Diversity of Xylanolytic Bacteria Isolated from Thai Sources

Twenty-three xylanolytic bacteria were isolated from soils, sediments and buffalo faeces collected in Thailand. They were divided into 10 groups based on the phenotypic and chemotaxonomic characteristics including 16S rRNA gene sequence analyses. Eleven isolates were Gram-positive, facultatively anaerobic, spore-forming, rod-shaped bacteria. They contained meso-diaminopimelic in cell wall peptidoglycan. Two isolates (Group IA) were identified as Bacillus subtilis, 4 isolates (Group IB) were B. licheniformis, 2 isolates (Group IC) were B. niabensis, one (Group ID) was B. nealsonii, and 2 isolates (Group IE) were B. cereus. Seven isolates were Gram-positive, non-spore-forming, rod-shaped bacteria and were identified as Isoptericola variabilis (2 isolates in Group II), as Jonesia denitrificans (2 isolates in Group III), as Microbacterium natoriense (2 isolates in Group IV), and one isolate as Nocardioides simplex (Group V). Five isolates were Gram-negative; facultatively anaerobic, non-spore-forming, rod-shaped bacteria and each of them were identified respectively as Acinetobacter junii (Group VI), Aeromonas enteropelogenes (Group VII), Pseudomonas stutzeri (Group VIII), Stenotrophomonas maltophilia (Group IX) and Zobellella denitrificans (Group X). The isolates produced xylanase activity ranged from 1.03 to 17.65±0.25 unit/ml.

xylanase-producing bacteria isolated from soils and related materials in Thailand based on their phenotypic and chemotaxonomic characteristics including 16S rRNA gene sequence similarity.

Isolation and Screening of Xylanase Activity
Twenty-three xylanolytic bacteria were isolated from soils, muddy shore sediments, hot spring sediments and buffalo faeces samples collected in Thailand (Table 1), by the spread plate method on XC agar medium as previous report (Kinengam et al., 2007).In this screening step for the thermotolerant strains, the agar plates were incubated at 40C for 2 days.Xylanase-producing capacity of the cultures was detected by using a Congo red overlay method, as reported previously (Teather & Wood, 1982;Ruijssenaars & Hartsmans, 2000).Isolates showing xylanase-producing capacity were transferred to C agar medium.This medium had the same composition of XC medium apart from the omission of the oat spelt xylan.They were assayed for xylanase activity by using dinitrosalicylic acid (DNS) method and using 1% oat spelt xylan as substrate (Miller, 1959).

Identification Methods
Cells grown on C agar medium were examined for their morphological and cultural characteristics, including cell shape, colony appearance, endospore formation and pigmentation, after incubation at 37C for 2 days.Physiological and biochemical characterization was performed using the API 20NE and API 50CH (combined with API 50CHB/E medium) strips (bioMérieux), in accordance with the manufacturer's directions.Catalase and oxidase; hydrolysis of casein, DNA, starch, Tween 80, L-tyrosine and urea; the methyl red/Voges-Proskauer (VP) reactions, indole production, citrate utilization and hydrogen sulfide (H 2 S) production were determined as described by Barrow and Feltham (1993).Growth at different pH (5, 6, 7, 8 and 9), in 3 and 5 % (w/v) NaCl and at different temperatures (10, 15, 20, 25, 30, 40, 45, 50, 55 and 60C) was tested by using C agar medium.All tests were carried out by incubating the cultures at 37C, except for investigations into the effect of temperature on growth.Diaminopimelic acid in the cell wall and quinone system were determined as described by Komagata and Suzuki (1987).DNA was prepared by the method of Saito and Miura (1963).DNA base composition was determined by reversed-phase HPLC (Tamaoka & Komagata, 1984).The 16S rRNA genes of the strains were amplifed by PCR with primers, 27F (5'-AGAGTTTGATCMTGGCTCAG-3') and 1492R (5'-TACCTTGTTACGACTT-3') and PCR products were purified and sequenced as described previously (Tanasupawat et al., 2004).The sequences of strains were aligned with selected sequences obtained from GenBank by using CLUSTAL_X version 1.83 (Thompson et al., 1997).The alignment was edited manually to remove gaps and ambiguous nucleotides prior to the construction of phylogenetic trees.The phylogenetic trees were constructed by using the neighbour-joining (Saitou & Nei, 1987) method in MEGA4 software (Tamura et al., 2007).The confidence values of branches of the phylogenetic trees were determined using bootstrap analysis (Felsenstein, 1985) based on 1000 resampled datasets.

