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Enhancement of Aeribacillus pallidus lipase production through optimization of medium composition using Box behnken design and its application in detergents formulations

Background : Alkaline, thermostable bacterial lipases are largely used in detergent applications, since they substitute the use of synthetic detergents which are known to cause substantial environmental problems. These enzymes based detergent are eco friendly and produce a waste water with low level of COD (Chemical Oxygen Demand). In the present study, we investigated a newly isolated Aeribacillus pallidus strain produces, without induction, a novel halophilous, thermoalkaline and detergent- tolerant lipase. Results: Considerable interest has been given to this lipase by the improvement of its catalytic activity through the optimization of the pH, the (C/N) ratio and the inoculums size, using the response surface methodology based on the Box-Behnken Design of experiments. A total of 16 experiments were conducted, and the optimized pH, (C/N) ratio and inoculums size were 10, 1 and 0.3 respectively. The results of the analysis of variance (ANOVA) test indicated that the established model was significant (p value < 0.05). Conclusions: The optimization of the production conditions leads to 6.68-fold of increase in the catalytic activity with a maximum of 68 U/mL. All in all, the lipase of Aeribacillus pallidus could be considered as a potential candidate to be incorporated in detergent formulations since it shows a good stability towards detergents and wash performance.

also they are used to modify flavours and produce fragrance compounds (Ferreira-Dias et al., 2015). Evenly, they are used in detergence as additives, providing that they are active and stable at high temperatures and alkaline pH, by the removal of oils and fats from cotton fabrics (Pandey et al., 1999;Veerapagu et al., 2013).
These hydrolytic enzymes are equally used in wastewater treatment by the biodegradation of oils (Boran et al., 2019), in leather industry by the elimination of fat from animal skin (Das et al., 2016).Further applications of lipases have been described such as medical applications (as new drugs for treatment of digestive aids and high cholesterol levels) (Hasan et al., 2006), textile industry (Hasan et al., 2006)… These enzymes are found in various microorganisms including yeast, fungi and bacteria (Nagarajan, 2012).
While, despite the high biotechnological potential of lipases in various fields, their catalytic activity is one of the most crucial factors to be improuved. Thus, the optimization of culture media is substantial, since lipase production is influenced mainly by the quality and concentration of the carbon source, nitrogen source and inducers . The optimization is usually conducted by varying one factor at a time leading to reach the optimal point (Strobel et Sullivan 1999).
Thus, recent studies intrested in optimizing the medium components for lipase activity have proved that using Plackett-Burman Design (PBD) and Response Surface Methodology (RSM) approaches, whose Box-Benkhen design is one among them (Box and Behnken 1960), were the most effective methods (Gupta et al. 2007;He and Tan, 2006), which are able to overcome the drawabcks of the one factor at time.
Infact, they create empirical model equations that correlate the relationship between variables and responses. Consequently, RSM has so many advantages, and 4 has successfully been applied to study and optimize the enzymatic processes (Soo et al.2004;Basri et al.2007) and enzyme production from microorganisms (Gaur et al.2008;Teng et Xu, 2008).
In the present investigation, we aimed to improve the lipase activity through optimization of the fermentation medium composition for a new thermostable lipase of Aeribacillus pallidus. Additionally, the enzyme showed excellent stability and compatibility with various commercial detergents suggesting its use potential as an additive in detergent formulations.

Preliminary optimization using "one-factor -at-a-time"
Aeribacillus pallidus produced about 24 U/mL of lipase in the production medium, at pH 10 and 65 °C in the presence of 1 mM sodium taurodeoxycholate (NaTDC) and 1 mM CaCl 2 after 22 h of incubation. Beyond that period, lipase activity began to decrease.
The improvement of microbial lipase activity is the aim of several investigations.
Thus, for maximizing lipase activity, each strain has its specific requirement in culture conditions as shown in table 1. Various components in the media such as carbon and nitrogen sources affect the carbon to nitrogen ratio (C/N) which consequently influence the catalytic activity of extracellular lipase enzyme (Jia et al. 2015). We discussed the effect of C/N ratio there in after.

