assessment of nitrogenase assembly and catalysis.


Assuntos
Azotobacter vinelandii/enzimologia , Metaloproteínas/biossíntese , Metaloproteínas/química , Nitrogenase/biossíntese , Nitrogenase/química , Amônia/metabolismo , Azotobacter vinelandii/genética , Azotobacter vinelandii/crescimento & desenvolvimento , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Biocatálise , Ferro/metabolismo , Metaloproteínas/genética , Metaloproteínas/isolamento & purificação , Molibdoferredoxina/química , Molibdoferredoxina/metabolismo , Nitrogênio/metabolismo , Nitrogenase/genética , Nitrogenase/isolamento & purificação , Conformação Proteica
8.
Appl Environ Microbiol ; 83(20)2017 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-28802272

RESUMO

Biological nitrogen fixation is accomplished by a diverse group of organisms known as diazotrophs and requires the function of the complex metalloenzyme nitrogenase. Nitrogenase and many of the accessory proteins required for proper cofactor biosynthesis and incorporation into the enzyme have been characterized, but a complete picture of the reaction mechanism and key cellular changes that accompany biological nitrogen fixation remain to be fully elucidated. Studies have revealed that specific disruptions of the antiactivator-encoding gene nifL result in the deregulation of the nif transcriptional activator NifA in the nitrogen-fixing bacterium Azotobacter vinelandii, triggering the production of extracellular ammonium levels approaching 30 mM during the stationary phase of growth. In this work, we have characterized the global patterns of gene expression of this high-ammonium-releasing phenotype. The findings reported here indicated that cultures of this high-ammonium-accumulating strain may experience metal limitation when grown using standard Burk's medium, which could be amended by increasing the molybdenum levels to further increase the ammonium yield. In addition, elevated levels of nitrogenase gene transcription are not accompanied by a corresponding dramatic increase in hydrogenase gene transcription levels or hydrogen uptake rates. Of the three potential electron donor systems for nitrogenase, only the rnf1 gene cluster showed a transcriptional correlation to the increased yield of ammonium. Our results also highlight several additional genes that may play a role in supporting elevated ammonium production in this aerobic nitrogen-fixing model bacterium.IMPORTANCE The transcriptional differences found during stationary-phase ammonium accumulation show a strong contrast between the deregulated (nifL-disrupted) and wild-type strains and what was previously reported for the wild-type strain under exponential-phase growth conditions. These results demonstrate that further improvement of the ammonium yield in this nitrogenase-deregulated strain can be obtained by increasing the amount of available molybdenum in the medium. These results also indicate a potential preference for one of two ATP synthases present in A. vinelandii as well as a prominent role for the membrane-bound hydrogenase over the soluble hydrogenase in hydrogen gas recycling. These results should inform future studies aimed at elucidating the important features of this phenotype and at maximizing ammonium production by this strain.


Assuntos
Compostos de Amônio/metabolismo , Azotobacter vinelandii/genética , Azotobacter vinelandii/metabolismo , Proteínas de Bactérias/genética , Fixação de Nitrogênio , Azotobacter vinelandii/enzimologia , Azotobacter vinelandii/crescimento & desenvolvimento , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Hidrogenase/genética , Hidrogenase/metabolismo , Família Multigênica , Nitrogênio/metabolismo , Nitrogenase/genética , Nitrogenase/metabolismo
9.
Appl Environ Microbiol ; 83(13)2017 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-28432097

