Optimization and strain improvement by mutation for enhanced cellulase production by Bacillus sp. (MTCC10046) isolated from cow dung

2.1 Isolation and screening of cellulase producing bacteria

The cow dung samples were collected from the village of Burdwan district, West Bengal, India, in plastic bags by sterilized spatula and stored in an ice box for approximately 12 h. It was brought to the laboratory for isolation of cellulolytic bacteria. One gram of cow dung sample was suspended with 100 ml of distilled water and was homogenized by constant shaking using an orbital shaker for 2 h at 180 rpm. Serial dilutions from 10⁻⁶ to 10⁻⁷ were prepared using sterilized distilled water. An aliquot of 100 μL of each dilution was spread plated onto Omeliansky’s agar medium (Omeliansky, 1902) [g/L (W/V), (NH₄)₂SO₄ 1; K₂HPO₄ 1; MgSO₄.7H₂O 0.5; NaCl traces; carboxymethyl cellulose (CMC) 1%, pH 7] and incubated at 37 °C. Morphologically dissimilar and discrete colonies were picked from different dilution plates and streaked on separate Omeliansky’s agar medium and incubated at 37 °C for 96 h. The replica plates were also prepared separately for staining. Cellulase producing bacteria were screened by congo red staining (Teather and Wood, 1982) and C1 strain was selected as potent cellulolytic bacteria (colony showing largest zone of decolorization).

2.2 Culture medium for production of cellulase

The fermentation medium is the same as the previously used medium (Omeliansky’s medium) during isolation with the only difference of addition of different carbon sources or different nitrogen sources and adjustment of pH for optimization media in different experiments. Different fermentable sugars or different nitrogen sources have been shown to either induce or inhibit cellulase production depending on individual species. To decipher the sugar effect or the effect of nitrogen sources on C1 strain cellulase activities of supernatant samples attained from cultures grown on either glucose, cellobiose, CMC, lactose, sucrose, or cellulose etc. as carbon sources and KNO₃, (NH₄)₂SO₄, NH₄NO₃, NH₄Cl, peptone etc. in medium were compared. Similarly different pH was adjusted during medium preparation to determine the optimum pH of C1 strain for cellulase production. All the ingredients were mixed proportionately except carbon source and the pH was adjusted by 0.2 N NaOH/0.1 N HCl. Then the medium was sterilized by autoclaving at 121 °C for 15 min. The carbon source was sterilized separately and added to the fermentation medium during inoculation. To check the maximum production of cellulase by the C1 strain in culture, the medium was enriched with the supplements step by step which individually increase the cellulase production.

2.3 Cultural conditions for cellulase production and preparation of crude enzymes

Two loops of C1 strain was inoculated into test tubes containing 5 ml of sterile water and shaken in a rotary shaker for mixing. Thereafter, 0.5 ml of culture was inoculated into 100 ml Erlenmeyer flask containing 20 ml of fermentation medium. Carbon sources are added after sterilization. The culture was incubated in a rotary shaker at 37 °C at 180 rpm for 10 days. Fermented broths were removed after 2, 4, 6, 8 and 10 day intervals and were centrifuged at 12,000g for 20 min at 4 °C. The cell free supernatants containing the crude enzyme were used for the estimation of cellulase enzymes. In some experiments like optimization pH, temperature, and suitable concentration of N-sources for cellulase production, fermented broths were removed after 8 day interval for crude enzyme preparation. The enzyme production by the C1 strain was determined by assay of cellulase enzymes using UV–visible spectrophotometer (Simadzu Model UV-190) at 540 nm.

2.4 DNA extraction and molecular phylogenetic analyses using 16S rRNA gene sequence

Genomic DNA was isolated from C1 strain following the method of Johnson (1994). The 16S rDNA genes was amplified with broadly conserved bacterial 16s rDNA primers SSU16s-F (5′-AAC TCC TAC GGG AGG CAG CAG-3′) and SSU16s-R (5′-AAG GAC TAC CAG GGT ATC TAA TCC-3′) which yielded PCR products of about 0.4 Kb (Wilmotte et al., 1993). The nucleotide sequences of the amplified small subunit rRNA genes were determined bidirectionally with same SSU 16s primers according to Shaikh and Tarr (2003). A continuous stretch of 415-nucleotide 16S rRNA gene sequences was used to search for similar sequences from RDP database site. After confirmation of generic affiliation, sequences from type strains of different species were retrieved from NCBI GenBank. A phylogenetic tree was constructed showing relationship between C1 strain and other reference strains by neighbor joining (NJ) method with Jukes and Cantor correction using TREECON software as described by Saha and Chakrabarti (2006).

