Correspondence: Vincenza Faraco, Department of Chemical Sciences, University of Naples ‘Federico II’, Complesso Universitario Monte S. Angelo, via Cintia, 4 80126 Napoli, Italy. Tel.: +39 081 674315; fax: +39 081 674313; e-mail: email@example.com
Ninety bacteria isolated from raw composting materials were screened for their cellulolytic activity on solid medium containing carboxymethylcellulose. The bacteria producing the highest cellulolytic activity levels were identified by 16S rRNA sequencing as Bacillus licheniformis strain 1, Bacillus subtilis subsp. subtilis strain B7B, Bacillus subtilis subsp. spizizenii strain 6, and Bacillus amyloliquefaciens strain B31C. Cellulase activity production by the most productive strain B. amyloliquefaciens B31C was optimized in liquid culture varying the carbon source. Comparison of growth curves of B. amyloliquefaciens B31C at temperatures from 28 to 47 °C indicated its thermotolerant nature. Moreover, analysis of time courses of cellulase activity production in this thermal range showed that increase of temperature from 28 to 37 °C causes an increase of cellulase activity levels. Investigating the enzymes responsible for cellulase activity produced by B. amyloliquefaciens B31C by proteomic analyses, an endoglucanase was identified. It was shown that the purified enzyme catalyzes carboxymethylcellulose's hydrolysis following Michaelis–Menten kinetics with a KM of 9.95 mg ml−1 and a vmax of 284 μM min−1. It shows a retention of 90% of its activity for at least 144 h of incubation at 40 °C and exhibits a range of optimum temperatures from 50 to 70 °C.
Second generation bioethanol produced from lignocelluloses represents one of the best alternatives to the fossil fuels. Despite its many advantages, cellulosic bioethanol is not industrially produced at competitive level yet mainly due to the high cost of cellulolytic enzymes (Lynd et al., 2008), and new more efficient and low cost cellulolytic enzymes should be developed.
Filamentous fungi are the major source of cellulases and hemicellulases (Baldrian & Valásková, 2008), but the production costs of these enzymes are very high. Bacteria, which have high growth rate as compared to fungi have good potential to be used in cellulase production. Various bacterial strains have the ability to produce cellulase complexes aerobically as well as anaerobically. However, the application of bacteria to cellulase production is not widely applied, yet.
Isolation of cellulolytic bacteria from soil (Elberson et al., 2000; Heck et al., 2002; An et al., 2005; Sangkharak et al., 2011; Kim et al., 2012), water (Heck et al., 2002), compost (Kang et al., 2007; Eida et al., 2012; Kim et al., 2012), invertebrates (Gupta et al., 2012), flour mill effluents (Kumar et al., 2009), decaying vegetables (Sakthivel et al., 2010) and animal waste slurry (Kim et al., 2012) have been so far reported.
The aim of this study was to isolate a new bacterium as a source of thermostable cellulases in order to identify and characterize its cellulolytic enzyme(s). Identification and characterization of the endoglucanase produced by the most productive selected strain Bacillus amyloliquefaciens B31C, isolated from raw composting materials, was reported.
Materials and methods
Cellulolytic bacteria isolation
Cellulolytic microorganisms were isolated from mature compost obtained from agro-industrial wastes collected in region Campania (Italy) and consisting of pomace with kernel (65%), liquid sewage sludge (22%) from industrial processing of vegetable (potatoes and carrotoes) and borland molasses (13%). Representative samples of 1 kg were taken from the external (right and left side of the pile, about 5–10 cm of depth) and internal central part (at about 40 cm of depth) of biomass. Microbial isolates were obtained in solid media following the method described by Hankin & Anagnostakis (1977) with some modifications. Initial compost suspensions were prepared by the addition of 20 g (w/v) of compost samples to 180 mL of quarter strength Ringer's solution (Oxoid, Ltd, Oxford, UK) in 250 mL Erlenmeyer flasks. After shaking, suitable dilutions were made in the same solution and were used to inoculate solid media of growth composed by 5 g L−1 carboxymethylcellulose (CMC; Sigma-Aldrich Chemie GmbH, Steinheim, Germany), 1 g L−1 (NH4)NO3, 1 g L−1 yeast extract, 50 mL L−1 standard salt solution, 1 mL L−1 trace elements solution, 0.02% Remazol Brilliant Blue R (Moore et al., 1979), 10 g L−1 bacteriological agar, at pH 7.0. After incubation at 28 °C for 7 days, the plates were flooded with a Remazol Brilliant Blue R solution to put better in evidence the presence of clear haloes around the cellulolytic colonies. Single colonies were picked and checked for purity by repetitive streaking on CMC solid medium.
