IntroductionLignin is considered one of the most prevalent polymers on earth. It naturally exists in plants as an integral part of lignocellulose to give them structure and rigidity1. Most technical lignins available today are byproducts of pulping processes, primarily kraft, soda, and sulfite methods2. Among them, the kraft process is the industry standard for paper and pulp production on a global scale. Each year, 130 million tons of kraft pulp are produced which may cause serious pollution to aquatic ecosystems upon direct disposal3. This abundant polymer can be broken down and transformed into molecules with higher value, allowing us to extract even more valuable products from it4.Lignin degradation can be accomplished by biological processes using entire cells (bacterial or fungal biomass) or ligninolytic enzymes5, offering an energy efficient, environmentally safe, and selective method6. While white-rot fungi produce powerful lignin degrading enzymes, their industrial applicability is limited7. In contrast, bacteria tolerate broader conditions and are easier for genetic modifications8,9.Laccase, lignin peroxidase (LiP), manganese peroxidase (MnP), versatile peroxidase (VP), and dye-decolorizing peroxidase (DyP) are examples of ligninolytic enzymes that can be produced by fungi and bacteria found in plant biomass and soil10,11. In biochemical research on ligninolytic bacteria, LiPs, MnPs, and VPs have not been observed. No homologs are discovered even when sequencing ligninolytic bacteria’s genomes or proteomes, implying fungi as their only known source. It has lately become obvious that DyPs, a different class of peroxidase, are quite abundant in bacteria. Although DyPs are structurally unrelated to the common fungal peroxidases, they do share several catalytic characteristics as well as redox potentials and reactivities12.The use of bacterial ligninolytic enzyme systems has been expanding rapidly over the last few decades due to their potential in industrial processes and environmental applications7,13. Recent research has focused on separating different ligninolytic bacteria for usage in a range of biotechnological processes, including bioremediation14, biofuel production15, and industrial enzyme development16. The ligninolytic enzymes hold great promise for industrial use in a variety of fields, such as food processing, cosmetics, aromatic compounds degradation, lignin valorization, and dye effluent decolorization17,18.The textile industry uses thousands of synthetic dyes to make fabrics. Over one-third of these are not absorbed by the fabric and end up in wastewater, threatening aquatic biodiversity and human health due to their toxicity and carcinogenicity19. Although physicochemical treatment techniques have been employed to eliminate dyes, they are not ecologically friendly nor commercially practical. On the other hand, biological methods are cheaper, effective, and sustainable for the environment20.Laccases and peroxidases, two ligninolytic enzymes, efficiently remove dyes, helping reduce textile-related water pollution21. Dye biodegradation entails intricate biochemical reactions that break down dye molecules into simpler, more biodegradable, and non-toxic byproducts20,22. Decolorization occurs when the dyes lose their chromophores as a result of repeated oxidation processes 23.Recent approaches emphasize the use of microbes or their enzymes to effectively treat textile dye effluents, incorporating different immobilization methods and bioreactor designs tailored for systems utilizing immobilized agents24. There are many reports on ligninolytic bacteria and their applications in dye degradation such as decolorization of methylene blue dye by a ligninolytic enzyme-producing Bacillus thuringiensis 25, multiple dye degradation by a newly isolated ligninolytic bacteria Bacillus paramycoides21, and decolorization of textile azo dyes by Streptomyces albidoflavus 3MGH26.Several studies have focused on optimizing the decolorization of various dyes, including Azure B (AB), methylene blue (MB), and Congo red (CR). Serratia liquefaciens, a lignin peroxidase-producing bacterium, demonstrated rapid decolorization of AB dye (100 mg/L) in mineral salt medium supplemented with glucose and yeast extract, achieving