Abstract
Biofuel is an important alternative source of fuel, as many countries are looking to decrease their dependence on fossil fuels. One of the critical steps in biofuel production is the conversion of lignocelluloses to fermentable sugars, and there is need for cheaper and more efficient enzymatic strategies. Consequently, lignocellulase genes from various organisms have been explored. Termites possess varied sets of efficient micro-scale lignocellulose degrading systems. In this study, bacteria that degraded cellulose and xylan were isolated from termite gastrointestinal tract. The isolate was identified as Cellulomonas sp. by 16S rRNA gene sequencing. The bacterial enzymes cellulase and xylanase showed the highest activity at 50 °C and pH 8.0. The agricultural substrates were hydrolyzed by cellulases and xylanases, and more sugar was released from corn stover (18.903+0.65 mM/L) than from rice straw or cotton stalk. After direct hydrolysis and fermentation of agricultural substrates, ethanol (0.425+0.035 g/L) and lactate (0.772+0.075 g/L) were the major end products. Thus, termite gut bacteria can efficiently hydrolyze hemicellulose and cellulose, and these bacteria also have the potential to convert these fermentable sugars into valuable secondary metabolites.
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Cellulomonas sp. Isolated from Termite Gut for Saccharification and Fermentation of Agricultural Biomass
Iram Batool,a,* Muhammad Gulfraz,a Muhammad Javaid Asad,a Faryal Kabir,a Sobia Khadam,a and Asma Ahmed b
Biofuel is an important alternative source of fuel, as many countries are looking to decrease their dependence on fossil fuels. One of the critical steps in biofuel production is the conversion of lignocelluloses to fermentable sugars, and there is need for cheaper and more efficient enzymatic strategies. Consequently, lignocellulase genes from various organisms have been explored. Termites possess varied sets of efficient micro-scale lignocellulose degrading systems. In this study, bacteria that degraded cellulose and xylan were isolated from termite gastrointestinal tract. The isolate was identified as Cellulomonas sp. by 16S rRNA gene sequencing. The bacterial enzymes cellulase and xylanase showed the highest activity at 50 °C and pH 8.0. The agricultural substrates were hydrolyzed by cellulases and xylanases, and more sugar was released from corn stover (18.903+0.65 mM/L) than from rice straw or cotton stalk. After direct hydrolysis and fermentation of agricultural substrates, ethanol (0.425+0.035 g/L) and lactate (0.772+0.075 g/L) were the major end products. Thus, termite gut bacteria can efficiently hydrolyze hemicellulose and cellulose, and these bacteria also have the potential to convert these fermentable sugars into valuable secondary metabolites.
Keywords: Cellulase; Xylanase; Termite; Saccharification; Cellulomonas
Contact information: a: Department of Biochemistry, University Institute of Biochemistry & Biotechnology, PMAS Arid Agriculture University Rawalpindi-46000, Pakistan; b: Institute of Molecular Biology and Biotechnology, University of Lahore, Lahore-54000, Pakistan; *Corresponding author: iram06ali@gmail.com
INTRODUCTION
In the last few decades, there has been a global effort to reduce the reliance on non-renewable resources. Lignocellulosic biomass is a source of energy that is renewable and available in large quantities, but the process of bioethanol production from cellulosic biomass is more complicated than from sugars. Technologies for the cost-effective conversion of lignocellulosic material into biofuel are in development (Ohgren et al. 2007; Hendriks and Zeeman 2009).
There is a need for low-cost raw materials, effective enzymes, and pretreatment methods to decrease the expenditure for bioethanol production (Sanchez and Cardona 2008). Cellulosic biomass is a low-cost, renewable, and abundantly available material throughout the world. These materials include wood chips, residues of crops, grasses, etc. (Binod et al. 2010). In terms of quantity, sugarcane bagasse, rice straw, corn stover, and wheat straw are the most accessible agricultural wastes (Kim and Dale 2004).
