Valorization of Agro-Industrial Waste for Pectinase Production by Bacillus subtilis SK16: A Step Toward Circular Bioeconomy

Valorization of Agro-Industrial Waste for Pectinase Production by Bacillus subtilis SK16: A Step Toward Circular Bioeconomy | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Valorization of Agro-Industrial Waste for Pectinase Production by Bacillus subtilis SK16: A Step Toward Circular Bioeconomy Sharmili Shivajirao Jagtap, Sangeetha P This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7020186/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 4 You are reading this latest preprint version Abstract Pectinase is an industrially important enzyme with wide-ranging applications in the food, textile, and biofuel industries. However, commercial production remains cost-intensive due to the use of high-purity substrates. In this study, a cost-effective and sustainable bioprocess was developed to enhance pectinase production using agro-industrial pectin-rich waste materials, in alignment with the United Nations Sustainable Development Goals 2030. Bacillus subtilis SK16 was identified as a potent pectinase producer with notable polygalacturonase activity. Additionally, B. subtilis SK16 demonstrated the ability to produce multiple industrially important extracellular enzymes, including amylase, xylanase, protease, cellulase, and lipase. In this study, pectinase produced by agro-industrial pectin-rich waste as a substrate. Among them orange peel (397 U/ml), wheat bran (330.97 U/ml), and apple peel (207.9 U/ml) were evaluated as individual substrates for enzyme production. To further enhance enzyme yield, a combinational substrate approach was applied. By adjusting the proportions of these waste components, the study aimed to determine the most effective combination for maximizing pectinase yield. A maximum pectinase activity of 615.90 U/ml was achieved with a substrate combination of 0.5% orange peel and 1.5% wheat bran, reflecting a 55% enhancement compared to the highest yield obtained from individual substrates. This study underscores the potential of utilizing agro-industrial wastes as sustainable substrates for enzyme production, promoting efficient solid waste bioconversion while offering an eco-friendly and economically viable solution for environmental waste reduction. The findings contribute to waste valorization, circular bioeconomy, and reduced reliance on synthetic media, thereby supporting environmental sustainability and industrial scalability. Pectinase Agro-industrial waste circular economy Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Highlights Utilized agro-industrial fruit peel wastes as sustainable substrates for eco-friendly pectinase production. Promoted solid waste bioconversion and waste valorization, aligning with circular bioeconomy principles. Enabled low-cost enzyme production by leveraging abundantly available organic waste materials. Supported SDG 2030 goals by fostering responsible waste management and sustainable industrial practices. Demonstrated a green biotechnological approach for transforming agri-waste into high-value bioproducts. 1. Introduction` India, as the world’s second-largest agro-based economy, generates substantial biomass due to rapid industrialization, urbanization, population growth, and intensified food production. According to the Indian Ministry of New and Renewable Energy (MNRE), approximately 500 million tons of agricultural waste are produced annually, of which 140 million tons remain unutilized and 92 million tons are burned, releasing an estimated 3.3 billion tons of CO₂ into the atmosphere ( Capanoglu et al., 2022 ). Mismanagement of this waste exacerbates climate change, pollution, and resource degradation, emphasizing the need for sustainable management practices. Agro-industrial residues are enriched with essential micro- and macronutrients, including carbohydrates, proteins, lipids, vitamins, and minerals, making them valuable feedstocks for the biotechnological recovery of bioactive compounds ( Hadj Saadoun et al. 2021 ). Their high carbon and nitrogen content further enhances their suitability as substrates for microbial fermentation ( Adriana et al. 2021 ). Considering that commercial substrates contribute 40–70% of total fermentation costs, the utilization of agro-wastes offers a cost-effective and sustainable alternative. Moreover, their valorization aligns with circular economy principles, enabling the transformation of waste into high-value products such as industrially important enzymes ( Pérez-Contreras et al. 2024 , Yusof et al. 2020 ). Among industrial enzymes, pectinase plays a pivotal role in a wide array of applications. Pectinase is an industrially important enzyme with a worldwide growing market demand, share about 25% of utilization in food industry ( Haile and Ayele 2022 , Mehmood et al. 2019 ; Mohammadi et al. 2020 ). As a result, the global pectinase market is expected to grow substantially, from $ 18.3 billion in 2022 to $ 26.6 billion by 2030, reflecting a compound annual growth rate (CAGR) of approximately 11.1% (Zion Market Research 2020) . However, its widespread application is hindered by high production costs, necessitating the development of cost-effective production methods. To address this challenge, researchers are exploring various strategies to reduce pectinase production costs. The utilization of low-cost agro-industrial wastes as substrates has become most preferable for pectinase production, addressing both economic and environmental concerns (Kaul et al. 2024 , Haile and Ayele 2022 , Ketipally and Ram 2018 ). In alignment with the United Nations Sustainable Development Goals (SDG 2030) particularly SDG 12 (Responsible Consumption and Production) and SDG 13 (Climate Action) there is a growing need to repurpose agro-industrial waste streams for productive use. Pectin-rich wastes such as fruit peels and cereal by-products are abundant and underutilized. Their valorization through microbial fermentation supports waste minimization, resource circularity, and the development of a circular bioeconomy, offering a dual benefit of waste reduction and bioproduct generation. Microbial pectinase is primarily sourced from bacteria, fungi, and yeast ( Haile and Ayele 2022 ) . In previous literature, most of the pectinolytic enzymes were produced from fungi ( Afzia et al. 2024 ), whereas there are fewer studies on bacterial pectinases compared to fungi. Fungal sources are prominent, with Aspergillus species being particularly noteworthy. Aspergillus spp . Gm was found to be a potent strain for pectinase production, showing optimal enzyme activity at 30°C and pH 5.8 ( KC et al. 2020 ). However, the fungal sourced pectinase has limited usage due to their narrow pH range, which is not suitable for applications at alkaline pH or neutral pH. Among bacterial sources, Bacillus subtilis has been identified as a potent pectinase producer. Alqahtani et al isolated and characterized a pectinase-producing B. subtilis strain with high specific activity and stability at pH 4.5 and 50°C ( Alqahtani et al. 2022 ). Interestingly, halophilic and halotolerant microorganisms from salty lakes have been explored as novel sources of salt-tolerant pectinases. Bacteria belonging to the genus Bacillus and extremely halophilic archaea of the genera Haloterrigena and Halostagnicola showed promising hydrolytic activities, including pectinase production ( Ruginescu et al. 2020 ). This diversity in bacterial sources allows for the production of pectinases with varied properties, suitable for different industrial applications. Among pectinase-producing microbes, Bacillus subtilis is recognized for its Generally Recognized as Safe (GRAS) status, high metabolic versatility, and ability to secrete multiple enzymes. In this study, a novel isolate, Bacillus subtilis SK16, was explored for its pectinase production capability using agro-industrial wastes, including orange peel, apple peel, and wheat bran. These substrates not only serve as nutrient sources but also exemplify the concept of waste valorization by turning local organic residues into industrially important enzymes ( Kuvvet et al., 2017 ). Various studies have demonstrated the successful utilization of these low-cost materials to enhance enzyme yields. For example, the utilization of orange peel and wheat bran as a substrate enabled Bacillus licheniformis to produce high quantities of pectinase in a simple medium, making it a promising option for large-scale commercial production ( Bibi et al. 2016 , Jahan et al. 2017 ). In addition, previous study reported that apple pomace, orange peels, and Satkara peel have been successfully used as low-cost substrates for pectinase production ( Ahmed et al. 2021 , Satapathy et al. 2021 , Qadir et al. 2020 ). This approach aligns with the growing emphasis on using agricultural biomasses as low-cost substrates for pectinase production (Shrestha et al. 2021 ). In addition to substrate selection, previous studies have primarily focused on optimizing pectinase production through various strategies such as strain improvement, statistical optimization of media composition, and fermentation parameter adjustments ( Ahmed et al. 2021 , Ortiz et al. 2017 ; Satapathy et al. 2021 ; Govindaraji and Vuppu 2020 ; Qadir et al. 2020 ). Techniques like Plackett–Burman design (PBD) and response surface methodology (RSM) have been widely used to enhance enzyme yields. For instance, statistical optimization of media components using orange pectin as a substrate resulted in a maximum pectinase activity of 170.05 U/mL ( Govindaraji and Vuppu 2020 ). Similarly, a combination of orange peel and coconut fiber (4:1) yielded a peak pectinase production of 3315 U/gds by Bacillus subtilis SAV-21 under optimized solid-state fermentation conditions ( Kaur and Gupta 2017 ). Bibi et al. further demonstrated that response surface methodology and central composite design could be used to optimize cultural parameters, achieving a maximum pectinase activity of 219 U/mL from Bacillus licheniformis ( Bibi et al. 2016 ). Additionally, some research has explored the use of multiple agro-waste substrates in combination to enhance enzyme yield. Rispoli and Shah ( Rispoli and Shah 2007 ) applied mixture design experiments to investigate the effect of glucose, yeast extract, starch, and magnesium sulfate on the production of cutinase enzymes by Colletotrichum lindemuthianum ( Rispoli and Shah 2007 ). While statistical optimization models and strain improvement approaches have contributed to enhancing enzyme production, they often involve high costs, complex optimization steps, and potential biosafety concerns associated with genetic modifications. In contrast, the combination of multiple agro-waste substrates presents a simple yet highly effective alternative for improving pectinase production ( Kaul et al. 2024 , Haile and Ayele 2022 ). Different agro-waste materials contribute a diverse range of nutrients that work synergistically to enhance microbial metabolism and enzyme synthesis. For example, fruit peels such as orange and apple serve as natural inducers due to their high pectin content, while wheat bran supplies essential proteins, amino acids, and trace elements that support microbial growth and metabolic activity. This synergistic effect leads to significantly higher enzyme yields compared to single-substrate fermentation, making it a promising strategy for industrial applications. Furthermore, this approach is cost-effective, scalable, and environmentally sustainable, as it utilizes abundant and renewable agro-industrial waste, reducing dependence on expensive pure carbon sources. Unlike statistical optimization models, which rely on mathematical predictions that may not always be applicable in large-scale fermentation processes, the combination substrate strategy ensures consistent and reproducible enzyme production across different batches, offering greater industrial feasibility. The novelty of the present study lies in two key aspects: (1) the identification of a novel Bacillus subtilis SK16 strain as a potent pectinase producer and (2) the development of a combinational agro-waste substrate strategy for enhanced enzyme production. In this study, nine different combinations of pectin-rich agro-waste substrates were evaluated to determine the most effective formulation for maximizing pectinase yield. The results demonstrated that a specific combination of orange peel, apple peel, and wheat bran significantly enhanced enzyme production compared to single-substrate media. By refining the ratio of these natural agro-waste components, our study highlights the synergistic effect of substrate combination, leading to improved pectinase production efficiency. This research contributes to sustainable biotechnology by promoting waste valorization, reducing dependency on costly synthetic carbon sources, and offering an economically viable alternative for industrial enzyme production. Furthermore, our findings address a critical gap in pectinase research by reinforcing the importance of strain selection and combinational substrate strategies for optimizing fermentation processes. This study aligns with the principles of the circular economy and SDG 2030 goals, making a significant contribution to the development of environmentally sustainable enzyme production technologies. 