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     

Fed‐batch fermentation of olive mill wastewaters for lipase production

In the Mediterranean basin countries, huge amounts of olive mill wastewaters (OMW) are produced by the olive oil industry. It constitutes a serious environmental problem, nevertheless its composition turns OMW into a potential growth medium to lipolytic microorganisms. The aim of this work was to study lipase production as well as OMW degradation in fed‐batch cultures of Candida cylindracea CBS 7869, Candida rugosa CBS 2275 and Yarrowia lipolytica W29 (ATCC 20460). Besides the improvement of lipase production, the fed‐batch approach enhanced the effluent degradation, since it led to good COD and lipids reduction, both higher than 50%. C. rugosa achieved the highest value of lipase productivity (130 U L^?1 h^?1), in parallel with highest lipids reduction (77%). This study demonstrates that OMW are becoming a competitive and valuable growth medium in fermentation processes with lipolytic microorganisms. The fed‐batch strategy used proved to be an efficient approach to enhance lipase production from OMW and to reduce significantly the final organic load of the medium. Copyright © 2012 Society of Chemical Industry     

Biodegradation and utilization of waste cooking oil by Yarrowia lipolytica CECT 1240

Biodegradation of waste cooking oil and its application as lipase production inducer in cultures of Yarrowia lipolytica CECT 1240 have been investigated, both in shake flasks and a bench‐scale bioreactor. The ability of this strain to degrade the spent oil was evaluated by monitoring COD throughout the cultures, and a remarkable decrease was recorded (almost 90% decrease in oil COD after 3 days in bioreactor). Moreover, the addition of waste cooking oil to the medium led to a significant augmentation in extracellular lipase production by the yeast, compared to oil‐free cultures. This confirms the suitability of the studied residue as an inducer of lipase biosynthesis, which is a very interesting fact, from an economic standpoint. These results were confirmed when a fed‐batch strategy was proposed. Finally, some properties of the crude enzyme were studied, and compared to the enzymes obtained when non‐used oil was added to the medium. Practical application : New strategies to valorize wastes from the food and agro industries are attracting a great scientific interest due to the important advantages offered from an economic and environmental point of view. For this reason, the yeast Yarrowia lipolytica CECT 1240 is proposed for degrading waste cooking oils. This approach entails also another benefit in terms of lipolytic enzyme synthesis, since the addition of used up oils has a lipase inducer effect. The enormous interest in lipases is reflected in the number of applications that they present. The process was successfully carried out both in shake flasks and a bench‐scale bioreactor, allowing producing high levels of lipolytic activity at the same time that the COD was diminished up to nearly 90%.     

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     

Biosynthesis of lipids and organic acids by Yarrowia lipolytica strains cultivated on glucose

The biochemical behavior of wild‐type or genetically modified (presenting decreased expression of intracellular acyl‐CoA oxidases) Yarrowia lipolytica strains cultivated on commercial glucose was studied. Flask nitrogen‐limited cultures were performed favoring the production of organic acids (and potentially the accumulation of lipid). Nitrogen depletion induced secretion of citric acid, while intracellular lipid was not produced in high quantities. Maximum total citric acid up to 49 g/L (yield 0.85 g/g glucose) was produced. In some of the wild‐type strains, an increase of glucose in the medium also induced noticeable production of acetic acid. Increasing the amount of added glucose led to an increase in the total lipid quantity (%) produced, although in the stationary growth phase the concentration of lipid declined, indicating lipid degradation even for the genetically modified strains. Total lipid amount did not exceed the value of 14%, while neutral fractions increased with increase in glucose concentration. In all cases, the total microbial lipids and major lipid fractions were composed of C₁₆ and C₁₈ (principally unsaturated) fatty acids. Finally, in several of the strains cultured in media containing a low glucose concentration, unicellular morphology was observed, while at high glucose concentrations mycelia were predominant.     

