A hybrid neural model (HNM) for the on‐line monitoring of lipase production by Candida rugosa

A mechanistic model was proposed by Gordillo for the representation of lipase production by Candida rugosa, with the bioreactor in batch and fed‐batch operation. However, the model was not able to represent the lipolytic activity. The objective of the present study is to propose an efficient hybrid neural‐phenomenological model (HNM) for this process. The experimental data used corresponded to fed‐batch operation with constant substrate feed rate at 2.8 × 10^?7; 5.6 × 10^?7 and 9.7 × 10^?7 kg s^?1. Artificial neural networks (ANNs) were trained to represent the aqueous and intracellular lipase activity and were further associated with a reduced version of the mechanistic model of the proposed HNM. When compared to the experimental data, the HNM exhibited higher accuracy. The HNM can be employed in process monitoring using only on‐line measurements of CO₂ and substrate feed rate to infer enzyme activities and also substrate and biomass concentrations. Copyright © 2007 Society of Chemical Industry     

The use of olive mill wastewater by wild type Yarrowia lipolytica strains: medium supplementation and surfactant presence effect

BACKGROUND: The aim of this work was to study the ability of two different wild type strains of the yeast Yarrowia lipolytica to grow on olive mill wastewater (OMW) and their potential to produce high‐value products such as lipases. Factors that affect cellular growth and OMW degradation were studied, such as nitrogen supplementation, cells concentration and surfactant addition. RESULTS: Both strains, W29 and IMUFRJ 50682, were able to grow in OMW with 19 g L^?1 of COD and approximately 800 mg L^?1 of total phenols concentration. The strain W29 presented the highest potential for extracellular lipase production in OMW medium. Lipase productivity was improved by the medium supplementation with ammonium sulphate up to 6 g L^?1, leading to 80% of COD degradation and 70% of total phenols reduction. The surfactant Tween 80 enhanced cell growth and COD degradation, but had a negative effect on lipase activity. CONCLUSIONS Y. lipolytica has a great potential for OMW valorisation by its use as culture medium for biomass and enzymes production. The operating conditions that favoured lipase production differ from the conditions that improve COD reduction. Copyright © 2008 Society of Chemical Industry     

Lipase production in continuous culture of Candida rugosa

The yeast Candida rugosa produces multiple extracellular lipases. The production of extra‐ and intracellular lipases was investigated in continuous cultures using a sole or different mixtures of carbon sources. Also, the effect of different C:N ratios was tested. Lipase productivity in continuous cultures increased by 50% compared with data obtained from batch fermentations and depended on the dilution rate applied. Maximum yields relative to consumed substrate were obtained with oleic acid at low dilution rate. It was found that during nitrogen limitation, lipase activity was suppressed. All carbon source mixtures tested allowed both cell growth and lipase production, but extra‐ and intracellular lipase activities were affected by the combination of substrates used. Maximum extracellular lipolytic productivity was attained with lactic and oleic acid mixtures, probably due to the non‐repressor effect of these carbon sources. The chemical composition of the biomass also depended on the type of substrate used and was related to the accumulation of lipidic compounds as intracellular inclusions, which were observed when oleic acid was used as the carbon source. The results obtained were compared with previous data from batch and fed‐batch cultures in order to select the best process strategies for the lipase production with C rugosa. The best lipase yields were obtained in fed‐batch fermentations using oleic acid. Copyright © 2003 Society of Chemical Industry     

Extraction of Candida rugosa lipase from aqueous solutions into organic solvents by forming an ion‐paired complex with AOT

Candida rugosa lipase was extracted from aqueous solutions into organic solvents by forming an ion‐paired complex with sodium bis(2‐ethylhexyl)sulfosuccinate (AOT). The optimal aqueous pH for lipase recovery was 4.5 and the optimal CaCl₂ concentration was 10 mmol dm^?3. The lipase recovery decreased with increasing aqueous enzyme concentration but increased with increasing AOT concentration in the organic phase. The presence of polar co‐solvents in the aqueous phase did not obviously improve the lipase recovery, which was also little influenced by the type of hydrophobic organic solvent used for solubilising AOT. Surprisingly, no detectable activity of the ion‐paired C. rugosa lipase was observed for both the esterification of lauric acid with 1‐propanol in isooctane and the hydrolysis of olive oil in isooctane containing an appropriate amount of water. The ion‐paired C. rugosa lipase mediated the enantioselective crystallisation of racemic ketoprofen in isooctane, indicating the feasibility of using it as a chiral mediator for the enantioseparation of hydrophobic racemic compounds in organic systems. Copyright © 2006 Society of Chemical Industry     

