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See detailInfluence of Homogenization Treatment on Physicochemical 4 Properties and Enzymatic Hydrolysis Rate of Pure 5 Cellulose Fibers
Jacquet, Nicolas ULg; Vanderghem, Caroline ULg; Danthine, Sabine ULg et al

in Applied Biochemistry and Biotechnology (in press)

The aim of this study is to compare the effect of different homogenization treat- 12 ments on the physicochemical properties and the hydrolysis rate of a pure bleached 13 cellulose. Results obtained show ... [more ▼]

The aim of this study is to compare the effect of different homogenization treat- 12 ments on the physicochemical properties and the hydrolysis rate of a pure bleached 13 cellulose. Results obtained show that homogenization treatments improve the enzymatic 14 hydrolysis rate of the cellulose fibers by 25 to 100 %, depending of the homogenization 15 treatment applied. Characterization of the samples showed also that homogenization had an 16 impact on some physicochemical properties of the cellulose. For moderate treatment inten- 17 sities (pressure below 500 b and degree of homogenization below 25), an increase of water 18 retention values (WRV) that correlated to the increase of the hydrolysis rate was highlighted. 19 Result also showed that the overall crystallinity of the cellulose properties appeared not to be 20 impacted by the homogenization treatment. For higher treatment intensities, homogenized 21 cellulose samples developed a stable tridimentional network that contributes to decrease 22 cellulase mobility and slowdown the hydrolysis process. [less ▲]

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See detailIn Vitro and In Vivo Characterization of Plant Growth Promoting Bacillus Strains Isolated from Extreme Environments of Eastern Algeria.
Ait-Kaki, Asma; Kacem-Chaouche, Noreddine; Ongena, Marc ULg et al

in Applied biochemistry and biotechnology (2014), 172

This report is to our knowledge the first to study plant growth promotion and biocontrol characteristics of Bacillus isolates from extreme environments of Eastern Algeria. Seven isolates of 14 (50 %) were ... [more ▼]

This report is to our knowledge the first to study plant growth promotion and biocontrol characteristics of Bacillus isolates from extreme environments of Eastern Algeria. Seven isolates of 14 (50 %) were screened for their ability to inhibit growth of some phytopathogenic fungi on PDA and some roots exudates. The bacteria identification based on 16S r-RNA and gyrase-A gene sequence analysis showed that 71 % of the screened isolates belonged to Bacillus amyloliquefaciens and the rest were closely related to B. atrophaeus and B. mojavensis. Most of them had high spore yields (22 x 108-27 x 108 spores/ml). They produced protease and cellulase cell wall-degrading enzymes while the chitinase activity was only observed in the B. atrophaeus (6SEL). A wide variety of lipopeptides homologous was detected by liquid chromatography-electrospray ionization-mass spectrometry analysis. Interestingly, some additional peaks with new masses were characterized, which may correspond to new fengycin classes. The isolates produced siderophores and indole-3- acetic acid phytohormone. The greenhouse experiment using a naturally infested soil with Sclerotonia sclerotiorum showed that the B. atrophaeus (6SEL) significantly increased the size of the chickpea plants and reduced the stem rot disease (P < 0.05). These results suggest that these isolates may be used further as bio-inoculants to improve crop systems. [less ▲]

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See detailIsolation and Cultivation of a Xylanolytic Bacillus subtilis Extracted from the Gut of the Termite Reticulitermes santonensis
Tarayre, Cédric ULg; Brognaux, Alison ULg; Brasseur, Catherine ULg et al

in Applied Biochemistry and Biotechnology (2013)

The aim of this work was the isolation of xylanolytic microorganisms from the digestive tract of the termite Reticulitermes santonensis. The reducing sugars released after the hydrolysis of xylans can be ... [more ▼]

The aim of this work was the isolation of xylanolytic microorganisms from the digestive tract of the termite Reticulitermes santonensis. The reducing sugars released after the hydrolysis of xylans can be further fermented to provide bioethanol. A xylanolytic strain of Bacillus subtilis was isolated from the hindgut of the termite and displayed amylase and xylanase activities. The bacterium was grown on media containing agricultural residues: wheat bran, wheat distiller’s grains, and rapeseed oil cake. Wheat bran led to the highest induction of xylanase activity, although the development of the strain was less fast than in the other media. It was possible to reach maximal xylanase activities of 44.3, 33.5, and 29.1 I.U./ml in the media containing wheat bran, wheat distiller’s grains, and rapeseed oil cake, respectively. Mass spectrometry identified a wide range of xylose oligomers, highlighting an endoxylanase activity. The enzyme was stable up to 45 °C and displayed an optimal pH close to 8. [less ▲]

