Caspian Journal of Applied Sciences Research, 1(12), pp. 105-111, 2012 Available online at http://www.cjasr.

com ISSN: 2251-9114, 2012 CJASR

Full Length Research Paper Preliminary Screening of Biosurfactant Producing Bacteria from Compost and Their Application in Composting Efficiency
Shahla Soltani Nejad
Department of Biology, Jiroft Branch, Islamic Azad University, Jiroft, Iran; Email: Shahlasoltani56@gmail.com
Received 2 September 2012; Accepted 14 October 2012

In this study biosurfactant producing bacteria which were isolated from compost, were selected during preliminary screening through blood agar hemolysis activity, drop collapse test and oil spreading technique. Isolates with at least more than one positive response to these three methods were subjected to complementary screening by measuring surface tension reduction as well as emulsification capacity. Using these screening methods, two biosurfactant producing isolates have been successfully selected that were able to reduce surface tension effectively. Biochemical analysis determined that these isolates belong to Bacillus and Streptomyces genera. The effect of cell biomass, cell-free supernatant, and a consortium of these two strains on compost quality were determined and specific parameters of compost were analyzed. The results showed that using these bacteria (or supernatants) in compost processing have slight stimulatory effect on bacterial population, surface tension reduction and reduction of pathogens. Key words: Compost, Biosurfactant, Bacteria

1. INTRODUCTION Composting is the aerobic process through which biodegrable organic materials undergo a partial mineralization and profound transformations due to the metabolism of a complex microbial population (Nakasaki et al., 2005). The result of such a process is a biologically stable and humified end product, the compost, which can be applied in agriculture (Xi et al., 2005). Biosurfactants are amphiphilic compounds produced by microorganisms, which either adhere to cell surfaces or are excreted extracellularly in the growth medium (Mulligan, 2005). Due to their amphiphilic structure, biosurfactants increase the surface area of hydrophobic water-insoluble substances, increase the water bioavailability of such substances and change the properties of the bacterial cell surface (Pociniczak et al., 2011). Surface activity makes surfactants excellent emulsifiers, foaming and dispersing agents (Pociniczak et al., 2011). In recent years, interest in biosurfactants has generated due to their possible applications in environmental protection, crude oil drilling, and in the pharmaceutical and food processing industries (Canter, 2004; Kuyukina et al., 2005; Wong, 2005). Many properties of microbial surface active compounds such as emulsification/de-

emulsification, dispersion, foaming, wetting and coating make them useful in physico-chemical and biological remediation technologies of both organic and metal contaminants (Pociniczak et al., 2011). Biosurfactants increase the bioavailability of hydrocarbon resulting in enhanced growth and degradation of contaminants by hydrocarbondegrading bacteria present in polluted soil (Pociniczak et al., 2011). In heavy-metal polluted soils biosurfactants form complexes with metals at the soil interface, which is followed by desorption of the metal and removal from the soil surface leading to the increase of metal ions concentration and their bioavailability in the soil solution (A et al., 2010). Thus, the main goal of this study was to isolate of biosurfactant producing bacteria from composting. 2. MATERIALS AND METHODS 2.1. Compost samples Compost samples were collected from different locations of Kerman composting factory, bottom (20 cm from bottom), core and surface (20 cm to surface), respectively. Each sample was homogenized by sterile hand-mixing.

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Soltani Nejad Preliminary Screening of Biosurfactant Producing Bacteria from Compost and Their Application in Composting Efficiency

