Soil samples from Eburru and Hell’s gate were separately suspended in physiological saline (0.85% NaCl) to suspend the cells. Aliquots of these preparations were inoculated in a selective medium containing 5g/l Carboxymethylcellulose (CMC), 2.2 g/LK₂HPO₄, 1.5g/L KH₂PO₄, 1.3 g/L (NH₄)₂SO₄, 0.1 g/L MgCl₂, 0.02 g/L CaCl₂ and 0.001 g/L FeSO₄. 7H₂O [19]. Incubation was done at 45^o C for 7 days. Further isolation was carried out by plating on CMC agar plates of selective medium (pH 6.9) supplemented with 0.1% yeast extract and 1.5% agar. Overnight incubation was done in a thermoshaker at 45^oC and 150 rpm.

Cellulolytic activity of the isolated bacteria was determined on a solid medium containing 5gL^-1 CMC, 2gL^-1 Yeast extract, 5gL^-1 Malt extract and 15gL^-1 Agar, pH 7.2 (modified fromDSMZ medium 65 without CaCO₃ and glucose) [20] and incubated for 3 days at 45^oC for secretion of cellulases. Cellulolytic activity was detected by soaking the plates with 1 mg/ml congo red for 15 minutes followed by destaining with 1M NaCl. Clear zones of hydrolysis were seen against the red color of Congo red stain indicating cellulose degradation.

Morphological characterization of the strain was carried out via Gram stain and determination of colony characteristics such as shape, color, surface elevation and margins. Temperature and pH tolerance tests were carried out at ranges of 4^oC to 60^oC and pH 3-12, respectively. Growth on selective media supplemented with 5 gL^-1 avicel and 5 gL^-1 cellobiose was determined.

Overnight cultures of identified bacterial isolates, inoculated in nutrient broth were used for DNA extraction according to procedure described by Kate Wilson [21] was used to harvest total genomic DNA from the bacterial cells. About 100 ng of DNA from each isolate was used as a template for 16S rRNA gene amplification using two bacterial specific primers: 16sF27, forward 5’…AGA GTT TGA TC (AC) TGG CTC AG…3’ and 16sR 1492, reverse 5’…TAC GG (CT) TAC CTT GTT ACG ACT T…3’. The amplification was performed in 50μl reaction volume containing 36.5μl PCR grade water, 5μl 10× PCR buffer, 4μl MgCl₂ (25mM), 1μl dNTP mix (10mM), 1μl of each primer (200μM), 0.5μl Taq polymerase (5U/μl) and 1μl of isolated DNA.

Polymerase chain reaction was performed in a TProfessionalthermocycler (Biometra, Germany). Conditions for the PCR were as follows; An initial denaturation step at 94ºC for 5 minutes followed by 35 cycles of 94ºC for 1 minute, 55ºC for 1 minute and 72ºC for 2 minutes and a final extension step of 7 minutes at 72ºC. The amplified products were electrophoresed on a 1% agarose gel in 1×TAE buffer containing 0.5 μg/ ml of ethidium bromide and visualized under UV light to determine the quality and size. The PCR products were then gel purified using MinElute Gel Extraction Kit (QIAGEN) according to manufacturer’s protocol and sent to International Livestock Research Institute (ILRI) Kenya for sequencing.

A consensus sequence for each isolate was generated using Chromas Lite software and the resulting sequences matched with known 16s rRNA gene sequences at NCBI using the BLAST algorithm.

The 16S rDNA sequences obtained were aligned using Jalview 2.8 to show sequence variation. Additionally these DNA sequences were used to retrieve related sequences from gene bank for determination of inferred relationship. All the sequences were then aligned in MUSCLE [22] and a phylogenetic tree constructed based on the nucleotide sequences with the Bayesian phylogenetic method in MrBayes software [23]. The trees were then visualised using fig tree software obtained at http://tree.bio.ed.ac.uk/

For enzyme production, the four isolates were separately inoculated in 100 mL of nutrient broth media supplemented with 1% CMC Modified [24], and cultured for 12 hours at 45^oC and 150 rpm. The culture was then centrifuged at 4000 rpm 4^oC for ten minutes and the cell free supernatant collected as the crude enzyme for assays. Protein concentration (mg/ml) of the cell free supernatant was determined using the Biuret method and the values obtained used to calculate specific activity of the crude enzyme in respective enzyme assays.

A quantitative Carboxymethyl cellulase (CMCase) assay for all four isolates was carried out using, 3, 5- Dinitrosalicylic acid (DNS) test [25]. A 1.0% (w/v) solution of carboxymethylcellulose (CMC) in 50mM Sodium acetate buffer (pH 5) was used as substrate. The reaction mixture (1 mL), containing 500μL crude enzyme and 500μL substrate, was incubated at 50^oC for 10 min. The amount of reducing sugars produced was then determined usingDNS test with a glucose standard. Heat inactivated (100^oC for 15 minutes) crude enzyme and substrate were used as controls.

Optimum time for CMCase production was determined by growing the isolate 3 in a minimum medium with 5gL^-1 of CMC as the sole carbon source and incubation was carried out for 10 days at 45^oC, pH 6.9 and 150 rpm. The Quantity of reducing sugars produced was assayed for ten days using the DNS test.