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See detailControlled Production of Exopolysaccharides from Enterobacter A47 as a Function of Carbon Source w ith Demonstration of Their Film and Emulsifying Abilities
Freitas, F; Alves, V.D.; Gouveia, A.R. et al

in Applied Biochemistry and Biotechnology (2013)

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See detailEffect of physicochemical characteristics of cellulosic substrates on enzymatic hydrolysis by means of a multi-stage process for cellobiose production
Vanderghem, Caroline ULg; Jacquet, Nicolas ULg; Danthine, Sabine ULg et al

in Applied Biochemistry and Biotechnology (2012), 166(6), 1423-1432

The effect of two types of cellulose, microcrystalline cellulose and paper pulp, on enzymatic hydrolysis for cellobiose production was investigated. The particle size, the relative crystallinity index and ... [more ▼]

The effect of two types of cellulose, microcrystalline cellulose and paper pulp, on enzymatic hydrolysis for cellobiose production was investigated. The particle size, the relative crystallinity index and the water retention value were determined for both celluloses. A previously studied multistage hydrolysis process that proved to enhance the cellobiose production was studied with both types of celluloses. The cellobiose yield exhibited a significant improvement (120% for the microcrystalline cellulose and 75% for the paper pulp) with the multistage hydrolysis process compared to continuous hydrolysis. The conversion of cellulose to cellobiose was greater for the microcrystalline cellulose than for the paper pulp. Even with high crystallinity, microcrystalline cellulose achieved the highest cellobiose yield probably due to its highest specific surface area accessible to enzymes and quantity of adsorbed protein. [less ▲]

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See detailPurification and characterisation of a 31 kDa chitinase from the Myzus persicae aphid, a target for Hemiptera biocontrol
Francis, Frédéric ULg; Saguez, Julien; Cherqui, Anas et al

in Applied Biochemistry and Biotechnology (2012), 166

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See detailEffect of Additives on Freeze-Drying and Storage of Yarrowia lipolytica Lipase.
Darvishi, Farshad; Destain, Jacqueline ULg; Nahvi, Iraj et al

in Applied Biochemistry and Biotechnology (2012)

The extracellular lipase of Yarrowia lipolytica presents numerous potentialities for biotechnological applications. This work describes the development and storage of powders obtained from supernatants ... [more ▼]

The extracellular lipase of Yarrowia lipolytica presents numerous potentialities for biotechnological applications. This work describes the development and storage of powders obtained from supernatants containing Y. lipolytica lipase by freeze-drying as downstream process that is important in obtaining a stable lipase powder with high enzymatic activity. Lipase was produced by Y. lipolytica U6 mutant strain in 20-L bioreactor. Non-concentrated cell-free culture supernatant samples were supplemented with different concentrations (0.5-1 %) of maltodextrin and glycerol as additives to freeze-drying. Effects of additives, temperature, pH, and storage time on lipase powders were determined. After addition of additives, freeze-drying yield increased 3.5-fold compared to supernatant without additive. Maltodextrin with 0.5 % concentration gave the best protection of lipase during dehydration treatment and its freeze-drying yield (77 %) is better than other formulations. Lipase powders were stored at 4 and 25 degrees C for 46 weeks without loss of lipase activity. A common impediment to the production of commercial enzyme is their low-stability aqueous solutions. The present study shows that freeze-dried lipase powders of Y. lipolytica have good stability for storage and various applications. [less ▲]

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See detailIsolation and identification of two new fungal strains for xylanase production
Bakri, Y.; Masson, M.; Thonart, Philippe ULg

in Applied Biochemistry and Biotechnology (2010), 162(6), 1626-1634

Fungi are well known for their ability to excrete enzymes into the environment. The aim of this work was to evaluate xylanase production by fungi isolated from soil. One hundred and thirty-six fungal ... [more ▼]

