Optimization of immobilized activated sludge performance in electro-sprayed matrices for treatment of cellulose industry wastewater

Optimization of immobilized activated sludge performance in electro-sprayed matrices for treatment of cellulose industry wastewater | 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 Article Optimization of immobilized activated sludge performance in electro-sprayed matrices for treatment of cellulose industry wastewater Farshad Ghafari-Arsoon, Omid Ramezani, Ali Partovinia This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6870463/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 01 Sep, 2025 Read the published version in Scientific Reports → Version 1 posted 10 You are reading this latest preprint version Abstract Today, the abundant use of cellulose industry products has led to an increase in production and, as a result, an increase in the volume of water consumed by this industry. On the other hand, the high volumetric flow rate of produced wastewater, chemical oxygen demand (COD), suspended solids and high turbidity of these wastewaters have caused many problems. In recent years, various methods, including physical, chemical and biological, have been used for wastewater treatment. In this study, firstly, the efficiency of the activated sludge collected from cellulose wastewater was investigated in freely suspended cell system in shake flask experiments at 150 rpm and 30 ̊C. Afterward, to investigate the performance of immobilized sludge, alginate and hybrid alginate-polyvinyl alcohol (PVA) polymers were used for microbeads production. In this survey, the electrospray technique and response surface methodology (RSM) were employed to produce microbeads and statistical optimization, respectively. In order to optimize the bioremediation process, three variables including electrospray voltage (0–12 KV), the volume of cell-polymer suspension (1–3 ml), and two type of carriers were selected. In order to analyze the wastewater, the results related to COD were evaluated. The optimization results showed that the maximum biodegradation and COD removal of 74% (from 6715 to 1736 mg/L) after 4 days was observed by the alginate-immobilized cells produced with the voltage and polymer-cell solution volume of 3 KV and 2.5 mL, respectively. Biological sciences/Biotechnology/Environmental biotechnology Earth and environmental sciences/Environmental sciences/Environmental chemistry/Pollution remediation cellulose wastewater chemical oxygen demand electrospray technique immobilized sludge microbeads Figures Figure 1 Figure 2 Figure 3 Introduction Pulp and paper production is among the important industries that lead to the creation of large amounts of wastewater (Kumar et al., 2021 ). The vast consumption of chemicals in the various processes of this industry has led to the production of highly polluted wastewater (Haq et al., 2020 ; Pazira, 2015 ). Therefore, the use of efficient methods for the treatment of these polluted wastewaters is essential and inevitable. There are various techniques including chemical and electro-coagulation, physical adsorption and biological treatment for removing pollutants from industrial wastewater. These methods can be used separately or in combination (Azimi et al., 2019 ; Boguniewicz-Zablocka et al., 2019 ; Haq et al., 2020 ; Kumar et al., 2021 ). Biological treatment is known as an effective method for removing organic matter with low molecular weight (Leiviskä et al., 2008 ). However, the high performance and cost-effectiveness of wastewater treatment was observed by microbial cells system, there is a possibility of microbial cells inactivation in direct contact with wastewater due to the presence of various inhibitors and toxicants (Partovinia and Rasekh, 2018 ). In recent studied reported that to prevent cell disruption as well as cell activity decrement, it is necessary to immobilized microbial cells to overcome these difficulties (Bouabidi et al., 2019 ; Khanpour-Alikelayeh et al., 2021 ; Paliwal et al., 2016 ; Sergio and Bustos, 2009 ). Immobilization technique is a process in which microbial cells attached or entrapped into a support while maintaining its activity. In general, carriers employed in biodegradation processes include natural and synthetic polymers (Bettmann and Rehm, 1984 ; Jeùrabkova et al., 1997 ; Leenen et al., 1996 ; Paje et al., 1998 ; Suzuki et al., 1998 ; Zhang et al., 2007 ). The natural polymers used mainly include alginate, carrageenan, chitosan, and agar, and the synthetic polymers include polyurethane and polyvinyl alcohol (Doria-Serrano et al., 2001 ; Jeùrabkova et al., 1997 ; Partovinia and Rasekh, 2018 ; Siripattanakul and Khan, 2010 ). Overall, among the natural