OPTIMIZATION TRENDS IN TOTAL LIPOPEPTIDE PRODUCTION BY Bacillus velezensis 0G REVEALED A SUSTAINABLE SUBMERGED FERMENTATION METHOD USING SWEET POTATO PEELS TENDÊNCIAS DE OTIMIZAÇÃO NA PRODUÇÃO DE LIPOPEPÍDEOS TOTAIS POR Bacillus velezensis 0G REVELARAM UM MÉTODO DE FERMENTAÇÃO SUBMERSO SUSTENTÁVEL USANDO CASCAS DE BATATA DOCE

Lipopeptide of the surfactin family are very potent biosurfactants with important applications for environmental remediation and chemical industries. In the present work, the complex production of lipopeptide (mostly surfactins) by Bacillus velezensis 0G, was evaluated under different growth conditions using factorial design 2 with central and axial points. All experiments were carried out based on sustainable submerged fermentation containing sweet potato peels. The response surface trends demonstrate a maximum biosurfactant production at inoculum volume of 32 mL; combined broth volume of 46 mL, and incubation time of 24h. These observations were based on the analysis of supernatant by HPLC.


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Revista Tecnológica -Universidade Estadual de Maringá -ISSN 1517-8048 DOI: 10.4025/revtecnol.v30i1.55243 ___________________________________________________________________________ 1 Introduction Lipopeptide of the surfactin family are very potent biosurfactants, with detergent action on biological membranes (JEMIL et al., 2017). Some of the main biological functions of lipopeptide are: bactericidal, fungicidal, antiviral, and antitumor (GARG; PRIYANKA; CHATTERJEE, 2018), with important applications for environmental remediation and chemical industries(SYED; CHINTHALA, 2015;VECINO et al., 2015). Lipopeptide biosurfactant molecules are produced by fermentative processes, characterized by low productivity and expensive downstream processing (DE et al., 2015). Surfactants produced by different strains of Bacillus, in addition to being obtained by less aggressive processes from the environmental point of view, showed significant effects on the biodegradation of aliphatic and aromatic hydrocarbons (DE et al., 2015;SOUSA et al., 2014).
The submerged fermentation process consists of introducing the microorganism into a liquid medium as inoculum. In this process, fermenters contain the growth medium, providing controlled agitation, aeration, pH levels, temperature and dissolved oxygen concentration, among other parameters(BAKRI; MEKAEEL; KOREIH, 2011). In this regard, chemometrics applied to microorganisms permitted a huge advance in metabolomics, so the nutrients and therefore the production of biomolecules, either from primary or secondary metabolism, can be optimized (BEZERRA et al., 2008;PEREIRA et al., 2013;PRADO et al., 2019;VAN DER GREEF;SMILDE, 2005). In order to minimize overall time, resources and waste, multivariate optimization tools are applied, such as experimental designs and response surface modeling (RAZA; AHMAD; KAMAL, 2014;TEÓFILO;FERREIRA, 2006).
Besides, considering the NEXUS approach for cities, where general efforts should focus on sustainable use of water, food and energy to ensure that there will be enough resources for future generations (DE AMORIM et al., 2018;KUMAR;SAROJ, 2014), a major concern about reuse of organic waste is part of those efforts to enhance sustainability of industrial processes, in order to generate value-added products (LIN et al., 2018). There has been a growing demand in recent decades for the use of agro-industrial waste, now called residual biomass (PELIZER; PONTIERI; MORAES, 2007). Particularly, sweet potato peel reportedly can be reused for metabolites production, ethanol and sugar (AKOETEY et al., 2017).
In this work, we have established the conditions to optimize Bacillus velezensis 0G submerged fermentation, using sweet potato skins (organic waste) as carbon source. A multivariate optimization was applied with 2³ factorial design with center point and axial points and response surface modeling. The three parameters studied were inoculum amount, broth volume and incubation time, optimized to increase surfactin production. The amount of surfactin produced was given by surface tension measurements, emulsification and HPCL analysis.
Phosphate buffer pH 8 was used (MORITA; ASSUMPÇÃO, 2007). All solutions were prepared with deionized water (> 18 MΩ cm, Milli-Q, Millipore). To ensure sterile conditions in liquid media, solid substrates the materials used are being autoclaved at 121 °C and 1.0 atm for 20 min before any experiment.
Non-contact mode topographic AFM images were obtained with a Thermo Microscope AutoProbe CP-Research. The ultra-low spring constant of its silicon nitride (Si 3 N 4 ) cantilever (ca. 0.03 N m -1 ), the use of small curvature tips (ca. 5 nm radius), and the scan rate of 1 to 4 Hz, allowed us to scan the of Bacillus structures without promoting any physical damage.