Effect of carbon source on lipase activity
The major factor affecting expression of lipase is the carbon source. Most of reports available, state that lipases are generally induced by natural oils (He and Tan 2006;Kaushik et al.2006;Abdel-Fattah 2002), and high levels of lipases activities were 5 reported from various thermophilic Bacillus sp (Eltaweel et al.2005;Bora and Kalita 2007;Lee et al.1999). Whereas the main observation from Box-Behnkhen study was the poor induction of GPL by oils. In fact, various carbon sources were tested at a final concentration of 1% and the resullts were analyzed independently ( Fig. 1): glycerol, Tween 80, olive oil, glucose, fructose and sucrose. Culture supernatants were sampled at different times during 24 h of growth and assayed for their lipase activity. Statistical analyses of the results showed that the highest lipase activity determined in the culture broth was obtained after 22 h of incubation. Therefore, only the results determined after this time of incubation were taken into account (fig 2). Statistical analysis of the results showed that lipase was significantly improved when glucose was used as the sole carbon source with 1.5 % (Fig. 2), to reach a better GPL activity with 32 U/mL. These results are in contradiction with those showing that carbon sources are used by bacteria to play an inhibitory role ( Lee et al.1999;Gowland et al 1987, Mates andSudakevitz 1973).
For further comparisons, the glucose effect on Staphylococcus xylosus lipase activity showed the same behaviour, with an optimal activity measured using 2.5% glucose (Ghribi et al.2009).

Effect of organic and inorganic nitrogen source on lipase activity
The source of nitrogen is an important parameter that affects the lipase activity.
Various organic and inorganic nitrogen sources were tested on lipase activity.
Generally, when organic nitrogen sources were used such as peptone and yeast extract, high level of lipase production was observed by various thermophilic Bacillus sp (Ghanem et al.2000;Sharma et al.2002;Sugihara et al.1991). So that, yeast extract is one of the most important organic nitrogen sources for high level lipase activity by different microorganisms (Bora and Kalita 2007), and this is in 6 accordance with our results. As shown in (Fig.3a), higher lipase activity was obtained when yeast extract was used as organic nitrogen source rather than using Soya Peptone, Tryptone, Pancreatic Digest Peptone and Casein Peptone. Thus, 0.05% yeast extract is enough to improve significantly lipase activity reaching 34 U/mL.
The requirement of the source of nitrogen depend on microorganisms, some bacteria show preference to organic sourses, while others prefer inorganic ones.
Some microorganisms required both organic and inorganic sourses. When we tested the effect of nitrogen sources, we observed that the combination of NH 4 Cl (inorganic nitrogene surce) with 0.05 % of yeast extract (organic nitrogen surce) improves the lipase activityto reach 36 U/mL ( Fig. 3.b).
Several investigations have shown that a combination of organic and inorganic nitrogen sources were used for lipase activity from Bacillus strain A30-1 (Wang et al.1995), Pseudomonas sp., (Dong et al.1999)and P. aeruginosa LP602 (Dharmsthiti and Kuhasuntisuk 1998) and it was also found that with higher concentration of ammonium chloride (2.5%), lipase activity was drastically increased by almost four fold.
GPL activity was increased by 6.68-fold using one variable at a time method. The variation of the concentration of inorganic nitrogen source (Ammonium chloride) in our study affects the C/N ratio. The results shown in table 2, statistically analyzed according to Duncan's test realized after ANOVA analysis, were obtained using 1.5% glucose, 0.05 %yeast extract and various C/N ratios corresponding to various ammonium chloride concentrations. Using the ratio C/N=1, whish correspond to 1.5% glucose and 2.3% NH 4 Cl, the optimal lipase activity reaches 36 U/mL. Up over this C/N ratio, a decrease of lipase activity was observed and reach 18 U/mL when 7 using C/N= 6.
In conclusion, using 0.5 g/l yeast extract with C/N ratio equal to 1, the optimal activity of GPL reaches 36 U/mL. These results show that to provide amino acids and vitamins necessary for metabolite production, a balance between organic and inorganic nitrogen sources and suitable values of C/N ratio must be taken into account.