Screening on solid and liquid media
Solid media composition, used for the screening of microbial isolates, was the same described above without Remazol Brilliant Blue R. The plates were incubated at 28 °C for 4 days. Afterwards, the strains were assayed for their ability to degrade CMC by incubation with 0.1% Congo red solution for 30 min followed by washing with 5 M NaCl (Kluepfel, 1988). All the strains with a clear halo around the colonies were chosen as positive. A comparison of the cellulase production was then carried out by agar spot method. After adjusting the turbidity of tested bacterial suspensions by comparison with McFarland Turbidity Standard at the value 0.5 (corresponding to about 1.5 × 108 CFU mL−1), in 25 mL of Ringer solution (Sigma-Aldrich), cells were spotted on agar medium in triplicate. Spots were incubated at 28 °C for 4 days and stained with 0.1% Congo red. Experiments were performed in duplicate.
The liquid medium adopted for analysis of cellulase production levels contained 1% CMC, 0.7% yeast extract, 4 g L−1 KH2PO4, 4 g L−1 Na2HPO4, 0.2 g L−1 MgSO4.7H2O, 0.001 g L−1 CaCl2.2H2O, 0.004 g L−1 FeSO4.7H2O (Abou-Taleb et al., 2009). When indicated, CMC was replaced with an equivalent amount of glucose or cellobiose (AppliChem, Germany).
Phenotypic characterization of microbial isolates
Morphological analysis of colony of each bacterial strain was carried out observing shape (regular/irregular/rhizoid/punctiform/filamentous), edge (entire/undulate), surface (dry/viscid/powdery), elevation (flat/raised) and colour of colony.
The presence of the enzyme catalase was detected evaluating the immediate appearance of effervescence after dissolving a single colony in 3% hydrogen peroxide (Hanker & Rabin, 1975).
The presence of the enzyme cytochrome oxidase was detected with commercial Oxidase strips following manufacturer's instructions (Oxoid Ltd).
Gram-positive and gram-negative microorganisms were distinguished by means of KOH test as described by Halebian et al. (1981).
The cellular morphology was studied with the optic microscope Eclipse E200 (Nikon).
Inoculum preparation and fermentation process
The bacterial strains were pre-inoculated dissolving a single colony in 3 mL of liquid medium having the composition described in the paragraph ‘'Screening on solid and liquid media'’ of section ‘Materials and methods’ and incubated over night at 28 °C. Fermentation was carried out in 250 mL plugged Erlenmeyer flasks, each containing 20 mL of medium and inoculated with volumes of pre-inoculum corresponding to 0.1 OD. Fermentations were incubated at 28 °C on rotary shaker at 225 rpm. When indicated, a higher temperature in the range 28–47 °C was adopted. From time to time, samples of liquid cultures were withdrawn and used for measurement of optical density (OD600 nm) and extracellular cellulase activity as below reported. The results of these analytical determinations reported in the figure and table correspond to mean values of the three replicates with a standard deviation lower than 10%.
