Development of a plate technique for screening of polysaccharide-degrading microorganisms by using a mixture of insoluble chromogenic substrates

Journal of Microbiological Methods 56 (2004) 375 – 382 www.elsevier.com/locate/jmicmeth

Development of a plate technique for screening of polysaccharide-degrading microorganisms by using a mixture of insoluble chromogenic substrates Leonid N. Ten a,b,1, Wan-Taek Im a, Myung-Kyum Kim a, Myung Suk Kang a, Sung-Taik Lee a,* a

Department of Biological Sciences, Korea Advanced Institute of Science and Technology, 371-1 Kuseong-Dong, Yuseong-Gu, Daejeon 305-701, Republic of Korea b National University of Uzbekistan, VUZ-gorodok, Tashkent, 700-174, Uzbekistan Received 8 October 2003; received in revised form 10 November 2003; accepted 10 November 2003

Abstract A plate assay based on the visible solubilization of small substrate particles and the formation of haloes on Petri dishes, containing a mixture of different dye-labelled polysaccharides as substrates, provides a specific, reliable and rapid simultaneous detection of corresponding polysaccharide-degrading microorganisms. It has potential for increasing the efficacy of screening of microorganisms, utilizing different polysaccharides, in large numbers of natural samples. Diversely colored insoluble forms of amylose, xylan and hydroxyethyl-cellulose (HE-cellulose) were prepared as chromogenic substrates by using the cross-linking reagent 1,4-butanediol diglycidyl ether and the dyes Brilliant Red 3B-A, Cibacron Blue 3GA and Reactive Orange 14. Using the method, the bacteria with amylase or xylanase or cellulase or a combination of these activities were screened from soil and sludge samples, selected and identified according to 16S rDNA sequencing. D 2003 Elsevier B.V. All rights reserved. Keywords: Plate assay; Chromogenic substrates; Polysaccharide-degrading bacteria; Xylan-Cibacron Brilliant Red 3B-A; Amylose-Cibacron Brilliant Red 3B-A; HE-Cellulose-Reactive Orange 14

1. Introduction Polysaccharide-degrading enzymes are widespread in nature. They can be found in every type of

* Corresponding author. Tel.: +82-42-869-2617; fax: +82-42863-5617. E-mail address: [email protected] (L.N. Ten), [email protected] (S.-T. Lee). 1 Tel.: +82-42-869-2657; fax: +82-42-863-5617. 0167-7012/$ - see front matter D 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.mimet.2003.11.008

organisms including mammals, plants, algae, molds, bacteria and phages (Oshima et al., 2002; Sutherland, 1999; Terra and Ferreira, 1994). For the production of polysaccharases, microorganisms are usually the most convenient; they can be obtained from various natural sources. For the screening of large numbers of microorganisms, efficient plate screening methods are a prerequisite. A number of plate screening methods for the detection of polysaccharide-degrading microorganisms have been described in the literature. These methods are based on the complex formation between