Optimization of medium components using response surface methodology
In order to optimize medium culture and to improve lipase activity by Aeribacillus pallidus, a statistical analysis based on experimental design were adopted. This experimental planification methodology is a valuable tool for optimizing the medium of lipase production which provide advantages: the use of multifactor effects, the obtaining of the optimum values and the developing of a system model with substantially less experimental requirements (Wu et al.2007).
Using this approach, several studies have shown an increase of lipase activity by several folds for various organisms (He and Tan 2006) such as Aspergillus niger (Soo et al.2004), Staphylococcus xylosus (Sharon et al 1998), Candida cylindracea (Jia et al.2015) and Geobacillus sp (Abdel-Fattah, 2002).

Data Analysis and Modeling
Based on the OFAT experiments, which indicated that glucose, ammonium chloride, inoculums size and pH are the most significant factors affecting the GPL activity, Box-Behnken Design was used to determine the optimum conditions for these significant factors (carbon to nitrogen (C/N) ratio (X1), pH (X2), inoculums size (X3)). The experimental design, experimental and predicted values for lipase activity obtained from the regression equation for 16 combinations were shown in 8 ( Table 1). The results demonstrated that there is a considerable variation in lipase activity produced by Aeribacillus pallidus. Indeed, the highest level of lipase activity was 68 U/mL (run3) and the lowest one was 30; 34 U/mL (run 6, 8 and 9).
Statistical analysis of variance (Table 3) were used to investigate the effectiveness of the model. The F-value (Fisher's statistical analysis) was used as a tool to evaluate the significance of the model and it was estimated in our study to be 87.18.
The coefficient of determination (R 2 ) is a measure of the quality of a linear's prediction regression. Generally, its determination and its prediction for the response is significant when it is close to 1 (Haaland, 1989;Kaushik et al.2006). In

3D Response Surface and contour Plots Analysis
Response surface and contour plots have been used in order to understand and define the effect of the studied variables within the experimental space. This technique was achieved by depicting the interactions between two variables while keeping the third at a constant level (Ghribi et al.2011). Thus, 3D responses facilated the visual determination of optimum levels of each parameter. Therefore, in the first time ( Fig. 5.a) we fixed the inoculum size at its zero-coded level OD600 = 0.3. So, the response was represented as function of the interaction between 9 liquid substrate ratio and pH level. It can be clear that lower than their central level, the carbon to nitrogen (C/N) ratio and pH significantly influenced the enzyme activity.
As shown in Fig.5.b and Fig. 5.c, the iso-responses are near parallel to the inoculum size axis. This suggests that neither an increase nor a decrease in inoculum size can affect significantly the lipase activity.
As analyzed in the present study, a successful and significant improvement from 30 U/mL to 68 U/mL in the catalytic activity of lipase was accomplished, when the respective values of carbon to nitrogen (C/N) ratio (X1), pH (X2), and inoculums size (X3) are 1, 10 and 0.3, respectively. In a recent study, an optimisation leads to 3fold increase of Pichia guilliermondii T1 lipase activity , (Ladidi et al.2017).

Stability and compatibility on GPL with laundry detergents
A detergent is a mixture of surfactants and oxidizing agents that show their cleaning efficiency at alkaline pH (9-11). Fig.6 proves that the lipase of Aeribacillus pallidus was highly stable and compatible with commercial solid detergents tested.
In fact, after an incubation period of 30 min, it retained 98%, 96%, 95 % and 90% of its initial activity with Dixan,OMO, Nadhif and Ariel, respectively,. Furthurmore, in the presence of liquid laundry detergents, GPL was founf to be highly stablesince it retained 100% of its initial activity in Dipex when incubated 1h at 50°C, while only 80% were retained when incubated in Ariel.
Enzyme activity of the control sample, which contained no additive and incubated under similar conditions, was taken as 100%. Each point represents the mean of three independent experiments.
These results are consistent with those reported for alkaline lipases from Bacillus stearothermophilus (Ben bacha et al.2015), Bacillus stratosphericus (Zin et al.2017). Our results shows clearly that the lipase of Aeribacillus pallidus can provide further support for its usefulness for future industrial application as a cleaning bioadditive in detergent compositions.

