M.D. Roldán

Alkaline cyanide degradation by Pseudomonas pseudoalcaligenes CECT5344 in a batch reactor. Influence of pH.

Water containing cyanide was biologically detoxified with the bacterial strain Pseudomonas pseudoalcaligenes CECT5344 in a batch reactor. Volatilization of toxic hydrogen cyanide (HCN) was avoided by using an alkaline medium for the treatment. The operational procedure was optimized to assess cyanide biodegradation at variable pH values and dissolved oxygen concentrations. Using an initial pH of 10 without subsequent adjustment allowed total cyanide to be consumed at a mean rate of approximately 2.81 mg CN(-) L(-1) O.D.(-1) h(-1); however, these conditions posed a high risk of HCN formation. Cyanide consumption was found to be pH-dependent. Thus, no bacterial growth was observed with a controlled pH of 10; on the other hand, pH 9.5 allowed up to 2.31 mg CN(-) L(-1) O.D.(-1) h(-1) to be converted. The combination of a high pH and a low dissolved oxygen saturation (10%) minimized the release of HCN. This study contributes new basic knowledge about this biological treatment, which constitutes an effective alternative to available physico-chemical methods for the purification of wastewater containing cyanide or cyano-metal complexes.

Chlorate and nitrate reduction in the phototrophic bacteriaRhodobacter capsulatus andRhodobacter sphaeroides

Chlorate or trimethylamine-N-oxide (TMAO) added to phototrophic cultures ofRhodobacter sphaeroides DSM 158 increased both the growth rate and the growth yield although this stimulation was not observed in the presence of tungstate. This strain, exhibited basal activities of nitrate, chlorate, and TMAO reductases independently of the presence of these substrates in the culture medium, and nitrate reductase (NR) activity was competitively inhibited by chlorate. Phototrophic growth ofRhodobacter capsulatus B10, a strain devoid of NR activity, was inhibited only by 100 mM chlorate. However, growth of the nitrate-assimilatingR. capsulatus strains E1F1 and AD2 was sensitive to 10mm chlorate, and their NR activities were not inhibited by chlorate. Both NR and chlorate reductase (CR) activities of strain E1F1 were induced in the presence of nitrate or chlorate respectively, whereas strain AD2 showed basal levels of these activities in the absence of the substrates. A basal TMAO reductase (TR) activity was also observed when these strains ofR. capsulatus were cultured in the absence of this electron acceptor. These results suggest that chlorate and TMAO can be used as ancillary oxidants byRhodobacter strains and that a single enzyme could be responsible for nitrate and chlorate reduction inR. sphaeroides DSM 158, whereas these reactions are catalyzed by two different enzymes inR. capsulatus E1F1 and AD2.

Cyanide metabolism of Pseudomonas pseudoalcaligenes CECT5344: role of siderophores

Cyanide is one of the most potent and toxic chemicals produced by industry. The jewelry industry of Córdoba (Spain) generates a wastewater (residue) that contains free cyanide, as well as large amounts of cyano-metal complexes. Cyanide is highly toxic to living systems because it forms very stable complexes with transition metals that are essential for protein function. In spite of its extreme toxicity, some organisms have acquired mechanisms to avoid cyanide poisoning. The biological assimilation of cyanide needs the concurrence of three separate processes: (i) a cyanide-insensitive respiratory chain, (ii) a system for iron acquisition (siderophores) and (iii) a cyanide assimilation pathway. Siderophores are low-molecular-mass compounds (600-1500 Da) that scavenge iron (Fe(3+)) ions (usually with extremely high affinity) from the environment under iron-limiting conditions. There are two main classes of siderophores: catechol and hydroxamate types. The catechol-type siderophores chelate ferric ion via a hydroxy group, whereas the hydroxamate-type siderophores bind iron via a carbonyl group with the adjacent nitrogen. In the presence of cyanide, bacterial proliferation requires this specific metal uptake system because siderophores are able to break down cyano-metal complexes. Pseudomonas pseudoalcaligenes CECT5344 is able to use free cyanide or cyano-metal complexes as nitrogen source. A proteomic approach was used for the isolation and identification, in this strain, of a protein that was induced in the presence of cyanide, namely CN0, that is involved in siderophore biosynthesis in response to cyanide. An overview of bacterial cyanide degradation pathways and the involvement of siderophores in this process are presented.