2. Materials and Methods 2.1 Microorganism and materials: Ammonium sulphate, Potassium di-hydrogen phosphate, Potassium phosphate dibasic, Magnesium sulphate, Sodium carbonate, Apple pectin and, Di-nitro salicylic acid (DNSA) were obtained from Hi-Media (India). Sodium potassium tartrate was purchased from SRL (India). Polygalacturonic acid, Xylan, was purchased from Sigma Chemicals (USA). Various agro-industrial residues, including wheat bran, and fruit peel wastes such as lemon, banana, mango, pomegranate, orange, and apples peels, were collected from local market. These residues were thoroughly washed with tap water to remove impurities, cut into small pieces, and dried in hot air oven for 6hrs at 55 o C. 2.2 Qualitative Screening of Pectinolytic Enzymes : Pectinolytic enzymes play a crucial role in the degradation of pectin, a structural polysaccharide in plant cell walls. These enzymes include pectinase, which hydrolyzes pectin into oligosaccharides; pectin lyase, which cleaves highly esterified pectin via a β-elimination mechanism; and polygalacturonase, which hydrolyzes glycosidic bonds in polygalacturonic acid. The qualitative screening of pectinolytic enzymes produced by best isolate SK16 was performed using iodine staining method. The bacterial strain was inoculated onto three different substrate-specific agar media: pectin agar (for pectinase activity), citrus pectin agar (for pectin lyase activity), and polygalacturonic acid agar (for polygalacturonase activity). The medium used for pectinase screening consisted of 1% pectin, 0.14% (NH₄)₂SO₄, 0.6% K₂HPO₄, 0.2% KH₂PO₄, 0.01% MgSO₄, and 2% agar-agar, adjusted to pH 7.0. Spot inoculation of bacterial isolates was performed on the respective media, followed by incubation at 37°C for 48 hours under aerobic conditions Following incubation, the plates were flooded with iodine solution, which interacts with intact pectin to form a dark-colored complex. The presence of clear hydrolysis zones around the bacterial colonies indicated enzymatic degradation of pectin, confirming enzyme production. The diameters of hydrolysis zones were measured to assess the relative activity of each enzyme. This method provided a rapid and effective preliminary screening of best isolate SK16’s pectinolytic potential. ( Cardoso et al. 2008 ; Ishihara et al. 2021 ) 2.3 Screening of Industrially Important Enzymes The enzymatic potential of SK16 strain was evaluated for the production of industrially significant enzymes, including xylanase, amylase, laccase, cellulase, lipase, and protease, using substrate-specific agar media. The strain was inoculated on xylan, carboxymethyl cellulose (CMC), starch, guaiacol, Victoria Blue R, and skim milk casein agar for the respective enzyme activities. Plates were incubated at 37°C for 48 hours. After incubation, enzyme activity was qualitatively assessed through substrate hydrolysis or specific staining techniques. Amylase production was confirmed by clear zones on starch agar after flooding with Lugol’s iodine (Luang-In et al. 2019 ). Xylanase and cellulase activities were detected using Congo red staining on xylan and CMC agar, with hydrolysis zones indicating degradation of xylan and cellulose (Abena et al. 2024; Luo et al. 2023 ; Islam et al. 2018; Patel et al. 2020). Protease and lipase activities were indicated by clear zones on skim milk casein agar and blue halos on Victoria Blue R agar, respectively. Laccase activity was confirmed by reddish-brown coloration due to guaiacol oxidation (Fu et al. 2013 ). These qualitative assays provided a preliminary assessment of the enzymatic potential of B. subtilis SK16. 2.4 Quantitative Assay for pectinase: Bacillus subtilis was inoculated into pectin broth and incubated at 37°C. after incubation period, the culture media was centrifuged at 7,500 rpm at 4°C. The resulting clear cell free supernatant was used as a crude pectinase source for the quantitative pectinase assay. Pectinase activity was measured based on the release of D-Monogalacturonic acid from pectin. The reaction mixture contained crude enzyme extract and 1% Apple pectin dissolved in 100 mM phosphate buffer at pH 7.0. The reaction was incubated at 37°C for 30 minutes, and the liberated D-galacturonic acid was quantified using the DNSA reagent method (Shet et al. 2022 ). One unit of pectinase was defined as the amount of enzyme required to liberate 1 µmol of D-galacturonic acid per minute under the assay conditions. 2.5 Molecular identification of Bacillus subtilis : The bacterial isolate exhibiting strong pectinolytic activity was subjected to molecular identification through 16S rRNA gene sequencing. Universal primers 27F (5′-AGA GTT TGA TCM TGG CTC AG-3′) and 1492R (5′-TAC GGY TAC CTT GTT-3′) were employed to amplify the 16S rRNA region from the extracted genomic DNA. The resulting amplicon sequences were compared with existing sequences in the NCBI database using the Nucleotide BLAST tool ( https://blast.ncbi.nlm.nih.gov/Blast.cgi ) to determine sequence similarity. For phylogenetic analysis, multiple sequence alignments were carried out using CLUSTALW, and evolutionary relationships were inferred through MEGA7 software, comparing the isolate’s sequence to those of closely related reference strains. 2.6 Production and optimization of Pectinase: Inoculum preparation and Processing of Agro-industrial wastes as a substrate: The inoculum was prepared by inoculating a loopful of Bacillus subtilis SK16 into 50 mL of sterile pectin broth in a 250 mL flask. The culture was incubated at 37°C with agitation at 200 rpm on a rotary shaker for 18 hours. Various agro-industrial residues, including wheat bran and fruit peel wastes (lemon, banana, mango, pomegranate, orange, and apple peels), were collected from the local market. These residues were washed to remove water-soluble impurities, cut into small pieces, and dried in a hot air oven for 6hrs at 55 o C. These dried substrates were used as a natural pectin source in media preparation for the production of pectinase. 2.7 Analysis of the parameter affecting Pectinase production: Pectinase production was carried out by submerged fermentation (Smf) using the basal medium with a pectin rich agro-waste (1%) as a substrate. Parameters influencing enzyme production, such as incubation period, pH, temperature, and natural substrates, and substrate concentration were analyzed using a one-factor-at-a-time approach, keeping all other variables constant. Each experiment was performed in triplicate. 2.8 Selection of Best agro waste for the production of pectinase: ​To identify the best natural pectin source for pectinase production, various natural substrates were selected based on their pectin content and local availability. These substrates include (pectin content for each is mentioned in the brackets) apple peel (12.5%), lemon peel (20.75%), mango peel (8.8%), orange peel (15.24%), banana peel (15–24%), wheat bran (significantly low), and pomegranate peel (6.80–10.1%). Additionally, different incubation times were assessed to determine the duration required for maximum enzyme production using Bacillus subtilis .​ 2.9 Effect of Substrate Concentration on pectinase yield: The influence of substrate concentration on pectinase production was evaluated using five different concentrations (0.5%, 1%, 1.5%, 2%, and 3%) of the three most effective pectin rich natural substrates at different incubation times. 2.10 Optimization of combination natural pectin sources: The study objective assumes that pectinase production is influenced by the composition of the natural pectin sources in the culture medium. In this study, the three best natural pectin sources such as 2% orange peel, 2% apple peel, and 1.5% wheat bran were used, collectively contributing to 2% (w/v) of the total pectin composition. Totally we have designed 9 different combinations for analyse the best combination for better pectinase production (Table 1). 3. Results 3.1 Microbial Strain and Molecular Identification The bacterial strain Bacillus subtilis SK16 was successfully isolated from alkaline soil and taxonomically identified through 16S rRNA gene sequencing (GenBank accession no. PQ206567). Phylogenetic analysis positioned the isolate within the Bacillus subtilis clade, showing close evolutionary relatedness to reference strains B. subtilis R8 (MH371779.1) and M124 (MH168996.1), supported by high bootstrap values. Primary screening revealed notable pectinolytic activity, with clear hydrolysis zones measuring 18 mm and 15 mm for polygalacturonase and pectinase, respectively, on pectin agar plates. However, no pectin lyase activity was observed, suggesting an absence of β-elimination cleavage of highly methylated pectin substrates (Fig. 1 and Table S1 ). 3.2 Screening of Industrially Important Enzymes Qualitative enzyme screening revealed that Bacillus subtilis SK16 produced multiple industrially relevant enzymes. Hydrolysis zones were observed for amylase (9 mm), protease (15 mm), lipase, xylanase (16 mm), and cellulase (9 mm), confirming extracellular enzyme production on selective media (Table S2). 3.3 Quantitative Pectinase Activity Pectinase activity in submerged fermentation reached a peak of 321.14 U/ml at 48 hours of incubation, as measured by the DNSA method. The crude enzyme extract effectively hydrolyzed pectin to release D-galacturonic acid under standard assay conditions (37°C, 30 min). 3.4 Pectinase Production Using Agro-Industrial Waste Agro-waste substrates including orange peel, apple peel, lemon peel, mango peel, banana peel, wheat bran, and pomegranate peel were tested for pectinase production. Orange peel supported the highest enzyme yield at 48 hours, surpassing even commercial pectin, followed by wheat bran and apple peel (Fig. 3 ). 3.5 Effect of Substrate Concentration The effect of substrate concentration (0.5–3%) was examined for orange peel, apple peel, and wheat bran. Maximum pectinase activity was observed at 2% apple peel and 2% orange peel at 48 hours (Fig. 4 and Fig. 5 ), and 1.5% wheat bran at 24 hours (Fig. 6 ). 3.6 Optimization of Combinational Agro-Waste Nine combinations of apple peel, orange peel, and wheat bran were tested. The highest pectinase activity (615.90 U/ml) was achieved using a combination of 0.5% orange peel and 1.5% wheat bran. This was followed by 0.5% apple peel and 1.5% wheat bran (496.59 U/ml). The triple combination (apple peel 0.6250%, orange peel 0.6250%, and wheat bran 0.75%) showed lower activity (68.26 U/ml), suggesting that dual-substrate systems were more effective than triple combinations. 