Utilization of olive mill wastewater for selective production of lipids and carotenoids by Rhodotorula glutinis.

Olive mill wastewater (OMW) is a zero-cost substrate for numerous value-added compounds. Although several studies on the production of lipids and carotenoids by Rhodotorula glutinis in OMW exist, none of them has specifically focused on the conditions for a target lipid or carotenoid. This study presents cultivation conditions that selectively stimulate the cell biomass, individual carotenoids and lipids. It was found that supplemental carbon and nitrogen sources as well as illumination affected cell biomass the most. High temperature, low initial pH, illumination, lack of urea and presence of glycerol stimulated the lipid synthesis. The highest total lipid content obtained in undiluted OMW supplemented with urea was 11.08 ± 0.17% (w/w) whilst it was 41.40 ± 0.21% (w/w) when supplemented with glycerol. Moreover, the main fatty acid produced by R. glutinis in all media was oleic acid, whose fraction reached 63.94 ± 0.58%. Total carotenoid yield was significantly increased with low initial pH, high temperature, illumination, certain amounts of urea, glycerol and cultivation time. Up to 192.09 ± 0.16 µg/g cell carotenoid yield was achieved. Torularhodin could be selectively produced at high pH, low temperature and with urea and glycerol supplementation. To selectively induce torulene synthesis, cultivation conditions should have low pH, high temperature and illumination. In addition, low pH, high temperature and urea supplementation served high production of ß-carotene. Up to 85.40 ± 0.76, 80.67 ± 1.40 and 39.45 ± 0.69% of torulene, torularhodin and ß-carotene, respectively, were obtained under selected conditions. KEY POINTS: • Cultivation conditions selectively induced target carotenoids and lipids • 41.40 ± 0.21% (w/w) lipid content and 192.09 ± 0.16 µg/g cell carotenoid yield were achieved • Markedly high selectivity values for torularhodin and torulene were achieved.

Parameters and kinetics of olive mill wastewater dephenolization by immobilized Rhodotorula glutinis cells.

Olive mill wastewater (OMW) with total phenol (TP) concentration range of 300-1200 mg/L was treated with alginate-immobilized Rhodotorula glutinis cells in batch system. The effects of pellet properties (diameter, alginate concentration and cell loading (CL)) and operational parameters (initial TP concentration, agitation rate and reusability of pellets) on dephenolization of OMW were studied. Up to 87% dephenolization was obtained after 120 h biodegradations. The utilization number of pellets increased with the addition of calcium ions into the biodegradation medium. The overall effectiveness factors calculated for different conditions showed that diffusional limitations arising from pellet size and pellet composition could be neglected. Mass transfer limitations appeared to be more effective at high substrate concentrations and low agitation rates. The parameters of logistic model for growth kinetics of R. glutinis in OMW were estimated at different initial phenol concentrations of OMW by curve-fitting of experimental data with the model.

Investigation of the simultaneous production of superoxide dismutase and catalase enzymes from Rhodotorula glutinis under different culture conditions.

The simultaneous production production of superoxide (SOD) and catalase (CAT) from Rhodotorula glutinis was studied. The effects of temperature, initial medium pH, and carbon source on the enzyme activities were investigated. Temperature and carbon sources were found to have significant effects on the enzyme activities. 10°C provided the highest specific CAT and SOD activities as 22.6 U/mg protein and 170 U/mg protein, respectively. Glycerol was found to be the best carbon source for enzyme activities, providing 113 U/mg protein for CAT and 125 U/mg protein for SOD, which were also the highest activities obtained in the present study.

Extracellular lipolytic enzyme activity of a newly isolated Debaryomyces hansenii.

A strain isolated from waste of a milk products plant and exhibited extracellular lipolytic activity was identified as Debaryomyces hansenii by 5.8S rRNA and 28S rRNA gene sequence analyses. Lipolytic activity was assayed spectrophotometrically by using p-nitrophenylpalmitate. Higher specific lipolytic activities were obtained in the presence of tristearin (0.68 U/mg prot), oleic acid (0.56 U/mg prot), and soybean oil (0.36 U/mg prot) than other triglycerides, fatty acids, and vegetable oils considered as carbon sources. Cheese whey appeared to be a good alternative to lipidic substances for lipolytic activity. Among various organic and inorganic nitrogen sources, soy flour was found to attain the lipolytic activity similar to that provided by universal yeast medium components. This work is the first report on the discussion of lipolytic activity enhancement by D. hansenii through modulating the cultivation medium. It also proposes low cost medium nutrients that could be of industrial value and could serve as basal nutrients for further optimization studies on the lipase production by D. hansenii.