over 90% decolorization within 48 h27. Similarly, Bacillus sp. MZS10 achieved 93.55% decolorization of AB dye within 14 h in a stirred-tank fermenter, with the process being dependent on cell density and optimized medium conditions28. Regarding MB, thermophilic Bacillus licheniformis B3-15 and Bacillus sp. s7s-1 showed the ability to decolorize and detoxify MB dye in aqueous solution using preformed biofilms on polypropylene perforated balls (BBs). At optimized initial conditions (10 mg L−1 MB, pH 5.2 for B3-15 or pH 4 for s7s-1), the two strains enhanced their decolorization potential, reaching 96 and 67%, respectively, showing significant detoxification potential29. In case of CR, Alcaligenes faecalis H77, bacterium isolated from textile wastewater, proved to be highly effective in biodegrading CR. Through optimization process, it decolorized 92.5% of CR (25 mg/L) in 72 h at pH 6.7 and 35 °C30.Additionally, some studies have employed genomic and transcriptomic approaches to identify ligninolytic enzymes in diverse bacterial strains. In this context, whole genome sequencing (WGS) has become a critical tool for uncovering bacterial ligninolytic genes, such as those encoding DyP-type peroxidases, multicopper oxidases (laccases), and accessory enzymes involved in aromatic compound metabolism31. This technique enhances the understanding of microbial degradation mechanisms and supports the development of effective bioremediation strategies32. For instance, the genome of Pseudomonas Hu109A revealed key lignin-degrading genes, including DyP-type peroxidases, laccase, and O-methyltransferases. It also carries gene clusters for both medium- and short-chain polyhydroxyalkanoates (PHA) synthesis, showing its ability to convert lignin into valuable products and its potential role for industrial applications33. In another study,Klebsiella spp. strains isolated from lignocellulose-rich environments exhibited over 20 genes linked to lignin degradation pathways (including catalases, peroxidases, glutathione S-transferases and thiol peroxidases), with strain P3TM.1 achieving 98% decolorization of MB within 48 h, indicating robust ligninolytic activity34.Ongoing research aims to discover novel dye-degrading strains, optimize degradation conditions, and create bacterial consortia to increase the efficiency of degradation35. Yet few studies link bacterial screening with genomic analysis to correlate genetic potential with actual bioremediation process.The aim of this study is detection and characterization of soil bacteria for a candidate with potential ligninolytic activity in industrial applications, especially in dye effluent decolorization. The study also aims to apply WGS as a powerful tool to identify lignin-metabolizing enzymes and gain deeper insights into their genetic and functional potential.Materials and methodsSource of bacterial isolatesThe bacterial isolates were recovered from 55 soil samples collected from various agricultural sites throughout Egypt including the Rakta Pulp and Paper Company’s effluent treatment facility in Eltalbia, Alexandria, Egypt, these sites are supposed to be rich in ligninolytic bacteria. The soil samples were gathered in sterile plastic bottles of 100 mL volumes. The samples were obtained from the higher layers of the soil, which contain a substantial amount of the microbial community. After collection, samples were kept at 4 °C for subsequent use afterward36.Isolation of bacteria with potential ligninolytic activitiesEnrichment and isolates’ recoveryFirstly, the enrichment method was used to allow the bacteria present in low numbers to multiply and reach detectable levels. This was done by the addition of 5 g of soil sample aseptically in a previously autoclaved 250 mL Erlenmeyer flask containing 95 mL minimal salt medium enriched with kraft lignin (KL). The enrichment culture was composed of 1 g/L of commercially available KL (Sigma-Aldrich, USA) and 0.1 g/L yeast extract in phosphate buffered (pH 7) minimal salt medium of the following ingredients (g/L): 4.55 K2HPO4, 0.53 KH2PO4, 0.5 MgSO4, and 5 NH4NO3. After soil sample addition, the flasks were placed in an orbital shaking incubator at 30 °C and 200 rpm for one week. Secondly, for isolation of potential ligninolytic bacteria, a series