4. Discussion The successful isolation of Bacillus subtilis SK16 from alkaline soil and its identification through 16S rRNA gene sequencing confirmed its taxonomic affiliation and enzymatic potential. The strain produces polygalacturonase (PG) and pectinase, but lacks pectin lyase activity, indicating substrate specificity for deesterified pectin (polygalacturonic acid). Such an enzyme profile is advantageous for degrading lowmethoxyl pectin in industrial applications such as fruit juice clarification, textile retting, and paper and pulp processing without the need for highly alkaline conditions. Moreover, recent studies report that B. subtilis strains optimized at neutral pH (7–7.4) can achieve high PG yields (345–630 U/mL) under mild fermentation conditions, and recombinant PG expressed in B. subtilis demonstrates improved efficiency for fiber-processing and juice systems under neutral–slightly acidic pH. This neutral-pH compatibility reduces chemical usage and enhances ecoefficiency in large-scale bioprocesses. The qualitative enzyme screening demonstrated that SK16 possesses a broad spectrum of extracellular enzymatic activities. Notably, high activities of xylanase and protease point to its robustness as an industrial workhorse, useful in sectors ranging from biofuel to detergent and food processing. The moderate cellulase and amylase activities further expand its biotechnological applicability. Quantitative analysis of pectinase production revealed a distinct time-dependent trend, with peak enzyme activity observed at 48 hours of incubation. This pattern is indicative of stationary phase-associated enzyme synthesis, consistent with typical bacterial growth kinetics and secondary metabolite secretion behavior. When exploring cost-effective substrates, orange peel emerged as a superior natural carbon source for pectinase production, outperforming commercial pectin. This can be attributed to its high native pectin content and additional nutrients that enhance microbial metabolism. Wheat bran, despite low pectin content, also significantly supported enzyme production, likely due to its cellulose and hemicellulose content that may serve as co-inducers or enhance metabolic activity. Interestingly, despite its low pectin content, wheat bran has been identified as an efficient substrate for pectinase production. The incubation time was a critical factor, with optimal enzyme production observed after 48 hours. Extending the incubation period beyond this did not significantly increase enzyme activity, suggesting 48 hours as the ideal incubation time for peak production. It indicates that pectinase production activity is correlated with incubation time (Guan et al. 2020 ). A few studies have shown that citrus peel, including orange peel, is a good source of enzyme production (Bibi et al. 2016 , Ahmed et al. 2016 ). Pili et al. demonstrated that pectin lyase production by Aspergillus brasiliensis was higher in the agro-waste residue containing media (orange peel, corn steep liquor, and parboiled rice water) compared to the synthetic media (Abena and Simachew 2024 ). Chiliveri et al reported that wheat bran was found to be an ideal substrate, and the maximum yield of pectate lyase (1371.25 U/gds) and polygalacturonase (85.45 U/gds) where, U/gds represents enzyme activity per gram of dry substrate (Chiliveri et al. 2016 ). Silva and Martin reported that the mixture of orange bagasse and wheat bran showed to be the best substrate for pectinase production in SSF using penicillium species (Silva et al. 2002 ). The differences in the amount of pectinase production using different agro-waste are due to the variation in compositions and the carbon/nitrogen content of agro-waste (Amin et al. 2018 ; Shrestha et al. 2021 ) From various literature sources, it has been established that fruit and vegetable pomace primarily contain pectin (1.50–13.40%), cellulose (7.20–43.60%), hemicellulose (4.26–33.50%), and lignin (15.30–69.40%) as their main constituents (Szymańska-Chargot et al. 2017 ). However, the pectin content varies significantly among different fruit peels. Substrate concentration had a pronounced effect on enzyme yield. For orange and apple peels, 2% concentration was optimal, while wheat bran performed best at 1.5%. These optimal levels balance nutrient availability without causing substrate inhibition or excessive viscosity, which could impede oxygen and nutrient diffusion. Optimization using mixed substrates demonstrated that combinations outperformed individual sources. The combination of orange peel and wheat bran yielded the highest enzyme activity, with more than a 55% increase compared to the best single substrate. This synergy may be attributed to complementary nutrient profiles pectin from fruit peel and polysaccharides from wheat bran that collectively support higher microbial enzyme output. The substantial increase in pectinase yield (615.90 U/ml) in combination substrate exceeding the best single-substrate yield by over 55%, reinforces the importance of substrate synergy in maximizing enzyme production. While extensive research exists on optimizing enzyme production, studies focusing on combinational agro-waste substrates remain limited. A study by Ozzeybek and Çekmecelioğlu optimized a mixture of apple pomace (20%), hazelnut shell (50%), and orange peel (30%) for bacterial pectinase and cellulase co-production, reporting pectinase activity of 8.27 U/ml and cellulase activity of 0.5 U/ml, demonstrating the viability of agro-waste-based enzyme production (Ozzeybek and Cekmecelioglu 2024 ). In contrast, the present study introduces a novel approach by demonstrating that wheat bran, despite being a low-pectin substrate, plays a pivotal role in enhancing enzyme yields when combined with fruit peels. This research contributes to sustainable biotechnology by valorizing agro-industrial waste, minimizing reliance on expensive pure carbon sources, and addressing waste management challenges. These findings provide a strong foundation for scaling up industrial pectinase production and highlight the effectiveness of combinational substrate strategies in improving fermentation efficiency and cost-effective bioprocessing Interestingly, the triple-substrate mix underperformed, possibly due to over-complexity or nutrient imbalance. These findings are consistent with earlier reports showing that specific binary mixtures (e.g., orange peel + rice water or wheat bran) favor microbial enzyme expression more than multiple-component systems. This study presents a novel approach to pectinase production through combinational substrate optimization, highlighting the underexplored potential of wheat bran as a co-substrate. The research supports the use of agro-industrial waste in bioprocessing, contributing to sustainable biotechnology by reducing reliance on costly commercial media and addressing environmental concerns associated with waste disposal. 5. Conclusion This study successfully demonstrated the isolation, identification, and characterization of Bacillus subtilis SK16 as an efficient pectinase-producing microorganism with notable polygalacturonase activity. The strain’s enzymatic profile, favoring de-esterified pectin substrates, along with its ability to produce multiple industrially important enzymes, highlights its broad biotechnological potential. Among various natural substrates tested, agro-industrial fruit peel wastes—particularly orange peel, wheat bran, and apple peel supported substantial enzyme production. Optimization studies revealed that 2% orange and apple peel, and 1.5% wheat bran, yielded maximal pectinase activity. Dual-substrate formulations, especially orange peel combined with wheat bran, enhanced enzyme production by over 55% compared to individual substrates. This study underscores the potential of utilizing agro-industrial wastes as sustainable substrates for enzyme production, promoting efficient solid waste bioconversion while offering an eco-friendly and economically viable solution for environmental waste reduction. Overall, the findings contribute to the advancement of sustainable biotechnology by transforming organic waste into value-added bioproducts and fostering greener industrial fermentation strategies. Declarations Acknowledgments The author sincerely thanks the Department of Microbiology for providing the essential laboratory facilities and continuous support during the course of this research. Special appreciation is extended to Angothu Shanker, Devayani, and Priyadharshini for their helpful assistance and contributions during the experimental phase of the study. Conflict of Interest The corresponding author declares, on behalf of all authors, that there are no conflicts of interest. Funding: This study was conducted without any financial support from government, commercial, or non-profit funding bodies. Author Contributions: Sangeetha P. was solely responsible for the conceptualization, experimentation, data analysis, manuscript drafting, and final revisions. Ethics Approval: Not applicable to this study. Consent to Participate: Not applicable. Consent for Publication: All authors consent to the publication of the manuscript.s Availability of Data and Materials: The datasets generated or analyzed during the study are available from the corresponding author upon reasonable request. Code Availability: Not applicable. References Abena T, Simachew A (2024) Production and characterization of acidophilic xylanase from wood degrading white rot fungus by solid-state fermentation of wheat straw. 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(2021) Simple stain-free screening method for pectinolytic microorganisms under alkalophilic conditions. Biotechnol Lett 43:1905–1911. https://doi.org/10.1007/s10529-021-03162-6 Islam F, Roy N (2018) Screening, purification and characterization of cellulase from cellulase producing bacteria in molasses. BMC Res Notes 11:1–6. https://doi.org/10.1186/s13104-018-3558-4 Jahan N, Shahid F, Aman A, et al. (2017) Utilization of agro waste pectin for the production of industrially important polygalacturonase. Heliyon 3(6):e00330. https://doi.org/10.1016/j.heliyon.2017.e00330 Kaul K, Rajauria G, Singh R (2024) Valorization of agro-industrial waste for pectinase production and its influence on circular economy. Food Bioprod Process 148:141–153. https://doi.org/10.1016/j.fbp.2024.09.008 Kaur SJ, Gupta VK (2017) Production of pectinolytic enzymes pectinase and pectin lyase by Bacillus subtilis SAV-21 in solid state fermentation. Ann Microbiol 67:333–342. https://doi.org/10.1007/s13213-017-1264-4 KC S, Upadhyaya J, Joshi DR, Lekhak B, Kumar Chaudhary D, et al. (2020) Production, characterization, and industrial application of pectinase enzyme isolated from fungal strains. Ferment 6(2):59. https://doi.org/10.3390/fermentation6020059 Ketipally R, Ram MR (2018) Optimization of pectinase production by Aspergillus oryzae RR 103. Curr Agric Res J 6(1):37. https://doi.org/10.12944/CARJ.6.1.05 Kumar D, Bhardwaj R, Jassal S, et al. (2021) Application of enzymes for an eco-friendly approach to textile processing. Environ Sci Pollut Res. https://doi.org/10.1007/s11356-021-16764-4 Kuvvet, C., Cekmecelioglu, D., & Uzuner, S. (2017). Improvement of Pectinase Production by Co-culture of Bacillus spp. Using Apple Pomace as a Carbon Source. Waste and Biomass Valorization, 10 (5), 1241–1249. https://doi.org/10.1007/s12649-017-0142-4 Luang-In V, Yotchaisarn M, Saengha W, et al. (2019) Isolation and identification of amylase-producing bacteria from soil in Nasinuan Community Forest, Maha Sarakham, Thailand. Biomed Pharmacol J 12(3):1061–1068. https://dx.doi.org/10.13005/bpj/1735 Luo AG, Wang YY, Xue SS, et al. (2023) Screening, identification, and optimization of enzyme-producing conditions for cellulose-degrading bacteria in distillery lees. Appl Sci 13(13):7693. https://doi.org/10.3390/app13137693 Mehmood T, Saman T, Irfan M, Anwar F, Ikram MS, Tabassam Q (2019) Pectinase production from Schizophyllum commune through central composite design using citrus waste and its immobilization for industrial exploitation. Waste Biomass Valor 10:2527–2536. https://doi.org/10.1007/s12649-018-0279-9 Mohammadi M, Mokarram RR, Shahvalizadeh R, Sarabandi K, Lim LT, Hamishehkar H (2020) Immobilization and stabilization of pectinase on an activated montmorillonite support and its application in pineapple juice clarification. Food Biosci 36:100625. https://doi.org/10.1016/j.fbio.2020.100625 Ortiz GE, Ponce-Mora MC, Noseda DG, et al. (2017) Pectinase production by Aspergillus giganteus in solid-state fermentation: optimization, scale-up, biochemical characterization and its application in olive-oil extraction. J Ind Microbiol Biotechnol 44(2):197–211. https://doi.org/10.1007/s10295-016-1873-0 Ozzeybek M, Cekmecelioglu D (2024) Formulation of apple pomace, orange peel, and hazelnut shell mix for co-production of bacterial pectinase and cellulase enzymes by mixture design method. Biomass Convers Biorefinery 14(2):1853–1861. https://doi.org/10.1007/s13399-022-02409-0 Patel K, Dudhagara P (2020) Optimization of xylanase production by Bacillus tequilensis strain UD-3 using economical agricultural substrate and its application in rice straw pulp bleaching. Biocatal Agric Biotechnol 30:101846. https://doi.org/10.1016/j.bcab.2020.101846 Pérez-Contreras S, La Cruz DA, Lizardi-Jiménez MA, Herrera-Corredor JA, Baltazar-Bernal O, Hernández-Martínez R (2024) Production of ligninolytic and cellulolytic fungal enzymes for agro-industrial waste valorization: Trends and applicability. Catalysts 15(1):30. https://doi.org/10.3390/catal15010030 Pili J, Danielli A, Nyari NL, et al. (2018) Biotechnological potential of agro-industrial waste in the synthesis of pectin lyase from Aspergillus brasiliensis . Food Sci Technol Int 24(2):97–109. https://doi.org/10.1177/1082013217733574 Qadir F, Ejaz U, Sohail M (2020) Co-culturing corncob-immobilized yeasts on orange peels for the production of pectinase. Biotechnol Lett 42:1743–1753. https://doi.org/10.1007/s10529-020-02897-y Rispoli FJ, Shah V (2007) Mixture design as a first step for optimization of fermentation medium for cutinase production from Colletotrichum lindemuthianum . J Ind Microbiol Biotechnol 34(5):349–355. https://doi.org/10.1007/s10295-007-0203-y Ruginescu R, Gomoiu I, Popescu O, et al. (2020) Bioprospecting for novel halophilic and halotolerant sources of hydrolytic enzymes in brackish, saline and hypersaline lakes of Romania. Microorganisms 8(12):1903. https://doi.org/10.3390/microorganisms8121903 Satapathy S, Soren JP, Mondal KC, et al. (2021) Industrially relevant pectinase production from Aspergillus parvisclerotigenus KX928754 using apple pomace as the promising substrate. J Taibah Univ Sci 15(1):347–356. https://doi.org/10.1080/16583655.2021.1978833 Shet AR, Muhsinah AB, Alhazmi AY, et al. (2022) Bioprocessing of agro-industrial waste for maximization of pectinase production by Aspergillus cervinus ARS2. Separations 9(12):438. https://doi.org/10.3390/separations9120438 Shrestha S, Khatiwada JR, Sharma HK, Qin W (2021) Bioconversion of fruits and vegetables wastes into value-added products. In: Adv Sci Technol Innov, pp 145–163. https://doi.org/10.1007/978-3-030-61837-7_9 Shrestha S, Rahman MS, Qin W (2021) New insights in pectinase production development and industrial applications. Appl Microbiol Biotechnol 105:9069–9087. https://doi.org/10.1007/s00253-021-11705-0 Silva D, Martins ED, Silva RD, Gomes E (2002) Pectinase production by Penicillium viridicatum RFC3 by solid state fermentation. Braz J Microbiol 33:318–324. https://doi.org/10.1590/s1517-83822002000400008 Szymańska-Chargot M, Chylińska M, Gdula K, et al. (2017) Isolation and characterization of cellulose from different fruit and vegetable pomaces. Polymers 9(10):495. https://doi.org/10.3390/polym9100495 Umar A, Ahmed S (2022) Optimization, purification and characterization of laccase from Ganoderma leucocontextum. Sci Rep 12(1):2416. https://doi.org/10.1038/s41598-022-06111-z Yusof AH, Dailin DJ, Low LZMI, Zaidel DN, El Enshasy H (2020) Potential application of pineapple waste as a fermentation substrate in yeast production. Int J Sci Technol Res 9:1933–1937. Zion Market Research (n.d.) Global Pectinase market: CAGR. https://www.zionmarketresearch.com/news/global-pectinase-market Table Table.1. Design of Experiment and pectinase activity analysis in carbon source mixtures. S.no Apple (%) Orange (%) WB (%) Pectinase (U/ml) 1 0.0000 0.5000 1.500 615.90 2 1.3125 0.3125 0.375 289.50 3 0.5000 0.0000 1.500 496.59 4 2.0000 0.0000 0.000 60.27 5 0.3125 1.3125 0.375 305.90 6 0.5625 0.3125 1.125 368.05 7 0.3125 0.5625 1.125 352.85 8 0.0000 2.0000 0.000 303.06 9 0.6250 0.6250 0.750 68.26 Supplementary Files 7.Graphicalabstract.png 6.Supplementarydata.doc Cite Share Download PDF Status: Under Review Version 1 posted Reviewers agreed at journal 15 Jul, 2025 Reviewers invited by journal 06 Jul, 2025 Editor assigned by journal 02 Jul, 2025 First submitted to journal 01 Jul, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7020186","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":481251726,"identity":"97005e60-aa3b-4652-9b51-8ca85ae8ec31","order_by":0,"name":"Sharmili Shivajirao Jagtap","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA/ElEQVRIiWNgGAWjYDACCRBhYMED5iQAMT+YUUBQiwRCi2QDiGFASAuUBAODA2AStw752c3PPt0okJBhkO4x3fCg5p6c8fnViR8eGDDI84sdwKrF4M4x49k5IIfJnDG7kXCs2NjsxtvNEkCHGc6cnYBdi0SCMTNYi0SO2Y3EhoTEbTfObgBpSTC4jV2L/Iz0z6haNs84u/kHPi0MN3LQbNnA37sNry0GN3KKwVrYZI6VAf2SYCxxg3ebRYKBBE6/AB22mTnnj409v3Tztps/ahLk+PvPbr75o8JGnl8ah8NggA0eNRJglRI4VSIAXA3/ASJUj4JRMApGwUgCAKt3WOUqgbOUAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0002-5421-6291","institution":"Pondicherry University","correspondingAuthor":true,"prefix":"","firstName":"Sharmili","middleName":"Shivajirao","lastName":"Jagtap","suffix":""},{"id":481251727,"identity":"f39590a3-5031-4299-9013-40a9b5d206e4","order_by":1,"name":"Sangeetha P","email":"","orcid":"","institution":"Pondicherry University","correspondingAuthor":false,"prefix":"","firstName":"Sangeetha","middleName":"","lastName":"P","suffix":""}],"badges":[],"createdAt":"2025-07-01 12:17:21","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7020186/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7020186/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":86525247,"identity":"8d76dc06-c972-4034-b8e0-bac0c5074dff","added_by":"auto","created_at":"2025-07-11 15:46:44","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":90406,"visible":true,"origin":"","legend":"\u003cp\u003eZone of Hydrolysis on Pectin-Containing Agar Plate by \u003cem\u003eBacillus subtilis SK16\u003c/em\u003e\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7020186/v1/ab2274b7a0b30aaa3904b39d.png"},{"id":86524670,"identity":"e343a433-d05b-4053-87a7-846d7103bb96","added_by":"auto","created_at":"2025-07-11 15:38:47","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":136084,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic tree of \u003cem\u003eBacillus subtilis \u003c/em\u003eSK16 based on 16S rRNA gene sequences, constructed using the [Neighbor-Joining/Maximum Likelihood/Maximum Parsimony] method. Bootstrap values are indicated at branch points.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7020186/v1/90b927c56f2a81506a141bc4.png"},{"id":86525282,"identity":"00a10a43-4c81-44d5-8d1b-b6513f0faaab","added_by":"auto","created_at":"2025-07-11 15:46:46","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":34654,"visible":true,"origin":"","legend":"\u003cp\u003eSelection of the Best Agro-Waste Carbon Source for Pectinase Production\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7020186/v1/4a915f7dc93b7f98e6b1ef29.png"},{"id":86524634,"identity":"a55eda2f-12cc-4d78-aa6c-d4eafe10c6fb","added_by":"auto","created_at":"2025-07-11 15:38:42","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":33036,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of Different Concentrations of Apple Peel on Pectinase Production\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7020186/v1/9fe4bf12fad245fb9bd1cd44.png"},{"id":86524651,"identity":"e7a72184-c5c4-47b3-83e3-9c9b23162736","added_by":"auto","created_at":"2025-07-11 15:38:45","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":35878,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of Different Concentrations of Orange Peel on Pectinase Production\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7020186/v1/4c5f79a1cb8a7933d41b7512.png"},{"id":86524640,"identity":"101b5e97-a12b-4529-afe1-5e8714eec3cf","added_by":"auto","created_at":"2025-07-11 15:38:43","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":31805,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of Different Concentrations of Wheat bran on Pectinase Production\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-7020186/v1/c1001f0144b0fcc7c4db9a47.png"},{"id":86525310,"identity":"46153026-a600-448d-88d6-83cd3209f750","added_by":"auto","created_at":"2025-07-11 15:46:52","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1290840,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7020186/v1/a67103cd-1dc2-42c9-8222-780c93b1d4ca.pdf"},{"id":86524656,"identity":"d0d77fe7-93a8-4142-97f7-7272d33d8d8a","added_by":"auto","created_at":"2025-07-11 15:38:45","extension":"png","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":3315008,"visible":true,"origin":"","legend":"","description":"","filename":"7.Graphicalabstract.png","url":"https://assets-eu.researchsquare.com/files/rs-7020186/v1/b4e7beea3d595b9958a6762f.png"},{"id":86524642,"identity":"09bc2b4e-7af7-4930-a36c-524ab03bb405","added_by":"auto","created_at":"2025-07-11 15:38:43","extension":"doc","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":39936,"visible":true,"origin":"","legend":"","description":"","filename":"6.Supplementarydata.doc","url":"https://assets-eu.researchsquare.com/files/rs-7020186/v1/53ee67cb59dd042d5ed479c0.doc"}],"financialInterests":"","formattedTitle":"Valorization of Agro-Industrial Waste for Pectinase Production by Bacillus subtilis SK16: A Step Toward Circular Bioeconomy","fulltext":[{"header":"Highlights","content":"\u003cul type=\"disc\"\u003e\n \u003cli\u003eUtilized agro-industrial fruit peel wastes as sustainable substrates for eco-friendly pectinase production.\u003c/li\u003e\n \u003cli\u003ePromoted solid waste bioconversion and waste valorization, aligning with circular bioeconomy principles.\u003c/li\u003e\n \u003cli\u003eEnabled low-cost enzyme production by leveraging abundantly available organic waste materials.\u003c/li\u003e\n \u003cli\u003eSupported SDG 2030 goals by fostering responsible waste management and sustainable industrial practices.\u003c/li\u003e\n \u003cli\u003eDemonstrated a green biotechnological approach for transforming agri-waste into high-value bioproducts.\u003c/li\u003e\n\u003c/ul\u003e"},{"header":"1. Introduction`","content":"\u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eIndia, as the world\u0026rsquo;s second-largest agro-based economy, generates substantial biomass due to rapid industrialization, urbanization, population growth, and intensified food production. According to the Indian Ministry of New and Renewable Energy (MNRE), approximately 500\u0026nbsp;million tons of agricultural waste are produced annually, of which 140\u0026nbsp;million tons remain unutilized and 92\u0026nbsp;million tons are burned, releasing an estimated 3.3\u0026nbsp;billion tons of CO₂ into the atmosphere (\u003c/span\u003eCapanoglu et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eMismanagement of this waste exacerbates climate change, pollution, and resource degradation, emphasizing the need for sustainable management practices.