Harnessing the potential use of cellulolytic Klebsiella oxytoca (M₂₁WG) and Klebsiella sp. (Z₆WG) isolated from the guts of termites (Isoptera)

Abstract

Background

For many years, denim-heavy quality cotton twill colored with indigo colors and with a well-worn/faded look has held a lot of appeal. Machine damage, drainage system blockage, and other issues come with the conventional usage of pumice stones for “stone-washing” denims. In view of the abovementioned information, a range of works has been done to investigate the economic prospects of bacterial cellulase enzymes for use in industrial processes, including biopolishing in the textile sector. Ethiopia has excellent termite diversity to isolate bacterial gut-associated cellulose enzymes for biostoning applications. The main purpose of this study was, therfore, to decipher how to isolate and characterize cellulase enzymes from termite (Isoptera) gut bacteria with the intention of employing it for biostoning of textiles.

Purpose

To use cellulolytic enzymes of Klebsiella oxytoca (M21WG) and Klebsiella sp. (Z6WG) isolated from termite guts in biostoning of textiles and improving garment quality.

Methods

Cellulase enzyme-producing bacteria were isolated and screened from the guts of worker termites sampled from Meki and Zeway termite mounds in the Central Rift Valley region of Ethiopia. Bacterial screening, biochemical, morphological, and 16S rRNA sequence identification techniques were employed to characterize the bacterial strains. In addition, the production, optimization, and purification of the associated cellulase enzymes were employed, and the potential application of the enzymes for biostoning of a textile was demonstrated.

Result

The isolated M21WG was found to be 99% identical to the Klebsiella oxytoca (MT104573.1) strain, while the isolated Z6WG showed 97.3% identity to the Klebsiella sp. strain (MN629242.1). At an ideal pH of 7, a temperature of 37 °C, a 72-h incubation time, and a substrate concentration of 1.5% carboxymethylcellulose sodium, the maximum activity of the crude cellulase extract from these bacteria was assessed. These bacteria produced cellulase enzymes that were moderately efficient. Consequently, it was determined that the cellulase enzymes were effective for biostoning of denim cloth.

Conclusion

It was determined that Klebsiella oxytoca (M21WG) and Klebsiella sp. (Z6WG) could be used as a doorway to better understand harnessing the use of these cellulase-producing bacteria from termite (Isoptera) guts. In this study, it was also attempted to assess the effectiveness of the two bacterial isolates in biostoning in anticipation of their potential application in the textile realm.

Background

Termites, a type of eusocial insect, have been known to create devastation on lignocellulosic materials such as wood and crops, as well as other man-made buildings (Eggleton, 2010; Nobre and Nunes, 2007). Termites depending on their evolutionary level, both in terms of behavior and anatomy, are divided into higher and lower categories (Ferbiyanto et al., 2015; Kakkar et al., 2015; Sreena et al., 2015; Sakolvaree and Deevong, 2016; Javaheri-Kermani and Asoodeh, 2019; Oberstet al., 2020; Kumar and Upadhyay, 2021). However, their commercial potential has been realized in the domain of microbial biotechnology for the exploration of biocatalysts derived from various symbiotic eukaryotes and prokaryotes, in particular, those that harbor the gut compartments of lower termites (Mattéotti et al., 2012; Ali et al., 2019b; Zhou et al., 2019; Arfah et al., 2019). As a social insect, a termite colony consists of three castes, i.e., reproductive, soldier, and worker castes. In their role of cellulose digestion, the worker termites use two sources of cellulolytic enzyme that include cellulase produced by the termite and the gut symbionts (Ferbiyanto et al., 2015; Egwuatu and Appeh, 2018; Sanchez-García et al., 2020; Ye et al., 2019; Zhao et al., 2021; Bezerra et al., 2021).

The symbiotic digestion of lignocellulose in the higher termite family (Termitidae) is carried out by an exclusively prokaryotic gut microbiota, which is influenced by microenvironmental factors such as pH, available substrates, and O₂ and H₂ gradients. This has prompted research into the functional roles of specific bacterial taxa in lignocellulose and humus digestion. Due to the thermodynamic inadequacy of acetogenesis in termite gut microbial fermentation, CO₂-reducing acetogenesis as a “H₂ sink” reaction is one of the most distinctive and perplexing aspects (Upadhyaya et al., 2012; Muwawaet al., 2016; Mikaelyan et al., 2018; Loh et al., 2021). The associated microorganisms represented by intestinal bacteria have repeatedly been shown to facilitate interactions between trees and wood feeders by supplementing essential nutrients or degrading complex dietary polymers (Sreena et al., 2015; Dar et al., 2021; Ge et al., 2021). Insects can be transiently or permanently infected with gut-associated microbes such as bacterial gut (Panthoea and some Erwinia species, which assisted in plant cell wall cellulose degradation), fungi, protozoa, and viruses, and this association can be useful or harmful (Kakkar et al., 2015; Bozorov et al., 2019).

