Ri-utilizzo dei reflui dei frantoi oleari per la produzione biotecnologica di lipasi microbiche
Author(s)
Brozzoli, Viviana
Date Issued
March 14, 2008
Type
Doctoral Thesis
Abstract
The olive mill wastewater (OMW) disposal is, currently, one of the main environmental problems in all olive-oil producing countries, especially in the Mediterranean area. In fact, for its high organic load, phenolic fraction with phytotoxic effects and antimicrobial activity, the OMW is a highly polluted agro-industrial effluent.
The biological treatment can be very difficult since solid residues, high organic load and phenols may strongly reduce the depuration efficiency. Consequently, a possible process should include several technological options, physical, chemical and biological, as well as combinations thereof, thus resulting in increased process costs.
At the moment, the Italian legislation allows land spreading of untreated olive mill wastewater that is the best economical solution. Application on agriculture soils is a practice which solves partially the problem of OMW disposal. Positive and negative effects on soil composition and fertility are still under study, so that OMW application must be strictly controlled. Land spreading, in fact, may cause serious negative environmental impact regarding, for instance, groundwater contamination.
In the last years, alternative solutions have been proposed in view of the use this waste as a source of valuable compounds. Several recent research studies have reported the possibility of OMW valorization to obtain products of actual or potential industrial interest.
The presence in OMW of a wide range of biomolecules such as organic acids, polyalcohols, simple and complex sugars and lipids makes it a potential basis for fermentation processes. In this way, OMW could be a putative candidate as a potentially suitable liquid growth medium for the production of microbial lipases by virtue of its residual lipid content.
For these reasons, the objective of the present PhD thesis was to assess the suitability of OMW as growth medium for the production of lipases and to set up a related fermentation process that might lead, at the same time, to a low polluting load final effluent.
A large number of microbial strains have been screened for lipase production belonging to several fungal genera, Candida and Penicillium in particular. In literature, numerous methods for lipolytic enzyme production are published and medium composition and cultural conditions have been fully optimised. Neverthless, the most frequently used medium is a chemical defined and complex one, significantly affecting the final product costs. Besides, in the last years, a whole range of microbial lipase preparations has been developed. Sigma, Amano, Roche, Novo Nordisk, etc., provide lipolytic preparations with various compositions and catalytic proprierties employed in areas such as detergent pharmaucetic and food industries and biodiesel production.
Our innovative approach consists in the trial of producing microbial lipases using an agroindustrial-waste based medium. Our basic idea, in fact, was that of developing a low cost production medium.
Firstly, 12 fungal strains belonging to well-known lypolytic species (Geotrichum candidum, NRRL 552, 553; Rhizopus sp, ISRIM 383; Rhizopus arrhizus, NRRL 2286; Rhizopus oryzae, NRRL 6431; Aspergillus oryzae, NRRL 1988, 495; Aspergillus niger, NRRL 334; Candida cylindracea, NRRL Y-17506; Penicillium citrinum, NRRL 1841, 3754, ISRIM 118) were screened for their ability to grow on undiluited OMW and to produce extracellular lipase activity. The highest lipase productions were obtained under non-optimized conditions after 168 h with Geotrichum candidum NRRL 553 (0.521 U/ml) and Candida cylindracea (0.460 U/ml). Interesting production levels were also achieved after 72 h with strains of Penicillium citrinum (0.365, 0.320 and 0.375 U/ml for NRRL 1841, NRRL 3754 and ISRIM 118, respectively). These strains were then selected to study the effect of culture conditions, such as OMW typology, nitrogen sources and inducers, on the enzyme production.
With regard to the lipase production by P. citrinum NRRL 1841, the enzyme activity was significantly influenced by nitrogen addition; on the other hand, the addition of oils resulted in a marked increase in biomass without affecting, however, lipase production.
Lipase production by C. cylindracea NRRL Y-17506 was significatly favored by ammonium salts and oil addition. This strain growth in OMW medium containing ammonium chloride and olive oil led to an activity peak of 9.48 U/ml after 264 hours of fermentation.