Removal of oil spot from cotton fabrics
To evaluate the performance of lipase of Aeribacillus pallidus in terms of its ability to remove oil spot, several pieces of stained white cotton were incubated at different conditions. As shown in Fig. 7, the limited washing performance was observed with detergent (Dixan) only, and its supplementation seems to improve the cleaning process as evidenced by oil spot removal when compared to detergent alone. Furthermore, the combination of this enzyme with the Dixan detergent resulted a complete oil spot removal.

Discussion
In the present study, we aimed to produce a high level of lipase activity of Aeribacillus pallidus, using the optimization of response surface methodology.
Results presented in this paper, showed that the highest production level of GPL was reached under aerobic conditions, at pH 10 and using inoculums size equal to 0.3. and when usingglucose at high concentrations. A high glucose concentrations and a combination between organic and inorganic nitrogen sources was found to be crutial to reach hight production level., The major factor affecting the lipase activity has always been carbon source, since lipases are generally inducible enzymes (Lotti et al. 1998). In fact, the addition of glucose as carbon source to the culture medium improve the biomass formation as well as the catalytic activity of the lipase (Fig.2), while its decrease in the presence of lipids such as tween, glycerol or olive oil. This observation can be explained by he fact that glucose is the most readily metabolized carbohydrates and a source of fast energy (Madigan et al. 2003).
After 22h of incubation, lipase activity began to decrease, might be due to the exhaustion of nutrients, accumulation of toxic, and the change in pH of the medium, or proteolysis of lipase by proteases which were produced simultaneously (Nouroozi et al. 2015). An excessive increase of the glucose concentration decrease both the lipase activity and cells formation (Fig. 2). This could be explained by the fact that the cells produced, at high glucose concentrations, are not physiologically able to synthesize lipases. This phenomenon can be observed in facultative anaerobic bacteria such as Bacillus thuringiensis (Al-mhanna, 2010).
Yeast extract increase the productivity for the most of microorganisms (Bora and Kalita 2007), thanks to its wealth of vitamins and trace elements for the growth of bacteria and increases their lipase activity (Gupta et al. 2007). Whereas, the decrease of lipase activity when using peptone as organic nitrogen source, could due to its complex composition which can cause toxic effects by one of its components (Sooch and Kauldhar 2013).
An other substantial factor for the productivity of lipase is the pH. The pH not only acts on enzymatic activity but also on the properties of the interface in a multiphasic system, on the solubility of the reagents in the medium, as well as the sharing of the enzyme between the aqueous phase and the interface. The optimum pH of lipase activity is usually around 7 while bacterial lipases generally have a slightly basic optimum pH (8-8.5) (Sharma et al. 2001); (Gargouri et al. 2008).
Most halophilic lipases showed maximal activity at pH alkalin and temperature up to 40° C (Esakkiraj et al. 2014); (Li et al. 2014). In the present work, we showed that Lipases incorporated in detergents must have a broad spectrum of substrates and must be able to withstand washing conditions such as pH values between 10 and 11, and a temperature that varies between 30 ° C and 60 ° C. Once the lipids are partially or fully hydrolyzed by the enzyme, it becomes easier to extract from the washed fabric. In fact, an alkaline lipase produced by Pseudomonas alcaligenes M-1was reported, , to be well adapted to remove fatty stains under wash conditions (Gerritse et al. 1998).
The performance of a good detergent lipase is defined by multiple parameters. The most relevant are the thermostability feature of lipases (Bora and Bora 2012), the ability to hydrolyze several types of triglycerides (short, medium and long chain) and its compatibility with other detergent components. The resulalts obtained in this study support the usefulness of lipase of Aeribacillus pallidus in future industrial applications as a cleaning bioadditive in detergent formulations.

