Mapping Salmonella typhimurium pathways using 13C metabolic flux analysis.

In the last years, Salmonella has been extensively studied not only due to its importance as a pathogen, but also as a host to produce pharmaceutical compounds. However, the full exploitation of Salmonella as a platform for bioproduct delivery has been hampered by the lack of information about its metabolism. Genome-scale metabolic models can be valuable tools to delineate metabolic engineering strategies as long as they closely represent the actual metabolism of the target organism. In the present study, a 13C-MFA approach was applied to map the fluxes at the central carbon pathways of S. typhimurium LT2 growing at glucose-limited chemostat cultures. The experiments were carried out in a 2L bioreactor, using defined medium enriched with 20% 13C-labeled glucose. Metabolic flux distributions in central carbon pathways of S. typhimurium LT2 were estimated using OpenFLUX2 based on the labeling pattern of biomass protein hydrolysates together with biomass composition. The results suggested that pentose phosphate is used to catabolize glucose, with minor fluxes through glycolysis. In silico simulations, using Optflux and pFBA as simulation method, allowed to study the performance of the genome-scale metabolic model. In general, the accuracy of in silico simulations was improved by the superimposition of estimated intracellular fluxes to the existing genome-scale metabolic model, showing a better fitting to the experimental extracellular fluxes, whereas the intracellular fluxes of pentose phosphate and anaplerotic reactions were poorly described.

Comparing the electrochemical degradation of the fluoroquinolone antibiotics norfloxacin and ciprofloxacin using distinct electrolytes and a BDD anode: evolution of main oxidation byproducts and toxicity.

The effects of the supporting electrolytes (SEs) Na2SO4, NaCl, Na2CO3, NaNO3, and Na3PO4 on the anodic oxidation of norfloxacin (NOR) and ciprofloxacin (CIPRO), assessed by the respective degradation kinetics and byproducts and electrolyzed solution antimicrobial activity, are compared. Galvanostatic anodic oxidations were performed in a filter-press flow cell fitted with a boron-doped diamond anode. Removal rates higher than the theoretical one for a process purely controlled by mass transfer were found for all SEs, indicative of contribution by indirect oxidation processes. However, the removal rates for NaCl were about tenfold higher, with the lowest energy consumption per order (EC O) of targeted pollutant removal rate (ca. 0.7 kW h m-3 order-1), a very competitive performance. The TOC removal rates were also affected by the SE, but not as markedly. The antimicrobial activity of the electrolyzed solutions against Escherichia coli showed distinct temporal profiles, depending on the fluoroquinolone and SE. For instance, when Na3PO4 was used, the antimicrobial activity was completely removed for NOR, but none for CIPRO; conversely, when NaCl was used, complete removal was attained only for CIPRO. From LC-MS/MS analyses of Na3PO4 electrolyzed solutions, rupture of the fluoroquinolone ring leading to byproducts with no toxicity against E. coli occurred only for NOR, whereas exactly the opposite occurred for the NaCl solutions. Clearly, the nature of both the SE and the fluoroquinolone influence the oxidation steps of the respective molecule; this was also evidenced by the distinct short-chain carboxylic acids identified in the degradation of NOR and CIPRO.

Optimization of the electrochemical degradation process of the antibiotic ciprofloxacin using a double-sided ß-PbO2 anode in a flow reactor: kinetics, identification of oxidation intermediates and toxicity evaluation.

The electrochemical degradation of ciprofloxacin-CIP (50 mg L-1 in 0.10 mol L-1 Na2SO4) was investigated using a double-sided Ti-Pt/ß-PbO2 anode in a filter-press flow reactor, with identification of oxidation intermediates and follow-up of antimicrobial activity against Escherichia coli. The effect of solution pH, flow rate, current density, and temperature on the CIP removal rate was evaluated. All of these parameters did affect the CIP removal performance; thus, optimized electrolysis conditions were further explored: pH = 10, qV = 6.5 L min-1, j = 30 mA cm-2, and θ = 25 °C. Therefore, CIP was removed within 2 h, whereas ~75% of the total organic carbon concentration (TOC) was removed after 5 h and then, the solution no longer presented antimicrobial activity. When the electrochemical degradation of CIP was investigated using a single-sided boron-doped diamond (BDD) anode, its performance in TOC removal was similar to that of the Ti-Pt/ß-PbO2 anode; considering the higher oxidation power of BDD, the surprisingly good comparative performance of the Ti-Pt/ß-PbO2 anode was ascribed to significantly better hydrodynamic conditions attained in the filter-press reactor used with this electrode. Five initial oxidation intermediates were identified by LC-MS/MS and completely removed after 4 h of electrolysis; since they have also been determined in other degradation processes, there must be similarities in the involved oxidation mechanisms. Five terminal oxidation intermediates (acetic, formic, oxamic, propionic, and succinic acids) were identified by LC-UV and all of them (except acetic acid) were removed after 10 h of electrolysis.

One-pot strategy for on-site enzyme production, biomass hydrolysis, and ethanol production using the whole solid-state fermentation medium of mixed filamentous fungi.