\u003c/span\u003e\u003c/p\u003e\u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eAgro-industrial residues are enriched with essential micro- and macronutrients, including carbohydrates, proteins, lipids, vitamins, and minerals, making them valuable feedstocks for the biotechnological recovery of bioactive compounds (\u003c/span\u003eHadj Saadoun et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eTheir high carbon and nitrogen content further enhances their suitability as substrates for microbial fermentation (\u003c/span\u003eAdriana et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eConsidering that commercial substrates contribute 40\u0026ndash;70% of total fermentation costs, the utilization of agro-wastes offers a cost-effective and sustainable alternative. Moreover, their valorization aligns with circular economy principles, enabling the transformation of waste into high-value products such as industrially important enzymes (\u003c/span\u003eP\u0026eacute;rez-Contreras et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2024\u003c/span\u003e, Yusof et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eAmong industrial enzymes, pectinase plays a pivotal role in a wide array of applications. Pectinase is an industrially important enzyme with a worldwide growing market demand, share about 25% of utilization in food industry \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e(\u003c/span\u003eHaile and Ayele \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2022\u003c/span\u003e, Mehmood et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Mohammadi et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). As a result, the global pectinase market is expected to grow substantially, from \u003cspan\u003e$\u003c/span\u003e18.3\u0026nbsp;billion in 2022 to \u003cspan\u003e$\u003c/span\u003e26.6\u0026nbsp;billion by 2030, reflecting a compound annual growth rate (CAGR) of approximately 11.1% \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e(Zion Market Research 2020)\u003c/span\u003e. However, its widespread application is hindered by high production costs, necessitating the development of cost-effective production methods. To address this challenge, researchers are exploring various strategies to reduce pectinase production costs. The utilization of low-cost agro-industrial wastes as substrates has become most preferable for pectinase production, addressing both economic and environmental concerns (Kaul et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2024\u003c/span\u003e, Haile and Ayele \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2022\u003c/span\u003e, Ketipally and Ram \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). In alignment with the United Nations Sustainable Development Goals (SDG 2030) particularly SDG 12 (Responsible Consumption and Production) and SDG 13 (Climate Action) there is a growing need to repurpose agro-industrial waste streams for productive use. Pectin-rich wastes such as fruit peels and cereal by-products are abundant and underutilized. Their valorization through microbial fermentation supports waste minimization, resource circularity, and the development of a circular bioeconomy, offering a dual benefit of waste reduction and bioproduct generation. Microbial pectinase is primarily sourced from bacteria, fungi, and yeast \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e(\u003c/span\u003eHaile and Ayele \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2022\u003c/span\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e)\u003c/span\u003e. In previous literature, most of the pectinolytic enzymes were produced from fungi \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e(\u003c/span\u003eAfzia et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), whereas there are fewer studies on bacterial pectinases compared to fungi. Fungal sources are prominent, with \u003cem\u003eAspergillus\u003c/em\u003e species being particularly noteworthy. \u003cem\u003eAspergillus spp\u003c/em\u003e. Gm was found to be a potent strain for pectinase production, showing optimal enzyme activity at 30\u0026deg;C and pH 5.8 \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e(\u003c/span\u003eKC et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). However, the fungal sourced pectinase has limited usage due to their narrow pH range, which is not suitable for applications at alkaline pH or neutral pH. Among bacterial sources, \u003cem\u003eBacillus subtilis\u003c/em\u003e has been identified as a potent pectinase producer. Alqahtani et al isolated and characterized a pectinase-producing \u003cem\u003eB. subtilis\u003c/em\u003e strain with high specific activity and stability at pH 4.5 and 50\u0026deg;C \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e(\u003c/span\u003eAlqahtani et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Interestingly, halophilic and halotolerant microorganisms from salty lakes have been explored as novel sources of salt-tolerant pectinases. Bacteria belonging to the genus \u003cem\u003eBacillus\u003c/em\u003e and extremely halophilic archaea of the genera \u003cem\u003eHaloterrigena\u003c/em\u003e and \u003cem\u003eHalostagnicola\u003c/em\u003e showed promising hydrolytic activities, including pectinase production \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e(\u003c/span\u003eRuginescu et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). This diversity in bacterial sources allows for the production of pectinases with varied properties, suitable for different industrial applications.\u003c/p\u003e\u003cp\u003eAmong pectinase-producing microbes, \u003cem\u003eBacillus subtilis\u003c/em\u003e is recognized for its Generally Recognized as Safe (GRAS) status, high metabolic versatility, and ability to secrete multiple enzymes. In this study, a novel isolate, \u003cem\u003eBacillus subtilis\u003c/em\u003e SK16, was explored for its pectinase production capability using agro-industrial wastes, including orange peel, apple peel, and wheat bran. These substrates not only serve as nutrient sources but also exemplify the concept of waste valorization by turning local organic residues into industrially important enzymes \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e(\u003c/span\u003eKuvvet et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eVarious studies have demonstrated the successful utilization of these low-cost materials to enhance enzyme yields. For example, the utilization of orange peel and wheat bran as a substrate enabled \u003cem\u003eBacillus licheniformis\u003c/em\u003e to produce high quantities of pectinase in a simple medium, making it a promising option for large-scale commercial production \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e(\u003c/span\u003eBibi et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2016\u003c/span\u003e, Jahan et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). In addition, previous study reported that apple pomace, orange peels, and Satkara peel have been successfully used as low-cost substrates for pectinase production \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e(\u003c/span\u003eAhmed et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2021\u003c/span\u003e, Satapathy et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2021\u003c/span\u003e, Qadir et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). This approach aligns with the growing emphasis on using agricultural biomasses as low-cost substrates for pectinase production (Shrestha et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In addition to substrate selection, previous studies have primarily focused on optimizing pectinase production through various strategies such as strain improvement, statistical optimization of media composition, and fermentation parameter adjustments \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e(\u003c/span\u003eAhmed et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2021\u003c/span\u003e, Ortiz et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Satapathy et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Govindaraji and Vuppu \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Qadir et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Techniques like Plackett\u0026ndash;Burman design (PBD) and response surface methodology (RSM) have been widely used to enhance enzyme yields. For instance, statistical optimization of media components using orange pectin as a substrate resulted in a maximum pectinase activity of 170.05 U/mL \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e(\u003c/span\u003eGovindaraji and Vuppu \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2020\u003c/span\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e).\u003c/span\u003e Similarly, a combination of orange peel and coconut fiber (4:1) yielded a peak pectinase production of 3315 U/gds by \u003cem\u003eBacillus subtilis\u003c/em\u003e SAV-21 under optimized solid-state fermentation conditions \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e(\u003c/span\u003eKaur and Gupta \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2017\u003c/span\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e).\u003c/span\u003e Bibi et al. further demonstrated that response surface methodology and central composite design could be used to optimize cultural parameters, achieving a maximum pectinase activity of 219 U/mL from \u003cem\u003eBacillus licheniformis\u003c/em\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e(\u003c/span\u003eBibi et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Additionally, some research has explored the use of multiple agro-waste substrates in combination to enhance enzyme yield. Rispoli and Shah \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e(\u003c/span\u003eRispoli and Shah \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2007\u003c/span\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e)\u003c/span\u003e applied mixture design experiments to investigate the effect of glucose, yeast extract, starch, and magnesium sulfate on the production of cutinase enzymes by \u003cem\u003eColletotrichum lindemuthianum\u003c/em\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e(\u003c/span\u003eRispoli and Shah \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2007\u003c/span\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e).\u003c/span\u003e While statistical optimization models and strain improvement approaches have contributed to enhancing enzyme production, they often involve high costs, complex optimization steps, and potential biosafety concerns associated with genetic modifications. In contrast, the combination of multiple agro-waste substrates presents a simple yet highly effective alternative for improving pectinase production \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e(\u003c/span\u003eKaul et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2024\u003c/span\u003e, Haile and Ayele \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2022\u003c/span\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e).\u003c/span\u003e Different agro-waste materials contribute a diverse range of nutrients that work synergistically to enhance microbial metabolism and enzyme synthesis. For example, fruit peels such as orange and apple serve as natural inducers due to their high pectin content, while wheat bran supplies essential proteins, amino acids, and trace elements that support microbial growth and metabolic activity. This synergistic effect leads to significantly higher enzyme yields compared to single-substrate fermentation, making it a promising strategy for industrial applications. Furthermore, this approach is cost-effective, scalable, and environmentally sustainable, as it utilizes abundant and renewable agro-industrial waste, reducing dependence on expensive pure carbon sources. Unlike statistical optimization models, which rely on mathematical predictions that may not always be applicable in large-scale fermentation processes, the combination substrate strategy ensures consistent and reproducible enzyme production across different batches, offering greater industrial feasibility.