Lipolytic enzyme production in batch and fed‐batch cultures of Ophiostoma piceae and Fusarium oxysporum

Lipase and esterase production by Ophiostoma piceae and Fusarium oxysporum were enhanced and extended by developing a fed‐batch process in stirred tank reactors. Fed‐batch strategy improved lipolytic enzyme production from Ophiostoma piceae in both 2 and 20 dm³ stirred tank reactors. However, fed‐batch fermentation of Fusarium oxysporum in the 2 dm³ reactor was more effective than both batch and fed‐batch fermentations in the 20 dm³ reactor. When a medium composed of only carbon and nitrogen source was intermittently fed to the cultures, the maximum specific lipase activity was improved by more than 80% and 35% in Ophiostoma piceae and Fusarium oxysporum cultures respectively. The maximum specific esterase activity was improved by 20% and 15% in Ophiostoma piceae and Fusarium oxysporum cultures respectively. The duration of production for both fungi extended from 144 to 216 h compared with a batch culture under the same condition. © 2000 Society of Chemical Industry     

Isolation of a yeast,Trichosporon cutaneum,able to use low molecular weight phenolic compounds: application to olive mill waste water treatment

This study deals with the degradation of phenolic compounds in olive oil mill waste; a highly polluting material in olive oil‐producing countries because of its abundance and the toxicity of its phenolic compound content. This investigation confirms the ability of an isolated yeast, identified as Trichosporon cutaneum, to degrade phenolic compounds extracted from olive mill waste water (OMW). The yeast was adapted to the OMW by an enrichment culture. The results of this biotransformation were a decrease in the phenolic content and hence a reduction in the phytotoxic effects of the effluent after the yeast treatment. The kinetic growth of the isolated yeast on phenol over a range of concentrations (0.3–3.0 g dm^?3) was studied. The ability of the strain to assimilate simple monomeric phenols and alkyl phenols, at a concentration of 1 g dm^?3, in a synthetic liquid medium used as the sole carbon source was investigated in a batch culture. The aromatic ring cleavage pathway occurred in the yeast through catechol oxidation. Using various concentrations of ethyl acetate extract from OMW as the sole carbon source, the yeast exhibited growth on the substrate up to 7 g dm^?3 equivalent of phenols. A significant reduction of COD after the treatment of the OMW extract by the yeast isolate was noticed. The removal of phenol and COD exceeded 80% of the original loading after 8 days of treatment, for extracts containing initial COD in the range 19 to 72 g dm^?3. Copyright © 2004 Society of Chemical Industry     

A comprehensive study of lipase production by Yarrowia lipolytica CECT 1240 (ATCC 18942): from shake flask to continuous bioreactor

BACKGROUND: Lipases are commercially important enzymes, and the development and optimization of their production processes are of great interest. The diversity of behaviours between strains stresses the need for research on this topic, especially when bioreactor culture is considered. The study of a continuous operating mode is especially attractive, since very scarce information is available on its application to microbial lipases production. RESULTS: Lipase production in submerged cultures of Yarrowia lipolytica CECT 1240 (ATCC 18 942) has been investigated. Significant lipolytic activity (over 700 U dm^?3), mostly extracellular and membrane‐bound, was obtained in shake flasks using medium supplemented with olive oil. The culture was carried out in air‐lift and stirred tank bench‐scale bioreactors and the latter was selected. The influence of aeration and agitation rates was assessed in batch cultures, and agitation from 400–700 rpm and low aeration rates (i.e. 0.2 vvm) are recommended. Batch, fed‐batch and continuous operation were investigated, and regular enzyme production (up to 600 U dm^?3) was achieved with the latter. CONCLUSION: Lipase production by the selected strain was successfully carried out in shake flasks and bench‐scale bioreactors. After studying batch, fed‐batch and continuous processes, continuous culture in a stirred tank bioreactor was found best in terms of regular enzyme production, exceptionally good operational stability and good fitting of the results to mathematical models. Copyright © 2009 Society of Chemical Industry     