During hydrolysis, monomeric sugars are generated via depolymerization of hemicelluloses and cellulose (Sarkar et al. 2012). The production of the cellulase enzyme accounts for about 40% of the entire cost of bioethanol synthesis (Gray et al. 2006). To decrease the cost of cellulase, numerous efforts have been made to optimize the hydrolysis conditions (Sarkar et al. 2012). The hydrolysis of lignocellulose depends on the synergy of the enzymatic system, including -glucosidase, -1,4-exoglucanase, -1,4-endoglucanase, (Alvira et al. 2010), and -1,4-endoxylanase. This enzyme cocktail is needed to establish a cost-effective technology, in addition to the lower price of biomass (Arantes and Saddler 2011).
Currently, there is no enzyme system that can be efficiently employed on such a vast scale. However, some organisms utilize wood as food, and those systems could be explored and applied to industry (Sanderson 2011). Termites damage billions of dollars of wood each year. Molecular phylogenetic analysis has revealed that termites harbor more than 200 species of symbiotic microorganisms, which produce enzymes that degrade cellulose and hemicelluloses (Brune 2007; Matsui et al. 2009). A study by Warnecke et al. (2007) revealed the occurrence of a huge and varied set of bacterial genes that encode hydrolytic enzymes for degradation of xylan and cellulose. Termites consume 50 to 100% of the deceased biomass in humid ecosystems, and they degrade about 65 to 87% of hemicelluloses and 74 to 99% of cellulose in cellulosic biomass (Ohkuma 2003). The gut of wood eating termite is a bioreactor where a number of microbes utilize cellulose and hemicellulose content of lignified plant materials and convert them to fermentable products. Without these microorganisms, termites are unable to hydrolyze cellulose, which is their main food (Matsui et al. 2009)
In this study, cellulomonas sp. was isolated from termite gut. The isolate was screened for cellulolytic and xylanolytic activity and identified by 16S rRNA sequencing. The crude enzyme activity was checked at different temperature and pH. The agricultural substrates were hydrolyzed with the enzymes produced by the isolate. The substrates were directly hydrolyzed and fermented with the isolate to find the end products.
EXPERIMENTAL
Materials
Termites (Microtermes obesi) were collected from putrefying trees of Acacia nilotica. Corn stover, cotton stalk, and rice straw were obtained from the National Agricultural Research Center in Islamabad, Pakistan. The agricultural substrates were ground and sieved through 20- and 40-mesh sized sieves (0.420 mm and 0.841 mm, respectively) to produce equal size particles.
Isolation and screening of bacteria
Termites were sterilized with 70% ethanol and under UV light for 5 to 10 min. The bodies of termites were ground and serially diluted with Milli Q water. The dilute sample was spread over nutrient agar media with 1% carboxymethyl cellulose (CMC) and 1% beechwood xylan provided by M. S. Traders (Sigma, Lahore, Pakistan) (Dheeran et al. 2012; Pourramezan et al. 2012). The plates were incubated at 30 C for 24 h.
The purified bacterial colonies were screened by the Congo red dye method using 0.2% CMC or 0.2% xylan separately (Dheeran et al. 2012). The bacteria were incubated at 30 C for 48 h. Clear zones around the bacterial colonies established their ability to degrade cellulose and xylan (Liang et al. 2014).
16S rRNA gene sequencing
For bacterial identification, PCR amplification (PCR Super Mix (Invitrogen™) ThermoFisher Scientific, Waltham, MA, USA) was directly performed by using bacterial colonies (Matteotti et al. 2011). Full length (1.5 kb) 16S rRNA fragment was amplified. The universal 16S rRNA gene primers 27F(5’-AGAGTTTGATCCTGGCTCA-3’) and 1492R(5’-ACGGCTACCTTGTTACGACTT-3’) were used. PCR products were sequenced at the Keck Center for Comparative and Functional Genomics, University of Illinois at Urbana-Champaign, IL, USA, and the sequence was submitted to NCBI under accession number KR902590. The BLASTN program of GeneBank was used to analyze gene sequences.
