Characterization of Cellulose and Starch Degradation by Extracellular Enzymes in Frankia Strains

Abstract

Annually, a considerable amount of agricultural waste is produced leading to serious environmental pollution if not managed effectively. A wide range of bio-decomposers, including fungi, bacteria, and actinomycetes may break down the complex agro-residues in an eco-friendly way through secreting many cellulolytic and amylolytic enzymes. The present study aimed at exploring the ability of Frankia to degrade cellulose and starch and identifying the cellulase and α-amylase genes in Frankia genomes for potential agricultural waste degradation. Frankia alni ACN14a and Frankia casuarinae CcI₃ produced clear zones around growing hyphae on carboxymethyl cellulose (CMC) and starch substrates. The hydrolytic index (HI) ranged from 1 to 2.14 reflecting variation in their degradation efficacy. Quantification of CMCase (carboxymethyl cellulase) production in strain ACN14a presented the maximum activity (0.504 U/mL) under 1% CMC after 16 days whereas strain CcI₃ produced a weak activity after 6 days from incubation. Besides, amylase activity in strain ACN14a reached the highest value (3.215 U/mL) after 4 days of growing with 1% starch, while strain CcI₃ had the superior production (3.04 U/mL) after 12 days from 1% starch condition. Data mining and genome blasting led to the identification of multiple genes related to cellulose and starch degradation. Two endoglucanases (celA1, FRAAL4955 and celA2, FRAAL4956), two glycosyl hydrolase family 16 (FRAAL6120 and FRAAL2663), and one glycosyl hydrolase family 16 (Francci3_3843) were predicted in the two genomes. Likewise, the α-amylase genes (FRAAL5900) from Frankia alni ACN14a and (Francci3_3679) from strain CcI₃ were identified. The gene expression of endo-1, 4-beta-glucanase (celA2, FRAAL4956) revealed the maximum increment in its mRNA abundance under 0.25% CMC exposure and showed a 3.3-fold increase. Frankia capability to degrade cellulose and starch represents a critical process in nutrient cycling and environment protection.

1. Introduction

Members of the Frankiaceae family are nitrogen-fixing actinobacteria that live in symbiotic nodules with Actinorhizal plants. In fact, these bacteria can form symbiotic relationships with more than 200 species of these plants (mainly trees and shrubs). The symbiotic relationship between Frankia and actinorhizal plants is crucial for both ecological and economic processes, such as crop protection, land reclamation, soil stability, and the production of fuel and lumber [1,2,3,4]. Frankia can degrade and resist a variety of environmental stressors such as herbicides [5,6,7,8], heavy metals [9,10,11,12,13], salinity [14,15,16,17], pH and temperature [18,19].

It’s long been thought that certain strains of Frankia sp. secrete hydrolytic enzymes like cellulases, pectinases, and proteases, which could aid in the penetration of plant roots and the development of symbiotic root nodules. These proteins are hypothesized to play a role in the molecular interactions between Frankia and their host plants [20]. Experiments with exponentially developing cultures have provided insights into a complex proteolytic system that includes extracellular DNAses, esterases, dehydrogenases, proteasomes, aminopeptidases and endo- extracellular proteinases [21]. Interestingly, strain CcI3 had a higher number of esterolytic, lipolytic, and proteolytic enzymes with signal peptides than polysaccharide-degrading enzymes [20]. In silico genome analyses of 21 Frankia genomes provide an insight into the lytic enzymes (i.e., cellulases) in genomes of Frankia cluster 1, whereas enzymes such as extracellular endoglucanases, pectate lyases were detected into cluster 3 genomes. Furthermore, chitinase genes were found in Frankia type strains F. inefficax and F. casuarinae [22,23].

