Morphological, physiological and biochemical characterizations of some soil isolates of Bacillus cereus group from Algeria

1 Laboratory of Food, Biomedical and Environmental Microbiology, Aboubakr Belkaid University, Tlemcen, 13000 Algeria. 2 Department of Biology, Faculty of Natural Sciences and Life, Tlemcen University, P. O. Box 119, 08000 Algeria. 3 Naama University, Algeria. 4 Department of Genetic and Bioengineering, Faculty of Engineering and Architecture, Yeditepe University, Kayısdagi, 34755 Istanbul, Turkey.


INTRODUCTION
In soil, microbes are especially active in the rhizosphere, which can contain more than a million microorganisms per gram of soil.Microbial community structures are variable and depend on factors such as temperature, pH, or geographic location, but are able to tolerate environmental change.Microorganisms found in this environment use energy substrates released by roots such as and necessary to their metabolism: sugars, amino acids, organic acids, hormones.Some of these microorganisms, mainly bacteria, are able to effectively colonize the root systems and stimulating plant growth and / or protecting against infections by plant pathogens.These rhizosphere bacteria are sometimes called Plant Growth-Promoting rhizobacteria (PGPR) (Kloepper et al., 1986).
The beneficial effects of rhizobacteria on plant growth are the result of either direct or indirect mechanisms by PGPR.Indirect mechanisms occur outside of the plant, while direct mechanisms are those that occur inside of the plant and directly affect their metabolism.These mechanisms can function simultaneously or sequentially at different stages of plant growth.Many strains of PGPR can affect plant growth directly by solubilizing phosphates, nitrogen fixation and mineral nutrients, making these foods available for the plant, by producing phytohormones such as indole-3-acetic acid (IAA), and repressing of soil pathogenic microorganisms (production of hydrogen cyanide, siderophores, antibiotics, and / or competition for nutrients).In addition, PGPR can contribute to the improvement of plant resistance to biotic and abiotic stress such as salinity, drought and heavy metal toxicity (Canbolat et al., 2006).
Bacillus cereus sensu lato (s.l.) are Gram-positive endospore forming bacteria that are abundant in different ecological environments.The growth temperature range varies from 5 to 50°C (Guinebretiere et al., 2008).This group of bacteria includes seven species that are genetically very similar.B. cereus sensu stricto (B.cereus s.s.) which is associated with food borne illness but is also involved in some cases of local super infections (Logan, 2012).Bacillus thuringiensis is an entomopathogen used with success in agriculture as a biocontrol agent against insects (De Maagd et al., 2001).Bacillus anthracis is the agent of anthrax (Mock and Fouet, 2001).This group (B.cereus sensu lato (s.l.) also species (Lechner et al., 1998).Bacillus cytotoxicus characterized by its high toxicity and its particular thermotolerance (Guinebretière et al., 2013), and Bacillus mycoides and Bacillus pseudomycoides species that are characterized by the formation of rhizoids colonies (Nakamura, 1998).
This study was aimed to characterized thirty six isolates of B. cereus from soil for a better appreciation for their potential plant growth promoting (PGP), that is, IAA production, phosphate solubilizer, starch hydrolysis, Bouali et al. 1095 proteolytic activity and biofilm formation.

Isolation of strains
Samples were isolated from several soil plots in the Naama region (South-Western region of Algeria) (Figure 1) over ten months (March till December 2014), from four different areas (about 100 m 2 each). 1 g of each sample of soil was added to a tube containing 9 mL of sterile distilled water.Serial dilutions were heated at 80°C for 10 min to eliminate vegetative cells and to select for spores.Each dilution (0.1 ml) was spread on Luria-Bertani (LB) agar plates.A total of thirty-six isolates of B. cereus group, were isolated according to the International Organization for Standardization (ISO, 1993).

