An investigation for exocellulase and endocellulase activities of thermotolerant Gram-positive bacteria from Al-Baha region, Saudi Arabia

 Corresponding author: Essam Hussein Abdel-Shakour Copyright © 2025 Author(s) retain the copyright of this article. This article is published under the terms of the Creative Commons Attribution License 4.0. An investigation for exocellulase and endocellulase activities of thermotolerant Gram-positive bacteria from Al-Baha region, Saudi Arabia Ahmed Mohammed Alghamdi 1, Yasser Abdullah Alghamdi 1 and Essam Hussein Abdel-Shakour 2, * 1 Department of Biology, College of Science and Arts, Albaha University, Baljurashi, Saudi Arabia. 2 Department of Botany and Microbiology, Faculty of Science, Al-Azhar University, Cairo, Egypt. World Journal of Advanced Research and Reviews, 2025, 28(02), 1155-1168 Publication history: Received on 04 October 2025; revised on 10 November 2025; accepted on 13 November 2025 Article DOI: https://doi.org/10.30574/wjarr.2025.28.2.3828 Abstract This study was designed to explore some bacterial genera with the ability to decompose cellulosic materials from different places in Al-Baha region. All the collected and prepared compost samples in Nawan, Almakhwah and Bani Zabyan, Al-Baha were used for bacteria isolation on cellulose powder and CMC substrates as sole sources of carbon. Six isolates were obtained in both types of media. All the twelve isolates were described as thermotolerant as the incubation temperature was 45 °C. Basic morphological and physiological description for all isolates showed a high degree of similarity with bacteria of the genera Bacillus, Amphibacillus, Paenibacillus in case of all the spore forming isolates, and Lactobacillus in case of the non-spore forming isolate A2. The accuracy of the description of the isolates was confirmed by PCR detection of the cellulase genes ß-glucosidase and 6-phospho-ß-glucosidase in bacteria like the isolates under study. This assay was performed in-silico through an online program using primers designed specifically for Bacilli and Lactobacilli. Growth curves for all isolates showed maximum growth activity at 24 to 30 hours from the start of incubation except for isolate B1, which reached the maximum growth at 24 hours. Application of the isolates for plant waste composting under laboratory conditions in consortia was conducted. Finally, a simple experiment for planting fenugreek seeds was conducted to test quality of the compost and to ensure that it is free of any toxic substances for plants. The results showed the quality of the compost produced in both natural and laboratory conditions. Keywords: Exocellulase; Endocellulase; Gram-positive; Bacteria; Compost 1. Introduction Microorganisms exist in nature in many environments. Soil is one of the most natural environments rich in microorganisms. There are many different types of microorganisms in the soil. Bacteria are the most dominant microbes in different types of soil [1]. Soil varies with its types in its content of bacteria in terms of abundance and species present, depending on the environmental conditions. One of the most famous natural environments associated with soil and its content of organic matter, especially plants, is the compost environment. Compost is a form of soil that is very rich in its content of microorganisms, especially bacteria. There are many taxonomically different bacterial groups in the soil of different types. Each bacteria group has a specific natural role according to the soil in which it lives and is abundant. Bacterial presence flourishes in the soil whenever the appropriate environmental conditions are available, as well as the availability of other organisms and multiple organic materials, especially plant ones. Based on this, we find that, for example, an environment such as compost, which is basically the product of the decomposition of plant organic matter, is one of the richest natural environments in terms of different bacterial species, both Gram positive and Gram negative. World Journal of Advanced Research and Reviews, 2025, 28(02), 1155-1168 1156 One of the most important and most famous bacterial groups in soil, especially in compost, is the group of Gram-positive bacteria with its different divisions. This difference comes from different environmental conditions and accordingly the roles played by one bacteria group differ from the other. Here, the importance of this diversity and the enzymatic activities of bacteria associated with it, according to the natural environment, its content of other living organisms, and the availability of specific organic materials. One of the most prevalent organic materials in the soil is the remains of plant origin rich in its content of cellulose. Bacteria, especially Gram-positive bacteria, have multiple roles in the decomposition of these plant residues. Here, the enzymatic activities of Gram-positive bacteria vary