Many insects’ ability to subsist on wood, leaves, and detritus has sparked interest in using their gut microbial flora as a bioresource for commercial conversion of low-cost cellulosic material into bioproducts (Li et al., 2021; Nelson et al., 2021). Higher termites, with the exception of some species that harbor cellulolytic amoeba, lack cellulolytic flagellates and instead have a prokaryotic microbiota in their hindgut that aids lignocellulose degradation by contributing bacterial celluloses and hemicellulose that work in tandem with endogenous cellulase secreted by the termites’ gut tissue (Ali et al., 2019a; Tokuda, 2021). Termites, as an insect group, have evolved symbiotic systems that efficiently digest lignocellulose meals, making them a good supply of cellulolytic enzyme (Aueret al., 2017; Ali et al., 2019b; Nandy et al., 2021).

Of pertinence is to mention that the expression of genes in caste differentiation and cellulose digestion has garnered tremendous research impetus in the niche of termite genomics and proteomics (Scharf and Tartar, 2008; Husseneder et al., 2012). As the termite gut mimics a fermenter chamber (Ngangi et al., 2019), both endogenous cellulases and the cellulases from symbiotic microbes confer excellent lignocellulose-degrading potency to termites (Duan et al., 2017). Needless to say, cellulases represent a group of complex biocatalysts that catalyze the hydrolysis of cellulose to fermentable sugar. The synergistic action of endo-β-glucanase (EC 3.2.1.4), cellobiohydrolase/exo-glucanase (EC 3.2.1.91/EC 3.2.1.176) (Guar et al., 2015; Reffas et al., 2016; Islam and Roy, 2018; Olowomofe et al., 2019; Regmi et al., 2020; Biswas et al., 2020), and β-glucosidase (E.C. 3.2.1.21) is bracketed together with the conversion of cellulose into glucose monomers (Gbenro et al., 2019; Javaheri-Kermani and Asoodeh, 2019; Liu et al., 2019b; Nazir et al., 2021). Bacteria are now being studied extensively for the production of cellulase due to their rapid growth, expression of multi-enzyme complexes, stability at extremes of temperature and pH, lower feedback inhibition, ability to colonize a wide range of environmental niches, and resistance to a variety of environmental stresses (Khatiwada et al., 2016; Rawway et al., 2018; Islam and Roy, 2019; Prabhakar et al., 2021).

The immense thrust received by cellulases over the years may be easily perceived in the context of their wide gamut of applications in the textile and laundry industry (Krik et al., 2002; Sreena etal., 2015; Sakolvaree and Deevong, 2016; Sankarraj and Nallathambi, 2018; Fasiku et al., 2020; Nursyirwani et al., 2020; Kabir and Koh, 2021), paper and pulp industry (Sondhi et al., 2015; Abu-Gharbia et al., 2018;Wang et al., 2020; Sepehri et al., 2020), wine-making and brewing (Sojitra et al., 2017, Irshad et al., 2017; Zahra et al., 2020), agriculture (Abdel-Azizet al., 2021; Fatmawati et al., 2021; Tomulescu et al., 2021), animal feed industry (Bharti and Sandhu, 2015; Phuong et al., 2015; Tamilanban et al., 2017; Luo et al., 2013; Ali et al., 2019a; Ali et al., 2019b; Tsegaye et al., 2019, Astolfi et al., 2019, Chauhan, 2020) and pharmaceutical domain (Gupta et al., 2013; Bharti and Sandhu, 2015; Obafemi et al., 2019; Barbosa et al., 2020) as well as in waste management (Gupta et al., 2013; Cui et al., 2014; Mahjabeen et al., 2016; Prasad et al., 2019; Ndlovu et al., 2019; Rehman et al., 2019; Karthika et al., 2020).

At this juncture, it is also worth noting that bacterial cellulase-mediated cellulose hydrolysis is associated to a number of beneficial properties, including increased stability, increased specific activity, and facilitated mass transfer (Maki et al., 2009; Islam and Roy, 2018; Prasad et al., 2019).

In the backdrop of the afore-stated, investigating and using the potential cellulase-producing gut bacteria of termites appears to be an intriguing topic of study. The goal of this research was to characterize cellulase-producing bacteria found in the guts of termites from Ethiopia’s Meki and Zeway central Rift Valley, Eastern part of Shawa Zone Oromia Regional State. We attempted to analyze the enzyme’s effectiveness in dye leaching after optimizing bacterial culture settings for maximum biocatalyst synthesis and partial purification of the enzyme in anticipation of its possible application in the textile realms.