Agricultural waste is a growing global concern in terms of environmental impact. It contaminates natural resources such as water and soil on a large scale [24]. The total generated waste globally reached 2.02 billion tons in 2016 and is expected to be 2.59 billion tons in 2030. Furthermore, agricultural waste represents about 998 million tons that are created yearly [25]. Saudi Arabia is considered one of the most producers of date palm fruit (with approximately 34 million trees and 15 cultivars) in the world [26,27]. Every year, the date palm produces a significant quantity of trash in the form of seeds, dried fruits, and fibrous components. The estimated loss of dates in Saudi Arabia reached 12.6% at the harvest time and part of this lost is due to parthenocarpic date palm fruit (Shees & Sish) [28,29]. In the early phases of fruit life, there is a high content of fiber composed of cellulose, hemicellulose, starch, lignin, and insoluble proteins [30]. It could be converted into added-value products by microbial biodegradation, which could match both environmental and economic needs such as minimizing land contamination with additional benefits from the output products. Agricultural wastes are classified as crop residues, animal wastes, food-related wastes and industrial wastes [24,31,32]. Cellulose, hemicellulose, lignin, and other inorganic materials represent the majority of biomass that compose the agricultural wastes [33]. Furthermore, the main constituent’s percentages of these substances are 40–50 from cellulose, 20–40 from hemicelluloses, 20–30 of lignin with different amounts of starch, protein and fats [34,35].

Many microorganisms including fungi, bacteria and protozoa (i.e., Actinobacteria, Trichoderma, Clostridium, Bacillus), could break down cellulose via depolymerizing cellulases enzymes that hydrolyze lignocelluloses in an ecofriendly way [36,37,38,39]. Conversion of agriculture wastes (categorized as lignocellulosic biomass) contributes to waste management and represents great potential as a bioenergy source leading to prevent environmental contamination and ensure energy security [40]. Under both aerobic and anaerobic circumstances, bacterial and fungal spores accelerate the breakdown of waste in a primary and effective decomposition technique. Through cellulose breakdown, phosphorous solubilization, and nitrogen fixing, microbial decomposition increases the nutritional content coming from the degraded product [41,42].

The key enzymes in this process are cellulases, which cleave the β-1,4 bond in the cellulose chain. β-glucosidases, endoglucanases, β-xylosidases, endoxylanases, mannosidases, and carbohydrate-binding modules are complex enzymatic systems that are functionally redundant for the breakdown of cellulose and hemicellulose [39]. The cellulases are generally divided into three categories: exocellulases that cleave at the ends of the cellulose chain, endocellulases which act inside the chain, and β-glucosidases, which convert cellobiose to glucose monomers [43].

Based on the availability of Frankia genomes and their capability to infect and penetrate the actinorhizal plant roots, the present study aimed at (1) Exploring the Frankia cellulolytic ability to degrade cellulose and starch components, (2) Assessing the degradation process through measuring carboxymethyl cellulase (CMCase) and amylase activities, (3) Determing if the cellulase genes are conserved in Frankia genomes, and (4) Evaluating the expression of cellulase genes into the selected Frankia genome.

2. Results

2.1. Screening Cellulose and Starch Degradation

Cellulose is considered the main component in the agriculture waste with up to 40–50% from their substances. Strains ACN14a and CcI₃ grown on MPN medium supplemented with 1% of either CMC or starch or grinded parthenocarpic date fruits showed activity around the hyphae. The cellulolytic and starch lysis activity confirmed by observed clear zone around the colonies (Figure 1). The cellulose and starch index ranged from 1 to 2.14, indicating CMCase and amylase production. The topmost activity assigned to strain ACN14a under cellulose treatmend (cellulolytic index reached 2.14, Figure 1A), followed by strain CcI₃ grown in presence of parthenocarpic date palm fruits (Figure 1D) whereas the miniumum efficiency had by strain ACN14a with parthenocarpic date palm fruits (hydrolytic index = 1, Figure 1C).

2.2. Quantification of Cellulase and Amylase Activities

The production of CMCase and amylase enzymes were assessed in ACN14a and CcI₃ cultures grown at 0.5 and 1% from CMC and starch. The CMCase production increased with incubation time and reached the highest level by strain ACN14a at 0.5% CMC after 16 days (0.386 U/mL), whereas the maximum CMCase activity under 1% CMC measured 0.504 U/mL at the same time (Figure 2A). Likewise, the amylase activity under the two concentrations from starch (0.5 and 1%) recorded after 4 days from the beginning of incubation and the enzyme activity displayed the topmost values at 0.5% starch and counted 0.678 U/mL. While at 1% starch, the activity measured 3.215 U/mL (Figure 2B). Finally, the amylase activity dropped at 6 and 8 days at 0.5 and 1% starch, respectively.

