Isolation of Endopyhtic and Xylanolytic Bacillus pumilus Strains from Zea mays

In this study, two endophytic xylanolytic bacteria, named M1 and M2, were isolated from surface sterilized Zea mays stern and leaf, respectively. Isolates were identified as Bacillus pumilus. Microorganisims showed different morphology on agar plates. Xylanase production level and profile varied between isolates, as well. Maximum xylanase production level of 188.0 ± 20.0 and 5.6 ± 1.1 U/ml were achieved by Bacillus pumilus M1 and Bacillus pumilus M2 in a liquid medium containing 3% corn cobs as a sole carbon source and inducer, respectively. Isolates produced very low level of cellulase in crude enzyme extract. B. pumilus M1 xylanase was partially characterized. Enzyme had a maximum activity at pH 8.0 and 65°C, seemed proper for pulp and paper industry, and required further characterization. In conclusion, this study indicated that inside part of Zea mays is a novel and good source for isolating xylanase producers. After detailed characterization, such enzymes could be used in various applications in paper and pulp, textile, food and feed industries.


Introduction
[3-1,4-Xylan, mainly found in the secondary walls of plants, represents one-third of the renewable biomass on the earth (Bakir 2005).Xylan polymer largely consists of a p-D-1,4linked D-xylose backbone substituted with acetyl, arabinosyl, and glucuronosyl side chains.Frequency and composition of the branches are dependent on the source of xylan.The complexity of the xylan molecules requires the action of the different hydrolytic enzymes.The most effective one is endo-[3-1,4-xylanase that cleaves the [3 -1,4 bonds of xylan backbone and produces xylooligosaccharides.
In this study, we screened stern and leaf parts of the Zea mays and isolated two xylanolytic Bacillus pumilus isolates.In addition, we determined the xylanase production level of isolates using agricultural byproduct, corn cobs and characterized enzyme partially.

Material and Method
Leaf and stern samples of Zea mays were collected from field of Ba şkent University Institute of Transplantation and Gene Sciences.Healthy samples having no disease symptoms were harvested (August, 2007) and subjected to microorganism isolation studies.

Isolation of xylanase producing bacteria and microbial maintenance :
Endopyhtic xylanase producer was isolated using the method of Suto et al. (2002) with some modifications.The leaf and stern samples were washed in running water for 10 min and cut into 0.5 cm pieces.Then, pieces were washed in sterile distilled water for extra 10 min and surface sterilized by soaking in 75% ethanol for 4 min to avoid surface originated microbial contamination.The sterilized samples, dried on sterile filter paper, were cut longitudinally into two pieces aseptically using sterile razor blade.Then, inside part of the stern and leaf samples were placed on a CRX agar (w/v: 0.5%; xylan, 0.5%; peptone, 0.5%; yeast extract, 0.1%; KH2PO4, 0.02%; MgSO4, 0.015%; Congo red, 0.01%; Na2CO3 and 2.0%; agar) and incubated at 35°C for 2 days.Due to enzymatic hydrolysis of xylan-Congo red complex in CRX agar, xylanase producing bacteria formed a clear zones in a red background.Then, xylanase positive isolates were subjected to the purification and characterization steps.
To make sure that growth on plate indicated the microorganism originally located inside the plant, surface of the samples were also contacted to surface of CRX and screened for microbial growth.

Identification of bacteria:
The pure isolates were characterized by means of conventional procedures (Sneath 1986).The test characterizing the isolates included endospore staining, Gram staining, motility, oxygen requirement and catalase test.The tests were performed in triplicate.Gram staining result was confirmed using McConkey agar plates.Endospore production was confirmed through heat test at 80°C for 10 min (Slepecky and Hemphill 1992).
Isolates were identified at genus level as a Bacillus according to Claus and Berkeley (1986) and Gupta et al. (1992), and then, they were further identified using the API 50 CH-AP1 50 CHB/E medium kit (BioWrieux).
Xylanase production: Xylanase production was performed in 250 ml shake-flask bioreactors containing 100 mI medium at 30°C, 175 rpm for 7 days.The medium contained 0.5% NaCI, 0.25% yeast extract, 0.1% KH2PO4, 0.02% MgSO4, 0.1% Na2CO3 and 3% steam hydrolyzed corn cobs powder, agricultural byproduct, as sole carbon source and inducer.Ground corn cobs were autoclaved for 30 min at 121°C and dried overnight at 100°C.The fermentation medium was centrifuged for 40 min at 11,000 x g and the supernatant was used as xylanase extract (crude enzyme extract).

