Sustainable bioconversion of sugarcane bagasse into valorizable riboflavin by Microbacterium proteolyticum BWBTDIPO1, characterization and in-silico mechanistic pathway prediction

Strategic bioconversion of lignocellulosic agro-wastes like sugarcane bagasse (SCB) into high-value Riboflavin is a rarely reported phenomenon. The present study explored a Riboflavin-producing bacterial strain, Microbacterium proteolyticum BWBTDIPO1 (GenBank Acc no.: PQ517523), isolated from the dumping area of Kolkata, West Bengal for its caliber to utilize SCB as a source of carbon and energy for the sustainable bio-production of the valuable nutraceutical under submerged fermentation (SmF) conditions. A strong association of bacterial growth (specific growth rate of μ = 0.325 h -1) with production of Riboflavin (yield of 397 ± 15.8 mgL - 1, which amounts to 19.85 ± 2.0 mg per gram of SCB after 78 h) was recorded. Yeast extract peptone mineral salt media (YPMSM) was found to be the most suitable medium for the production. UV-Vis spectrophotometry and thin-layer chromatography (TLC) with an R f value of 0.83 confirmed that the metabolite was Riboflavin. The biochemical assays revealed that the strain utilized SCB components, including lignin, cellulose, and hemicellulose. Further confirmation of biomass deconstruction was assessed via FTIR, XRD, and FESEM analysis, where reports ensured successful deconstruction of SCB by the bacterial isolate. To complement the experimental findings, KEGG-based in silico pathway analysis was performed to explore potential metabolic routes associated with lignocellulosic biomass utilization and carbon metabolism. The predicted pathways provide a hypothetical framework for understanding biomass deconstruction and require experimental validation. This is the first report of Riboflavin production from waste SCB via bacterial treatment (M. proteolyticum), indicating the novel nature of the strain and the methodology employed. Valorizable Riboflavin obtained from waste SCB can be a green alternative to chemical Riboflavin synthesis as well as bulk SCB waste management, promoting the concept of microbe-mediated waste-to-wealth conversion, thus contributing to circular bioeconomy.

The rapid expansion of agricultural industries has resulted in the accumulation of billions of tons of agro-waste worldwide in the last 2 decades (Sharma et al., 2022). Lignocellulosic wastes have a significant impact on global pollution levels and pose global waste management concerns. India is one of the leading producers of sugarcane, as it is a major cash crop, with an expected annual production of 421,559.43 thousand tonnes by the end of 2030 (Supriya et al., 2024). Sugarcane bagasse (SCB), which is primarily composed of lignin, cellulose, and hemicellulose, is one of the most abundant, nutrient-rich biomass materials that can serve as an excellent substrate and carbon and energy source for microbial fermentation (Jin et al., 2023). Versatile microorganisms have been reported to deconstruct the recalcitrant LCBs through enzymatic hydrolysis of the complex polymers present. These include several species like Pseudomonas putida, Serratia marcescens, Planococcus sp., etc., that use enzymes like lignin peroxidase, manganese peroxidase, laccase, etc., for the purpose (Zhou et al., 2025; Majumdar et al., 2020; Priyadarshinee et al., 2017). The bioconversion of SCB into high-value metabolites using microbes has attracted scientific attention for its eco-friendly, cost-effective applicability across industries. One of the value-added nutraceuticals is Vitamin B2 (Riboflavin). The production of Riboflavin by microorganisms has remained underexplored and less reported under natural conditions, except for some strains like Lactobacillus plantarum, Bacillus subtilis, etc. (Perez Visnuk et al., 2022; Chu et al., 2022). In 2025 Microbacterium proteolyticum was reported to produce high yield of Riboflavin of up to 459.31 ± 22.79 mg/L without any genetic modifications (Paul et al., 2025). Most cases of bacterial Riboflavin production reportedly involve genetically modified organisms for the overproduction of the metabolite, like E coli, Corynebacterium glutamicum (Liu and Sun, 2025; Brito et al., 2025). The applications of Riboflavin are wide-ranging, from pharmaceutical, nutraceutical, cosmetic, food, and feed industries.

Despite enormous commercial demand, the production of Riboflavin still remains challenging, specifically from organic sources. The conventional chemical synthesis of Riboflavin involves complex, multistep processes that require chemical treatment, expensive catalysts, and energy-intensive operations (Liu et al., 2023). As a consequence of the drawbacks of conventional processes, biosynthesis from microbial sources via sustainable alternatives has gained popularity. Yet the production of Riboflavin from lignocellulosic wastes, such as SCB, has not yet been explored and warrants further attention. Hunting for microbes with the required cellulolytic and ligninolytic metabolic potential and enzymatic machineries is a need of the hour (Zhang et al., 2025). There exists a significant research gap when it comes to the production of Riboflavin from SCB biomass. Efficient coupling of biomass deconstruction with commercially important Riboflavin production can be achieved through advanced microbial bioprocesses (Bashandy et al., 2022).

The present investigation presents a green approach to integrate microbial fermentation for SCB biomass valorization and bioconversion into commercial Riboflavin. This includes suitable media selection, bacterial growth kinetics, production time kinetics, physicochemical characterization of the produced metabolite and deconstructed biomass, followed by predictive bioinformatics-based pathway analysis. The pathway analysis was incorporated to provide a mechanistic framework for interpreting the observed biomass deconstruction and to identify potential metabolic routes that may facilitate the utilization of lignocellulosic components and support Riboflavin biosynthesis. The strain was explored for Riboflavin production in our previous work (Paul et al., 2025). To the best of our knowledge, this is the first report of SCB utilization by M. proteolyticum for the simultaneous deconstruction of SCB and the production of valuable Riboflavin. This contributes to the development of sustainable production processes from waste agrobiomass, thus bringing new insights into the phenomenon of waste-to-wealth and circular bioeconomy. This work has potential applicability of bacterial LCB bioconversion for industrial and biorefinery processes.