\u003c/p\u003e\u003cp\u003eThe novelty of the present study lies in two key aspects: (1) the identification of a novel \u003cem\u003eBacillus subtilis\u003c/em\u003e SK16 strain as a potent pectinase producer and (2) the development of a combinational agro-waste substrate strategy for enhanced enzyme production. In this study, nine different combinations of pectin-rich agro-waste substrates were evaluated to determine the most effective formulation for maximizing pectinase yield. The results demonstrated that a specific combination of orange peel, apple peel, and wheat bran significantly enhanced enzyme production compared to single-substrate media. By refining the ratio of these natural agro-waste components, our study highlights the synergistic effect of substrate combination, leading to improved pectinase production efficiency. This research contributes to sustainable biotechnology by promoting waste valorization, reducing dependency on costly synthetic carbon sources, and offering an economically viable alternative for industrial enzyme production. Furthermore, our findings address a critical gap in pectinase research by reinforcing the importance of strain selection and combinational substrate strategies for optimizing fermentation processes. This study aligns with the principles of the circular economy and SDG 2030 goals, making a significant contribution to the development of environmentally sustainable enzyme production technologies.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Microorganism and materials:\u003c/h2\u003e\u003cp\u003eAmmonium sulphate, Potassium di-hydrogen phosphate, Potassium phosphate dibasic, Magnesium sulphate, Sodium carbonate, Apple pectin and, Di-nitro salicylic acid (DNSA) were obtained from Hi-Media (India). Sodium potassium tartrate was purchased from SRL (India). Polygalacturonic acid, Xylan, was purchased from Sigma Chemicals (USA). Various agro-industrial residues, including wheat bran, and fruit peel wastes such as lemon, banana, mango, pomegranate, orange, and apples peels, were collected from local market. These residues were thoroughly washed with tap water to remove impurities, cut into small pieces, and dried in hot air oven for 6hrs at 55\u003csup\u003eo\u003c/sup\u003eC.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2 Qualitative Screening of \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003ePectinolytic Enzymes\u003c/span\u003e:\u003c/h2\u003e\u003cp\u003ePectinolytic enzymes play a crucial role in the degradation of pectin, a structural polysaccharide in plant cell walls. These enzymes include pectinase, which hydrolyzes pectin into oligosaccharides; pectin lyase, which cleaves highly esterified pectin via a β-elimination mechanism; and polygalacturonase, which hydrolyzes glycosidic bonds in polygalacturonic acid.\u003c/p\u003e\u003cp\u003eThe qualitative screening of pectinolytic enzymes produced by best isolate SK16 was performed using iodine staining method. The bacterial strain was inoculated onto three different substrate-specific agar media: \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003epectin agar (for pectinase activity), citrus pectin agar (for pectin lyase activity), and polygalacturonic acid agar (for polygalacturonase activity). The medium used for pectinase screening consisted of 1% pectin, 0.14% (NH₄)₂SO₄, 0.6% K₂HPO₄, 0.2% KH₂PO₄, 0.01% MgSO₄, and 2% agar-agar, adjusted to pH 7.0. Spot inoculation of bacterial isolates was performed on the respective media, followed by incubation at 37\u0026deg;C for 48 hours under aerobic conditions\u003c/span\u003e\u003c/p\u003e\u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eFollowing incubation, the plates were flooded with iodine solution, which interacts with intact pectin to form a dark-colored complex. The presence of clear hydrolysis zones around the bacterial colonies indicated enzymatic degradation of pectin, confirming enzyme production. The diameters of hydrolysis zones were measured to assess the relative activity of each enzyme. This method provided a rapid and effective preliminary screening of best isolate SK16\u0026rsquo;s pectinolytic potential. (\u003c/span\u003eCardoso et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Ishihara et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2021\u003c/span\u003e)\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3 Screening of Industrially Important Enzymes\u003c/h2\u003e\u003cp\u003eThe enzymatic potential of SK16 strain was evaluated for the production of industrially significant enzymes, including xylanase, amylase, laccase, cellulase, lipase, and protease, using substrate-specific agar media. The strain was inoculated on xylan, carboxymethyl cellulose (CMC), starch, guaiacol, Victoria Blue R, and skim milk casein agar for the respective enzyme activities. Plates were incubated at 37\u0026deg;C for 48 hours.\u003c/p\u003e\u003cp\u003eAfter incubation, enzyme activity was qualitatively assessed through substrate hydrolysis or specific staining techniques. Amylase production was confirmed by clear zones on starch agar after flooding with Lugol\u0026rsquo;s iodine (Luang-In et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Xylanase and cellulase activities were detected using Congo red staining on xylan and CMC agar, with hydrolysis zones indicating degradation of xylan and cellulose (Abena et al. 2024; Luo et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Islam et al. 2018; Patel et al. 2020). Protease and lipase activities were indicated by clear zones on skim milk casein agar and blue halos on Victoria Blue R agar, respectively. Laccase activity was confirmed by reddish-brown coloration due to guaiacol oxidation (Fu et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). These qualitative assays provided a preliminary assessment of the enzymatic potential of \u003cem\u003eB. subtilis\u003c/em\u003e SK16.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4 Quantitative Assay for pectinase:\u003c/h2\u003e\u003cp\u003e\u003cem\u003eBacillus subtilis\u003c/em\u003e was inoculated into pectin broth and incubated at 37\u0026deg;C. after incubation period, the culture media was centrifuged at 7,500 rpm at 4\u0026deg;C. The resulting clear cell free supernatant was used as a crude pectinase source for the quantitative pectinase assay. Pectinase activity was measured based on the release of D-Monogalacturonic acid from pectin. The reaction mixture contained crude enzyme extract and 1% Apple pectin dissolved in 100 mM phosphate buffer at pH 7.0. The reaction was incubated at 37\u0026deg;C for 30 minutes, and the liberated D-galacturonic acid was quantified using the DNSA reagent method (Shet et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). One unit of pectinase was defined as the amount of enzyme required to liberate 1 \u0026micro;mol of D-galacturonic acid per minute under the assay conditions.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5 Molecular identification of \u003cem\u003eBacillus subtilis\u003c/em\u003e:\u003c/h2\u003e\u003cp\u003eThe bacterial isolate exhibiting strong pectinolytic activity was subjected to molecular identification through 16S rRNA gene sequencing. Universal primers 27F (5\u0026prime;-AGA GTT TGA TCM TGG CTC AG-3\u0026prime;) and 1492R (5\u0026prime;-TAC GGY TAC CTT GTT-3\u0026prime;) were employed to amplify the 16S rRNA region from the extracted genomic DNA. The resulting amplicon sequences were compared with existing sequences in the NCBI database using the Nucleotide BLAST tool (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://blast.ncbi.nlm.nih.gov/Blast.cgi\u003c/span\u003e\u003cspan address=\"https://blast.ncbi.nlm.nih.gov/Blast.cgi\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) to determine sequence similarity. For phylogenetic analysis, multiple sequence alignments were carried out using CLUSTALW, and evolutionary relationships were inferred through MEGA7 software, comparing the isolate\u0026rsquo;s sequence to those of closely related reference strains.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.6 Production and optimization of Pectinase:\u003c/h2\u003e\u003cp\u003eInoculum preparation and Processing of Agro-industrial wastes as a substrate:\u003c/p\u003e\u003cp\u003eThe inoculum was prepared by inoculating a loopful of \u003cspan type=\"ItalicUnderline\" class=\"ItalicUnderline\" name=\"Emphasis\"\u003eBacillus subtilis\u003c/span\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eSK16\u003c/span\u003e into 50 mL of sterile pectin broth in a 250 mL flask. The culture was incubated at 37\u0026deg;C with agitation at 200 rpm on a rotary shaker for 18 hours. Various agro-industrial residues, including wheat bran and fruit peel wastes (lemon, banana, mango, pomegranate, orange, and apple peels), were collected from the local market. These residues were washed to remove water-soluble impurities, cut into small pieces, and dried in a hot air oven for 6hrs at 55\u003csup\u003eo\u003c/sup\u003eC. These dried substrates were used as a natural pectin source in media preparation for the production of pectinase.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e2.7 Analysis of the parameter affecting Pectinase production:\u003c/h2\u003e\u003cp\u003ePectinase production was carried out by submerged fermentation (Smf) using the basal medium with a pectin rich agro-waste (1%) as a substrate. Parameters influencing enzyme production, such as incubation period, pH, temperature, and natural substrates, and substrate concentration were analyzed using a one-factor-at-a-time approach, keeping all other variables constant. Each experiment was performed in triplicate.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e2.8 Selection of Best agro waste for the production of pectinase:\u003c/h2\u003e\u003cp\u003e​To identify the best natural pectin source for pectinase production, various natural substrates were selected based on their pectin content and local availability. These substrates include (pectin content for each is mentioned in the brackets) apple peel (12.5%), lemon peel (20.75%), mango peel (8.8%), orange peel (15.24%), banana peel (15\u0026ndash;24%), wheat bran (significantly low), and pomegranate peel (6.80\u0026ndash;10.1%). Additionally, different incubation times were assessed to determine the duration required for maximum enzyme production using \u003cem\u003eBacillus subtilis\u003c/em\u003e.​\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e2.9 Effect of Substrate Concentration on pectinase yield:\u003c/h2\u003e\u003cp\u003eThe influence of substrate concentration on pectinase production was evaluated using five different concentrations (0.5%, 1%, 1.5%, 2%, and 3%) of the three most effective pectin rich natural substrates at different incubation times.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e2.10 Optimization of combination natural pectin sources:\u003c/h2\u003e\u003cp\u003eThe study objective assumes that pectinase production is influenced by the composition of the natural pectin sources in the culture medium. In this study, the three best natural pectin sources such as 2% orange peel, 2% apple peel, and 1.5% wheat bran were used, collectively contributing to 2% (w/v) of the total pectin composition. Totally we have designed 9 different combinations for analyse the best combination for better pectinase production (Table\u0026nbsp;1).