Results

In this study, cellulolytic gut bacteria from termites (Isoptera) were isolated from worker termites, screened, and identified based on morphological, biochemical, and genetic properties for potential textile industry application. The soil (mound-building) interface-feeding higher termite samples were obtained in the central Rift Valley, in the eastern part of Shawa Zone, Oromia Regional State, 121.4 km from Addis Ababa, Ethiopia. The high specificity of bacterial populations in the gut compartment, as well as the convergence of higher termite gut arrangements in a homologous region, provided significant evidence of differentiation from other microorganisms.

Bacterial colonies were initially produced on nutrient agar and then cultured on the CMC-Na medium. The bacterial isolates were re-streaked on CMC-Na agar plates after enrichment to achieve pure colonies, which were then transferred to broth culture for further use and study.

Screening for cellulolytic isolates

The isolated bacterial strains in this study successfully demonstrated the expected clean zone. The pure colony isolates that were predicted to produce cellulase were cultured on CMC-supplemented agar plates containing Congo red, and the cultures were examined for clear zones surrounding the colonies caused due to the breakdown of CMC.

For the evaluation of enzyme efficiency between different bacterial strains, the widths of the clear zones created by colonies were employed. As a result, six of the twenty-seven termite gut isolates (M₂₁WG, M₂₂WG, M₂₂YG, Z₅WG, Z₆WG, and Z₆YG) were effective and yielded the effectively positive result indicated below (Fig. 1).

Fig. 1

Cellulose-degradation/CMC/ by M21WG [A] and Z6WG [B] bacterial isolates /reresentaives/

Among them, twenty-seven bacterial isolates, two M₂₁WG, and Z₆WG were considered as the most remarkable in their colony characteristics and cellulase producing potential with yellow color hydrolytic zone while red color was non-hydrolytic zone. The diameter of the bacterial colony was 4 mm and the halo 36 ± 0.65 mm and 29 ± 0.81 mm while the diameters of the clear zone were 32 ± 0.62 mm and 25 ± 0.73 mm, respectively, for each bacterium. The maximum halo zone diameter minus bacterial colony/colony diameter ratio was 8 ± 0.155 mm and 6.25 ± 0.183 mm (Table 1). The other remaining bacterial isolates did not show clear zones similar to the control plate and were not considered as cellulase-producing bacterial candidates.

Table 1 Hydrolytic capacity value of cellulase degrading bacteria

The M₂₁WG and Z₆WG cellulolytic bacterial isolates were screened as described above. The isolate M₂₁WG showed the clearest zone, and it was chosen for further analysis. In general, the presence of a halo zone around the bacterial colony on agar culture plates indicated cellulose hydrolyzing activity (on the basis of zone of clearance after staining the plates with Congo red dye was the highest halo zone). As a result, the bacteria found in termite guts may have the ability to produce cellulase, an essential enzyme that could be used in the cellulose degradation process.

Morphological characterization of the selected isolates

The best gut bacteria isolates were provisionally identified using morphological and cultural characterization, then purified using the streak plate method and restreaked until a single bacterial colony or pure culture was obtained (Fig. 2). Colony morphological characteristics such as colony formation, color, colony surface roughness and microscopic appearance, and cell arrangement were documented for comparative analysis of the selected isolates.

Fig. 2

Colony morphology of the isolated bacteria, M21WG, and Z6WG isolates

The two selected bacterial isolates, M₂₁WG and Z₆WG, were then analyzed for their biochemical attributes (Table 2). Both of the isolates were positive for glucose and lactose fermentation, as well as indole test. Pertinently, fermentation potency was exhibited to show positive nature.

Table 2 Morphological and cultural characters of the most efficient bacterial isolates, M₂₁WG and Z₆WG

From Table 2, the bacterial isolates, M₂₁WG and Z₆WG, were negative for gram staining tests. Both were found to be gram-negative (G-) and rod-shaped.

Biochemical characteristics of isolated bacteria

The two selected bacterial isolates, M₂₁WG and Z₆WG, were considered for further investigation and their biochemical tests are presented in Table 3. The creation of a bubble in Table 3 suggested that the isolates were catalase-positive and could produce the enzyme cellulase, which basically transformed hydrogen peroxide into water and oxygen. Furthermore, the isolates were found to be negative for motility, indole, and citrate tests. The results of carbohydrate utilization tests are also shown in Table 3.

Table 3 Biochemical characteristics of the two most efficient isolated bacterial isolates, M₂₁WG and Z₆WG

Molecular identification of isolates using 16S rRNA sequencing

The molecular phylogenetic history of the bacterial isolates M₂₁WG and Z₆WG was then determined using 16S rRNA gene sequences. About 1500 bp of 16S rRNA gene was PCR amplified from the contig region from genomic DNA of both M₂₁WG and Z₆WG bacterial isolates. Those unclear (ambiguous) positions were removed for each sequence pair which was called the pair-wise deletion option. The BLAST algorithm (https://blast.ncbi.nlm.nih.gov/Blast.cgi) using the online NCBI Genbank showed that M₂₁WG isolate has 99% maximum identity with sequences of Klebsiella oxytoca strain (MT104573.1), and algorithm (https://blast.ncbi.nlm.nih.gov/Blast.cgi) Z₆WG isolate has 97.30% similarity with Klebsiella sp. strain (MT429262.1).