Materials

All the chemicals used were of analytical grade and purchased from Sigma-Aldrich (USA), Hi-Media (Germany), and Merck (Germany). The glassware and plastic consumables used were from Borosil, Tarsons, and Nest. Kerosene and other oils were purchased from the local market. Sugarcane bagasse was collected from local vendors and sugarcane juice sellers.

Bacterial culture maintenance

The bacterial isolate used in the present study was isolated from a waste-dumping zone of Barasat Regent Garment Factory, dating back to 2025 (Paul et al., 2025). Identified as M. proteolyticum BWBTDIPO1 (GenBank Acc no.: PQ517523). The bacterial culture was preserved on nutrient agar plates (stored at 2 °C–8 °C) and in 50% Glycerol (stored at −80 °C) for further use during the investigation.

Pretreatment and processing of sugarcane bagasse (SCB)

In the current investigation, SCB was employed as an agro-industrial substrate. SCB was purchased from sugarcane juice vendors in the local market. As described by Chindaprasirt and Rattanasak (Chindaprasirt and Rattanasak, 2020). Fresh SCB with a white-yellow pale appearance, devoid of any visible fungal spotting, was carefully selected for the experiments. The SCB was thoroughly washed with distilled water three times to remove dust particles and superficial impurities. The washed substrate was oven–dried at 70 °C for 24 h and ground to obtain a finely powdered, ready-to-use material for further experiments (Charoensopa et al., 2024). A sieve of 0.5 mm mesh size to confirm uniform particle size (Thite and Nerurkar, 2019). The pretreated SCB was utilized for further experimentation. This approach is categorized as milling pretreatment (grinding), which aims to reduce particle size for increased bioavailability (Chakraborty et al., 2024; Kumar et al., 2022). The ground biomass was subjected to autoclave sterilization, which can also be referred to as a thermal pretreatment (121 °C for 15 min). The pretreated biomass was utilized for further experimentation and analysis.

Media selection for growth and riboflavin production

The evaluation of the most suitable media for the production of Riboflavin under submerged fermentation (SmF) conditions was carried out using different mineral salt media (MSM) compositions. Basic MSM composition consisted of KH 2 PO 4 1.2 gl −1, K 2 HPO 4 4.8 gl −1, MgSO 4 0.25 gl −1, FeSO 4 ·7H 2 O0.0025 gl −1, CaCl 2 ·2H 2 O 0.026 gl -1, and NaCl 5 gl −1 (Paul et al., 2022). The pretreated SCB biomass was incorporated into the prepared MSM as the sole carbon source. Different nitrogen sources were tested in the medium formulations, namely yeast extract (5% w/v) and peptone (5% w/v), individually and in combination (Lal et al., 2022). So, the four different types of media were prepared: MSM (mineral salt media), YMSM (Yeast extract MSM), PMSM (Peptone MSM), and YPMSM (Yeast extract peptone MSM). Prepared media, along with the supplemented biomass (5% w/v), were autoclaved to eliminate any microbial contamination. Sterile media were cooled to room temperature and aseptically inoculated with an overnight activated bacterial culture of M. proteolyticum BWBTDIPO1. Submerged fermentation was carried out for 5 days under optimized conditions (120 rpm, 30 °C) to facilitate proper growth and Riboflavin biosynthesis. Samples were periodically withdrawn to monitor growth kinetics and assess the rate of Riboflavin production. Viable cell counts were taken after 24 h by measuring colony-forming units (CFUs) using the serial dilution method (Ramadhani et al., 2023). Riboflavin production was quantified spectrophotometrically at 266 nm (λ max). The best media for growth and production were chosen for further studies (LabMan Scientific Instruments Pvt. Ltd., India Model No.: LMSP-UV1900).

Bacterial growth kinetics

The evaluation of bacterial growth kinetics during SmF was conducted to determine the behavior of the microbial strain when using SCB as a primary lignocellulosic substrate for Riboflavin production. An inoculation of 4% (v/v) from an actively growing overnight culture was done under aseptic conditions, followed by SmF, in yeast-extract peptone mineral salt media (YPMSM) with pretreated SCB, under optimized incubation conditions (120 rpm, 30 °C). Serial dilution and spread plating on nutrient agar media were performed for the CFU mL -1 count. CFU values were plotted against time to obtain the bacterial growth curve, which was further analyzed to identify its distinct phases (Oliveira et al., 2023). The specific growth rate (µ) of the strain was calculated using the traditional growth kinetic model following the differential equation stated below:

Where X represents viable cell count (CFU mL -1), T is the fermentation time, μ is the specific growth rate constant (h -1), K represents cellular decay coefficient (h -1). Since the value of decay constant is negligible during the exponential phase of growth, this equation is simplified as below:

Rearrangement and integrating the above equation, we get:

Where X f and X i correspond to final and initial CFU measured (mL -1), respectively. While, t f and t i are the final and initial times of fermentation.

These kinetic parameters, calculated from this expression, were subsequently used to correlate with Riboflavin production profiles to simultaneously understand growth and production kinetics.

Riboflavin production kinetics and yield calculation in YPMSM

Quantitative estimation of Riboflavin production under SmF conditions was performed spectrophotometrically using a standard curve prepared from commercially purchased Riboflavin. YPMSM was used as the media in this study. Fermentation broth was aseptically withdrawn at regular intervals for the purpose (Lal et al., 2022). The collected broth was filtered through Whatman filter paper to separate debris and SCB biomass (Namnuch et al., 2021). The filtrate was centrifuged at 7,000 rpm for 15 min, and the resulting clear supernatant was used for estimation of the final product yield in mg mL -1. For spectrophotometry, 0.8 mL of culture supernatant was mixed with 0.2 mL of NaOH to stabilize the chromophore. This reaction mixture was neutralized using 2.5 mL of 0.1 M potassium phosphate buffer (pH 6.0). Analytical-grade Riboflavin was procured from the market, and standard solutions were prepared within the concentration range 0.1–1.0 mg mL -1. Absorbance was recorded at 266 nm (λ max), and a calibration curve was prepared for the determination of Riboflavin yield (Jiménez-Nava et al., 2023). Production kinetics was carried out by Riboflavin concentration calculation at frequent intervals, simultaneously with the growth curve analysis as described in Section 2.5. Yield was calculated as substrate conversion efficiency, i.e., yield per Gram of SCB.