polymers, alginate is used more often in the entrapment technique due to the mild fabrication condition (Bettmann and Rehm, 1984 ; Leenen et al., 1996 ; Sergio and Bustos, 2009 ). Moreover, alginate due to the cost-effectiveness, biocompatibility and biodegradability introduced as an appropriate choice for industrial applications (Partovinia and Rasekh, 2018 ; Siripattanakul and Khan, 2010 ). An and Lo ( 2001 ) successfully employed the PVA-alginate for the activated sludge immobilization. According to their results, 10-12.5% of PVA was determined as the optimum polymer concentration for sludge immobilization. They also suggested that the presence of 1% alginate in the microbeads prevent their aggregation (An and Lo, 2001 ). In the previous researches, various approaches such as emulsification technique using a homogenizer and ultrasonic method was employed to reduce the microbeads size which helps to overcome to mass transfer limitation (Ausheva et al., 2008 ). Recently, to produce smaller-sized beads, the electrospray method has been used to produce beads with a size of 100–200 µm (Barron and He, 2017 ; Partovinia and Vatankhah, 2019 , 2023 ). Previous studies demonstrate the ability of activated sludge in pulp and paper effluent treatment (Ince et al., 2011 ; Paris and Blondeau, 1999 ; Sharma et al., 2023 ). Partovinia and Vatankhah ( 2023 ) applied immobilized activated sludge to investigate the effect of beads particle size on the biodegradation efficiency of phenol and microbial growth rate. They used electrospray technique to immobilize activated sludge in a hybrid matrix of alginate and polyvinyl alcohol. According to their results, free cell and immobilized cell systems performed similarly at low pollutant concentrations, while the immobilized cell system showed superior biodegradation at higher concentrations like 2000 mg/L. Furthermore, immobilized cells in smaller beads was shown the higher degradation capacity (Partovinia and Vatankhah, 2023 ). According to best of our knowledge, until now the effect of alginate and PVA beads as well as their hybrid in the biological treatment of cellulose industry wastewater has not been investigated. It is noteworthy that alginate beads have not always been completely successful in the biodegradation studies (Jeùrabkova et al., 1997 ; Paje et al., 1998 ; Suzuki et al., 1998 ). Therefore, the stability and activity of microbeads in cellulose industry wastewater, which includes various chemical compounds and toxicants, can be one of the main topics in this research. On the other hand, the immobilized bead size is very crucial in its performance and the bead size reduction lead to increase of mass transfer and biodegradation capacity (Ogbonna et al., 1991 ). Besides, the effect of the immobilized activated sludge content (volume of polymer-cell suspension), the size of the produced beads and the type of polymer (alginate and hybrid alginate-PVA) in the biological treatment of cellulose industry wastewater and COD reduction will be evaluated. Finally, the performance of free and immobilized activated sludge in wastewater treatment will be compared. Materials and Methods Cellulose effluent and materials In this survey, the effluent of the activated sludge pond was selected from the Pouya Ayesh Mazand (Cellulose Group, Mazandaran, Iran). Concentrated sulfuric acid (98%), potassium dichromate, mercury sulfate, silver sulfate, and potassium hydrogen phthalate (KHP) were obtained from Merck, Germany. In this study, a thermal reactor (CR2200 model, Germany) and spectrophotometer (Jenway model 6300, England) was used for the digestion process and COD measurement, respectively. Chemical Oxygen Demand (COD) Analysis Method Chemical oxygen demand is one of the most important indicators for measuring wastewater pollution. To measure the COD, digestion solution, catalyst solution, and standard solution are required. To prepare the digestion solution, 1.5 g of potassium dichromate, 84 mL of concentrated sulfuric acid, and 16.7 g of mercury sulfate were added to 250 mL of deionized distilled water and the volume was adjusted to 500 mL after cooling. Catalyst solution was prepared by adding 2.2 g of silver sulfate into 0.409 kg of concentrated sulfuric acid and placed on a stirrer until completely mixed. Potassium hydrogen phthalate (KHP) as the standard solution was prepared by dissolving 0.425 g in 500 mL of double-distilled water, and 1 mL of this solution contains 1 mg COD (1 mg/1 mL). In this research, to measure the COD quantity, 2.5 mL of sample (e.g. wastewater, deionized water as a control sample or standard solution), 1.5 mL of digestion solution, and 3.5 mL of catalyst solution were added to Pyrex glass vials and