Also, in this study, the identification of closest relatives via phylogenetic analysis helped to determine the possible affiliation of the isolates. Based on the taxonomic analysis of the 16S rRNA gene sequences, phylogenetic trees were constructed by using multiple sequence alignment programs to understand their genetic and evolutionary relationships (Tamura et al, 2004; Tamura et al, 2021). The isolates were identified based on the percentage similarity with the known species sequences in the database (Fig. 3).

Fig. 3

Phylogenetic tree for the two bacterial isolates, Klebsiella oxytoca (M21WG) and Klebsiella sp. (Z6WG)

Production of crude cellulase and some factors that affect culture condition for cellulase production

In this study, the isolates Klebsiella oxytoca (M₂₁WG) and Klebsiella sp. (Z₆WG) were examined to have a promising potential for cellulase activity of 287.33 ± 0.67 U/mL and 236.67 ± 0.89 U/mL, respectively. The study on the effect of pH on cellulase activity of both enzymes of the isolates has demonstrated that the maximum cellulase production was found to be at values between pH 6 and 8, while the activity of 267 ± 1.38 U/mL was recorded for Klebsiella oxytoca (M₂₁WG) at pH 7 and 220 ± 1.83 U/mL for Klebsiella sp. (Z₆WG) was recorded at pH 7, as the pH approaches the ideal activity level and the manufacturing activity steadily increased (Fig. 4a).

Fig. 4

a Effect of pH, b temperature (°C), c incubation time (h), and d substrate CMC-Na concentration (%) by Klebsiella oxytoca M21WG and Klebsiella sp. Z6WG on cellulase activity

The influence of temperature on cellulase activity was fairly strong in the temperature range of 35–45 °C for both isolates, with the best activity reported at 37 °C. Klebsiella oxytoca M₂₁WG enzyme activity was at 231 ± 1.11 U/mL, while Klebsiella sp. Z₆WG enzyme activity was around 203 ± 1.66 U/mL (Fig. 4b). The effect of incubation time on cellulase activity was investigated by incubating the production medium with the test isolate at different time intervals (24–108 h). The highest cellulase production (257 ± 0.41 U/mL) occurred at 72 h for Klebsiella oxytoca M₂₁WG, and a relatively lower production enzyme activity of 214 ± 0.85 U/mL occurred at the same incubation period for Klebsiella sp. (Fig. 4c).

Klebsiella oxytoca (M₂₁WG) 242 ± 1.111 U/mL and Klebsiella sp. (Z₆WG) 210 ± 1.36 U/mL had the greatest influence of substrate CMC-Na concentration on cellulase activity in this investigation at a substrate concentration of 1.5%. Cellulase production increased as the initial concentration increased from 0.1 to 1.5%, with further increases in concentration gradually lowering the products. Cellulase production was revealed to be dependent on the carbon sources that supplemented in the culture media (Fig. 4d).

Partial purification of cellulase enzyme by ammonium sulfate precipitation and dialysis

The partial purification of cellulase isolated from Klebsiella oxytoca (M₂₁WG) and Klebsiella sp. (Z₆WG termite guts) had been the difference between crude cellulase, specific activity, protein fold, and yield or concentration of salting in and salting out of purification of protein 80% yield of ammonium sulfate precipitation. The specific activity and protein fold while total protein and yield was decreased from the below (Table 4).

Table 4 The purification step of cellulase isolate from Klebsiella oxytoca (M₂₁WG) and Klebsiella sp. (Z₆WG)

In the enzyme fermentation process, the crude extracts contain different mixtures of proteins and undesirable products as organic acids and other metabolites. So, purification of the required favorable product must take place by different purification methods. The maximum specific activity of crude (0.33 ± 0.27, 0.32 ± 0.74 U/mg), partially purified using ammonium sulfate precipitation (7.45 ± 0.63, 3.57 ± 0.57 U/mg), and dialysis (14.12 ± 0.58 and 13.61 ± 67 U/mg) of Klebsiella oxytoca (M₂₁WG) and Klebsiella sp. (Z₆WG) estimated.

Assessment of biostoning potential of the partially purified cellulase for prospective in the textile industry

Over the years, denim-heavy grade cotton twill, dyed with indigo colors and well-worn look, has churned commendable popularity. Conventional use of pumice stones (with or without oxidizing agent like potassium permanganate) for “stone washing” of denim suffers from numerous practical snags including impairment of machine parts, blockage of the drainage system, issues of removal of residues on the pumice stones, requisite for a large amount of stones for even small batches, and the possibility of excessive abrasion that may damage the fabric. The stoned denim swatches by the respective cellulase at different dosages and the untreated piece are attesting (visual) the efficacy of the biocatalysts.