Extraction of riboflavin, its biochemical and physicochemical characterization

The extraction of Riboflavin was carried out using a standardized protocol described in our previous study, with slight modifications to accommodate the requirements of the present fermentation systems. Cultured broth was filtered through Whatman filter paper to remove residual biomass contents. Filtrate was centrifuged at 7,000 rpm for 15 min to obtain a clear metabolite-containing supernatant. The clear supernatant was used for acidified ethanol extraction (Paul et al., 2025). As a preliminary biochemical characterization technique, UV-Visible spectrophotometry was performed to confirm the chemical nature of the metabolite. The extracted Riboflavin was then dissolved in ethanol and scanned between 200–700 nm. The obtained spectra were analyzed for the presence of Riboflavin by a comparative study with existing reports on Riboflavin.

To further confirm the physicochemical nature of the metabolite, Thin Layer Chromatography (TLC) was performed. Extracted metabolite and commercially purchased Riboflavin were individually spotted on the pre-coated silica gel plates. Chromatographic separation was performed using a mobile phase composed of methanol: water (70:30, v/v) (Sikdar et al., 2024). After solvent migration, the developed chromatograms were dried and visualized. Comparative evaluation of the R f values revealed the nature of the extracted metabolite. Details of the biochemical and physicochemical characterization employed in the previous investigation were retained in the current investigation to ensure comparability between the methodologies.

Assessment of lignocellulosic biomass (LCB) deconstruction

The capability of M. proteolyticum BWBTDIPO1 to deconstruct LCB was determined by evaluation of the major structural components of LCB, namely, lignin, cellulose, hemicellulose, and reducing sugar fractions. Following SmF, the treated and untreated fractions were compared after incubation under optimized conditions for 96 h. Samples were collected after incubation and extensively washed with distilled water to remove residual media components, soluble metabolites, and attached bacterial biomass. The washed biomass was dried and subjected to compositional analysis.

Determination of alkali lignin degradation

Alkali lignin degradation was evaluated by the alkaline extraction of lignin from both treated and untreated SCB samples. The biomass was boiled in water for 4 h to remove water-soluble impurities and other extractives. The insoluble residue obtained was filtered and treated with a 15% (w/v) NaOH solution at a biomass-to-solvent ratio of 1:15–1:18. This mixture was allowed to stand for 4 h to facilitate the solubilization of alkali lignin. On successful dissolution of lignin fractions, a dark coloration is observed in the filtrate (Wu et al., 2022). The absorbance of the extracted alkali lignin was recorded at 465 nm in a UV-Vis spectrophotometer. The percentage degradation of alkali lignin was calculated by comparison of the optical density (OD) values of treated and untreated SCB samples following the given equation:

Determination of acid-soluble and acid-insoluble lignin degradation

Acid hydrolysis was done for the quantitative assessment of acid-soluble lignin (ASL) and acid-insoluble lignin (AIL) fractions within the SCB (Zhang et al., 2022). 1 g of dried biomass was treated with 10 mL of sulfuric acid (72%) containing ammonium nitrate and was maintained at room temperature for 6 h. This reaction mixture was gently agitated at regular intervals of 30 min to ensure uniform hydrolysis of the LCB components. After the primary hydrolysis treatment, 84 mL of dH 2 O was added to dilute the acid concentration (Bellary et al., 2023). This mixture was autoclaved at 121 °C to achieve complete hydrolysis of the components, specifically the structural polysaccharides. After cooling down to room temperature, this was filtered using Whatman filter paper. The filtrate was analyzed spectrophotometrically at 320 nm to determine the acid-soluble lignin fraction (Ruwoldt et al., 2022). On the other hand, analysis of the solid residue weight during drying revealed the amount of acid-insoluble material present. A comparative analysis between treated and untreated samples indicated the degradation efficiency. The ASL and AIL were calculated using the following expressions:

Here, W denotes the dry weight of lignin fractions.

Determination of hemicellulose degradation

The percentage of hemicellulose degradation was evaluated using a standardized methodology with slight modifications. 1 g of biomass was treated with 150 mL of NaOH solution (500 mmol L -1) and boiled for 3 h to solubilize the hemicellulosic fraction. After the treatment, the mixture was cooled to room temperature and filtered through Whatman filter paper (Zhang et al., 2024). The remaining residue was repeatedly washed with distilled water till the pH was set to neutral. The biomass was then dried in hot air oven at 105 °C till a constant weight was achieved. The difference in weight between the biomass of treated and untreated samples was calculated to determine the degradation of hemicellulose (Hamin et al., 2023).

Determinations of reducing sugar content by the DNS assay

The difference in the reducing sugar content of SCB following bacterial treatment is an indirect estimation of cellulose and hemicellulose utilization. Given the fact that cellulose and hemicellulose are polysaccharides of non-reducing sugars, the hydrolysis of these polymers can result in the release of monomers (monosaccharides) like glucose, xylose, arabinose, mannose, etc. Hence, quantifying reducing sugars provides direct insight into microbial depolymerization and utilization of these carbohydrates in LCB (Pratami et al., 2022; Saha et al., 2025). The estimation of reducing sugars was performed using the DNS (3,5-dinitrosalicylic acid) assay. DNS reagent preparation was done according to the standardized formulation (1 g of 3,5-dinitrosalicylic acid, 30 g of sodium potassium tartrate, 0.5 g of sodium sulfate, and 2 g of sodium hydroxide in 100 mL of distilled water). Standard solutions of glucose were prepared for plotting the calibration curve in the range 0.2–1.0 mg mL -1. 1 mL of hydrolyzed sample (SCB) was mixed with 1 mL of DNS reagent and treated in a boiling water bath for 10 min until the development of a reddish-brown color (Kininge and Gogate, 2025). After cooling to room temperature, 10 mL of distilled water was added to the reaction mixture, and absorbance was measured at 540 nm in a spectrophotometer. Reduced sugar concentrations were calculated from the standard curve.