The efficient use of renewable lignocellulosic feedstocks to obtain biofuels and other bioproducts is a key requirement for a sustainable biobased economy. This requires novel and effective strategies to reduce the cost contribution of the cellulolytic enzymatic cocktails needed to convert the carbohydrates into simple sugars, in order to make large-scale commercial processes economically competitive. Here, we propose the use of the whole solid-state fermentation (SSF) medium of mixed filamentous fungi as an integrated one-pot strategy for on-site enzyme production, biomass hydrolysis, and ethanol production. Ten different individual and mixed cultivations of commonly used industrial filamentous fungi (Aspergillus niger, Aspergillus oryzae, Trichoderma harzianum, and Trichoderma reesei) were performed under SSF and the whole media (without the extraction step) were used in the hydrolysis of pretreated sugarcane bagasse. The cocultivation of T. reesei with A. oryzae increased the amount of glucose released by around 50%, compared with individual cultivations. The release of glucose and reducing sugars achieved using the whole SSF medium was around 3-fold higher than obtained with the enzyme extract. The addition of soybean protein (0.5% w/w) during the hydrolysis reaction further significantly improved the saccharification performance by blocking the lignin and avoiding unproductive adsorption of enzymes. The results of the alcoholic fermentation validated the overall integrated process, with a volumetric ethanol productivity of 4.77 g/L.h, representing 83.5% of the theoretical yield. These findings demonstrate the feasibility of the proposed one-pot integrated strategy using the whole SSF medium of mixed filamentous fungi for on-site enzymes production, biomass hydrolysis, and ethanol production. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:671-680, 2018.

The effect of the supporting electrolyte on the electrooxidation of enrofloxacin using a flow cell with a BDD anode: Kinetics and follow-up of oxidation intermediates and antimicrobial activity.

The role of the supporting electrolyte - SE (Na2SO4; NaCl; Na2CO3; NaNO3; Na3PO4 - 0.1 M ionic strength) in the galvanostatic (10 mA cm-2) electrochemical degradation of the fluoroquinolone antibiotic enrofloxacin (ENRO; 100 mg L-1) using a filter-press flow cell with a boron-doped diamond anode was investigated (flow rate, solution volume, and temperature were kept fixed at 420 L h-1, 1.0 L, and 25 °C, respectively). The electrochemical degradation performance with the different SEs was assessed by following up [ENRO], total organic carbon concentration (TOC), oxidation intermediates (detected by LC and LC-QqTOF), and antimicrobial activity towards Escherichia coli as the electrolyses progressed. With NaCl as SE, complete removal of ENRO was attained ∼10 times faster than with the other salts. The determination of terminal oxidation intermediates (short-chain carboxylic acids) produced during the electrolyses allowed concluding that their nature and number is indeed affected by the salt used as SE, most probably due to distinct electrogenerated oxidants. With NaCl, the antimicrobial activity of the electrolyzed solution decreased gradually (to ∼20%) from 8 to 16 h of electrolysis due to the cleavage of the fluoroquinolone structure. On the other hand, with Na2SO4, Na2CO3 and NaNO3 as SEs the growth of Escherichia coli cells was observed only after ∼14 h, whereas it was completely inhibited with Na3PO4. Clearly, the electrooxidation and mineralization of ENRO is strongly affected by the SEs used, which determine the degradation mechanism and, consequently, the removal rates of the solution's organic load and antimicrobial activity.

Electrochemical mineralization of cephalexin using a conductive diamond anode: A mechanistic and toxicity investigation.

The contamination of surface and ground water by antibiotics is of significant importance due to their potential chronic toxic effects to the aquatic and human lives. Thus, in this work, the electrochemical oxidation of cephalexin (CEX) was carried out in a one compartment filter-press flow cell using a boron-doped diamond (BDD) electrode as anode. During the electrolysis, the investigated variables were: supporting electrolyte (Na2SO4, NaCl, NaNO3, and Na2CO3) at constant ionic strength (0.1 M), pH (3, 7, 10, and without control), and current density (5, 10 and 20 mA cm-2). The oxidation and mineralization of CEX were assessed by high performance liquid chromatography, coupled to mass spectrometry and total organic carbon. The oxidation process of CEX was dependent on the type of electrolyte and on pH of the solution due to the distinct oxidant species electrogenerated; however, the conversion of CEX and its hydroxylated intermediates to CO2 depends only on their diffusion to the surface of the BDD. In the final stages of electrolysis, an accumulation of recalcitrant oxamic and oxalic carboxylic acids, was detected. Finally, the growth inhibition assay with Escherichia coli cells showed that the toxicity of CEX solution decreased along the electrochemical treatment due to the rupture of the ß-lactam ring of the antibiotic.

Salmonella typhimurium and Escherichia coli dissimilarity: Closely related bacteria with distinct metabolic profiles.

Live attenuated strains of Salmonella typhimurium have been extensively investigated as vaccines for a number of infectious diseases. However, there is still little information available concerning aspects of their metabolism. S. typhimurium and Escherichia coli show a high degree of similarity in terms of their genome contents and metabolic networks. However, this work presents experimental evidence showing that significant differences exist in their abilities to direct carbon fluxes to biomass and energy production. It is important to study the metabolism of Salmonella to elucidate the formation of acetate and other metabolites involved in optimizing the production of biomass, essential for the development of recombinant vaccines. The metabolism of Salmonella under aerobic conditions was assessed using continuous cultures performed at dilution rates ranging from 0.1 to 0.67 h(-1), with glucose as main substrate. Acetate assimilation and glucose metabolism under anaerobic conditions were also investigated using batch cultures. Chemostat cultivations showed deviation of carbon towards acetate formation, starting at dilution rates above 0.1 h(-1). This differed from previous findings for E. coli, where acetate accumulation was only detected at dilution rates exceeding 0.4 h(-1), and was due to the lower rate of acetate assimilation by S. typhimurium under aerobic conditions. Under anaerobic conditions, both microorganisms mainly produced ethanol, acetate, and formate. A genome-scale metabolic model, reconstructed for Salmonella based on an E. coli model, provided a poor description of the mixed fermentation pattern observed during Salmonella cultures, reinforcing the different patterns of carbon utilization exhibited by these closely related bacteria.