Conversions of olive mill wastewater‐based media by Saccharomyces cerevisiae through sterile and non‐sterile bioprocesses

BACKGROUND: Olive mill wastewaters (OMWs) are an important residue and several methods have been proposed for their treatment. RESULTS: Remarkable decolorization (~63%) and phenol removal (~34% w/w) from OMW was achieved. In glucose‐based flask sterile cultures, enrichment with OMWs increased ethanol and biomass production compared with cultures without OMWs added. Flask sterile and un‐sterilized cultures demonstrated similar kinetic results. Batch‐bioreactor trials performed showed higher ethanol and lower biomass quantities compared with the respective shake‐flask experiments, while cultures used under un‐sterilized conditions revealed equivalent results to the sterile ones. In non‐sterile bioreactor cultures, OMWs addition enhanced biomass production in comparison with culture with no OMWs added, whereas ethanol biosynthesis was not affected. The maximum ethanol quantity achieved was 52 g L^?1 (conversion yield per sugar consumed of 0.46 g g^?1) in a batch bioreactor non‐sterilized trial with OMW–glucose enriched medium used as substrate, that presented initial reducing sugars concentration at ~115 g L^?1. Fatty acid analysis of cellular lipids demonstrated that in OMW‐based media, cellular lipids containing increased concentrations of oleic and linoleic acid were produced in comparison with cultures with no OMWs added. CONCLUSIONS: S. cerevisiae simultaneously produced bio‐ethanol and biomass and detoxified OMWs, under non‐sterile conditions. © 2012 Society of Chemical Industry     

Xylitol production by Candida tropicalis from corn cob hemicellulose hydrolysate in a two‐stage fed‐batch fermentation process

BACKGROUND: Xylitol, a sugar alcohol widely used in food and pharmaceutical industries, can be produced through biological reduction of xylose present in hemicellulose hydrolysates by Candida tropicalis. However, the aeration rate and by‐products originating from hemicellulose hydrolysis strongly inhibit the production of xylitol in a fermentation process. A two‐stage fed‐batch fermentation system was developed to reduce these inhibitory effects and to improve xylitol production from corn cob hemicellulose hydrolysates by C. tropicalis. RESULTS: Results of batch fermentations indicated that high xylitol production could be obtained from C. tropicalis at an initial xylose concentration of 80 g L^?1 in corn cob hydrolysate medium at an aeration rate of 0.4 vvm at the micro‐aeration stage. In the two‐stage fed‐batch fermentation process, 96.5 g L^?1 xylitol was obtained after 120 h, giving a yield of 0.83 g g^?1 and a productivity of 1.01 g L^?1 h^?1, which were 12.16% and 65.57% higher than those in a batch fermentation. CONCLUSION: High xylitol production can be achieved in a two‐stage fed‐batch fermentation process, in which the negative effects of aeration rate and inhibitory compounds on xylitol formation can be considerably reduced. Copyright © 2011 Society of Chemical Industry     

Study of a Lipase from Candida rugosa Diddens and Lodder

Lipasic system of Candida rugosa (CBS 613) strain was studied. The enzyme was purified in one step by hydrophobic chromatography. The properties of this lipase were determined. It is an oligomeric enzyme composed of five identical monomers of 46 kg · mol^?1. Its optimum reaction conditions are pH = 7 and temperature = 40°C. This enzyme presents a rapid thermal denaturation and then a more stable form. It is a cell-bound lipase which is induced by triacyl glycerols. This enzyme presents a high specificity for external positions on glycerol.     

