Antifungal and antibacterial properties of surfactin isolated from Bacillus subtilis growing on molasses

1 Institute for Ecology of Industrial Areas, Department of Environmental Microbiology, 40-844 Katowice, Poland. 2 Silesian University of Technology, Department of Environmental and Safety Management, 41-800 Zabrze, 66, De Gaulle, Poland. 3 University of Life Sciences, Department of Phytopathology and Mycology, 20-069 Lublin, Poland. 4 "Strzegowa" Water Company, Research Accredited Laboratory, 63-500 Ostrzeszów, Piastowska 105, Poland.


INTRODUCTION
Gram-positive bacteria, including Bacillus spp., produce a variety of antibacterial and antifungal metabolites, antibiotics such as zwittermicin-A and kanosamine and cyclic lipopeptides (LPs) from the surfactin, iturin and fengycin families (Emmert and Handelsman, 1999;Jacobsen et al., 2004;Ongena and Jacques, 2007).Cyclic lipopeptides have well potential in biotechnological applications because of their biosurfactant properties.Biosurfactants have numerous beneficial qualities including non-toxic, non-hazardous, biodegradable, environmental friendly, selective, effective under extreme conditions, numerous industrial applications, and unique surface-active properties.In spite of these beneficial properties, their higher production cost compared to synthetic surfactants is a major drawback.Biosurfactants could potentially replace synthetic surfactants if cost of production was lowered substantially.One of the ways to reduce substrate cost for biotechnology at present is to use recycled agricultural wastes at the right balance and high concentrations of nutrients to support microbial growth and biosurfactants production.
So far, several renewable substrates including various  agricultural and industrial by-products and waste materials have been intensively studied for microorganism cultivation and biosurfactant production on the laboratory scale.These include olive oil mill effluent, waste frying oil, oil refinery wastes, soapstock, molasses, whey, starch wastes, cassava-flour processing effluent and distillery waste (Deleu and Paquot, 2004;Makkar and Cameotra, 2002).Value added products or benefits can improve the economics of such bioprocesses including microbial waste reduction.In our study, the antifungal and antibacterial properties of surfactin produced by Bacillus subtilis cultured on molasses were assessed.

Microbial strains
The bacterial strain named T'-1 used in this study were isolated from 100-year-old oil refinery sludge in Czechowice-Dziedzice (Poland) as described by Berry et al. (2006).The isolate was identified on the previous study by the following method: 16S rRNA gene sequence analysis, fatty acids analysis (FAME) and BIOLOG™ system (Płaza et al., 2012).

Culture conditions for isolation of biosurfactant
Bacterial suspensions of B. subtilis, obtained from a nutrient agar slant incubated for 24 h at 30°C, in the liquid Standard Methods medium of the following composition (g x cm -3 ): peptone -8, yeast extract -2.5, glucose -1 were adjusted to OD600nm 0.65 (ca.107-108 CFU x dm -3 ).Then, three ml of the bacterial suspension was inoculated in 300 ml Erlenmeyer flasks containing 100 ml of the sterilized 10% (v/v) of molasses as nutrients sources.The characteristics of the molasses are presented in Table 1.The cultures were grown aerobically at 30°C for 96 h with constant shaking (110 rpm).After incubation, the cultures were centrifuged at 10000 g for 20 min.Then, the supernatants were used to isolate biosurfactant.

Isolation of surfactin
Biosurfactant was isolated as described Joshi et al. (2008).Biosurfactant was precipitated by adjusting the pH of the cell-free supernatant to 2.0 using concentrated HCl, then the acidified supernatant was stored overnight at 4°C.The precipitate was centrifuged at 9000 g for 15 min.Then, the biosurfactant pellet was suspended in the redistilled water and adjusted to pH 7.0 using 2 N NaOH.The surfactin concentration was expressed as described by Sheppard and Mulligan (1987).

In vitro antagonism experiment
Biosurfactant isolated from Bacillus sp. were tested for the ability to inhibit the growth of fungal plant pathogens in Petri dishes on potato dextrose agar (PDA) medium as described by Velmurugan et al. (2009) with some modifications.200 µl of the surfactin solution were added drop by drop in wells made with a sterile cork borer at 1 cm diameter in gelified medium.Fungal growth inhibition was evaluated after plate incubation at 25°C for 3 or 10 days, depending on the pathogens growth.Sterilized molasses was used as the controls.Antifungal index (mycellial growth inhibition) was calculated as the follows:

A index antifungal
where, A = diameter of fungal growth in the treatment samples B = diameter of fungal growth in the controls The ability to inhibit the growth of E. coli isolates by biosurfactant was performed in Petri dishes on standard method agar (SMA) medium.200 µl of the surfactin were dispensed in wells made with a sterile cork borer at 1 cm diameter in the centre of gelified medium.The critical micelle concentration (CMC) value determined according to the method described by Sheppard and Mulligan (1987), was 0.127 mg/liter.The isolates from the solid medium were inoculated around the wells.Bacterial growth inhibition was evaluated after plate incubation at 37°C by 48 h.The experiments were carried out in triplicate.

