Laurent Mignot

Exopolysaccharide production by free and immobilized microbial cultures

Abstract The batch production of different exopolysaccharides (alginate, xanthan, pullulan, dextran) by free and immobilized microbial cultures was investigated. First, conventional free-cell cultures were performed to obtain control fermentation parameters and macromolecular characteristics of exopolysaccharides. Then microbial cultures were immobilized in composite agar layer/microporous membrane structures and tested for polysaccharide production. The immobilized-cell system proved unsuitable for xanthan and pullulan production. Owing to the fouling of the microporous membrane by the polysaccharide, dextran production by immobilized Leuconostoc mesenteroides also was inefficient. More promising results have been obtained with immobilized Azotobacter vinelandii cultures. The amount of alginate produced by immobilized A. vinelandii represented about 60% of that recovered from a free-cell culture, whereas the polysaccharide yield reached 35% instead of 9% for the free counterpart. These results are compared to the macromolecular characteristics of exopolysaccharides.

Photoproduction of molecular hydrogen by Rhodospirillum rubrum immobilized in composite agar layer/microporous membrane structures

SummaryViable cells of Rhodospirillum rubrum were immobilized by entrapment in a planar agar matrix bounded by a microporous membrane filter and were tested for H2 photoproduction in synthetic waste water provided with malate and glutamate. Optimum H2 production was obtained at 15 klx for a C/N ratio of 7–8. Production rates as high as 565 mm3 H2 · h-1 per cubic centimetre of agar were recorded. The composite structures, however, suffered from high diffusion limitations which increased with the population of immobilized bacteria. The H2-evolving activity could be maintained over several months by periodically incubating the biocatalytic structures in a rich nutrient broth. No contamination of the nutrient broth due to leakage of photosynthetic organisms from the gel appeared during incubation of the structures.

Cell immobilization in composite agar layer microporous membrane structures: growth kinetics of gel-entrapped cultures and cell leakage limitation by a microporous membrane

Agar discs containing different amounts of viable Escherichia coli cells (from 10 to 106 organisms·g−1 agar) were incubated in a nutrient medium and the growth of agar-entrapped bacteria and free (released) cells was monitored. The study was repeated with composite immobilized-cell structures obtained by placing a microporous membrane filter between the gel matrix and the incubation medium. In both cases, immobilized cells grew exponentially and reached a peak concentration an order of magnitude higher than that of free (suspended) cell cultures. The maximum specific growth rates of entrapped bacteria, ranging between 0.0115 min−1 and 0.0145 min−1, i.e., slightly higher than that of control free cultures (0.011 min−1), showed no clear dependence on the initial cell loading (ICL). The microporous filter proved efficient in limiting cell leakage since it noticeably lengthened the leakage time at a given ICL. This efficiency, however, decreased at high ICL and high growth rate of immobilized organisms.

Diffusion in immobilized-cell agar layers: influence of microbial burden and cell morphology on the diffusion coefficients ofl-malic acid and glucose

SummaryThe diffusivities ofl-malic acid and glucose in an agar membrane entrapping small amounts ofEscherichia coli orRhodospirillum rubrum whole cells were measured using time lag (TL) and steady state (SS) methods. Diffusivities were overestimated by the SS method. For concentrations of immobilizedR. rubrum cells ranging between 104 and 109 organisms cm−3 agar (20 ng-2 mg dry weight cm−3 agar), the diffusion coefficient ofl-malic acid, determined by both methods, was related to the logarithm of the membrane cell content by a decreasing linear relationship. The diffusion coefficient of glucose obtained by TL analysis was not significantly affected by the presence in the membrane of 3 ng-0.3 mg dry wt.E. coli cm−3 agar. However, values arising from the SS method decreased linearly as a function of the amount of immobilized organisms. Membranes containingR. rubrum cells offered higher diffusional resistance tol-malic acid and glucose than those loaded with the same amount ofE. coli cells.