2.2. Isolation and enrichment of biosurfactantproducing microorganisms Direct isolation of the microorganisms was carried out using serial dilution (up to 10-7) of composting samples in 0.85 % sterile saline. Using the spreading method on nutrient agar plates, total bacterial count was measured after incubation at 30C for 24 hours. Morphologically distinct colonies were isolated and purified by replicating on the same solid medium to obtain pure cultures (Nasr et al., 2009). 2.3. Preliminary screening of biosurfactant producing bacteria Pure isolates were cultured in E-medium at 30C and 200 rpm for 72 h (Youssef et al., 2004). The broth cultures were centrifuged at 22000g for 45 min (Tabatabaee et al., 2005). The supernatant was subsequently subjected to the preliminary screening methods using oil spreading, oil collapse methods and hemolytic activity as below. 2.4. Oil spreading test The selected strains were compared by measuring of the diameter of the clear zones occurred when a drop of a biosurfactant-contain solution is placed on an oil-water surface. The 50 ml of distilled water was added to a large Petri dish (15 cm diameter) followed by the addition of 20 l of crude oil to the surface of water and 10 l of supernatant of culture broth. The diameter of clear zones of triplicate assays from the sample were determined and compared to 10 l of distilled water as negative control (De Acevedo and McInnerny, 1996). Each test was run in triplicate and the data were shown as mean standard deviation. 2.5. Oil collapse method A modified oil collapse method was carried out using 96 well microtiter- plates containing 100 l mineral oil which was equilibrated for an hour at room temperature. Ten microliter of supernatant of culture broth was added to the surface of a well and the picture captured after 1 minute using 10 objective lens of microscope. Biosurfactant production was considered positive when the drop diameter was at least 0.5 mm larger than those produced by distilled water and also by culture medium as negative controls (Tugrul et al., 2005; Safary et al., 2010).

2.6. Hemolytic activity Isolates were screened on blood agar plates containing 5% (v/v) sheep blood and incubated at 37C for 48 h. Hemolytic activity was detected as the presence of a clear zone around bacterial colonies (Nasr et al., 2009; Youssef et al., 2004; Liu et al., 2011). Each test was performed in triplicates. 2.7. Complementary screening Isolates which were positive for at least more than one of preliminary methods were subjected to the complementary screening to verify their ability to produce biosurfactant. 2.8. Surface tension measurements Surface tension reduction was measured using Krss Hamburg Nr2215 Tensiometer (Pavitran et al., 2004). The results were compared to distilled water and medium composition (as negative control) and Tween 20 (as a positive control). The criterion used for selecting biosurfactant-producing agents was the ability of the isolates to reduce surface tension below 40 mN/m (Pavitran et al., 2004; Tabatabaee et al., 2005; Youssef et al., 2004). Each test was performed in triplicates. 2.9. Emulsification index (E24) The emulsifying capacity was evaluated by an emulsification index (E24). The E24 of culture samples was determined by adding 2 ml of crude oil and 2 ml of the cell-free broth in test tube, vortexed at high speed for 2 min and allowed to stand for 24h. The E24 index is given as percentage of the height of emulsified layer (cm) divided by the total height of the liquid column (cm). The percentage of emulsification index calculated by using the following equation (Banat, 1993; Yakimov et al., 1995; Liu et al., 2011).
E24 = Height of emulsion formed 100 / Total height of solution

2.10. Biochemical analysis of superior isolates Morphological and biochemical identification tests were also performed following directions in Bergeys Manual of Systematic Bacteriology (Sneath et al., 1986).

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2.11. Preparation of the inoculums Cell biomass of two biosurfactant producers (B and S strains), cell-free supernatants, and a consortium of these two strains were used as the inoculums and effect of them on compost quality (surface tension, temperature, E. coli, coliform, Salmonella and total bacterial populations) were determined. 2.12. Temperature measurements A thermometer was used to record the temperature inside each sample. Temperature was measured daily for up to 30 days. 2.13. Determination populations of total bacterial

Salmonella populations were determined through plating 1 ml aliquots of the sample dilutions on E. coli/coliform Petrifilm and SS agar medium, respectively and incubated at 37oC for 24 h. Blue colonies surrounded with gas bubbles on Petrifilm were counted as E. coli, whereas all colonies producing gas were considered as coliforms. 3. RESULTS Compost samples were screened for biosurfactantproducing microorganisms. The initial isolation yielded a total of 50 pure isolates, which were grown on nutrient agar. Among them, 15 isolates gave positive response to hemolytic activity; 6 positive results were obtained for oil spreading and 4 positive responses were obtained when oil collapse method was used; and in total 12 isolates were obtained with positive responses to more than one of the above preliminary screening methods. The putative biosurfactant producing isolates were screened in complementary stage using two methods. Results from these experiments indicate that the surface tension varies from 39.25 mN/m to 62 mN/m and the emulsion activity ranging from 10 to 75% (Table 1).