Physicochemical characterization of deconstructed LCB

LCB biomass, like SCB, undergoes significant changes following bacterial treatment, and evaluating these structural modifications is necessary to understand the microbial strain’s capacity to deconstruct the recalcitrant LCB matrix. Structural characterization of treated and untreated LCB in YPMSM was carried out using three major analytical approaches. FTIR (Fourier Transform Infrared) spectroscopy, XRD (X-ray Diffraction), and FESEM (Field Emission Scanning Electron Microscopy) were performed for this purpose (Majumdar et al., 2020). For FTIR, untreated and bacterially treated samples were thoroughly dried and pulverized into a fine powder. This powdered biomass was mixed homogeneously with spectroscopic-grade KBr (Potassium bromide) in a standardised ration 10:90 and compressed into pellets for analysis. FTIR spectra were recorded over the 400–4,000 cm -1 range using a standardized procedure. Comparative interpretation of the spectra obtained was used to identify alterations in glycosidic, hydroxyl, carbonyl, and aromatic ether linkages associated with microbial deconstruction.

XRD was performed to observe modifications in the crystallographic structure of the LCB. XRD patterns were recorded using the Xpert PRO Diffractometer. Diffractograms were recorded over the scanning range corresponding to the cellulose diffraction peaks at a scanning rate of 6° min -1. The crystallinity index (CrI) of SCB samples was calculated using the Segal method, which follows the given expression (Khan et al., 2026):

Where CrI represents the crystallinity index (%), I 002 denotes the maximum intensity of the crystalline lattice plane, approximately 22.5°, and I am represents the intensity of the amorphous region at around 19°.

Additionally, the crystallite size (t, nm) of cellulose can be estimated using the Scherrer equation with respect to the 200 crystallographic plane records:

Here, K stands for the shape of the correction factor (0.91), λ is the X-ray radiation wavelength, β 1 / 2 represents the full width at half maximum intensity (radians) and θ is the diffraction angle.

Surface morphology and relative changes in SCB after bacterial treatment were observed via FESEM (The morphological features revealed visualization of physical disruptions of LCB surface. FESEM analysis was carried out following standard protocol (Gupta et al., 2021).

In-silico prediction of metabolic pathway for LCB deconstruction

Metabolic pathway analysis of lignocellulosic biomass degradation was performed using KEGG global metabolic pathway mapping to identify potential metabolic routes that may be involved in lignocellulosic biomass utilization (Li et al., 2024). The resulting pathways represent predictive annotations based on database information and literature reports and should not be considered experimentally validated metabolic mechanisms. The metabolic network map was analyzed to identify pathways potentially associated with degradation of lignin, cellulose, and hemicellulose by M. proteolyticum. Relevant metabolic regions corresponding to lignin-derived aromatic compound metabolism, cellulose hydrolysis and glycolysis, and hemicellulose-associated pentose metabolism were manually highlighted using image-based annotation in Python/Google Colab (Kanehisa et al., 2017). The highlighted pathways were categorized into three major biomass conversion routes: (i) lignin → aromatic monomers → TCA cycle, (ii) cellulose → cellobiose → glucose → glycolysis, and (iii) hemicellulose → xylose/arabinose → pentose phosphate pathway (Moraes et al., 2018).

Statistical analysis

All data presented in the investigation are the mean ± SD of triplicate measurements (n = 3). The results were interpreted and represented through Microsoft Excel 365. Probability tests were conducted to determine the degree of difference between the means. p-value < 0.05 was considered statistically significant.

Media selection for growth and riboflavin production

The selection of the most suitable fermentation medium plays a crucial role in determining the final output capacity of the bioconversion process. In the current study, among the four formulations of mineral salt media used, yeast extract peptone MSM was found to be the most suitable medium for bacterial growth and Riboflavin production under SmF conditions. Intermediate growth and production were observed in the other media types, all of which were supplemented with SCB. The superior performance might be due to the synergistic nutritional benefits of yeast extract and peptone, which support flavin biosynthesis required for Riboflavin production. This has been observed in case of natural pigment production from agro-industrial waste by Penicillium brevicompactum (Basto et al., 2022). Compared with MSM, the poor performance of MSM indicated that SCB alone could not support adequate growth and production. Similar observations have been previously reported by Mahajan and Nikhanj in 2025, where biopigment production using potato waste agrowaste was boosted in the presence of yeast extract and peptone supplements (Mahajan and Nikhanj, 2025). Nitrogen availability is an important factor affecting the synthesis of enzymatic machinery required for the deconstruction of LCB, as reported earlier by Berikashvili in 2025, where yeast extract and peptone were found to play a major role in increasing the availability of nitrogen in the deconstruction process (Berikashvili et al., 2025). The superior performance of YPMSM can be attributed to the readily available sources of vitamins, nucleotides, peptides, and essential growth factors present in it that help in the synthesis of essential biomolecules, enhanced metabolic activity, and microbial growth (Chen et al., 2022). Different microbes have shown the capability to degrade lignin and participate in LCB deconstruction with similar nutrition requirements. Inotnotus obliquus was found to degrade wheat straw to produce methionine and crude proteins as valuable products. (Zang et al., 2022). Pseudomonas stutzeri was found to degrade crop residues, specifically lignin fractions, using a medium supplemented with yeast extract and peptone (Minhas et al., 2025). Exploring the strain further and determining its growth and production characteristics was necessary for developing an understanding of optimal parameters for large-scale industrial production.

