Introduction

The term “cellulases” refers to a group of enzymes that catalyze the hydrolysis of cellulose into sugars. Cellulolytic microorganisms play an important role in the biosphere by recycling cellulose, the most abundant carbohydrate produced by plants [1]. Cellulolytic enzymes from microorganisms also have many potential biotechnologic and industrial applications. Cellulases are required in large quantities because of their application in many industries, such as textiles, detergent, food, animal feed, bio-fuel, paper and pulp, pharmaceutical, and waste management [2–5, 8, 11, 12]. The first step in the development of an industrial process for the production of an enzyme is to isolate the potential strains. Isolation and screening of microbes for cellulases is of immense importance keeping in view the demand for new enzymes and the improvement of their biotechnologic applications. An easy, fast, and environmentally friendly qualitative method is described for screening microorganisms producing extracellular cellulase on agar plate.

Material and Methods

Soil Samples and Microorganisms

Soil samples were collected from different locations of western Himalaya (Himachal Pradesh, India) for the isolation of bacteria-producing cellulases. Samples were transported to the laboratory and stored at 4ºC until used. Ten-fold serial dilutions of each soil sample were prepared in sterilized distilled water, and 0.1 ml diluted sample was spread on the surface of nutrient agar medium (0.3% beef extract, 0.5% peptone, 0.5 % NaCl, and 1.7% agar [pH 7.0]) for bacteria; potato dextrose agar (20% potatoes, 2% dextrose,1.7% agar) (HiMedia, India) was used for fungi. Plates were incubated at 28°C for 48 hours for bacteria and 96 hours for fungi. Morphologically different colonies appearing on the plates were purified on the respective medium for bacteria and fungi. The purified isolates were preserved at 4ºC and used during the course of study. The actinomycete Streptomyces sannanensis MTCC 6637 was obtained from the Microbial Type Culture Collection & Gene Bank, Institute of Microbial Technology, India.

Screening and Identification of Cellulase Producers

Five microlitres of overnight grown culture was spot plated on CMC agar (0.2% NaNO3, 0.1% K2HPO4, 0.05% MgSO4, 0.05% KCl, 0.2% carboxymethylcellulose (CMC) sodium salt, 0.02% peptone, and 1.7% agar) (HiMedia, India). Plates incubated at 28ºC for 48 hours were flooded with 1% hexadecyltrimethyl ammonium bromide (HAB) for 30 to 40 minutes [6]. From cellulase-producing microorganisms, two potential bacterial strains RK3 (Gram +ve) and MN34 (Gram –ve) and one fungal strain RS2 were selected for identification and further studies. Genomic DNA from bacterial strains was isolated and amplified by using universal 16S rRNA primers [9], and fungal DNA was isolated and amplified by using internal transcribed spacer 1 (ITS 1) and internal transcribed spacer 4 (ITS 4) primers [15]. The amplified gene products were purified from agarose gel using Qiaquick Gel Extraction Kit (Qiagen, Germany). Nucleotide sequencing of the genes was done by using Big Dye Terminator Cycle Sequencing Kit (Applied Biosystems) and 3130xl Genetic Analyzer (Applied Biosystems). The BLASTN program (http://www.ncbi.nlm.nih.gov/BLAST/; National Center for Biotechnology Information, Bethesda, MD) was used for homology searches with the standard program default.

Comparison of Cellulase Screening Methods

Screening of cellulase producers was done on CMC agar and CMC Congo red agar (0.05% K2HPO4, 0.025% MgSO4, 0.188% CMC sodium salt, 0.02% Congo red, 1.5% agar, 0.2% gelatin, and 10% soil extract). Five microlitres of cellulase-producing bacteria were spot plated on CMC agar in three sets and on CMC Congo red agar. The plates were incubated at 28 °C for 48 hours. The first set of plates was flooded with 1% HAB for 30 to 40 minutes [6]; the second set of plates was flooded with 0.1% Congo red for 15 to 20 minutes and then with 1 M NaCl for 15 to 20 minutes [14]; and the third set of plates was flooded with Gram’s iodine (2.0 g KI and 1.0 g iodine in 300 ml distilled water) for 3 to 5 minutes (new method). After incubation, CMC Congo red plates were also observed for zone of clearance around the colony [7]. In another set of experiments, the standard cellulase from Aspergillus niger was used (Sigma-Aldrich) for validation of results. Ten microlitres of enzyme solution (10 mg/mL) was poured into the wells in CMC plates, which were made with a gel cutter, and the plates were incubated for 24 hours at 28ºC and flooded individually with 1% HAB for 30 to 40 minutes, 0.1% Congo red for 15 to 20 minutes, followed by 1 M NaCl for 15 to 20 minutes, and Gram’s iodine for 3 to 5 minutes.