Ten g of each sample was combined with 90 ml of Phosphate Buffer Saline (PBS) and homogenized. Aliquots of the homogenized samples were used for the enumeration of total bacterial and E. coli/coliform populations. Sample homogenates were serial diluted (1:10) using PBS, plated on Plate Count Agar and incubated at 30oC for total bacterial populations. E. coli, coliform and

Table 1: Detection of biosurfactant producing isolates by preliminary and complementary screening methods
Preliminary methods Isolate Hemolytic activity Oil collapse Oil spreading(cm) Complementary methods Surface tension(mN/m) E24 (%)

A B C D E F G H K L M S Culture medium

++* +++ + ++++ ++ +++ ++++ _ _ +++ ++ _ _

_ +++ _ _ +++ _ _ + _ _ _ +++ _

1.80.57 4.50.05 2028 2.20.57 1.90 00 00 2.80.05 20.28 1.50.05 2.70 4.200.28 00

520.28 39.250.28 540.62 500.89 490 600.05 620 550.28 480 54 0.57 610.05 40.2 0 66 0.05

600.57 750.28 300.57 100.57 170.62 160.05 190.28 550 150.62 300.57 400.28 720 770

water Tween 20

_ _

_ _

00 _

72.5 0.05 35.5 0

1000 _

- : negative results, +: positive results, : Standard deviation * - : not hemolysis, +: incomplete hemolysis, ++ complete hemolysis with diameter < 1 cm, +++ complete hemolysis with diameter < 3 cm. ++++ complete hemolysis with diameter > 3 cm. -: round drop, +: flat drop

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Soltani Nejad Preliminary Screening of Biosurfactant Producing Bacteria from Compost and Their Application in Composting Efficiency

Following complementary screening, two potential biosurfactant producing strains were isolated and were further characterized.

Identification tests determined that they belong to Bacillus and Streptomyces genera.

Table 2: Biochemical characterization of superior biosurfactant producing isolates Characteristics B strain S strain Cell morphology Bacilli Bacilli Gram reaction Spore forming Catalase Oxidase Motility Formation of Indole Growth at 55 C Fermentation of glucose - : negative result, +: positive result 3.1. Temperature measurements The changes of temperature values in the compost through 30 days of composting are shown in figure 1. Adding of biosurfactant producing strains and supernatant contained biosurfactant, increases temperature of compost. + + + _ + _ + + + _ + _ _ _ _ +

50 45 Temprature (C) 40 35 30 25 0 3 5 7 9 30 Days of incubation

B strain biomass Consortium of B and S biomass S strain biomass S strain supernatant B strain supernatant

Fig. 1: Temperature profiles of the composting 3.2. Surface tension reduction The results regarding the surface tension measurements are indicated in figure 2. The results show that a consortium of B and S strains has caused the most decrease in surface tension.

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Caspian Journal of Applied Sciences Research, 1(12), pp. 105-111, 2012

80 Surface tension (mN.m-1)

B strain biomass Consortium of B and S strain S strain biomass S strain supernatant B strain supernatant 0 2 4 6 8 10 Days of incubation

60

40

20

Fig. 2: Curve changes of surface tension during composting 3.3. Determination of bacterial populations According to table 3, the numbers of Salmonella spp. in all treatments are the same, except control.
Salmonella spp. (CFU.4g-1) 10 1.2 10 1.2 10 1.2 10 1.2 10 1.2 10 1.5

The number of total Coliforms in sample of treatment with biosurfactant producing strains is less than sample of treatment with supernatant.