Enzyme production and activity assays
Two enzyme production media containing nutrient broth with 1% CMC and 1% xylan, pH 6.8 to 7.2 (Dheeran et al. 2012; Bashir et al. 2013) were prepared. The media were inoculated with termite gut bacteria 31 (TGB31) and incubated at mild rotation for 48 h at 30 C.
The enzyme activities of CMCase and xylanase of the TGB31 were studied by using CMC and xylan as substrates, respectively. The effect of a various range of temperatures, 30, 40, 50, and 60 C, and also a pH at 5.0, 6.0, 7.0, 8.0, and 9.0 was assessed using crude enzymes. CMC (1%) and xylan (1%) were prepared in diverse buffers for different range of pH according to Rastogi et al.(2009). The reaction time for CMC was 60 min because it was slower to hydrolyze, and 30 min for xylan. Buffers with substrates only and no enzyme were used as controls. The p-hydroxybenzoic acid hydrazide (PAHBAH) method was used to determine the sugar content released during the reaction. Sodium citrate (100 mM) and 0.6 M NaOH was made and stored on ice. To determine sugar content 10 mg of p- hydroxyl benzahydride was added to 10 mL of the above solution. Then 150 µL of the working solution and 50 µL of the samples were mixed in 96-well microplates. The mixtures were boiled for 10 min and brought to room temperature. Absorbance was measured at 410 nm (Moretti and Thorson 2008). One unit (U) of enzyme activity was defined as the amount of enzyme that released 1 µmol of reducing sugars per min during the reaction.
Saccharification of corn stover, cotton stalk, and rice straw
First the contents of cellulose, hemicelluloses (Agblevor et al. 2003), and lignin (Anwar et al.2012) were determined for corn stover, cotton stalk, and rice straw.
Then corn stover, cotton stalk, and rice straw were taken 5% by dry weight, which means 5 g in 100 mL of distilled water (w/v). The ratio of crude enzymes of TGB31 (CMCase and xylanase) to substrates was 1:1, means 100 mL of crude enzymes were added. With mild rotation, the reaction mixture was placed at 50 C for 24 h. The combined effect of enzymes was also studied. Agricultural substrates treated with distilled water were used as controls.
End product analysis
The agricultural substrates were directly treated with bacterial isolates for saccharification and fermentation without any chemical treatment. Corn stover, cotton stalk, and rice straw were used at 5% dry weight (w/v) and supplemented (with, in g/L of H2O: KH2PO4 1.5, MgSO4 0.3, NaCl 0.01, CaCl2 0.1, FeSO4 7, H2O 0.005, NH4Cl 0.3, and yeast extract 0.05) (Rastogi et al. 2009). The agricultural substrates were inoculated with 1% of cultured isolates (1 mL of culture up to 100 mL of 5% substrate). The reaction mixture was incubated at 30 C for 5 days at mild rotation and microaerophilic conditions. The cell viability was determined by protein estimation using the Bradford method.
The fermentative medium from corn stover and rice straw was centrifuged at 14,000 rpm for 20 min and 4 C to remove the remaining substrates and dead bacterial cells. The supernatant was filtered through 0.22 µm membranes, and the filtrate was stored at -20 C for high-performance liquid chromatography analysis (Protea, model: RID-10A, Shimadzu, Bucharest, Romania). The filtrate was later injected into HPX 87 H columns with a refractive index detector. The mobile phase was 5 mM sulfuric acid with a flow rate of 0.4 mL/min at 25 C. Acetate, ethanol, formate, and lactate were tested as end products to find the efficiency of the isolates for secondary metabolites production.
Statistical analysis
The outcomes for enzymatic pretreatment and end products were analyzed by analysis of variance (ANOVA) using MSTAT-C software (Michigan State University, East Lansing, USA). GraphPad Prism 5.0 software (GraphPad Software, Inc., San Diego, USA) was used to determine the standard deviation.
