Development and improved selected markers to biosurfactant and bioemulsifier production by Rhizopus strains isolated from Caatinga soil

1 Biological Sciences Post-Graduation Program, Federal University of Pernambuco, 50670-420 Recife-PE, Brazil. 2 Northeast Network for Biotechnology, Federal Rural University of Pernambuco, 52171-900 Recife-PE, Brazil. 3 Agronomic Institute of Pernambuco (IPA), 50761-000 Recife-PE, Brazil. 4 Post-Doctorate National Program-CAPES, Catholic University of Pernambuco, 50050-900 Recife-PE, Brazil. 5 Department of Mycology, Federal University of Pernambuco, 50670-901 Recife-PE, Brazil. 6 Nucleus of Research in Environmental Sciences and Biotechnology, Catholic University of Pernambuco, 50050-590, Recife-PE, Pernambuco, Brazil.

40°C, intense insolation, scanty water resources, and the annual rainfall in the area is estimated to be lower than 1000 mm, leading to prolonged periods of serious drought.Moreover, the predominantly shallow soils present low natural fertility (Menezes et al., 2012;Silva et al., 2015).These environmental conditions of the semiarid region of Caatinga biome have a direct influence on soil microbial life.Thus, microorganisms that survive in these stressful conditions do develop adaptive mechanisms of response, synthesizing appropriate metabolites (Gorlach-Lira and Coutinho, 2007;Silva et al., 2015).
Studies aiming to explore the biotechnological potential of genus Rhizopus have demonstrated that species of this genus are able to produce different types of compounds of an enormous industrial importance, namely enzymes (Freitas et al., 2014), organic acids (Abe et al., 2007), chitin, and chitosan (Berger et al., 2014) including biosurfactant (Freitas Silva et al., 2012).
Biosurfactants are products of the metabolism of living cells especially of bacteria, yeasts, and filamentous fungi that may be produced extracellularly or as part of cell membranes (Mulligan, 2005).
Structurally, biosurfactants are amphipathic molecules possessing hydrophobic and hydrophilic domains as basic information according to Desai and Banat (1997).Their complex structural organization gives them important physico-chemical properties such as lowering surface and interfacial tensions between immiscible phase systems, promoting the formation of micelles through which hydrophobic compounds can be solubilized in water or vice-versa (Fracchia et al., 2015).In addition, these compounds are known to be efficient dispersing and emulsifying agents, exhibit high foaming and wetting abilities, and display low critical micelle concentration (Mnif and Ghribi, 2015).These properties make biosurfactants molecules with a wide range of practical applications in the bioremediation of contaminated environments, enhanced oil recovery, as ingredients in the food processing industry, cosmetics and pharmaceutical industry (Makkar et al., 2011;Mnif and Ghribi, 2015).
The natural origin of these molecules turn them more interesting compounds, along with non/low toxicity, high biodegradability, effectiveness at extreme conditions (pH, temperature, and salinity), biocompatibility, and specificity in their function (Makkar et al., 2011).
Due to their advantages and numerous possible uses in different areas, microbial surfactants have been the central point of diverse studies aiming to identify potential microorganism producers of these molecules (Walter et al., 2010).However, the majority of screening studies have been carried out using bacteria (Kebbouche-Gana et al., 2009;Nwaguma et al., 2016;Joy et al., 2017;Batool et al., 2017), and outgrows by far those evaluating the fungi producing potential (Sari et al., 2014;Lodha et al., 2016).Till date however, the need for discovery of biosurfactant producing microorganisms capable of inhabiting environments featured by adverse typical conditions such as extreme salinity, higher temperatures, and scanty humidity is still enormous (Techaoei et al., 2007;Kebbouche-Gana et al., 2009;Kiran et al., 2010).Hence, the objective of the current study was to evaluate the potential of Rhizopus strains in the production of biosurfactants using different screening methods (Kiran et al., 2010;Sari et al., 2014).

Micro-organisms
Rhizopus strains from the Caatinga soil used were: Rhizopus arrhizus var.arrhizus UCP 1295 and Rhizopus microsporus var.chinensis UCP 1296 and var.microsporus, UCP 1304 and were kindly provided by the culture collection, UCP (Catholic University of Pernambuco), Recife-PE, Brazil which is registered in the World Federation for Culture Collections (WFCC).