Table 3: Prevalence of coliforms, E. coli, and Salmonella spp.in compost

Treatment B strain biomass Consortium of B and S strains S strain biomass S strain supernatant B strain supernatant Control Coliforms(CFU. g-1) 104 4.4 104 4.4 104 4.4 104 7.3 104 7.3 105 1.3 E.coli (CFU. g-1) 103 1.4 103 1.4 103 1.4 103 2.4 103 2.4 103 2.2

4. DISCUSSION Biosurfactants are amphiphilic molecules with great diversity, environmental acceptability and broad spectrum of functions and industrial applications, which make them interesting bioproducts. Compost is an organic fertilizer containing primary nutrients as well as trace minerals, humus and humic acids, in a proportion that almost exactly matches plant requirements. Pure isolates were cultured in biosurfactant production medium and following centrifugation, supernatants were used for preliminary screening since excretion type bacteria that release biosurfactants to the culture medium are more interesting from the industrial point of view than bacteria with adherent biosurfactants due to the simplicity and economical aspects of the recovery process (Nasr et al., 2009; Liu et al., 2011). The primary screening of biosurfactant producing bacteria was carried out using hemolytic activity, oil collapse and oil spreading techniques. Selection of these methods was due to their strong advantages including simplicity, low cost, quick

implementation and use of relatively common equipment that is accessible in almost every microbiological laboratory; however, as expected, these methods are not perfect or flawless. In the hemolytic method, there are many bio-products that can cause red blood cell lysis which do not necessarily have to be surface actives molecules (Plaza et al., 2006; Youssef et al., 2004; Liu et al., 2011).). The drop collapse method depends on the principle that a drop of liquid containing a biosurfactant collapses and spreads over the oily surface. There is a direct relationship between the diameter of the sample and concentration of the biosurfactant and in contrast, the drop lacking biosurfactant remains beaded due to the hydrophobicity of the oil surface that cause aggregation of droplets (Nasr et al., 2009) but this method is not sensitive in detecting low levels of biosurfactant production. The results of our experiments indicate, 30% of total isolates were positive for hemolytic activity, 12% were considered positive based on oil spreading and 8% were positive for oil collapse and since these methods have shown differences, the isolates with

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Soltani Nejad Preliminary Screening of Biosurfactant Producing Bacteria from Compost and Their Application in Composting Efficiency

more than one positive response were exposed to complementary screening. The latter screening stage included surface tension and emulsion activity measurements. Sixteen percent of positive isolates in preliminary screening have shown reduction of surface tension below the standard index (40 mN/m). Amiriyan et al. (2004) suggested that emulsifier activity depends on the affinity of bioemulsifier for hydrocarbon substrates, which involves a direct interaction with hydrocarbon itself rather than an effect on surface tension of the medium (Amiriyan et al., 2004). Adding of biosurfactant producing strains and supernatant contained biosurfactant, increases temperature of compost. This increase of temperature shows the rate of microorganism activities. When the compost is immobilized, there is no extra change in temperature. A consortium of B and S strains has caused the most decrease in surface tension. Xi et al (2005) explained adding of biosurfactant producing strains has the most applications because of agreement with compost compare with direct adding of biosurfactant in order to decrease of surface tension. Salmonella is very sensitive against temperature and little humidity. The presence of Salmonella spp. in compost shows that compost has not been in suitable temperatures or it may be polluted by external materials (eg. animals such as mice) or equipment. Probably microbial competition can be the reason of decrease in Salmonella spp. population or increase of temperature in samples of treatment with biosurfactant, helps the decrease of the number of Salmonella spp. The biosurfactant shows antimicrobial effects, but the decrease of bacterial population is not because of antibacterial effects and more studies should be down. In the present study, two superior isolates S and B with biosurfactant-producing ability and the former with emulsion capacity were isolated from compost. Their ability to reduce surface tension and emulsion capacity makes them potential candidates for biosurfactant and bioemulsion production. Further studies have been initiated to identify their properties and consequently determine the potential of their different industrial applications. ACKNOWLEDGEMENTS This work was financially supported by Islamic Azad University, Jiroft branch, Iran.

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