of tenfold serial dilutions were made by adding 1 ml of enriched soil suspension to 9 ml of sterile normal saline and vortexing the mixture followed by transferring 100 μL aliquots of the produced dilutions to surface inoculation of minimal salt medium agar plates containing 1 g/L KL and 0.05 g/L nystatin (to inhibit fungal growth). The plates were then incubated at 30 °C until appearance of colonies, up to one week37. The grown colonies were suspected to have potential ligninolytic activity since KL was the sole carbon source in the medium.Purification and storage of bacterial isolatesBased on the variation of colony morphology, individual colonies were picked up and purified using the streak plate technique on minimal salt medium agar plates containing KL. Multiple subcultures were performed to obtain colonies that are pure and distinct38. The recovered isolates were kept in 25% glycerol at −80 °C for further investigation.Assessment of the recovered isolates for their ligninolytic activities using dye decolorization assaysSolid phase dye decolorization assayThis assay was performed by using some indicator dyes to evaluate the ability of the bacterial isolate to decolorize the dye. Luria–Bertani (LB) agar plates containing dye to final concentrations of 25 mg/L for each of Azure B (AB) and methylene blue (MB) or 50 mg/L for congo red (CR) were prepared. All of the indicator dyes were filter sterilized before being aseptically added to the autoclaved media39. Pure colonies of each tested isolate were streaked onto the LB agar plates containing dye. Following that, the plates were incubated for five days while being checked each day for the appearance of decolorization zones and/or stained growth (dye adsorption by the growth).Liquid phase dye decolorization assayThe bacterial isolates which had the ability to decolorize all the indicator dyes used in the previous assay, were subjected to the liquid phase dye decolorization assay. For each test isolate, single colonies from the growth obtained on LB plate were used for separate inoculation of 250 mL conical flasks containing 25 mL aliquots of LB broth. The flasks were placed in an orbital shaking incubator at 30 °C and 200 rpm until reaching the exponential phase, then pre-filtered sterilized dyes were aseptically added separately to the flasks at final concentrations of 6 mg/L for each of AB and MB or 40 mg/L for CR. Incubation was resumed for an additional 2 days under the same conditions. Control flasks without bacterial inoculation were included to account for spontaneous dye decolorization. After the completion of the experiment, samples were collected and subjected to centrifugation at 15,000 rpm for 10 min. The absorbance of each sample was estimated spectrophotometrically (Jenway 6800 UV/Vis spectrophotometer, UK) and the produced decolorization of the test dye was calculated as a percentage of its λmax absorbance using the equation shown below (Eq. 1). AB was measured at 650 nm and MB at 665 nm while CR at 470 nm.$$\mathbf{D}\mathbf{e}\mathbf{c}\mathbf{o}\mathbf{l}\mathbf{o}\mathbf{r}\mathbf{i}\mathbf{z}\mathbf{a}\mathbf{t}\mathbf{i}\mathbf{o}\mathbf{n}(\mathbf{\%})=\frac{{\varvec{A}}{\varvec{b}}{\varvec{s}}{\varvec{o}}{\varvec{r}}{\varvec{b}}{\varvec{a}}{\varvec{n}}{\varvec{c}}{\varvec{e}}\left({\varvec{c}}{\varvec{o}}{\varvec{n}}{\varvec{t}}{\varvec{r}}{\varvec{o}}{\varvec{l}}\right)-\mathbf{A}\mathbf{b}\mathbf{s}\mathbf{o}\mathbf{r}\mathbf{b}\mathbf{a}\mathbf{n}\mathbf{c}\mathbf{e}(\mathbf{t}\mathbf{e}\mathbf{s}\mathbf{t})}{{\varvec{A}}{\varvec{b}}{\varvec{s}}{\varvec{o}}{\varvec{r}}{\varvec{b}}{\varvec{a}}{\varvec{n}}{\varvec{c}}{\varvec{e}}({\varvec{c}}{\varvec{o}}{\varvec{n}}{\varvec{t}}{\varvec{r}}{\varvec{o}}{\varvec{l}})}\times 100$$Additionally, the visual examination of the pellet’s color after centrifugation was checked to determine whether the dye had been adsorbed to the cells rather than being degraded. The experiment was carried out in duplicate40.Bacterial identification by 16S rRNAThis was carried out for the bacterial isolates which exhibited the highest percentage of dye decolorization in liquid phase assay. The purified cultures of the tested isolates were sent to Biotech Serve Co. (Giza, Egypt), for DNA extraction, amplification of