Sustainability analysis and benchmarking of olive mill wastewater treatment methods

A large number of publications are available in the literature regarding olive mill wastewater treatment methods. However, none of the proposed methods can be considered as a best available method in terms of its effectiveness, and its environmental and economic impact. Using a literature survey, data were collected and evaluated in order for a sustainability and benchmarking analysis to be developed. Physicochemical, biological and advanced oxidation methods were evaluated and judged in terms of their effectiveness, environmental impact and cost. Effectiveness of each method was estimated in terms of COD and phenolic compounds reduction, environmental impact in terms of CO₂ production, while for the economic impact the operational costs were taken into account. Finally, a procedure is suggested for selection of the most appropriate method based on user preferences (in terms of effectiveness, environmental impact and cost). The present analysis showed that the most effective processes in terms of organics reduction are membrane filtration, electrolysis, supercritical water oxidation and photo‐Fenton. Lower environmental impact was found with anaerobic digestion, coagulation and lime processes, while the lowest cost category involves biocomposting and membrane filtration, thanks to the exploitation of byproducts (biocompost and phenolic compounds, respectively). © 2013 Society of Chemical Industry     

Improvement of Yarrowia lipolytica Lipase Enantioselectivity by Using Mutagenesis Targeted to the Substrate Binding Site

Enhanced enantioselectivity : The resolution of 2‐bromo‐arylacetic acid esters by Lip2p lipase from Yarrowia lipolytica was improved through mutagenesis of the substrate binding site. Position 232 was identified as crucial for the discrimination. Saturation of this position led to the identification of variant V232S, which has a tremendously increased activity and E value as compared to the parental enzyme.

     

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     

Electrochemical treatment of olive mill wastewater

Background Olive mill wastewater (OMW) constitutes a very strong agro‐industrial wastewater posing severe environmental threats in olive oil producing countries. The main objective of this study was to treat olive mill wastewater by electrochemical oxidation. The variables studied included the type and concentration of electrolyte solutions, voltage and time applied. Results: The electrolyte type and concentration significantly affected the degradation efficiency of the electrochemical oxidation. Optimal conditions for NaCl concentration were 3% (w/v) and 16 V. At these conditions chemical oxygen demand (COD) removal reached 70.8% after 8 h of electrochemical treatment, while color and turbidity were completely removed after short periods of treatment. However, bio‐assays indicated that the ecotoxicity of the treated wastewater remained unchanged, possibly due to the formation of chlorinated by‐products. Na₂SO₄ did not demonstrate sufficient efficiency. The simultaneous use of FeCl₃ and NaCl contributed to electro‐coagulation of OMW. After settlement, two separate phases were formed: the supernatant phase and the settled solids. Under optimal conditions (2% Na₂SO₄ + 1% FeCl₃; 24 V), the removal efficiency of COD reached 85.5% at the supernatant phase. Conclusion: NaCl was an effective electrolyte for OMW treatment. The electro‐coagulation process was also a successful process, but as in the case of NaCl the remaining acute toxicity of treated OMW was high. Copyright © 2007 Society of Chemical Industry     

A full‐scale system for aerobic biological treatment of olive mill wastewater

Olive mill wastewater (OMW) is a major environmental problem in the Mediterranean basin. Although many methods for OMW treatment have been developed, only a few have been adopted in pilot‐ or full‐scale applications. A full‐scale system for aerobic biological treatment of OMW was developed. The system consists of a trickling filter and a recirculation tank. Continuous recirculation of the wastewater was used to provide oxygen concentrations from 0.7 to 1.2 mg L^?1. Low ambient temperatures did not affect system performance since the raw wastewater was warm enough. Nutrient addition was not necessary as raw wastewater contained sufficient nitrogen and phosphorous concentrations. Indigenous olive pulp bacteria proved to be resistant to full‐scale conditions. Feed chemical oxygen demand and phenolic concentrations were about 43 000 and 9500 mg L^?1, respectively. The system reduced more than half of the organic load under continuous operation and a hydraulic retention time of 24 h. The efficiency of this method could be improved by combining it with another technology to further reduce the organic load. The absence of mechanical aeration and the very low hydraulic retention time denotes that the proposed system could be viable and attractive. Copyright © 2011 Society of Chemical Industry     

Electrochemical treatment of olive mill wastewaters: Removal of phenolic compounds and decolourization

Olive mill wastewaters (OMW) characterized by their dark brown colour and their very high content of phenolic compounds constitute an environmental concern, particularly in Mediterranean countries where the production of olive oil is important. In this investigation, OMW were electrolyzed at platinized expanded titanium electrodes in a modified Grignard reactor divided into two cylindrical compartments separated by a cation-exchange membrane. Results show better than 90% decolourization and nearly 85% removal of phenols, as established by gas chromatography/mass spectrometry, at high current intensity. This process can easily be monitored by cyclic voltammetry at a boron-doped diamond electrode. A significant reduction of chemical oxygen demand and total organic carbon was observed. The electrodes are not passivated and the ohmic potential drop is very low. Electrochemical oxidation in a modified Grignard reactor is a promising process for the destruction of phenolic compounds present in OMW.     

