Optimization of CO fermentation by Clostridium carboxidivorans in batch reactors: Effects of the medium composition.

OBJECTIVES: The objective of this study was to investigate the effects of medium composition on CO fermentation by Clostridium carboxidivorans. The focus was to reduce the medium cost preserving acceptable levels of solvent production. METHODS: Yeast extract (YE) concentration was set in the range of 0-3 g/L. Different reducing agents were investigated, including cysteine-HCl 0.6 g/L, pure cysteine 0.6 g/L, sodium sulphide (Na2S) 0.6 g/L, cysteine-sodium sulphide 0.6 g/L and cysteine-sodium sulphide 0.72 g/L. The concentration of the metal solution was decreased down to 25 % of the standard value. Fermentation tests were also carried out with and without tungsten or selenium. RESULTS: The results demonstrated that under optimized conditions, namely yeast extract (YE) concentration set at 1 g/L, pure cysteine as the reducing agent and trace metal concentration reduced to 75 % of the standard value, reasonable solvent production was achieved in less than 150 h. Under these operating conditions, the production levels were found to be 1.39 g/L of ethanol and 0.27 g/L of butanol. Furthermore, the study revealed that selenium was not necessary for C. carboxidivorans fermentation, whereas the presence of tungsten played a crucial role in both cell growth and solvent production. CONCLUSIONS: The optimization of the medium composition in CO fermentation by Clostridium carboxidivorans is crucial for cost-effective solvent production. Tuning the yeast extract (YE) concentration, using pure cysteine as the reducing agent and reducing trace metal concentration contribute to reasonable solvent production within a relatively short fermentation period. Tungsten is essential for cell growth and solvent production, while selenium is not required.

Continuous H-B-E fermentation by Clostridium carboxidivorans: CO vs syngas.

Leveraging renewable carbon-based resources for energy and chemical production is a promising approach to decrease reliance on fossil fuels. This entails a thermo/biotechnological procedure wherein bacteria, notably Clostridia, ferment syngas, converting CO or CO2 + H2 into Hexanol, Butanol and Ethanol (H-B-E fermentation). This work reports of Clostridium carboxidivorans performance in a stirred tank reactor continuously operated with respect to the gas and the cell/liquid phases. The primary objective was to assess acid and solvent production at pH 5.6 by feeding pure CO or synthetic syngas under gas flow differential conditions. Fermentation tests were conducted at four different dilution rates (DL) of the fresh medium in the range 0.034-0.25 h-1. The fermentation pathways of C. carboxidivorans were found to be nearly identical for both CO and syngas, with consistent growth and metabolite production at pH 5.6 within a range of dilution rates. Wash-out conditions were observed at a DL of 0.25 h-1 regardless of the carbon source. Ethanol was the predominant solvent produced, but a shift towards butanol production was observed with CO as the substrate and towards hexanol production with synthetic syngas. In particular, the maximum cell concentration (0.5 gDM/L) was obtained with pure CO at DL 0.05 h-1; the highest solvent productivity (60 mg/L*h of total solvent) was obtained at DL 0.17 h-1 by using synthetic syngas as C-source. The findings highlight the importance of substrate composition and operating conditions in syngas fermentation processes. These insights contribute to the optimization of syngas fermentation processes for biofuel and chemical production.

In vivo immobilized carbonic anhydrase and its effect on the enhancement of CO2 absorption rate.