\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003e3.1 Microbial Strain and Molecular Identification\u003c/h2\u003e\u003cp\u003eThe bacterial strain \u003cem\u003eBacillus subtilis SK16\u003c/em\u003e was successfully isolated from alkaline soil and taxonomically identified through 16S rRNA gene sequencing (GenBank accession no. PQ206567). Phylogenetic analysis positioned the isolate within the \u003cem\u003eBacillus subtilis\u003c/em\u003e clade, showing close evolutionary relatedness to reference strains \u003cem\u003eB. subtilis\u003c/em\u003e R8 (MH371779.1) and M124 (MH168996.1), supported by high bootstrap values. Primary screening revealed notable pectinolytic activity, with clear hydrolysis zones measuring 18 mm and 15 mm for polygalacturonase and pectinase, respectively, on pectin agar plates. However, no pectin lyase activity was observed, suggesting an absence of β-elimination cleavage of highly methylated pectin substrates (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003e3.2 Screening of Industrially Important Enzymes\u003c/h2\u003e\u003cp\u003eQualitative enzyme screening revealed that \u003cem\u003eBacillus subtilis SK16\u003c/em\u003e produced multiple industrially relevant enzymes. Hydrolysis zones were observed for amylase (9 mm), protease (15 mm), lipase, xylanase (16 mm), and cellulase (9 mm), confirming extracellular enzyme production on selective media (Table S2).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003e3.3 Quantitative Pectinase Activity\u003c/h2\u003e\u003cp\u003ePectinase activity in submerged fermentation reached a peak of 321.14 U/ml at 48 hours of incubation, as measured by the DNSA method. The crude enzyme extract effectively hydrolyzed pectin to release D-galacturonic acid under standard assay conditions (37\u0026deg;C, 30 min).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003e3.4 Pectinase Production Using Agro-Industrial Waste\u003c/h2\u003e\u003cp\u003eAgro-waste substrates including orange peel, apple peel, lemon peel, mango peel, banana peel, wheat bran, and pomegranate peel were tested for pectinase production. Orange peel supported the highest enzyme yield at 48 hours, surpassing even commercial pectin, followed by wheat bran and apple peel (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003e3.5 Effect of Substrate Concentration\u003c/h2\u003e\u003cp\u003eThe effect of substrate concentration (0.5\u0026ndash;3%) was examined for orange peel, apple peel, and wheat bran. Maximum pectinase activity was observed at 2% apple peel and 2% orange peel at 48 hours (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e), and 1.5% wheat bran at 24 hours (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003e3.6 Optimization of Combinational Agro-Waste\u003c/h2\u003e\u003cp\u003eNine combinations of apple peel, orange peel, and wheat bran were tested. The highest pectinase activity (615.90 U/ml) was achieved using a combination of 0.5% orange peel and 1.5% wheat bran. This was followed by 0.5% apple peel and 1.5% wheat bran (496.59 U/ml). The triple combination (apple peel 0.6250%, orange peel 0.6250%, and wheat bran 0.75%) showed lower activity (68.26 U/ml), suggesting that dual-substrate systems were more effective than triple combinations.\u003c/p\u003e\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThe successful isolation of Bacillus subtilis SK16 from alkaline soil and its identification through 16S rRNA gene sequencing confirmed its taxonomic affiliation and enzymatic potential. The strain produces polygalacturonase (PG) and pectinase, but lacks pectin lyase activity, indicating substrate specificity for deesterified pectin (polygalacturonic acid). Such an enzyme profile is advantageous for degrading lowmethoxyl pectin in industrial applications such as fruit juice clarification, textile retting, and paper and pulp processing without the need for highly alkaline conditions. Moreover, recent studies report that B. subtilis strains optimized at neutral pH (7\u0026ndash;7.4) can achieve high PG yields (345\u0026ndash;630 U/mL) under mild fermentation conditions, and recombinant PG expressed in \u003cem\u003eB. subtilis\u003c/em\u003e demonstrates improved efficiency for fiber-processing and juice systems under neutral\u0026ndash;slightly acidic pH. This neutral-pH compatibility reduces chemical usage and enhances ecoefficiency in large-scale bioprocesses.\u003c/p\u003e\u003cp\u003eThe qualitative enzyme screening demonstrated that SK16 possesses a broad spectrum of extracellular enzymatic activities. Notably, high activities of xylanase and protease point to its robustness as an industrial workhorse, useful in sectors ranging from biofuel to detergent and food processing. The moderate cellulase and amylase activities further expand its biotechnological applicability.\u003c/p\u003e\u003cp\u003eQuantitative analysis of pectinase production revealed a distinct time-dependent trend, with peak enzyme activity observed at 48 hours of incubation. This pattern is indicative of stationary phase-associated enzyme synthesis, consistent with typical bacterial growth kinetics and secondary metabolite secretion behavior.\u003c/p\u003e\u003cp\u003eWhen exploring cost-effective substrates, orange peel emerged as a superior natural carbon source for pectinase production, outperforming commercial pectin. This can be attributed to its high native pectin content and additional nutrients that enhance microbial metabolism. Wheat bran, despite low pectin content, also significantly supported enzyme production, likely due to its cellulose and hemicellulose content that may serve as co-inducers or enhance metabolic activity. Interestingly, despite its low pectin content, wheat bran has been identified as an efficient substrate for pectinase production. The incubation time was a critical factor, with optimal enzyme production observed after 48 hours. Extending the incubation period beyond this did not significantly increase enzyme activity, suggesting 48 hours as the ideal incubation time for peak production. It indicates that pectinase production activity is correlated with incubation time (Guan et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). A few studies have shown that citrus peel, including orange peel, is a good source of enzyme production (Bibi et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2016\u003c/span\u003e, Ahmed et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Pili et al. demonstrated that pectin lyase production by Aspergillus brasiliensis was higher in the agro-waste residue containing media (orange peel, corn steep liquor, and parboiled rice water) compared to the synthetic media (Abena and Simachew \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Chiliveri et al reported that wheat bran was found to be an ideal substrate, and the maximum yield of pectate lyase (1371.25 U/gds) and polygalacturonase (85.45 U/gds) where, U/gds represents enzyme activity per gram of dry substrate (Chiliveri et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Silva and Martin reported that the mixture of orange bagasse and wheat bran showed to be the best substrate for pectinase production in SSF using \u003cem\u003epenicillium\u003c/em\u003e species (Silva et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). The differences in the amount of pectinase production using different agro-waste are due to the variation in compositions and the carbon/nitrogen content of agro-waste (Amin et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Shrestha et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2021\u003c/span\u003e)\u003c/p\u003e\u003cp\u003eFrom various literature sources, it has been established that fruit and vegetable pomace primarily contain pectin (1.50\u0026ndash;13.40%), cellulose (7.20\u0026ndash;43.60%), hemicellulose (4.26\u0026ndash;33.50%), and lignin (15.30\u0026ndash;69.40%) as their main constituents (Szymańska-Chargot et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). However, the pectin content varies significantly among different fruit peels. Substrate concentration had a pronounced effect on enzyme yield. For orange and apple peels, 2% concentration was optimal, while wheat bran performed best at 1.5%. These optimal levels balance nutrient availability without causing substrate inhibition or excessive viscosity, which could impede oxygen and nutrient diffusion.\u003c/p\u003e\u003cp\u003eOptimization using mixed substrates demonstrated that combinations outperformed individual sources. The combination of orange peel and wheat bran yielded the highest enzyme activity, with more than a 55% increase compared to the best single substrate. This synergy may be attributed to complementary nutrient profiles pectin from fruit peel and polysaccharides from wheat bran that collectively support higher microbial enzyme output.\u003c/p\u003e\u003cp\u003eThe substantial increase in pectinase yield (615.90 U/ml) in combination substrate exceeding the best single-substrate yield by over 55%, reinforces the importance of substrate synergy in maximizing enzyme production. While extensive research exists on optimizing enzyme production, studies focusing on combinational agro-waste substrates remain limited. A study by Ozzeybek and \u0026Ccedil;ekmecelioğlu optimized a mixture of apple pomace (20%), hazelnut shell (50%), and orange peel (30%) for bacterial pectinase and cellulase co-production, reporting pectinase activity of 8.27 U/ml and cellulase activity of 0.5 U/ml, demonstrating the viability of agro-waste-based enzyme production (Ozzeybek and Cekmecelioglu \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIn contrast, the present study introduces a novel approach by demonstrating that wheat bran, despite being a low-pectin substrate, plays a pivotal role in enhancing enzyme yields when combined with fruit peels. This research contributes to sustainable biotechnology by valorizing agro-industrial waste, minimizing reliance on expensive pure carbon sources, and addressing waste management challenges. These findings provide a strong foundation for scaling up industrial pectinase production and highlight the effectiveness of combinational substrate strategies in improving fermentation efficiency and cost-effective bioprocessing\u003c/p\u003e\u003cp\u003eInterestingly, the triple-substrate mix underperformed, possibly due to over-complexity or nutrient imbalance. These findings are consistent with earlier reports showing that specific binary mixtures (e.g., orange peel\u0026thinsp;+\u0026thinsp;rice water or wheat bran) favor microbial enzyme expression more than multiple-component systems.\u003c/p\u003e\u003cp\u003eThis study presents a novel approach to pectinase production through combinational substrate optimization, highlighting the underexplored potential of wheat bran as a co-substrate. The research supports the use of agro-industrial waste in bioprocessing, contributing to sustainable biotechnology by reducing reliance on costly commercial media and addressing environmental concerns associated with waste disposal.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eThis study successfully demonstrated the isolation, identification, and characterization of \u003cem\u003eBacillus subtilis\u003c/em\u003e SK16 as an efficient pectinase-producing microorganism with notable polygalacturonase activity. The strain\u0026rsquo;s enzymatic profile, favoring de-esterified pectin substrates, along with its ability to produce multiple industrially important enzymes, highlights its broad biotechnological potential. Among various natural substrates tested, agro-industrial fruit peel wastes\u0026mdash;particularly orange peel, wheat bran, and apple peel supported substantial enzyme production. Optimization studies revealed that 2% orange and apple peel, and 1.5% wheat bran, yielded maximal pectinase activity. Dual-substrate formulations, especially orange peel combined with wheat bran, enhanced enzyme production by over 55% compared to individual substrates.\u003c/p\u003e\u003cp\u003eThis study underscores the potential of utilizing agro-industrial wastes as sustainable substrates for enzyme production, promoting efficient solid waste bioconversion while offering an eco-friendly and economically viable solution for environmental waste reduction. Overall, the findings contribute to the advancement of sustainable biotechnology by transforming organic waste into value-added bioproducts and fostering greener industrial fermentation strategies.