stirred well for sufficient mixing. To perform the digestion process, the vials were placed in a thermo-reactor at 150°C for 120 minutes. At the end of this stage, the vials were cooled and the optical density was measured at a wavelength of 600 nm. Finally, the COD level of the samples was calculated using the standard curve (Boyles, 1997 ; Partovinia et al., 2022 ; Rice et al., 2012 ). Activated sludge immobilization in alginate and alginate-PVA hybrid beads Polymer preparation and microbial cell immobilization in alginate Alginate microbeads were prepared by dissolving sodium alginate powder in distilled water (1% w/w) on a stirrer for 30 min at 60 ºC. In order to immobilize the cells, at first 40 mL of activated sludge solution (MLSS = 15000 mg/Lit) was centrifuged at 3000 rpm for 3 min. Then, the aqueous phase was decanted and microbial cells were added to 10 mL of alginate polymer solution and gently mixed for 1 hour to cells were uniformly dispersed in the solution for the electrospray process. Then, 1 ml of the polymer-cell solution was transferred to a 1 mL syringe with a steel needle of 19 G. An electric field with a voltage of 0–12 KV was employed and the solution transferred dropwise with the rate of 2.5 mL/h by a syringe pump into the calcium chloride solution (crosslinking agent). The fabricated beads were left 2 h in the crosslinking solution for hardening process and then washed with distilled water to remove un-immobilized cells on the beads surface (Partovinia and Vatankhah, 2023 ). Polymer preparation and microbial cell immobilization in alginate-PVA hybrid To fabrication of hybrid alginate-polyvinyl alcohol micro-carriers, first alginate 1% (w/v) and PVA 10% (w/v) solutions were prepared, mixed at a ratio of 20:80 (Alginate: PVA), then electrospray process was employed. Biological wastewater treatment using experimental design approach The biological experiments were carried out in 150 mL Erlenmeyer flasks with a working volume of 30 mL in shaker incubator with 150 rpm and 30 ºC for 4 days. Statistical optimization and modelling were carried out by response surface methodology (RSM) based on central composite design (CCD) using three variables. These factors and ​​related ranges are given in the Table 1 . Table 1 Main factors and their coded levels used in the experimental design Factors Range and levels (coded) -2 -1 0 1 2 A: Voltage (KV) 0 3 6 9 12 B: Cell-polymer suspension volume (mL) 1 1.5 2 2.5 3 C: Carrier type Alginate Hybrid Alginate-PVA The total number of trials in the CCD method is calculated as 2 k + 2k + n, where k and n are the number of main factors and central points, respectively. In this study, k and n are 2 and 5, respectively, resulting in 13 experiments for alginate and 13 experiments for hybrid matrix (Montgomery, 2017 ). Statistical analysis method In this study, Design Expert software (version 7) was used for analysis of variance and statistical optimization. Response surface methodology with central composite design was employed to statistically evaluate the main and interactive effects of selected variables and optimize biological treatment. Results and Discussion Cellulose wastewater treatment by freely cell suspended system The initial COD for the selected cellulose industry effluent was measured 6715 mg/L. According to the results, measured COD of control system without inoculation and treatment by freely cell suspension of activated sludge was 6560 and 2320 mg/L after 96 h, respectively. Therefore, it can be concluded that the activated sludge with 65% COD removal after 4 days had a significant role in reducing COD and the organic pollution load of the wastewater. Similarly, Tyagi et al. ( 2014 ) employed combination of two bacterial species Bacillus subtilis and Micrococcus luteus and a fungus Phanerochaete crysosporium to treat paper mill wastewater. Their results showed that microbial combination was able to reduce 94.7% COD after 9 days (Tyagi et al., 2014 ). Haq et al. ( 2016 ) showed that the strain Serratia liquefaciens LD-5 successfully reduced 85% COD of pulp and paper effluent after 144 h of treatment at 30°C, pH 7.6 and 120 rpm (Haq et al., 2016 ). Tiku et al. ( 2010 ) used three bacteria as the consortium exhibited high efficiency to reduce the COD of the effluent, about 76% reduction in COD within 10 h (Tiku et al., 2010 ). Gholami et al. ( 2022 ) showed that the maximum COD removal in recycled paper and cardboard mill wastewater treatment, by Citrobacter freundii AT-4 was 79.54% after 10 days of incubation. In the case of Bacillus subtilis AT-5 and Klebsiella pneumoniae AT-1, the maximum COD removal were 70.08% and 71.26%, respectively (Gholami et al., 2022 ). Cellulose wastewater treatment by