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polysaccharides and dyes (Teather and Wood, 1982), the solubility characteristic of polysaccharides (Hankin and Anagnostakis, 1977), the gel-forming properties of polysaccharides (Kennedy and Sutherland, 1994), and on the use soluble and insoluble dyelabelled polysaccharides (Castro et al., 1995; Lee and Lee, 1997; McCleary, 1988). In the last case, when the chromogenic substrate is hydrolyzed, dyelabelled oligosaccharides diffuse from the colony zone that becomes visible as a halo in which the label is (nearly) absent. Reactive dyes are used for labelling polysaccharides; for example, Cibacron Blue 3GA was coupled to xylan (Lee and Lee, 1997), dextran and starch (Dhawale et al., 1982), and Remazol Brilliant Blue R (RBB) was covalently linked with xylan (Biely et al., 1985), inulin (Castro et al., 1995), amylose and starch (Rinderknecht et al., 1967). Currently, various dyed polysaccharides are commercially available, but most are produced as blue substrates and usually each substrate is available only in a single colored form. The plate screening methods with chromogenic substrates provide an array of relatively straightforward and simply applicable tools for specific detection of polysaccharide-degrading microorganisms. However, usually, only one chromogenic substrate is used as a supplement to the agar medium, thus enabling microorganisms to be selected according to a single parameter. Screening of microorganisms with a set of degrading activities or with a specific combination of degrading activities is labor-intensive and time-consuming, especially for large-scale searching programs. An increase of the efficacy of plate screening techniques can be achieved, particularly by simultaneous detection of microorganisms that produce diverse polysaccharide-degrading enzymes. The aims of this study were as follows: firstly, the synthesis of insoluble dye-labelled xylan, amylose and hydroxyethyl-cellulose (HE-cellulose) in different colored forms and their use as chromogenic substrates in a plate assay for simultaneous detection of diverse polysaccharide-degrading microorganisms; secondly, to test the developed plate technique in the screening and selection of bacteria that reveal high xylan-degrading, amylose-degrading and cellulose-degrading activities or specific combination of them.

2. Materials and methods 2.1. Chemicals Oat spelt xylan, amylose from potato, Cibacron Blue 3GA and Reactive Orange 14 were purchased from Sigma (St. Louis, USA). Hydroxyethyl-cellulose (Cat. No. 43,496-5), hydroxyethyl-cellulose (Cat. No. 43,497-3), 1,4-butanediol diglycidyl ether and Cibacron Brilliant Red 3B-A were obtained from Aldrich (Milwaukee, USA). 2.2. Synthesis of dye-labelled substrates Xylan-red, a cross-linked xylan covalently coupled with Cibacron Brilliant Red 3B-A, was prepared by the method reported previously (Lee and Lee, 1997) with some modifications. To a xylan suspension (2 g in 30ml distilled water), 10 ml of 2 M NaOH, 1.9 g of Cibacron Brilliant Red 3B-A and 1.2 ml of 1,4-butanediol diglycidyl ether were added, stirred for 5 min and left standing at room temperature. After 48 h, the mixture solidified into a gel. The gel was mixed with 100 ml of distilled water and was ground by a blender for 15 s. To remove the unbound dye, the ground particles were washed with boiling water and filtered (Whatman, type 1) repeatedly until the filtrate was colorless. The wet (highly hydrated) product was used as a supplement to agar medium. The content of the chromogenic substrate in the product was 0.89%. For analyses of the synthesized substrate, the particles were successively washed with 25%, 50% and 100% (v/v) ethanol and pure acetone and then air-dried at 25 jC. The same method was applied for the preparation of red cross-linked amylose (Amylose-red) using Cibacron Brilliant Red 3B-A and of blue cross-linked amylose (Amylose-blue) using Cibacron Blue 3GA. However, in both cases, the volume of 1,4-butanediol diglycidyl ether was increased to 3 ml; otherwise, the gel that formed was not solidified enough. The content of the dyed amylose was 0.80% in wet Amylose-blue and 0.76% in wet Amylose-red when used as supplement to the agar medium. Insoluble blue HE-cellulose (HE-cellulose-blue) and orange HE-cellulose (HE-cellulose-orange) were prepared from HE-cellulose (average m.w. ca. 720,000) by the same procedure using the dyes Cibacron Blue 3GA and Reactive Orange 14, respectively. The content of the dyed HE-cellulose was

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around 0.70% in both wet products. Xylan-Cibacron Blue (Xylan-blue) was synthesized as previously described (Lee and Lee, 1997). The content of the chromogenic substrate in the wet product was 0.91%.

(0.7 g/l). Samples were taken from the culture, analyzed and stored at 20 jC.