Conclusion
Experimental planning methodology was used in our study as a tool to significantly increase the catalytic activity of GPL and predict the optimal values of the important influent factors. A Box-Behnken design with three optimal factors was applied (C/N ratio, pH value and inoculums size). A high degree of similarity was obtained between the predicted and experimental values suggesting the significance of the regression model describing the response. This novel lipase showed excellent stability and compatibility with various commercial detergents, 13 suggesting its potential use as an additive in detergent formulations.

Source of strains
Strains were previously isolated from the production water (an oil/water mixture) of the oil-field managed by Thyna Petroleum Services (TPS), located at 11 km northwest of Sfax city, Tunisia (Mnif et al.2014). The formation water deposit from depths of 1300m at a temperature of 78 °C, a salinity of 100 g /L and a pH of 7.6.
Samples were directly collected in sterile bottles and stored in dark at 4 °C until use.

Lipase production at shake flask scale
The thermoalkaline lipase from Aeribacillus pallidus (GPL) was produced using the optimized medium which is composed of (g/1): 30 g NaCl, 1 g KH2PO4, 0.4 g NH4Cl, 1 g MgSO4.6H2O, 1g CaCl2, 0.5g yeast extract and 1mL trace-element solution (Jaouadi et al.2009) in 1 L of distilled water. The pH was adjusted with 4M KOH solution to 7.4. Aliquots of 50 mL were dispensed into flasks and sterilized by autoclaving at 121 °C for 20 min. These flasks were incubated at 55 °C under aerobic conditions for 22 h with shaking at 200 rpm. Before each assay, the microbial cell debris were removed by centrifugation at 13000 rpm for 30 min. Next, the obtained clear supernatant was used as a crude enzyme preparation.

Lipase activity
The lipase activity was measured titrimetrically at pH 10 and 65°C with a pH-stat under standard conditions, using TC4 (250µl) in 30 mL of buffer containing 2.5 mM Tris-HCL,150 mM NaCl pH 10, 1mM CaCl 2 and 1 mM sodium taurodeoxycholate (NaTDC) or olive oil emulsion whose obtained by mixing 10 mL of olive oil in 90 mL 14 of 10 % (w/w) gum Arabic (for 3x 30s) in a Waring blender. One unit of lipase activity corresponds to 1 μmol of fatty acid released per minute under the assay conditions used.

Medium optimization for maximum lipase activity by Aeribacillus pallidus using Statistical Procedure
Lipase production optimization was carried out using experimental planification methodology through the response surface methodology. Preliminary studies through one factor at a time have proved that ratio C/N, pH and inoculum size affect significantly lipase activity. Therefore, in order to determine the optimum levels of these three significant variables, to predict the possible interaction between them and to enhance lipase activity, a Box-Benkhen design for three independent variables was adopted. It was generated using NemrodW version 2007 software (LPRAI,Marseille, France).

One-factor-at-a-time (OFAT)
Firstly, the classical one-factor-at-a-time (OFAT) approach was employed to evaluate the influential parameters on the lipase activity. In the first step of culture conditions optimization, we have started with optimizing the carbon source, the organic and inorganic nitrogen source, and their concentrations, the inoculums size and pH. These parameters were optimized by keeping all factors at a constant level in the basal medium, except the one under study and each subsequent factor was examined after taking into account the previously optimized factor(s).