In order to optimise lipase production by P. citrinum in OMW-based medium, the combined effect of three variables (i.e, concentration of NH4Cl, yeast extract and initial pH) was assessed using a multi-factorial design with ‘optimizer’ function of ‘Modde 5.0’ program. The optimised combination by the model was as follows: pH 6.15, 2.7 g/l NH4Cl e 1.1 g/l extract yeast. The maximum lipase activity was 1.242 U/ml after 192 hour of fermentation. To gain information on the possible up-scaling of the process, further experiments were performed in 3-l laboratory-scale reactors. Specifically, pneumatically agitated (Airlift) and mechanically agitated (STR) reactors were employed using the optimised OMW-based medium. In both cases, the extracellular lipase peaked 192 h after inoculation. Howewer, the maximum activity was significatly higher in STR with respect to the Airlift (0.700 vs 0.420 U/ml, respectively).
Of all strains, C. cylindracea appeared to be particularly interesting and was, therefore, used as the model microorganism to further investigate the feasibility of an OMW substrate. Firstly, the optimisation of medium composition was assessed in shaken cultures. In particular, the effects on the lipase production of olive oil concentration (1, 3, 5 e 10 g/l), glucose (5 g/l), Tween 80 (0,5 g/l) and several growth nutrients (yeast extract, malt extract and peptone) addition were studied. The best medium composition was as follows: diluited OMW (1:2), olive oil 3 g/l, NH4Cl 2.4 g/l and yeast extract 0.5 g/l. The glucose and Tween 80 addition negatively affected the production of lipolytic enzyme.
Lipase production by C. cylindracea on OMW-optimized medium was subsequently assessed in mechanically agitated bioreactor (STR). To study the agitation influence on enzyme production, a set of experiments was carried out at three impeller speed, 300, 500 and 700 rpm; moreover, an additional experiment was carried out at dissolved oxygen DO > 20% saturation (agitation speed automatically controlled between 300 and 800 rpm). To evaluate the effect of pH, three conditions were compared: free pH; fixed pH (6.5) maintained constant by addition of HCl 4.0 M and NaOH 4.0 M; pH lower than 6.5 controlled with addition of HCl 4.0 M. The maximum lipase productions were obtained with the pH left free to vary, 500 rpm costant agitation speed (18.5 U/ml) and variable agitation speed between 300 and 800 rpm to ensure a dissolved oxygen value upper to 20% (18.7 U/ml); in the latter thesis the onset of enzyme activity was anticipated thus leading to increased bioprocess productivity. At 300 e 700 rpm agitation speed, the maximum lipase productions were 2.54 and 11.65 U/ml, respectively.
Finally, to set up the bioprocess of lipase production by C. Cylindracea grown on OMW-based medium, the isoenzymatic profiles of the raw sample was evaluated. This aspect appears to be very interesting since it is known that commercial C. rugosa lipase is a mixture of 3 isoenzymes namenly Lip 1, Lip2 and Lip 3 but the yeast is able to produce up to seven different isoenzymes (Lip 1-Lip 7). Moreover isoenzymatic profiles can depend on media composition and fermentation conditions. With this aim, a set of analitycal isoelectrofocusing experiments were carried out. In the gels, a sample of commercial lipase (Type VII, Sigma) and two raw samples of lipase by C. cylindracea grown on OMW-optimized medium and corresponding to two lipolytic activity peaks (1st and 2nd peak, 48esime and 192esime hour of fermentation, respectively) obtained in STR, were loaded. The results suggest that the raw samples were constituted of more lipolytic isoenzymes with the isoenzymatic profile having only one band in common with that of the commercial lipase (assigned pI 4.7). The sample corresponding to the 1st activity peak showed a strong band at pI 5.1 and a triplette of weak bands at pIs 5.06, 5.0 e 4.9. Moreover, the isoenzymatic profiles changed during fermentation; in fact, the bands at pIs 5.1, 5.0 and 4.9 disappeared and a new strong band at pI 4.5 formed. Finally, in both raw samples a band at pI 3.8 was observed.
OMWs valorisation by its use as growth medium for lipase production by C. cylindracea NRRL Y-17506 and P. citrinum NRRL 1841 appears to be possible and promising. Moreover, the investigation for further up-scaling is need to evaluate the economic fattibility of the bioprocess.
Additional information
Dottorato di ricerca in Biotecnologie degli alimenti
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