CMCase assay on solid medium
A preliminary analysis of levels of cellulase production in liquid medium was performed observing CMC hydrolysis on solid medium. Culture supernatants from different growth times (4–9, 14, 15, 17, 20 and 24 h) were assayed on solid CMC medium (Lynd et al., 2008) by 0.1% Congo red staining after 1 h incubation at 50 °C.
endo-1,4-β-Glucanase activity produced in liquid or submerged culture was assayed by using Azo-CMC (Megazyme, Ireland) as substrate, following supplier's instructions.
Intracellular protein extraction
Intracellular crude protein extract was obtained by using a French press (Constant System, UK). Pellets obtained after 7 and 15 h of incubation were resuspended in Na phosphate 50 mM pH6.5, before applying a pressure of around 2.5 kbar.
16S rRNA gene partial sequence
Total genomic DNA of selected strains was extracted and purified using InstaGene™ Matrix (Bio-Rad Laboratories, Hercules, CA) according to the supplier's recommendations. Two synthetic oligonucleotide primers at the 5′ and 3′ end of the 16S rDNA gene, described by Weisburg et al. (1991), fD1 (5′-AGAGTTTGATCCTGGCTCAG-3′) and rD1 (5′-AAGGAGGTGATCCAGCC-3′; Escherichia coli positions 8–17 and 1540–1524, respectively), were used to amplify the 16S rRNA gene for all the bacteria, using previously reported conditions (Blaiotta et al., 2002). The PCR amplification fragment was sequenced by Primm srl (Milan, Italy). The sequences were analyzed by MacDNasis Pro v3.0.7 (Hitachi Software Engineering Europe S. A., Olivet Cedex, France) and compared to the GenBank nucleotide data library using the Blast software at the National Centre of Biotechnology Information website (http://www.ncbi.nlm.nih.gov), in order to determine their closest phylogenetic relatives.
The partial 16S rDNA gene sequences of the isolates 1, B7B, 6 and B31C have been submitted to EMBL, and the accession numbers are HE590856, HE590857, HE590858 and HE585988, respectively.
Strain deposition in culture collection
The characterized strain B31C used in this study was deposited in the publicly accessible culture collection DSMZ, and it was assigned to the collection number DSM 25043 Bacillus sp. B31C.
Determination of protein concentration
Protein concentration of crude enzyme preparation was determined by Bradford method using Biorad reactive (München, Germany) following the procedure suggested by the supplier. Bovine serum albumin was used to set up the standard curve.
Proteins secreted by Bacillus sp. B31C were precipitated from the cultures by the addition of ammonium sulphate up to 80% saturation, after removing cells by centrifugation. Precipitated proteins were brought in 20 mM Tris–HCl pH 7, by dialysis through ultrafiltration devices with a cut-off of 10 kDa (Millipore S.p.A., Vimodrone, Italy).
Semi-denaturing gel electrophoresis was carried out loading non-denatured and not-reduced samples on a SDS polyacrylamide gel (Laemmli, 1970). Proteins showing cellulolytic activity were visualized following a modified version of the method reported by Beguin (1983). After electrophoresis, the gel was soaked in 20 mM Tris–HCl pH 7 and gently shaken to remove SDS and allow renaturation of the proteins in the gel. The gel was then laid on the top of a thin sheet of 1.5% agar containing 1% CMC. After 1 h incubation at 40 °C, zones of CMC hydrolysis were revealed by staining the agar replica with 0.1% congo red.
Protein identification by mass spectrometry was performed on slices of interest from the non-denaturing PAGE as previously described (Lettera et al., 2010).
Proteins secreted by Bacillus sp. B31C precipitated with 80% ammonium sulphate and brought in 20 mM Tris–HCl pH 7 as above described, were loaded on HiTrap Phenyl FF high sub (GE Healthcare, Uppsala, Sweden) equilibrated in buffer A (0.02 M Tris–HCl, 1.2 M (NH4)2SO4, pH 7.5), and the proteins were eluted isocratically with buffer B (0.02 M Tris–HCl pH 7.5). Fractions containing activity were combined and concentrated on an Amicon PM-10 membrane and analyzed by SDS-PAGE.