RESUMO

Overcoming the inhibitory effects of excess environmental ammonium on nitrogenase synthesis or activity and preventing ammonium assimilation have been considered strategies to increase the amount of fixed nitrogen transferred from bacterial to plant partners in associative or symbiotic plant-diazotroph relationships. The GlnE adenylyltransferase/adenylyl-removing enzyme catalyzes reversible adenylylation of glutamine synthetase (GS), thereby affecting the posttranslational regulation of ammonium assimilation that is critical for the appropriate coordination of carbon and nitrogen assimilation. Since GS is key to the sole ammonium assimilation pathway of Azotobacter vinelandii, attempts to obtain deletion mutants in the gene encoding GS (glnA) have been unsuccessful. We have generated a glnE deletion strain, thus preventing posttranslational regulation of GS. The resultant strain containing constitutively active GS is unable to grow well on ammonium-containing medium, as previously observed in other organisms, and can be cultured only at low ammonium concentrations. This phenotype is caused by the lack of downregulation of GS activity, resulting in high intracellular glutamine levels and severe perturbation of the ratio of glutamine to 2-oxoglutarate under excess-nitrogen conditions. Interestingly, the mutant can grow diazotrophically at rates comparable to those of the wild type. This observation suggests that the control of nitrogen fixation-specific gene expression at the transcriptional level in response to 2-oxoglutarate via NifA is sufficiently tight to alone regulate ammonium production at levels appropriate for optimal carbon and nitrogen balance.IMPORTANCE In this study, the characterization of the glnE knockout mutant of the model diazotroph Azotobacter vinelandii provides significant insights into the integration of the regulatory mechanisms of ammonium production and ammonium assimilation during nitrogen fixation. The work reveals the profound fidelity of nitrogen fixation regulation in providing ammonium sufficient for maximal growth but constraining energetically costly excess production. A detailed fundamental understanding of the interplay between the regulation of ammonium production and assimilation is of paramount importance in exploiting existing and potentially engineering new plant-diazotroph relationships for improved agriculture.


Assuntos
Azotobacter vinelandii/enzimologia , Proteínas de Bactérias/genética , Deleção de Genes , Glutamato-Amônia Ligase/genética , Fixação de Nitrogênio , Compostos de Amônio/metabolismo , Azotobacter vinelandii/genética , Azotobacter vinelandii/crescimento & desenvolvimento , Azotobacter vinelandii/fisiologia , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Glutamato-Amônia Ligase/metabolismo
10.
J Biol Inorg Chem ; 22(1): 161-168, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-27928630

RESUMO

The alternative, vanadium-dependent nitrogenase is employed by Azotobacter vinelandii for the fixation of atmospheric N2 under conditions of molybdenum starvation. While overall similar in architecture and functionality to the common Mo-nitrogenase, the V-dependent enzyme exhibits a series of unique features that on one hand are of high interest for biotechnological applications. As its catalytic properties differ from Mo-nitrogenase, it may on the other hand also provide invaluable clues regarding the molecular mechanism of biological nitrogen fixation that remains scarcely understood to date. Earlier studies on vanadium nitrogenase were almost exclusively based on a ΔnifHDK strain of A. vinelandii, later also in a version with a hexahistidine affinity tag on the enzyme. As structural analyses remained unsuccessful with such preparations we have developed protocols to isolate unmodified vanadium nitrogenase from molybdenum-depleted, actively nitrogen-fixing A. vinelandii wild-type cells. The procedure provides pure protein at high yields whose spectroscopic properties strongly resemble data presented earlier. Analytical size-exclusion chromatography shows this preparation to be a VnfD2K2G2 heterohexamer.


Assuntos
Azotobacter vinelandii/enzimologia , Molibdênio/farmacologia , Nitrogenase/biossíntese , Nitrogenase/isolamento & purificação , Azotobacter vinelandii/efeitos dos fármacos , Azotobacter vinelandii/crescimento & desenvolvimento , Azotobacter vinelandii/metabolismo , Biocatálise , Meios de Cultura/química , Relação Dose-Resposta a Droga , Nitrogenase/metabolismo
11.
PLoS One ; 11(4): e0153266, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27055016

RESUMO

Azotobacter vinelandii is a soil bacterium that undergoes a differentiation process that forms cysts resistant to desiccation. During encystment, a family of alkylresorcinols lipids (ARs) are synthesized and become part of the membrane and are also components of the outer layer covering the cyst, where they play a structural role. The synthesis of ARs in A. vinelandii has been shown to occur by the activity of enzymes encoded in the arsABCD operon. The expression of this operon is activated by ArpR, a LysR-type transcriptional regulator whose transcription occurs during encystment and is dependent on the alternative sigma factor RpoS. In this study, we show that the two component response regulator GacA, the small RNA RsmZ1 and the translational repressor protein RsmA, implicated in the control of the synthesis of other cysts components (i.e., alginate and poly-ß-hydroxybutyrate), are also controlling alkylresorcinol synthesis. This control affects the expression of arsABCD and is exerted through the regulation of arpR expression. We show that RsmA negatively regulates arpR expression by binding its mRNA, repressing its translation. GacA in turn, positively regulates arpR expression through the activation of transcription of RsmZ1, that binds RsmA, counteracting its repressor activity. This regulatory cascade is independent of RpoS. We also show evidence suggesting that GacA exerts an additional regulation on arsABCD expression through an ArpR independent route.