Regarding strain CcI₃, weak CMC degradation was observed on the MPN agar medium supported with 1% CMC. The same trend was found in liquid medium designated for CMCase quantification and a low enzyme activity (0.003 U/mL) was established after 6 days. Besides, strong amylase activity was manifested at 0.5 and 1% from starch and valuable activities were presented at 6 and 8 days whereas the highest activity was displayed after 12 days from culture growing. The activity was measured at 1.89 and 3.04 U/mL under the two conditions, respectively (Figure 3). It could be concluded that strain ACN14a excels in cellulase degradation whereas both strains (ACN14a and Ccl₃) exhibited high activity in starch conversion.

2.3. Genomes Analysis and Phylogenetic Tree

To find possible genes linked to cellulose and starch degradation, the Frankia strains ACN14a and CcI₃ genomes were examined and analyzed. The published Frankiaceae genomes (14 genomes) were searched for homologues at protein level using a number of known cellulose and starch degradation genes and motifs. For cellulose degradation, four genes were identified in ACN14a strain: endoglucanase (celA1, FRAAL4955, accession YP_715138), endoglucanase (celA2, FRAAL4956, accession YP_715139), glycosyl hydrolase family 16 (FRAAL6120), and glycosyl hydrolase family 16 (FRAAL2663), in addition to one gene in strain CcI₃: glycosyl hydrolase family 16 (Francci3_3843, accession: YP_482922). The deduced amino acid sequence of cellulase from Streptomyces sp. NPDC046977 (WP_360906084.1) shared identities and positives reached 51% and 59%, respectively with celA1 (FRAAL4955) whereas 52% and 62% were detected with celA2 (FRAAL4956), respectively. The constructed phylogenetic tree showed two distinct clusters, one cluster (cluster I) involved the genes related to endoglucanase (Endo-1, 4-beta-glucanase) and glycosyl hydrolase family 16 genes from all tested Frankia strains and Streptomyces. While cluster II contained the endo-beta-1,3-1,4 glucanase from Bacillus, Streptococcus and Stutzerimonas (Figure 4).

Likewise, the α-amylase genes were identified in the Frankia genomes using the deduced amino acid sequence of the alpha amylase gene (ADB81848.1) from Bacillus sp. B-5. Identity and similarity counted 27% and 59% were found with alpha-amylase from Frankia alni ACN14a (FRAAL5900, accession: YP_716044), respectively. Furthermore, the same α-amylase gene from Bacillus sp. B-5 displayed 27% identity and 55% similarity with the putative alpha-amylase α-gene from strain CcI₃ (Francci3_3679, accession: YP_482760), respectively. This high similarity most likely reflects an identical function for these genes. The phylogenetic tree was created by comparing the inferred amino acid sequences of putative α-amylase from the fourteen selected Frankia genomes (including ACN14a and CcI₃) with other alpha amylase genes from other organisms that were acquired from the GenBank (Figure 5). As illustrated in Figure 5, eleven alpha amylase genes were identified in the analyzed Frankia genomes and separated in one group (group I), whereas the other identified genes in the literature categorized in the second group (group II). Besides, alpha-1,4-glucan:maltose-1-phosphate maltosyltransferase genes from Arthrobacter agilis (AOY13650.1), was considered as out of group.

2.4. Expression of Identified Genes

Applying qRT-PCR, the expression of identified cellulase genes were assessed using the specific primers listed in Table 1. Change in mRNA level were evaluated in strain ACN14a after 10 days of growing with CMC. Both putative signal peptide (FRAAL4954) gene and endo-1,4-beta-glucanase (celA1, FRAAL4955) gene showed no significant change in their expression under CMC exposure. Besides, only the endo-1, 4-beta-glucanase (celA2, FRAAL4956) displayed an increase in its mRNA abundance under CMC exposure (0.1% CMC) reaching a 2.1-fold change (Figure 6). Under 0.25% CMC, the gene expression continued to increase and recorded a 3.3-fold change whereas the expression decreased with higher concentration of CMC (0.5%) and showed a 1.6-fold change. This decrease in FRAAL4956 gene expression may be due to time-dependent treatment.