The increase in the dosage for both the test cellulase was found to bear a direct correspondence to the gray value for the selected areas in contrast to the untreated counterparts. For cellulase from M₂₁WG, a maximal gray value of 142 was obtained for the swatch treated with 4 ECU dosages against 27.1 for the untreated (control) sample while the corresponding values for the biocatalyst from Klebsiella sp. Z₆WG were 150 and 22.8, respectively (Fig. 5a, b). In a similar vein, the corresponding color histograms of the M₂₁WG cellulase treatment, the rMean (red), gMean (green), and bMean (blue) values for the untreated samples increased from 52.44, 41.74, and 39.79 (for 1 ECU dosage) to 159. 03, 133.52, and 115.37 for 4 ECU treatments, respectively. While for Z₆WG treatment, the corresponding values were found to escalate from 82.83, 65.82, and 60.06 to 166.78, 144.35, and 127.57, respectively.

Fig.5

A (i) Plot profile attesting (visual) inspection and (ii) depict the 3D surface profile of color decolorization the efficacy of the biocatalysts by treatment with isolated Klebsiella oxytoca (M₂₁WG). B (i) plot profile attesting (visual) inspection and (ii) depict the 3D surface profile of color decolorization the efficacy of the biocatalysts by treatment with isolated Klebsiella sp. (Z₆WG)

It is difficult to control the quantity of abrasion the fabric was exposed to, and acid washing of jeans caused several environmental problems and a novel method of stoning jeans was required to keep jeans charming and comfortable. The biostoning was found to bear cellulolytic enzymes, supporting other previous studies that indicated the applicability in the textile industry.

Discussions

The isolated bacteria were found to allow cellulolytic enzymes, supporting other previous studies that revealed the selected to produce cellulase. The findings of the current investigation revealed a large amount of information about the cellulase-producing bacteria found in termite guts and their roles in the textile industry. Cellulolytic enzymes were found to be allowed by the isolated bacteria, supporting prior research that demonstrated cellulolytic bacteria utilize cellulose as a carbon and energy source (Dar et al., 2018; Liu et al., 2019a). A distinct halo zone around the microbial colonies is created by the positive bacterial isolate. The cellulose hydrolysis capacity (HC) of the bacterial isolate was determined using colonies that formed distinct halo zones (Phuong et al., 2015; Shindeet al., 2017b; Katiyar et al., 2018).

Egwuatu and Appeh (2018) studied the isolation and characterization of filter paper-degrading bacteria from the guts of Coptotermes formosanus by biochemical tests Klebsiella oxytoca and Klebsiella terrigena that is coherent in our investigation. Dar et al. (2018) studied and explored the gut of Helicoverpa armigera for cellulose-degrading bacteria and evaluated a potential strain for lignocelluloses biomass (cotton bollworm) deconstruction. Klebsiella spMD21 (MG367463) is identified based on 16S rRNA gene sequencing. Klebsiella sp. CCFM8384 (KJ803941) in India related coherence to this finding Klebsiella sp. MT429262.1 Z₆WG indicate percent bootstrap values based on 1000 replicates to decide the redundancy of the get sequence with the reference classifications. Dantur et al. (2018) studied that Klebsiella michiganensis/oxytoca Kd70 was isolated from the intestine of the larvae of Diatraeasaccharalis and that independent development of infecting urinary tract in mice and human/animal pathogenicity could have occurred originally from a plant/soil ancestor in Argentina (Dantur et al, 2015). The optimum pH of fermentation for maximum cellulase production was monitored at pH 7 for bacteria species including Bacillus subtilis K-18, Enterobacter species, Klebsiella species, Pseudomonas species, and Serratias pecies and able to survive in both alkaline environments and acidic conditions (Chandran et al., Bai et al, 2012, 2021; Shinde et al., 2017a).

The ability of bacteria to grow in a wide range of pH involving extremes pH ranges; the plasma membrane of microbes might create damage or distracting to the membrane transport protein and also inhibits enzymatic activity.