Presence and level of exoglucanase and β-glucosidase activities in addition to CMCase activities for isolate 3 were determined. Assay preparations included 500μl of crude enzyme, and 1% Avicel, cellobiose and CMC prepared in 50mM Sodium acetate buffer pH 5 respectively and incubation carried for 30 minutes at 60^oC. Quantity of reducing sugar (mg/ml) produced was determined using the DNS test.

Effect of temperature on CMCase activity was determined from 30^oC - 90^oC at intervals of 10 ^oC. Reactions were prepared as follows; 1% CMC in 50mM sodium acetate buffer pH 5 and incubated in a water bath for 60 minutes for the selected temperatures. Heat inactivated crude enzyme and substrate were included. One enzyme unit (U) was defined as the amount of enzyme producing 1 μmol of reducing sugar measured as glucose per minute per mL.

Effect of pH on enzyme activity was determined by mixing 500μL of the crude enzyme extract in each case with 500μl of 1% (W/V) CMC broth substrate, with pH values ranging from 3 -11.

The buffers used for pH adjustment included Glycine-HCl (pH 3.0), HAc-NaAc (pH 4.0 - 5.0), Na₂HPO₄-NaH₂PO₄ (pH 6.0 - 8.0) and Glycine-NaOH (pH 9.0 - 11.0) buffers at a final concentration of 50mM (modified [24] the reaction mixture was incubated in a water bath at pre-determined optimum temperature of 60^oC for 1 hour. Specific enzyme activity was then calculated.

To determine the stability of the enzyme extracts, reaction mixtures at the predetermined optimum pH and temperature values (pH 5 and 60^oC) were prepared. The quantity of reducing sugars was determined from 0 to 8 hours at 1 hour intervals.

All experiments were carried out in triplicate and the experimental data were analyzed using Statistical Package for Social Sciences version 16.0. Post hoc test (Tukey’s test) was used to evaluate differences among mean values for treatments at P<0.05.

Optimum temperature for Bacillus licheniformis strain Vic was recorded at 60^oC with a specific enzyme activity of 0.0698U/mg for the crude enzyme extract (Fig. 5). There was no significant difference in endoglucanase activity at 70^oC (P>0). This implies that industrial application of this isolate’s CMCase would yield optimum results at both 60^oC and 70^oC. Enzyme activity at both 80^oC and 90^oC was higher than that observed at 30^oC. Cellulases of this isolate can thereby be considered to be thermotolerant.

It was noted that optimum temperature for the crude enzyme activity is higher than the optimum temperature for growth of the isolate (Table 1 & Fig. 5). This implies that for industrial applications requiring high temperatures it would be advisable to use the extracellularly excreted enzymes rather than the whole organism.

Fig. (5). Specific enzyme activities of isolate 3 at different temperatures. Optimum activity was recorded at 60oC.

Several studies have reported the optimum temperature above 50^oC for CMCase activity. An optimum temperature of 65^oC for Bacillus licheniformis M9 has been reported [10] while Bacillus licheniformis JKF and Bacillus DUSELR13 have higher optima of 70 and 75^oC, respectively [16, 14]. Optimum CMCase activities have been recorded at temperatures ≤ 50^oC. For instance, 50^oC Bacillus sp. [13], 40^oC for Bacillus thuringiensis strains [24] and 30^oC for Bacillus licheniformis C108 [9].

At the predetermined optimum temperature (60^oC), the optimum pH was found to be 5 with a specific enzyme activity of 0.08214U/mg implying that it’s an acidic cellulase (Fig. 6). Acidic cellulases act at a pH range of 3.8-5.8 [27]. Bacillusstrain M-9 [10] was found to have similar optimum pH. A wide range of optimum pH has been noted with reports of values as high as pH 10 for B. licheniformis C108 endoglucanase [9] and pH 4 for B.thuringiensis var. israelensis (BtI) and B.thuringiensis var. thompsoni (BtH) [24].

Fig. (6). Specific enzyme activities of isolate 3 at different pH values. The optimum activity was recorded at pH 5.

From the time stability study at the predetermined optimum pH and temperature, the isolate’s CMCase activity was found to be stable throughout the study period with 92.2% activity retained after eight hours of incubation (Fig. 7). From the curve, enzyme activity at 6, 7 and 8 hours appears to have reached a platue. Post hoc analysis (Tukey’s test) show that there is no significant difference in the enzyme activity (P>0.05) at this time range. This phenomenon can be attributed to end product inhibition. Cellulase activity can be inhibited by accumulation of cellobiose and glucose [28]. To reduce end product inhibition, supplementation of β- glucosidases during hydrolysis or removal of reducing sugars by ultrafiltration or simultaneous saccharification and fermentation (SSF), can be done [28].

Fig. (7). Reducing sugar concentration for enzyme assay up to eight hours. Optimum activity was obtained after five hours. The crude enzyme extract retained 92.2 % of its activity at the eighth hour of incubation at pH and 60oC.

Cellulases from B.licheniformis strain vic can be used to break down cellulose at a high temperature and are stable for relatively long periods of time. This in addition to the extracellular production of the three enzyme components of a cellulase system implies that the isolate can be solely used to produce fermentable products. Bacillus species have the Generally Recognised as Safe (GRAS) status, high growth rate and ability to secrete proteins extracellularly.