Production of biosurfactants by Mucoralean fungi isolated from Caatinga bioma soil using industrial waste as renewable substrates

In this work it was investigated the potential of Mucorales fungi isolated from the Caatinga of Pernambuco state for production of biosurfactants using renewable substrates. The strains (Mucor circinelloides UCP 0005, M. circinelloides UCP 0006 and Rhizopus arrhizus UCP 1609) were cultivated in alternative culture media consisting of instant noodle waste (INW), corn steep liquor (CSL) and post-frying soybean oil (PFSO), according to conditions established by a 2 full-factorial design (FFD). The production of biosurfactants was evaluated by determining surface tension and emulsification index (EI24) and statistical analysis was performed using Pareto diagram. The presence of the main sources of carbon and nitrogen in production medium was confirmed by FTIR spectroscopy. According to the results, the three fungi evaluated were able of produce biosurfactant in media containing renewable sources. However, the strain that showed the greatest reduction in surface tension (72 to 27 mN/m) was M. circinelloides UCP 0006 in condition 3 of the FFD (1% INW and 4% CSL, in absence of PFSO). The infrared analysis of the INW showed the presence of carbohydrates, fatty acids and proteins, proving that this is a suitable substrate for the cultivation of fungi. The biosurfactants produced by M. circinelloides UCP 0005 and M. circinelloides UCP 0006 were able to form water-in-oil emulsions and the biosurfactant from R. arrhizus UCP 1609 formed oil-in-water emulsions. The present study demonstrated that the three Mucorales fungi tested were able to produce biosurfactants from renewable sources, with emphasis on M. circinelloides UCP 0006.


Introduction
The Caatinga is biome unique of Brazil that has a wide variety of species and is the target of growing interest of industries for therapeutic purposes (Sá filho et al, 2021). Occupies about 10% of the national territory, located between the Atlantic forest and the savannah, it is present in 9 northeastern states. With the characteristic semi-arid climate, it has a highly diverse resident microbiota that presents peculiar characteristics, being able to produce bioproducts with innovative properties of high added value and industrial interest (Santos et al, 2021).
There are few records in literature with microorganisms isolated from the Caatinga bioma for the production of biosurfactants. Among the microorganisms, fungi have biotechnological potential due their extensive reproduction capacity, fast and easy adaptation (Riordon et al, 2019).
Biosurfactants are secondary metabolites produced by several microorganisms such as filamentous fungi, yeasts and bacteria (Araújo et al, 2019). Structurally, the molecule has amphipathic characteristics, that is it non-polar (hydrophobic portion) and polar (hydrophilic portion soluble in water) in same molecule (Uzoigwe, 2015). The main properties of potent biosurfactants are emulsifying and solubilizing capacity, reduction of surface tension and interfacial activity. These properties are already widely applied in industrial area as wetting, solubilizing and foaming substances, among others (Antunes et al, 2013).
The advantage of using biosurfactants in relation to the chemical surfactants is the low toxicity, biodegradability and synthesis from renewable and low-cost substrates (Pacwa-Plociniczak et al., 2011). In this context, the ability of microorganisms in bioconvert industrial residues for the production of biosurfactants is a sustainable alternative, as it meets the environmental demand by reuse of industrial residues reducing the process costs, making the process attractive and easy to industrial employ (Rivera et al, 2019;Oliveira et al, 2020).
In this context, the present study aims to evaluate the capacity of different isolates of Mucorales fungi to production of biosurfactants of high industrial interest using industrial waste as alternative substrates.

Renewable substrates
The renewable substrates used in this study were previously established by Andrade et al., (2018) Table 1.

Inoculum preparation
To prepare the inoculum, 100 mL sterile water were added to the Erlenmeyer flasks and young spores of different fungal isolates were added to the Erlenmeyer flasks. Then, it was performed the count in Neubauer chamber until 10 7 spores/mL. 5% this suspension was used as inoculum in the production media.

Biosurfactant production
The production was carried out in 250 mL-Erlenmeyers flasks containing 100 mL of the production media, consisting of renewable substrates (INW, CSL and PFSO), at concentrations established by a 2 3 full-factorial design (FFD). The pH of the media was adjusted to 5.5, and then, they were sterilized in autoclave and inoculated with 5% spore solution. Fermentations were carried out under orbital shaking at 150 rpm and 28°C, for 96 h. After this period, the cultures were subjected to filtration and centrifugation, in order to separate the biomass from the metabolic liquids. Cell-free metabolic liquids were used to determination of surface tension and emulsification index.

Factorial design
A 2 3 FFD was carried out in order to investigate the influence of concentration of each low-cost substrate (INW, CSL and PFSO), as well as the interaction between them, on surface tension as response variable. Table 2 shows the levels studied for each independent variable of FFD. A set of eight assays with four replicates at the central point was performed. The data obtained from the experiments were subjected to statistical analysis by Statistica® software, version 12.0 (StatSoft Inc., USA) and the significance of the results was tested at p < 0.05 level. Post-frying soybean oil (%) 0 0.5 1 Source: Authors.

Determination of surface tension
Surface tension was measured in triplicate on cell-free metabolic liquids using Du Noüy ring method in an automatic tensiometer model Sigma 70 (KSV Instruments Ltd., Finland), at temperature of 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).

