Impact of microbial inoculum storage on dark fermentative H2 production.

Complex organic substrates represent an important and relevant feedstock for producing hydrogen by Dark Fermentation (DF). Usually, an external microbial inoculum originated from various natural environments is added to seed the DF reactors. However, H2 yields are significantly impacted by the inoculum origin and the storage conditions as microbial community composition can fluctuate. This study aims to determine how the type and time of inoculum storage can impact the DF performances. Biochemical Hydrogen Potential tests were carried out using three substrates (glucose, the organic fraction of municipal solid waste, and food waste), inocula of three different origins, different storage conditions (freezing or freeze-drying) and duration. As a result, H2 production from glucose with the differently stored inocula was significantly impacted (positively or negatively) and was inoculum-origin-dependent. For complex substrates, hydrogen yields with the stored inocula were not statistically different from the fresh inocula, offering the possibility to store an inoculum.

Fed-batch anaerobic valorization of slaughterhouse by-products with mesophilic microbial consortia without methane production.

This work aimed at setting up a fully instrumented, laboratory-scale bioreactor enabling anaerobic valorization of solid substrates through hydrogen and/or volatile fatty acid (VFA) production using mixed microbial populations (consortia). The substrate used was made of meat-based wastes, especially from slaughterhouses, which are becoming available in large amounts as a consequence of the growing constraints for waste disposal from meat industry. A reconstituted microbial mesophilic consortium without Archaebacteria (methanogens), named PBr, was cultivated in a 5-L anaerobic bioreactor on slaughterhouse wastes. The experiments were carried out with sequential fed-batch operations, including liquid medium removal from the bioreactor and addition of fresh substrate. VFAs and nitrogen were the main metabolites observed, while hydrogen accumulation was very low and no methane production was evidenced. After 1,300 h of culture, yields obtained for VFAs reached 0.38 g/g dry matter. Strain composition of the microbial consortium was also characterized using molecular tools (temporal temperature gradient gel electrophoresis and gene sequencing).

Production of oils from acetic acid by the oleaginous yeast Cryptococcus curvatus.

The feasibility of the conversion of acetic acid, a metabolite commonly obtained during anaerobic fermentation processes, into oils using the yeast Cryptococcus curvatus was reported. This microorganism exhibited very slow growth rates on acetate as carbon source, which led to design a two-stage cultivation process. The first consisted of cell growth on glucose as carbon source until its complete exhaustion. The second step involved the use of acetate as carbon source under nitrogen limitation in order to induce lipid accumulation. A typical experiment performed in a bioreactor involved a preliminary yeast growth with a glucose initial concentration of 15 g/L glucose. Further additions of acetate and nitrogen source allowed a final lipid accumulation up to 50% (w/w). These promising results demonstrated the suitability of the technique proposed.

Characterization of monoterpene biotransformation in two pseudomonads.

AIMS: To study the metabolic profile of Pseudomonas rhodesiae and Pseudomonas fluorescens in water-organic solvent systems using terpene substrates for both growth and biotransformation processes and to determine the aerobic or anaerobic status of these degradation pathways. MATERIALS AND METHODS: Substrates from pinene (alpha-pinene, alpha-pinene oxide, beta-pinene, beta-pinene oxide, turpentine) and limonene (limonene, limonene-1,2-oxide, orange peel oil) families were tested. For the bioconversion, the terpene-grown biomass was concentrated and used either as whole cells or as a crude enzymatic extract. CONCLUSION: Pseudomonas rhodesiae was the most suitable biocatalyst for the production of isonovalal from alpha-pinene oxide and did not metabolize limonene. Pseudomonas fluorescens was a more versatile micro-organism and metabolized limonene in two different ways. The first (anaerobic, cofactor-independent, noninducible) allowed limonene elimination by synthesizing alpha-terpineol. The second (aerobic, cofactor-dependent) involved limonene-1,2-oxide as an intermediate for energy production through a beta-oxidation process. SIGNIFICANCE AND IMPACT OF THE STUDY: Enzymatic isomerization of beta- to alpha-pinene was described for the first time for both strains. Alpha-terpineol production by P. fluorescens was very efficient and appeared as a promising alternative for the commercial production of this bioflavour.

Spores of Aspergillus niger as reservoir of glucose oxidase synthesized during solid-state fermentation and their use as catalyst in gluconic acid production.

