Screening of Microorganisms and Raw Materials for Lipase Production by Solid-State Fermentation

The production of biodiesel from vegetable oils using eco-friendly processes is a hot topic actually. These processes are based on enzymatic biocatalysts, namely lipases, and present many advantages over classical processes i.e. they do not require the use of sodium hydroxide, nor huge quantities of water. Lipases are widespread in nature, being produced by many microorganisms. However, fungal lipases have benefits over bacterial lipases due to their low cost of production, thermal and pH stability, substrate specificity and activity in organic solvents. These low cost production processes rely, most of the time, on solid-state fermentation (SSF). The aim of this research was to select microorganisms for their ability to secrete lipolytic enzymes and to grow on a solid support compatible with SSF. Thirtyfive yeast and mold strains were tested in term of growth rate and extracellular lipase production. Different solid support such as vermiculite, crushed wheat husk, cacao seed-husk and carbon sources such as soy oil, sunflower oil, olive oil or sucrose were also tested for their ability to support cell growth and lipase production.


INTRODUCTION
Lipases are among the most widely used catalyst in enzyme technology, due to their ability to recognize a wide variety of substrates and to catalyze different type of reactions, such as hydrolysis, esterification, interesterification, trans-esterification, but also peroxidation, and epoxidation [1,2].The lipase promiscuity makes that they have been used in many different applications, pharmaceuticals and drugs synthesis [1] but also for biodiesel production [3,4].Compared to classical processes, lipase based processes yield lower cost of production by suppressing complex downstream processes, including the removal of the catalyst and solid salt formed and the use of huge quantities of water for removing the glycerol [3,4].Additionally, lipase-based biodiesel synthesis make it possible to reduce the production of glycerol as byproduct of the reaction [5] increasing by this mean the yield of wanted molecules in the final product.Moreover, life cycle analysis showed that biodiesel synthesis by the biological route is more environmentally friendly than chemical route.Moreover, it has advantages in terms of simplified purification process and energy savings [6].
It has been demonstrated that fungal lipases are produced at higher yield by solid-state fermentation (SSF) [1,7,8].By this way of production, lipase exhibit benefits over bacterial lipases due to higher catalytic efficiency [9], lower cost of extraction, thermal and pH stability, substrate specificity and activity in organic solvents.The most well-known lipase producing organisms from the fungal kingdom are from Aspergillus sp., Candida sp., Mucor sp. or Rhizopus sp.[8,10,11].
The efficacy of the production of enzyme by SSF rely on different factors including strain type, raw material composition of the growth medium and culture conditions [12,13].The first two factors constitute a base for the feasibility of the industrial production of any bioproduct.A proper design of an industrial process for lipase production requires testing different microorganisms and raw materials using conceptual or qualitative design before deciding the optimum concentration of each raw material.Surprisingly, this step is frequently omitted in the literature in the field.Therefore, the aim of this research is to design the base of an industrial production of lipase starting from different fungi strains and raw materials.Camaguey, Cuba) and from ICIDCA (La Habana, Cuba) strains collections.A letter-number code was used for denomination of strains not fully identified.The composition of medium A was as follow (g/L): oil (from olive, sunflower or soy), 40; Yeast extract, 1; sugar (sucrose or glucose), 20; K 2 HPO 4 ,2; MgSO 4 , 1; Urea, 4; CaCl 2 , 0.2; NaCl, 0.  , 0.8.Future experiments will consider the reduction of these salts if possible in order to reduce the medium cost.The composition of medium B is similar to that of medium A, except that all component concentrations were doubled.YNBT solid medium was as described elsewhere [14].SSF were performed in 250 mL erlenmeyers flasks containing 10 g of solid support sterilized at 121°C for 20 minutes and moisturized with 20 mL of liquid medium.Cultures were inoculated with a spore suspension at an initial concentration of 1.5x10  spores/mL and performed in duplicate at 30°C for 96 h.Samples were collectedevery 12 hours for pH, dry cell weight (DCW) and the lipase activity determination.
The solid supports tested were vermiculite (0.5-2 mm size, Sigma-Aldrich), wheat husk and cocoa seed-husk particles, as shown Figure 1.In this case wheat husk and cocoa seed-husk were triturated to obtain particles size between (0.5-2 mm).These natural supports were washed three times with distilled water and dried at 80°C for 6 h until reaching constant weight before utilization.
The compound vermiculite is basically formed of aluminum silicates, magnesium and iron.Their natural form is that of mica, of brown color and has a plane sheet geometry.Their chemical composition is (Mg.Ca) 0.7 (Mg.Fe.Al) 6.0 The cocoa seed-husk is dry, crispy and of brown color composed of carbohydrates, fats, water, protein, nicotiamide, lignin and minerals such as (Ca), (Na), (K), (Mg), (Fe), (Cu), (Zn) and (Mn).Wheat husk is composed of hemicelluloses and cellulose fiber, fats acid, minerals and vitamins.