the 16S rRNA gene, and sequencing using the Sanger dideoxy sequencing method. The two primers used for PCR amplification of the 16S rRNA gene were universal bacterial primers 27F (5′-AGA GTT TGA TCM TGG CTC AG-3′) and 1492R (5′-TAC GGY TAC CTT GTT ACG ACT T-3′). The NCBI BLAST (BASIC LOCAL ALIGNMENT SEARCH TOOL; http://blast.ncbi.nlm.nih.gov/Blast.cgi) was used to assess DNA similarity and sequence alignment. The sequences with the highest identity score were then retrieved and aligned using multiple sequence alignment tool with the CLUSTAL W of MEGA software (Version 11.0.13). Then, the aligned sequences were used for the preparation of a phylogenetic tree using the same software (MEGA) following the maximum likelihood method41.Estimation of extracellular and intracellular ligninolytic activity for the test isolateThe dye was incubated with either the culture supernatant or crude cell lysate of the isolate that exhibited the highest activity in the previous assay to assess extracellular and intracellular activity, respectively. The experiment was carried out by culturing the isolate in both LB broth and lignin containing medium to induce the lignin degrading/dye-decolorizing enzymes40. Two pure colonies of the bacterial isolate were used for separate inoculation of 250 mL flasks containing 25 mL of either LB broth or Mineral Salt Medium supplied with 600 mg/L KL (MSM-KL), 10 g/L glucose, and 3 g/L peptone. The MSM was composed of the following components in g/L: K2HPO4, 2.0; Na2HPO4, 2.4; MgSO4.7H2O, 0.01; CaCl2, 0.01 and a trace element solution of 1.0 mL/L36. After that, the flasks were placed in an orbital shaking incubator at 30 °C and 200 rpm for 3–4 days. At the end of the incubation period, the contents of each flask were centrifuged at 4 °C and 7000 rpm in a cooling centrifuge for separation of culture biomass from culture supernatant. The biomass was subjected to sonication for the preparation of crude cell lysate. Both cell-free culture supernatant and crude cell lysate were used for determination of extracellular and intracellular ligninolytic activities, respectively as shown below.Testing extracellular decolorizing activity of the test isolateThe decolorizing activity was carried out using the cell-free culture supernatant42. The reaction mixture was composed of 0.5 mL of the culture supernatant, 0.5 mL of 0.160 mM AB, and 1.5 mL of 125 mM sodium tartrate buffer (pH 3.0) and the assay was carried out in test tubes. To start the reaction, 0.5 mL of 2 mM hydrogen peroxide was added to each test tube43. Static and shaking conditions were both carried out for the reaction over a period of 2 days. Control flasks were included to account for spontaneous dye decolorization. The absorbance of each sample was estimated spectrophotometrically at λmax (650 nm) and the produced decolorization activity was calculated as a percentage relative to control using Eq. (1).Testing the intracellular decolorizing activity of the test isolate using the crude cell lysateAfter harvesting the cell biomass from each flask, the cells were washed using phosphate buffered saline (PBS) and resuspended in 5 mL of PBS, sonicated by using an ultrasonic processor with an amplitude of 50% for 5–10 min, in cycles of 30 s on and 30 s off, while maintaining the sample on ice to prevent overheating. After that, the sample was centrifuged at 15,000 rpm for 5 min. The supernatant was collected, recentrifuged under the same condition and this process was repeated several times to remove any cell debris. The obtained clear supernatant representing the crude cell lysate was used for measuring the decolorization activity. The assay was carried out in test tubes and the reaction mixtures were composed of certain volumes of crude cell lysate (0.1, 0.5, 1, and 2 mL). To each reaction mixture, 0.5 mL of 0.160 mM AB was added, and the volume was completed with PBS to 2.5 mL. To start the reaction, 0.5 mL of 2 mM hydrogen peroxide was added to each reaction mixture. Static and shaking conditions were both carried out for the reaction over a period of 2 days. Control flasks were included to account for spontaneous dye decolorization. The assay was completed as mentioned earlier.Estimation of decolorizing activity of the test isolate under growth