Fungus isolation
The new Rhizopus species strains were isolated from Caatinga soil sample collected in Natal, Rio Grande do Norte state, Northeast of Brazil and were used in the following media (g/L): wheat germ agar medium (wheat germ 15, glucose 5, and agar 15, supplemented with chloramphenicol 0.1), malt extract agar (MEA; malt extract 20 and agar 20), Sabouraud dextrose agar (SDA; peptone 10, glucose 40, and agar 20).The isolation of the fungus was carried out by soil sprinkling technique according to Benny (2008).Briefly, 5 mg of soil sample was weighed using a precision balance and then were spread onto wheat germ agar medium plates and incubated at 28°C until sporulation.Afterwards, using a sterile syringe mature sporangiospores, they were transferred directly from the colonies to MEA plates and then incubated at 28°C for 7 days.Pure culture of the isolate was maintained on SDA slants and stored at 4°C in a refrigerator.Transfers were done to fresh SDA slants, each three months to maintain the isolate viable.

Morphological and molecular identification
The macroscopic and microscopic identification was conducted according to Zheng et al. (2007).The macroscopic morphology (colony size, aspect and color) was attained by naked eye examination of 5 to 7 days old culture grown on potato dextrose agar (PDA) medium (g/L peeled potato 200, dextrose 20, and agar 15).The microscopic morphology was observed by light microscopy using Lactophenol Cotton Blue staining and distilled water.

Primary screening: Hemolysis and parafilm M tests
Preliminary identification of the potentially biosurfactant-producing Rhizopus strains was performed by the hemolytic activity test (Satpute et al., 2010).Spores of Rhizopus strains were inoculated on the central part of the agar plate containing 5% (v/v) of defibrinated sheep blood and incubated at 28°C for four days.The experiments were monitored for observation of hemolytic activity which was detected by appearance of clear zone on blood agar plate.
Parafilm M assay is a rapid and simple test that does not require specialized equipment and can be carried out with small sample volumes.The test consisted of placing 25 μL of mycelia-free metabolic liquid on hydrophobic surface of the parafilm M strip.The shape of the drops was examined after 1 min and its diameters were measured using a caliper.The presence of the surface active compounds in the mycelia-free metabolic liquid was detected by the flat shape of the drop, while the semispherical shape indicates the absence of biosurfactant/bioemulsifier.The medium without microorganism was used as control (Sari et al., 2014).

Biosurfactant/Bioemulsifier production
The strains R. arrhizus var.arrhizus UCP 1295, R. microsporus var.chinensis UCP 1296, R. microsporus var.microsporus UCP 1304, and Rhizopus spp.UCP 1607 were grown on PDA for 96 h at 28°C.Spore suspensions were prepared in the sterile water and adjusted to 10 7 spores/mL, and 5% of suspensions were inoculated in Erlenmeyer flasks containing 100 mL of the medium constituted by soybean post-frying oil (5% v/v), sodium glutamate (1% w/v), and salt solution (g/L): (NH4NO3 1.0, KH2PO4 0.2, and MgSO4.7H2O0.2), and the pH was adjusted to 5.5.The flasks were incubated in orbital shaker at 150 rpm, at 28°C during 96 h.The net metabolic liquid containing biosurfactant was obtained by filtration followed by centrifugation (10.000 ×g for 15 min), and was used for secondary screening.

Surface tension determination
The measurement of surface tension in the mycelia-free broth was performed using an automatic Tensiometer (model Sigma 70 KSV Ltd, Finland) utilizing the Du Nouy ring method as described by Kuyukina et al. (2001).The results were reported as the average of measurements in triplicate.

Oil spreading assay
In order to determine the biosurfactant dispersing ability, oil spreading test was applied (Andrade Silva et al., 2014).Distilled water (40 mL) was inserted in a Petri dish (15 cm of diameter), and this was followed by addition of 1.0 mL of burnt motor oil onto water layer surface.After that, 0.5 mL of metabolic liquid (A), 0.5 mL of commercial detergent (B), 0.5 mL of chemical surfactant SDS (C), and 0.5 mL of distilled water (D) were placed in the center of the oil film.The presence of the biosurfactant/bioemulsifier in the myceliafree broths was detected by the spreading of oil resulting in the formation of oil displacement areas.The clear zone diameters were measured and the respective oil displacement areas (ODA) were determined and expressed in cm 2 using the equation that follows.The experiments were performed in triplicate.ODA = 3.14 × r 2

Emulsification index (E24)
The emulsifier properties of the biosurfactant in crude extracts produced by Rhizopus strains were evaluated by emulsification index assay.For determination of emulsification index, 1.0 mL of mycelia-free metabolic liquid containing biosurfactant and 1.0 mL of burnt motor oil were mixed together in a test tube, and then homogenized vigorously for 2 min at room temperature (25°C).After 24 h, measurements were performed through the equation: where He = emulsion height; Ht = mixture total height (Liu et al., 2013).