Media formulation using complex organic nutrients for improved activity, productivity, and yield of Candida rugosa lipase and esterase enzymes.

Candida rugosa is an excellent source of multiple lipase and esterase enzymes; therefore, it is of technological importance to formulate the medium that provides high activity for each enzyme. In this work, the cultivation medium comprising complex nutrients that provided the highest activity, productivity, and yield of C. rugosa enzymes individually was formulated. Time courses of the extracellular and intracellular lipase and esterase activities of C. rugosa were represented and the role of protease in the cultivation progress was discussed. Urea, soy-peptone, yeast extract, a mixture of soy-peptone and yeast extract, cheese whey, and wheat mill bran were tested for their lipolytic and esterasic activities. Urea provided considerably higher extracellular lipase activity when compared to other nitrogen sources; however, soy-peptone provided the highest extracellular esterase activity. Hazelnut, olive, sesame, soybean, and flax seed oils affected the enzyme activities to different extents related to their fatty acid compositions. Hazelnut oil and olive oil provided the highest extracellular lipase and esterase activities, respectively, whereas sesame oil produced the highest biomass. High C(18) and C(16) ester contents of vegetable oils promoted high lipase and esterase productions, respectively. A temperature of 30 degrees C yielded the highest extracellular and intracellular lipase and esterase activities; however, 35 degrees C produced the highest biomass.

Impact of inoculation strategy on the progress of Candida rugosa cultivation.

Batch cultivation of Candida rugosa for the production of lipase and esterase enzymes was carried out by employing two different inoculation strategies. The effects of triolein (2, 3 and 5 g/l) and oleic acid (3 g/l) as carbon sources of the enzyme production medium on the activity, productivity, and yield of enzymes were also compared for both strategies. Inoculation of the cells into the enzyme production medium either directly or after cultivation in a pre-culture medium rich in glucose affected the activity and yield of esterase more than those of lipase for both carbon sources. In both strategies, triolein and oleic acid yielded the same lipase activity (16.67 U/ml) whereas triolein provided higher esterase activity (0.0035 U/ml). Time courses of the extracellular and intracellular lipase and esterase enzymes indicated that lipase activity was growth-associated and the cells secreted esterase into the medium after a considerable level of extracellular lipase activity was reached. The role of protease in the enzyme activities was also discussed.

Effects of alcohol and buffer treatments on the activity and enantioselectivity of Candida rugosa lipase.

Several treatments were employed on Candida rugosa lipase (CRL) to improve its biocatalytic performance. Besides conventional alcohol treatment conditions, the effects of pH of the buffer solution used in the treatment as well as the changes in stirring, dialysis, and centrifugation steps of the treatment procedure were investigated for the first time for the resolution of racemic naproxen methyl ester. The highest enantioselectivity and conversion in S-naproxen production were achieved by CRL treated with pH 7.5 buffer solution. The elimination of the centrifugation step resulted in an increase in the enantioselectivity, whereas alcohol treatment of CRL was found to be inconvenient for S-naproxen production. Higher activity for p-nitrophenyl acetate was achieved when 20% butanol and pH 4 buffer solution were used, and when dialysis and stirring times were shortened.

Effects of lipidic carbon sources on the extracellular lipolytic activity of a newly isolated strain of Bacillus subtilis.

