Study of drop and bubble sizes in a simulated mycelial fermentation broth of up to four phases

The mean sizes and size distributions of air bubbles and viscous castor oil drops were studied in a salt-rich aqueous solution (medium), first separately, and then simultaneously as a three-phase system. The dispersion was created in a 150-mm-diameter stirred tank equipped with a Rushton turbine, and the sizes were measured using an advanced video technique. Trichoderma harzianum biomass was added in some experiments to study the effect of a solid phase under unaerated and aerated conditions to give either three-or four-phase systems. In all cases, the different dispersed phases could be clearly seen. Such photoimages have never been obtained previously. For the three phases, air-oil-medium, aeration caused a drastic increase in Sauter mean drop diameter, which was greater than could be accounted for by the reduction in energy dissipation on aeration. Also, as in the unaerated case, larger drops were observed as the oil content increased. On the other hand, mean bubble sizes were significantly reduced with increasing oil phase up to 15% with bubbles inside many of the viscous drops. With the introduction of fungal biomass of increasing concentration (0.5 to 5 g L(-1)) under unaerated conditions, the Sauter mean drop diameter decreased. Finally, in the four-phase system (oil [10%]-medium-air-biomass) as found in many fermentations, all the phases (plus bubbles in drops) could clearly be seen and, as the biomass increased, a decrease in both the bubble and the drop mean diameters was found. The reduction in size of bubbles (and therefore increase in interfacial area) as the oil and bio- mass concentration increased provides a possible explanation as to why the addition of an oil phase has been reported to enhance oxygen transfer during many fermentations.     

Oil and air dispersion in a simulated fermentation broth as a function of mycelial morphology

The culture conditions of a multiphase fermentation involving morphologically complex mycelia were simulated in order to investigate the influence of mycelial morphology (Trichoderma harzianum) on castor oil and air dispersion. Measurements of oil drops and air bubbles were obtained using an image analysis system coupled to a mixing tank. Complex interactions of the phases involved could be clearly observed. The Sauter diameter and the size distributions of drops and bubbles were affected by the morphological type of biomass (pellets or dispersed mycelia) added to the system. Larger oil drop sizes were obtained with dispersed mycelia than with pellets, as a result of the high apparent viscosity of the broth, which caused a drop in the power drawn, reducing oil drop break-up. Unexpectedly, bubble sizes observed with dispersed mycelia were smaller than with pellets, a phenomenon which can be explained by the segregation occurring at high biomass concentrations with the dispersed mycelia. Very complex oil drops were produced, containing air bubbles and a high number of structures likely consisting of small water droplets. Bubble location was influenced by biomass morphology. The percentage (in volume) of oil-trapped bubbles increased (from 32 to 80%) as dispersed mycelia concentration increased. A practically constant (32%) percentage of oil-trapped bubbles was observed with pelleted morphology at all biomass concentrations. The results evidenced the high complexity of phases interactions and the importance of mycelial morphology in such processes.     

Maximization of bioconversion of castor oil into ricinoleic acid by response surface methodology

In this study, response surface methodology was applied to optimize process variables like temperature, pH, enzyme concentration (mg/g oil), and buffer concentration (g/g oil) for hydrolysis of castor oil using Candida rugosa lipase. A 2⁴ full factorial central composite design was used to develop the quadratic model that was subsequently optimized and the optimal conditions were as follows: temperature 40 °C, pH 7.72, enzyme concentration 5.28 mg/g oil, buffer concentration 1 g/g oil and there was 65.5% conversion in 6 h. These predicted optimal conditions agreed well with the experimental results. This is the first report on the application of response surface methodology in castor oil hydrolysis using C. rugosa lipase with higher percentage conversion in 6 h.     