Production of organic solvent tolerant lipase by Staphylococcus caseolyticus EX17 using raw glycerol as substrate

BACKGROUND: In this work we used Plackett–Burman statistical design and central composite design in order to optimize culture conditions for lipase production by Staphylococcus caseolyticus strain EX17 growing on raw glycerol, which was obtained as a by‐product of the enzymatic synthesis of biodiesel. The stability of lipase was verified over several organic solvents, such as methanol, ethanol and n‐hexane. RESULTS: Optimal culture conditions for lipase production were found to be 36 °C, initial pH 8.12, glycerol 30 g L^?1, olive oil 3.0 g L^?1, and soybean oil 2.5 g L^?1, with 145.8 U L^?1 of enzyme activity. When commercial glycerol was substituted by the raw glycerol from biodiesel synthesis, lipolytic activity was 127.3 U L^?1. Experimental validation of enzyme production matched values predicted by the mathematical model, which was 138.3 U L^?1. Stability tests showed that lipase from S. caseolyticus EX17 was stable in methanol, ethanol, and n‐hexane. CONCLUSIONS: Results obtained in this work suggest that raw glycerol can be used for lipase production by S. caseolyticus EX17 and that this enzyme has a potential application in the synthesis of biodiesel. Copyright © 2008 Society of Chemical Industry     

Citric acid,biomass and cellular lipid production by Yarrowia lipolytica strains cultivated on olive mill wastewater‐based media

BACKGROUND: Olive mill wastewaters (OMWs) are an important residue and several physico‐chemical and/or biotechnological methods have been proposed for their treatment. RESULTS: The ability of three Yarrowia lipolytica strains to grow on and convert glucose‐enriched OMWs into added‐value compounds in carbon‐ and nitrogen‐limited shake‐flask cultures was assessed. Remarkable decolorization (up to 63%) and non‐negligible removal of phenolic compounds (up to 34%, w/w) occurred. In nitrogen‐limited cultures, the accumulation of cellular lipids was favored by OMW addition into the medium. In contrast, although remarkable quantities of citric acid (Cit) were produced in control experiments (cultures without OMW addition), in which Cit up to 18.9 g L^?1 was produced with yield of Cit synthesized per sugar consumed ～0.73 g g^?1), adaptation of cultures to media supplemented with OMWs reduced the final Cit quantity and conversion yield values achieved. In OMW‐based media, the highest concentration of citric acid produced was 18.1 g L^?1, with conversion yield ～0.51 g g^?1. In carbon‐limited cultures, despite the presence of inhibitory compounds in the medium (e.g. phenols), biomass production was enhanced with the addition of OMWs. The highest biomass concentration achieved was 12.7 g L^?1, with biomass conversion yield per sugar consumed ～0.45 g g^?1. Fatty acid analysis of cellular lipid produced demonstrated that adaptation of all strains in OMW‐based media favored the synthesis of cellular lipids that contained increased concentrations of cellular oleic acid. CONCLUSIONS: The Y. lipolytica strains tested can be regarded as possible candidates for simultaneous OMWs remediation and production of added‐value compounds. Copyright © 2011 Society of Chemical Industry     

In‐line mixing for production of citric acid by Candida lipolytica grown on n‐paraffins

This study reports on the effects of internal fermenter and external in‐line agitation and fed‐batch mode of operation on citric acid production from Candida lipolytica using n‐paraffin as the carbon source. An optimum range of fermenter agitation speeds in the range 800–1000 rpm corresponding to Reynolds numbers of 50433–62947 (based on initial batch conditions) seemed to give the best balance between substrate utilization for biomass growth and citric acid production. Proof of concept evidence is presented that indicates that an external in‐line agitator could be used in place of high speed internal agitation to increase citric acid production. However, more work is required to optimize the external agitator concept. Application of multiple fed‐batch feedings can be used to extend the batch fermentation and increase final citric acid concentrations and product yield. Experiments were conducted implementing a three‐cycle fed‐batch process which increased overall citric acid yields to 0.8–1.0 g citric acid g^?1 n‐paraffin, approximately 200% improvement from those found in the normal batch process. The three‐cycle fed‐batch mode of operation also increased the final citric acid concentration to 42 g dm^?3 from about 6 g dm^?3 for normal batch operation. Increased citric acid concentrations in three‐cycle fed‐batch mode was achieved at longer fermentation times. Copyright © 2004 Society of Chemical Industry     

Daily doses of light in relation to the growth of Scenedesmus obliquus in diluted three‐phase olive mill wastewater