Optimization of nutritional parameters
In one's element, the name of Aeribacillus pallidus has been reassigned to Geobacillus pallidus (Minana Galbis et al.2010), owing to DNA composition level , fatty acid composition and the polar lipid profile (Chamkha et al.2008). This strain is a Gram-positive bacterium, aerobic, thermophilic, halotolerant (Minana Galbis et al.2010).
To select the best carbon source that maximizes lipase activity for this strain, various carbon sources were tested, as, glycerol, Tween 80, Olive oil, Glucose, Fructose and Sucrose at a final concentration of 1%. The concentration of the selected carbon source was then varied in the range of 0-2% to work out the optimum concentrations.
The effects of nitrogen sources were evenly evaluated, with various organic and inorganic nitrogen sources at the same final nitrogen concentration. For organic sources, we have already used (yeast extract, peptone, soya peptone, casein peptone and tryptone) when the Ammonium chloride (NH 4 Cl), ammonium nitrate (NH 4 NO 3 ), ammonium sulphate ((NH 4 ) 2 SO 4 ), ammonium molybdate ((NH 4 ) 6 Mo 7 O 24 and HgN 2 O 4 T) were applied in addition to 0.5 % w/v as inorganic nitrogen sources.
After selecting yeast extract as the best organic nitrogen source, it was considered as a constant level in the basal medium.

Optimization of physicochemical parameters
Similarly, the effects of pH (6-10) and the inoculums size (OD 0.1-0.6) were already evaluated on strain growth and lipase activity, as the physical parameters before being subjected to statistical optimization. Then, fermentation was performed in 250 mL shake flasks with 50mL medium and incubated at 55°C in a shaker (200 rpm) for about. All components were analyzed independently, and every test was performed in triplicate. The influential factors and levels for the enzyme activity were evaluated.

Optimization of medium components by response surface methodology
To analyze the experimental design data and to determine the optimum conditions for lipase activity by Aeribacillus pallidus, response surface methodology (RSM) was applied. Box-Behnken Design (BBD) is one of the response surface methodology, with a three-level factorial design was used as the experimental design, to optimize the concentrations of three significant factors namely ratio C/N, pH and inoculums size for enhancing lipase activity. The remaining factors were maintained at fixed concentration. The independent variables were studied at three different levels, low (−1), medium (0) and high (+1). All the experiments were carried out in triplicate and the average of lipase production obtained was taken as the response (Y).
The significance of the coefficients is evaluated by multiple regression analysis based upon the F-test with unequal variance (P<0.05). To check the compatibility of the proposed model with the obtained experimental data, we performed an analysis of variance.
In order to check the GPL's stability and compatibility with detergents, the mentioned commercial detergents were diluted in tap water to obtain a final concentration of 7 mg/mL (to simulate washing conditions). The endogenous lipolytic enzymes present in these laundry detergents were inactivated by heating the diluted detergents for 1 h at 90 °C, prior to the addition of the purified enzymes. 15 U/mL of lipase of Aeribacillus pallidus was shake-incubated with each laundry detergent for 30 min at different temperature 30, 40, 50 and 60°C, and residual activity was determined at pH 10 and 65 °C using TC4 as a substrate.
The enzyme activity of a control (without any detergent), incubated under similar conditions, was taken as 100%.

Removal of oil spot from cotton fabrics
New white cotton cloth pieces (5 × 5 cm) were speckled with lubricating oil and used to simulate the washing conditions and determine the efficiency of the GPL as a biodetergent additive compared. The endogenous lipases found in Dixan liquid laundry detergent were inactivated by heating the diluted detergents for 1 h at 90°C prior to the addition of the purified tested enzyme. The stained cloth pieces were shake-incubated (220 rpm) in different wash treatments at 50 °C for 30 min in Erlenmeyer 250 mL containing a total volume of 100 mL of: tap water, Dixan detergent (7 mg/mL in tap water), and detergent added with GPL (15 U/mL). After treatment, the cloth pieces were taken out, rinsed with water, dried and submitted 18 to visual observation to examine the stain removal effects of the enzymes. The

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All the data or materials used during this study are available from the corresponding author upon reasonable request.
Bacillus stratosphericus. AMB Expr 7:131.     Effect of C/N ratio and pH on lipase activity: response surface plot (right) and its contour plo Washing performance analysis test of GPL in the presence of the commercial detergent Dixan