Sustainable production of biosurfactant by Issatchenkia orientalis UCP 1603 using renewable substrates

Biological surfactants are amphipathic molecules produced by microorganisms and are considered multifunctional compounds of the 21st century. The current work aimed to use low-cost renewable substrates for economic production of biosurfactant by Issatchenkia orientalis UCP 1603. Fermentations were carried out at 28°C and 150 rpm for 72 h, using agro-industrial by-products (cassava wastewater, corn steep liquor and post-frying soybean oil) as substrates, according to a 2 full-factorial design (FFD) to identify their influence on biosurfactant production. The results showed the ability of the yeast to produce biosurfactant in all conditions of FFD, standing out the condition 4 due to the greatest reduction of surface tension (from 72 to 29.7 mN/m). The statistical analyses evidenced the significative influence of cassava wastewater and corn steep liquor on biosurfactant production. The tensoactive properties of the biomolecule were confirmed by parafilm test and emulsification index. This study evidenced I. orientalis as promising biosurfactant-producing yeast, with excellent ability and higher biotechnological potential for transformation of agroindustrial by-products.


Introduction
The growing search for natural surfactants or biosurfactants is related to their biodegradability properties, low toxicity, and lower environmental impact, when compared to those of synthetic origin. Biosurfactants are metabolites of an amphipathic nature, produced mainly by bacteria and yeasts, and more rarely by filamentous fungi. They have promising and multifunctional properties, such as stability to different environmental conditions of temperature, pH, and salinity (Santos, et al., 2016;Araújo, et al., 2019;Muñoz, et al., 2022).
Although biosurfactants have promising industrial applications, chemical surfactants derived from petroleum are the most available on the market. The large-scale production of microbial surfactants is still limited, considering the high cost of production, mainly caused by the value of nutrients in conventional culture media. In this context, agro-industrial by-products and waste can be used as renewable sources to ensure an economic and sustainable production process (Santiago, et al., 2021;Valencia, et al., 2021).
The yeast Issatchenkia orientalis (syn. Pichia kudriavzevii) has been isolated from different substrates as described from cocoa bean (Dandi, et al., 2013), as well as corns talk, sweet sorghum stalk, and rice straw (Kwon, et al., 2011). In addition, it was described as fermenter of monosaccharides as glucose, fructose and mannose as substrates for ethanol production (Hisamatsu, et al., 2006), and ethanol from rice straw via simultaneous saccharification (Oberoi, et al., 2012). The first report of biosurfactant production by I. orientalis SR4 using xylene in the medium composition as unique carbon source was described by Katemai, et al. (2008). The authors were isolated and characterized of the biosurfactant produced by I. orientalis using the emulsification activity test surface activity and suggest composition as an authentic oleic acid.
In this context, this study is the first to formulate a new culture medium based on agro-industrial residues to evaluate the production of biosurfactant by the yeast Issatchenkia orientalis UCP 1603, isolated from the Caatinga biome (Pernambuco, Brazil). In addition, this is a biotechnological strategy that will contribute to the reduction of production costs, as well as an important path to environmental protection and sustainability, in order to replace the widely commercialized chemical surfactants.

Microorganism and maintenance
The microorganism used in this study was the yeast I. orientalis UCP 1603, isolated from Caatinga soil of the state of Pernambuco, Brazil, and identified by morphological, biochemical, and molecular methods. The strain was deposited on Culture Collection UCP (Universidade Católica de Pernambuco), which is registered at number 927 at the World Federation for Culture Collection (WFCC). The yeast was maintained at 5°C in Yeast Malt Agar (YMA) medium, with the following composition (w/v): yeast extract 0.3%, malt extract 0.3%, tryptone 0.5%, D-glucose 1% and agar 5%, dissolved in distilled water (100 ml) and pH 6.0. The subculture was performed every four months to maintain cell viability.

Agro-industrial substrates
In this study, three agro-industrial by-products was used for the formulation of biosurfactant production medium.
Cassava wastewater (CWW) was kindly supplied by indigenous village of Pankará, in Carnaubeira da Penha, Pernambuco, Brazil, and was obtained from cassava press for the manufacture of powder flour. Corn steep liquor (CSL), a residue from the corn processing industry, was kindly provided by Ingredion Industries Ltd, municipality of Cabo de Santo Agostinho, Pernambuco, Brazil. Post-frying soybean oil (PFSO) was kindly provided by a local food trade in the city of Recife, Pernambuco, Brazil,

Biosurfactant production
Fermentations were carried out in 250 ml-Erlenmeyer flasks containing 100 ml of saline solution (KH2PO4 0.2 g/L and MgSO4.7H2O 0.2 g/L, pH 5.0) supplemented with different concentrations of CWW, CSL and PFSO, according to a 2³ full-factorial design (FFD). Production media were sterilized in autoclave and then, inoculated with a cell suspension (10 7 cells/ml). The flasks were incubated in an orbital shaker at 150 rpm and 28°C for 72 h. After this period, the cultures were subjected to centrifugation at 6000 g for 15 min to obtain cell-free metabolic liquids, which were used to perform the following analyses: determination of surface tension and emulsification index, as well as parafilm test.

Full-factorial design (FFD)
A 2 3 FFD was carried out in order to determine the effect of each independent factor (concentration of CWW, CSL and PFSO) and the interaction between them, using surface tension as response variable. A set of eight assays with four replicates in the central points was performed, and each independent variable was investigated at high (+1), center (0) and low (−1) levels, according to Table 1. Statistical analysis of the data obtained from the experiments was executed using STATISTICA software package version 10.0 (StatSoft Inc., Tulsa, OK, USA), and the significance of the results was determined (p ≤ 0.05). Table 1. Variables and levels of the 2 3 full-factorial design (FFD) applied for biosurfactant production by I. orientalis UCP 1603.