Cultivation of Bacillus velezensis. Pre-inoculum
A pre-inoculum of Bacillus velezensis 0G was cultivated in modified Landy medium (LANDY et al., 1948). Following the proportion indicated on Table 1, a final volume of 2 mL was placed in 10 mL Erlenmeyers (covered with cotton, gauze, aluminum foil and autoclaved). Stock solutions were prepared in non-sterile medium and then -sterilized. Each Erlenmeyer received 500 µL of a glycerol stock of Bacillus velezensis OG, and then incubated with shaking (250 rpm) for 24h at 37 °C. The resulting broth was used as inoculum. The base medium, without potato peel, was prepared in a 250 mL Erlenmeyer and autoclaved. The proportion was (w/v) for 100 mL solution was: 0.76% peptone, 0.06% sodium chloride and 3% glucose, in sodium phosphate buffer, pH 8.0. Then 1 mL of preinoculum was inoculated into the medium. The Erlenmeyers with the inoculum solution, containing Bacillus velezensis were placed in the shaker at a temperature of 37 ° C and 250 rpm. Revista Tecnológica -Universidade Estadual de Maringá -ISSN 1517-8048 DOI: 10.4025/revtecnol.v30i1.55243 ___________________________________________________________________________
2.5 Factorial design 2³ with central and axial points for screening the best conditions for total lipopeptide production using Bacillus velezensis 0G by submerged fermentation The factorial design of experiments 2 3 with central and axial points was performed. Table 2 presents the factors, or variables to be studied: amount of broth, amount of inoculum and incubation time. There are also levels that in the upper row of the table are coded from -1.68 to +1.68. In the table rows the values of these levels are decoded into experimental values.  Table 3 shows the matrix signals for a 2³ factorial design with center and axial points, where 19 trials were generated. The assays 1 to 14 were done in duplicate with their respective control (i.e. with all fermentation elements minus the inoculum addition). Assays 15-19 are the replicates of the central point. Table 3. Signal level matrix for 2³ factorial design with center and axial points.

Assay
Broth Amount of inoculum Source: Authors The set of assays was performed in 3 batches, randomly chosen, in duplicate. After completion of the assigned Bacillus incubation time, the solutions were transferred to 50 mL Falcon® flasks and centrifuged at 9000 rpm for 10 min. The biomass was separated from the supernatant and stored. Supernatant were used for HPLC, emulsification and surface tension measurements.

Emulsification Index Measurements
Following the methodology of Cooper and Goldenberg (COOPER; GOLDENBERG, 1987) 6mL of toluene hydrocarbon and 4mL of supernatants from each assay were distributed in test tubes. Each tube was placed in vortex for 2 minutes. They were allowed to stand at room temperature and the emulsion´s height and total liquid height were measured after 24 hours from the moment of homogenization.

Determination of surface tension
For surface tension determination, the Cole-Parmer tensiometer, Surface Tensiomat® 21 was used. The equipment consists of a steel wire attached to a movable gear that enables its controlled twisting. At half the length of the wire is perpendicularly attached an aluminum rod that follows the twisting movement performed. At the other end of the rod is the measuring element. It is a platinum ring. Thus when used, with the application of a torsional force, the ring submerged in the liquid rises parallel to the liquid surface, until the meniscus rupture, indicating the value on display the surface tension.

HPLC measurements and data analysis
For HPCL measurements several runs were performed in a reverse phase C18 column, with isocratic mobile phase made of acetonitrile:water:trifluroacetic acid (80%:20%:0.1%). Total volume of 20mL was injected for each assay, keeping the supernatant sample: mobile phase proportion to 1:1. The run lasted 15 minutes: the first 10 minutes performed at 1 mL/min and the last five minutes at 1.5 mL/min. HPLC curves were obtained using Chromera software. As the signal required treatment, baseline removal and peak area integration were performed using Origin™. Statistics were calculated using Excel (MS Office). Results analysis and response surface plots were built in Design Expert® version11 software. Revista Tecnológica -Universidade Estadual de Maringá -ISSN 1517-8048 DOI: 10.4025/revtecnol.v30i1.55243