Enzyme assay:
Xylanase activity was determined according to Bailey et al. (1992) using 1% Birchwood xylan in 50mM phosphate buffer at pH 7.0 and 40°C.Cellulase (EC 3.2.1.4)activity was assayed in 50 mM phosphate buffer at pH 7.0 and 40 °C using1 x 6 cm VVhatman No.1 filter paper strips as substrate (Miller et al. 1959).Dinitrosalicylic acid (DNSA) method was used to determine reducing sugar concentration in both assays by using either xylose or glucose as standard.Initial reaction rates were taken into account in activity measurements.One unit of xylanase (U) was defined as the quantity of enzyme necessary to produce 1 gmole of xylose/glucose equivalents per min at 40°C under the giyen conditions.All the experiments were performed at least two times and the averages were giyen.

Effect of pH and temperature on xylanase activity:
To determine the pH-dependance of the xylanase activity, the following buffers (50 mM) were used over the pH range 5.0-9.0 at 40 °C by standard activity assay; citric acid-Na2HPO4 (pH 5.0-6.0),sodium phosphate (pH 7.0-8.0),glycine-NaOH (pH 9.0) and carbonate-bicarbonate (pH 11.0).The effect of temperature on the xylanase activity was determined at 40-80 °C in 50 mM phosphate buffer at pH 7.0 using the standard xylanase assay.