Isolation, phenotypic and molecular identification
The isolation of Rhizopus spp.UCP 1607 from the Caatinga semi-arid region soil sample was accomplished on basis of colony morphology (Figure 1).The isolate Sporangiospores ovoid, the mostly regular in shape and size, 5 to 7 × 4 to 6.5 µm, light gray the solitary (D).The fungus was identified as belonging to R. arrhizus (Zheng et al., 2007).Species of Rhizopus are worldwide distributed, inhabiting different environments (Ribes et al., 2000), so they may be isolated from soil, dung, decaying organic material and mature fruits (Santiago et al., 2013), and a variety of food products (Abdel-Hafez, 1984).Some species of this genus live as pathogens causing diseases in humans, animals and plants (Santiago et al., 2013).
There are various studies on isolation and assessment of diversity of tropical areas filamentous fungi including Brazil (Siqueira and Brussaard, 2006;Cavalcanti et al., 2006).However, only few reports referred to isolates from semi-arid environments (El-Said and Saleem, 2008;Grishkan and Nevo, 2010).In this context, little is known about filamentous soil mycota of the Caatinga biome.
Considering this fact, Cavalcanti et al. ( 2006) studied the diversity of soil filamentous fungi in Xingó, state of Bahia, a region with typical Caatinga ecosystem.Among Zygomycota, two Rhizopus spp.were isolated and identified as R. microsporus var.chinensis and R. microsporus var.microsporus.Santiago et al. (2013) worked from soil samples of three different semi-arid areas of the state of Pernambuco to evaluate the distribution of Mucorales order.These authors reported the R. microsporus var.microsporus (10.19%) and R. arrhizus var.arrhizus (7.41%) as one of the most frequent genus in the three areas.In this study, Rhizopus stolonifer and R. microsporus var.chinensis were isolated as well.Oliveira et al. (2013) assessed the diversity of filamentous fungi from soil in the same state and identified same species of Rhizopus (R. microsporus var.microsporus and R. arrhizus).
It was observed that molecular identification of the isolate, using the nucleotide sequence found was compared to those deposited in National Center for Biotechnology Information (NCBI) website using the BLAST program.The results identified homology of 95% of similarity to R. arrhizus.In the current study, the phenotypic characteristics of the R. arrhizus matched with the molecular analysis for the definitive identification of the fungus.Different rDNA regions have been frequently used for the identification of Mucorales.Moreover, the 18S regions of the small ribosomal subunit, the region of the internal transcribed spacer (ITS), and the large ribosomal subunit (LSU) region are the most commonly used as markers (Kasai et al., 2008;Bellemain et al., 2010;Yang et al., 2016;Ziaee et al., 2016).

Detection of biosurfactant-producing Rhizopus strains
According to Thavasi et al. (2011), microorganisms with positive hemolytic activity for production of biosurfactants show a clear zone in the blood agar plates.In this context, only the strain Rhizopus spp.UCP 1607 produced extracellular compound during the radial growth on the blood agar and formed a higher clear zone diameter (40 mm), after 72 h of incubation.The hemolytic activity demonstrated by the strain was superior to that presented by Aspergillus species MSF1 that caused the appearance of a clear zone with a diameter of 7 mm in blood agar medium as described by Kiran et al. (2010).The hemolytic activity was employed by several authors as initial criterion for selection of biosurfactant producers (Batool et al., 2017;Nwaguma et al., 2016).However, Satpute et al. (2010), recommend the use of more than one screening method for detection of biosurfactantproducing microorganisms.
Parafilm test and surface tension determination are both physical methods widely applied for identification of biosurfactant-producing microorganisms (Sari et al., 2014;Korayem et al., 2015).The results from parafilm M assay showed that Rhizopus strains tested in this work were able to produce biosurfactants with different surface-active properties, since the droplet diameter of the metabolic liquid of each strain was larger, compared with fresh culture medium (Table 1).However, the best   2).The surface tension of the cell free broth from R. arrhizus UCP1607 reached the lowest value (31.8 mN/m).Correlations between the drop diameters and the reduction of surface tension and the drop spreading value from Rhizopus strains were observed (Table 1).Sari et al. (2014) evaluated the capability of isolates of Pseudozyma strains for biosurfactants production, and found the surface tensions varying from 35.8 to 44.3 mN/m.Therefore, it was concluded that the biosurfactants produced by all Rhizopus strains were their primary metabolite, due to the production of growthassociated biosurfactant.
According to Sharma et al. (2016), a microorganism is considered a good surface-active compounds producer if its net metabolic liquid is able to reduce the surface tension of water from 72 to 35 mN/m or below this value.Similar criterion for biosurfactants-producing microorganism detection was applied by Ariech and Guchi (2015), considering surface-active potential biomolecule reduced the surface tension below 40 mN/m.