___________________________________________________________________________ 3 Results and Discussion
Usually, biosurfactant production is indirectly quantified. Methods based on measuring changes in the surface properties of biosurfactant water solutions have been validated and utilized. Thus surface tension measurements (SVENSSON; GUDMUNDSSON;ELIASSON, 1996), emulsification, foaming (PETKOVA et al., 2020) and turbidometric methods (MUKHERJEE; DAS; SEN, 2009). However, these methods can only be used as semi-quantitative techniques. In an attempt to optimize direct biosurfactant analysis with HPLC, Biniarz made an interesting study employing solvent modification for the organic samples (BINIARZ; ŁUKASZEWICZ, 2017), which was adapted to this study. Following are the results with indirect and direct lipopeptide production quantification.
The growth of Bacillus was visualized by AFM measurements of residual biomass, as indicated in Figure 1. The maximum height measured for one unit is 0.7 µm and the FWHM for lines "1" and "2" are 1.30 µm and 2.50 µm, respectively.
The Bacillus unit observed on gold substrate has a maximum height of 0.7 µm and the FWHM for lines "1" and "2" are 1.30 µm and 2.50 µm, respectively. Table 4 shows the 24-hour emulsification values (E24) and the surface tension of the duplicates of the 19 trials, central points and axial points of factorial design. Unfortunately the software could not build a reliable response surface, indicating that these indirect surfactin concentration measurements were not exact. There is a linear behavior of absorbance vs. retention times for 3.73 min, 4.17 min, 4.54 min and 5.44 min. But for the analyzed samples, there are contributions to total concentration only for the retention times of 2.94min and 3.2 the remaining peaks at longer retention times had no significant height, comparing to the peaks of standard surfactin shown in figures 3 and 4, respectively  Figure 5 shows the A peak area calculated for each sample was summed production was obtained.  The appropriateness of a given experimental technique for an application of interest can be evaluated by multivariate analysis, as it is a powerful tool to turn data into information (BEEBE; KOWALSKI, 1987). In this case, the production of lipopeptides, mostly surfactin, evaluated by both indirect and direct concentration measurements gave us a surprising result for the indirect concentration measurement techniques: both surface tension and emulsification results could not be used for response surface modeling. Those analyses generated non-reproducible results. Surface tension gave no trends for an optimum cultivation, while emulsification, being performed by observing the foam height, presented some poor results for quantitative discussions. A possible explanation for such difficulty is given by Petkova et al. Both foaming and emulsification process involving ionic surfactants are very sensitive to lower surface coverage, and simpler characteristics, such as total surfactant concentration, which do not account for the dynamic surface properties of the surfactants, cannot be used to explain the results of foaming or emulsification (PETKOVA et al., 2020).

Results of emulsification index (24h) and surface tension
On the other hand, the direct concentration measurement by HPLC could provide enough information for the response surface plot. It was modeled to find the best conditions for fermentation, that is, the best amount of inoculum, broth and incubation time in order to provide the higher total lipopeptide production. A Quartic model could be calculated, as shown in table 5.  Figure 6. Response surface between the ideal region around -1.32 (X1) and +1.23 (X2). Figure 6 shows the r Broth". In this graph it is possible to observe Warmer colors (orange, red) graph was pointed as being an Figure 7 shows the r Surprisingly the optimum small incubation time and small amount of broth or amount of broth. But due to data given from figure 6, only the 1.64 (X3). shows the response surface between "Amount of inoculum In this graph it is possible to observe trends in optimum lipopeptide Warmer colors (orange, red) indicate higher lipopeptide production. Considering this, in thi pointed as being an ideal the region around -1.32 (X1) and +1.23 (X2). shows the response surface between incubation time and amount of the optimum condition for total lipopeptide concentration is time and small amount of broth or B) great incubation time and greater amount of broth. But due to data given from figure 6, only the option A) surface between Broth (X1) and Incubation time (X3). The best these combined factors indicates X1 of -1.51 and X3 of -1.64.

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Universidade Estadual de Maringá -ISSN 1517-8048 10.4025/revtecnol.v30i1.55243 ___________________________________________________________________________ noculum amount (X2). Red area shows of inoculum" and "quantity of lipopeptide production Considering this, in this 1.32 (X1) and +1.23 (X2). esponse surface between incubation time and amount of broth. condition for total lipopeptide concentration is given for both A) great incubation time and greater option A) can be consideredsurface between Broth (X1) and Incubation time (X3). The best level for Revista Tecnológica

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The 3D plots are given in figure inoculum vs. quantity of Broth for the incubation time and Broth. Incubation time is an important parameter, but as the concavity of the surface in figure inoculum plays a major role in the fermentation process. Figure 8. 3D response surface between broth (X1) and Inoculum amount (X2). The best levels for these combined factors Figure 9. 3D response surface between Broth (X1) and Incubation time (X3). The best levels for these combined factors were A combined analysis made it possible to identif near -1.30, for the inoculum amount these factor values into experimental values, it can be concluded that the best parameters for Revista Tecnológica -Universidade Estadual de Maringá DOI: 10.4025/revtecnol.v ___________________________________________________________________________ D plots are given in figure 8 and 9. The 3D response curve for Broth can be compared to Figure 9, showing the 3D response curve for the incubation time and Broth. Incubation time is an important parameter, but as the surface in figure 9 is smaller than the one observed in Figure 8 inoculum plays a major role in the fermentation process.
analysis of the 2 factor graphs and the 3D plots on each response surface identify some trends for best parameter levels: for the amount of , for the inoculum amount around +1.23 and incubation time into experimental values, it can be concluded that the best parameters for 119 Universidade Estadual de Maringá -ISSN 1517-8048 10.4025/revtecnol.v30i1.55243 ___________________________________________________________________________ 3D response curve for the Amount of the 3D response curve for the incubation time and Broth. Incubation time is an important parameter, but as the observed in Figure 8, so amount of 3D response surface between broth (X1) and Inoculum amount (X2). The best 3D response surface between Broth (X1) and Incubation time (X3). The best levels on each response surface, : for the amount of broth about -1.64. Decoding into experimental values, it can be concluded that the best parameters for