3. Discussion

Agriculture generates a large amount of waste composed of cellulose, hemicellulose, lignin, chitin, and pectin, which can only be broken down naturally by fungi, bacteria, and protozoa [31,43,44].

Cellulases are divided into groups based on the similarity of their sequences or their manner of action. Exoglucanases (they are also known as cellobiohydrolases) are hydrolytic enzymes that work on the end of glycan chains. The cellodextrins (cellobiose, cellotriose, and cellotetraose) are released into solution when they cleave two, three, or four glucose units at a time. Endoglucanases function at a location inside the glycan chain.

Frankia are able to colonize actinorhizal plants and form a symbiotic relationship with them. Frankia must penetrate plant roots to establish symbiosis and form symbiotic root nodules so that many hydrolytic enzymes such as cellulases and pectinases can be secreted [20]. Frankia strains ACN14a and CcI3 showed cellulolytic and amylolytic activity confirmed by the clear zones around the growing colonies. Cellulase and amylase activities were demonstrated in Frankia plate cultures with indices ranged from 1 to 2.14 reflecting their promising ability in degrading cellulose and starch in comparison with other microorganisms. On the other hand, nine bacterial strains were isolated from sago (Metroxylon sago) waste products (five bacterial species with amylolytic activity and four with cellulolytic property). Serratia liquefaciens bacteria had the best amylolytic activity with hydrolysis index which reached 3.08, whereas Acinetobacter iwoffii showed cellulose hydrolysis index counted 2.01 [45]. Besides, three fungus strains and sixty-three bacterial strains were isolated from the soil of the tobacco rhizosphere and the surface of the tobacco leaves by Zhang et al. [46]. For the following stages, 14 strains with the largest hydrolysis zones were assessed, and had hydrolytic indices higher than 1.5.

Frankia ACN14a displayed the maximum CMCase activity under 1% CMC and produced 0.504 U/mL after two weeks of incubation since cellulose contains β 1→4 linkage that is hard to degrade. The maximum starch degradation, which contain α 1→4 linkages that are easier to degrade by amylase, was after 4 days with activities reaching 3.215 U/mL. This may reflect earlier amylase and late cellulase activities. In strain CcI₃, the amylase activity measured up to 3.04 U/mL. Guder and Krishna [47] isolated twenty bacterial strains from the rumen of sheep that break down cellulose. Cellulose-degrading bacteria were screened using the Whatman filter paper degradation test and the CMC (carboxyl methylcellulose) hydrolytic test, which showed a clear zone greater than 10 mm surrounding the colony on CMC agar. The cellulase enzyme activity ranged from 0.225 U/mL to 1.652 U/mL with the maximum productivity assigned to Enterobacter pecies strain. Trichoderma afroharzianum degraded filter paper with cellulase activity reached 0.91 U/mL [48], whereas β-glycoside hydrolase (Bgl-16A) from Bacillus subtilis 1AJ3 had an enzymatic activity of 365.29 U/mg [49]. Additionally, Saratale et al. [50] evaluated cellulase and hemicellulase production under solid state fermentation using various agricultural wastes. The optimal circumstances resulted in the production of maximal activities of glucoamylase (505.0 U/gds (enzyme unit per gram of initial dry solid substrate), cellobiase (244.60 U/gds), endoglucanase (188.66 U/gds), exoglucanase (24.22 U/gds), and filter paperase (30.22 U/gds). Furthermore, the isolated Bacillus subtilis from silkworm displayed the enzyme activity of endoglucanase (CMCase), the filter paper (FPase), and exoglucanase (CXase) reached 11.91 U/mL, 15.40 U/mL, and 20.61 U/mL, respectively [51]. On the other hand, Bacillus amyloliquefaciens SS35 showed high enzyme activity of 0.079U/mL when grown with CMC as a sole carbon source [52]. It could be concluded that Frankia strains (ACN14a and CcI3) had a medium CMCase activity in comparison with the other discussed microbes above.