2.3. Agar plate with chromogenic substrates

The production of polysaccharide-degrading extracellular enzymes in the culture broth were measured after centrifugation of the broth at 15,000  g for 20 min at 4 jC and overnight dialysis of the supernatant at 4 jC against distilled water. Oat spelt xylan was used as the substrate for xylanase activity. Xylan (1 g) had been previously treated with 20 ml of 1.0 M NaOH and 20 ml of 1.0 M HCl, and the volume was brought to 100 ml with 100 mM of potassium phosphate buffer, pH 6.0. The mixture was then stirred for 1 h at 25 jC. The insoluble xylan was removed by centrifugation for 20 min in a bench top instrument and the soluble fraction was used for xylanase assay. Xylanase activity was determined by mixing 1 ml of soluble oat spelt xylan in 100 mM potassium phosphate buffer, pH 6.0, with 1 ml of original or diluted samples of the culture supernatant at 40 jC for 30 min. Three parallel replicates were used for each sample. The release of reducing sugar was measured using the dinitrosalicylic (DNS) reagent method (Miller, 1959). Amylase activity was measured in a 1-ml reaction mixture containing 0.5 ml of 1% (w/v) amylose in 0.1 M phosphate buffer (pH 7.0) and 0.5 ml of original or diluted samples of the culture supernatant. Three parallel replicates were used for each sample. The reaction was carried out for 30 min at 30 jC and the reducing sugar produced was determined by the DNS reagent method with glucose as standard. Cellulase activity was measured by incubating of 0.5 ml of 2% (w/v) HE-cellulose (average m.w. ca. 90,000) in 0.1 M potassium phosphate buffer, pH 6.0, with 0.5 ml of original or diluted samples of the culture supernatant at 30 jC for 30 min. Three parallel replicates were used for each sample. The amount of reducing sugar produced was measured by the DNS reagent method. One unit (U) of xylanase, amylase and cellulase activity was defined as the amount of protein that produced reducing sugars corresponding to 1 Amol of xylose or glucose equivalents from soluble xylan, amylose or HE-cellulose, respectively, per minute under the assay condition. In all case, average values from three replicates and standard deviation were calculated.

In principle, any suitable agar medium can be used as a basal medium. For screening polysaccharidedegrading bacteria, plates were prepared by adding a mixture of synthesized chromogenic substrates to nutrient agar medium (23 g/l) (Difco, USA). The mixtures were composed of the following: (a) Xylan-red, 25.0 g/l, and Amylose-blue, 75.0 g/l; (b) Xylan-blue, 25.0 g/l, and Amylose-red, 75.0 g/l; (c) Xylan-red, 25.0 g/l, and HE-cellulose-blue, 75.0 g/l; (d) Xylan-red, 25.0 g/l, Amylose-blue, 75.0 g/l, and HE-cellulose-orange, 100.0 g/l. To keep the particles dispersed, autoclaved substrate suspension was agitated gently while being poured into plates. 2.4. Screening procedure The samples were obtained from different sources: the anaerobic sludge from a sewage treatment plant in Daejeon (South Korea) and the soil samples from the Daejeon area. Appropriate serial dilutions of soil or sludge suspension in sterile water were spread on nutrient agar plates and the plates were incubated at 30 jC for 1– 5 days. The plate screening procedure was done with a nutrient agar medium supplemented with a mixture of various chromogenic substrates. The isolated colonies were purified by being streaked on nutrient agar plates that contained a mixture of corresponding chromogenic substrates. Any colony that showed a solubilization of one or more substrates and a corresponding distinct halo or haloes that exceeded the colony diameter by a factor of two or more was considered as polysaccharide-degrading enzyme producer and was consequently selected. 2.5. Culture conditions Isolated bacteria were grown at 30 jC for 36 h with rotary shaking (140 rpm) in 500-ml Erlenmeyer flasks containing 150 ml of a medium composed of nutrient broth (8 g/l) (Difco, USA), xylane (0.25 g/l), amylose (0.6 g/l) and HE-cellulose (average m.w. ca. 720,000)