Analytical Techniques
The lipase crude extract was prepared by mixing 1 g solid SSF samples with10 ml of 100mM phosphate buffer (pH 7) containing 100mM NaCl for 30 minutes at 220 rpm and 30°C.After centrifugation of ten min at 18000 xg, the lipase activity in liquid phase was determined by the titrimetric method as described elsewhere [15][16][17][18].The enzymatic activity was calculated according equation ( 1) Where: Lipolytic activity on YNBT plates was estimated by measuring every 24 h the dynamics of the tributyrin hydrolysis halo formed around the colony over a period of seven days [14].The lipolytic activity was calculated through the relative size of the hydrolysis' halo around the colony by means of equation ( 3): Where: LAC-lipolytic activity coefficient (dimensionless) H-Diameter of the colony + halo diameter of hydrolysis (mm) B-Diameter of the colony (mm) C-Diameter of the halo of hydrolysis (mm) Lipase productivity was calculated by dividing the maximum lipase activity by the culture time at which sample was collected.Total soluble sugars were quantified by a modified phenol-sulfuric acid method [19] using sucrose solutions (0 to 1 g/L) as standard.The pH was determined according to a specific methodology adapted to solid media [20].Briefly, one g of solid medium was weighted in a 250 ml flask and mixed with 10 mL of distilled water in an orbital shaker at 30°C and 220 rpm during 30 min.Then after, the solid is separated from the liquid by filtration and the supernatant used to measure pH using conventional pH-meter.Oleic acid concentration was determined by the phosphor-vanillin colorimetric method as described elsewhere [21].Specific growth rate of strain 7, 10 and 32 was estimated from a semi-log representation of the diameter of the colony on plate against time.

Statistical Data Processing
The Statgraphics software package [22] was used for processing the experimental data gathered during this research.Analysis of variance (ANOVA) in combination with the Fisher Least Significant Difference test (LSD) [23] were used for inferring the experimental data.

Selection of the Best Lipase-Producing Strains
The 35 strains listed in Table 1 were grown on tributyrin-agar plates and the lipase activity-coefficient (LAC) were determined every 24-hours over a period of 120h (data not shown).From ANOVA and LSD tests performed on LAC, three strains, namely Penicilium expansum (refered as 7), Rhizopus stolonifer (referred as 10) and Aspergillus niger Van Tieghem.H/6-24-6 (referred as 32) showed a significantly higher LAC and thus a higher lipase productivity (Figure 2, Table 1).However, comparison of mean differences between these three strains indicated that there are no significant differences in terms of LAC between them (data not shown).Beside this, the specific growth rate of strain 7, 10 and 32 was estimated as 0.014, 0.019 and 0.012 h -1 , respectively (data not shown).Strain A.
niger Van Tieghem.H/6-24-6 was selected for further characterization since it possesses the highest ability to produce lipase.

Evaluation of Lipase Production in SSF using Different Solid Support
Lipase production was further investigated for strain A. niger Van Tieghem.H/6-24-6 growing in media A and B. containing olive oil and sucrose as carbon source, respectively.Different solid support for SSF were tested, namely vermiculite, wheat husk and cocoa seed-husk, for their ability to support lipase production.As shown in Figure 3, during the SSF on vermiculite the consumption kinetic of total soluble reducing sugars (TSR) and olive oil were correlated.The correlation coefficients were equal to 0.980 and 0.883 for medium A and B, respectively.This highlights that the two substrates were consumed simultaneously and that no carbon-catabolite repression phenomenon seems to occur in those conditions.As shown in Figure 3, lipase production started from the early stage of the SSF, leading to the hydrolysis of triolein, the main triglyceride of olive oil, and thus to the release of oleic acid in the medium.In those conditions, these fatty acids could be consumed together with TSR and simultaneously by the cell.Lipase production was clearly induced by the growth medium during the first 48 hours, and no further increase could be observed then after (Figure 3).The maximum level of lipase activity ranged from 18.14 IU/g DW and 18.85 IU/g DW for medium A and B, respectively, corresponding to a productivity of 0.378 and 0.393 IU h -1 gDW -1 .These lipase production values are in similar to those reported in the literature.Of 19 reports accounting for lipase production on SSF more than 50 % were under 37 IU/gDW and almost 74% were under 57 IU/gDW (1).For instance, a lipase production of 30 IU/g DW and 5 IU/g DW were obtained with Penicilium restrictum and Rhizomucor pusillus grown on babassu oil cake and sugarcane bagasse, respectively (1).Although the concentrations of constitutive components of both media were different, the resulting dynamics of cultures in both conditions were similar.Indeed, the p-value of 0.9405 (F-rate 0.0059) calculated from ANOVA on lipase dynamics in both media indicated the absence of statically significant differences between both results.Therefore, the higher concentrations of components in B medium did not allow further increase in lipase production and thus of process profitability in these experimental conditions.In those conditions the pH ranged between 6.5-7 during all the culture.
The dynamics of olive oil and TRS consumption during SSF on wheat husk (WH) were similar to those observed with vermiculite (Figure 4).However, the lipase production obtained was higher, with a maximal lipase activity of 35.30IU/gDW obtained after 72 h of growth in B medium.This means that a maximum enzyme productivity of 0.49 IU h -1 gDW -1 was obtained in these conditions.It is noticeable that after 48 hours of culture, lipase activity on WH and vermiculite were of the same order of magnitude (16-18 IU/gDW) but with WH, lipase activity continued to increase then after, probably due to the availability of some organic component present in the rich composition of wheat seeds.Indeed, while vermiculite is a synthetic and inert solid, WH that is of biologic origin, contains carbohydrates, proteins and other natural components that could promote cell growth and lipase production.
According to these observations, the utilization of WH as solid support instead of vermiculite could yield to a 25% increase of lipase productivity, contributing, thus, to improve process profitability.Beside this, with WH there is a significant difference in lipase production between medium A and B compared to vermiculite.The improvement of lipase production (Δ≈10 IU/gDW) could also be associated to the chemical composition of WH, which allows cells to utilize more efficiently nutrients present in the concentrated medium.Using WH as solid support, the pH drops from 7 to 4.2 after 24 h and remain constant then after.
For SSF using cacao seed husk (CSH) as solid support, the dynamic of olive oil and TRS consumption in medium A and B was similar to those obtained with vermiculite and WH (Figure 5).Similarly, to WH, CSH particles are a source of carbon and other nutrients that could explain the similar behavior of these two natural solid supports regarding lipase production.After 48 hours of SSF, the activity was 16 IU/gDW and then increased to a maximal value of 26.72 IU/gDW after 70 h for B medium.Therefore, lipase productivity (0.371 IU h -1 gDW -1 ), was lower compared to that observed in WH (0.490) and even a slightly lower than that obtained with vermiculite (0.393).Using CSH as solid support, the pH drops from 6 to 5.5 after 24 h and remain constant then after.For all the solid support tested, the pH of the medium was in the range of those compatible with lipase production [1].