Growth kinetics of Microbacterium proteolyticum BWBTDIPO1

The growth kinetics of M. proteolyticum BWBTDIPO1 were evaluated in the most suitable media, which was found to be YPMSM supplemented with SCB (pretreated). As depicted in Figure 1, the bacterial growth profile exhibited a sigmoidal pattern, which is typically observed in batch fermentation systems, hence indicating the successful utilization of SCB as a carbon source. Distinct lag, exponential, and stationary phases were observed in the curve. The bacterial population remained relatively stable until the 16th hour, indicating a long lag phase, which might have resulted from adaptation of the strain to SCB as the carbon source. SCB is a complex organic source and is recalcitrant due to the presence of lignin, polymeric cellulose, and hemicellulose, requiring the secretion of hydrolytic enzymes for its deconstruction. A pronounced exponential phase was observed from the 18th to the 40th hour of fermentation, during which rapid growth occurred, indicating the phase of maximum metabolic activity and active cellular growth, reflecting the efficient deconstruction of the SCB biomass components. The specific growth rate was calculated to be 0.325 h -1, with a generation time of 2.13 h. Although M. proteolyticum has not yet been reported to utilize SCB as a carbon source, this is the first report to reveal this rarely or not yet explored potential. Value-added products, such as lipase, have been extracted from this species, with implications for biodiesel production (Tripathi et al., 2014). This novel strain was the first of its kind reported to participate in Riboflavin biosynthesis (Paul et al., 2025). The growth entered the stationary phase after 40 h and continued until the 60th hour, indicative of reduced nutrient availability but sustenance to the complex microenvironment. It is often observed that metabolites continue to be produced during the stationary phase, even if there is a lack of overall growth, and cell density remains constant. A complete understanding of the comparative kinetics of growth vs. production is required to evaluate the key production phases during the fermentation process, as this is essential information for large-scale industrial production. These give the necessary understanding of the accurate timing of cell harvesting and product recovery within the manufacturing timeline. A bacterial growth curve for Riboflavin production on SCB has not yet been reported by any other group; hence, no reference exists for this typical fermentation process. But a similar pattern of growth was observed on SCB, while production of other metabolites like beta-carotene, biosurfactant, etc., Was produced by different bacterial species utilizing SCB as a carbon source (Gupta et al., 2026; Díaz-Ruiz et al., 2024). Yield or the production rate plays a major role in deciding the economic suitability of the process. Correct estimation of yield is an aspect that must be accurate and reliable for upscaling operations.

Riboflavin production kinetics and yield estimation under SmF conditions

The Riboflavin production kinetics of M. proteolyticum BWBTDIPO1 under SmF conditions revealed a progressive increase in the accumulation of the metabolite throughout the fermentation period (Figure 1). A comparative analysis of growth and production demonstrated a close association between biomass formation and Riboflavin synthesis, specifically during the active phase of growth and metabolic activity. Production remained relatively low during the first 12 h, which corresponded with the lag phase. This phase, which generally is meant for cellular adaptation and physiological acclimatization, activates different pathways meant for LCB deconstruction. After approximately 18 h of fermentation, a significant increase in the production of Riboflavin was observed, which continued till 48th hour, indicating enhanced metabolic activity towards flavin biosynthesis. The steep increment in production also indicated the bacterial utilization of SCB as its energy substrate. Decline in production was observed after 48 h but the production of Riboflavin went on till late exponential and early stationary phases, reaching the highest yield at 78th hour (397 ± 15.8 mgL - 1, which amounts to 19.85 ± 2.0 mg per gram of SCB). This also indicated the presence of growth-associated as well as non-growth-associated production of Riboflavin. The overall production profile suggested a strong capacity of the current strain to utilize waste SCB biomass for bioconversion into Riboflavin. Compared to the yield obtained in the previous study, where the medium was Luria Bertini (LB) broth, the maximum yield of 459.31 ± 22.79 mg/L was achieved, the yield was found to be less in the current investigation (Paul et al., 2025). The composition of SCB as a carbon source is much more complex in comparison to the glucose present within the LB broth. Although taking into consideration the cost of production of Riboflavin has been controlled in this study, since zero cost waste agro biomass SCB has been used instead of synthetic medium. Other species such as Bacillus subtilis and Novosphingobium panipatense have been reported to produce high amounts of Riboflavin by utilizing waste produced after yeast fermentation (Bashandy et al., 2022). Novosphingobium panipatense was reported to give a high yield of Riboflavin (497.12 mgL -1) under optimized conditions and other influencing factors like amino acids, carbon source and nitrogen sources playing major role in the yield enhancement (Mahmoud and Bashandy, 2021). The present study required no external supplementation of cofactors, amino acids, etc., making the current process economically suitable. Species like Escherichia coli have been genetically modified to overexpress Riboflavin, resulting in up to 74.66% increase in production from 182.65 ± 9.04 mgL -1 to 319.01 ± 20.65 mgL -1 (Fu et al., 2023). No genetic modification was required in the present strain, and the yield of Riboflavin obtained was significant (397 ± 15.8 mg L - 1), thereby making the process more sustainable. Optimizing process parameters, media composition, and the addition of cofactors, growth regulators, etc., can further increase the yield of this train and is an area to explore in future research.