2.6. Analytical procedures

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2.7. 16S rDNA sequencing The 16S rDNA was enzymatically amplified by Taq DNA polymerase by using a universal eubacterial primer set, 9F (5V-GAGTTTGATCCTGGCTCAG-3V) and 1512R (5V-ACGG(H)TACCTTGTTACGACTT) according to previously described method (William et al., 1991). After purifying the PCR product with an AccuPrepk PCR Purification Kit (BioNeer, Korea) the resulting PCR product was sequenced with an ABI Prism BigDye Terminator cycle sequencing ready reaction kit (Applied Biosystems, Foster City, CA, USA) and an automatic DNA sequencer (model 3700, Applied Biosystems). The primers used for the sequencing were 9F [5V-GAGTTTGATCCTGGCTCAG-3Vpositions 9– 27 (Escherichia coli 16S rRNA numbering)]. The 16S rDNA sequences of all the isolates were aligned with NCBI BlastN database.

3. Results and discussion Insoluble red amylose and xylan were synthesized using the dye Cibacron Brilliant Red 3B-A and the

Fig. 1. Detection of amylose-degrading and xylan-degrading bacteria in nutrient agar plate, containing Amylose-blue and Xylan-red as chromogenic substrates (incubation: 36 h, 30 jC). The colony (a) reveals only xylanase activity; the colonies (b) and (c) produce xylanase and amylase; the colonies (d), (f) and (g) have only amylase activity; and the colonies (h), (k), (m) and (n) do not reveal any degrading activities.

Fig. 2. Detection of xylan-degrading and cellulose-degrading bacteria in nutrient agar plate, containing Xylan-red and HEcellulose-blue as chromogenic substrates (incubation: 36 h, 30 jC). The colonies (a), (b) and (c) produce xylanase and cellulase, but (b) and (c) have lower cellulase activity compare with (a); the colonies (d) and (f) reveal only cellulase activity; and the colonies (g), (h), (k) and (m) have no degrading activities.

cross-linking reagent 1,4-butanediol diglycidyl ether. The same cross-linking reagent and Cibacron Blue 3GA were used for a preparation blue xylan, amylose and HE-cellulose. Reactive Orange 14 dye was used in the preparation of orange HE-cellulose. The dye content, the stability and other properties of the chromogenic substrates resemble those described for Cibacron Blue-xylan (data not shown) (Lee and Lee, 1997). The commercially available blue xylan, amylose and HE-cellulose were described elsewhere (Rinderknecht et al., 1967; Biely et al., 1985; Lee and Lee, 1997), but references to orange HE-cellulose and red xylan and amylose were not found in the literature. To increase the efficacy of plate screening techniques, a mixture of different chromogenic substrates was used as a supplement to the agar medium. To screen xylanase-producing or amylase-producing bacteria or both, Xylan-red and Amylose-blue were incorporated into a plate medium. The concentration of the substrates was adjusted for two reasons: firstly, to enable the small particles inside the medium to be observed; and secondly, to obtain colored halo or haloes surrounding the colonies through the diffusion

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of dye-labelled oligosaccharides, produced by the enzymatic hydrolysis of the colored polysaccharides. A picture of the agar plate (back side) is presented in Fig. 1. Ten bacterial colonies can be seen on the plate. The particles of Xylan-red were degraded by colony 1-a; a red halo surrounds the colony; and the unchanged particles of Amylose-blue can be seen inside the halo zone. This phenomenon indicates that bacterium 1-a has produced xylanase, but it does not reveal amylase activity. Around the bacterial colonies 1-b and 1-c Xylan-red and Amylose-blue particles were solubilized; red and blue haloes surround the colonies and a clearing zone appears around the colonies. Bacteria 1-b and 1-c have produced both xylan-degrading and amylose-degrading enzymes. Degradation of Amylose-blue particles and the blue halo, surrounding colonies 1-d, 1-f and 1-g, indicates that the bacteria have amylase activity but not produced xylanase. The colonies 1-h, 1-k, 1-m and 1-n belong to bacteria that have no xylanase and amylase activities. The similar result was observed when a mixture of Xylan-blue and Amylose-red was used, but the picture of substrate degradation was less clear at the same substrate concentrations. Nutrient agar plates supplemented with Xylan-red and HE-cellulose-blue were successfully used to