according to the various natural conditions such as temperature, humidity, and air availability. Each genus of Gram-positive bacteria has specific enzymatic activities in the decomposition of natural substances, especially cellulose. We find here that the difference of groups and genera is of great importance, as the different genera of Gram-positive bacteria play a part in the complete decomposition in nature of polymers such as cellulose. Gram-positive bacteria have important roles in the process of decomposing cellulose in nature through their production of various enzymes responsible for this decomposition. The most famous of which are Bacilli and Lactobacilli groups. The genus Bacillus is distinguished from other bacteria mainly by its ability to form resting endospores [2]. Cellulose polymer consists of crystalline and non-crystalline parts [3]. Cellulase enzymes are distinguished from other glycoside hydrolases by their ability to hydrolyze the beta 1,4 linkages to liberate glucose units [4]. ß-glucosidase and 6-phospho ß-glucosidase are important cellulase enzymes required to complete the process of hydrolysis to glucose [5]. Completed hydrolysis of cellulose is facilitated by the action of three major types of enzymes namely endocellulases, exocellulases, and β-glucosidases according to the location action in cellulose. Endocellulases act internally on cellulose chain, exocellulases act on chain ends to produce glucose or cellobiose, and ultimately β-glucosidases act on cellobiose units to produce glucose [6]. Several unique cellulase enzymes with hydrolytic activity of crystalline and amorphous cellulose moieties have been discovered in species of the genus Bacillus and Paenibacillus. The endocellulase enzyme works on the amorphous parts, and therefore it can be detected by the growth of bacteria on a medium such as CMC agar, while a medium such as cellulose powder agar is used to detect the exocellulase enzyme, which works on the crystalline parts [7]. Many of the basics of the process of consumption of cellulose by microbes and the production of cellulase and its various applications are detailed in many references such as [4, 3, 8, 6]. This study was designed to explore some bacterial genera with the ability to decompose cellulosic materials from different places in Al-Baha region. The study aimed to isolate these bacteria from soil and compost samples on media containing cellulose powder or carboxymethylcellulose (CMC) as sole carbon and energy sources. A qualitative screening to these isolates for detection of exocellulase and endocellulase activities on agar media was conducted. The obtained isolates were subjected to morphological and physiological characterization. The detection of specific cellulase genes in the closest bacteria to our isolates by in silico PCR also was considered. The study ended with an investigation for potential of these cellulolytic bacterial isolates in degradation of some natural cellulose substrates and subsequent detecting the characteristics of the resulting compost and its suitability for plant cultivation. 2. Materials and methods 2.1. Sample collection locations The areas were determined according to altitude, and compost samples were collected from a designated place in Nawan, Almakhwah (location coordinates: 19°33'27.5"N 41°07'29.8"E) at 24 meters above sea level. Compost samples were also collected from Bani Zabyan between Baljurashi and Al-Baha (location coordinates: 19°57'55.5"N 41°28'56.1"E) from a designated place in a home garden at 2,200 meters above sea level. In this last place, an experiment was designed to manage some plant organic waste and follow it up for a month (June-July 2022) and then use it for isolation as well. The experiment was designed on four treatments as follows: 1Mixing the residues with the addition of agricultural soil 2Mixing the residues without adding any soil 3Mixing the residues with the addition of previously prepared compost in Bani Zabyan 4Mixing the residues with the addition of previously prepared compost in Nawan, Almakhwah . All these samples were used for bacteria isolation in the laboratory as follows. World Journal of Advanced Research and Reviews, 2025, 28(02), 1155-1168 1157 2.2. Culture medium and method of isolation The following medium (PH 6.5-7.5) was used [9], consisting of (g/L): Cellulose substrate (Cellulose powder, SDFCL Company) or carboxymethylcellulose, CMC, LOBA CHEMIE Company), 20; KCl, 0.5; MgSO4.7H2O, 0.5; K2HPO4 (Anhydrous), 1.0; NaNO3, 2.0; Potable Water, 1000 ml, in liquid or solid by adding 2% agar. The samples were first stimulated (1 g in 25 ml) by culturing them in a cellulose liquid medium and incubated aerobically within a rotary incubator (120 rpm) for 1 day at 40 ° C, then cultured on cellulose solid medium and continued incubation for 3-5 days at temperature of 45° C. The purity of the isolates was confirmed on the same agar medium and preserved on agar slopes. 