Genome analysis of published and finished Frankiaceae genomes (14 genomes) exhibited many genes may contribute to cellulose and starch degradation, subsequently, may participate in agriculture waste management. The main found genes were endoglucanase and glycosyl hydrolase family 16. Similarly, bacterial strain CP22 related to genus Micromonospora with ability to produce cellulase, had 47 glycoside hydrolase domains for cellulase hydrolysis (9 encode for β-glucosidases, 8 produce endoglucanases and 3 for exoglucanases) [53]. However, Bacillus paralicheniformis with strong cellulase activity owned 10 cellulase genes that were classified into three groups involved exoglucanase, endo-β-1,4-glucanase and beta-glucosidase [54]. Streptomyces diastaticus strain CS1801 possesses 103 glycoside hydrolase family genes, that aid in the breakdown of chitin and cellulose [55]. Meanwhile, Streptomyces argenteolus M178 and Streptomyces thermocarboxydus C42 were chosen based on their capacity to degrade carboxymethylcellulose (CMC). The probable four cellulase genes (cel5AM and cel12BM from the M178 strain and cel9AC and cel5AC from the C42 strain) were discovered by shotgun cloning studies of cellulase genes from these genomic DNAs using Streptomyces lividans as a host [56].

Applying real-time quantitative-PCR will help in identifying gene function related to specific trait when bacteria grow under specific stress. Frankia strain ACN14a exhibited up-regulation in endo-1, 4-beta-glucanase (celA2, FRAAL4956) gene expression reflecting its possible role in CMC degradation. The transcriptional assays were performed after 10 days (at 0.1, 0.25 and 0.5% from CMC) whereas the enzymatic assays were performed up to 16 days (at 0.5 and 1% from CMC). The highest enzymatic activity was observed on days 12 and 16 under 0.5 and 1% CMC whereas the highest gene expression was observed on day 10 under 0.25%. Expression may increase with prolonged incubation period (i.e., 12 and 16 days) which could explain these differences. In the basidiomycete Phanerochaete chrysosporium, qRT-PCR was used to analyze the transcription of the cellobiose dehydrogenase gene (cdh) and the b-glucosidase gene (bgl) in connection with cellobiose metabolism. Expression levels of both transcripts were significantly reduced when glucose was added to the cellulose-degrading culture, whereas cdh transcripts recorded 2.3-fold higher than those in glucose medium [57]. Notably, Pseudomonas stutzeri had cellulase gene (PST_2494 gene) that measured higher gene expression reached 4 folds [58]. Otherwise, gene expression of cellulase genes in cellulose degradation process varies based on the growth stages of microorganisms. In Auricularia heimuer (the wood-rotting edible mushroom), two strains (g5372 and g7270) showed higher levels from cellulase gene expression during the mycelium phase, whilst g9664 and g10234 strains exhibited higher levels during the fruiting phase [59]. In Clostridium cellulovorans, cellulases and hemicellulases were analyzed at the mRNA level using cells grown under varied culture conditions. When cellulose, pectin and xylan, were present, the majority of abundant genes under study (cbpA-exgS, engH, engE, hbpA, manA, engM, pelA, and xynA) showed extensive expression [60].

The majority of agricultural wastes are valuable byproducts with excellent nutritional qualities. Some wastes may be turned into food items and biofertilizers, and some are used as animal feed, i.e., fruit peel and green walnut husks are natural antimicrobials and compost as a source of nutrients for plants. Frankiaceae can fix nitrogen in a symbiotic relationship with actinorhizal plants and with its ability to degrade cellulose and starch, this will make it a valuable microorganism with a dual role. By fixing nitrogen and recycling agricultural waste, Frankiaceae will be a significant microbe as a source of nutrients for plants and potential biofuel producers. Some waste has the potential to be used as raw materials for the production and conversion of other valuable goods because they include high levels of proteins, carbohydrates, and minerals [61].

4. Materials and Methods

4.1. Frankia Strains and Culture Conditions

Frankia alni strain ACN14a (DSM 45986) and Frankia casuarinae strain CcI₃ (DSM 45818) were chosen for the current research. Frankia strains were grown and maintained in MPN medium containing (g/L), 4.2 MOPS (20 mM 3-(N-morpholino) propane sulfonic acid), 1.7 K₂HPO₄ and 0.267 NH₄Cl, with adjusted to pH 6.8. For each 100 mL from MPN buffer, 1.7 mL from metal mix, in addition to 5 mM propionate and 5 mM succinate as a carbon source were added (see Tisa et al. [62] and Rehan et al. [12]).