Physicochemical characterization of extracted riboflavin

Preliminary physicochemical characterization and confirmation of the extracted metabolite were done via UV-Vis spectrophotometry and thin-layer chromatography (TLC). Given their reliability and time-efficiency, these methodologies have been widely accepted for the initial identification of Riboflavin (Tetik et al., 2026). The presence of an isoalloxazine chromophore with distinct UV absorption maxima allows tentative confirmation of Riboflavin. Since the detailed biochemical and structural characterization has already been established in the previous investigation, the current study deals with the primary confirmatory analysis to validate the identity of the produced metabolite. The extracted metabolite was dissolved in acidified ethanol and analyzed by UV-Vis spectrophotometry over 200–700 nm. The absorption peaks at 226 and 266 nm corroborated with the previous report of Riboflavin, thus indicating the successful production of Riboflavin during SmF conditions in YPMSM supplemented with SCB (Paul et al., 2025) (Figure 2a). Performing only UV-Visible spectrophotometric analysis cannot serve as a confirmatory test for further experimentation, and more advanced analytical methods are needed to validate the product. Further confirmatory results were obtained by TLC where commercially procured Riboflavin was used as a reference standard. Visualization after movement of the solvent front revealed similar retention values for both samples, indicating the presence of Riboflavin within the extract. The Rf value for both was calculated to be 0.83 (Figure 2b). Similar TLC profiles of Riboflavin were reported by other groups (Kher et al., 2022; Anwar et al., 2024). Mobile phases of different types were examined by Hemlatha and Devi, in 2022 with compositions in different ratios (Butanol: benzene: acetic acid: water (8:7:5:3) and butanol: acetic acid: water (9:4:5)), and obtained Rf values of 0.4, thus defining the significance of Mobile phases in the process (Hemalatha and Subathra Devi, 2022). The earliest experimentations by Diaz et al., dating back in 1993 used a similar mobile phase, i.e. methanol: water in the ratio of 70:30 as the mobile phase, and demonstrated that Rf values of the Riboflavin molecule to be 0.86 which is similar to the results found in our study (Díaz et al., 1993).

Assessment of SCB deconstruction

Proper deconstruction of LCB is an essential prerequisite for sustainable bioconversion processes, since the highly recalcitrant structure often restricts microbial accessibility and substrate utilization. In the present study, the SCB biomass was assessed by evaluating its different fractions, including alkali lignin, acid-soluble lignin (ASL), acid-insoluble lignin (AIL), and reducing sugars (Mota et al., 2023). These analytical approaches together provide insight into the structural destabilization of the lignocellulosic matrix and release of monomeric carbohydrates. Comparative analysis of treated and untreated biomass reveals the specific impacts of microbial treatment on SCB components (Tahmasebi et al., 2024) (Figure 3).

Alkali lignin

The alkali lignin fraction present within the biomass is one of the most recalcitrant components of the LCBs (Woiciechowski et al., 2020). One of the most efficient ways to understand the status of alkali lignin present within the biomass is through alkaline pretreatment, a process that involves the participation of NaOH, which primarily targets the ester links and ether links present between the individual components of the biomass, i.e., lignin, cellulose, and hemicellulose. This treatment solubilizes the alkali lignin portion of the biomass and the solubilization capacity also describes the bioavailability or the stability of the lignin fraction (Long et al., 2024). There was noticeable difference between the colour intensity and subsequent OD values of the solubilized lignin fractions between the treated and untreated SCB biomass (Sun et al., 2025). There was a reduction of lignin solubilization in the untreated fraction resulting in lower value of OD at 465 nm (0.359) compared to the treated biomass with OD value of 0.443. The initial deconstruction that was initiated with the pretreatment processes, primarily mechanical milling for particle size reduction, induced enhancement in the bioavailability of the recalcitrant components of the LCB, as described by Chakraborty et al., in 2024 (Chakraborty et al., 2024). Thermal pretreatment, as described by Kumar et al. in 2024, closely resembles the sterilization via autoclaving, where LCB is subjected to high steam heat (121 °C for 15 min) (Kumar et al., 2024). High temperature accompanied by high pressure can lead to the deconstruction of LCB components, thus reducing structural complexity, bioavailability, and solubility of the components. This helps the microbial attack occur more easily. Further, the increased solubilization in treated biomass is a resultant of the microbial attack on the bonds between the lignin and carbohydrate complexes present, specifically hemicellulose. This loosening of biomass components is generally observed when there is any enzymatic hydrolysis of such bonds. Improved ligin release have been observed with other microbes like Sutcliffiella sp., Burkholderia sp., Klebsiella sp., Pseudomonas sp., etc., (Xu et al., 2023; Sumranwanich et al., 2024), This is the first report on LCB deconstruction by Microbacterium proteolyticum, where results indicate that this strain is capable of altering and destabilizing the lignin-rich matrix of SCB.

Acid-soluble and acid-insoluble lignin

The two-step hydrolysis approach using sulfuric acid (H 2 SO 4) in order evaluate the extent of lignin deconstruction. This method ensures fractionation and quantification of acid-soluble lignin (ASL) and acid insoluble lignin (AIL) components of the LCB (Xu et al., 2023). Quantification of ASL was done spectrophotometrically by. Measuring absorbance at 320 nm. A very minute difference in OD was observed between the treated (0.59) and untreated (0.65) fractions, indicating a negligible impact on this lignin fraction. In contrast, a substantially greater impact was observed in the AIL fraction. The dry residual biomass after acid hydrolysis in bacterially treated samples weighed 0.26 ± 0.01 g, while untreated biomass weighed 0.75 ± 0.03 g, which accounts for a 65.33% ± 3.4% reduction in the AIL fraction. These results indicate the removal of recalcitrant lignin from the biomass. Since AIL is a highly condensed and structurally resistant component, this significant reduction strongly suggests microbial deconstruction of the LCB framework present in SCB biomass during the process of SmF (Morales et al., 2020). Thus results indicate successful utilization of the lignin component of the SCB by the current bacterial strain. A comparatively higher impact on the AIL fraction suggests selective ligninolytic potential of the present strain and can be an effective strategy for any future implications for bacterial pretreatment strategies, hence can be used as an efficient tool for industrial bioconversion strategies.