l-Sorbose Oxidase from Trametes sanguinea

A crude inhibitor for pancreatic lipase was extracted from soybean seeds. The lipase activity decreased curvilinearly with an increase in inhibitor concentration. At a low inhibitor concentration, enhanced inhibition was observed by the co-existence of protein such as bovine serum albumin in the reaction mixture. The lipase activity was inhibited immediately after the addition of inhibitor which did not cause the significant destraction of substrate emulsion. The lipase activities of Aspergillus niger, Rhizopus delemar and castor bean seeds were also inhibited. The inhibition was observed when various oil substrates such as soybean oil, linseed oil, olive oil emulsions and Ediol were used, and the extent of inhibition varied among them. Column chromatography of inhibitor on Sephadex G–100 showed that the molecular weight of a main peak of inhibitor was estimated as about 80,000.     

Lipases in the storage tissues of peanut and other oil seeds during germination

The castor-bean endosperm-the best-studied material of reserve lipid hydrolysis in seed germination-was previously shown to have an acid lipase and an alkaline lipase having reciprocal patterns of development during germination. We studied oil seeds from 7 species, namely castor bean (Ricinus communis L.), peanut (Arachis hypogaea L.), sunflower (Helianthus annus L.), cucumber (Cucumis sativus L.), cotton (Gossypisum hirsutum L.), corn (Zea mays. L.) and tomato (Lycopersicon esculentum Mill.). The storage tissues of all these oil seeds except castor bean contained only alkaline lipase activity which increased drastically during germination. The pattern of acid and alkaline lipases in castor bean does not seem to be common in other oil seeds. The alkaline lipase of peanut cotyledons was chosen for further study. On sucrose gradient centrifugation of cotyledon homogenate from 3-d-old seedlings, about 60% of the activity of the enzyme was found to be associated with the glyoxysomes, 15% with the mitochondria, and 25% with a membrane fraction at a density of 1.12 g cm^-3. The glyoxysomal lipase was associated with the organelle membrane, and hydrolyzed only monoglyceride whereas the mitochondrial and membrane-fraction enzymes degraded mono-, di- and triglycerides equally well. Thus, although the lipase in the glyoxysomes had the highest activity, it had to cooperate with lipases in other cellular compartments for the complete hydrolysis of reserve triglycerides.     

An Antibody to the Castor Bean Glyoxysomal Lipase (62 kD) also Binds to a 62 kD Protein in Extracts from Many Young Oilseed Plants

An antibody raised against purified glyoxysomal lipase (triacylglycerol hydrolase EC 3.1.1.3.) from castor bean (relative molecular weight of 62,000) also binds to a protein with a relative molecular weight of 62,000 in extracts of food reserve tissues from many young oilseed plants. These plants include Brassica napus L., Zea mays L., Arachis hypogaea L., Glycine max L., Gossipium hirsutum L., Cucurbita pepo L., Helianthus annuus L., Pisum sativum L., and Cicer arietinum L. The antibody caused inhibition of triacylglycerol hydrolysis by the lipases in extracts from seedlings of corn, oilseed rape, castor bean, soybean, and peanut. The pattern of antilipase binding to the 62 kilodalton protein in subcellular fractions from these other seedlings was consistent with the patterns of lipase activity reported in the literature and it is suggested that lipases from these oil seeds all have a subunit with a molecular weight of 62,000. The protein was only found in the food reserve tissues and was not present in extracts of roots and leaves of mature plants. In addition, the immunoreactive 62 kilodalton polypeptide was not detectable in lima beans and only at very low levels in kidney beans. Both these seeds are known to contain very little storage lipid and would not be expected to contain lipase. With the exception of the acid lipase of castor bean, ungerminated seeds do not generally contain active lipases. The immunoreactive 62 kilodalton protein could not be detected in the ungerminated seeds of most plants and only at very low low levels in others.     