BACKGROUND: The use of olive‐oil mill wastewater (OMW) from a three‐phase centrifugation process used in the olive‐oil industry, has been studied in relation to the production of the microalga Scenedesmus obliquus CCAP 276/3A. The chemical characteristics of OMW indicated nitrogen deficiency. RESULTS: S. obliquus is able to assimilate nutrients present in a culture medium (water‐OMW 5%) and grow at its maximum specific growth rate of 0.026 h^?1, both under mixotrophic as well as heterotrophic conditions. The different daily doses of light (DDL) used, in the range 0–36 E m^?2 d^?1, determined light‐limited and light‐inhibited cultures. The light‐inhibited mixotrophic cultures bore characteristics similar to those of the heterotrophic cultures, and became more so when the dose of light received was higher. The low protein yield (258 mg g^?1) and high percentage of carbohydrates of the biomass (65.8%) confirmed a nutritional‐stress situation associated with nitrogen limitation. CONCLUSION: The similarity between the fatty‐acid composition of the heterotrophic and mixotrophic cultures strongly inhibited by light appeared to indicate the cancelling of the photosynthetic behaviour of the cells at high DDL values. The biomass generated can be used for biofuels. The maximum elimination of biological oxygen demand (BOD₅) per unit of biomass was achieved in the heterotrophic cultures. Copyright © 2009 Society of Chemical Industry     

Characterization of Candida rugosa Lipase Immobilized on Poly(N‐methylolacrylamide) and Its Application in Butyl Butyrate Synthesis

Candida rugosa lipase was immobilized on poly(N‐methylolacrylamide) by physical adsorption. The biocatalyst performance (immobilized lipase) was evaluated in both aqueous (hydrolysis) and organic (butyl butyrate synthesis) media. In the first case, a comparative study between free and immobilized derivatives was provided in terms of pH, temperature and thermal stability following the olive oil hydrolysis, establishing new optimum values. In the second case, the influence of temperature, biocatalyst concentration and acid/alcohol molar ratio was simultaneously studied according to a 2³ full experimental design. The highest molar conversion (96 %), volumetric productivity (1.73 g L^–1 h^–1) and specific esterification activity (1.00 μM mg^–1 min^–1) were obtained when working at the lowest level of temperature and butyric acid in excess. Under these conditions, repeated batch use of the immobilized enzyme was performed and half‐life time (t_1/2) was found to be 145 h.     

Performance evaluation of batch and unsteady state fed‐batch reactor operations for the production of a marine microbial surfactant

BACKGROUND: Biosurfactants are microbially derived surface‐active and amphipathic molecules produced by various microorganisms. These versatile biomolecules can find potential applications in food, cosmetics, petroleum recovery and biopharmaceutical industries. However, their commercial use is impeded by low yields and productivities in fermentation processes. Thus, an attempt was made to enhance product yield and process productivity by designing a fed‐batch mode reactor strategy. RESULTS: Biosurfactant (BS) production by a marine bacterium was performed in batch and fed‐batch modes of reactor operation in a 3.7 L fermenter. BS concentration of 4.61 ± 0.07 g L^?1 was achieved in batch mode after 22 h with minimum power input of 33.87 × 10³ W, resulting in maximum mixing efficiency. The volumetric oxygen flow rate (K_La) of the marine culture was about 0.08 s^?1. BS production was growth‐associated, as evident from fitting growth kinetics data into the Luedeking‐Piret model. An unsteady state fed batch (USFB) strategy was employed to enhance BS production. Glucose feeding was done at different flow rates ranging from 3.7 mL min^?1 (USFB‐I) to 10 mL min^?1 (USFB‐II). USFB‐I strategy resulted in a maximum biosurfactant yield of 6.2 g l^?1 with an increment of 35% of batch data. The kinetic parameters of USFB‐I were better than those from batch and USFB‐II. CONCLUSION: Comparative performance evaluation of batch and semi‐continuous reactor operations was accomplished. USFB‐I operation improved biosurfactant production by about 35% over batch mode. USFB‐I strategy was more kinetically favorable than batch and USFB‐II. © 2012 Society of Chemical Industry     

Effect of lipids and surfactants on extracellular lipase production by Yarrowia lipolytica