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe author sincerely thanks the Department of Microbiology for providing the essential laboratory facilities and continuous support during the course of this research. Special appreciation is extended to Angothu Shanker, Devayani, and Priyadharshini for their helpful assistance and contributions during the experimental phase of the study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest\u003c/strong\u003e\u003cbr\u003e\u0026nbsp;The corresponding author declares, on behalf of all authors, that there are no conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e\u003cbr\u003e\u0026nbsp;This study was conducted without any financial support from government, commercial, or non-profit funding bodies.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions:\u003c/strong\u003e\u003cbr\u003e\u0026nbsp;Sangeetha P. was solely responsible for the conceptualization, experimentation, data analysis, manuscript drafting, and final revisions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics Approval:\u003c/strong\u003e\u003cbr\u003e\u0026nbsp;Not applicable to this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to Participate:\u003c/strong\u003e\u003cbr\u003e\u0026nbsp;Not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for Publication:\u003c/strong\u003e\u003cbr\u003e\u0026nbsp;All authors consent to the publication of the manuscript.s\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of Data and Materials:\u003c/strong\u003e\u003cbr\u003e\u0026nbsp;The datasets generated or analyzed during the study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCode Availability:\u003c/strong\u003e\u003cbr\u003e\u0026nbsp;Not applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAbena T, Simachew A (2024) Production and characterization of acidophilic xylanase from wood degrading white rot fungus by solid-state fermentation of wheat straw. 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J Taibah Univ Sci 15(1):347\u0026ndash;356. https://doi.org/10.1080/16583655.2021.1978833\u003c/li\u003e\n\u003cli\u003eShet AR, Muhsinah AB, Alhazmi AY, et al. (2022) Bioprocessing of agro-industrial waste for maximization of pectinase production by \u003cem\u003eAspergillus cervinus\u003c/em\u003e ARS2. Separations 9(12):438. https://doi.org/10.3390/separations9120438\u003c/li\u003e\n\u003cli\u003eShrestha S, Khatiwada JR, Sharma HK, Qin W (2021) Bioconversion of fruits and vegetables wastes into value-added products. In: Adv Sci Technol Innov, pp 145\u0026ndash;163. https://doi.org/10.1007/978-3-030-61837-7_9\u003c/li\u003e\n\u003cli\u003eShrestha S, Rahman MS, Qin W (2021) New insights in pectinase production development and industrial applications. Appl Microbiol Biotechnol 105:9069\u0026ndash;9087. https://doi.org/10.1007/s00253-021-11705-0\u003c/li\u003e\n\u003cli\u003eSilva D, Martins ED, Silva RD, Gomes E (2002) Pectinase production by \u003cem\u003ePenicillium viridicatum\u003c/em\u003e RFC3 by solid state fermentation. Braz J Microbiol 33:318\u0026ndash;324. https://doi.org/10.1590/s1517-83822002000400008\u003c/li\u003e\n\u003cli\u003eSzymańska-Chargot M, Chylińska M, Gdula K, et al. (2017) Isolation and characterization of cellulose from different fruit and vegetable pomaces. Polymers 9(10):495. https://doi.org/10.3390/polym9100495\u003c/li\u003e\n\u003cli\u003eUmar A, Ahmed S (2022) Optimization, purification and characterization of laccase from \u003cem\u003eGanoderma leucocontextum.\u003c/em\u003e Sci Rep 12(1):2416. https://doi.org/10.1038/s41598-022-06111-z\u003c/li\u003e\n\u003cli\u003eYusof AH, Dailin DJ, Low LZMI, Zaidel DN, El Enshasy H (2020) Potential application of pineapple waste as a fermentation substrate in yeast production. Int J Sci Technol Res 9:1933\u0026ndash;1937.\u003c/li\u003e\n\u003cli\u003eZion Market Research (n.d.) Global Pectinase market: CAGR. https://www.zionmarketresearch.com/news/global-pectinase-market\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Table","content":"\u003cp\u003eTable.1. Design of Experiment and pectinase activity analysis in carbon source mixtures.\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"659\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003eS.no\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 139px;\"\u003e\n \u003cp\u003eApple (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 155px;\"\u003e\n \u003cp\u003eOrange (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 125px;\"\u003e\n \u003cp\u003eWB (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 154px;\"\u003e\n \u003cp\u003ePectinase (U/ml)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 139px;\"\u003e\n \u003cp\u003e0.0000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 155px;\"\u003e\n \u003cp\u003e0.5000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 125px;\"\u003e\n \u003cp\u003e1.500\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 154px;\"\u003e\n \u003cp\u003e615.90\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 139px;\"\u003e\n \u003cp\u003e1.3125\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 155px;\"\u003e\n \u003cp\u003e0.3125\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 125px;\"\u003e\n \u003cp\u003e0.375\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 154px;\"\u003e\n \u003cp\u003e289.50\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 139px;\"\u003e\n \u003cp\u003e0.5000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 155px;\"\u003e\n \u003cp\u003e0.0000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 125px;\"\u003e\n \u003cp\u003e1.500\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 154px;\"\u003e\n \u003cp\u003e496.59\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 139px;\"\u003e\n \u003cp\u003e2.0000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 155px;\"\u003e\n \u003cp\u003e0.0000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 125px;\"\u003e\n \u003cp\u003e0.000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 154px;\"\u003e\n \u003cp\u003e60.27\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 139px;\"\u003e\n \u003cp\u003e0.3125\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 155px;\"\u003e\n \u003cp\u003e1.3125\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 125px;\"\u003e\n \u003cp\u003e0.375\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 154px;\"\u003e\n \u003cp\u003e305.90\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 139px;\"\u003e\n \u003cp\u003e0.5625\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 155px;\"\u003e\n \u003cp\u003e0.3125\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 125px;\"\u003e\n \u003cp\u003e1.125\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 154px;\"\u003e\n \u003cp\u003e368.05\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 139px;\"\u003e\n \u003cp\u003e0.3125\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 155px;\"\u003e\n \u003cp\u003e0.5625\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 125px;\"\u003e\n \u003cp\u003e1.125\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 154px;\"\u003e\n \u003cp\u003e352.85\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 139px;\"\u003e\n \u003cp\u003e0.0000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 155px;\"\u003e\n \u003cp\u003e2.0000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 125px;\"\u003e\n \u003cp\u003e0.000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 154px;\"\u003e\n \u003cp\u003e303.06\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 139px;\"\u003e\n \u003cp\u003e0.6250\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 155px;\"\u003e\n \u003cp\u003e0.6250\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 125px;\"\u003e\n \u003cp\u003e0.750\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 154px;\"\u003e\n \u003cp\u003e68.26\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"international-journal-of-environmental-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"IJER","sideBox":"Learn more about [International Journal of Environmental Research](https://www.springer.com/journal/41742)","snPcode":"41742","submissionUrl":"https://www.editorialmanager.com/ijer/default2.asp...\n","title":"International Journal of Environmental Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Pectinase, Agro-industrial waste, circular economy","lastPublishedDoi":"10.21203/rs.3.rs-7020186/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7020186/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePectinase is an industrially important enzyme with wide-ranging applications in the food, textile, and biofuel industries. However, commercial production remains cost-intensive due to the use of high-purity substrates. In this study, a cost-effective and sustainable bioprocess was developed to enhance pectinase production using agro-industrial pectin-rich waste materials, in alignment with the United Nations Sustainable Development Goals 2030. \u003cem\u003eBacillus subtilis\u003c/em\u003eSK16 was identified as a potent pectinase producer with notable polygalacturonase activity. Additionally, \u003cem\u003eB. subtilis\u003c/em\u003eSK16 demonstrated the ability to produce multiple industrially important extracellular enzymes, including amylase, xylanase, protease, cellulase, and lipase. In this study, pectinase produced by agro-industrial pectin-rich waste as a substrate. Among them orange peel (397 U/ml), wheat bran (330.97 U/ml), and apple peel (207.9 U/ml) were evaluated as individual substrates for enzyme production. To further enhance enzyme yield, a combinational substrate approach was applied. By adjusting the proportions of these waste components, the study aimed to determine the most effective combination for maximizing pectinase yield. A maximum pectinase activity of 615.90 U/ml was achieved with a substrate combination of 0.5% orange peel and 1.5% wheat bran, reflecting a 55% enhancement compared to the highest yield obtained from individual substrates. This study underscores the potential of utilizing agro-industrial wastes as sustainable substrates for enzyme production, promoting efficient solid waste bioconversion while offering an eco-friendly and economically viable solution for environmental waste reduction. The findings contribute to waste valorization, circular bioeconomy, and reduced reliance on synthetic media, thereby supporting environmental sustainability and industrial scalability.\u003c/p\u003e","manuscriptTitle":"Valorization of Agro-Industrial Waste for Pectinase Production by Bacillus subtilis SK16: A Step Toward Circular Bioeconomy","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-11 15:38:27","doi":"10.21203/rs.3.rs-7020186/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2025-07-15T22:31:36+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-07-06T10:42:25+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-07-02T18:06:46+00:00","index":"","fulltext":""},{"type":"submitted","content":"International Journal of Environmental Research","date":"2025-07-01T08:16:16+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"international-journal-of-environmental-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"IJER","sideBox":"Learn more about [International Journal of Environmental Research](https://www.springer.com/journal/41742)","snPcode":"41742","submissionUrl":"https://www.editorialmanager.com/ijer/default2.asp...\n","title":"International Journal of Environmental Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"d9cf9e03-8fc4-4fe9-b953-9194b4137b8c","owner":[],"postedDate":"July 11th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-01T08:45:58+00:00","versionOfRecord":[],"versionCreatedAt":"2025-07-11 15:38:27","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7020186","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7020186","identity":"rs-7020186","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}