Fungi are well known for their ability to excrete enzymes into the environment. The aim of this work was to evaluate xylanase production by fungi isolated from soil. One hundred and thirty-six fungal isolates were screened for xylanase production. Two xylanase producing isolates, FSS117 and FSS129, were identified on the basis of analyses of 5,8S gene sequencing. The closest phylogenetic neighbors according to 5,8S gene sequence data for the two isolates were Aspergillus tubingensis and Aspergillus terreus, respectively. When birchwood xylan or corn cob hulls was used as a substrate for 5 days under submerged culture cultivation, xylanase production from A. terreus FSS129 was 113 and 174 IU ml -1, respectively. The pH and temperature for optimum xylanase activity were 8 and 65∈°C. © 2010 Springer Science+Business Media, LLC. [less ▲]

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See detailAn enhanced process for the production of a highly purified extracellular lipase in the non-conventional yeast Yarrowia lipolytica
Turki, S.; Ayed, A.; Chalghoumi, N. et al

in Applied Biochemistry and Biotechnology (2010), 160(5), 1371-1385

Yarrowia lipolytica LgX64.81 is a non-genetically modified mutant that was previously identified as a promising microorganism for extracellular lipase production. In this work, the development of a fed ... [more ▼]

Yarrowia lipolytica LgX64.81 is a non-genetically modified mutant that was previously identified as a promising microorganism for extracellular lipase production. In this work, the development of a fed-batch process for the production of this enzyme in this strain was described. A lipolytic activity of 2,145 U/mL was obtained after 32 h of batch culture in a defined medium supplemented with 10 g/L of tryptone, an enhancer of lipase expression. To maximize the volumetric productivity, two different fed-batch strategies had been investigated. In comparison to batch process, the intermittent fed-batch strategy had not improved the volumetric lipase productivity. In contrast, the stepwise feeding strategy combined with uncoupled cell growth and lipase production phases resulted in a 2-fold increase in the volumetric lipase productivity, namely, the lipase activity reached 10,000 U/mL after 80 h of culture. Furthermore, this lipase was purified to homogeneity by anion exchange chromatography on MonoQ resin followed by gel filtration on Sephacryl S-100. This process resulted in an overall yield of 72% and a 3.5-fold increase of the specific lipase activity. The developed process offers a great potential for an economic production of Lip2 at large scale in Y. lipolytica LgX64.81. © 2009 Humana Press. [less ▲]

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See detailA multistage process to enhance cellobiose production from cellulosic materials
Vanderghem, Caroline ULg; Boquel, Pascal; Blecker, Christophe ULg et al

in Applied Biochemistry and Biotechnology (2010), 160(8), 2300-2307

Cellobiose, a disaccharide, is a valuable product that can be obtained from cellulose hydrolysis. In this study, a simple methodology is presented to enhance the production and improve the selectivity of ... [more ▼]

Cellobiose, a disaccharide, is a valuable product that can be obtained from cellulose hydrolysis. In this study, a simple methodology is presented to enhance the production and improve the selectivity of cellobiose during enzymatic hydrolysis of cellulose. The approach consisted of a multistage removal of filtrate via vacuum filtration and resuspension of the retentate. By this process, the remaining solid was further hydrolyzed without additional enzyme loading. Compared to the continuous hydrolysis process, the production of cellobiose increased by 45%. Increased selectivity of cellobiose is due to the loss of beta-glucosidases in the filtrate, while enhanced productivity is likely due to mitigated product inhibition. [less ▲]

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See detailThe utilization of gum tragacanth to improve the growth of Rhodotorula aurantiaca and the production of γ-decalactone in large scale
Alchihab, Mohamed ULg; Destain, Jacqueline ULg; Aguedo, Mario ULg et al

in Applied Biochemistry and Biotechnology (2010), 162(1), 233-241

The production of γ-decalactone and 4-hydroxydecanoic acid by the psychrophilic yeast R. aurantiaca was studied. The effect of both compounds on the growth of R. aurantiaca was also investigated and our ... [more ▼]