Lipase applications in oil hydrolysis with a case study on castor oil: a review

Lipase (triacylglycerol acylhydrolase) is a unique enzyme which can catalyze various types of reactions such as hydrolysis, esterification, alcoholysis etc. In particular, hydrolysis of vegetable oil with lipase as a catalyst is widely studied. Free lipase, lipase immobilized on suitable support, lipase encapsulated in a reverse micelle and lipase immobilized on a suitable membrane to be used in membrane reactor are the most common ways of employing lipase in oil hydrolysis. Castor oil is a unique vegetable oil as it contains high amounts (90%) of a hydroxy monounsaturated fatty acid named ricinoleic acid. This industrially important acid can be obtained by hydrolysis of castor oil. Different conventional hydrolysis processes have certain disadvantages which can be avoided by a lipase-catalyzed process. The degree of hydrolysis varies widely for different lipases depending on the operating range of process variables such as temperature, pH and enzyme loading. Immobilization of lipase on a suitable support can enhance hydrolysis by suppressing thermal inactivation and estolide formation. The presence of metal ions also affects lipase-catalyzed hydrolysis of castor oil. Even a particular ion has different effects on the activity of different lipases. Hydrophobic organic solvents perform better than hydrophilic solvents during the reaction. Sonication considerably increases hydrolysis in case of lipolase. The effects of additives on the same lipase vary with their types. Nonionic surfactants enhance hydrolysis whereas cationic and anionic surfactants decrease it. A single variable optimization method is used to obtain optimum conditions. In order to eliminate its disadvantages, a statistical optimization method is used in recent studies. Statistical optimization shows that interactions between any two of the following pH, enzyme concentration and buffer concentration become significant in presence of a nonionic surfactant named Span 80.     

Novel extremely acidic lipases produced from Bacillus species using oil substrates

The extremely acidophilic microorganisms Bacillus pumilus and Bacillus subtilis were isolated from soil collected from the commercial edible oil and fish oil extraction industry. Optimization of conditions for acidic lipase production from B. pumilus and B. subtilis using palm oil and fish oil, respectively, was carried out using response surface methodology. The extremely acidic lipases, thermo-tolerant acidic lipase (TAL) and acidic lipase (AL), were produced by B. pumilus and B. subtilis, respectively. The optimum conditions for B. pumilus obtaining the maximum activity (1,100 U/mL) of TAL were fermentation time, 96 h; pH, 1; temperature, 50 °C; concentration of palm oil, 50 g/L. After purification, a 7.1-fold purity of lipase with specific activity of 5,173 U/mg protein was obtained. The molecular weight of the TAL was 55 kDa. The AL from B. subtilis activity was 214 U/mL at a fermentation time of 72 h; pH, 1; temperature, 35 °C; concentration of fish oil, 30 g/L; maltose concentration, 10 g/L. After purification, an 11.4-fold purity of lipase with specific activity of 2,189 U/mg protein was obtained. The molecular weight of the extremely acidic lipase was 22 kDa. The functional groups of lipases were determined by Fourier transform-infrared (FT-IR) spectroscopy.     

Production enhancement of Rhizopus arrhizus lipase by feeding oleic acid

A strategy for Rhizopus arrhizus lipase production enhancement by feeding oleic acid was developed. The oleic acid was proved to have strong inducing effect on lipase production, but high concentration oleic acid could repress lipase production. The decrease rate of oleic acid concentration using peanut oil as initial carbon source was figured out according to the change of oleic acid concentration in the fermentation broth. Our feeding strategy designed based on the decrease rate of oleic acid could avoid the repression of lipase production that is caused by high concentration of oleic acid in the fermenting liquor, and this strategy worked as a new feeding method showing excellent performance. The maximum lipase activity was gained by feeding dilute oleic acid every 12 h starting at 60 h, which maintained the oleic acid concentration around 18 mg/L, and the lipase activity was 31% higher than that of no feeding.     