4.2. Primary Screening of Cellulose and Starch Degradation

The qualitative screening of Frankia strains to degrade both cellulose and starch was performed using the MPN agar medium supplemented with 1% of carboxymethyl cellulose (CMC), or soluble starch or grinded date palm parthenocarpic fruits (prepared by putting the parthenocarpic fruits at 70 °C overnight and used as a source for cellulose and starch). Frankia strains ACN14a and CcI₃ (50 µL from homogenized cultures) were inoculated on the agar medium at 30 °C for two weeks. The enrichment plates were flooded with Congo red (0.1%) for 30 min at room temperature, followed by washing with 1 M NaCl for several times [63]. Degradation of cellulose is indicated by the development of a distinct zone surrounding a colony. For starch degradation detection, iodine solution (1 gm of iodine crystal and 2 gm of potassium iodide in 100 mL of dH₂O₂) was implemented to cover the growing cells and positive colonies demonstrated clear zone of hydrolysis [64]. Cellulase production was assessed using the ratio of colony diameter to hydrolysis clear zone diameter. The hydrolytic index (HI) of cellulose and starch was measured as a ratio between the diameter of clear zone and the bacterial growing colonies (D/d) [51,65].

$$\mathrm{H}\mathrm{I}={\displaystyle \frac{\mathrm{D}}{\mathrm{d}}}$$

D = Hydrolytic zone diameter (mm)

d = colony diameter (mm)

4.3. Carboxymethyl Cellulase (CMCase) and α-Amylase Activity

Frankia strains were grown in liquid MPN medium supplemented with 0.5 and 1% from CMC or starch at 30 °C for two weeks. Samples (2 mL) withdrawn gradually after 4, 6, 8, 12, and 16 days. The collected samples centrifuged at 12,000 rpm for 8 min to discard the mycelium whereas the adequate filtrate (crude enzyme extract) used for CMCase and α-Amylase activities. The 3,5-dinitrosalicylic acid (DNS) method was applied to evaluate the liberated reducing sugars from CMC as recommended by the International Union of Pure and Applied Chemistry (IUPAC) [66] with modifications. Briefly, the reaction mixture (200 μL) involved 100 μL of 50 mM phosphate-Citrate Buffer (P4809, Sigma-Aldrich, MO, USA, pH 5.0 containing 1% CMC (w/v)) and 100 μL of culture filtrate (crude enzyme). The mixture is incubated at 50 °C for 1 h, followed by adding 600 μL of DNS reagent (potassium sodium tartrate tetrahydrate (30 g), 3,5-dinitrosalicylic acid (1 g) and sodium hydroxide (2 g) to prepare 100 mL solution) and boiled at 100 °C for 10 min. Then and after cooling at room temperature, the intensity of color in the mixture measured spectrophotometrically at 540 nm by the plate reader (BioTek Epoch 2 Microplate Spectrophotometer, VT, USA) and glucose standard graph was used to calculate the amount of reducing sugars [67]. Under conventional test conditions, one unit of cellulase and amylase activity is defined as the amount of enzyme needed to release 1 µmol glucose per minute. All samples were measured in triplicated against un-inoculated culture as control.

4.4. Bioinformatic Analysis and Phylogenetic Tree Construction

The Integrated Microbial Genomes System from the Joint Genome Institute (https://img.jgi.doe.gov/, provided the amino acid sequences for the Frankia genomes [68]. A blastp search of Frankia ACN14a, CcI₃, and other finished Frankia genomes (14 genomes) at JGI (Accessed at 5 April 2025) was conducted using the discovered deduced amino acid sequences of numerous reported known cellulase and starch degradation genes which were published in the literature [69,70,71,72,73,74,75,76] as a query sequence seeking homologues at the protein level [77]. Specific blastp parameters were applied: standard search in nr database, max E-value: 1 × 10⁻¹, matrix: BLOSUM62. For phylogenetic construction, the identified cellulase (endoglucanase and glycosyl hydrolase) and α-amylase amino acid sequences in 14 finished and published Frankia genomes with their similar amino acid sequences in the GenBank database (https://www.ncbi.nlm.nih.gov/genbank/, accessed in 8 April 2025) were aligned by ClustalW [78] and their relationship inferred with MEGA 12 software [79] with the following parameters: Maximum Likelihood and Jones-Taylor-Thornton (JTT) model- were constructed from 1000 bootstrap replicates.