The production of γ-decalactone and 4-hydroxydecanoic acid by the psychrophilic yeast R. aurantiaca was studied. The effect of both compounds on the growth of R. aurantiaca was also investigated and our results show that γ-decalactone must be one of the limiting factors for its production. The addition of gum tragacanth to the medium at concentrations of 3 and 4 g/l seems to be an adequate strategy to enhance γ-decalactone production and to reduce its toxicity towards the cell. The production of γ-decalactone and 4- hydroxydecanoic acid was significantly higher in 20-l bioreactor than in 100-l bioreactor. By using 20 g/l of castor oil, 6.5 and 4.5 g/l of γ-decalactone were extracted after acidification at pH 2.0 and distillation at 100 °C for 45 min in 20- and 100-l bioreactors, respectively. We propose a process at industrial scale using a psychrophilic yeast to produce naturally γ-decalactone from castor oil which acts also as a detoxifying agent; moreover the process was improved by adding a natural gum. [less ▲]

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See detailSurvival of Freeze-dried Leuconostoc mesenteroides and Lactobacillus plantarum Related to Their Cellular Fatty Acids Composition during Storage
Coulibaly, Ibourahema ULg; Yao, Amenan Anastasie ULg; Lognay, Georges ULg et al

in Applied Biochemistry and Biotechnology (2009), 157(1), 70-84

Lactic acid bacteria strains Lactobacillus plantarum CWBI-B534 and Leuconostoc ssp. mesenteroïdes (L. mesenteroïdes) Kenya MRog2 were produced in bioreactor, concentrated, with or without cryoprotectants ... [more ▼]

Lactic acid bacteria strains Lactobacillus plantarum CWBI-B534 and Leuconostoc ssp. mesenteroïdes (L. mesenteroïdes) Kenya MRog2 were produced in bioreactor, concentrated, with or without cryoprotectants. In general, viable population did not change significantly after freeze-drying (p>0.05). In most cases, viable population for cells added with cryoprotectants was significantly lower than those without (p<0.05). Cellular fatty acids (CFAs) from the two strains in this study were analyzed before and after freeze-drying. Six CFAs were identified, namely, palmitic (C16:0), palmitoleic (C16:1), stearic (C18:0), oleic (C18:1), linoleic (C18:2), and linolenic (C18:3) acids were identified. Four of them, C16:0, C16:1, C18:0, and C18:1, make up more than 94% or 93% of the fatty acids in L. mesenteroides and L. plantarum, respectively, with another one, namely, C18:3, making a smaller (on average 5–6%, respectively) contribution. The C18:2 contributed very small percentages (on average≤1%) to the total in each strain. C16:0 had the highest proportion at most points relative to other fatty acids. Moisture content and water activity (a w) increased significantly during the storage period. It was observed that C16:1/C16:0, C18:0/C16:0 and C18:1/C16:0 ratios for freeze-dried L. mesenteroides or L. plantarum, with or without cryoprotectants, did not change significantly during the storage period. According to the packaging mode and storage temperatures, C18:2/C16:0 and C18:3/C16:0 ratios for freeze-dried L. mesenteroides and L. plantarum with or without cryoprotectants decreased as the storage time increased. However, a higher C18:2/C16:0 or C18:3/C16:0 ratio for L. mesenteroides and L. plantarum was noted in the freeze-dried powder held at 4 °C or under vacuum and in dark than at 20 °C or in the presence of oxygen and light [less ▲]

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See detailXylanase Production by Penicillium canescens on Soya Oil Cake in Solid-State Fermentation.
Assamoi, Antoine; Destain, Jacqueline ULg; Thonart, Philippe ULg

in Applied biochemistry and biotechnology (2009), (2),

There is an increasing interest for the organic residues from various sectors of agriculture and industries over the past few decades. Their application in the field of fermentation technology has ... [more ▼]

There is an increasing interest for the organic residues from various sectors of agriculture and industries over the past few decades. Their application in the field of fermentation technology has resulted in the production of bulk chemicals and value-added products such as amino acid, enzymes, mushroom, organic acids, single-cell protein, biologically active secondary metabolites, etc. (Ramachandran et al., Bioresource Technology 98:2000–2009, 2007). In this work, the production of extracellular xylanase by the fungus Penicillium canescens was investigated in solid-state fermentation using five agro-industrial substrates (soya oil cake, soya meal, wheat bran, whole wheat bran, and pulp beet). The best substrate was the soya oil cake. In order to optimize the production, the most effective cultivation conditions were investigated in Erlenmeyer flasks and in plastic bags with 5 and 100 g of soya oil cake, respectively. The initial moisture content, initial pH, and temperature of the culture affected the xylanase synthesis. The optimal fermentation medium was composed by soya oil cake crushed to 5 mm supplemented with 3% and 4% (w/w) of casein peptone and Na2HPO4.2H2O. After 7 days of incubation at 30 °C and under 80% of initial moisture, a xylanase production level of 18,895±778 U/g (Erlenmeyer flasks) and 9,300± 589 U/g (plastic bags) was reached. The partially purified enzyme recovered by ammonium sulfate fractionation was completely stable at freezing and refrigeration temperatures up to 6 months and reasonably stable at room temperature for more than 3 months. [less ▲]