2.5 Cellulase enzyme assay

The CMCase activity was measured by incubating 0.5 ml of culture supernatant with 0.5 ml of 1% CMC prepared in 0.05 M sodium acetate buffer, pH 4.8 at 40 °C for 1 h. The reducing sugars liberated were estimated by the 3,5-dinitrosalicylic acid (DNS) method (Miller, 1959). The enzyme reaction was stopped by the addition of 3 ml DNS reagent (dinitrosalicylic acid 1 g, NaOH, 16 g, potassium sodium tartrate 300 g, and distilled water up to 1 L) to the above 1 ml reaction mixture, boiled in capped glass tubes for 5 min, cooled and then optical density was measured at 540 nm. The CMCase activity was determined using a calibration curve for d-glucose. One unit of CMCase activity was defined as the amount of enzyme that released 1 μmol of reducing sugars as glucose equivalents min⁻¹ and the specific activity is the number of units of enzyme activity per milligram of enzyme protein.

Avicelase activity was determined under same conditions i.e., by incubating 0.5 ml of culture supernatant with 0.5 ml of 1% Avicel (microcrystalline cellulose) prepared in 0.05 M sodium acetate buffer, pH 4.8 at 40 °C for 1 h. After incubation, released reducing sugars were measured by the DNS method. One unit of Avicelase activity was defined as the amount of enzyme that released 1 μmol of reducing sugars as glucose equivalents min⁻¹ and the specific activity is the number of units of enzyme activity per milligram of enzyme protein.

The filter paper (FPase) activity was measured according to the method of Ghosh (1987). 50 mg (1 × 6 cm² strip) of Whatman No. 1 filter paper were added in 0.5 ml of 0.05 M sodium acetate buffer (pH 4.8) and 0.5 ml of enzyme solution. The mixture was incubated at 40 °C for 1 h and then the reducing sugar liberated was measured by the DNS method. One unit of FPase activity was defined as the amount of enzyme that released 1 μmol of reducing sugars as glucose equivalents min⁻¹ and the specific activity is the number of units of enzyme activity per milligram of enzyme protein.

β-glucosidase (or cellobiase) activity was measured by incubating 0.5 ml of culture supernatant with 0.5 ml of 1% (w/v) salicin prepared in 0.05 M sodium acetate buffer, pH 4.8 at 40 °C for 1 h. After incubation, released reducing sugars were measured by the DNS method. One unit of β-glucosidase activity was defined as the amount of enzyme that released 1 μmol of reducing sugars as glucose equivalents min⁻¹ and the specific activity is the number of units of enzyme activity per milligram of enzyme protein.

The presence of glucose was also estimated by glucose oxidase method (Baker and Panow, 1991) as the latter gives true glucose concentration eliminating interference by other reducing sugars to detect the effect of lactose on cellulase production.

2.6 Protein concentration

The soluble protein concentration was determined according to the method of Lowry et al. (1951).

2.7 Mutagenesis by N-methyl-N-nitro-N-nitrosoguanidine (NTG)

The wide type cell grown in CMC medium at 50 °C for 24 h was harvested at logarithmic phase by centrifugation (10,000g, 20 min) at 4 °C and washed twice with McIlvaine’s buffer (containing 0.1 M citric acid and 0.2 M phosphate buffer) pH 5.0. The cell was resuspended in buffer at a concentration of 5.8 × 10⁸ cell/ml. and NTG (1 mg/ml) was added into the cell suspension. After incubation for 1 h at 37 °C in incubation shaker at 100 rpm, the cell was centrifuged and washed immediately with buffer. The treated sample was transferred into CMC plates (Xu et al., 2011) and incubated at 50 °C for 48 h. The cellulolytic activity was assayed using congo red followed by Teather and Wood (1982).

2.8 Statistical analysis

The statistical analyses (Zar, 1999) were performed using the SPSS version 11 software (Kinnear and Gray, 2000) using 3 replicates. It was performed by using a two-way factorial ANOVA, multivariate ANOVA, and three-way factorial ANOVA. In all cases, a post hoc Tukey test was carried out t y test was carried out to determine the significant differences within the variables.