Optimum temperature and temperature resistance
To determine the optimum temperature of the purified enzyme, the incubation (10 min) with the substrate Azo-CMC dissolved in 100 mM sodium acetate buffer pH 4.8 was performed at different temperatures. The thermo-resistance of CelB31C was studied by incubating the purified enzyme in 100 mM sodium acetate buffer pH 4.8, at 30–70 °C. The samples withdrawn were assayed for residual Azo-CMCase activity.
The results of the experiments reported in the text and figures correspond to mean values of the three replicates with a standard deviation lower than 10%.
Determination of vmax and KM
For the experiments of enzyme kinetics characterization, cellulase activity was assayed in the total reaction mixture of 1 mL containing 0.5 mL of suitably diluted enzyme and 0.5 mL of 2% (w/v) CMC solution in 50 mM citrate buffer at pH 4.8. This mixture was incubated at 50 °C for 30 min. The release of reducing sugars was determined by the 3,5-dinitrosalicylic acid method (Miller, 1959). One unit of cellulase activity was defined as the amount of enzyme that liberated 1 μmol reducing sugar per minute from substrate.
The values of Michaelis–Menten constants (KM and vmax) of purified CelB31C were identified by linear regression plots of Lineweaver and Burk. The enzyme was incubated at 50 °C with the substrates of different concentrations of CMC ranging from 0.5 to 50 mg mL−1 in 50 mM citrate buffer at pH 4.8.
The results of the experiments reported in the text and figures correspond to mean values of the three replicates.
Results and discussion
Screening of cellulolytic microorganisms on solid medium
By screening of 90 microorganisms isolated from mature compost on CMC solid medium (Moore et al., 1979; Kluepfel, 1988), 31 cellulolytic microorganisms showing the presence of a clear halo around the colonies were selected. A finer screening of these isolates was performed in normalized growth conditions (4 days at 28 °C), and 15 cellulolytic bacteria with a halo diameter from 6 to 17 mm were selected for further studies.
Phenotypic characterization of the selected microorganisms
The 15 microorganisms selected on solid medium were characterized from a phenotypic point of view by analysis of colony and cell morphology, gram reaction, and the presence of catalase and oxidase activities. On the basis of these results, the analyzed microorganisms were grouped in five phenotypes (Table 1).
Table 1. Phenotypes of the selected microorganisms; *net diameter measured from the edge of the colony to the outer edge of the halo, after 4 days of growth
1; 7; B23; B35
6; 8; 34; 38
14-5 A; B7B; B9A; B31B2; E4
Irregular, rhizoid flat, dry, yellow
Irregular, flat dry, yellow
Irregular, flat, viscid, yellow
Irregular, flat, transparent
Regular, flat, viscid, light yellow
Rod-shaped endospore-forming bacteria
Rod-shaped endospore-forming bacteria
Rod-shaped endospore-forming bacteria
Rod-shaped endospore-forming bacteria
Rod-shaped no endospore-forming bacteria
Halo size* (mm)
Screening of cellulolytic microorganisms in liquid medium
A further screening of the 15 selected microorganisms was performed by cultivating them in liquid growth medium and assaying culture samples for cellulase production by plate method on CMC. The strains showing a significant halo intensity were chosen for the further analyses by quantitative CMCase assay with the substrate Azo-CMC. In the Table 2, the values of the maximum Azo-CMCase activity measured for each strain are reported. All the analyzed strains exhibited low activity levels in the earlier growth stages (starting from the fifth hour), and achieved a maximum AZO-CMCase level between the 14th and 24th hour, corresponding to the stationary growth phase.
Table 2. Maximum value of Azo-CMCase activity measured for each strain and the corresponding time of production
Maximum value of AZO-CMCase activity (U mL−1)
Molecular identification of selected cellulolytic microorganisms
The cellulolytic strains 1, B7B, 6 and B31C were identified by sequencing of 16S rRNA gene. 16S rRNA gene sequence analysis showed that the closest relative species (showing a 99% identity) for the strains 1, B7B, 6 and B31C are Bacillus licheniformis, Bacillus subtilis subsp. subtilis, Bacillus subtilis subsp. spizizenii and B. amyloliquefaciens, respectively.