Assuntos
Azotobacter vinelandii/metabolismo , Proteínas de Bactérias/metabolismo , Membrana Celular/metabolismo , Regulação Bacteriana da Expressão Gênica , Fosfolipídeos/metabolismo , Resorcinóis/química , Transdução de Sinais , Azotobacter vinelandii/crescimento & desenvolvimento , Proteínas de Bactérias/genética , Ensaio de Desvio de Mobilidade Eletroforética , Resorcinóis/análise
12.
Mikrobiol Z ; 78(3): 78-87, 2016.
Artigo em Russo | MEDLINE | ID: mdl-30141855

RESUMO

The features of the growth of Azotobacter vinelandii IMV V-7076 and Bacillus subtilis IMV V-7023 in optimized environments in single and mixed culture at deep cultivation to simulate the accumulation stage (capacity) biomass biotechnology creation of complex bacterial preparation for crop production. When growing Bacillus subtilis IMV B-7023 in monoculture specific growth rate of the bacteria was higher than that of Azotobacter vinelandii IMV B-7076. Therefore, by cultivating a mixed culture medium in the fermenter was inoculated with the bacterial suspension A. vinelandii + B. subtilis in a ratio of 10 : 1. The greatest biomass accumulation was observed after 24 hours of cultivation of mixed cultures. Spent submerged culture A. vinelandii IMV B-7076 and B. subtilis IMV B-7023 in the fermenter can be recommended for suspension of high-viable cells of both strains in biotechnology industrial manufacturing complex bacterial preparation for crop production.


Assuntos
Azotobacter vinelandii/crescimento & desenvolvimento , Bacillus subtilis/crescimento & desenvolvimento , Técnicas de Cocultura , Biomassa , Meios de Cultura , Fermentação , Microbiologia Industrial
13.
Mikrobiol Z ; 77(2): 15-21, 2015.
Artigo em Russo | MEDLINE | ID: mdl-26036028

RESUMO

It is shown that the bacteria Bacillus subtilis B-7023 IMV produce indole-3-acetic acid and amino acids in the liquid medium Knoop. Processing cucumber seed suspension containing 10(7) cfu/ml as bacilli, and Azotobacter vinelandii IMV V-7076, resulted in a decrease in the length of the roots of plants. Reduction of bacterial load bacilli to 10(6) cfu/ml followed by reduction of indole-3-acetic acid in the medium, and to an increase in the length of roots, shoots and total plant mass. During the cultivation of Bacillus subtilis IMV V-7023 with ciliates Colpoda steinii reduced the amount of free forms of auxin in the medium to 5.5 times, and the related--to trace amounts. The content of histidine, phenylalanine, tyrosine, methionine and lysine significantly reduced.


Assuntos
Azotobacter vinelandii/metabolismo , Bacillus subtilis/metabolismo , Cilióforos/fisiologia , Cucumis sativus/microbiologia , Raízes de Plantas/microbiologia , Sementes/microbiologia , Aminoácidos/biossíntese , Antibiose , Azotobacter vinelandii/crescimento & desenvolvimento , Bacillus subtilis/crescimento & desenvolvimento , Carga Bacteriana , Cucumis sativus/efeitos dos fármacos , Cucumis sativus/crescimento & desenvolvimento , Meios de Cultura/química , Germinação/efeitos dos fármacos , Ácidos Indolacéticos/metabolismo , Ácidos Indolacéticos/farmacologia , Reguladores de Crescimento de Planta/biossíntese , Reguladores de Crescimento de Planta/metabolismo , Reguladores de Crescimento de Planta/farmacologia , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/crescimento & desenvolvimento , Sementes/efeitos dos fármacos , Sementes/crescimento & desenvolvimento
14.
Environ Toxicol Chem ; 34(4): 887-97, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25556815