Reactive absorption into aqueous solutions promoted by carbonic anhydrase (CA, E.C. 4.2.1.1.) has been often proposed as a post-combustion CO2 capture process. The state of the art reveals the need for efficient biocatalyst based on carbonic anhydrase that can be used to further develop CO2 capture and utilization technologies. The present study is focused on the use of a thermostable CA-based biocatalyst. The carbonic anhydrase SspCA, from the thermophilic bacterium Sulfurihydrogenibium yellowstonense, was in vivo immobilized as membrane-anchored protein (INPN-SspCA) on the outer membrane of Escherichia coli cells. The dispersed biocatalyst, made by cell membrane debris, was characterized in terms of its contribution to the enhancement of CO2 absorption in carbonate/bicarbonate alkaline buffer at operating conditions relevant for industrial CO2 capture processes. The amount of immobilized enzyme, estimated by SDS-PAGE, resulted in about 1 mg enzyme/g membrane debris. The apparent kinetics of the biocatalyst was characterized through CO2 absorption tests in a stirred cell lab-scale reactor assuming a pseudo-homogeneous behaviour of the biocatalyst. At 298 K, the assessed values of the second-order kinetic constant ranged between 0.176 and 0.555 L∙mg-1∙s-1. Reusability of the biocatalyst after 24 h showed the absence of free enzyme release in the alkaline solvent. Moreover, the equilibration of dispersed cell membrane debris against the alkaline buffer positively affected the performances of the heterogeneous biocatalyst. These results encourage further studies on the in vivo immobilized SspCA aimed at optimizing the enzyme loading on the cell membrane and the handling of the biocatalyst in the CO2 absorption reactors.

Butanol production by bioconversion of cheese whey in a continuous packed bed reactor.

Butanol production by Clostridium acetobutylicum DSM 792 fermentation was investigated. Unsupplemented cheese whey was adopted as renewable feedstock. The conversion was successfully carried out in a biofilm packed bed reactor (PBR) for more than 3 months. The PBR was a 4 cm ID, 16 cm high glass tube with a 8 cm bed of 3mm Tygon rings, as carriers. It was operated at the dilution rate between 0.4h(-1) and 0.94 h(-1). The cheese whey conversion process was characterized in terms of metabolites production (butanol included), lactose conversion and biofilm mass. Under optimized conditions, the performances were: butanol productivity 2.66 g/Lh, butanol concentration 4.93 g/L, butanol yield 0.26 g/g, butanol selectivity of the overall solvents production 82 wt%.

Bifurcational and dynamical analysis of a continuous biofilm reactor.

A dynamical model of a continuous biofilm reactor is presented. The reactor consists of a three-phase internal loop airlift operated continuously with respect to the liquid and gaseous phases, and batchwise with respect to the immobilized cells. The model has been applied to the conversion of phenol by means of immobilized cells of Pseudomonas sp. OX1 whose metabolic activity was previously characterized (Viggiani, A., Olivieri, G., Siani, L., Di Donato, A., Marzocchella, A., Salatino, P., Barbieri, P., Galli, E., 2006. An airlift biofilm reactor for the biodegradation of phenol by Pseudomonas stutzeri OX1. Journal of Biotechnology 123, 464-477). The model embodies the key processes relevant to the reactor performance, with a particular emphasis on the role of biofilm detachment promoted by the fluidized state. Results indicate that a finite loading of free cells establishes even under operating conditions that would promote wash out of the suspended biophase. The co-operative/competitive effects of free cells and immobilized biofilm result in rich bifurcational patterns of the steady state solutions of the governing equations, which have been investigated in the phase plane of the process parameters. Direct simulation under selected operating conditions confirms the importance of the dynamical equilibrium establishing between the immobilized and the suspended biophase and highlights the effect of the initial value of the biofilm loading on the dynamical pattern.

Differential oncogenic potential of activated RAS isoforms in melanocytes.

RAS genes are mutated in approximately 30% of all human cancers. Interestingly, there exists a strong bias in favor of mutation of only one of the three major RAS genes in tumors of different cellular origins. NRAS mutations occur in approximately 20% of human melanomas, whereas HRAS and KRAS mutations are rare in this disease. To define the mechanism(s) responsible for this preference in melanocytes, we compared the transformation efficiencies of mutant NRAS and KRAS in immortal, non-transformed Ink4a/Arf-deficient melanocytes. NRAS mutation leads to increased cellular proliferation and is potently tumorigenic. In contrast, KRAS mutation does not enhance melanocyte proliferation and is only weakly tumorigenic on its own. Although both NRAS and KRAS activate mitogen-activated protein kinase signaling, only NRAS enhances MYC activity in these cells. Our data suggest that the activity of specific RAS isoforms is context-dependent and provide a possible explanation for the prevalence of NRAS mutations in melanoma. In addition, understanding this mechanism will have important implications for cancer therapies targeting RAS pathways.