RESULTS AND DISCUSSION
Isolation and Screening of Bacteria
In this study, the termite gut was explored to identify bacteria producing enzymes that degrade cellulose and hemicelluloses and to determine the role that these bacteria play in this small ecological niche. Termites harbor microbes that produce cellulases and hemicellulases, which hydrolyze lignocellulosic material (Scharf and Tartar 2008; Zhang et al. 2009). Approximately 53 colonies of bacteria were isolated from the termite gut. Only one bacterial isolate (TGB31) was considered for further study based on the screening method. The bacterial isolate degraded both CMC and xylan (Fig. 1), producing hydrolysis zones with diameters of 2 mm for CMC and 3 mm for xylan.
Fig. 1. Congo red screening method to show zone of clearance due to enzymatic hydrolysis produced by bacterial isolate TGB31 on (a) CMC media and (b) xylan media
16S rRNA gene sequencing
The bacterial isolate was identified by PCR amplification of the 16S rRNA gene. The BLASTN program was used to compare the sequence with GenBank data. The TGB31 isolate (GenBank accession no. KR902590) belongs to phyla Actinobacteria, and the genus was Cellulomonas. Based on the similarity analysis, isolate TGB31 is closely related to Cellulomonas denverensis.Fall et al. (2007) and Wenzel et al. (2002) also isolated and characterized Cellulomonas bacteria from the termite gut.
Enzyme activity assays
Figure 2 illustrates that TGB31 showed maximum endoglucanase activity at 50 C and pH 8 (Fig. 3).
Fig. 2. Optimization of temperature for CMCase (cellulase) activity U/mL of TGB31
Fig. 3. Optimization of pH for CMCase (cellulase) activity U/mL of TGB31
Immanuel et al. (2006) observed that Micrococcus, Bacillus, and Cellulomonas species obtain maximum cellulase activity at neutral pH and 40 °C. Generally, cellulases isolated from microbes from mesophilic environments have an optimum pH of 4.0 to 8.0 and optimum temperature of 40 to 50 °C (Dutta et al. 2008).
Xylanase showed highest activity at 50 C (Fig. 4). Lisov et al. (2017) found that the temperature optima for xylanases from Cellulomonas flavigena are 40 °C by CFXyl1 and 50 °C for CFXyl4, CFXyl3, and CFXyl2. Xylanases are stable below 60 °C and degrade rapidly at 65 to 70 °C (Amaya et al. 2010). Figure 5 shows that the best pH for xylanase was 8.0. The optimal pH of xylanase from C. flavigena was determined by Amaya et al. (2010) to be 6.5 and 5.7 in C. fimi by Chen et al. (2012). Xylanases show the highest stability at pH 8 to 10 (Lisov et al. 2017).
Fig. 4. Optimization of temperature for xylanase activity U/mL of TGB31
Fig. 5. Optimization of pH for xylanase activity U/mL of TGB31
Saccharification of corn stover, cotton stalk, and rice straw
Table 1 shows the content of cellulose hemicelluloses and lignin content in the agricultural substrates. Figure 6 demonstrates that both of the enzymes (xylanase and CMCase) hydrolyzed the agricultural substrates with diverse efficiency. Corn stover was a potential substrate for these enzymes. Saha and Cotta (2006) reported that the cellulose and hemicelluloses contents in corn stover were 42.6 and 21.3%, respectively. The contents of cellulose and hemicelluloses are high in corn stover as compared to rice straw and cotton stalk. Therefore the sugar contents produced by corn stover are higher than other substrates. The rice straw has contents of 32 and 19%, respectively, for cellulose and hemicelluloses (Karimi et al. 2006). The degradation of rice straw is very slow in soil, and also high mineral content is observed in rice straw (Vlasenko et al. 1997). It is possible that minerals interfere with enzyme activity. The least amount of sugar content was released from cotton stalk. The lignin content in cotton stalk is high enough, about 30%, and also the holocellulose (cellulose and hemicellulose) content is 41.8% (Silverstein et al. 2007).