AIMS: To exploit conidiospores of Aspergillus niger as a vector for glucose oxidase extraction from solid media, and their direct use as biocatalyst in the bioconversion of glucose to gluconic acid. METHODS AND RESULTS: Spores of A. niger (200 h old) were shown to fully retain all the glucose oxidase synthesized by the mycelium during solid-state fermentation (SSF). They acted as catalyst and carried out the bioconversion reaction effectively, provided they were permeabilized by freezing and thawing. Glucose oxidase activity was found retained in the spores even after repeated washings. Average rate of reaction was 1.5 g l(-1) h(-1) with 102 g l(-1) of gluconic acid produced out of 100 g l(-1) glucose consumed after approx. 100 h reaction, which corresponded to a molar yield close to 93%. These results were obtained with permeabilized spores in the presence of a germination inhibitor, sodium azide. CONCLUSIONS: Spores of A. niger served as efficient catalyst in the model bioconversion reaction after permeabilization. SIGNIFICANCE AND IMPACT OF THE STUDY: To our knowledge, this is the first detailed study on the ability of A. niger spores to act as reservoir of enzyme synthesized during SSF without its release into solid media. Use of this material served as an innovative concept for enzyme extraction and purification from a solid medium. Moreover, this approach could compete efficiently with the conventional use of mycelial form of the fungus in gluconic acid production.

Optimization of isonovalal production from alpha-pinene oxide using permeabilized cells of Pseudomonas rhodesiae CIP 107491.

Optimization studies on the synthesis of isonovalal from alpha-pinene oxide by Pseudomonas rhodesiae CIP 107491 operated in a biphasic medium are presented. Three key parameters are identified. The first is the need for a permeabilization of cells by freezing them and then treating the thawed material with an organic solvent such as chloroform, toluene or diethyl ether. This operation allows both enzyme release into the aqueous phase outside the cells and an improvement in the transport properties of both substrate and product across the cell membrane, strongly increasing reaction rates. The second is that the enzyme alpha-pinene oxide lyase, which exhibits an irreversible inactivation by isonovalal (or a by-product), presents a constant turn-over, i.e., the total product synthesis is proportional to the biomass loading and is close to 108 mmol (16.4 g) isonovalal l(-1) g(-1) biomass. The third phenomenon is that the biphasic system used is not phase-transfer-limited, a feature attributed to the spontaneous formation of an oil-in-water emulsion. It is thus possible to carry out a very efficient process, allowing the recovery of 2.63 mol isonovalal l(-1) (400 g l(-1)) from 25 g biomass l(-1) in 2.5 h, corresponding to an average reaction rate as high as 0.70 mmol min(-1) g(-1) cells (160 g l(-1) h(-1)).

Direct analysis of fluoxetine and norfluoxetine in plasma by gas chromatography with nitrogen-phosphorus detection.

A quantitative method for the simultaneous GC resolution and detection of fluoxetine and his metabolite norfluoxetine in human plasma was developed. The procedure required 1.0 ml of plasma, extraction with a mixed organic solvent and injection into a capillary gas chromatograph with an OV-1 fused-silica column coupled to a nitrogen-phosphorus detector. The calibration curves were linear over the range 5-3000 ng/ml. The detection limits were 0.3 and 2 ng/ml for fluoxetine and norfluoxetine, respectively. The assay is suitable for routine analysis.

[Compared diagnostic performances of CKMB measurements by immuno-inhibition and three immunoenzyme methods for the early diagnosis of myocardial infarction]. / Performances diagnostiques comparées des mesures de la CKMB par immuno-inhibition et par trois méthodes immunoenzymologiques pour le diagnostic précoce de l'infarctus du myocarde.

In 98 patients consecutively admitted in a medical intensive care unit, an aliquot taken from the blood sample withdrawn for the cardiac enzyme admission request has been frozen. After thawing of these 98 aliquots total CK and the creatine kinase MB isoenzyme were measured on the same day. For this last determination, four methods were used and compared: an immunoinhibition method (Merck) and three immunoenzymatic assays (Abbott on IMX; Baxter on Stratus II; Hybritech on single use Icon cylinder). In 19 out of the 98 patients studied the diagnosis of myocardial infarction was made retrospectively by a cardiologist. This diagnosis was established according to the criteria defined by the WHO. The clinical performances (sensitivity, specificity, positive predictive value, negative predictive value) have been calculated for each test according to the following criteria: on the one hand, a cut-off of 8% (reference range of our laboratory) for the immunoinhibition technique; on the other hand, a cut-off defined by the manufacturer together with a cut-off obtained from the ROC curves for the three immunoenzymatic assays. Our results clearly demonstrate that the clinical performances of the three immunoenzymatic CKMB assays are very comparable and appear to be much better than the immunoinhibition method which should be abandoned.