3.1 Isolation of cellulase producing bacteria from cow dung

After pouring congo red solution (0.75%) and washing it with 2% NaCl, a light colored clear zone was found around the streak. But whole plate becomes deep red in color. This indicates that the strains were cellulase producing. The cellulase producing C1 strain was isolated from cow dung and was identified to be a species of Bacillus sp. with the help of partial 16S rDNA sequence homology of C1 strain (Fig. 1).

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Figure 1

Neighbor-joining phylogenetic tree is showing relationship of C1 strain with different spp. of the Bacillus 100% similarities taken from blast analysis. The tree was generated using TREECON software (Van de Peer and De Wachter, 1997) with Jukes and Cantor’s correction. Bootstrap values of 1000 replications are shown at the nodes. Sequence of Geobacillus stearothermophilus (AB02196) was taken as out group. Bar 0.1 substitutions per site.

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3.2 Bacterial growth and cellulase production with various saccharides

Cellulase production was found to be dependent upon the nature of the carbon source used in culture media. To maximize cellulase yield, the effects of different carbon sources (1% W/V) in production medium have been examined and are presented in Table 1. The results were taken after 8 days of incubation. These findings showed that Bacillus sp. utilized all carbon sources for bacterial growth. The CMC was the best among the tested carbon sources for this strain, as highest CMCase, Avicelase, FPase and β-glucosidase were recorded which proved the powerful induction of extracellular cellulase activity (Table 1). After CMC, lactose was the next better water-soluble carbon source for cellulase production by this strain. To maximize cellulase yield, the effects of different saccharides as carbon sources in the production medium have been examined and are presented in Table 1. The results of a two way ANOVA revealed significant differences in the specific activity for different carbon sources (between Carbon source, CS: F_(1)8,72 = 1104.808; P < 0.001) and enzyme types (between enzyme types, ET: F_(1)3,72 = 11.58; P < 0.001) and their interactions (between CS and ET: F_(1)24,72 = 14.46; P < 0.001).

3.3 Effect of days on cellulase production

Incubation time is an important parameter for optimal production of enzymes. For determining the optimum incubation days, a range of days (2–10 days) was tested, keeping the incubation temperature constant at 37 °C and pH at 7.0. It was observed that the strain gradually raised cellulase synthesis and reached maximum activity (Fig. 2) at 8 days (0.73 U/mg CMCase, 0.77 U/mg Avicelase, 0.84 U/mg FPase and 0.93 U/mg β-glucosidase) after that enzyme activity slowly decreased.

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Figure 2

Time course of CMCase, Avicelase, FPase and β-glucosidase of crude enzyme produced by culture medium containing 1% CMC at 37 °C by Bacillus sp.

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3.4 Optimum concentration of CMC

We find that among different carbon sources CMC was the best carbon source. Among different concentrations of CMC, 8% CMC was optimum for cellulase production of this strain (1.114 U/mg CMCase, 1.065 U/mg Avicelase, 1.117 U/mg FPase and 1.041 U/mg β-glucosidase) (Fig. 3) revealed by a two way ANOVA followed by Tukey test (Fig. 3). The results of a two way univariate ANOVA using CMC concentrations and the enzyme types as variables were tested to justify the differences in the specific activity exhibited by Bacillus sp. (strain C1). All P values are significant at P < 0.001 level.

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Figure 3

CMCase, Avicelase, FPase and β-glucosidase of crude enzyme produced by culture medium containing different concentrations of CMC at 37 °C after 8 days by Bacillus sp.

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3.5 Effect of temperature on cellulase production

The medium was adjusted to pH 7.0 during the optimization of incubation temperature. For determining the optimum temperature, a range of temperatures (35 –55 °C) was tested, keeping the incubation days constant at 8 days and using 8% CMC. It was observed that the levels of CMCase, Avicelase, FPase and β-glucosidase were highest at 50 °C (2.05 U/mg CMCase, 2.4 U/mg Avicelase, 2.02 U/mg FPase and 2.6 U/mg β-glucosidase) (Fig. 4), after that cellulase production slowly decreased. The results of a two-way ANOVA substantiate the differences in the specific activities at different temperatures for the enzymes .The significant F value for the temperature and enzyme interactions is suggestive of the fact that the peak activity of the enzymes vary in their temperature ranges.

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Figure 4

Production of cellulases by Bacillus sp. in broth culture containing 8% CMC after 8 days of incubation at different temperatures.

