Ana G. Población

A periplasmic D-malate:cytochrome c oxidoreductase (DMCO) was identified in Ectopseudomonas oleovorans CECT5344, utilizing 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyl tetrazolium chloride (INT) as an artificial electron acceptor. The assay was adapted for a spectrophotometric or native polyacrylamide gel electrophoresis (PAGE) analysis. The DMCO-encoding gene (BN5_4044) was cloned and expressed in Escherichia coli, enabling a partial purification and biochemical characterization. In addition to D-malate, the enzyme oxidizes D-2-hydroxyglutarate and, to a lesser extent, D-lactate, with cytochrome c also serving as an electron acceptor. DMCO requires Zn²⁺ for activity and exists as a dimer, as determined by gel filtration. The in vitro reconstitution of the electron transfer from D-malate to oxygen was achieved using spheroplasts, enriched periplasmic fractions, and cytochrome c. This extracytoplasmic respiration, unique among homologs of this protein, may eliminate the need for a dedicated inner membrane transporter, thereby avoiding potential upstream respiratory bottlenecks. In the context of bioremediation, and particularly regarding the cyanide metabolism, this D-malate oxidation to oxaloacetate facilitates detoxification by forming the corresponding cyanohydrin, which can be subsequently assimilated for growth.

Most cyanide-containing industrial effluents also contain other cyano-derivatives and high amounts of metals and metal-cyanide compounds. For this reason, the biotreatment of these wastes requires the use of microorganisms capable to degrade all these different cyano-compounds and to tolerate metals. Pseudomonas pseudoalcaligenes CECT 5344 is a cyanotrophic bacterium capable of metabolize cyanide in its free form, but it is not very efficient at degrading metal-cyanide complexes. Therefore, for the optimization of the cyanide biodegradation process it is essential to find and characterize new bacterial strains, capable of assimilating metal cyanide-complexes, to complement the capacities of P. pseudoalcaligenes CECT 5344.

The effect of pH on bacterial cell-growth and the evolution of extracellular pH triggered by bacterial growth has been monitored for three bacterial strains, Escherichia coli ATCC 25922 and Pseudomonas putida KT2440 as reference strains, and Pseudomonas pseudoalcaligenes CECT 5344 because of its capacity to assimilate cyanide as the sole nitrogen source under alkaline conditions. In a first instance, the influence of the initial pH in the growth curve has been texted in LB-medium adjusted to pH 6, 7 and 8, for E. coli and P. putida, and 7.5, 8.25 and 9 for P. pseudoalcaligenes. Although the initial pH were different, the pH of the extracellular medium at the end of the stationary phase converged to a certain pH that is specific for each bacterium. Similar experiments were carried out in minimal medium with glucose as the carbon source. In this case, the pHs of the culture of both Pseudomonadaceae strains were almost constant, whereas it suddenly dropped during the exponential growth phase of E. coli. When the initial pH was 6 the extracellular pH fell sharply to 4.5, which irreversibly prevented further cellular growth. Nevertheless, at higher initial pH values subsequent cellular growth of E. coli restored the medium to the initial pHs values. Finally, in all cases the evolution of the pH has been shown to depend on the carbon source used. Among the sources used, cellular growth with glucose or glycerol did not affect the extracellular pH, whereas citrate caused the alkalinization of the media. This phenotype is in concordance with computational predictions, at least in the case of the genome-scale metabolic model of Pseudomonas putida KT2440.

Furfural and 5-hydroxymethylfurfural (HMF) are degradation products of lignocellulose during pretreatment operations. Furfural compounds are a group of chemical compounds whose common thread is an aldehyde group attached to a furan ring, and they constitute a problem for the development of second-generation biofuels because they act as fermentation inhibitors of the lignocellulose hydrolysates. Up to date, very few bacteria have been described to be able to eliminate them. The objective of this work was to isolate and characterize bacterial strains able to use, as the sole carbon source, 5-(hydroxymethyl)-furfural (HMF) and furan derivatives.