conditionsSingle colonies were used for separate inoculation of 250 mL conical flasks containing 25 mL aliquots of either LB broth or MSM-KL supplied with 10 g/L of glucose, and 3 g/L of peptone36. The flasks were placed in a shaking incubator for 3 days, and then pre-filtered sterilized AB dye was aseptically added to the flasks at final concentrations of 6 mg/L. Incubation was resumed for an additional one day. Control flasks without bacterial inoculation were included to account for spontaneous dye decolorization40. Samples were withdrawn at various time intervals (0, 4, and 24 h) following dye addition and subjected to centrifugation at 15,000 rpm for 10 min. The absorbance of each sample was estimated spectrophotometrically at λmax (650 nm) and the produced decolorization activity was calculated as a percentage relative to control using Eq. (1).Effect of different factors on growth and decolorizing activity of the test isolate using one-factor-at-a-time approach (OFAT)Preparation of seed and dye biodegradation flasksFresh seed culture (1–3 × 106 CFU/mL) was prepared for each parameter under investigation and used to inoculate dye biodegradation flasks containing 25 mL MSM-KL per 250 mL flask. In each experiment, after the predetermined incubation period, prefiltered AB dye was added at 6 mg/L and the flasks were re-incubated under the same growth conditions for 1 additional day. Samples were withdrawn at different time intervals (0, 4, and 24 h) following AB addition for determination of dye decolorization activity and microbial growth by dry cell weight (DCW) method. The decolorizing activity was performed by the quantitative assay and expressed as a percentage following the previously mentioned Eq. (1). The uninoculated medium was used as a control in all cases. All experiments were done in duplicate.Bacterial growth estimation by DCWTo determine the DCW, 1.5 mL aliquots were collected in pre-weighed centrifuge tubes and centrifuged, then the supernatants were carefully aspirated out while the cell pellets were dried at 70 °C till constant weight. After that, each tube was weighed to determine the dry weight of the cells by subtracting the initial weight from the final weight of each centrifuge tube44,45. The DCW was calculated and expressed as g/L.Studying the effect of nutritional and environmental factorsIn the conventional scaling-up approach, various nutritional and physical parameters were optimized by maintaining all factors at a constant level in the basal medium, except the one under study. Each subsequent factor was examined after taking into account the previously optimized factor(s)46. The impact of several parameters on the decolorizing activity of the selected isolate was investigated. The factors and their ranges were tested as follows:Inoculum size (% v/v): 1, 1.5, 2, and 2.5.Incubation time (day): 1, 2, 3, 4, 5, and 6.Agitation speed (RPM): 120, 160 and 200.Temperature (ºC): 25, 30, and 35.Carbon source (0.4% w/v): glucose, sucrose, starch, and the effect of no addition of extra carbon source was also assessed.Lignin concentration (mg/L): 200, 400, 600, 800, and 1000.Nitrogen source (0.4% w/v): tryptone, urea, peptone, ammonium nitrate, and yeast extract.Initial pH of the fermentation medium: 6, 8, 10, and 12.Optimization of dye decolorizing activity using design expert softwareResponse surface methodology (RSM), central composite design (CCD) was applied to optimize the environmental factors for the dye decolorization assay. Because the OFAT approach to optimization is time-consuming and tedious, RSM with CCD was selected for appropriate variation of key components. The model for decolorization of AB was implemented with the identification of likely interactions and optimal operating parameters. The effects of four independent factors, including lignin concentration (A), tryptone concentration (B), pH (C), and incubation time (D), each with three levels (low, medium, and high), on one response % decolorization of the dye as a dependent variable were examined in a total of 30 experiments. To determine the percentage of dye decolorization after four hours of dye addition, the combined impacts of four factors were examined in the designated ranges including lignin concentration (400–600 mg/L), tryptone