基因拷贝数和甲醇浓度对重组毕赤酵母产华根霉脂肪酶的影响

将华根霉脂肪酶基因克隆到甲基营养型毕赤酵母中表达,以甲醇利用快型菌株为宿主,在7 L发酵罐水平对脂肪酶基因拷贝数分别为3、5、6的3株基因重组菌——XY RCL-3、XY RCL-5、XY RCL-6进行高密度发酵调控,同时研究了甲醇浓度对表达华根霉脂肪酶的影响。结果表明,XY RCL-5在相同条件下发酵产酶能力高于XY RCL-6和XY RCL-3,最适甲醇诱导浓度控制在0.1%±0.02%时,酶活可达到12 500 U/mL,菌体干重达到204 g/L,蛋白浓度也能达到8.02 g/L。     

Interfacing Pichia pastoris cultivation with expanded bed adsorption

For improved interfacing of the Pichia pastoris fed-batch cultivation process with expanded bed adsorption (EBA) technique, a modified cultivation technique was developed. The modification included the reduction of the medium salt concentration, which was then kept constant by regulating the medium conductivity at low value (about 8 mS/cm) by salt feeding. Before loading, the low conductivity culture broth was diluted only to reduce viscosity, caused by high cell density. The concept was applied to a one-step recovery and purification procedure for a fusion protein composed of a cellulose-binding module (CBM) from Neocallimastix patriciarum cellulase 6A fused to lipase B from Candida antarctica (CALB). The modified cultivation technique resulted in lower cell death and consequently lower concentration of proteases and other contaminating proteins in the culture broth. Flow cytometry analysis showed 1% dead (propidium-stained) cells compared to 3.5% in the reference process. During the whole process of cultivation and recovery, no proteolysis was detected and in the end of the cultivation, the product constituted 87% of the total supernatant protein. The lipase activity in the culture supernatant increased at an almost constant rate up to a value corresponding to 2.2 g/L of CBM-CALB. In the EBA process, no cell-adsorbent interaction was detected but the cell density had to be reduced by a two-times dilution to keep a proper bed expansion. At flow velocity of 400 cm/h, the breakthrough capacity was 12.4 g/L, the product yield 98%, the concentration factor 3.6 times, the purity about 90%, and the productivity 2.1 g/L x h.     

Bioreactor operated production of lipase: castor oil hydrolysis using partially-purified lipase.

A highly stable lipase from Pseudomonas aeruginosa KKA-5 was produced by batch cultivation technique employing shake flask and 5 L-bioreactor. The bioreactor was run at different airflow rates. Low airflow rates (1 and 3 L/min), did not lead to effective growth and lipase production. Growth increased by about one order and lipase production increased by about 6 times, at an airflow rate of 5 L/min. Lipase production occurred during decelerated cell growth. A highly stable lipase was produced which retained its activity in the running bioreactor, even after a period of one month. This stable lipase was partially-purified using ammonium sulphate precipitation technique. Castor oil was hydrolyzed using 300U crude and partially-purified lipase, each. Approximately 21-fold, partially-purified lipase could hydrolyze 81% castor oil within a period of 96 hr, where as only 63% hydrolysis was obtained, in 216 hour, when crude lipase was used.     

Plant Growth, Metabolism and Adaptation in Relation to Stress Conditions IV. Effects of Salinity on Certain Factors Associated with the Germination of Three Different Seeds High in Fats

Seeds of flax, cotton and castor beans were germinated in twosalt concentrations (0.5 and 1.0 per cent NaCl) over a periodof 12 d. The controls showed a continual increase in percentagegermination and in water content with time in all three seeds,while there was a continual decrease in dry matter, and in oilcontent. Salt at the lower concentration caused a marked decreasein each case, the decrease was even more marked at the higherconcentration. With the other parameters investigated, namely respiration,glycerol content and lipase activity, there was an increaseto a maximum and then a decrease in all controls with time.On the whole, the two salt concentrations caused marked decreasesin all these parameters except for respiration in flax whenthe lower salt concentration caused a significant increase;and for lipase activity which showed a significant increaseat the lower salt concentration in both cotton and in castorbean. Seed germination, salinity, growth, metabolic changes     