Microbial and toxic evaluation of raw and treated olive oil mill wastewaters

BACKGROUND: Olive oil mill wastewaters (OMW) from a three‐phase system were treated in a laboratory‐scale continuous stirred tank reactor (CSTR) for hydrogen production. Microbiological and physicochemical characterizations of raw and anaerobically treated olive oil mill effluents were performed. Toxicity assessment was also conducted in order to determine the impact of residual waters on the aquatic environment. RESULTS: Pseudomonas aeruginosa that has the ability to utilize phenolic compounds was isolated in raw OMW, along with several gram‐negative bacteria (Serratia odorifera, Enterobacter cloacae and Aeromonas hydrophila). The dominant species in the untreated effluent appears to be Enterobacter cloacae, whereas in the treated effluent it is Citrobacter braakii. Phenols, pH, NO^?₂, total dissolved solids (TDS) and conductivity were the main parameters related to the observed toxicity. CONCLUSION: A significant differentiation in the microbiological flora was detected between treated and untreated effluents whereas the microbial communities were correlated to the toxicity test results, pointing to the need for further research regarding identification of the microbial flora. This can improve the treatment processes to optimize management of OMW for environmentally safe discharge. Copyright © 2010 Society of Chemical Industry     

Anaerobic digestion of Aegean olive mill effluents with and without pretreatment

BACKGROUND: Olive oil production is an important economical activity in the Aegean region of Turkey. However, the effluents of the olive oil producing mills with their high organic loads and toxic compounds are causing serious environmental problems. The anaerobic biological treatment of olive mill wastewater (OMWW) using the treatment plants of the regional industries could be a method of choice and within the scope of this study floccular and granular sludges were investigated in batch mode for their success in the treatment of OMWW while producing biogas. The major limitation of this treatment is the inhibition of methanogenic bacteria by the phenolic compounds in OMWW. Thus an integrated solution was suggested in which a pre‐treatment step (dephenolization) was also introduced before biological step. RESULTS: The effluents of 27 olive mills out of 47 were found to have total phenolics (TP) less than 3 g L^?1 and could be treated anaerobically after simple dilution. The biogas production for the untreated OMWW was higher for floccular sludge than for the granular sludge (68.5 mL and 45.7 mL respectively). Combined pre‐treatment experiments, first coagulation with polyaluminum chloride, followed by flocculation with cationic polyelectrolyte and finally Fenton's oxidation, could remove 80% of TP and 95% of the total suspended solids. CONCLUSION: OMWW having TP values less than 3 g L^?1 can be treated anaerobically using floccular sludge after simple dilution and biogas can be produced. For OMWW samples having higher TP values pre‐treatment is necessary and the pre‐treatment given in this study may be used effectively. Copyright © 2010 Society of Chemical Industry     

Kinetic study of an anaerobic fluidized bed system used for the purification of fermented olive mill wastewater

The anaerobic digestion of olive mill wastewater (OMW) in a fluidized bed, pretreated with Geotrichum candidum, has been studied. The bioreactor used (volume = 3.5 dm³; biomass concentration = 11.5 g VSS dm^?3) maintained satisfactory operation for 4 to 35 days, in terms of hydraulic retention time, and removed 92% of the initial COD. The system was used to develop and test a kinetic model which was subsequently employed to determine growth yield and maintenance coefficient. From the results obtained, the Michaelis-Menten equation accurately described the substrate uptake (i.e. COD removal) in the anaerobic fluidized bed system. Pretreatment of the OMW was found to increase the rate of substrate uptake by a factor of 3.2 when compared to untreated OMW.