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See detailIsolation and biomass production of a Saccharomyces cerevisiae strain binding copper and zinc ions
Stroobants, Aurore ULg; Delroisse, Jean-Marc; Delvigne, Frank ULg et al

in Applied Biochemistry and Biotechnology (2009), 157(1),

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See detailStudy on mass transfer of isopropylbenzene and oxygen in a two-phase partitioning bioreactor in the presence of silicone oil.
Aldric, Jean-Marc ULg; Lecomte, Jean-Paul; Thonart, Philippe ULg

in Applied Biochemistry and Biotechnology (2009), 153

A two-phase partitioning bioreactor (TPPB) to treat gas effluents polluted by volatile organic compound (VOC) has been developed. In this work, both the mass transfer of isopropylbenzene (IPB) and oxygen ... [more ▼]

A two-phase partitioning bioreactor (TPPB) to treat gas effluents polluted by volatile organic compound (VOC) has been developed. In this work, both the mass transfer of isopropylbenzene (IPB) and oxygen have been considered in relation to their influence on the hydrodynamics of the reactor and the type of silicone oils used as a second phase. The synergistic effect of silicone oil and stirrer speed on the global oxygen mass transfer coefficient (KLa) and gas-hold-up (up to 12%) have been investigated. The addition of 10% of low viscosity silicone oil (10 centistokes) in the reactor does not significantly affect the oxygen transfer rate. The very high solubility of IPB in the silicone oil leads to an enhancement of driving force term, especially for high fraction of silicone oil. However, it does not seem useful to exceed a volume fraction of 10% since KLaIPB decreases sharply at higher proportions of silicone oil. KLaIPB and KLa O2 evolve in the same way with the proportion of silicone oil. These results confirm the potentialities of our bioreactor to improve both the oxygen and pollutant gas transfer in the field of the treatment of gaseous pollutants, even for highly concentrated effluents. [less ▲]

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See detailProduction of γ-Decalactone by a Psychrophilic and a Mesophilic Strain of the Yeast
Alchihab, Mohamed ULg; Destain, Jacqueline ULg; Aguedo, Mario ULg et al

in Applied Biochemistry and Biotechnology (2009), 158

Among 18 psychrophilic strains isolated near the Antarctic Station, the psychrophilic <br />strain Rhodotorula aurantiaca A19 was selected for its ability of growth and γ- <br />decalactone production at ... [more ▼]

Among 18 psychrophilic strains isolated near the Antarctic Station, the psychrophilic <br />strain Rhodotorula aurantiaca A19 was selected for its ability of growth and γ- <br />decalactone production at low temperatures. The effects of temperature, initial pH, and castor <br />oil concentration on the growth and γ-decalactone production by a psychrophilic and a <br />mesophilic strain of R. aurantiaca were investigated. The highest γ-decalactone production <br />in flasks (5.8 g/l) was obtained with the strain A19 at 14 °C and initial pH 7.0 in medium <br />containing 20 g/l castor oil. On the other hand, these factors did not affect the production of <br />γ-decalactone by the mesophilic strain. In fermentor, a γ-decalactone concentration of 6.6 g/l <br />was reached with the strain A19, whereas a maximum of 0.1 g/l was obtained with the <br />mesophilic strain. Our results suggest that the ability to synthesize γ-decalactone is a <br />particularity of the strain A19, since the mesophilic strain (no. 30645) produced small amounts, <br />and the other (no. 31354) did not exhibit this property. It is, to our knowledge, the first report of γ-decalactone production by R. aurantiaca and furthermore by a psychrophilic yeast strain. <br />Moreover, the amount of γ-decalactone obtained in fermentor with the strain 19 was on the <br />order of concentrations usually described in patents. [less ▲]