In situ lipolysis of castor oil in homogenised castor seeds

Summary Castor oil was lipolysedin situ in homogenised castor seeds to the extent of 87% in 2 hr at pH 4.8 and 25–37°C, without recourse to isolation of lipase and oil from castor seeds.IICT Communication No. 2562     

γ-decalactone production by Yarrowia lipolytica and Lindnera saturnus in crude glycerol

Flavor compounds are commonly obtained from chemical synthesis or extracted from plants. These sources have disadvantages, such as racemic mixture generation, more steps to yield the final product, low yield, and high cost, making the microbial fermentation an alternative and potential way to obtain flavor compounds. The most important lactone for flavor application is γ-decalactone, which has an aroma of peach and can be obtained by ricinoleic acid biotransformation through yeast peroxisomal β-oxidation. The aim of this work was to use crude glycerol, a residual biodiesel industry, for the production of bioaroma from two different yeasts. Yarrowia lipolytica CCMA 0357 and Lindnera saturnus CCMA 0243 were grown at different concentrations (10, 20, and 30% w/v) of substrates (castor oil and crude glycerol) for γ-decalactone production. L. saturnus CCMA 0243 produced higher concentration of y-decalactone (5.8?g/L) in crude glycerol, whereas Y. lipolytica CCMA 0357 showed a maximum production in castor oil (3.5?g/L). Crude glycerol showed better results for γ-decalactone production when compared to castor oil. L. saturnus CCMA 0243 has been shown to have a high potential for γ-decalactone production from crude glycerol.     

Quantitative studies of cell-bubble interactions and cell damage at different pluronic F-68 and cell concentrations

Pluronic F-68 (PF-68) is routinely used as a shear-protection additive in mammalian cell cultures. However, most previous studies of its shear protection mechanisms have typically been qualitative in nature and have not covered a wide range of PF-68 and cell concentrations. In this study, interactions between air bubbles along with the associated cell damage were investigated using the novel adenovirus-producing cell line PER.C6, a human embryonic retinoblast transfected with the adenovirus type 5 E1 gene. A wide range of PF-68 and cell concentrations (approximately 3 orders of magnitude) were used in these studies. At low PF-68 concentrations (0.001 g/L), cells had a very high affinity for bubbles, indicated by a more than 10-fold increase in cell concentration in the foam layer liquid versus the bulk liquid. At high PF-68 concentrations ( approximately 3 g/L), however, the cell concentration in the foam layer liquid was only approximately 40% of that in the bulk cell suspension. The number of cells associated with each bubble decreased from approximately 1000 cells at 0.001 g/L PF-68 to approximately 120 cells at 3 g/L PF-68. Despite the lower cell affinity for bubbles at a high PF-68 concentration, at high cell concentrations (10(7) cells/mL and 1 g/L PF-68) significant cell entrapment occurred in the foam layer, on the order of 1000 cells/bubble. For the cells carried by the bubbles, quantitative cell damage data revealed that the probability of cell death from bubble rupture was independent of bulk cell concentration but was affected by PF-68 concentration. These quantitative studies further indicated that even at a low PF-68 concentration of 0.03 g/L, approximately 30% of the attached cells were killed during the bubble rupture process. At the same time, at low PF-68 concentration (<0.1 g/L), significant cell death occurred prior to bubble rupture. On average, a bubble disrupted more cells in the bulk liquid and/or foam layer than during rupture. For both mechanisms, the number of cells damaged by each bubble increased with decreasing PF-68 concentration and increasing bulk cell concentration.     