RESUMO

Proper characterization of nanoparticle (NP) interactions with environmentally relevant bacteria under representative conditions is necessary to enable their sustainable manufacture, use, and disposal. Previous nanotoxicology research based on planktonic growth has not adequately explored biofilms, which serve as the predominant mode of bacterial growth in natural and engineered environments. Copper nanoparticle (Cu-NP) impacts on biofilms were compared with respective planktonic cultures of the ammonium-oxidizing Nitrosomonas europaea, nitrogen-fixing Azotobacter vinelandii, and denitrifying Paracoccus denitrificans using a suite of independent toxicity diagnostics. Median inhibitory concentration (IC50) values derived from adenosine triphosphate (ATP) for Cu-NPs were lower in N. europaea biofilms (19.6 ± 15.3 mg/L) than in planktonic cells (49.0 ± 8.0 mg/L). However, in absorbance-based growth assays, compared with unexposed controls, N. europaea growth rates in biofilms were twice as resilient to inhibition than those in planktonic cultures. Similarly, relative to unexposed controls, growth rates and yields of P. denitrificans in biofilms exposed to Cu-NPs were 40-fold to 50-fold less inhibited than those in planktonic cells. Physiological evaluation of ammonium oxidation and nitrate reduction suggested that biofilms were also less inhibited by Cu-NPs than planktonic cells. Furthermore, functional gene expression for ammonium oxidation (amoA) and nitrite reduction (nirK) showed lower inhibition by NPs in biofilms relative to planktonic-grown cells. These results suggest that biofilms mitigate NP impacts, and that nitrogen-cycling bacteria in wastewater, wetlands, and soils might be more resilient to NPs than planktonic-based assessments suggest.


Assuntos
Bactérias/efeitos dos fármacos , Bactérias/crescimento & desenvolvimento , Biofilmes/crescimento & desenvolvimento , Cobre/toxicidade , Poluentes Ambientais/toxicidade , Nanopartículas Metálicas/toxicidade , Fixação de Nitrogênio , Plâncton/microbiologia , Compostos de Amônio/metabolismo , Azotobacter vinelandii/efeitos dos fármacos , Azotobacter vinelandii/crescimento & desenvolvimento , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Nitratos/metabolismo , Nitrosomonas europaea/efeitos dos fármacos , Nitrosomonas europaea/crescimento & desenvolvimento , Oxirredução , Paracoccus denitrificans/efeitos dos fármacos , Paracoccus denitrificans/crescimento & desenvolvimento
15.
Bioprocess Biosyst Eng ; 37(6): 1133-40, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24173209

RESUMO

The oxygen transfer rate (OTR) was evaluated as a scale-up criterion for alginate production in 3- and 14-L stirred fermentors. Batch cultures were performed at different agitation rates (200, 300, and 600 rpm) and airflow rates (0.25, 0.5, and 1 vvm), resulting in different maximum OTR levels (OTRmax). Although the two reactors had a similar OTRmax (19 mmol L(-1) h(-1)) and produced the same alginate concentration (3.8 g L(-1)), during the cell growth period the maximum molecular weight of the alginate was 1,250 kDa in the 3-L stirred fermentor and 590 kDa in 14-L stirred fermentor. The results showed for the first time the evolution of the molecular weight of alginate and OTR profiles for two different scales of stirred fermentors. There was a different maximum specific oxygen uptake rate between the two fermenters, reaching 8.3 mmol g(-1) h(-1) in 3-L bioreactor and 10.6 mmol g(-1) h(-1) in 14-L bioreactor, which could explain the different molecular weights observed. These findings open the possibility of using [Formula: see text] instead of OTRmax as a scaling criterion to produce polymers with similar molecular weights during fermentation.


Assuntos
Azotobacter vinelandii/crescimento & desenvolvimento , Reatores Biológicos , Oxigênio/metabolismo , Alginatos , Ácido Glucurônico/biossíntese , Ácidos Hexurônicos
16.
Int Microbiol ; 16(1): 35-44, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24151780

RESUMO

The alginate biofilm-producing bacterium Azotobacter vinelandii aerobically fixes nitrogen by oxygen-sensitive nitrogenases. Here we investigated the bacterial response to nitrogen/oxygen gas mixtures. A. vinelandii cells were cultured in nitrogen-free minimal media containing gas mixtures differing in their ratios of nitrogen and oxygen. The bacteria did not grow at oxygen concentrations >75% but grew well in the presence of 5% nitrogen/25% oxygen. Growth of wild-type and alginate-deficient strains when cultured with 50% oxygen did not differ substantially, indicating that alginate is not required for the protection of nitrogenases from oxygen damage. In response to decreasing nitrogen levels, A. vinelandii produced greater amounts of alginate, accompanied by the formation of blebs on the cell surface. The encystment of vegetative cells occurred in tandem with the release of blebs and the development of a multilayered exine. Immunoelectron microscopy using anti alginate-antibody revealed that the blebs contained alginate molecules. By contrast, alginate-deficient mutants could not form blebs. Taken together, our data provide evidence for a novel bleb-dependent polysaccharide export system in A. vinelandii that is activated in response to low nitrogen gas levels.