Cellulose-degrading bacteria from paper and pulp mill dumpsites the ability of bacteria Klebsiella michiganensis optimum at pH 7 cellulase activity of 35.6 U/mL/min (Olowomofe et al., 2019). Shinde et al. (2017a) studied the molecular characterization of cellulolytic bacteria derived from termite gut and the optimization of bacterial growth at different temperatures and optimum ranges (30 to 45 °C). The temperature specificity of most isolates grew well between 30 and 40 °C. For example, Klebsiella variicola (isolate Z) showed luxuriant growth at 30 °C and other isolates such as Klebsiella pneumoniae (isolates B2, C1, C3, and B1 plate). According to Sakthivel et al. (2010), the effect of parameters on cellulase product was maximum after 72 h (0.72 U/mL), pH at 7 (0.80 U/mL), and substrate concentration of CMC 1.0% (1.15 U/mL) for Cornybacterium lipophiloflavum isolated from a decaying vegetable. According to the study of Olowomofe et al. (2019), Klebsiella michiganensis had optimum cellulase activity after 72 h (38.5 U/mL/min) after 48 h. These isolates recorded the lowest enzyme activity after 96 h. Islam and Roy (2018) studied the effect of carbon source-high substrate specificity by CMC 1.0% (w/v) which showed to be cellulase of Paenibacillus sp. C1 has the highest activity against CMC while low activity of Bacillus sp. C2, and Aeromonas sp. C3 when compared to lactose, rice xylan, wheat bran, xylose, and oat spelt xylan.

The ability to resist on substrate might be useful for many industrial applications. Purwanto (2016) previously studied that the maximum specific activity and protein fold of enzymes at 80% of ammonium sulfate precipitation and dialysis was 0.0107, 0.0160, 1.24, and 1.84 respectively. Among others, cellulases have received considerable interest in the textile industry for mercerization, scouring, bio-polishing, laundering, and biostone finishing (Shah, 2013).

Microorganisms like Klebsiella, Pseudomonas, Desulphovibrio, Bacillus, Sphingomonas, Streptococcus, Escherichia, Aeromonas, Proteus, Schewanella, Alcaligenes, and Citrobacter are among the bacteria that successfully decolorize textile wastewater (Mustafa et al., 2021). The termite (Coptotermes curvignathus) gut revealed ligninolytic activity based on the decolorization of all four lignin-like synthetic dyes, including Azure B, phenol red, methylene blue, and Remazol Brilliant Blue (Simol al., 2021). In the event of bacterial monoculture, the decolorization percentage of bacterial species ranged from percent (C.I. Reactive Red 195 decolorized by Klebsiella pneumoniae strain RY10302) to 92% (C.I. Reactive Blue 194 decolorized by Enterobacter sp. strain RY11902).

The decolorization percentage for a mixed culture of four bacteria ranged from 58% (C.I. Reactive Blue 19) to 94% (C.I. Reactive Black 1), suggesting that these bacteria could be used to biologically decolorize dyeing wastewater (Imran et al., 2015; Yadav and Chandra, 2015; Liu et al., 2017; Parveen et al., 2017; Gaur et al., 2018; Ozyurek and Bilkay, 2018; Chantarasiri, 2020; Kumar et al., 2021; Mustafa et al., 2021; Sharma et al., 2021; Tahir and Yasmin, 2021). Within 72 h, strain AB-PR achieved maximum decolorization and Cr (VI) removal efficiency of 90 and 89 percent, respectively, whereas individual decolorization of AB-113 by strain AB-PR remained greater than the co-removal of two contaminants. Under saline circumstances, Klebsiella sp. strain AB-PR can remove significant amounts of azo dyes as well as heavy metals (Saxena and Gupta, 2021; Emadi et al., 2021, Muhamad et al., 2021, Padhan et al., 2021; Prabhakar et al., 2021).

In the textile industry, cellulase-based solutions such as DeniMax® (Novozymes) and ValumaxA 838 have made it simple and cost-effective to create new hues and finishes (Agrawal, 2017). Cellulase can be utilized for a variety of procedures in the textile industry, including denim finishing, cotton softening, biopolishing, biostone washing, and so on (Singh et al., 2021). According to Bhattacharjee et al. (2019), the reduction of indigo dye takes place under controlled conditions with the presence of hydrosulfite in caustic soda as the medium. Because the indigo dye is carcinogenic, poisonous, and emits fumes during the activation process, it is exceedingly dangerous to the environment and human health.

Conclusion

The goal of this study was to harness the use of certain cellulase-producing bacteria from termite (Isoptera) guts in Meki and Zeway areas in the Central Rift Valley of Ethiopia. After optimizing the culture parameters of two isolated bacterial strains, Klebsiella oxytoca (M₂₁WG) and Klebsiella sp. (Z₆WG) for maximum biocatalyst production, it generally consumed less energy, released less chemical into the textile effluents, improved fabric quality, and partially purified the cellulase enzymes. Some environmental conditions influenced the bacteria’s ability to produce this enzyme. In general, this study has demonstrated that both isolates have the potential to produce cellulase enzymes for prospective use as environmentally friendly dye-leaching/biostoning product in the textile industry.