Reducing sugar

Reducing sugars present in the fermentation medium were quantified using the 3,5-dinitrosalicylic acid (DNS) assay, a well-known colorimetric method for measuring fermentable monosaccharides produced during bacterial treatment (Chettri and Verma, 2024). There was a significant shift in absorption between treated (0.617 ± 0.08 mg mL -1) and untreated samples (0.205 ± 0.02 mg mL -1). The untreated samples showed lower reducing sugar concentrations, which are a result of thermal pretreatment methods performed as part of the fermentation pre-processing (Tsegaye et al., 2018). The large shift in absorbance in the treated sample is a direct indication of microbe-mediated deconstruction of LCB biomass, a process in which carbohydrate monomers are released from the polysaccharide and lignin complex matrix. Thus, these results support the current strain’s LCB-degrading capability (Abbas et al., 2023).

Physicochemical characterization of deconstructed LCB

To evaluate the structural and compositional differences induced in the SCB biomass components during submerged fermentation by Microbacterium proteolyticum BWBTDIPO1, physicochemical characterization was performed. In the present study, Fourier transform infra-red spectroscopy (FTIR), X-ray diffraction (XRD) and field-emission scanning electron microscopy (FESEM) were used as primary analytical techniques to investigate the chemical, crystallographic, and surface morphology of the SCB biomass after bacterial treatment. Similar analytical approaches were followed while working with several strains of Pseudomonas sp. and Bacillus sp. (Bhardwaj et al., 2025; Ratheesh et al., 2025).

FTIR

FTIR is one of the most widely accepted analytical techniques for evaluating chemical and structural alterations within a given material (Alokika and Singh, 2020). Significant modifications in functional groups and bonding patterns were observed on comparing the spectral profiles of the untreated and bacterially treated SCBs, as shown in Figures 4a,b. Peaks in the range of 2,918–2,919 cm -1 and 2,850–2,852 cm -1 correspond to aldehydes, carboxyl groups associated with C-H stretches. Slight shift of peaks from 2,918.47 cm -1 to 2,919.48 cm -1 and from 2,850.42 cm -1 to 2,852.57 cm -1 indicates rearrangements within aliphatic hydrocarbon chains, which have been previously reported to be associated commonly with bacterial attacks on lignin-carbohydrate complexes. Very strong alterations were observed in the peak near 1736 cm -1, which is directly correlated with carbonyl-associated regions and C=O stretching in acetyl and ester linkages (Ejaz et al., 2024). These are hallmark changes resulting from active bacterial enzymatic hydrolysis. Notable shifts in the peak from 1,634.09 to 1,607.31 cm -1 in untreated and treated biomasses indicate a high likelihood of altered aromatic structures, commonly present in lignin structural networks, hinting at depolymerization, ring modifications, cleavage, etc., associated with ligninolytic activity driven by microbial enzymatic machineries (Kathiresan et al., 2025). The disappearance of the peak at 1,317.43 cm -1 is correlated with crystalline cellulose-associated CH 2 wagging, suggesting possible changes in cellulose organization. Hemicellulose-lignin associations disruptions via. aryl-alkyl ether linkages and acetyl groups present might be associated with the appearance of a new peak at 1,244.16 cm -1 after bacterial treatment. Disruption of glycosidic linkages is linked with peaks at the 1,158 cm-1 and 1,026 cm-1 region (referring to C-O and C-O-C bond disruptions). Collectively, these results suggest that bacterial treatment and the current strain’s enzymatic activity significantly impact LCB biomass. The list of alterations and associated shifts, along with their significance, is presented in Table 1.

XRD

Lignocellulosic biomasses contain both amorphous and crystalline domains, which are primarily associated with lignin, cellulose microfibrils, and hemicellulose. Any microbial degradation of this lignocellulosic matrix can induce significant alterations in its diffraction intensities, peak intensities, and crystallinity patterns (Abdelwahab and Shukry, 2015). Comparative X-ray profiling between treated and untreated biomass revealed modifications in the crystalline architecture, thereby supporting the claim for microbial biodegradability (Figure 5). The crystallographic plane of native cellulose, which yields a peak in the range 2θ = 21°–23°, was observable in both samples. In the untreated samples, a broader diffraction peak with lower intensity was observed, indicating the presence of a compact lignin-hemicellulosic matrix surrounding cellulose microfibrils (Kathiresan et al., 2025). The bacterially treated SCB showed a more intense, relatively sharper diffraction peak in the same region, suggesting structural reorganization following microbial treatment (Boyetey et al., 2023; Siddiqui et al., 2022). This also indicates changes in the amorphous fractions, which are mostly associated with lignin and hemicellulose. Cellulose, which is mostly responsible for imparting crystallinity to the structure of LCB, appears to be present via the crystalline peaks in the XRD diffractogram. Delignification can increase crystallinity, as reflected in this study’s treated samples. Reduced diffuse scattering throughout the region between 2θ = 15°–19°, suggesting these alterations and these results are very consistent with FTIR observations. These results suggest that the current bacterial strain can deconstruct recalcitrant amorphous fractions, thereby exposing the cellulose domains, which are reflected in the XRD analysis.

FESEM

To investigate the surface morphological alterations, FESEM was performed with untreated SCB and bacterially treated SCB (Ghai et al., 2023). Comparative study between these samples revealed significant surface deterioration and structural disruptions, thus confirming microbial destruction of the LCB framework by the strain. In the untreated SCB, a dense packed intact fibrillar structure with a smooth surface could be seen (Figure 6). The fibers are well organised and minimal surface ruptures were detected. On the other hand, the bacterially treated SCB demonstrated substantial disruption of the ultrastructure of the biomass, with clear signs of erosion, fibrillar disorganisation, fragmentation, pore formations, ruptures, pronounced cracking in some regions (Siddiqui et al., 2022). These results clearly indicate the impact of bacterial treatment on the structural deconstruction of the LCB.