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See detailSolid-state fermentation of xylanase from Penicillium canescens 10-10c in a multi-layer packed bed reactor
Assamoi, antoine; Destain, Jacqueline ULg; Delvigne, Frank ULg et al

in Applied Biochemistry and Biotechnology (2008), 145

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See detailComparison Of Yarrowia Lipolytica Lipase Immobilization Yield Of Entrapment, Adsorption, And Covalent Bond Techniques
Alloue, Wam.; Destain, Jacqueline ULg; El Mejdoub, Thami ULg et al

in Applied Biochemistry and Biotechnology (2008), 150(1),

The purpose of this study was to immobilize lipase from Yarrowia lipolytica using three methods including inclusion, adsorption, and covalent bond to study enzyme leaching, storage, and catalytic ... [more ▼]

The purpose of this study was to immobilize lipase from Yarrowia lipolytica using three methods including inclusion, adsorption, and covalent bond to study enzyme leaching, storage, and catalytic properties. Sodium alginate and chitosan were the polymers selected to immobilize lipase by inclusion. The beads of each polymer were dried by freeze drying and fluidization. The results show that chitosan was more adapted to the inclusion of lipase. Even though freeze dried, bead activity was low compared to that of fluidized beads. The freeze-drying process seems to produce suitable beads for storage at 4 and 20 °C. The immobilization by adsorption was carried out on both celite and silica gel. Maximum immobilization yield of 76% was obtained with celite followed by 43% in silica gel. The enzyme adsorbed on the two supports exhibited greater stability at a certain temperature (50 °C) and in no polar solvents (Isooctane, n-heptane, and n-hexane). In addition, the lipase immobilized by covalent bond retained residual activity equitable to 70%. It was demonstrated that the enzyme immobilized by covalent bond showed greater activity (80%) after 5 months of storage. [less ▲]

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See detailFoam control in fermentation bioprocess: from simple aeration tests to bioreactor.
Etoc, A.; Delvigne, Frank ULg; Lecomte, Jean-Paul et al

in Applied Biochemistry and Biotechnology (2006), 130(1-3), 392-404

In this article, we describe the development of a simple laboratory test for the effective screening of foam control agents on a selected fermentation system, the mass production of Yarrowia lipolytica ... [more ▼]

In this article, we describe the development of a simple laboratory test for the effective screening of foam control agents on a selected fermentation system, the mass production of Yarrowia lipolytica. Aeration testing is based on sparging air in the foaming medium allowing partial reproduction of the gas-liquid hydrodynamic encountered in bioreactors. "Dynamic sparge test," for which measurements are made during foam formation, was used to compare the capacity of three antifoams, based on different technologies, to control the foam produced in the fermentation broth. The selected foam control agents were: (1) an organic antifoam (TEGO AFKS911), (2) a silicone-based emulsion containing in situ treated silica (DC-1520) and (3) a silicone/ organic blend silica-free formulation. The testing results demonstrated dramatic differences among them and showed that the capacity of TEGO AFKS911 and DC-1520 to control the foam generated in the fermentation broth decreases as a function of fermentation time. This occurred to a much lesser extent for the silicone/ organic blend formulation. These results were correlated with the change of the foam nature and the increase of foam stability of the fermentation broth with culture time. The increase in protein content as a function of growth time was correlated with an increase in foam stability and antifoam consumption. A "synthetic fermentation broth" was also developed, by adding both proteins and microorganism to the culture medium. This allowed us to mimic the fermentation broth, shown by the similar antifoams behaviour, and is therefore a simple methodology useful for the selection of appropriate antifoams. [less ▲]

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See detailUtilization of methyloleate in production of microbial lipase
Destain, Jacqueline ULg; Fickers, Patrick ULg; Weekers, F. et al

in Applied Biochemistry and Biotechnology (2005), 121(Spring), 269-277

In this article, we report the development and optimization of an industrial culture medium for the production of extracellular lipase in the yeast Yarrowia lipolytica. Until now olive oil in combination ... [more ▼]

In this article, we report the development and optimization of an industrial culture medium for the production of extracellular lipase in the yeast Yarrowia lipolytica. Until now olive oil in combination with glucose was used as the carbon source and inducer for the production of lipase. Our results demonstrate that methyloleate, a cheap hydrophobic compound, could efficiently substitute olive oil as the inducer and carbon source for lipase production. A new process of lipase production was developed yielding a twofold increase in the level of production compared with the levels in previous reports. [less ▲]

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