Results and Discussion

The two bacterial isolates producing cellulase were identified as Bacillus sp. RK3 (AM943531) and Pseudomonas sp. MN34 (AM992885) based on partial 16S rRNA gene sequencing. The cellulase-producing fungal-isolate strain RS2 was identified as Penicillium chrysogenum RS2 (AM948960) by sequencing of its ITS1–5.8S–ITS2 region [15]. These three cultures and one actinomycete S. sannanensis MTCC 6637 were used for further studies. The observation on the development of cellulose clearance zone showed Gram’s iodine to be more effective compared with HAB, and Congo red plus 1 M NaCl for screening and qualitative estimation of cellulase production by microorganisms on CMC agar plates. The plates flooded with HAB or with Congo red plus 1 M NaCl as well as the CMC Congo red plates showed low intensity of clearance zone (Figs. 1 and 2). There was difficulty in differentiating the enzyme-lysed zone from the CMC-containing area in the plates flooded with these reagents. Moreover, there was a waiting period of at least 30 to 40 minutes for the development of clearance zones [6, 14]. Interestingly, the plates flooded with Gram’s iodine showed distinct, clear, and prominent zones of clearance around the colonies showing cellulase production with bluish-black colouration in the nonhydrolysed part of the medium (Fig. 3). It took only 3 to 5 minutes for the development of clearance zones after flooding. The plates with Congo red added to the medium before inoculation also showed low intensity for zone of clearance (Fig. 4). Moreover, Congo red (“Direct Red 28” CI 22120, empiric formula C32H22N6O6S2Na2) is a benzidine-based dye and expected to metabolize to benzidine, which is a known carcinogen [10, 13]. There is a possibility that the added Congo red could have affected growth and cellulase production by the isolates because of its harmful effects. However, a sharp increase in the colour contrast of the hydrolyzed zone and nonhydrolysed portion of the medium is obtained on flooding with Gram’s iodine because it further enhanced the appearance of already sharply discernible clearance zone by producing bluish-black complex with cellulose (polysaccharide) and not with the glucose (monosaccharide). This new method for assaying has the double advantage of being quick as well as based on nontoxic chemicals.

Fig. 1
figure 1

Effect of HAB on the cellulolytic zone in CMC agar plates. (i) Uninoculated. (ii) Inoculated with Bacillus sp. RK3. (iii) Pseudomonas sp. MN34. (iv) S. sannanensis MTCC 6637. (v) P. chrysogenum RS2

Full size image
Fig. 2
figure 2

Effect of Congo red and NaCl on the cellulolytic zone in CMC agar plates. (i) Uninoculated. (ii) Inoculated with Bacillus sp. RK3. (iii) Pseudomonas sp. MN34. (iv) S. sannanensis MTCC 6637. (v) P. chrysogenum RS2

Full size image
Fig. 3
figure 3

Effect of Gram’s iodine on cellulolytic zone in CMC agar plates. (i) Uninoculated. (ii) Inoculated with Bacillus sp. RK3. (iii) Pseudomonas sp. MN34. (iv) S. sannanensis MTCC 6637. (v) Penicillium chrysogenum RS2

Full size image
Fig. 4
figure 4

Cellulase production in CMC Congo red agar plates. (i) Uninoculated. (ii) Inoculated with Bacillus sp. RK3. (iii) Pseudomonas sp. MN34. (iv) S. sannanensis MTCC 6637. (v) Penicillium chrysogenum RS2

Full size image

Furthermore, the method was also validated by using the standard cellulase form A. niger (Fig. 5). It was observed that plates flooded with Gram’ iodine after incubation also showed distinct, clear, and prominent zone in 3 to 5 minutes, whereas the plates flooded with HAB or with Congo red plus 1 M NaCl showed hazy zones after 30 to 40 minutes of incubation. The new method worked well with bacteria, actinomycetes, and fungi; hence, it can be employed for screening a large number of microorganisms with greater throughput.

Fig. 5
figure 5

Detection of cellulase activity for standard cellulase from A. niger. Carboxymethylcellulose agar plates were inoculated with 10 μl cellulase and after 24 hours flooded with (i) HAB, (ii) Congo red and NaCl, and (iii) Gram’s iodine

Full size image

Conclusion

The present studies showed that the use of Gram’s iodine remarkably enhances the sharpness of the clearance zone, making the process for screening cellulase-producing microorganisms easy, efficient, and rapid. Although the new method is a qualitative assay, the greatest advantage lies in its simplicity, quickness of performance, and effectiveness for screening a large number of microorganisms. Moreover, it avoids the use of toxic chemicals.