In-silico prediction of metabolic pathway mapping for LCB deconstruction

In silico metabolic pathway prediction and analysis were performed to understand the biochemical mechanisms underlying LCB deconstruction and assimilation by the present strain. It is evident that the complex nature of the substrate requires the microbe to utilize multiple metabolic pathways for its deconstruction (Qin et al., 2026; Duan et al., 2022). Metabolic pathway mapping suggested the potential involvement of Microbacterium proteolyticum in lignocellulosic biomass degradation through interconnected central carbon metabolic pathways. These proposed pathways are hypothetical and intended to provide a conceptual framework for interpreting the observed lignocellulosic biomass deconstruction (Kanehisa et al., 2017).

Since direct molecular evidence regarding lignocellulose-degrading pathways in M. proteolyticum BWBTDIPO1 is currently unavailable, bioinformatics-based pathway mapping was employed as a complementary approach to interpret the experimental observations. The analysis was intended to identify plausible metabolic routes involved in substrate assimilation and carbon flux rather than to demonstrate experimentally validated biochemical mechanisms.

Lignin-associated pathways indicated the conversion of aromatic intermediates into metabolites entering the tricarboxylic acid (TCA) cycle. Cellulose degradation pathways showed potential hydrolysis into cellobiose and glucose, followed by glycolytic metabolism (Liu et al., 2026). Similarly, hemicellulose degradation pathways were associated with the conversion of pentose sugars, such as xylose and arabinose, into intermediates of the pentose phosphate pathway. These predicted pathways indicate potential metabolic capabilities that may support the utilization of diverse lignocellulosic substrates; however, experimental validation is required to confirm their activity (Figure 7).

The predicted pathways identified in the present study are consistent with lignocellulose degradation strategies previously reported in bacteria and microbial consortia. Lignin degradation is commonly associated with the conversion of lignin-derived aromatic compounds into central metabolic intermediates that subsequently enter the TCA cycle through aromatic catabolic pathways (Linger et al., 2014; Grgas et al., 2023). Similarly, cellulose degradation generally involves the hydrolysis of cellulose into cellobiose and glucose, followed by glycolytic metabolism, a mechanism widely reported in cellulolytic microorganisms (Fu et al., 2025; Zhang et al., 2026). Hemicellulose-derived pentose sugars, including xylose and arabinose, are typically assimilated through pathways linked to the pentose phosphate pathway, thereby contributing to central carbon metabolism (Elmore et al., 2020; Wynands et al., 2026; Weng et al., 2023). The predicted pathway architecture observed for M. proteolyticum BWBTDIPO1, therefore, suggests potential metabolic capabilities that could support the utilization of lignin, cellulose, and hemicellulose fractions of sugarcane bagasse. Furthermore, the integration of these pathways with central carbon metabolism provides a plausible explanation for the conversion of biomass-derived carbon into cellular metabolites and the subsequent biosynthesis of value-added products such as Riboflavin, which has previously been reported for this strain (Paul et al., 2025). Nevertheless, these pathways are based on KEGG-guided prediction and literature-supported annotation and should be regarded as hypothetical until validated through genomic, transcriptomic, proteomic, metabolomic, or enzyme activity studies.

The integrated pathway map further suggests possible interactions among lignin, cellulose, and hemicellulose utilization pathways that may contribute to lignocellulosic biomass assimilation by the strain. These predicted metabolic routes provide a conceptual framework for understanding how biomass-derived carbon could potentially be redirected toward cellular metabolism and value-added Riboflavin biosynthesis. The pathway for Riboflavin biosynthesis in this strain has previously been reported by Paul et al. (2025).

The present study demonstrates the valorization of SCB, an abundantly available agro-waste residue, into high-value Riboflavin via microbial submerged fermentation using Microbacterium proteolyticum BWBTDIPO1. Yeast extract peptone mineral salt media (YPMSM) was found to support bacterial growth (μ = 0.325 h -1) along with the best production with a maximum yield of 397 ± 15.8 mgL - 1, which amounts to 19.85 ± 2.0 mg per gram of SCB after 78 h of fermentation. Biochemical and physicochemical assays confirmed that the metabolite was Riboflavin. Detailed physicochemical characterization by FTIR, XRD, and FESEM revealed significant deconstruction of SCB during fermentation. This was evident from the increased alkali lignin solubilization, substantial reduction of acid-insoluble lignin, and increased amount of reducing sugars on bacterial treatment. These findings establish that M. proteolyticum BWBTDIPO1 is not only efficient in utilizing SCB but also actively participates in the production of Riboflavin. Furthermore, in silico pathway prediction suggested potential interconnected metabolic networks involved in lignocellulosic biomass utilization. The inclusion of predictive pathway analysis provided a preliminary mechanistic perspective linking lignocellulosic biomass utilization with central carbon metabolism and potential Riboflavin biosynthesis, thereby complementing the experimental findings. However, these pathways remain hypothetical and require validation through genomic, transcriptomic, proteomic, metabolomic, and enzyme-based investigations. To the best of our knowledge, based on the literature survey, this is the first report of M. proteolyticum -mediated LCB deconstruction, simultaneous with high-value Riboflavin production. Future studies should focus on the metabolic engineering of selective strains after identifying key genes regulating Riboflavin production and LCB degradation. Whole-genome sequencing followed by in silico analysis to generate mechanistic insights can pave the way for new avenues of exploration. Techno-economic approaches, life cycle assessments, and systems biology are essential components of future investigations. Besides, process optimization of the bioconversion process must be done to enhance yield and minimize cost during large-scale industrial operations. This work bridges the boundaries between waste management and strategic sustainable Riboflavin biosynthesis, hence bringing new insights to the concept of micro-mediated waste-to-wealth conversion, contributing to Sustainability Development Goal and circular bio-economy.