concentration (1–7 g/L), pH (8–10), and incubation period (24–72 h).Table 1 also displays the experimental design matrix produced from the CCD model using DESIGN EXPERT (DESIGN EXPERT Software, v. 13.0.5.0, Stat-Ease Inc., Statistics Made Easy, Minneapolis, MN, USA) which contributed to the final model equation with observed and predicted responses.Table 1 The central composite design (CCD) runs in design expert Software for three selected levels of the four tested factors including lignin conc, tryptone conc, initial pH, and incubation time.Full size tableThe dye biodegradation media (MSM) of the RSM model-based experiments were prepared and distributed as 25 mL aliquots in 250 mL Erlenmeyer flasks. They were inoculated at 1.5% v/v mL of the seed culture and then incubated at the previously found optimum agitation speed and temperature. After incubation, samples were collected following four hours of dye addition to be tested for dye decolorization activity of the test isolate.Statistical and graphical investigationsTo determine how well the model fits the data, analysis of variance (ANOVA) was examined which included estimation of p-values, lack of fit F-values, adjusted and predicted R2. The model reduction was also implied to improve model statistics. Only factors with p-values lower than 0.05 were considered significant. To ensure model fitness, lack of fit p-value must be greater than 0.1. To illustrate the distinct and combined effects of parameters on the response, 3D response surface and contour graphs were displayed.Validation of the applied model dataThe final model’s equation and an examination of RSM plots were used to figure out and predict the maximum response values (highest decolorization percentages). Two optimized confirmation experiments were run, and the findings were compared with the values predicted by the equation to assess the model’s accuracy. The results obtained under optimum conditions and those obtained under non-optimized conditions were also compared.Detection of genes responsible for ligninolytic activityGenomic DNA extractionDNA extraction was carried out using Quick-DNA™ Bacterial Miniprep kit according to the manufacturer’s instructions.Quality control pre-testsThe genomic extract of the test isolate was then sent to Beijing Genomics Institute (BGI) Hongkong Tech Solution NGS Lab (Tai Po, New Territories, Hong Kong) by DHL to perform the whole genome sequencing process (WGS). Upon arrival, some quality control pretests were done such as determination of sample concentration, purity, and integrity. Concentration was detected by a fluorometer (e.g., Qubit Fluorometer, Invitrogen). Sample integrity and purity were detected by agarose gel electrophoresis (concentration of agarose gel: 1% voltage: 150 V, electrophoresis time: 40 min).Genome sequencing and assemblyThe genomic DNA extract of the selected test isolate was sequenced using a DNBSEQ G400 PE150 (MGI Tech Co., Ltd). The raw reads of low quality from paired-end sequencing (those with consecutive bases covered by fewer than five reads) were discarded. The sequenced reads were then assembled using Spades v 3.15.2 software.Bioinformatic analysis for detection of genes responsible for ligninolytic activityThe bioinformatics analysis was proceeded for annotation and detection of genes responsible for ligninolytic activity. Genome annotation is the process of identifying functional elements along the sequence of a genome. The FASTA file of clean reads after assembly was uploaded to BACTERIAL AND VIRAL BIOINFORMATICS RESOURCE CENTER BV-BRC (https://www.bv-brc.org/) for annotation of genes. The annotation service available in BV-BRC uses a modular, updated version of rapid annotation using subsystem technology (RAST)47 that is called the RAST toolkit (RASTtk)48. Genes related to lignin degradation and dye decolorization activity were identified and manually annotated by performing a BLASTP search against the ‘nr’ database.Submission of the next generation sequencing (NGS) reads to the national center for biotechnology information (NCBI)The FASTQ files of Next Generation Sequencing (NGS) readings were submitted to the National Center for Biotechnology Information (NCBI) to SRA database (https://submit.ncbi.nlm.nih.gov/about/sra/).ResultsIsolation