A NOVEL BACILLUS PUMILUS STRAIN FOR ALKALINE XYLANASE PRODUCTION AT AND ABOVE 40

The objective of the study was to select a bacterial strain which can produce xylanase at alkaline pH and temperatures above 40C and to characterize the strain. Among the 45 bacterial strains, isolated from different sources, five strains, which were expected to produce alkaline xylanase, above 40C, isolated from open media containing xylan as carbon source (GS7, GS15, GS17, GS20 & GS*), were selected for the study. Xylanase production by the strains GS17 and GS*, was less affected by pH changes between pH 8.0 and 9.5 than the strains GS7, GS15 and GS20. Strains GS17 and GS* produced xylanase at pH 10.0 while the others did not produce. Therefore strains GS17 and GS* were selected. Xylanase production, by GS17 at 39 hours was 1.2, 1.3, 1.2 and 3.2 times higher at 40, 45, 50 and 55C than by GS*. At 60C, GS17 produced 4.25 UmL (30h) of xylanase activity while GS* did not. As GS17 produced xylanase at and above pH 9.0 and 40 to 60C, it was selected. The xylanase of GS17 showed activity in the temperature range from 40 to 95C showing highest activity at 60C. Strain GS17 is non-branching, gram-positive, sporulating, motile, aerobic, catalase positive, β hemolytic, oxidase positive long rods. Hence, it belongs to the genus Bacillus. Slightly oval shaped terminal and subterminal endospores were observed in 24h old cultures. When the culture became old, formation of spores was very much reduced. Further, based on the shape and arrangement of spores, ability to produce acid from glucose, xylose & mannose; growth temperature; inability to hydrolyze starch and tyrosine; inability to produce urease and indole; inability to reduce nitrate; ability to grow in NaCl and ability to grow in different carbohydrate sources, the strain, GS17 was identified as Bacillus pumilus. Thus, Bacillus pumilus which was locally isolated can grow and produce xylanase at and above 40C and pH 9.0.


INTRODUCTION
Xylanases are widely used in industries to bleach craft pulp, to increase the brightness in paper industry, to improve the digestibility of animal feed and to clarify the juices in the food industry (Nakamura et al., 1993).When Trichoderma harzianum was grown in SSF on sugarcane bagasse, the peak of xylanase activity (287Ug -1 DM) was obtained at 7 th day (Barbosa et al., 2001).Although many bacteria and fungi have been studied for xylanase production, several xylanases commercially available are active at neutral or acidic pH and their optimum temperatures for activity are below 45ºC.Enzymes which are active under alkaline conditions and at higher temperatures have great potential for industrial applications for bleaching process to avoid changes in pH or temperatures.Different strains of Bacillus, which are able to produce xylanase under alkaline conditions have been isolated and identified (Duarte, 2000).Among them, the strains that can produce xylanase up to 40ºC were selected and identified using the differences in morphology and biochemical properties.
The objective of the study is to select a bacterial strain which can produce xylanase at alkaline pH and temperature above 40 o C from strains isolated from different sources and to characterize the strain by microscopic, biochemical and cultural tests.

Analytical methods
The growth of the organism was measured by measuring the optical densities at 600 nm against the respective fresh fermentation media.Enzyme was diluted with Tris aminomethane buffer (pH-8.5).Enzyme assay was carried out at pH 8.5 and 60 o C by preincubating the enzyme extract with 10gL -1 xylan in 0.01M Tris aminomethane buffer (pH-8.5)for 5 min.Then 0.5 mL of the enzyme was mixed with 0.5 mL substrate and incubated for 5 min.at 85 o C. Reducing sugar was measured by DNS method (Miller, 1959).One unit of xylanase activity is defined as the amount of enzyme that produces 1 µmol of reducing sugar in 1 minute at pH 8.5 and 60 o C with 20 gL -1 xylan.

Effect of pH on the growth and xylanase production
Strains of GS 7 , GS 15 GS 17 & GS 20 and GS* activated in the activation media at different pH levels (7.5, 8.0, 8.5, 9.0 & 10.0) and at 40 o C, were inoculated to the fermentation medium at the respective pH values (7.5, 8.0, 8.5, 9.0 & 10.0) and incubated at 40 o C and at 100 rpm.The pH was maintained in the test samples and not in the control samples.Growth and the xylanase production were monitored.

Effect of temperature on xylanase production
The bacterial strains GS 17 and GS* (18 h old; 20%, v/v) activated at optimum pH value and at different temperatures (35, 40, 45, 50, 55 & 60 o C) at 100 rpm, were transferred into the fermentation medium at optimum pH value and incubated at the respective temperatures (35, 40, 45, 50, 55 & 60 o C) at 100 rpm while maintaining the pH.The experiment was proceeded as said above.