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3.6 Effect of pH on cellulase production

For determining the optimum pH, a range of pH (6.0–8.5) was tested, keeping the incubation temperature constant at 50 °C and 8 days and using 8% CMC. It was observed that the strain gradually raised cellulase synthesis and reached maximum activity (Fig. 5) at pH 7.0 (2.26 U/mg CMCase, 2.45 U/mg Avicelase, 2.05 U/mg FPase and 2.84 U/mg β-glucosidase) after that enzyme activity slowly decreased. Like the temperature effects on cellulase production, effects of pH and enzyme interactions in the ANOVA were also noted (Fig. 5).

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Figure 5

Production of cellulases by C1 in broth culture containing 8% CMC after 8 days of incubation at 50 °C at different pH.

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3.7 Effect of nitrogen sources on cellulase production

The enzyme production is affected significantly under different concentrations of the organic and inorganic nitrogen sources (Table 2). For determining the suitable nitrogen source, all optimized conditions are used. The results of a two way ANOVA revealed significant differences in the specific activity for different nitrogen sources (between Nitrogen source, NS: F_(1)6,56 = 13272.808; P < 0.001) and enzyme types (between enzyme types, ET: F_(1)3,56 = 194.58; P < 0.001) and their interactions (between NS and ET: F_(1)18,56 = 323.941; P < 0.001). Among the different nitrogen sources tested, the enzyme activity was higher with NH₄NO₃ (Table 3). To find out the suitable concentration of NH₄NO₃, different concentrations of NH₄NO₃ were tested, among which 0.175% NH₄NO₃ was optimum for this strain (5.6 U/mg CMCase, 5.75 U/mg Avicelase, 5.4 U/mg FPase and 5.62 U/mg β-glucosidase) (Fig. 6).

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Figure 6

CMCase, Avicelase, FPase and β-glucosidase of crude enzyme produced by culture medium containing different concentrations of NH₄NO₃ at 37 °C after 8 days by Bacillus sp.

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3.8 Optimum concentration of lactose

We find that lactose was the penultimate best carbon source for cellulase production. Therefore, effects of two different concentrations of lactose have been tested (Figs. 7A and B) and it was observed that 1% lactose was better for cellulase production of this strain (Fig. 7A). A repeated measures ANOVA justified the differences on the supplement added to the medium, the time (in days) of incubation and the activity of the different enzyme by this Bacillus sp. strain C1.

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Figure 7A

Time course of CMCase, Avicelase, FPase and β-glucosidase of crude enzyme produced by culture medium containing 1% lactose at 37 °C by Bacillus sp.

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Figure 7B

Time course of CMCase, Avicelase, FPase and β-glucosidase of crude enzyme produced by culture medium containing 2% lactose at 37 °C by Bacillus sp.

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3.9 Effect of lactose on cellulase production

CMC was the optimum carbon source for cellulase production among the sources tested (Table 1). We noticed that the effect of disaccharide lactose on cellulase synthesis significantly differed. The specific activity of CMCase, Avicelase, FPase and β-glucosidase recorded highest when 8% CMC was supplemented with 1.0% lactose (Table 3). Activities of the same enzymes under similar conditions were comparatively lower with 7% CMC with 1% lactose or with any other combination percentage of them as substrate (Figs. 8A, B).

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Figure 8A

Time course of CMCase, Avicelase, FPase and β-glucosidase of crude enzyme produced by culture medium containing (A) 7% CMC + 1% lactose at 37 °C by Bacillus sp.

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Figure 8B

Time course of CMCase, Avicelase, FPase and β-glucosidase of crude enzyme produced by culture medium containing (B) 8% CMC + 1% lactose at 37 °C by Bacillus sp.

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3.10 Effect of mutagenic treatment on cellulose production

The wild type Bacillus sp. strain C1 was subjected to mutagenic treatment using NTG for strain improvement. After mutagenesis, following C1M¹¹, C1M¹⁶, C1M²³, C1M²⁶, C1M²⁹ mutant colonies were obtained based on the ratio of diameter between the clearing zone and colony on the CMC-congo red medium. The cellulase activity of clones that displayed the largest clearing zones was assessed after 60 h of cultivation. The five best isolates were selected and cultivated in the CMC agar medium (Table 4). Mutant strain C1M²⁶ exhibited the highest CMCase activity at 16.30 U/mg protein followed by the mutant strain C1M¹⁶ (14.75 U/mg proteins) (Table 4).