Levels
Source: Authors.

Determination of surface tension
Surface tension was measured in triplicate on cell-free metabolic liquids using an automatic tensiometer model Sigma 70 (KSV Instruments Ltd., Finland), by the Du Noüy ring method at room temperature (± 28°C) (Kuyukina, et al., 2001). The measurement of surface tension on distilled water was used as control (surface tension of water = 72 mN/m).

Parafilm M test
Parafilm M test was carried out placing the cell-free metabolic liquid (25 μl) of on hydrophobic surface of the parafilm M strip. The shape of the drops on the surface were examined after 1 min and their diameters were measured using a caliper. The spreading of drops on the hydrophobic surface was considered an indicator of the presence of the biosurfactant (Hasani, et al., 2018). Distilled water and SDS 2% were used as negative and positive control, respectively.

Emulsification index
Emulsification index (EI24) was determined in triplicate using the methodology described by Nitschke & Pastore (2004). Briefly, 2 ml of the cell-free metabolic liquid from each condition of FFD were mixed separately with 2 ml of hydrophobic compound (canola oil, soybean oil, PFSO and burned motor oil) in a test tube and vortexed thoroughly for 2 min at room temperature (28°C). EI24 was determined after 24 h as the percentage of the height of the emulsion layer divided by the total height of the liquid column.

Biosurfactant production
The determination of surface tension has often been used as a rapid method to detect the BS production in the culture medium (Araújo, et al., 2019;Sharma, 2021). Promising biosurfactant-producing microorganisms are considered those that reduce surface tension to values less than 40 mN/m (Rahman, et al., 2019). In this context,  Previously, research on the production of biosurfactant by yeasts of the genus Issatchenkia have been performed (Thaniyavarn, et al., 2008;Johny, 2013;Aragã, et al., 2014). However, only the work of Nwaguma, et al. (2019) reported the production of this biomolecule by strains of I. orientalis isolated from palm saps grown in olive oil and yeast extract. However, these authors only presented results of rapid methods for detection of biosurfactant such as hemolytic activity, oil displacement and emulsifying activity. Hence, the present work shows innovation on the production of biosurfactant by I. orientalis isolated from the soil of the Caatinga biome, Brazil, and cultivated in agro-industrial substrates. Other yeasts have been investigated for their ability to produce biosurfactants in media based on alternative substrates and by-products, as summarized in Table 3.

Effects of agro-industrial substrates on biosurfactant production
In this study, the production of biosurfactant by I. orientalis UCP 1603 was improved using the 2 3 FFD proposed in Table 1, to determine the relationship and influence between independent and dependent variables on the process. To analyze the mathematical models, adjustments to the points were made by nonlinear regression methods and Table 2 shows the experimental and predicted values of surface tension of the model. According to Figure 1, the obtained experimental values of surface tension were distributed close to the straight line, which indicates that such values were close to predicted values in all conditions evaluated. In addition, the ANOVA revealed that the regression model had a high coefficient of determination (R 2 =0.969), indicating that 96.9% of the variation in the process was explained by the independent variables (concentration of agro-industrial substrates) and that only 3.1% was not explained by the model. Reproducibility of the experimental data was confirmed by the low pure error (0.043) and value of the adjusted determination coefficient (Adj. R 2 =0.9173). Thus, the model demonstrated to be suitable to predict the biosurfactant production under the experimental conditions.  In addition, surface tension was used as response variable, and the effects of independent variables (CWW, CSL and PFSO concentrations), and the interactions between them, were analyzed by estimated effects represented in Pareto diagram ( Figure 2). According to it, only CSL, CWW, the interaction between them as well as the interaction between CSL and PFSO showed negative influence on surface tension. This means that an increase in concentrations of these substrates led to lower surface tension, suggesting the production of biosurfactant in the culture medium. Previously, several studies reported the effectiveness of CSL and CWW as inductors for microbial surfactants production (Maia, et al., 2018;Araújo, et al., 2019;Cândido, et al., 2022). The elemental composition of these agro-industrial by-products (Table 4) confirms their suitability as alternative substrates to the conventionally used carbon and nitrogen sources, guaranteeing microbial growth and the production of biosurfactants.

Parafilm test
The parafilm test is commonly used as a preliminary assay to select biosurfactant-producing microorganisms (Yaraguppi, et al., 2020;Shatila, et al., 2021). In current study, it was used to compare with the results of surface tension and to confirm the surfactant properties in the metabolic liquids obtained in the FDD (Table 2)

Emulsification index
Emulsifying property commonly is evaluated by the determination of EI24, as the ability to maintain at least 50% of the original emulsion after 24 hours of formation (Lima, et al., 2017). Therefore, the biomolecule produced by I. orientalis UCP 1603 demonstrated promising emulsifying property with burned motor oil, with EI24 of 70-73% obtained in central point of the FFD (Table 2). Previously, in the study carried out by Marcelino, et al. (2019), strains of the genus Issatchenkia cultivated in hydrolysate of detoxified bagasse reached an emulsification index in kerosene of 44.7-57.8%. In addition, lowest values of EI24 were obtained with canola oil (13-52%), soybean oil (34-62.5%) and PFSO (35-59%) (data not shown), suggesting that the emulsifying activity depends on the affinity of the bioemulsifier/biosurfactant with the hydrocarbon substrates.

Conclusion
This study showed the biotechnological potential of Issatchenkia orientalis UCP 1603 as a new biosurfactant producer using low-cost agro-industrial by-products as alternative and renewable substrates. The biosurfactant production by the yeast was associated with higher levels of organic carbon and nitrogen sources. The results obtained were promising for several applications of this biomolecule and gives a broader strategy in the process media-optimization, using statistic methodology.