Assuntos
Azotobacter vinelandii/fisiologia , Proteínas de Bactérias/metabolismo , Nitrogênio/farmacologia , Oxigênio/farmacologia , Polissacarídeos Bacterianos/metabolismo , Alginatos , Azotobacter vinelandii/efeitos dos fármacos , Azotobacter vinelandii/crescimento & desenvolvimento , Azotobacter vinelandii/ultraestrutura , Proteínas de Bactérias/genética , Ácido Glucurônico/metabolismo , Ácidos Hexurônicos , Mutação
17.
Enzyme Microb Technol ; 53(2): 85-91, 2013 Jul 10.
Artigo em Inglês | MEDLINE | ID: mdl-23769307

RESUMO

The transcription of genes involved in alginate polymerization and depolymerization, as well as the alginase activity (extracellular and intracellular) under oxygen-limited and non oxygen-limited conditions in cultures of A. vinelandii, was studied. Two levels of dissolved oxygen tension (DOT) (1% and 5%, oxygen-limited and non-oxygen-limited, respectively) strictly controlled by gas blending, were evaluated in a wild type strain. In cultures at low DOT (1%), in which a high molecular weight alginate (1200 kDa) was synthesized, the transcription levels of alg8 and alg44 (genes encoding alginate polymerase complex), and algX (encoding a protein involved in polymer transport through periplasmic space) were considerably higher as compared to cultures conducted at 5% DOT, under which an alginate with a low MW (42 kDa) was produced. In the case of genes encoding for intracellular and extracellular alginases, the levels of these transcripts were higher at 1% DOT. However, intracellular and extracellular alginase activity were lower (0.017 and 0.01 U/mg protein, respectively) in cultures at 1% DOT, as compared with the activities measured at 5% DOT (0.027 and 0.052 U/mg protein for intracellular and extracellular maximum activity, respectively). The low alginase activity measured in cultures at 1% DOT and the high level of transcription of genes constituting alginate polymerase complex might be mechanisms by which oxygen regulates the production of alginates with a high MW.


Assuntos
Alginatos/metabolismo , Azotobacter vinelandii/metabolismo , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Consumo de Oxigênio , Polissacarídeo-Liase/metabolismo , Alginatos/química , Azotobacter vinelandii/efeitos dos fármacos , Azotobacter vinelandii/genética , Azotobacter vinelandii/crescimento & desenvolvimento , Proteínas de Bactérias/efeitos dos fármacos , Proteínas de Bactérias/genética , Biomassa , Microbiologia Industrial , Peso Molecular , Oxigênio/farmacologia , Polimerização , Polissacarídeo-Liase/efeitos dos fármacos , Polissacarídeo-Liase/genética , Transcrição Genética
18.
J Ind Microbiol Biotechnol ; 40(7): 715-23, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23640429

RESUMO

Alginates are polysaccharides that may be used as viscosifiers and gel or film-forming agents with a great diversity of applications. The alginates produced by bacteria such as Azotobacter vinelandii are acetylated. The presence of acetyl groups in this type of alginate increases its solubility, viscosity, and swelling capability. The aim of this study was to evaluate, in glucose-limited chemostat cultivations of A. vinelandii ATCC9046, the influence of dissolved oxygen tension (DO) and specific growth rate (µ) on the degree of acetylation of alginates produced by this bacterium. In glucose-limited chemostat cultivations, the degree of alginate acetylation was evaluated under two conditions of DO (1 and 9 %) and for a range of specific growth rates (0.02-0.15 h⁻¹). In addition, the alginate yields and PHB production were evaluated. High DO in the culture resulted in a high degree of alginate acetylation, reaching a maximum acetylation degree of 6.88 % at 9 % DO. In contrast, the increment of µ had a negative effect on the production and acetylation of the polymer. It was found that at high DO (9 %) and low µ, there was a reduction of the respiration rate, and the PHB accumulation was negligible, suggesting that the flux of acetyl-CoA (the acetyl donor) was diverted to alginate acetylation.