Characteristics of immobilized lipase-catalyzed hydrolysis of olive oil of high concentration in reverse phase system

Candida rugosa lipase immobilized by adsorption on swollen Sephadex LH-20 could almost completely hydrolyze 60% (v/v) olive oil in isooctane. Kinetic analysis of the lipase-catalyzed hydrolysis reaction was found to be possible in this system. Amount of fatty acids produced was linearly proportional to the enzyme concentration of 720 mug/g wet gel. The specific enzyme activity was 217 units/mg protein at 60% (v/v) olive oil concentration. When the initial rate is plotted versus concentration of olive oil, this system did not follow Michaelis-Menten kinetics. Maximum activity was obtained at pH 7, but optimum temperature shifted towards higher one with the increase of olive oil concentration. Among the various chemical compounds tested, Hg(2+) and Fe(2+) inhibited the lipase seriously. As the concentration of olive oil increased, the rate of the hydrolysis also increased, but degree of the hydrolysis was observed to decrease. The supply of water from the inside of the gel to the surface of the gel was the main factor for the control of the rate of hydrolysis in batch hydrolysis. The immobilized lipase was used to hydrolyze olive oil two times. Achievement of chemical equilibrium took a longer time with the addition of water and the degree of hydrolysis decreased in the second consecutive trial. After the second hydrolysis trial, the gels were regenerated in a packed column first by eluting out both residual fatty acids around the gel particles and the accumulated glycerol with ethanol and then with 0.05M phosphate buffer, pH 7. The immobilized lipase on the regenerated gel showed the same hydrolysis activity as the original one.     

A Rapid Rosin-Celloidin-Paraffin method for Embedding Tissues

Pieces of tissue of various sizes or tissue fragments are dehydrated in 95% alcohol, cleared, washed with ether and infiltrated with a solution containing parloidin 9.6 g., camphor 3.0 g., absolute alcohol 200.0 ml., ether 200.0 ml., rosin 45.0 g. and castor oil 10 drops. After evaporation to the desired consistency, the mass is hardened with chloroform vapor, trimmed, passed through 3 changes of xylene to remove the rosin, embedded in paraffin, sectioned serially, stretched and stained as for paraffin methods. Methods of defatting tissues and detailed procedure for embedding fragments of bone marrow by this method are given.     

Optimization of extracellular lipase production from the biocontrol fungus Nomuraea rileyi

Lipases are important cuticle-degrading enzymes that hydrolyze the ester bonds of waxes, fats and lipoproteins during the infection of insects by the fungus Nomuraea rileyi. Lipase production by the N. rileyi strain MJ was optimized by varying environmental and nutritional conditions in culture medium containing different vegetable oils at various concentrations with shaking at 150 rpm for 8 days at 25°C. The maximum lipase production was obtained using castor oil (30.5±0.6 U mL^?1), followed in order by coconut oil (20.8±0.4 U mL^?1), olive oil (20.8±0.4 U mL^?1) and cottonseed oil (20.6±0.4 U mL^?1). The highest lipase activity (37.7±0.4 U mL^?1) was obtained when castor oil was used at a concentration of 4% (v/v) of basal medium. When the surfactant Tween 80 was added at the fourth day rather than at the beginning of incubation, a maximum lipase activity of 44.9±3.5 U mL^?1 was obtained. The optimal temperature and pH for lipase production were 25°C and pH 8.0, respectively. This is the first report on lipase production by the biocontrol fungus N. rileyi.     

One-pot conjugated linoleic acid production from castor oil by Rhizopus oryzae lipase and resting cells of Lactobacillus plantarum

Conjugated linoleic acid (CLA) has attracted as novel type of fatty acids having unusual health-promoting properties such as anticarcinogenic and antiobesitic effects. The present work employed castor oil as substrate for one-pot production of CLA using washed cells of Lactobacillus plantarum (L. plantarum) and lipases as catalysts. Among the screened lipases, the lipase Rhizopus oryzae (ROL) greatly assisted resting cells to produce CLA. Mass spectral analysis of the product showed that two major isomers of CLA were produced in the reaction mixture i.e. cis-9, trans-11 56.55% and trans-10, cis-12 43.45%. Optimum factors for CLA synthesis were found as substrate concentration (8 mg/mL), pH (6.5), washed cell concentration (12% w/v), and incubation time of 20 h. Hence, the combination of ROL with L. plantarum offers one pot production of CLA selectively using castor oil as a cost-effective substrate.