of bacterial isolates with potential ligninolytic activityA total of 177 possible ligninolytic bacteria were retrieved from 55 soil samples that were collected from different locations in Egypt. Coded numbers were assigned to soil samples and bacterial isolates (Table 2).Table 2 Isolates recovered from the collected soil samples and their assessment for ligninolytic activity using solid phase dye decolorization assay.Full size tableAssessment of the recovered isolates for their ligninolytic activities using dye decolorization assaysSolid phase dye decolorization assayDye decolorization assay in solid agar plates revealed that 46 (25.9%), 60 (33.8%), and 51 (28.8%) of bacterial isolates showed decolorization zones with AB, MB, and CR, respectively (Fig. 1 and Table 2). As demonstrated in Table 2, some isolates on AB and MB plates showed dye adsorption, while others did not. However, all the tested isolates in the CR assay had adsorbed the CR dye on the growth surface.Fig. 1Decolorization zones of some representative isolates with potential ligninolytic activity on LB-dye agar plates. (a) AB (Azure B) for isolates 434, 304, 164, and 391; (b) MB (methylene blue) for isolates 434,304, 164, and 422; (c) CR (Congo red) for isolates 434, 111, 414, and 422.Full size imageLiquid phase dye decolorization assayLiquid phase assays were carried out for a total of 16 bacterial isolates which gave positive results on the three different types of dyes in solid phase assay (Table 3). The liquid assay of AB showed that isolates 434 and 304 have the highest decolorization capacity with percentages of 98.5 and 72.5%, respectively, and of 86.3 and 81%, respectively in MB assay after 2 days of incubation with the dye. However, in the CR assay, isolate 391 showed the highest decolorization activity by 29.2% (Fig. 2). Dye adsorption on the pellets’ surface after centrifugation was also monitored and the results are shown in (Fig. 2). Similar to the results obtained in solid phase dye decolorization assay, all the tested isolates in CR assay showed adsorbed dye on the pellets’ surface.Table 3 Summarized results showing bacterial isolates with decolorization capacity on all methylene blue-, Azure B- and Congo red- LB agar plates.Full size tableFig. 2Decolorization percentages produced by the tested isolates in LB broth containing Azure B (AB), methylene blue (MB), or Congo red (CR) as determined in liquid phase assay. (a) Isolates showed adsorption of dye on pellets’ surface after centrifugation.Full size imageMolecular identification of bacterial isolatesThe 16S rRNA gene was sequenced to identify two selected isolates (434 and 304) that had the highest decolorization activity for the tested dyes. Isolate 434 could be identified as Streptomyces intermedius since it showed 99.86% homology of the 16S rRNA nucleotide sequences with Streptomyces intermedius strain DSM 40372 (NR_119347.1). While isolate 304 could be identified as Streptomyces griseorubens with 99.93% homology of the 16S rRNA nucleotide sequence of Streptomyces griseorubens strain NBRC 12780 (NR_041066.1). The partial nucleotide sequences of 16S rRNA genes of the two isolates (434 and 304) were submitted and deposited in the NCBI GenBank database under the accession numbers OQ928560 and OQ928561, respectively. According to the phylogenetic tree (Fig. 3), S. intermedius and S. griseorubens are the closest strains in similarity to the tested isolates, 434 and 304, respectively.Fig. 3The phylogenetic tree of isolates 434, and 304 based on 16S rRNA genes sequence.Full size imageExtracellular and intracellular ligninolytic activity for the test isolateExtracellular decolorizing activity of the test isolateNone of the culture supernatants from either LB or lignin containing medium grown culture exhibited significant dye-decolorizing ability (not more than 10% decolorization could be detected) for either static or shaking assay conditions after 2 days of incubation with the dye.Intracellular decolorizing activity of the test isolate using the crude cell lysateThe results for the crude cell lysate obtained from LB culture showed no decolorizing activities after 2 days of incubation with the dye, while the corresponding results of lignin containing medium’s culture