Results
Xylanolytic microorganism isolation: Surface sterilized, longitudinally cut stern and leaf samples from healthy Zea mays were placed on CRX agar and screened for successive xylanase positive colonies.To make sure that there was no external bacterial contamination, exterior part of the surface sterilized samples were also displayed for microbial growth.Since there was no surface originated microbial growth on CRX plates, xylanase positive colonies were considered to be from inside of the plant.
Therefore, two xylanolytic bacteria, M1 and M2, were isolated from inside of the stern and leaf part of Zea mays, respectively.They were identified at genus level.They were rod shaped, Gram positive, aerobic, motile, catalase positive and formed endospores (Table 1).Gram and endospor staining results were confirmed by growth test on McConkey agar and heat test (Slepecky and Hemphill 1992).On the basis of these properties, M1 and M2 were classified as strains of the Bacillus genus (Claus and Berkeley 1986).The identification was confirmed by utilization the method described by Gupta et al. (1992), as well.Then, isolates were identified further as a Bacillus pumilus strains according to API 50 CH-API 50 CHB/E medium kit (BioWrieux).Therefore, isolates will be referred as of this point as B. pumilus M1 and B. pumilus M2.
Isolates presented different morphology on agar plate as presented in Table 2. Mi colonies were 5-7 mm in size, irregular, lobate, swarm and shiny.
However, colonies of B. pumilus M2 were rather bigger (10-12 mm) than B. pumilus M1, curled, unlobate, spread and opaque.Morphology comparison results pointed to possible strain difference between two isolates.
Xylanase production: In addition to qualitative determinations, xylanase production performance of isolates were quantitatively determined and comparatively evaluated.The production of xylanase was performed in shake flask fermentation media containing 3% of ground corn cobs as sole a carbon source and inducer.days fermentation, respectively (Figures 1 and 2).Moreover, isolates had very low level of cellulase activity (0.002-0.003U/m1) in crude enzyme extract.
As shown in Figures 1 and 2, xylanase production profı les resembled the bacterial growth profile, and so we may conclude that there was a possible correlation between growth and the activity of the xylanase enzyme.A low level of xylanase activity was detected in the earlier stages of fermentation and the enzyme activity steadily reached its highest value.Then, probably because of the enzyme degradation by proteinase activity and xylanase inactivation, there was a decrease in the xylanase titers.Since B. pumilus M1 produced xylanase at reasonable rate without any optimization, its enzyme was partially characterized.The pH and temperature dependence of B. pumilus M1 xylanase was giyen in Figures 2 and 3, respectively.Enzyme had maximum activities at both pH 6.5 and 8.0, which may indicate the production of multiple, at least two, xylanases with acidic and basic pH optima, as stated form many microorganisms (Avc ı oğ lu et al. 2005).Enzyme had also maximum activity at 65°C.Most of the mesophilic xylanases are optimally active at temperature below 50°C and act in acidic and neutral pH ranges.Only few xylanases are presented to be active and stable at alkaline pH and high temperature (Collins et al. 2005).
Figurel.Xylanase production profiles of B. pumilus M1 on 3% corn cobs.(The fermentation medium contained 0.5% NaCI, 0.25% yeast extract, 0.1% KH2PO4, 0.02% MgSO4, 0.1% Na 2CO3 and 3% corn cobs.The fermentations were carried out in an incubator shaker at 35 °C and 175 rpm.; • :xylanase activity; ♦ : cell dry weight).It is also noteworthy to mention that existence of B.pumilus M1 and B. pumilus M2 within plant was found to be tissue selective which can be explained by xylan composition and structure variations through tissue to tissue within a plant (Coughlan and Hazlewood 1993) and nature of the microbial xylanolytic system such as substrate specifity and type of xylanolytic enzymes (Liab et al. 2000 andErsay ı n 2006).
Discussion: Many bacterial and fungal endophytic microorganisms have been isolated from various types of vegetation such as cotton (Mclnroy et al. 1995), native tree species from national park in Thailand (Lumyong et al. 2002), banana (Cao et al. 2004) and rice (Naik et al. 2007).Roots and leaves of maize (Zea mays L.) were screened for endophytic actinomycetes (Mclnroy et al. 1995, De Aroujo et al. 1999).
Endopyhtes were mainly considered as producers of bioactive compounds (Strobel et al. 1996, andStrobel andLong 1998) and protective agent of plant against disease and insect (Bultman andBell 2003, Cao et al. 2004).However, there are few studies reported on utilization of plants as sources of endophytic xylanase producer (Burke andChairney 1997, Suto et al. 2002) and no work presented on xylanolytic endophyte isolation from maize.This is the first report presenting the screening of maize for xylanolytic bacteria isolation.
When compared to the data presented in literature, screening and isolation of xylanase producer from Zea mays (plant) was easier and quicker than that from soil (Panbangred et al. 1983, Avcioglu et al. 2005, Rawashadeh et al. 2005, Xu et al. 2005, Kinegam et al. 2007).In this study, we isolated two xylanase producers B. pumilus isolates.Together with morphological difference, due to variations in xylanase production profile and titer, B. pumilus M1 and B. pumilus M2 were considered to be two different isolates.In the literature, different xylanase activities have been reported over a wide range, usually 2-1000 U/mi for Bacillus species (Bak ı r et al. 20005).Clearly the xylanase activity of our isolate M1, 188.0±20.0U/ml, was not very high, but not low either.After optimization of enzyme production conditions, it is possible to increase xylanase production level.Significant xylanase production level increase (-50-80 folds) with enzyme production condition optimization was reported for B. pumilus (Poorna and Prema 2006), Rhizopus oryzae (Bakir et al. 2001) and Aspergillus oryzae (Szendefy et al. 2006).Accordingly, xylanase production by the our isolates may possibly be further increased by optimization of the enzyme production conditions such as nitrogen sources of medium, air flow, pH and stirring rate.
Xylanases from fungi, active in neutral or acidic pH, but bacterial xylanases generally have higher optimal pH and are stable at alkaline pHs which makes bacterial xylanases being more suitable for applications in the paper and pulp industry (Porna and Prema 2006) In conclusion, we have shown that plants can be considered as a source of xylanolytic microorganism and indigenous leaf and stern part of the Zea mays could serve as a source for xylanolytic microorganism isolation.After detailed characterization, such enzymes could be used in various applications in paper and pulp, textile, food and feed industries.Due to wide range of application area, isolation of new xylanase producers is a great value for both researchers and industry.

Figures 1
Figures 1 and 2 present the xylanase production profile of organisms on 3% corn cobs.Both M1 and M2 were able to produce xylanase from low-cost byproduct (corn cobs), but xylanase production level and profile varied between isolates.When compared to B. pumilus M1, B. pumilus M2 produced very low level of xylanase and maximal xylanase activities of 188.0 ± 20.0 U/ml and 5.6 ± 1.1 U/ml were achieved by B. pumilus M1 and B. pumilus M2 strains after 3 and 4 A., F. I. ŞAHIN ve M. HABERAL, "Isolation of Endopyhtic and Xylanolytic Bacillus pumilus Strains 377 from Zea mays"
. The most studied xylanase producers among bacterial sources are Bacillus species, due to their high yield and stability at alkaline pHs (Avc ı oğ lu et al. 2005, Duarte et al. 2000, Ersay ı n 2006).Having maximum activity at pH 8.0 and 65°C with no cellulase activity, B. pumilus M1 xylanase was thought to be a promising for paper and pulp industry.After detailed characterization, possible application area of B. pumilus M1 xylanase can be investigated.

Table 2 .
Characteristics of M1 and M2 isolates on agar plate.