Oxprenolol hydrochloride: pharmacology, pharmacokinetics, adverse effects and clinical efficacy.

Oxprenolol is a nonselective beta-adrenergic blocking agent that also possesses intrinsic sympathomimetic activity (ISA) and membrane stabilizing effects. Oxprenolol undergoes first pass metabolism with only 30% of an oral dose reaching the systemic circulation. The drug is approximately 80% protein bound and is eliminated primarily by glucuronidation in the liver. Less than 4% of oxprenolol is excreted unchanged in the urine. Oxprenolol may reduce the heart rate and prolong the effective and functional atrioventricular nodal refractory period. Oxprenolol has less negative inotropic and chronotropic effects than propranolol. Plasma renin activity is reduced; however, changes in plasma aldosterone level are not significant. Long term metabolic effects require further study. Oxprenolol appears to be comparable to other beta blockers in the treatment of hypertension and angina pectoris with no additional adverse reactions. If its partial agonist effect proves useful, it may have an advantage over other agents in treating patients with borderline cardiac reserve. Because of limited data, the use of oxprenolol for the treatment of arrhythmias, migraine, thyrotoxicosis, anxiety, and glaucoma cannot be recommended at this time.

Nafcillin-induced platelet dysfunction and bleeding.

This paper describes two cases of nafcillin-induced platelet dysfunction, with positive rechallenge data for one patient. Nafcillin resulted in abnormal bleeding times in both patients and a clinically apparent bleeding episode in one of the cases. Platelet function tests were performed on one patient during the initial therapy and after rechallenge with nafcillin. Platelet aggregation showed abnormal responses to ADP, collagen, and epinephrine. Platelet count and morphology were normal. Nafcillin should be recognized as another antibiotic which causes platelet function abnormalities and clinical bleeding episodes.

Piperacillin distribution into bile, gallbladder wall, abdominal skeletal muscle, and adipose tissue in surgical patients.

The concentrations of piperacillin in serum, bile, gallbladder wall, abdominal skeletal muscle, and adipose tissue were measured simultaneously at various times after the intravenous administration of a single 5-g dose to each of 14 patients undergoing biliary tract surgery. Piperacillin concentrated in the bile with peak levels exceeding 4,000 micrograms/ml. In a single patient with cystic duct obstruction, trace gallbladder bile piperacillin levels were measured. Gallbladder wall concentrations of piperacillin tended to be higher than corresponding serum concentrations, with a correlation observed between tissue values and the degree of acute gallbladder inflammation and gallbladder bile piperacillin concentrations. Mean peak muscle and adipose tissue piperacillin concentrations of 31 and 27 micrograms/g, respectively, were reached at between 2 and 3 h after the start of infusion. These concentrations exceeded the minimum inhibitory concentration for a majority of susceptible organisms. A single 5-g dose of piperacillin achieved therapeutic levels in gallbladder wall, intraabdominal skeletal muscle, and adipose tissue and concentrated in the bile of patients with patent biliary tracts.

Assessment of quinidine gluconate for nonlinear kinetics following chronic dosing.

Two different chronic dosing regimens of quinidine gluconate were administered to each of four healthy volunteers in a pilot study to evaluate quinidine for nonlinear pharmacokinetics. Analysis of plasma quinidine levels following the last dose indicates that disproportionate increases in steady-state plasma concentrations can occur in some subjects as the daily dose increases. Measurement of 2'-oxoquinidinone (2'-OXO) and 3-hydroxyquinidine (3-OH) metabolites revealed that the formation of 2'-OXO is proportional to the availability of quinidine base. Hydroxylation was a more variable process. Rate-limited hydroxylation was documented in one subject, and an apparent increase in hepatic microsomal enzyme-mediated hydroxylation was shown in a second subject who ingested large amounts of caffeine daily. By using a highly selective high-performance liquid chromatography assay technique, the total body clearance of quinidine was found to be greater than previously published data. Our results suggest that some individuals may exhibit dose-dependent elimination of quinidine and that the variability in quinidine's pharmacokinetics is related in part to its hydroxylation. Future studies must use highly specific quinidine assays and control for variables that may influence this route of metabolism.