Bacillus spp. have been widely reported as cellulolytic microorganisms (Robson & Chambliss, 1984; Fukumori et al., 1985; Kawai et al., 1988; Abou-Taleb et al., 2009). Bacillus subtilis, B. megaterium and B. amyoliquefaciens from soil, Bacillus pumilis from rot biomass, Paenibacillus polymyxa and Brevibacillus sp. from compost are among the species studied for cellulase production (Lynd et al., 2002; Lee et al., 2008; Tamaru et al., 2010).
Among the manuscripts so far reported on characterization of native cellulases from Bacillus spp. (Robson & Chambliss, 1984; Fukumori et al., 1985; Kawai et al., 1988; Singh et al., 2004; Kotchoni et al., 2006; Lee et al., 2008; Liang et al., 2009; Afzal et al., 2010; Annamalai et al., 2011; Zhu et al., 2011; Rawat & Tewari, 2012), only one regards a strain of B. amyoliquefaciens (Lee et al., 2008).
Optimization of culture conditions of B. amyloliquefaciens B31C for cellulase production
Cellulase production by the most productive strain B. amyloliquefaciens B31C was analyzed in liquid cultures varying the carbon source and growth temperature. The effect of carbon source was analyzed by replacing CMC with an equivalent amount of each one of the alternative tested carbon sources, glucose and cellobiose. Both the tested sugars stimulated B. amyloliquefaciens B31C growth, reaching a maximum of c. 4 OD mL−1 vs. the c. 3 OD mL−1 reached in CMC medium. The order of growth levels reached with the different sugars glucose > cellobiose > CMC is in agreement with previous results (Liang et al., 2009) for Brevibacillus sp.
CMC was shown the most effective carbon source for cellulase production by B. amyloliquefaciens B31C, with a maximum value of 0.06 U mL−1, that was 3- and 9- fold higher than maximum cellulase activity production in the presence of cellobiose and glucose, respectively. Similar results have been reported for Bacillus sp. (Paul & Varma, 1993; Abou-Taleb et al., 2009). In B. amyloliquefaciens B31C, glucose causes a decrease of cellulase production suggesting its role of catabolite repressor. Cellobiose and glucose substrates have been shown to be inducers of cellulase production for some cellulolytic microbes (Robson & Chambliss, 1984; Paul & Varma, 1990), whilst glucose has been also reported as a catabolite repressor (Abou-Taleb et al., 2009).
Effects of growth temperature on cellulase activity production of B. amyloliquefaciens B31C were also analyzed, culturing the microorganism at 28, 37 and 47 °C. Both the growth and the activity levels were enhanced by increasing temperature from 28 °C (Fig. 1). Growth rate and activity levels for the different temperatures follow the order 37 °C > 47 °C > 28 °C. Ray et al. (2007) reported that the minimum cellulase yield by B. subtilis and Bacillus circulans was observed when fermentation was carried out at 45 °C, while the maximum level was obtained at 40 °C. Immanuel et al. (2006) also reported a maximum endoglucanase activity in Cellulomonas, Bacillus and Micrococcus sp. at 40 °C.
Comparison of growth curves of B. amyloliquefaciens B31C at temperatures from 28 to 47 °C indicated its thermotolerant nature as expected as a consequence of its adaptation to adverse environmental conditions. Intracellular extracts from bacteria harvested after 7 and 15 h at 37 °C were also assayed for AZO-CMCase activity but no activity was detected.