Rhizopus strains
The oil displacement assay requires no specialized equipment, and also the method is rapid and simple which can be undertaken with small volumes of sample (Walter et al., 2010).Table 2 shows the results for dispersing ability of the crude biosurfactant extracts produced by Rhizopus strains.Thus, significant oil displacement activities were demonstrated by biosurfactants produced by R. microsporus var.microsporus UCP1304 and R. microsporus var.chinensis UCP1296 corresponding to 39.6 and 56.7 cm 2 of oil displacement areas, respectively.However, the biosurfactant produced by R. arrhizus UCP1607 exhibited excellent potential in the dispersion of burnt motor oil on water surface that resulted in 66.4 cm 2 of oil displacement area (ODA).The results showed that the oil displacement area of the biosurfactant produced by R. arrhizus UCP1607 (Figure 3A) was superior to dispersion induced by commercial detergent (44.2 cm 2 ) (Figure 3B).Synthetic surfactant dodecyl sulphate (SDS) showed the best dispersion (75.4 cm 2 ) (Figure 3C) as positive control, as well as the negative control in which the burnt motor oil with distilled water was used (Figure 3D).
All results obtained in this study were higher than the biosurfactants produced by Aspergillus niger CF12 (18.5%) and Rhizopus nigricans CF3 (21.66%), as described by Lodha et al. (2016).
In addition, the current study suggests that the importance of the screening methods mainly based on primary and secondary assays led to isolation of biosurfactant and bioemulsifying agents using myceliafree broths, in particular from filamentous fungi.Those assays considered the important properties of higher dispersion oil activity, surface tension of the tension active agent, and bioemulsification.According to Uzoigwe et al. (2015), emulsification index test is a suitable screening method for detection of bioemulsifier producing microorganisms.
Most of the surfactants compounds are chemically synthesized.However, the main drawback for the biosurfactants commercialization is associated with noneconomical production and is not yet competitive with the synthetics products.The renewable substrates used, especially from industrial wastes as soybean post-frying oil, supplemented with sodium glutamate showed excellent results to biosurfactant and bioemulsifier production at an experimental scale.The agro-industrial waste soybean post-frying oil is considered as the promising substrate for reduction of the cost of production to tenso-active and emulsifier molecules (Andrade Silva et al., 2014;Freitas et al., 2014).

Conclusion
In this work, four biosurfactant/bioemulsifier-producing strains were isolated from Caatinga soil of Brazil, namely R. arrhizus var.arrhizus UCP 1295, R. microsporus var.chinensis UCP 1296, R. microsporus var.microsporus UCP 1304 and Rhizopus spp.UCP1607, collectively identified as R. arrhizus.The experimental result showed that among the four strains, the best biosurfactant activity was achieved in R. arrhizus UCP 1607 strain.The biosurfactant produced by R. arrhizus UCP 1607 strain had a large hemolytic activity, parafilm drop, oil-spreading diameter, and low surface tension, while the best emulsifying activity was observed using R. microsporus var.microsporus UCP1304.The screening methods mainly based on surface tension determination have led in many cases to the elimination of bioemulsifying agents.The promising physico-chemical results showed that evaluation of emulsifying activities from mycelia-free broths demonstrated great possibility for production of bioemulsifiers with powerful potential to induce stable emulsion.The better surface active properties with its great effectiveness confirmed the lowering of surface tension and oil dispersion in water by the new strain of R. arrhizus UCP 1607.Further studies are under way to scale up growth conditions and to optimize biosurfactant and bioemulsifier productions.

Figure 2 .
Figure 2. Tensoactivity of crude biosurfactants of the Rhizopus strains on parafilm M hydrophobic surface.

Table 1 .
Evaluation of tensio-active produced by Rhizopus strains using parafilm M test and surface tension determination.

Table 2 .
Oil displacement area by biosurfactants/bioemulsifiers produced by different Rhizopus atrains compared with chemical surfactant and commercial detergent.