Isolation and Screening of Xylanase Activity
Twenty-three isolates showed xylanase clear zone with 1.0-12.0mm in diameter, surrounded their colonies.The xylanase producing bacteria of Group I isolates showed clear zone with 1.7-7.5 mm in diameter and produced xylanase activity ranged from 1.03 to 3.89±0.31unit/ml while Group II to Group X isolates showed clear zone with 1.0-12.0mm in diameter and produced xylanase activity ranged from 1.16 to 17.65±0.25unit/ml.It was found that the isolate CR2-1 in Group II was produced biggest clear zone with 12.0 mm in diameter and had highest xylanase activity (17.65±0.25 unit/ml) as shown in Table 1.In this study, the isolates showed a wide ranges of xylanolytic activity and were better than as reported in the non spore-forming, Gram-positive irregular rods (0-0.13 units/ml) and the isolates of Gram-positive spore-forming rods; the isolates of Gram-negative rods; and isolate of Gram-positive rods/cocci (0-0.17units/ml) by Kinengam et al. (2007).
Their phenotypic characteristics are almost the same as J. denitrificans ATCC 14870 T (data not shown).The isolates FXN1-1B (922 nt) and PHX2-5 (983 nt) were closely related to each other with 99.8% 16S rRNA gene sequence similarity and to J. denitrificans ATCC 14870 T with 99.2 and 99.1% sequence similarity, respectively (Figure 2).The isolate PHX2-5 contained MK-9 of menaquinone and 58.4 mol% of DNA G+C content.Variable characteristics were shown in Table 3.Based on the phenotypic properties, chemotaxonomic characteristics and 16S rRNA gene sequence, isolates FXN1-1B and PHX2-5 were identified as J. denitrificans (Rocourt et al., 1987).
Recently, Kinengam et al. (2007)   B. nealsonii isolates are found in soils and B. licheniformis isolates are distributed in soil and muddy shore sediment while B. cereus isolates are found in muddy shore sediment and buffalo faeces.In addition, the xylanolytic bacteria of I. variabilis from hot spring sediment, J. denitrificans, M. natoriense, N. simplex, Ac.Junii, A. enteropelogenes, P. stutzeri, St. matophila and Z. denitrificans from soils, muddy shore sediment and buffalo faeces are firstly isolated.

Conclusion
The xylanolytic bacteria isolated from various samples collected in Thailand were identified based on the analysis of 16S rRNA gene sequence.B. subtilis, B. niabensis and B. nealsonii isolates were found in soils.B. licheniformis isolates were distributed in soil and muddy shore sediment and B. cereus isolates were found in muddy shore sediment and buffalo faeces.This study, we reported the new finding of the xylanolytic bacteria of I. variabilis from hot spring sediment, J. denitrificans, M. natoriense, N. simplex, Ac. junii, A. enteropelogenes, P. stutzeri, St. matophila and Z. denitrificans from soils, muddy shore sediment and buffalo faeces.These isolates are the most likely source of enzymes and constitute a heterogeneous group of xylanase producing bacteria belonging to different genera.The isolated bacteria that be able to produce extracellular enzymes will provide the possibility to have optimal activities at different temperature and pH.Thus, the applications of the isolates are required for further study.

Table 1 .
Location, sample, isolate number, group, xylanase activity and identification of the isolates One unit of xylanase activity was defined as 1 µmol of xylose released per min under the condition assayed. *

Table 2 .
Differential characteristics of Bacillus isolates in Group I (A to E) reports that the isolates of Microbacterium barkeri, Bacillus niabensis, B. funiculus, B. megaterium, Pseudoxanthomonas suwonensis, Cupriavidus gilardii, and Rhodococcus rhodochrous strains isolated from soil samples collected in Nan Province, Thailand could produce xylanase.They are found to be diverse species.M. barkeri strain is found in soils collected in Viengsa district while M. barkeri, B. niabensis, B. funiculus, B. megaterium, Px. suwonensis, C. gilardii, and R. rhodochrous strains are distributed in soils samples collected in Muang district.However, in this study, the xylanolytic bacteria, B. subtilis, B. niabensis and

Table 3 .
Differential characteristics of the isolates in Group II to Group X