Morphological, physiological and biochemical characterizations of isolates
It is possible to differentiate B. cereus from other Bacillus species because they share similar morphology mobility, hemolytic activity, inability to utilize mannitol, and the production of an extremely active lecithinase , on Mossel culture media (MYP agar) B. cereus forming rough and dry colonies with a violet pink background surrounded by egg yolk precipitation.Hemolytic activity is tested using on Columbia blood agar incubated at 30°C for 24 h and is manifested by a characteristic lysis area near the bacterium (Fricker et al., 2008).To characterize the effect of temperature on the growth of Bacillus cereus group, isolates, were incubated on LB plates at 10, 15, 20, 30, 37, 45 and 50°C for 72 h.For long term conservation, obtained isolates were stored in Eppendorf tubes containing LB with 20% glycerol at -20ºC (Guinebretiere et al., 2008).Other biochemical characterizations of isolates are tested using the API 20E.

Extraction and analysis of fatty acid methyl ester (FAME) profiles
We used gas chromatography (HP6890, Hewlett Packard, Palo Alto, CA, USA) to separate FAME.We identified FAME profiles of each bacterial strain by comparing the commercial databases (TSBA 6) with the MIS software package (Sherlock 6.0 MIDI, Inc., Newark, DE, 2005).Each samples is processed in a single test tube; a summary of the processing activities associated with each step includes: -Harvest bacteria from third quadrant.
-Saponification: Combine water and methanol.Add NaOH pellets to the solution while stirring and stir until the pellets dissolve.
-Methylation: add acid to methanol while stirring.
-Extraction: add the methyl tert-butyl ether (MTBE) to the hexane and stir well.
-Wash: add NaOH pellets to the water while stirring and stir the pellets are dissolved.*Corresponding author.E-mail: wafaa_bio@yahoo.fr.
Author(s) agree that this article remains permanently open access under the terms of the Creative Commons Attribution License 4.0 International License

Production of Indole acetic acid
40 μg/ml of L-tryptophan was added to LB medium to assure growth of our isolates.They were incubated at 30°C for 48 h.Cultures of bacteria were centrifuged for 15 min at 10000 rpm, and 1 ml of culture filtrate was mixed with 1 ml of Salkowski's reagent and the mixture incubated at room temperature for 30 min.A pink color was indicative to indole acetic acid production (Gordon and Weber, 1951).

Phosphate solubilization
Prepared GYA according to Beneduzi et al. (2008): 10 μL aliquot of each culture was plate on GYA and incubated at 28ºC for 7 days, presence of clear halos around their colonies indicate that isolate can solubilize phosphate.

Starch hydrolysis
This test was performed by cultivating the isolates on a nutrient agar containing 1% starch.After incubation at 30° for 48 h, cultures bacteria were covered with Lugol's solution.Hydrolysis of starch is well demonstrated by the appearance of a clear zone around the colony when placed against a negative result revealing a brown color around culture (Gupta et al., 2004).

Proteolytic activity: Search for caseinase
The hydrolysis of casein was studied on an agar medium containing 5% skim milk.After incubation at 30°C for 48 h, a clear halo around the streak indicated the hydrolysis of casein by against a negative result showed no hydrolysis zone around culture (Marchand et al., 2009).

Formation of biofilm in microtiter plate
Polystyrene microtiter plates were filled with 200 µl cultures grown in LB and inoculated.The plates were incubated at 30°C.After 24 h, we washed wells three times with 200 µl of phosphate buffered saline, and stained with 0.1% (wt/vol) crystal violet for 30 min to color biofilm.The cultures were distained twice with 200 µl sterile deionized water.The remaining crystal violet was dissolved in 200 µl 96% ethanol, and we measured at 595 nm the absorbance (Djordjevic et al., 2002).

Biofilm formation in tube
A loopful of isolates was inoculated in 10 mL LB medium in test tubes at 30ºC for 24 h.After incubation, tubes were decanted and washed with phosphate buffer saline and dried.Tubes were then colored with crystal violet (0.1%).Excess isolate was washed with deionized water.Tubes were dried in inverted position.Isolates which showed a visible film lined the wall of the tube were considered as positive biofilm formation (Christensen et al., 1982).