2.3. Cellulase enzyme detection Qualitative detection of the ability of the isolates to produce the enzyme and hydrolyzing of the cellulose in the medium was carried out by the method of [10] by adding the iodine reagent to the plates. The appearance of the decomposition areas around the bacterial growth were recorded as clear halos. 2.4. Description of the isolates The culture characterization tests were carried out on the nutrient agar medium and incubated aerobically. Gram stain was conducted for the isolates. The motility and growth of the isolates at different temperatures were tested, according to the instructions mentioned in [11]. 2.5. PCR detection of some components of the cellulase enzyme genes in bacteria like the isolates under study This inspect was performed using an online program (www.in-silico.com) for the complete genomes of many different bacteria [12] and was determined using primers specially designed for Bacilli and Lactobacilli bacteria according to [5]. These primers are pre-designed to amplify the cellulase genes ß-glucosidase and 6-phospho-ß-glucosidase, two of the cellulase enzymes important to complete the hydrolysis process. The primers were (5’-TTTGCTGAAATGGG-3’), the forward, and (5’-GGATCAATTTGCCANCCCC-3’), the reverse. PCR was performed for all available species of Bacillus (91 species) and Lactobacillus (57 species) in the program. Only the representative species of all genera that gave positive results were selected, and they were checked for their registration in the GenBank, and all their data were recorded. To ensure the accuracy of the program's work, it was also used to detect the well-known 16s rRNA gene using general primers F27 (5′-AGAGTTTGATCCTGGCTCAG-3′) the forward, and R1492 (5′- GGTTACCTTGTTACGACTT-3′) the reverse, that yield around 1500 bp PCR amplicons. 2.6. Temperature tolerance of the isolates A culture of isolates was carried out to test their ability to consume either type of cellulose media in each case, namely CMC or cellulose powder. This was done with solid media and incubation at increasing temperatures included 30, 45, 50, and 55 then followed up for three days and the results were recorded. 2.7. Creation of the growth curve for the isolates The isolates were cultured separately with the same liquid medium on which they were first isolated (CMC and cellulose powder). Incubation was carried out at 45°C and growth was measured at intervals for each isolate (6 hours or multiples thereof) for two days to construct a growth curve for each isolate. Optical density measurements were taken with a spectrophotometer Model 6715 JENWAY at a wavelength of 600 (OD 600). Inoculations were made first with a volume of 2 ml of each isolate in each medium with an initial optical density of 0.002. 2.8. Application of the isolates in plant waste composting under laboratory conditions The ability of the isolates was tested under laboratory conditions to hydrolyze mixture of plant organic residues, cotton linters and filter papers. The mixture was prepared homogeneously, distributed in 1L flasks (100 grams each) and sterilized. The water used was also sterilized to ensure that the mixtures were saturated and did not dry out during the incubation period. The isolates were cultured collectively, where all isolates growing on CMC were injected into one treatment, and all isolates growing on cellulose powder were injected in a second treatment. The organisms were first cultured for activation in liquid medium for 24 hours and then injected with a volume of 2 ml of each isolate in each case. The incubation was carried out at a temperature of 45 °C for 6 days and followed up to ensure the stirring and mixing well. On the sixth day, 10 grams of the soil mixture and the compost used first in the isolation process were added to each treatment and the incubation continued for 24 hours, to help ripen the resulting compost by other thermophilic microbes other than the isolates under study. The resulting compost characteristics were recorded and saved for later use. World Journal of Advanced Research and Reviews, 2025, 28(02), 1155-1168 1158 2.9. Use of the prepared compost in seed planting A planting test was conducted for fenugreek (Trigonella foenum-graecum) seeds (20 seed per treatment) to test the quality of the compost and to ensure that it is free of any toxic substances for plants. This procedure was done by using the compost mixture produced first in treatments 3 and 4 to which compost was added under natural conditions, and then the produced compost under laboratory conditions was added to it. The first treatment used in isolation, to which soil was added, was used here as control without any additions. 