Effect of temperature on xylanase activity
The effect of temperature on the xylanase activity was determined by incubating the appropriately diluted crude enzyme (0.01M Tris buffer at pH 8.6) with 0.5 mL of soluble xylan (2gL -1 ) at different temperatures, ranging from 40 to 95 o C.

Biochemical tests
Oxygen requirement test, test for anaerobic growth, Catalase test, Oxidase test, Triple Sugar Iron Agar test and Lactose Fermentation test were done on strain GS 17 (Theivendrarajah, 1990 &Barrow andFeltham, 1993).

Identification of genus of selected strain
Colony morphology such as form, elevation, margin, opacity, diameter (in mm) after 40 h, colour and surface of the strain GS 17 , were studied to identify the genus of the selected bacterial strain.

Determination of species of strain GS 17
Shape and arrangement of endospore were observed under oil-immersion microscope after Grams staining.Production of acid from different carbohydrates such as glucose, xylose and mannose were tested.Production of urease, hemolysis of blood agar, indole test, nitrate reduction test, decomposition of tyrosine, hydrolysis of starch, citrate utilization test and voges-proskauer (vp) test were done to the selected strain (Theivendrarajah, 1990 &Barrow andFeltham, 1993).Growth of the selected strain was tested at 5, 15,25,35,40,45,50,55 and 60 o C at pH 9.0, and 100rpm.Effect of different concentrations of NaCl on the growth of the selected strain was tested at 600nm.

Effect of pH on growth and xylanase production
Five bacterial strains which were isolated from open xylan agar plate as the potential xylanase producers were selected for this study.
When the pH was maintained at 8.0 and 8.5, GS 15 showed the highest growth, while GS 17 showed highest growth when the pH was maintained at 9.0, 9.5 and 10.0.Most of the organisms showed better growth in the pH nonmaintained media than in pH maintained media (Tables 1 and 2) except GS 7 (at pH 8.0), GS 15 (at pH 8.0), GS 17 (at pH 8.5, 9.0 and 9.5) and GS* (at pH 8.5).Strains GS 17 and GS* were able to grow at pH 10.0.Therefore, among the five strains selected, GS 17 and GS* were able to grow in alkaline media, up to pH 10.0.Since the strains GS 17 and GS* were able to grow and produce xylanase under alkaline conditions, they were selected for further studies.
All the organisms produced xylanase in parallel with their growth.When the enzyme production decreased, all the selected organisms stopped growing.This indicated that the organism was unable to survive beyond that level due to the non-production of xylanase.The enzyme production was maximum, when the cell population entered into stationary phase, suggesting the enzyme production is not growth associated.Xylanase production by strains GS 17 and GS* was less affected than strains GS 7 , GS 15 and GS 20 when the pH of the medium was changed from 8.0 to 9.5.Xylanase production by strains GS 17 and GS* was better than by strains GS 7 , GS 15 and GS 20 when the pH of the medium was maintained at 8.0, 8.5, 9.0 and 9.5 during fermentation.Xylanase was produced by strains GS 17 and GS* when the pH of the medium was maintained at 10.0 but strains GS 7 , GS 15 and GS 20 did not produce xylanase.Therefore, strains GS 17 and GS* were selected as the best alkaline xylanase producers from the selected strains.

Effect of temperature on xylanase production
In order to select the bacterial strains which are capable of producing xylanase at high temperatures, the effect of different temperatures on xylanase production, was studied at pH 9.0 and the results are summarized in Table 3.
Strains GS 17 and GS* started to produce xylanase at 14 h at all the tested temperatures.At the commencement of the xylanase production, GS 17 produced higher activity (8.85UmL -1 ) at 40 o C than GS*.GS* produced higher activity at 35 o C (6.25UmL -1 ).Both the strains GS 17 and GS* produced xylanase activity at 14h of fermentation (1.21 and 0.16 UmL -1 respectively) at 55 o C. At 60 o C, strain GS 17 produced 0.22UmL -1 of xylanase activity while the strain GS* did not produce the enzyme.Both the strains GS 17 , and GS* produced highest xylanase activity [(24.52UmL - (42h) and 19.26 UmL -1 (48h)] at 40 o C. With the increase in temperature above 40 o C, the enzyme production also decreased.Both strains produced xylanase activity up to 55 o C. Though both strains GS 17 and GS* produced xylanase at higher temperatures, xylanse production by strain GS 17 at 39 hours and at pH 9 was 1.21, 1.22, 1.31, 1.17 and 3.20 times higher at 35, 40, 45, 50 and 55 o C respectively, than that produced by strain GS*.The strain GS* did not produce xylanase at 60 o C and at pH 9.0 while the strain GS 17 produced 2.12 UmL -1 (39h) of xylanase activity at 60 o C. As strain GS 17 gave a higher xylanase activity than strain GS*, at alkaline pH and higher temperatures, GS 17 was selected for further studies.