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screen the bacteria that reveal xylanase and cellulase activities. One of the obtained agar plates is presented in Fig. 2. The bacterium 2-a produced both enzymes, and the particles of both substrates are consequently degraded around the colony; red and blue haloes surround the colony. In the same way, bacteria 2-b and 2-c reveal high xylanase and low cellulase activity; while bacteria 2-d and 2-f produced only cellulase; the colonies 2-g, 2-h, 2-k and 2-m belong to bacteria without the activities. In the last case, the colony bodies are visible as colored spots on a redblue background due to the absorbance of transmittance light by the bodies, but the effect is not connected with substrate degradation. According to Fig. 1, the xylan-degrading activity of bacterium 1-c is approximately identical to that of bacterium 1-a, but its amylose-degrading activity is higher than that of the bacteria 1-d, 1-f and 1-g. According to Fig. 2, the cellulose-degrading activity of bacteria 2-b and 2-c is much lower than that of bacterium 2-a. This estimation was confirmed by determination of the enzyme production in the liquid culture of the selected colonies. The data are presented in Table 1. The correlation between the halo diameter and the level of enzyme production is correct for all used chromogenic substrates. However, the produc-

Table 1 Polysaccharide-degrading activities of selected bacteria Enzymatic activities (U/ml)a

Colonies

Halo diameter (mm) Xyl

Amyl

Cel

Xylanase

Amylase

Cellulase

1-c 2-a 3-a 3-b 3-d 3-e 3-k 1-a 2-b 1-d 1-f 2-f 2-d 1-m 2-m

24 32 28 28 33 30 33 22 29 0 0 0 0 0 0

20 –b 24 23 26 24 25 0 –b 14 14 –b –b 0 –b

–b 28 17c 15c 18c 15c 7c –b 6 –b –b 13 13 –b 0

2.15 F 0.03 5.03 F 0.06 4.83 F 0.06 4.24 F 0.04 6.83 F 0.08 5.72 F 0.07 7.08 F 0.09 1.94 F 0.03 3.91 F 0.04 0 0 0 0 0 0

6.17 F 0.07 8.34 F 0.09 5.41 F 0.06 5.40 F 0.05 7.15 F 0.09 5.91 F 0.08 6.75 F 0.08 0 0 2.04 F 0.04 1.79 F 0.03 0 0 0 0

0.57 F 0.01 4.23 F 0.06 8.13 F 0.10 6.83 F 0.08 10.13 F 0.12 6.03 F 0.09 0.76 F 0.02 0 0.23 F 0.01 0 0 1.53 F 0.03 1.32 F 0.04 0 0

Xyl: Xylan-red, Amyl: Amylose-blue, Cel: HE-cellulose-blue or HE-cellulose-orange. a Each value is an average of three replicates, F indicated standard deviation from mean value. b The substrate was not applied as a supplement to medium. c Diameter of the clearing zone.

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tion of the same enzyme can be compared only when incubation occurs under the same conditions and only among colonies, located on agar plates that contain the same medium. If the chromogenic substrate composition is different in the compared plates, the correlation must be used carefully. One of the main parts of our screening program has included the selection of bacteria that simultaneously have high amylase, xylanase and cellulase activities. Successful application of the mixture of two chromogenic substrates has led to the use of agar plates that contain three different-colored substrates. The orange HE-cellulose was deliberately synthesized and used together with Xylan-red and Amylose-blue as a supplement to the agar medium. A picture of one plate is presented in Fig. 3. According to the formation of haloes and the solubilization of substrate particles, the colonies 3-a, 3-b, 3-d, 3-e and 3-k produce cellulose-degrading, xylandegrading and amylose-degrading enzymes, but the 3-k colony reveals low amylase activity. Other colonies reveal one (3-m) or two (3-f, 3-g) polysaccharide-