Identification and characterization of bacterial strains
We obtained thirty six B. cereus group isolates from various localities in Naama south-western Algeria.All isolates showed typical colony morphology with a zone of precipitation.All of the isolates were Gram positive, rod shaped, endospore forming, catalase positive, lecithinase positive and hemolytic (Figure 2).All isolates were able to grow in the range of temperature tested 15 to 45°C (Table 1).To clarify the existing diversity of B. cereus group and its special ability to adapt to widely diverse habitats, Guinebretière et al. (2008) showed that the genetic structure of B. cereus group belong the same phylogenetic group, describe more that found.All isolates of this study affiliated to group III.This genetic group includes isolates whose growth temperature is between 15 to 45°C.Temperature is one of the most important environmental factors to which microorganisms tolerate different kinds of environmental changes.In the API 20 E, all isolates were VP, Citrate, ADH, Gelatine positive, except three isolates were ADH negative, however the results given by other tests are always negative (Table 1).

FAME analysis
Ten bacterial isolates were examined in this study (Figure 3).They were classified into B. cereus groups.The characterization studies based on FAME analysis showed that total 31 different FAMEs were present in 10 bacterial isolates tested in the present study (Table 2).The data of fatty acid analysis showed that three isolates (bact3, bact4, bact7) were B. cereus Subgroup A, remaining isolates were B. cereus Subgroup B.
According to the results based on fatty acid profiles we can deduce a low diversity among the isolates.The ten samples were isolated from several soil plots, both the origin of the isolates and the pressure exerted by  Ain Sefra environmental conditions in the soil can explain the low diversity identified by our results, it has been reported that temperature is assumed to select some bacterial groups (Von Stetten et al., 1999).Major environmental variables that may influence bacterial-community composition include vegetation type and temperature, plant species variables influence the composition of bacterial communities (Kuske et al., 2002), agricultural growing practice (Buckley and Schmidt 2001), temperature (Ward et al., 1998), nutrient status (Broughton and Gross, 2000), salinity (Nubel et al., 2000), and other environmental variables.

Plant growth promotion activities for isolates
Five isolates of B. cereus showed IAA production (Table 3).According to Mirza et al. (2001), IAA by the production of microorganisms may vary between different species and strains of the same species.The culture conditions and substrate stage growth conditions may also cause variations in production.Also, Idris et al. (2007) showed that reduction in IAA concentration in B. amyloliquefaciens FZB42 was caused by inactivating of gene responsible for IAA biosynthesis consequently causing in decrease plant growth promotion activity.The level of IAA production was affected also by two genetic factors, the location of auxin biosynthesis genes in the bacterial genome and the mode of expression.
Among environmental stress factors, acidic pH, osmotic and matrix stress, and carbon limitation modulate the IAA biosynthesis in different bacteria (Spaepen et al., 2007).The ability to produce heat resistant endospores is one of the main features of Bacillus, tailoring it to design and marketing, since these microorganisms can be stored for a longer period and may stay longer in the soil (Kokalis-Burelle et al., 2006).The isolation efficient of bacteria depends on some factors of the interaction of plant-soilclimatic conditions (Chagas-Junior et al., 2012).There are few studies related to quantification of IAA by bacteria of the species B. thuringiensis and B. cereus.Gomes et al. (2003) demonstrated that both bacteria, B. thuringiensis and B. pumilus, isolated from the cabbage plant, increased the growth of lettuce in the greenhouse.B. cereus is known for its ability to produce gibberellic acid, IAA, zeatin and gibberellins (Karadeniz et al., 2006).In addition, Tilak et al. (2006) confirmed the involvement of B. cereus in promoting the growth of Cajanus cajan (L.) Mill sp., while Bullied et al. (2002) found that this promotes the growth of bacteria soybean (Glycine max).Three isolates showed capability to solubilize inorganic phosphate (Table 3).Bacillus species have been reported for their capacity to increase availability of phosphorus in soil (Bhattacharyya and Jha, 2012) and From the present study, pH of soil isolates was alkaline (8.5), Hwangbo et al. (2003) reported that the inverse relationship between pH and soluble phosphate.However, P-solubilizing bacteria (PSB) are widely distributed in soil; environmental stress factors can affect establishment and performances of these bacteria (Ahemad and Khan, 2012).
Furthermore, obtained results showed that all Bacillus cereus in our study were starch hydrolysis and proteolytic activity (Figure 4A and 4B).There are many reports (Sellami-Kamoun et al., 2008) indicating that Bacillus sp. were able to produce a large variety of extracellular enzymes, such as amylases and proteases are the most significant industrial.It has been confirmed that variation of amylases and proteases not only in type but also in pH and optimum temperature (Gupta et al., 2003).