Effect of temperature on xylanase activity
The supernatant xylanolytic activities were assayed at different temperatures ranging from 40 o C -95 o C at pH of 9.0 with 20gL -1 xylan.When the temperature was varied from 40 to 95 o C, the activity of xylanase increased upto 60 o C (25.65 UmL -1 ).Xylanase from the isolate exhibited a temperature profile with a sharp peak of maximal activity at 60 o C and activity between 40 -95 o C. The optimum temperature for xylanase enzyme activity was 60 o C at pH 9.0.When the enzyme was kept at room temperature, 84% (21.45UmL -1 ) of the original activity was observed for more than 5 min.The optimum temperature for activity depends on the type of organism.

Microscopic studies
Microscopic study was carried out to identify the genus of the strain.The strain GS 17 was stained as blue-violet rods with spores indicating that it is a gram positive rod.Strain GS 17 moved rapidly across the microscopic field with twisting and this indicated the true motility.The hanging drop method used here is a type of wet mount slide preparation that permits the observation of living, unstained cells in a fluid medium.Gram-positive motile non-branching spore forming rods belong to Family Bacillaceae (Ananthanarayan et al., 1997;Prescott, 1996).Thus, the strain GS 17 may belong to the Family Bacillaceae.

Biochemical tests
Biochemical tests were carried out to confirm the genus of the strain and to identify the species.The strain GS 17 had shown good growth under aerobic condition but did not grow under anaerobic condition (in anaerobic jar).This indicated that the strain GS 17 is a strict aerobe.The strain GS 17 produced O 2 from H 2 O 2. This showed that the strain GS 17 is a catalase producer.If the bacterium oxidizes tetramethylp-phenylenediamine dihydrochloride, it will turn purple, indicating that the organism can produce cytochrome oxidase.No change in colour indicates that there is no production due to lack of cytochrome oxidase.The strain GS 17 did not bring about a colour change.Therefore, it does not produce cytochrome oxidase.Strain GS 17 ferment lactose, sucrose and glucose.The strain inoculated slants showed pink red butt and yellow slope.These results indicated that strain GS 17 ferments glucose.When Mac Conkey agar medium was inoculated with strain Gs 17 it did not change colour into red.This indicated that strain Gs 17 does not ferment lactose.

Identification of the genus of selected strain
The colonies of the strain GS 17 have a circular form with an entire margin, white in colour, moist and shiny with a convex elevated surface.After 40 h of growth, the colony diameter was 1.5 to 2.0 mm.The strain produced opaque single colonies.Based on studies so far carried out, it can be confirmed that the strain GS 17 belongs to the genus Bacillus.Bacillus is distinguished from the other endosporeforming bacteria on the basis of being a strict or facultative aerobe, rod-shaped, and (usually) catalase-positive.According morphological, cultural and biochemical tests carried out so far, GS 17 belongs to genus Bacillus.Its species has been identified, in the following experiments.