Fig. 3. Detection of amylose-degrading, xylan-degrading and cellulose-degrading bacteria in nutrient agar plate, containing Amyloseblue, Xylan-red and HE-cellulose-orange as chromogenic substrates (incubation: 36 h, 30 jC). The colonies (a), (b), (d), (e) and (k) produce amylase, cellulase and xylanase; the colonies (f) and (g) have xylanase and cellulase activities; the colony (m) reveals only xylanase activity; and the colonies (c), (h) and (n) have no degrading activities.

degrading activities; and 3-c, 3-h and 3-n colonies have not produced the polysaccharide-degrading enzymes. In this case, the orange halo around the corresponding colonies is not clear enough to compare with the other haloes. Nevertheless, cellulase production can be detected by visible solubilization of HEcellulose-orange particles and it is usually enough for primary screening. The production of three polysaccharide-degrading enzymes by the above-mentioned bacteria was confirmed by determination of the enzyme activities in their culture broths. The data is presented in Table 1. For screening of polysaccharide-degrading microorganisms, soluble as well as insoluble chromogenic substrates are used (Castro et al., 1995; Lee and Lee, 1997; McCleary, 1988). In our opinion, insoluble substrates are more convenient if used in mixture form as supplements to the agar medium. Two parameters are applicable for a determination of the enzyme production: solubilization of substrate particles and halo formation. Moreover, the condition of separated substrate particles is easy to observe if the particle size is not very small, and just a few separated particles are enough to find nearby growing polysaccharidedegrading colony. This parameter cannot be used if soluble chromogenic substrates are applied as a supplement to the agar medium. Currently, chromogenic substrates for the assay of proteinases are commercially available (AZCL-casein, AZCL-collagen; Megazyme, Ireland). It is most likely plate method described here can also be applied to simultaneous screening of microorganisms that produce polysaccharide-degrading and protein-degrading enzymes. In the screening program, bacterial representatives with different polysaccharide-degrading activities were selected (Figs. 1– 3) in order to construct our own strain library. The strains that reveal one, two or three activities were isolated, purified and stored. Their amylase, xylanase and cellulase production in liquid medium was measured and correlated with the corresponding halo diameters on plate assays (Table 1). The strains were used for the primary comparative estimation of the enzyme production by selected colonies at the large-scale screening program. Another part of the screening program has included the selection of bacteria that reveal high amylase, xylanase and cellulase activities. Using the plate

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screening technique with Xylan-red, Amylose-blue and HE-cellulose-orange, the most active bacterial strains were selected from among more than a hundred isolated colonies namely Bacillus sp. ACX53, Bacillus sp. ACX71 and Cellulomonas sp. ACX95. According to the diameter of the clearing zones, the selected strains are much more active as producers of the polysaccharide-degrading enzymes than the strains presented in Table 1. All isolated strains were identified using 16S rDNA sequencing and their similarities to most close type strains are presented in Table 2. Among bacteria, Bacillus and Cellulomonas species produce a number of extracellular polysaccharidedegrading enzymes (Chaudhary et al., 1997; Priest, 1977). Most of isolated strains (Table 2), which reveal high amylase, cellulase and xylanase activities, belong to these species. Bacteria and other microorganisms having a number of polysaccharide-degrading activities can be successfully used for microbial utilization of agricultural by-products or wastes (Jecu, 2000), for production of ruminant feed enzymatic additives (Beauchemin et al., 2003), and in other fields of