The production of extracellular lipase in submerged cultures of Yarrowia lipolytica CECT 1240 has been investigated. Several compounds have been added to the culture medium, in order to assess their efficiency as inducers of lipase production. First, the effect of triglycerides (olive oil, sunflower oil, tributyrin) and fatty acids (oleic acid) has been studied. The highest activity level was obtained with sunflower oil (58 U cm^?3), followed by olive oil (49 U cm^?3). The cultures with tributyrin and oleic acid attained similar activities (33 U cm^?3). Then, several surfactants (Tween 80, Triton X‐100, gum arabic, polyethylene glycol 200) were added to the cultures with sunflower oil, in an attempt to increase the levels of extracellular lipase activity. The obtained activities were slightly lower than those achieved without surfactants. The assay of a wide range of surfactant concentrations in the case of PEG‐200 (with which the highest activity levels had been attained) did not improve the results. This strain secreted lipase concentrations two‐fold higher and showed significantly different behaviour towards the presence of surfactants in the culture medium, compared with other wild‐type Yarrowia lipolytica strains. Copyright © 2003 Society of Chemical Industry     

Invert emulsion as a medium for fungal lipase activity

An extracellular lipase from the fungusPythium ultimum was active in an invert [water-in-oil] emulsion consisting of 4% water emulsified into edible oils with taurocholic acid as the surfactant. The pH range for optimum lipolytic activity was 7.5–8.5, and the optimum temperature for activity was 45°C. Specific activity of the purified lipase was 919.5 μmol/min/mg protein in the invert emulsion. Water content of the invert emulsion influenced activity of the lipase differently, depending on the substrate. The rate of olive oil hydrolysis with thePythium lipase decreased with time, possibly due to inactivation of the enzyme and inhibition by free fatty acid products of the reaction. Total hydrolysis of olive oil by thePythium lipase was compared with that by lipases fromCandida rugosa andRhizopus arrhizus in the invert emulsion. Hydrolysis essentially ceased within 24 h or less for the lipases from each source. However, the addition of aqueous solution at 8 h from the beginning of incubation stimulated hydrolysis byC. rugosa andR. arrhizus lipases by 1.8-fold and 2.5-fold, respectively, but not by theP. ultimum lipase, over corresponding controls after 48 h.     

Effect of lipase‐producing yeast on the oily fraction of microbiologically debittered table olives

This study was carried out to evaluate the influence of the lipase‐producing yeasts, present in the brine, on the quality of table olive oil. The oily fraction of olives processed microbiologically using the β‐glucosidase‐positive, lipase‐negative yeast Candida wickerhamii 1542 showed an excellent quality, such as the typical extra‐virgin olive oil. In contrast, the quality of the oily fraction of olives debittered microbiologically with the β‐glucosidase‐ and lipase‐producing yeast Williopsis californica 1639 worsened dramatically to such a point that the acidity of the oily fraction increased more than 90%. The microbiological analysis of brines showed that the lipase‐producing yeasts are part of the natural yeast population of the brine, and when their presence is high, it can damage significantly the quality of table olives. The lipase‐producing yeast W. californica 1639 in the brine showed the maximum lipolytic activity at pH 6, while the NaCl of the brine, at the maximum concentration of 12%, inhibited lipase activity by 70%. The concentration of the polyphenols present in the brine was not able to control the lipolytic activity of W. californica 1639 during the microbiological debittering process of table olives.     

Lovastatin production by Aspergillus terreus in a two‐staged feeding operation

BACKGROUND: Lovastatin is known to inhibit its own synthesis in the fungus Aspergillus terreus. Therefore, the use of a fermentation strategy that continuously removes some of the lovastatin produced from the bioreactor can enhance its productivity. This paper reports on the effects of dilution rate and the composition of the feed medium on lovastatin production by A. terreus. RESULTS: The feeding strategy consisted of an initial batch/fed‐batch phase and a semi‐continuous culture phase in which the pelleted biomass was retained inside a slurry bubble column. A nitrogen‐free medium was fed at various fixed dilution rates in the semi‐continuous phase. In experiments that were designed to assess the effects of the composition of the medium, the dilution rate was held at 0.42 d^?1, but different feed media were used in separate runs. The best two‐staged production strategy was shown to consist of a 96 h batch/fed‐batch phase that used a nutritionally complete medium. This was followed by a semi‐continuous operation using a medium that was free of both nitrogen and carbon sources. CONCLUSION: Semi‐continuous operation enhanced productivity of lovastatin by 315% compared with a conventional batch operation. The optimal dilution rate in semi‐continuous operation was about 0.42 d^?1. Copyright © 2008 Society of Chemical Industry