Determination of species of strain GS 17
To determine the species of a bacterial strain, it is important to know its endospore formation, acid production from sugars, urea hydrolysis, blood hemolysis, indole production, nitrate reduction, tyrosine utilization, starch hydrolysis, citrate utilization, glucose fermentation, growth temperature and growth in NaCl.
When serum-water glucose medium, serumwater xylose medium and serum-water mannose medium were inoculated with strain GS 17 and incubated at 37 o C for 24h, the colour changed to red.This indicated that strain GS 17 can produce acid from glucose, xylose and mannose.
If the strain is a urease-producer, the enzyme will hydrolyze urea by releasing ammonia and carbon dioxide.Ammonia released will change the phenol red indicator to red pink colour due to the change in pH of the medium to alkaline conditions.Medium inoculated with Strain GS 17 did not show any colour change.This indicated that strain GS 17 is not a urease producer.
When blood agar plate was inoculated with strain GS 17 , around the bacterial colony a transparent clear zone was observed.This indicated that GS 17 has broken down the haem in hemoglobin present in blood.Thus, strain GS 17 is a β-hemolytic organism.
When tryptophan is metabolised by an organism using the enzyme tryptophanase, indole is produced and the presence of indole can be detected using Kovac's reagent.Kovac's reagent is yellow in colour.It contains 4(p)dimethylaminobenzaldehyde, which reacts with indole and produces a red colour on the surface of the reagent.Light red or pink colour at the border of Kovac's indole reagent and tryptophan solution (tryptone water) indicates the formation of indole (Theivendrarajah, 1990).When tryptone water medium was inoculated with strain GS 17 and mixed with Kovac's indole reagent, no colour change was observed.This indicated that strain GS 17 does not utilise tryptophan and produce indole.
Reduction of nitrate to nitrite by an organism can be tested by incubating one loopful of inoculum in a broth containing nitrate and after 4 h adding sulphanilic acid reagent to the broth.If nitrite is present the acid reagent is diazotized and forms a pink-red compound with alpha napthalamine.The strain GS 17 did not show any colour change, indicating that it does not have the ability to produce nitrate reductase.
If the organism decomposes tyrosine crystals there should be a clear zone under and around the bacterial growth.When the tyrosine-agar plates were inoculated with strain GS 17 , no clear zone was observed around and under the colonies.This indicated that the strain GS 17 did not utilize tyrosine.
If the organism produces starch hydrolysing enzymes it can hydrolyse starch into monosaccharides.To check whether the organism has utilized starch, after the growth of the organism I 2 has to be added.If there is blue colour formation, it indicates the production of starch hydrolyzing enzyme (Theivendrarajah, 1990).When starch-agar medium was inoculated with strain GS 17 and I 2 /KI was added after 48 h of incubation, a blue colour was formed.This indicated that the strain GS 17 did not produce starch-hydrolyzing enzymes.
Bacteria that utilize citrate can extract nitrogen from the ammonium phosphate incorporated in the medium, resulting in the production of ammonia, which combines with water to form NH 4 OH.These reactions in combination produce an alkaline pH, resulting a colour change in the indicator from green to blue.If the organism does not utilize citrate, the medium remains green in colour.When citrate phosphate broth was inoculated with strain GS 17 , the medium remained green.This indicated that the strain GS 17 did not utilize citrate as the carbon source.
If acetoin is produced by the organism, in the presence of sodium hydroxide acetoine is oxidised to diacetyl.The diacetyl will give a pink compound with creatine.GS 17 was cultured in glucose-phosphate-peptone water and incubated at 37 o C for 48 h.When creatine and sodium hydroxide were added the culture and exposed to air, a pink colour was obtained.This indicated that GS 17 had produced acetoin by the fermentation of glucose.When different concentrations of NaCl were added to the fermentation medium, the growth (OD at 600nm) of the strain GS 17 was increased from 0 to 1.678 at 42 h in the media with NaCl concentration up to 10gL -1 and beyond that concentration growth of the strain decreased.In the presence of 70gL -1 NaCl, very low growth (OD 600 nm 0.021) was observed (Figure 2).

Final confirmation of species of identified strain Bacillus GS 17
Characteristics of the selected strain GS 17 was compared with 13 species of Bacillus.If the character of strain GS 17 is similar to the known species its score would be 1.If the character is not similar and variable, it will not get any score.Total score was counted, divided by total characteristics and it was multiplied by 100 and presented as a percentage.
Based on these morphological findings and biochemical studies, the strain GS 17 got the highest score of 86% showing similarities with Bacillus pumilus, followed by Bacillus subtilis (81%, Table 4).The strain GS 17 showed clear characteristics of Bacillus pumilus than the other Bacillus species.As the strain GS 17 got the highest score, it was identified as Bacillus pumilus.Summary of the identification studies of the strain GS 17 is present in Table 4.

CONCLUSION
Selected bacterial strain GS 17 can produce xylanase at pH 9.0 and above and temperatures above 40 o C. Based on the microscopic, biochemical and culture studies, the strain GS 17 was identified as Bacillus pumilus.

Figure 1 .
Figure 1.Growth of strain GS 17 at 20 h in the activation medium at pH 9.0 and at different temperatures and at 100rpm.

Figure 2 .
Figure 2. Growth of strain GS 17 at 24 h in fermentation medium containing different concentrations of NaCl at pH 9.0 and 40 o C while shaking at 100 rpm.

Table 3 . The effect of temperature on the xylanase production by the strains GS 17 and GS* grown at different temperatures at 100rpm in fermentation medium
.(The pH of the medium was maintained at 9.0, by the manual addition of 0.1N NaOH.) 17 was grown at varying temperatures, ranging from 5 to 60 o C in activation medium.The temperature range for growth of strain GS 17 was 25 to 55 o C. GS 17 grows at 55 o C but no growth at 5 and 15 o C (Figure1).Strain GS 17 grew well at temperatures between 25-55 o C. Strain GS 17 did not grow at or below 15 o C. At 60 o C less growth (OD 600nm 0.14) was observed.