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biotechnology (Bhat, 2000). Developed plate technique provides unique possibility for a selection of these microorganisms straight in the primary screening. At the screening by using a mixture of two or three chomogenic substrates four colonies showed high xylan-degrading activity but they did not have cellulase activity, namely Bacillus sp. HX10, Streptomyces sp. MX31, Bacillus sp. HX18 and Bacillus sp. AX12. These strains can be potential producer of cellulasefree xylanases, the interest in those has markedly increased during the last few years due to the potential use of these enzymes in the pulp and paper industry (Techapun et al., 2003). Cellulase produced simultaneously with xylanases in microbial-based processes could destroy the structure of cellulose and diminish pulp quality therefore cellulase-free xylanases are required. For screening naturally occurring microorganisms, which produce totally cellulose-free xylanases, described plate technique is very applicable. Not one in existing methods (Breccia et al., 1995; Hankin and Anagnostakis, 1977; Kennedy and Sutherland, 1994; McCleary, 1988; Teather and Wood, 1982) provides same target selection of poly-

Table 2 The similarity of selected bacteria with the type strains Colonies

Selected strains

Most close type strains

Sequence similarity % (of bp)

2-a 3-b 1-c 3-a 3-d 3-e 3-k 1-a 2-b 1-d 1-f 2-f 2-d 1-m 2-m – – – – – – –

Bacillus sp. ACX1 Cellulomonas sp. ACX2 Bacillus sp. ACX6 Bacillus sp. ACX18 Bacillus sp. ACX7 Cellulomonas sp. ACX46 Bacillus sp. ACX26 Bacillus sp. CX2 Bacillus sp. CX3 Aeromonas sp. A14 Aeromonas sp. A17 Flexibacter sp. C32 Flexibacter sp. C30 Klebsiella sp. NA8 Klebsiella sp. NA9 Bacillus sp. ACX53 Cellulomonas sp. ACX95 Bacillus sp. ACX71 Bacillus sp. HX18 Streptomyces sp. MX31 Bacillus sp. HX10 Bacillus sp. AX12

Bacillus subtilis IAM 12118T Cellulomonas cellasea DSM 20118T Bacillus subtilis IAM 12118T Bacillus subtilis IAM 12118T Bacillus subtilis IAM 12118T Cellulomonas flavigena DSM 20109T Bacillus subtilis IAM 12118T Bacillus pumilus NCDO 1766T Bacillus pumilus NCDO 1766T Aeromonas hydrophila ATCC 7966T Aeromonas eucrenophila ATCC 23309T Flexibacter sancti IFO 15057T Flexibacter sancti IFO 15057T Klebsiella planticola DSM 3069T Klebsiella planticola DSM 3069T Bacillus subtilis IAM 12118T Cellulomonas cellasea DSM 20118T Bacillus subtilis IAM 12118 T Bacillus vallismotris DSM 11031T Streptomyces somaliensis DSM 40267T Bacillus pumilus NCDO 1766T Bacillus vallismotris DSM 11031T

99.9 97.9 100.0 100.0 100.0 99.4 100.0 99.6 99.8 99.5 100.0 97.3 97.8 100.0 100.0 100.0 97.8 100.0 99.5 99.1 99.7 99.7

(529) (593) (534) (576) (548) (619) (564) (567) (566) (609) (635) (599) (600) (532) (564) (505) (608) (538) (551) (530) (650) (578)

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saccharide-degrading microorganisms already in the primary screening. In general, novel approach in plate technique provide unique information straight in the primary screening when a target of a selection is microorganisms having a set of required polysaccharide-degrading activities or specific combination of them. In conclusion, the major advantages of the screening technique developed in the present work can be summarized as follows: diverse polysaccharidedegrading colonies can be detected simultaneously by using a mixture of corresponding insoluble chromogenic substrates as medium supplements. Two visible parameters (the solubilization of substrate particles and the formation of haloes) are used for the detection. The plate assay is simple, rapid and well adapted for screening of large number of samples. The diameter of the halo zone is very useful for predicting the enzyme yield as an aid to select strains with a high level of polysaccharide-degrading activities.

Acknowledgements This work was supported by the Korea Science and Engineering Foundation through the Brain Pool Program (2003) and the 21C Frontier Microbial Genomics and Applications Center Program (Grant MG02-0101-001-2-2-0), Ministry of Science and Technology, Republic of Korea.

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