An isolate exhibiting high extracellular lipolytic activity was identified as Bacillus subtilis by 16S rRNA gene sequence analysis. The enzyme activity of the isolate was improved by using different concentrations of lipidic carbon sources such as vegetable oils, fatty acids and triglycerides. Lipolytic activity was assayed spectrophotometrically using p-nitrophenyl palmitate. One percent (v/v) of sesame oil provided the highest activity with 80 and 98% enhancements with respect to 1% (v/v) concentrations of linoleic acid and triolein as the favored fatty acid and triglyceride, respectively. Glucose presented a repressive effect on lipase production. Lipase secreted by B. subtilis was partially purified by ultrafiltration and anion exchange chromatography; and the purified enzyme was tested for its residual activity in the presence of EDTA, SDS, Triton X-100, Tween 20, Tween 80 and protease. The present work reports, for the first time, that the lipolytic activity of a B. subtilis strain can be improved by using inexpensive vegetable oils; and also that B. subtilis lipase is suitable for use in detergents.

A parametric study on biphasic medium conditions for the enantioselective production of naproxen by Candida rugosa lipase.

A parametric study to increase the enantioselectivity of Candida rugosa lipase (CRL) toward S-Naproxen production by the hydrolysis of racemic Naproxen methyl ester in an aqueous-organic biphasic batch system was carried out. Effects of organic solvent type, aqueous phase/organic solvent volume ratio, agitation rate, concentrations of the substrate and the enzyme, pH of the aqueous phase, and temperature on the enantiomeric excess for the product (eep), on the enantiomeric ratio (E) and on the conversion (x) were evaluated. Employing isooctane as the solvent resulted in higher eep, E, and x than those obtained in hexane, cyclohexane, and toluene. The higher the volume ratio of aqueous phase/organic solvent employed, the higher the conversion and enantioselectivity achieved. The increase in agitation rate increased the hydrolysis rate. Higher concentration of racemic Naproxen methyl ester than 10 mg/mL decreased both the conversion and enantioselectivity. The increase in crude CRL concentration resulted in enhancement of x, but the decrease of eep and E. Acidic pH led to higher conversion and enantioselectivity than the medium and alkaline pH values. A further increase in temperature to over 45 degrees C decreased the conversion and enantioselectivity. The highest enantiomeric ratio achieved in the S-Naproxen production was E = 171.1, with x = 49.8% and eep = 95.7%.

Isolation of lipase producing Bacillus sp. from olive mill wastewater and improving its enzyme activity.

The bacteria that could grow on media containing olive mill wastewater (OMW) were isolated and their lipase production capacities were investigated. The strain possessing the highest lipase activity among 17 strains grown on tributyrin agar medium was identified as Bacillus sp. The effect of initial pH on the lipase activity was investigated in tributyrin medium and pH 6 was found to be the optimal. The liquid medium composition was improved by replacing tributyrin with various carbon sources. Among the media containing different compositions of triolein, trimyristin, trilaurin, tricaprin, tricaprylin, tributyrin, triacetin, Tween 80, OMW, glucose, and whey; the medium contained 20% whey +1% triolein was found to give the highest lipase activity. Cultivation of Bacillus sp. in the optimal medium at pH 6 and 30 degrees C for 64h resulted in the extracellular and intracellular lipase activities of 15 and 168U/ml, respectively.

Effect of ionic environments on the adsorption and diffusion characteristics of serine alkaline protease enzyme in polyethersulfone ultrafiltration membranes.

Static adsorption of serine alkaline protease (SAP) enzyme on hydrophobic polyether sulfone (PES) ultrafiltration membranes in different ionic environments was investigated. The amount of SAP adsorbed on membranes was the lowest at its isoelectric point (IEP) where the maximum adsorption was obtained below the IEP of the enzyme. The extent of SAP adsorption in the phosphate buffer solutions including different salts followed the order: (NH4)2HPO4 > KH2PO4 > Na2HPO4-NaH2PO4 (buffer) > CaCl2 > ((NH4)2HPO4 + H2PO4 + CaCl2), which was consistent with the Hofmeister series. The zeta potentials of membranes contacted with the ionic species were calculated by streaming potential measurements and found that the increase in ionic strength decreased the electrical double layer thickness leading to a decrease in adsorption. A model based on mass balance was developed to calculate the diffusion coefficient of SAP in PES membranes. Employing experimental data evaluated in a diffusion cell along with the data of adsorption isotherms, diffusion coefficients of SAP in PES membranes in the presence of different ionic species were calculated. To detect the structural changes occurred, membrane surfaces were analysed by Fourier transform infrared-attenuated total reflectance (FTIR-ATR) measurements.