Evaluation of Different Substrates for Inducing Lipase Production in SSF
Lipase production is known to be modulated by the nature of the carbon source used.Therefore, different carbohydrate (glucose, sucrose) and oil (from soy,  sunflower and olive) were tested for their ability to induce lipase production.Two different samples of CSH were gathered and used as solid support.The CSH samples were denominated as CSH1 and CSH2.Strain A. niger Van Tieghem.H/6-24-6 was grown in all contrasted combinations using medium A concentrations reported in material and section.In all these media, the concentration of sugar and oil remained the same as reported for medium A but components were substituted accordingly for each experimental combination shown in Table 3 ) was monitored over 120 h (data not shown) and results were then statistically analyzed with a multifactorial analysis of variance at 95% of confidence level (Table 3).The type of medium on lipase production was found significant (F-rate = 3.75 at α< 0.0246, data not shown).Table 3 shows that sunflower oil + CSH1 and sucrose significantly prevails over the others carbon and oil source.

CONCLUSION
From the 35 strains screened for their efficiency to support lipase production, Aspergillus niger strain Van Tieghem.H/6-24-6 was selected as the most promising.
For this latter, lipase productivity was evaluated in regards to the solid support used for SSF and the nature of carbon source used for lipase production.Our results clearly highlight that wheat-husk particles are the solid support of choice to develop a cheap process of lipase production by SSF in combination with sunflower oil and sucrose as energy source.Experiments are in progress to validate our finding at higher scale.

V
CS and V CC -volume of NaOH consumed by the sample and the control respectively (L) C NaOH -concentration of NaOH used (mol/L) c f -conversion factor (1x10 6 µmol/mol) d f -dilution factor [10] m ws -mass of wet sample used (g) Δt-period of time where the hydrolisis of the triglyceride takes place.(15 minutes) Y ws -fraction of water in the wet sample.

Figure 1 :
Figure 1: From left to right, samples of vermiculite, cocoa seed-husk, and wheat husk.

Figure 2 :
Figure 2: Results of the test of multiple ranks for lipase activity coefficient for variable: type of strain.

Figure 3 :
Figure 3: Evolution of olive oil (Oil-A, Oil-B), total reducing sugars (TRS-A, TRS-B) and lipase activity (LA-A and LA-B) during SSF using vermiculite particles as solid support in media A and B.

Figure 4 :
Figure 4: Evolution of olive oil (Oil-A, Oil-B), total reducing sugars (TRS-A, TRS-B) and lipase activity (LA-A and LA-B) during SSF using wheat husk (WH )particles as solid support in media A and B.

Figure 5 :
Figure 5: Evolution of olive oil (Oil-A, Oil-B), total reducing sugars (TRS-A, TRS-B) and lipase activity (LA-A and LA-B) during SSF using cocoa seed-husk (CSH) particles as solid support in media A and B.