Table 1. Percentage Composition of Cellulose Hemicelluloses and Lignin in Agricultural Substrates
Fig. 6. Sugar concentration (mM/L) using CMCase (cellulase), xylanase, mix (CMCase, xylanase) to hydrolyze corn stover (C.S), cotton stalk (Co.S), and rice straw (R.S)
Xylanases released more sugar content from all substrates than the CMCases because the hemicellulose is easier to hydrolyze than cellulose (Cardona et al. 2009). The analysis of variance tested for both of the enzymes showed that the difference in the sugar yields among the agricultural substrates were highly significant (p=0.000). When the mixture of both enzymes was used, the released sugar fell between the values released by both enzymes separately. Sugar content released from corn stover was 18.903+0.6506 mM/L when treated with crude xylanase as compared to sugar content released by treatment with CMCase (14.442+0.724 mM/L). Lisov et al. (2017) hydrolyzed rye, wheat, and oat with the xylanases isolated from Cellulomonas flavigena and obtained the highest yield from rye (approximately 1.3mM/L). Bacillus sp. andListeria sp. isolated from leaf litter hydrolyzed pure cellulose, and the sugar concentration obtained was 0.0721 and 0.0772 mM/L, respectively (Gunathilake et al. 2013).
End product analysis
It was reported by Dermoun et al. (1988) that Cellulomonas uda ferments the hydrolyzed sugars into acetate, formate, ethanol, and lactic acids as primary end products. The genes for alcohol dehydrogenase (ADH) in C. fimi were observed to be more in number than the genes of ADHS of C. thermocellum and Z. mobilis, combined (Christopherson et al. 2013). So, it was designed to study different end products, specifically the ethanol. The end products from cotton stalk were not analyzed because there was no growth of bacterial isolates. Ethanol and lactate were the major end products of the experiment (Fig. 7). Poulsen et al. (2016) observed that formate was the dominant end product and lactate was the least product fermented by C. uda. Most Cellulomonas sp. does not encode pyruvate decarboxylase, which is necessary for homoethanol production (Christopherson et al. 2013). End products produced by isolate TGB 31 have shown statistically significant (p=0.000) results obtained from both of the agricultural substrates. Figure 7 also illustrates that more ethanol (0.425+0.035 g/L) was produced when corn stover was used as the substrate. This result confirmed that more sugar was released from corn stover. It was determined by Millati et al. 2005 that S. cerevisiae produced 0.42 g ethanol/g of glucose but cannot utilized xylose for fermentation. Metabolically engineered yeast, which was developed for xylose utilization, produced 0.24 and 0.28 g ethanol/g xylose (Fujitomi et al. 2012; Kato et al. 2013).
Fig. 7. Concentration of end products (g/L) of TGB31using corn stover (C.S) and rice straw (R.S)
Over all, the production of ethanol and other secondary metabolites is low. The production of sugars and end products can be increased by optimizing the different conditions to achieve the maximum potential of the bacterial isolate.
CONCLUSIONS
- Termite gut bacteria were able to degrade hemicellulose and cellulose.
- These enzymes have the potential to hydrolyze pure substrates and degrade agricultural substrates without any chemical pretreatment.
- Cellulomonas sp. isolated from termite gut directly hydrolyzed agricultural substrates into valuable secondary metabolites.
ACKNOWLEDGMENTS
The authors are grateful for the support of the HEC (Higher Education Commission) Pakistan, Grant No. 112-25295-2BM1-280.
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Article submitted: September 14, 2017; Peer review completed: November 12, 2017; Revised version received and accepted: November 24, 2017; Published: December 1, 2017.
DOI: 10.15376/biores.13.1.752-763