3. Results 3.1. Sample collection locations Compost samples were collected from Nawan, Almakhwah at 24 m above sea level. Compost samples were also collected from Bani Zabyan between Baljurashi and Al-Baha at 2,200 m above sea level. In this last place, an experiment was designed to manage some plant organic waste and followed up for a month (June-July 2022) and then used also for isolation. The experiment was designed on four treatments as follows: 1Mixing the residues with the addition of agricultural soil 2Mixing the residues without adding any soil 3Mixing the residues with the addition of previously prepared compost in Bani Zabyan 4Mixing the residues with the addition of previously prepared compost in Nawan, Almakhwah. 3.2. Culturing and isolation All the samples mentioned above were used for bacteria isolation on cellulose powder and CMC substrates as sole sources of carbon in the liquid or agar media at temperature of 45° C. During the isolation process, one bacterial colony was chosen among the very similar in appearance. Six isolates were obtained on the cellulose powder agar medium, and the same number was obtained on the CMC agar medium. All the sources, locations, and isolation media for the isolates are listed in (Table 1). 3.3. Cellulase enzyme detection Qualitative detection of the ability of the isolates to produce exocellulase on cellulose powder agar medium was carried out by adding the iodine reagent to the plates. The appearance of the decomposition areas around the growth of the isolates were recorded as clear halos. The same test was performed concerning the ability of the isolates to produce endocellulase on CMC agar medium by adding the iodine reagent to the plates and recording the results (Figures 1 and 2). Table 1 The sources, locations, and isolation media for the isolates Isolate No. Isolate Source Location Isolation medium Isolation temperature M1 Plant residues mixture with agricultural soil Bani Zabyan AlBaha Cellulose powder 45° C M2 Plant residues mixture without soil Bani Zabyan AlBaha Cellulose powder 45° C M3 Plant residues mixture with compost from Bani Zabyan Bani Zabyan AlBaha Cellulose powder 45° C M4 Plant residues mixture with compost from Nawan Bani Zabyan AlBaha Cellulose powder 45° C A1 Compost Nawan, Almakhwah Cellulose powder 45° C A2 Compost Nawan, Almakhwah Cellulose powder 45° C N1 Plant residues mixture with agricultural soil Bani Zabyan AlBaha CMC 45° C N2 Plant residues mixture without soil Bani Zabyan AlBaha CMC 45° C World Journal of Advanced Research and Reviews, 2025, 28(02), 1155-1168 1159 N3 Plant residues mixture with compost from Bani Zabyan Bani Zabyan AlBaha CMC 45° C N4 Plant residues mixture with compost from Nawan Bani Zabyan AlBaha CMC 45° C B1 Compost Nawan, Almakhwah CMC 45° C B2 Compost Nawan, Almakhwah CMC 45° C Figure 1 Growth of the Isolates which utilize cellulose powder on nutrient agar (left pic for each isolate), and their exo-cellulolytic activity (middle and right pic for each isolate), shown on cellulose powder agar before and after adding iodine solution Figure 2 Growth of the isolates that utilized CMC, on nutrient agar (left pic for each isolate), and their endocellulolytic activity (middle and right pic for each isolate), shown on CMC agar before and after addition of the iodine solution World Journal of Advanced Research and Reviews, 2025, 28(02), 1155-1168 1160 3.4. Description of the isolates The culture characteristics were recorded on the nutrient agar medium (Figures 1 and 2). Gram’s stain was conducted for the isolates (Figure 3), and all were Gram positive, rod shaped, and spore former bacteria except one only, the isolate A2. The motility was tested and recorded positive results for all the isolates except A2 also. Growth of the isolates at different temperatures was also carried out (Table 2). According to these characteristics collectively for all isolates and by matching them with the characteristics of Gram-positive bacteria in [11], it appeared that these isolates may be largely like the following bacterial genera: Bacillus, Amphibacillus, Paenibacillus in case of all the spore forming isolates, and Lactobacillus in case of the non-spore forming isolate A2. Figure 3 Photomicrographs of the isolates under oil immersion lens 1000x Table 2 The morphological and physiological characteristics of the isolates Test M1 M2 M3 M4 A1 A2 N1 N2 N3 N4 B1 B2 Cell shape Rod Rod Rod Rod Rod Rod Rod Rod Rod Rod Rod Rod Endospores production + + + + + - + + + + + + Motility + + + + + - + + + + + + Gram’s stain reaction + + + + + + + + + + + + Aerobic or Facultative anaerobic + + + + + + + + + + + + Growth at 5 oC - - - - - - - - - - - - Growth at 55 oC - - - - - - - - - - - - Growth at 30 oC + + + + + + + + + + + + World Journal of Advanced Research and Reviews, 2025, 28(02), 1155-1168 1161 Growth at 40 oC + + + + + + + + + + + + Growth at 50 oC - - - - - - - - - - - - (+) Positive result (-) Negative result 3.5. PCR detection of some components of the cellulase enzyme genes in bacteria like the isolates under study This assay was performed using an online program (www.in-silico.com) and