Assuntos
Alginatos/química , Alginatos/metabolismo , Azotobacter vinelandii/crescimento & desenvolvimento , Azotobacter vinelandii/metabolismo , Glucose/metabolismo , Oxigênio/metabolismo , Acetilação , Azotobacter vinelandii/efeitos dos fármacos , Biomassa , Glucose/farmacologia , Hidroxibutiratos/metabolismo , Oxigênio/farmacologia , Consumo de Oxigênio/efeitos dos fármacos , Poliésteres/metabolismo , Solubilidade , Viscosidade
19.
J Microbiol Biotechnol ; 23(4): 572-8, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23568214

RESUMO

Azotobacter vinelandii, a strict aerobic nitrogen-fixing bacterium, has been extensively studied with regard to the ability of N2-fixation due to its high expression of nitrogenase and fast growth. Because nitrogenase can also reduce cyanide to ammonia and methane, cyanide degradation by A. vinelandii has been studied for the application in the bioremediation of cyanide-contaminated wastewater. Cyanide degradation by A. vinelandii in NFS (nitrogen-free sucrose) medium was examined in terms of cell growth and cyanide reduction, and the results were applied for cyanide-contaminated cassava mill wastewater. From the NFS medium study in the 300 ml flask, it was found that A. vinelandii in the early stationary growth phase could reduce cyanide more rapidly than the cells in the exponential growth phase, and 84.4% of cyanide was degraded in 66 h incubation upon addition of 3.0 mM of NaCN. The resting cells of A. vinelandii could also reduce cyanide concentration by 90.4% with 3.0 mM of NaCN in the large-scale (3 L) fermentation with the same incubation time. Finally, the optimized conditions were applied to the cassava mill wastewater bioremediation, and A. vinelandii was able to reduce the cyanide concentration by 69.7% after 66 h in the cassava mill wastewater containing 4.0 mM of NaCN in the 3 L fermenter. Related to cyanide degradation in the cassava mill wastewater, nitrogenase was the responsible enzyme, which was confirmed by methane production. These findings would be helpful to design a practical bioremediation system for the treatment of cyanide-contaminated wastewater.


Assuntos
Azotobacter vinelandii/crescimento & desenvolvimento , Azotobacter vinelandii/metabolismo , Cianetos/metabolismo , Águas Residuárias/química , Águas Residuárias/microbiologia , Poluentes Químicos da Água/metabolismo , Azotobacter vinelandii/enzimologia , Biodegradação Ambiental , Meios de Cultura/química , Metano/metabolismo , Nitrogenase/metabolismo , Oxirredução
20.
Environ Sci Technol ; 47(4): 2061-8, 2013 Feb 19.
Artigo em Inglês | MEDLINE | ID: mdl-23339336

RESUMO

The acquisition of essential metals, such as the metal cofactors (molybdenum (Mo) and iron (Fe)) of the nitrogenase, the enzyme responsible for the reduction of dinitrogen (N(2)) to ammonium, is critical to N(2) fixing bacteria in soil. The release of metal nanoparticles (MNPs) to the environment could be detrimental to N(2) fixing bacteria by introducing a new source of toxic metals and by interfering with the acquisition of essential metals such as Mo. Since Mo has been reported to limit nonsymbiotic N(2) fixation in many ecosystems from tropical to cold temperate, this question is particularly acute in the context of Mo limitation. Using a combination of microbiology and analytical chemistry techniques, we have evaluated the effect of titanium (Ti) and tungsten (W) oxide nanoparticles on the diazotrophic growth and metals acquisition in pure culture of the ubiquitous N(2) fixing bacterium Azotobacter vinelandii under Mo replete and Mo limiting conditions. We report that under our conditions (≤10 mg·L(-1)) TiO(2) NPs have no effects on the diazotrophic growth of A. vinelandii while WO(3) NPs are highly detrimental to the growth especially under Mo limiting conditions. Our results show that the toxicity of WO(3) NPs to A. vinelandii is due to an interference with the catechol-metalophores assisted uptake of Mo.


Assuntos
Azotobacter vinelandii/efeitos dos fármacos , Molibdênio/metabolismo , Nanopartículas/toxicidade , Titânio/toxicidade , Tungstênio/toxicidade , Azotobacter vinelandii/crescimento & desenvolvimento , Azotobacter vinelandii/metabolismo
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