Alkaline cyanide biodegradation by Pseudomonas pseudoalcaligenes CECT5344

Pseudomonas pseudoalcaligenes CECT5344 uses cyanide, cyanate, beta-cyanoalanine, and other cyanoderivatives as nitrogen sources under alkaline conditions, which prevents volatile HCN (pK(a) 9.2) formation. The cyanide consumed by this strain is stoichiometrically converted into ammonium. In addition, this bacterium grows with the heavy metal, cyanide-containing waste water generated by the jewellery industry, and is also a cyanide-resistant strain which induces an alternative oxidase and a siderophore-based mechanism for iron acquisition in the presence of cyanide. The detection of cyanase and beta-cyanoalanine nitrilase activities in cyanide-induced cells suggests their implication in the cyanide degradation pathway.

The assimilatory nitrate reduction system of the phototrophic bacterium Rhodobacter capsulatus E1F1

The phototrophic bacterium Rhodobacter capsulatus E1F1 assimilates nitrate under anaerobic phototrophic growth conditions. A 17 kb DNA region encoding the nitrate assimilation (nas) system of this bacterium has been cloned and sequenced. This region includes the genes coding for a putative ABC (ATP-binding cassette)-type nitrate transporter (nasFED) and the structural genes for the enzymes nitrate reductase (nasA), nitrite reductase (nasB) and hydroxylamine reductase (hcp). Three genes code for putative regulatory proteins: a nitrite-sensitive repressor (nsrR), a transcription antiterminator (nasT) and a nitrate sensor (nasS). Other genes probably involved in nitrate assimilation are also present in this region. The sequence analysis of these genes and the biochemical properties of the purified nitrate, nitrite and hydroxylamine reductases are reviewed.

Molecular and Regulatory Properties of the Nitrate Reducing Systems of Rhodobacter

Abstract. Phototrophic bacteria of the genus Rhodobacter possess several forms of nitrate reductase including assimilatory and dissimilatory enzymes. Assimilatory nitrate reductase from Rhodobacter capsulatus E1F1 is cytoplasmic, it uses NADH as the physiological electron donor and reduced viologens as artificial electron donors, and it is coupled to an ammonium-producing nitrite reductase. Nitrate reductase induction requires a high C/N balance and the presence of nitrate, nitrite, or nitroarenes. A periplasmic 47-kDa protein facilitates nitrate uptake, thus increasing nitrate reductase activity. Two types of dissimilatory nitrate reductases have been found in strains from Rhodobacter sphaeroides. One of them is coupled to a complete denitrifying pathway, and the other is a periplasmic protein whose physiological role seems to be the dissipation of excess reducing power, thus improving photoanaerobic growth. Periplasmic nitrate reductase does not use NADH as the physiological electron donor and is a 100-kDa heterodimeric hemoprotein that receives electrons through an electron transport chain spanning the plasma membrane. This nitrate reductase is regulated neither by the intracellular C/N balance nor by O2 pressure. The enzyme also exhibits chlorate reductase activity, and both reaction products, nitrite and chlorite, are released almost stoichiometrically into the medium; this accounts for the high resistance to chlorate or nitrite exhibited by this bacterium. Nitrate reductases from both strains seem to be coded by genes located on megaplasmids.

Draft whole genome sequence of the cyanide‐degrading bacterium Pseudomonas pseudoalcaligenes CECT5344

Pseudomonas pseudoalcaligenes CECT5344 is a Gram-negative bacterium able to tolerate cyanide and to use it as the sole nitrogen source. We report here the first draft of the whole genome sequence of a P. pseudoalcaligenes strain that assimilates cyanide. Three aspects are specially emphasized in this manuscript. First, some generalities of the genome are shown and discussed in the context of other Pseudomonadaceae genomes, including genome size, G + C content, core genome and singletons among other features. Second, the genome is analysed in the context of cyanide metabolism, describing genes probably involved in cyanide assimilation, like those encoding nitrilases, and genes related to cyanide resistance, like the cio genes encoding the cyanide insensitive oxidases. Finally, the presence of genes probably involved in other processes with a great biotechnological potential like production of bioplastics and biodegradation of pollutants also is discussed.