showed slightly decolorizing activities after 2 days of incubation with the dye (less than 5% decolorization).Decolorizing activity of the test isolate under growth conditionsAfter 72 h growth age of S. intermedius isolate, the addition of AB dye resulted in decolorization capacity in LB medium of 0.002, 0.06, and 30% and in MSM-KL of 6.8, 21, and 41% following dye addition and re-incubation under the same growth conditions for 0, 4, and 24 h, respectively.Effect of different factors on growth and decolorizing activity of the test isolate using one-factor-at-a-time approach (OFAT)Effect of different inoculum sizesThe result of the decolorization assay at 4 and 24 h following the addition of dye to 72 h age culture and re-incubation under the same growth conditions showed that the highest decolorization capacity was 22.69 and 39.18% upon using 1.5% v/v inoculum size, respectively (Fig. 4A).Fig. 4Effect of different inoculum sizes (A) and different incubation times (B) on decolorization percentage of AB after 0, 4, and 24 h of dye addition to the grown culture of S. intermedius test isolate. Lines above columns indicate standard deviation values.Full size imageEffect of incubation timeTwo days of incubation growth period produced the maximum decolorization activity, which was 24.7 and 44.92% after 4 and 24 h following the addition of dye and re-incubation under the same growth conditions, respectively (Fig. 4B).Effects of agitation speed and incubation temperatureAs demonstrated in Fig. 5A,B, the maximum decolorization activity was obtained at 160 rpm and 30 ºC which was 33.58 and 47.18% at 4 and 24 h following dye addition to 2 days age culture and re-incubation under the same conditions, respectively.Fig. 5Effect of agitation speed (A) and different temperatures (B) on decolorization percentage of AB after 0, 4, and 24 h of dye addition to the grown culture of S. intermedius test isolate. Lines above columns indicate standard deviation values.Full size imageEffect of carbon sourceThe maximum activity for dye decolorization was observed by no addition of extra carbon source to MSM-KL. These maximum activities were 25.65 and 50.84% at 4 and 24 h following the addition of dye to 2 days age culture and re-incubation under the same growth conditions, respectively (Fig. 6A).Fig. 6Effect of carbon source (A) and lignin concentrations (B) on decolorization percentage of AB after 0, 4, and 24 h of dye addition to the grown culture of S. intermedius test isolate. Lines above columns indicate standard deviation values.Full size imageEffect of lignin concentrationThe ability to decolorize AB by 19.07 and 54.92% at 4 and 24 h following dye addition, respectively, was demonstrated by a lignin concentration of 600 mg/L (Fig. 6B).Effect of nitrogen sourceThe highest activity was obtained when using tryptone as a nitrogen source by 38.69, and 55.17% followed by peptone and yeast extract as shown in Fig. 7A at predetermined assay time measurements.Fig. 7Effect of some nitrogen sources (P: Peptone, T: Tryptone, YE: Yeast extract, AN: Ammonium nitrate, and U: Urea) (A) and different pH (B) on decolorization percentage of AB after 0, 4, and 24 h of dye addition to the grown culture of S. intermedius test isolate. Lines above columns indicate standard deviation values.Full size imageEffect of initial pHAfter 4 and 24 h of dye addition, pH 8 and 10 showed close decolorization activity by 34.27, 56.59, and by 34.92, 56.76%, respectively (Fig. 7B).Statistical optimization of process parameters, analysis, and validationRSM (CCD) was used to perform comprehensive research on dye decolorization by the selected S. intermedius test isolate. The design included 16 factorial points, 8 axial points, and 6 repetitions at the center point for the estimation of the pure error sum of squares. Analysis of variance (ANOVA) for percentage decolorization given in Table 4 revealed that the quadratic model is highly statistically significant, as indicated by the model F value of 24.64 which means that there is only a 0.01% chance that the value is significant by chance. the p- value is a measure of a test’s statistical significance; a value below 0.05 denotes a test parameter’s significance at the 5% level, and multiple model variables were significant (p