Comparative review of two new wide-spectrum penicillins: mezlocillin and piperacillin.

The antimicrobial spectra, pharmacokinetics, tissue penetration, side effects, clinical trials and indications, dosage, and cost of mezlocillin (Mezlin) and piperacillin (Pipracil), two new semisynthetic beta-lactam penicillins, are reviewed. Both mezlocillin and piperacillin are active against a wider range of bacteria than previously available penicillins, but their spectra are not identical. Piperacillin is more active than mezlocillin against Pseudomonas aeruginosa; their activities against Klebsiella pneumoniae, Streptococcus faecalis, and Bacteroides fragilis are similar to one another. Neither drug is absorbed orally; both are well absorbed (60-70%) after i.m. injection. Following i.v. infusion or injection, both drugs distribute rapidly (distribution half-life = 10-20 min); neither is protein bound substantially. Both drugs are primarily excreted unchanged in the urine by glomerular filtration and tubular secretion. Elimination half-lives of both drugs are slightly prolonged in renal-failure patients. However, the half-life of mezlocillin in renal failure is longer then the half-life of piperacillin because of dose-dependent kinetics of mezlocillin at low glomecular filtration rates. Probenecid alters the disposition of both drugs. Both drugs are widely distributed throughout the body. Reported side effects are similar to those of other penicillins. Mezlocillin and piperacillin may be used to treat susceptible organisms causing the following conditions: complicated and uncomplicated urinary-tract infections, septicemia, uncomplicated gonococcal urethritis, and lower respiratory-tract, intra-abdominal, gynecologic, skin, and skin-structure infections. Piperacillin is also effective for bone and joint infections. Dosages of both antibiotics should be adjusted based on patients' clinical condition and renal status. Both agents are relatively expensive in comparison with older penicillins and cephalosporins; their daily costs are similar to third-generation cephalosporins, carbenicillin, and ticarcillin. The potential benefits of mezlocillin and piperacillin are in their extended in vitro spectra of activity and minimal toxicities. More comparative clinical trials are needed to support any claims of clinical superiority of these drugs over older, less expensive regimens.

Gentamicin inactivation by piperacillin or carbenicillin in patients with end-stage renal disease.

Possible antibiotic inactivation was studied in 12 subjects with end-stage renal disease who were undergoing thrice-weekly hemodialysis. The study was a randomized three-way crossover. Subjects received (i) gentamicin as a single intravenous dose of 2 mg/kg, (ii) 4 g of piperacillin intravenously every 12 h for four doses or 2 g of carbenicillin intravenously every 8 h for six doses, and (iii) gentamicin as described in (i) plus piperacillin or carbenicillin as described in (ii). Subjects were studied on their off-dialysis days, and each treatment phase was separated by a 3-week wash-out period. Gentamicin was inactivated to a greater extent by carbenicillin than by piperacillin (P less than 0.05). In the six subjects in the carbenicillin group, the terminal elimination-phase half-life (t 1/2 beta) of gentamicin was 61.6 h when gentamicin was administered alone, and it was 19.4 h when gentamicin was administered with carbenicillin. In six subjects in the piperacillin group, the mean t 1/2 beta of gentamicin when gentamicin was given alone was 53.9 h, and it was 37.7 h when gentamicin was given with piperacillin. The inactivation rate constant (ki) of gentamicin was 0.0251/h for the carbenicillin group and 0.0064/h for the piperacillin group, demonstrating that carbenicillin inactivated gentamicin for time faster than did piperacillin. No inactivation of either beta-lactam could be measured. Control samples verified that no in vitro inactivation occurred.

The pathophysiology of epilepsy.