A new type of immobilized-cell photobioreactor with internal illumination by optical fibres

A prototype of an immobilized-cell photobioreactor based on a composite agar layer/microporous membrane structure is described. This photobioreactor has been tested for hydrogen-gas production using viable cells ofRhodospirillum rubrum and a phosphate buffer supplemented with malate and glutamate as nutrient medium. The major problem was the high diffusional resistance of the immobilized-cell layer at high cell population. The device has been patented and might be readily applied to other light-dependent bioreactions having more short-term economic interest than hydrogen photoproduction.

Continuous marennin production by agar-entrapped Haslea ostrearia using a tubular photobioreactor with internal illumination.

Abstract The marine diatom Haslea ostrearia was immobilized in a tubular agar gel layer introduced into a photobioreactor of original design with internal illumination for the continuous synthesis of marennin, a blue-green pigment of biotechnological interest. Marennin was produced for a long-term period (27–43 days) and the volumetric productivity was maximum (18.7 mg day−1 l−1 gel) at the highest dilution rate (0.25 day−1) and lowest agar layer thickness (3 mm). Heterogeneous cell distribution in the agar layer revealed diffusional limitation of light and nutrients. However, the 3 mm gel thickness led to a more homogeneous cell distribution during incubation and to an increase of the whole biomass in the agar gel layer.

Influence of the oxygenation level on d-xylose fermentation by free and agar-entrapped cultures of Candida shehatae

This paper investigates the effects of the oxygenation level on the performance of d-xylose alcoholic fermentation by free- and immobilized-cell batch cultures of Candida shehatae (ATCC 22984). Yeast cells were immobilized in composite agar layer/microporous membrane structures. Fermentations were performed under varying oxygenation levels corresponding to different O2 flow rates (OFRs). Low OFRs enhanced the fermentation performance of free and immobilized yeasts. The best ethanol yield coefficient, obtained at an OFR of 5 mmol O2 h−1 dm−3 for both culture modes, was slightly higher (0.425 g g−1) for immobilized cultures than for their free counterparts (0.39 g g−1). More sustained aeration inhibited ethanol production by free and immobilized organisms. However, this inhibition was more pronounced for agar-entrapped cultures. Xylitol production of free cultures normally decreased as the OFR increased. At high OFR, however, immobilized organisms surprisingly produced more xylitol than at lower OFR or in anaerobiosis. This effect is discussed by referring to the mass transfer limitations that occur inside the immobilized-cell structures. Gel-entrapped cultures displayed higher specific and volumetric production rates of ethanol and xylitol than free-cell cultures.

Diffusion in immobilized-cell agar layers: influence of bacterial growth on the diffusivity of potassium chloride

SummaryThe diffusitivity of potassium chloride in composite agar slab/microporous membrane structures loaded with various amounts of Escherichia coli whole cells was determined using both time-lag and steady-state methods. The diffusion coefficient of KCl decreased linearly with the logarithm of the immobilized-cells content. The effect exerted by bacterial growth inside the immobilization matrices on KCl diffusivity was then investigated. The diffusion coefficient of KCl obtained by time-lag analysis decreased during incubation of the immobilized-cell structures, whereas less consitent results arose from the steady-state method. An apparent doubling time for immobilized E. coli, increasing with the initial cell content of the gel, was obtained from the calibration relationship between KCl diffusivity and the number of organisms in agar.

Microbial sensor based on lipoic acid reduction

Abstract In this paper, we propose the principle and illustrate the feasibility of a simple and original potentiometric biosensor for the determination of various substrates of bacterial metabolism. The biosensor consists of a gold electrode coated by a carbohydrate polymer matrix contaning viable bacteria and lipoic acid (LA) and bounded by a microporous membrane. In the presence of a substrate (S), immobolized microorganisms reduce LA into dihydrolipoic acid (LAH 2 ) as a consequence of S metabolism. The appearance of LAH 2 inside the polymer layer causes the gold electrode potential to fall rapidly. When the S content of the sample has been exhausted by bacterial consumption, the potential rises again as a consequence of LAH 2 reoxidation by oxygen. Low amounts of S can be related to the amplitude of the potential shift. We have tested this microbial sensor using Escherichia coli cells entrapped in agar with glucose as substrate. Glucose concentrations ranging from 20 μM to 400 μM in 20 cm 3 samples can be determined within a few minutes with satisfactory reproducibility. The biosensor is stable over two months. Further improvements are discussed.