Role of hydrophobicity of spores in attachment
The hydrophobicity of B. cereus spores was determined and expressed a percentage and illustrated in Table 4. Surface hydrophobicity of untreated B. cereus spores(ranged between 18.5 and 57%).In this study, it is interesting to note that hydrophobicity of spores varied among the analyzed B. cereus soil isolates which displayed either a hydrophilic or moderate hydrophobic character.Three isolates were moderately hydrophobic, adherence to hexadecane range between 43, 37 and  57%.Remaining isolates were hydrophilic character, with hydrophobicity values between 18.5% and 36%.According to the literature, spores of B. cereus groups generally considered hydrophobic.On the other hand, the hydrophilic character of the soil isolates of B. cereus could also be related to an adaptation to alkaline pH conditions.This notice is an accordance with those mentioned by Seale et al. (2008).They stated that hydrophobicity increased at acidic pH while it decreased at alkaline pH (Giotis et al., 2009).Decreased hydrophobicity was observed for the spores of a strain of B. cereus alkali-tolerant (Bernardes et al., 2010).These data are confirmed by the results of Hamadi et al. (2004) who found that the hydrophilicity of strains of S. aureus  and E. coli is low apparent for neutral to alkaline pH (> 5 to 11) so that the hydrophobicity is more apparent at acidic pH (2-3).In our results, pH of soil isolates was alkaline between (8 to 9) and hydrophobicity associated with very moderate hydrophobicity is an argument for adaptation at pH alkaline conditions.

Biofilm formation
In our study, we use a tube method and microtiter plate assay to test the capacity of thirty six isolates to form biofilms.The tube test showed significantly different results from the results obtained by the standard microtiter-plate test.All isolates did not form a biofilm in the microtiter plate assays, while in the Tube assays, formation of biofilm took place preferentially at the point of the interface between the liquid and air for all B. cereus (Figure 5).According (Deighton and Balkau, 1990)   observations and reported that B. cereus ATCC14579 did not produce biofilm in the microtiter plate.The dynamic condition had negative effect not only on attachement of B. cereus cells to polystyrene surface but also on biofilm development, where all tested isolates were no biofilm producer.According to Carpentier and Cerf (2011), the conditions for growth are not always favorable for the adhesion of bacteria.The choice of material in which these microtiter are produced, also remains imperative to avoid negative interference with the adhesive properties of the tested microorganisms.This illustrates the need for reliable and reproducible techniques to culture and biofilm development.It is recognized that biofilms are formed in response to environmental signals which differ from one species to another.These signal nutrient availability and quorumsensing involved in the regulation of maturation of the biofilm (Stanley and Lazazzera, 2004).Factors which influence structure and development of biofilm, and the composition of the microbial community include nutrient availability and type of sugar provided (Stoodley et al., 2001).Finally, the results indicate that the bacteria tested in this study may be promising in promoting plant growth.

Table 1 .
Biochemical and physiological characteristic of 36 B. cereus isolates.

Table 4 .
Hydrophobicity percentage of the ten Bacillus cereus selected isolates.
the adherence capability of Staphylococci was influenced by many factors include different mechanisms of adherence for plastic and glass surfaces.Number of spores attached to the polystyrene surface was dramatically affected by hydrophobicity.Auger et al. (2006) assured same