RESULTS AND DISCUSSION
Currently, the main drawback for the widespread use of biosurfactants is the disadvantageous economics of their production.Biosurfactants could potentially replace synthetic surfactants if cost of their production is lowered substantially.Achieving this goal requires finding alternative inexpensive substrates and highly efficient microorganisms for biosurfactant production.The choice of inexpensive raw materials is important to the overall economics of the process because they account for 50% of the final product cost.The best way to reduce substrate cost for biotechnology at present is to use wastes with the right balance of carbohydrates and lipids to support optimal bacterial growth and biosurfactants production, and which are either free and carry a cost credit for environmental benefit (Makkar and Cameotra, 2002).
As known, millions of tons of hazardous and non-hazardous wastes are generated each year throughout the world.There is a great need for better management of these wastes via the concept: reduce, reuse, and recycle (Makkar and Cameotra, 2002).The 16S rRNA gene sequencing could not clearly assign isolate T'-1 to any species of Bacillus as isolate showed > 99% similarity to two distinct species B. subtilis and Bacillus amyloliquefaciens.The metabolic profile of 94 biochemical tests as measured by the BIOLOG™ system, showed identification matches for B. subtilis ss spizenii with SIM value of 0.567 utilizing 68 substrates.However, FAME analysis showed that this strain was B. subtilis.The results identified that the surface active properties of free-cell supernatant were the best when bacteria grew on molasses.Replacing traditional microbiological media with agro-industrial wastes as substrates for biosurfactant production holds great potential.Moreover, this will reduce many management problems of processing industrial waste and cost of biosurfactant production.
On the other hand, among the E. coli isolates high inhibition caused by surfactin was noted (Table 3 and Figure 2).Ghribi et al. (2012) evaluated the antimicrobial activity of the biosurfactant produced by B. subtilis SPB1 growing on traditional microbiological medium against bacteria, fungi and yeast.The biosurfactant produced by B. subtilis SPB1 was characterized by an important antifungal activity, especially, against Penicillium notatum, Penicillium italicum, and Aspergillus niger and lower activity against Rhizopus oryzae and Aspergillus oryzae.This activity was clearly negative against Alternaria alternata, Puccinia allii and Peronospora destructor.Moreover, it was clear that this compound presented also an important antiyeast activity, mainly, against Candida albicans.The compound showed important antimicrobial activity against microorganisms with multidrug-resistant profiles.Its activity was very effective against Staphylococcus aureus, Staphylococcus xylosus, Enterococcus faecalis, Klebsiella pneumonia.The compound showed also higher activity against Grampositive bacteria than against Gram-negative bacteria.
Bacteria of the genus Bacillus are known as producers of different number of secondary metabolites with the properties against bacteria, fungi and yeast growing on traditional microbiological media (Ongena et al., 2007;Arguelles-Arias et al., 2009;Perez-Garcia et al., 2011).Jacques (2011) described four main lipopeptides families from the Bacillus species: the surfactins, the iturins, the fengycins (or plipastatins) and the kurstakins.Joshi et al. (2008) described the antifungal compound produced by B. subtilis 20B cultured on LB medium.The growth inhibition of Chrysosporium indicum, Alternaria burnsii, Fusarium oxysporium, Fusarium udum, Trichoderma herzanium and Rhizoctonia bataticola was observed.Whereas, no inhibition of Aspergillus niger and Penicillium chrysogenum was observed.B. subtilis producing iturin A and surfactin was shown to be effective for the control of damping-off caused by R. solani in tomato plants (Asaka and Shoda, 1996).Ongena et al. (2005) describe the ability of B. subtilis strain GA1 to protect wounded apple fruits against grey mould disease caused by Bacillus cinerea.Treatment with endospores and vegetative cells from the strain GA1 was evaluated.The results confirm the potential of B. subtilis species for the control of post-harvest diseases for other fruits (Perez-Garcia et al., 2011).
The toxicity of iturins to fungi has been found to rely on their ability to permeate membranes (Jacques, 2011).Also, fengycins have fungitoxic activity but more speci-fically against filamentous fungi.The fengycins action is less known compared to the other lipopeptides.Several studies present that lipopeptides are co-produced and active in synergistic way, for example surfactin with iturin, surfactin with fengycin and iturin with fengycin (Jacques, 2011;Ongena et al., 2007).Some of these species have more than antifungal properties, they have also antibacterial activity (Basurto-Cadena et al., 2012;Chen et al., 2012;Ghribi et al., 2012).Our results indicate that biosurfactant identified as surfactin produced by Bacillus strain cultured on molasses is capable of antifungal and antibacterial activities against a wide variety of phytopathogenic fungi and E. coli bacteria.

Conclusions
Most of biosurfactants applications depend on their specific properties.In addition, several biosurfactants have  clear advantages compared with their synthetic counterparts, the production of microbial surfactants on a commercial scale up to now has not been realized because of their low yields and high production costs.One of the strategy is to use cheaper and waste substrates to lower the initial raw material costs involved in the biosurfactant production.

Figure 2 .
Figure 2. Growth inhibition of E. coli strains caused by surfactin produced by Bacillus T'-1 cultured on molasses; A -surfactin; B control.

Table 1 .
Selected parameters of molasses as medium for Bacillus growth.

Table 3 .
Growth inhibition of E. coli isolates by surfactin produced by Bacillus sp.cultured on molasses E. coli isolates. E.

coli isolate Growth inhibition (after 48 h)
reported to have manifold biological activities like antibiotics, fungicides, insecticides, antiviral and antitumor agents or specific toxins and enzyme inhibitors.Developing knowledge of the biological properties is a key factor for introducing biosurfactants in high-added value products in different industries.Biosurfactants capable of suppressing pathogens and maintaining their population by competing against deleterious microorganisms could be successfully utilized as biopesticides for sustainable organic farming.The present research has importance in order to minimize the use of synthetic surfactants and fungicide, thus contributing to the environmental protection.The results here indicate that the B. subtilis growing on molasses produced surfactin which has antifungal and antibacterial activities.Despite possessing many commercially attractive properties and been