Recent developments in the understanding of the cellular events in focal seizures are described. This is followed by a discussion of the pathogenesis of neuronal hyperexcitability, the mechanism of spread of the seizure discharge and the mechanisms involved in seizure termination. The pathophysiology of generalized seizures is described in terms of theories of the site of origin of the seizure discharge and possible mechanisms of the clinical phenomena associated with this discharge.

Gentamicin and ticarcillin in subjects with end-stage renal disease. Comparison of two assay methods and evaluation of inactivation rate.

Eight subjects with end-stage renal disease were given gentamicin alone (single dose) and in combination with ticarcillin (multiple doses) to determine to what extent ticarcillin inactivates gentamicin in vivo and when this inactivation becomes significant. Gentamicin serum concentrations were performed by both a rapid radioimmunoassay (1 1/2 hours) and a conventional bioassay (8 hours) to determine whether the bioassay results would be falsely lowered by ticarcillin. The results of the study showed that ticarcillin inactivates gentamicin in vivo with an inactivation rate of 0.01 hours(-1). This was demonstrated by a decrease in serum gentamicin levels which became significant at 12 hours after gentamicin was given, and a decrease in gentamicin half-life from 46 to 28 hours during ticarcillin administration. Ticarcillin did not interfere with the bioassay method of measuring gentamicin levels. Although ticarcillin inactivates gentamicin in subjects with end-stage renal disease, the serum level data of gentamicin suggest that supplemental doses of the aminoglycoside are not necessary for 48 hours after a single loading dose of gentamicin. Widely used microbiological assay procedures are reliable in determining gentamicin levels and do not appear to be falsely lowered by concurrent ticarcillin administration.

Piperacillin pharmacokinetics in subjects with chronic renal failure.

The pharmacokinetic parameters of piperacillin sodium were studied in eight volunteer subjects with chronic renal failure. Subjects were given a single 30-min intravenous infusion of 70 mg/kg (lean body weight) on their off-dialysis day. Blood was drawn from the contralateral arm at 15 and 30 min and 1, 3, 6, 9, and 12 h from the start of the infusion. Kinetic parameters were determined during the elimination phase with a one-compartment open model for linear kinetics. The following pharmacokinetic parameters (mean +/- standard deviation) were determined for the eight subjects: elimination half-life = 3.33 +/- 0.99 h, elimination rate constant = 0.22 +/- 0.06 h-1, apparent volume of distribution = 0.18 +/- 0.05 liters per kg, and total body clearance = 0.041 +/- 0.019 liters per kg/h. The mean peak serum concentration was 372 +/- 125 microgram/ml, and mean trough at 12 h was 39 +/- 27 microgram/ml. A dose of 70 mg/kg (lean body weight) or a dose of 4 g appears to provide adequate serum concentrations against susceptible organisms for a 12-h interval. No adverse reactions were noted in any subject throughout the study.

Penicillin-aminoglycoside inactivation: another possible mechanism of interaction.

A case of a carbenicillin-tobramycin interaction resulting in laboratory test reports of subtherapeutic serum tobramycin levels in a 71-year-old man with renal failure is reported. The patient's Pseudomonas aeruginosa infection was treated with carbenicillin, 2 g every eight hours, and tobramycin, 80 mg after daily hemodialysis. The serum antibiotic levels were monitored using a microbiologic assay and a radioimmunoassay technique. At 30 minutes and five hours after the tobramycin was administered, microbiologic assay of serum levels indicated negligible tobramycin concentrations (2 micrograms/ml). Radioimmunoassay of tobramycin levels showed markedly higher concentrations (4.1 micrograms/ml at 30 minutes after infusion). The difference in assay results was attributed to greater inactivation occurring with the microbiologic assay, which was the less rapid technique. In vitro and in vivo factors influencing the occurrence and extent of the carbenicillin-tobramycin interaction are reviewed. When using an aminoglycoside-penicillin combination in patients with renal failure, it is important to use a rapid assay technique or one that inactivates one of the antibiotics before bioassay, and to be aware of the patient-related factors that can alter assay results.