Materials and methods

Description of the sampling area

The sampling for the study purpose was made from termite castes in Rift Valley, eastern part of Shawa Zone Oromia Regional State 121.4 km away from Addis Ababa, Ethiopia. Worker termites were sampled from the termite castes found around Meki and Zeway areas in the Central Rift Valley. Meki is the capital of Dugda District, and Zeway is the capital city of Adami Tulu Jido Kombolcha District. The site was purposively selected because termite’s caste mound where high-density epigeal occurs and diversity located in farmland that had been under cultivation for a long time and damage the agricultural products (Debelo and Degaga, 2014; Debelo, 2018).

Isolation of bacteria from the termite guts

The termites were taken from farmland areas in Ethiopia, where there is a high density of epigeal mounds/nests. Thirty-six healthy worker termites were chosen from the colony and surface sterilized by rubbing the entire body of each termite with a cotton applicator soaked in 70% ethanol, then washing in sterile distilled water and air-drying for 1 min. The head and thorax of an externally sterilized termite were crushed using sterile forceps under sterile conditions. Another set of forceps was sterilized and used to take the termite’s complete abdomen away from its body. The abdomen was inserted in 1 mL of phosphate buffer solution after the head and thorax were discarded (PBS). The remaining nine termites went through the same procedure. The bodies were dissected with a sharp blade and a magnifying hand lens after the heads were removed using forceps (Egwuatu and Appeh, 2018).

The intestine was eventually extracted using fine-tipped forceps under the Laminar Air Hood MN20 (Microbiology Safety Cabinet), based on a modest modification of previously described extraction techniques (Phuong et al., 2015; Devaraj and Kesti, 2019). The gut contents were then homogenized in 5 mM sterile phosphate buffer saline (1× PBS), pooled, and transferred to a 1-mL microcentrifuge tube containing PBS solution, where they were centrifuged for 10 s at 4000 rpm at 4 °C. Serial dilutions of the supernatant were performed (Sreeremya et al., 2015).

By adding 0.9 mL of phosphate buffer solution to another sterile microcentrifuge tube and transferring 0.1 mL of the original homogenate into it, ten-fold serial dilutions of termite abdomen homogenate were generated, mixing carefully after each transfer. Using sterile disposable pipettes, those three 10-fold serial dilutions were done to obtain concentrations of 10⁻², 10⁻⁴, and 10⁻⁶ using sterile disposable pipettes (VanDexter and Boopathy, 2019). Then, the diluted termite gut contents were inoculated in Nutrient Broth agar (Nutrient Broth 9 g/L and agar 15 g/L) and CMC medium (NaNO₃ 2.5 g; KH₂PO₄ 2 g; MgSO₄ 0.2 g; NaCl 0.2 g; yeast extract 0.2 g; CaCl₂.6H₂O 0.1 g in a liter) containing 0.5% carboxymethyl cellulose (CMC) as a sole carbon source; the pH of the medium is adjusted to ± 7.2 with 1 M NaOH and HCl. The sterilization of the prepared medium took 15 min in an autoclave at 121 °C. The medium was sterilized before being placed onto sterile Petri dishes and allowed to solidify. The termite gut bacteria were then cultured in this culture media, and plates were incubated at 37 °C for 48 h before being stored at 4 °C with some modifications (Ali et al., 2019a; Peristiwatiet al., 2018).

Screening of cellulolytic bacteria

Cellulolytic bacteria were screened from among the pure colonies using 1% carboxymethyl cellulose (CMC)-supplemented agar plates based on the methodology reported by Sharma et al. (2015). Post-incubation at 30 °C for 24 h, the plates were flooded with 0.1% Congo red for 15 min, followed by de-staining with 1 M NaCl solution for 15 min and measurement of the clear zones, attesting the CMC hydrolysis (Phuong et al., 2015; Javaheri-Kermani and Asoodeh, 2019) based on the following equations:

$$\mathrm{Diameter}\ \mathrm{of}\ \mathrm{clear}\ \mathrm{zone}=\mathrm{Diameter}\ \mathrm{of}\ \mathrm{halo}\ \mathrm{zone}-\mathrm{Diameter}\ \mathrm{of}\ \mathrm{colony}$$

(1)

$$\mathrm{Hydrolytic}\ \mathrm{capacity}=\frac{\mathrm{Diameter}\ \mathrm{of}\ \mathrm{clear}\ \mathrm{zone}\ \left(\mathrm{CZ}\right)}{\mathrm{Diameter}\ \mathrm{of}\ \mathrm{bacterial}\ \mathrm{colony}\ \left(\mathrm{CD}\right)}$$

(2)

Morphological and biochemical characteristics of the bacterial isolates

Morphological features were determined using standard methods. Pertinently, the bacterial isolates from the termite guts were able to grow under aerobic conditions. The biochemical tests encompassed by means of catalase activity test, urease test, citrate test, indole test, motility test, and methyl red test, fermentation tests for glucose and lactose. This procedure was repeated across treatments for each pure colony which served as biological replicates. The various reagents for these tests were procured from Hi-Media (Waghmare et al., 2014; Guder and Krishna, 2019).