Determination of emulsification index (EI24)
The ability of the biosurfactant in form emulsions was verified after 24 h of homogenization, according to Cooper and Goldenberg (1986). The hydrophobic substrate used was burnt engine oil burned in ratio of 1:1 and in triplicate. The emulsification index (EI24) was evaluated according with following equation: EI24 (%) = Emulsion height (EH) / Total height (TA) x 100 (Eq. 1)

Microscopic analysis of emulsions
The type of emulsion (water in oil/oil in water) was determined after the formation of emulsion by homogenization of the metabolic liquid (containing the biosurfactant) and the hydrophobic substrate (burnt engine oil). Then, a drop of the emulsion was transferred with a Pasteur pipette to slide and visualized in optical microscope with increase of 40x. From the image, the emulsion formed was classified according to the type and formation of bubbles.

Identification of functional groups of industrial waste
INW used in production medium was subjected to Fourier-transform infrared (FTIR) spectroscopy, in order to identify the functional groups in its composition. The functional groups of CSL and PFSO were identified according with Naumann et al, (2000) and Forato et al (2013), respectively.

Production of biosurfactant by Mucorales fungi
The three Mucorales fungi used in this study were able of metabolize agro-industrial residues for production of biosurfactants, as shown in Table 3. The strain that showed the greatest reduction in surface tension (72 to    Penicillium sclerotiorum, also using renewable substrates. It corroborates the ability of filamentous fungi to use different sources of carbon and nitrogen to produce biosurfactants, justifying the importance and necessity of investing in the researches with these microorganisms.   (2018) by releasing the chemical amino group, making it acidic (Marcelino et al. al, 2020). Thus, in the present study, the production of the biosurfactant by the Mucorales fungi was favored by the amino acids present in CSL.

Influence of carbon and nitrogen sources for biosurfactant production by Mucor circinelloides and
The influence of concentrations of carbon sources (INW and PFSO) and nitrogen source (CSL), as well as their interactions in production of biosurfactants by M. circinelloides UCP 0005, M. circinelloides UCP 0006 and R. arrhizus UCP 1609, were statistically evaluated by Pareto diagram (Figure 1).   Figure 1C demonstrates that for production of biomolecule by R. arrhizus UCP 1609, PFSO and CSL were the components of the production medium that most contributed with the reduction of surface tension. Therefore, INW, in concentration used, did not statistically influence the reduction of surface tension, requiring the increase of this concentration.

Chemical characterization of waste used for production of biosurfactants by Mucorales fungi.
The chemical composition (functional groups) of the residues used was identified by infrared spectroscopy. According with results obtained by Forato et al (2013), CSL has the presence of different amino acids (protein source) in its composition (peaks between 1700 and 1000 cm -1 ), confirming its function in production medium as the main nitrogen source. Moreover, CSL also shows the versatility of its composition with the presence of fatty acids (peaks between 3100 and 2800 cm -1 ) and oligo and polysaccharides (peaks between 1200 and 1000 cm -1 ) (Naumann et al, 2000, Forato et al 2013, also proving to be an excellent source of carbon for microorganisms. On the other hand, INW ( Figure 2) demonstrates a more intense spectral band in 1014 cm -1 (Figure 3), evidencing the presence of oligo and polysaccharides (1151 cm -1 and 1078 cm -1 ) confirming the presence of carbohydrates. In addition to the presence of carbohydrates in composition of INW, it was also evidenced the presence of fatty acids (3296 to 2800 cm -1 ) and proteins (1742 cm -1 ) in its composition (Naumann et al, 2000). Source: Authors.

Potential of the biosurfactant obtained from Mucorales fungi for formation of stable emulsions
The emulsifying capacity of a biomolecule is an important parameter that expresses the versatility of its application in different areas (Zargar et al, 2022). The literature indicates that a biosurfactant with the potential to form stable emulsions is one that has an emulsification index (EI24) above 50% (Ferreira et al, 2020). Thus, according to Figure   Research, Society andDevelopment, v. 11, n. 2, e13411225332, 2022 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v11i2.25332 9 Figure 4 shows the optical microscopy of emulsions formed by biosurfactant from M. circinelloides UCP 0005 ( Figure 2A), M. circinelloides UCP 0006 ( Figure 2B) and R. arrhizus UCP 1609 ( Figure 2C). Figure 2A and Figure 2B show the formation of oil-in-water emulsions by the biosurfactant of M. circinelloides UCP 0005 and M. circinelloides UCP 0006, respectively. With a greater number of globular and homogeneous droplets between the phases, there are fewer empty spaces between the bubbles, characteristic of oil drops from a dispersed phase suspended in a continuous or aqueous phase (Souza et al, 2016). On the other hand, Figure 2C shows the formation of water-in-oil emulsions formed by the biosurfactant of R.

Conclusion
In this study, the maximum efficiency in production of biosurfactant from renewable sources was evidenced by M.
circinelloides UCP 0006. However, the isolates M. circinelloides UCP 0005 and R. arrhizus UCP 1609 also showed potential to produce biomolecule with tensoactive and emulsification properties of high industrial interest. The production of biosurfactant by Mucorales fungi was influenced by favorable constitution of alternative production medium rich in hydrophilic carbon source (starch present on INW) and hydrophobic carbon source (PFSO), as well as is rich in source of nitrogen (amino acids present in CSL). Future studies can be carried out using a new factorial design with alteration of the amounts of residues used, considering the possibility of contribution of INW on biosurfactant production by M. circinelloides UCP 0005.