Complete genome sequence of the cyanide-degrading bacterium Pseudomonas pseudoalcaligenes CECT5344

Pseudomonas pseudoalcaligenes CECT5344, a Gram-negative bacterium isolated from the Guadalquir River (Córdoba, Spain), is able to utilize different cyano-derivatives. Here, the complete genome sequence of P. pseudoalcaligenes CECT5344 harboring a 4,686,340bp circular chromosome encoding 4513 genes and featuring a GC-content of 62.34% is reported. Necessarily, remaining gaps in the genome had to be closed by assembly of few long reads obtained from PacBio single molecule real-time sequencing. Here, the first complete genome sequence for the species P. pseudoalcaligenes is presented.

Interactions Between Nitrate Assimilation and 2,4-Dinitrophenol Cometabolism in Rhodobacter capsulatus E1F1

The phototrophic, nitrate-photoassimilating bacterium Rhodobacter capsulatus E1F1 cometabolizes 2,4-dinitrophenol (DNP) by photoreducing it to 2-amino-4-nitrophenol under anaerobic conditions. DNP uptake and nitrate metabolism share some biochemical features, and in this article we show that both processes are influenced by each other. Thus, as was demonstrated for nitrate assimilation, DNP uptake requires a thermolabile periplasmic component. Nitrate assimilation is inhibited by DNP, which probably affects the nitrite reduction step because neither nitrate reductase activity nor the transport of nitrate or nitrite is inhibited. On the other hand, DNP uptake is competitively inhibited by nitrate, probably at the transport level, because the nitroreductase activity is not inhibited in vitro by nitrate, nitrite, or ammonium. In addition, the decrease in the intracellular DNP concentration in the presence of nitrate probably inactivates the nitroreductase. These results allow prediction of a negative environmental effect if nitrate and DNP are released together to natural habitats, because it may lead to a lower rate of DNP metabolism and to nitrite accumulation.

DNA microarray analysis of the cyanotroph Pseudomonas pseudoalcaligenes CECT5344 in response to nitrogen starvation, cyanide and a jewelry wastewater.

Pseudomonas pseudoalcaligenes CECT5344 is an alkaliphilic bacterium that can use cyanide as nitrogen source for growth, becoming a suitable candidate to be applied in biological treatment of cyanide-containing wastewaters. The assessment of the whole genome sequence of the strain CECT5344 has allowed the generation of DNA microarrays to analyze the response to different nitrogen sources. The mRNA of P. pseudoalcaligenes CECT5344 cells grown under nitrogen limiting conditions showed considerable changes when compared against the transcripts from cells grown with ammonium; up-regulated genes were, among others, the glnK gene encoding the nitrogen regulatory protein PII, the two-component ntrBC system involved in global nitrogen regulation, and the ammonium transporter-encoding amtB gene. The protein coding transcripts of P. pseudoalcaligenes CECT5344 cells grown with sodium cyanide or an industrial jewelry wastewater that contains high concentration of cyanide and metals like iron, copper and zinc, were also compared against the transcripts of cells grown with ammonium as nitrogen source. This analysis revealed the induction by cyanide and the cyanide-rich wastewater of four nitrilase-encoding genes, including the nitC gene that is essential for cyanide assimilation, the cyanase cynS gene involved in cyanate assimilation, the cioAB genes required for the cyanide-insensitive respiration, and the ahpC gene coding for an alkyl-hydroperoxide reductase that could be related with iron homeostasis and oxidative stress. The nitC and cynS genes were also induced in cells grown under nitrogen starvation conditions. In cells grown with the jewelry wastewater, a malate quinone:oxidoreductase mqoB gene and several genes coding for metal extrusion systems were specifically induced.