4.5. Gene Expression of Cellulase Enzyme

TRIzol™ LS reagent (Invitrogen, Carlsbad, CA, USA) was used to extract total RNA from Frankia alni ACN14a grown in 0, 0.25, 0.5 and 1% from CMC for 6 and 10 days as described in the manufacturer’s instructions. The extracted RNA (approximately 500 ng) was treated with Dnase I (ezDNase, Invitrogen) and then reverse-transcribed into complementary DNA (cDNA) using the SuperScript IV VILO Master Mix Kit (Cat. No. 11766050, Invitrogen). QuantiTect SYBR Green PCR Kit (Cat. No. 204143, Qiagen, Hilden, Germany) was utilized to perform quantitative real-time PCR (qRT-PCR) on the Real-Time PCR System (7500, Applied Biosystems, Thermo Fisher Scientific, CA, USA) as recommended by the manufacturer. The relative expression (fold change) calculated through the comparative 2^−ΔΔCT method [80] against untreated Frankia cells and rpsA as a normalized gene. The applied primers are listed in Table 1.

Table 1. Primers Sequence used for quantitative real-time RT-PCR.

Genes	Gene Locus_Tag	Forward	Reverse
putative signal peptide	FRAAL4954	5′-CCACTGGCTCGATCAGTTC-3′	5′-GGCGTGCTGAAGGTGAC-3′
Endo-1,4-beta-glcanase	FRAAL4955	5′-GGCGGTCGATATGCTCTTT-3′	5′-GACACACGACCTCCGAATG-3′
Endo-1,4-beta-glcanase	FRAAL4956	5′-GTACATGGTCTACGCCATCC-3′	5′-GACCTGACGGGTGAACTG-3′
rpsA	FRAAL1781	5′-GCAGTCGACAAGACGATCAA-3′	5′-CTCGGTCTTGTAACCGATGTC-3′

Table 1. Primers Sequence used for quantitative real-time RT-PCR.

Genes	Gene Locus_Tag	Forward	Reverse
putative signal peptide	FRAAL4954	5′-CCACTGGCTCGATCAGTTC-3′	5′-GGCGTGCTGAAGGTGAC-3′
Endo-1,4-beta-glcanase	FRAAL4955	5′-GGCGGTCGATATGCTCTTT-3′	5′-GACACACGACCTCCGAATG-3′
Endo-1,4-beta-glcanase	FRAAL4956	5′-GTACATGGTCTACGCCATCC-3′	5′-GACCTGACGGGTGAACTG-3′
rpsA	FRAAL1781	5′-GCAGTCGACAAGACGATCAA-3′	5′-CTCGGTCTTGTAACCGATGTC-3′

4.6. Statistical Analysis

Data from all groups were compared using the t-test of independent samples, and statistical significance was evaluated using a one-way ANOVA analysis of variance. Statistical significance was indicated with a p-value of 0.05 [81].

5. Conclusions

Conversion of agricultural wastes (containing cellulose and starch) into a range of value-added products such as organic fertilizers will contribute to waste management and prevent environmental contamination. Frankia (gram-positive actinobacteria) produces many cellulases enzymes to penetrate the actinorhizal plants roots. Frankia strains ACN14a and CcI₃ generated clear zones around growing cells in the presence of CMC, starch and shees (parthenocarpic date fruits) with hydrolysis index ranged between1 to 2.14. The main identified genes related to cellulose and starch degradation in the genomes were endoglucanase (celA1 and celA2) and glycosyl hydrolase family 16. Under cellulose stress, the expression of endo-1, 4-beta-glucanase (celA2, FRAAL4956) showed increment in its mRNA abundance and recorded 3.3-fold change under 0.25% CMC. Eventually, Frankia may play a key role in agricultural waste bioconversion and biofuel potential.