was determined using primers specially designed for Bacilli and Lactobacilli bacteria. These primers are pre-designed to amplify the cellulase genes ßglucosidase and 6-phospho-ß-glucosidase, two of the cellulase enzymes important to complete the hydrolysis process. PCR was performed for all available species of Bacillus, Amphibacillus, and Paenibacillus (91 species) and Lactobacillus (57 species) in the program. Only the representative species of all genera that gave positive results were selected and listed in (Table 3) with all their detailed information in the GenBank. To ensure the accuracy of the program's work, it was also used to detect the well-known 16s rRNA gene using general primers F27 and R1492 that recorded positive results in (Table 4) and confirmed the results of PCR detection of the cellulase genes. 3.6. Temperature tolerance of the isolates The isolates in each case, CMC or cellulose powder, were tested for their ability to grow at increasing temperatures included 30, 45, 50, and 55 °C. The results were recorded, and all isolates were able to grow in each case in temperatures 30 and 45 °C. No growth was recorded on both types of cellulose media at the rest of the tested temperatures. 3.7. Growth curves for the isolates The isolates were cultured separately with the same liquid medium on which they were first isolated, CMC and cellulose powder. Incubation was carried out at 45°C and growth was measured at intervals for each isolate as mentioned in methods for two days to construct a growth curve for each isolate. Optical density measurements were recorded, and the curves were constructed (Figures 4 and 5). The curves showed that all isolates reach their maximum growth activity at 24 to 30 hours from the start of incubation except for isolate B1, which recorded the maximum growth at 24 hours from the start of incubation. Table 3 The representative Gram-positive bacterial genera and species which gave positive bands (812-839 bp) upon amplification using the cellulase primers No. Gram-positive bacteria Fragment size In base pairs (bp) Product enzyme GenBank Protein accession GenBank Sequence accession Sequence Start position Sequence End position 1 Bacillus subtilis BSn5 815 aryl-phosphobeta-dglucosidase ADV94750 CP002468 2039584 2040398 2 Bacillus pumilus SAFR-032 818 glycoside hydrolase family 1 protein (betaglucosidase - 6phospho-betaglucosidase) ABV64287 CP000813 3608869 3609686 3 Bacillus licheniformis ATCC 14580 818 Glycoside Hydrolase Family 1 protein QCY01555 CP034569 776108 776925 4 Bacillus coagulans 2-6 (Weizmannia coagulans 2-6) 830 6-phospho-betaglucosidase AEH52551 CP002472 534021 534850 5 Bacillus amyloliquefaciens CC178 815 6-phospho-betaglucosidase AGZ58492 CP006845 3736578 3737392 World Journal of Advanced Research and Reviews, 2025, 28(02), 1155-1168 1162 6 Amphibacillus xylanus NBRC 15112 821 6-phospho-betaglucosidase BAM48390 AP012050 2393887 2394707 7 Paenibacillus polymyxa CR1 812 6-phospho-betaglucosidase AIW41298 CP006033 4577300 4578111 8 Lactobacillus plantarum 16 830 6-phospho-betaglucosidase AGO08864 CP006033 2325006 2325835 9 Lactobacillus johnsonii DPC 6026 839 6-phospho-betaglucosidase AEB92527 CP002464 223101 223939 10 Lactobacillus rhamnosus ATCC 8530 818 family 1 glycosyl hydrolase (arylphospho-beta-Dglucosidase BglH) AER63304 CP003094 450553 451370 Table 4 Testing the accuracy of the in-silico program by amplifying a known 16S rRNA gene for the bacteria representing the isolates, and the selected here were Bacillus subtilis BSn5 and Lactobacillus plantarum 16 Gram-positive bacteria PCR Fragment size In base pairs GenBank Sequence accession Sequence Start position Sequence End position Bacillus subtilis BSn5 1511 bp CP002468 2311298 2312808 Lactobacillus plantarum 16 1529 bp CP006033 1884668 1886196 Figure 4 Growth curves for the isolates growing on cellulose powder liquid medium World Journal of Advanced Research and Reviews, 2025, 28(02), 1155-1168 1163 Figure 5 Growth curves for the isolates growing on CMC liquid medium 3.8. Application of the isolates in plant waste composting under laboratory conditions Figure 6 Application of bacterial isolates for growth on a mixture of plant residues (The far left of the picture for each case, followed by the compost form after the end of the incubation). The treatments were (1) Treatment inoculated with all 6 isolates initially isolated on Cellulose powder (2) Treatment inoculated with all 6 isolates initially isolated on CMC Isolate N1 Isolate N2 Isolate N3 Isolate N4 Isolate B1 Isolate B2 0 0.1 0.2 0.3 0.4 0 6 12 24 30 36 48 OD (600 nm) Time (hrs) 0 0.05 0.1 0.15 0.2 0.25 0.3 0 6 12 24 30 36 48 OD (600 nm) Time (hrs) 0 0.1 0.2 0.3 0.4 0 6 12 24 30 36 48 OD (600 nm) Time (hrs) 0 0.05 0.1 0.15 0.2 0.25 0 6 12 24 30 36 48 OD (600 nm) Time (hrs) 0 0.1 0.2 0.3 0.4 0 6 12 24 30 36 48 OD (600 nm) Time (hrs) 0 0.05 0.1 0.15 0.2 0.25 0.3 0 6 1224303648 OD (600 nm) Time (hrs)