Identification of the cellulolytic enzymes produced by the selected B. amyloliquefaciens B31C
Proteins responsible for cellulase activity of the most productive strain B. amyloliquefaciens B31C were tentatively identified after a fractionation on a semi-denaturing SDS-PAGE where samples from the supernatant of the cell cultures after ammonium sulfate precipitation were loaded without any denaturing treatment. The resulting gel was laid over another gel containing CMC as substrate for cellulase activity detection (Fig. 2). Five protein bands, in correspondence to the area visualized for cellulase activity were excised from the SDS-PAGE and subjected to protein identification after in situ digestion and LC-MS/MS analysis of the peptide mixtures. Raw data were used to search protein databases with the MS/MS ion search program on a MASCOT server against the whole unreviewed set of protein entries (3201) that are present in the UniProtKB/TrEMBL database for B. amyloliquefaciens strain FZB42.
Interestingly, in one of the protein bands (band 4 in Fig. 2), three peptides matched corresponding peptides of the GH5 family endoglucanase BglC from B. amyloliquefaciens FZB42 (UniProt entry: A7Z597), covering 9% of its protein sequence.
Apparently, none of the other proteins identified in the excised bands seems to be responsible for the cellulase activity, although the presence of proteins in B. amyloliquefaciens B31C, other than the identified one, with cellulase activity, cannot be ruled out.
Characterization of the enzyme CelB31C
The enzyme CelB31C purified to apparent homogeneity (Fig. 3) was subjected to characterization. The estimated molecular weight deduced from SDS-PAGE was shown 55 000 Da. These results are close to those of Afzal et al. (2010) and Zhu et al. (2011) reporting Bacillus spp. purified CMCases with a molecular weight of 65 and 64 kDa, respectively and very different from those of Kotchoni et al. (2006), Singh et al. (2004) and Rawat & Tewari (2012) who have reported the molecular mass of purified CMCases from Bacillus spp. of around 185, 170 and 183 kDa, respectively.
A range of optimum temperatures from 50 to 70 °C was identified for CelB31C (Fig. 4a). An optimum temperature of 60 °C was reported for the other purified CMCases from Bacillus spp. (Singh et al., 2004; Afzal et al., 2010; Zhu et al., 2011; Annamalai et al., 2011; Rawat & Tewari, 2012).
The enzyme showed a higher thermoresistance (Fig. 4b) than the other cellulases so far characterized from Bacillus spp. (Singh et al., 2004; Afzal et al., 2010; Zhu et al., 2011; Annamalai et al., 2011; Rawat & Tewari, 2012) and from B. amyloliquefaciens DL-3 (Lee et al., 2008).
CelB31C follows a Michaelis–Menten kinetic towards CMC: the KM for this substrate is 9.95 ± 1.4 mg mL−1 similar to those reported by Rawat & Tewari (2012) and Afzal et al. (2010), and the vmax 710.7 ± 3.5 mg mL−1 min−1 similar to that reported by Rawat & Tewari (2012) and 10-fold higher than that of the cellulase described by Afzal et al. (2010).
The strain B. amyloliquefaciens B31C isolated from compost produces extracellular proteins exhibiting higher cellulase activity levels than the other analyzed strains. An endoglucanase matching with the endoglucanase BglC from B. amyloliquefaciens FZB42 (UniProt entry: A7Z597) was identified in B. amyloliquefaciens B31C. The cellulase purified from B31C, CelB31C, shows a significant thermo-resistance and a range of optimum temperatures from 50 to 70 °C. Retention of 90% of activity for at least 144 h of incubation of the purified enzyme CelB31C at 40 °C and the observation that its activity was not affected by increasing the assay temperature up to 70 °C, highlight its potential for cellulose conversion. CelB31C could be therefore an interesting candidate as biocatalyst in pretreatment stage for second generation bioethanol production.
This work was supported by grant from the Ministero dell'Università e della Ricerca Scientifica Industrial Research Project ‘Integrated agro-industrial chains with high energy efficiency for the development of eco-compatible processes of energy and biochemicals production from renewable sources and for the land valorization (EnerbioChem)’ PON01_01966, funded in the frame of Operative National Programme Research and Competitiveness 2007–2013 D. D. Prot. n. 01/Ric. 18.1.2010.