Molecular identification of bacterial isolates by 16S rRNA sequencing

Based on the initial screening results, the genomic DNA (gDNA) of two potential cellulase-producing bacteria designated as M₂₁WG and Z₆WG was extracted, and their 16S rRNA genes were PCR amplified by using universal bacterial-specific primers E9F (5′-GAGTTTGATCCTGGCTCAG-3′) and U1510R (5′-GGTTACCTTGTTACGACTT-3′) (Farrelly et al., 1995). Finally, the PCR products were sent to YAAZH Xenomics, Coimbatore, India, for Sanger sequence; the obtained sequence data was compared with known sequences in the Genbank using the basic local alignment search tool of the national center for biotechnology information (NCBI) (Saitou and Nei, 1987; Felsenstein, 1985; Kimura, 1980; Kovtunovych et al., 2003; Younis et al., 2017).

Cellulase assay

Post-plotting of the standard glucose concentration curve, the cellulase (CMCase) activity was quantified based on the DNS method (Lowry, 1951).

Yan et al. (2019) encompassed a protocol of the addition of 0.5 mL of crude enzyme solution to 1.5 mL of 1% CMC-Na solution, followed by incubation of the enzyme-substrate mixture, post-shaking well at 50 °C for 30 min in a water bath and, the addition of 1.5 mL of DNS reagent and shaking, boiling for around 10 min, subsequent cooling, and eventual recording of absorbance value at 540 nm in the UV-visible spectrophotometer (Biochrom, England). The following equation was used to calculate the CMCase activity:

$$\mathrm{CMCase}\ \mathrm{or}\ \mathrm{Cellulase}\ \mathrm{activity}\left(\frac{\mathrm{U}}{\mathrm{mL}}\right)=\frac{A\times 1000}{30\times 0.5}$$

(3)

where A is the glucose content from the sample (mg/mL), 30 refers to 30 min of the reaction time, 1000 is the dilution factor, and 0.5 indicates the volume of enzyme used in the analysis.

Determination of optimal process parameters for cellulase production

Process parameters such as pH, temperature, incubation time, and substrate concentration were optimized for maximal cellulase production by the two potential isolates M₂₁WG and Z₆WG. For the determination of the optimum pH and temperature, the two isolates were cultured in respective 250 mL Erlenmeyer flasks containing CMC-Na broth under a range of pH (4.0–10.0), temperature (27–52 °C), incubation time (24–108 h), and substrate concentration (0.1–2.5%) carbon source at 120 rpm for 3 days (Javaheri-Kermani and Asoodeh, 2019; Sakolvaree and Deevong, 2016; Khatiwada et al., 2016).

Partial purification of cellulase and produced under optimal conditions

Post-culturing of the bacterial isolates under the respective optimal conditions, as determined in the previous step, we resorted to partial purification of the crude enzyme. The crude enzyme, corresponding to each of the isolates, was harvested post centrifugation of the respective culture at 10,000 rpm for 20 min to eliminate cells and residual medium; 100 mL of crude enzyme-rich culture supernatant was subjected to overnight precipitation with ammonium sulfate (80% saturation) at 4 °C followed by centrifugation for 20 min at 4 °C. This was followed by re-suspension of the pellet in 100 mM phosphate buffer (pH 7.0) and overnight dialysis against the sample buffer using a cellulose membrane tube (12 kDa cutoff). The partially purified enzyme was used for subsequent enzyme assay by Biochrom, England (Potprommanee et al., 2017).

Assessment of biostoning potency of the partially purified cellulase for prospective in the textile industry

The purpose of this investigation was to see how effective partly purified cellulase from the selected bacterial isolates M21WG and Z6WG were at bleaching denim fabric. In 250-mL Erlenmeyer flasks with 10 steel balls, the textile samples (in three repetitions) were treated (enzymomechano treatment) with the partly purified enzyme (harvested from each of the isolates) (diameter 0.7 cm). Denim swatches were immersed in 20 mL of 100 mM sodium phosphate buffer (pH 7) at a solid: liquid ratio of 1:10 and incubated at 40 °C for 1 h at 250 rpm. An HP Scanjet G4050 scanner was used to scan the control and treated denim swatches at 300 dpi resolution.

The Fiji (Win 32) software was used to analyze the BMP images that were captured as a result of the scanning. Demarcate a rectangular area (50 w × 20 h) on each of the denim images. The image of each of the denim swatch, plot profile, 3D surface plot, and color histogram (RGB) were generated, and the results were analyzed to assess the bleaching potency of the enzyme, isolated from each of the isolates some modified (Gautam and Sharma, 2012; Costa et al., 2021).