immobilized activated sludge The results of biological wastewater treatment using the immobilized activated sludge after 96 hours are given in the Table 2 . As can be shown in Table 1 , the highest COD removal observed in experiment, in which the activated sludge was immobilized in alginate polymer and the voltage and polymer-cell solution volume were 6 KV and 2 mL, respectively. In this case, the highest COD removal was achieved almost 78%, which is higher than the 65% COD removal by free cells. On the other hand, according to the results, the lowest COD removal equal to 40% was observed for immobilized cells in hybrid alginate-polyvinyl alcohol polymer at voltage and polymer-cell solution volume of 3 KV and 1.5 mL, respectively. In most previous studies, it has also been shown that immobilized microbial cells generally have a higher biodegradation rate than free cells (Paliwal et al., 2015 ; Paliwal et al., 2016 ; Partovinia and Rasekh, 2018 ). The reason for this can be attributed to various factors such as the protective role of the carrier material against toxic compounds, the quorum sensing and cooperative effect of microorganisms which live together, the reduction of the inhibitory effect of toxicants, and controlled mass transfer. Paliwal et al. ( 2015 ) reported that co-immobilization of bacterial consortium in corncobs as the agro-industrial residue enhanced the decolorization efficacy of these consortium by retaining the ligninolytic enzymes production (Paliwal et al., 2015 ). Table 2 Results of COD content in treated wastewater using central composite experimental design matrix for actual variables. Run no. Factor 1 Factor 2 Factor 3 Response A:Voltage B:Cell-Polymer suspension volume C:Type of Polymer Chemical Oxygen Demand KV mL mg O 2 /L 1 3 1.5 Alginate 3140 2 9 1.5 Alginate 1521 3 3 2.5 Alginate 2318 4 9 2.5 Alginate 2492 5 0 2 Alginate 1719 6 12 2 Alginate 1521 7 6 1 Alginate 3792 8 6 3 Alginate 1903 9 6 2 Alginate 2270 10 6 2 Alginate 1458 11 6 2 Alginate 3164 12 6 2 Alginate 3130 13 6 2 Alginate 2396 14 3 1.5 PVA:ALG 4043 15 9 1.5 PVA:ALG 2140 16 3 2.5 PVA:ALG 2260 17 9 2.5 PVA:ALG 2502 18 0 2 PVA:ALG 2101 19 12 2 PVA:ALG 2473 20 6 1 PVA:ALG 3705 21 6 3 PVA:ALG 3797 22 6 2 PVA:ALG 2700 23 6 2 PVA:ALG 1956 24 6 2 PVA:ALG 2656 25 6 2 PVA:ALG 2656 26 6 2 PVA:ALG 2072 The analysis of variance for biological wastewater treatment The ANOVA results of wastewater treatment by the immobilized activated sludge are presented in the Table 3 . The obtained p-values for voltage, cell-polymer suspension volume, and polymer type were 0.3321, 0.0962, and 0.1589, respectively, which indicates that among the three selected variables, only the cell-polymer suspension volume, possibly have an effective role on COD reduction. Also, the values obtained for P AB , P AC , and P BC (0.0245, 0.7421, and 0.3987, respectively) revealed that just interaction of voltage and cell-polymer suspension volume has a significant effect on biological wastewater treatment and COD reduction. Table 2 ANOVA results of the quadratic model for cellulose wastewater treatment using immobilized cell system. Analysis of variance table Source Sum of Squares Degree of freedom Mean Square F Value p-value Significance status Model 7.976E+06 8 9.970E+05 3.13 0.0227 Significant A:Voltage 3.169E+05 1 3.169E+05 1.00 0.3321 Not significant B:Cell-Polymer suspension volume 9.866E+05 1 9.866E+05 3.10 0.0962 Possibly significant C:Type of Polymer 6.905E+05 1 6.905E+05 2.17 0.1589 Not significant AB 1.938E+06 1 1.938E+06 6.10 0.0245 Significant AC 35574 1 35574 0.11 0.7421 Not significant BC 2.384E+05 1 2.384E+05 0.75 0.3987 Not significant A^2 6.911E+05 1 6.911E+05 2.17 0.1587 Not significant B^2 2.092E+06 1 2.092E+06 6.58 0.0201 Significant Residual 5.407E+06 17 3.180E+05 Lack of Fit 2.895E+06 9 3.217E+05 1.02 0.4917 Not significant Pure Error 2.511E+06 8 3.139E+05 Cor Total 1.338E+07 25 By applying multiple regression analysis, quadratic equation 1 was found to be the best fit for experimental data of COD in immobilized cell system: COD (mg O 2 /Lit) = 2304.6–114.9 A − 202.7 B + 462.2 AB + 248.5 B 2 (1) where A and B represent actual values for voltage and cell-polymer suspension volume, respectively. The effect of main variables of biodegradation and COD removal Figure 1 (a) shows that with increasing the voltage and decreasing the size of the alginate beads, the COD level decreased, but in general, no significant difference was observed in the COD reduction results. Similarly, Partovinia and Vatankhah ( 2023 ) showed the phenol biodegradation using the cell immobilization technique was higher in smaller beads size of alginate (Partovinia and Vatankhah, 2023 ). Figure 1 (b) shows that with increasing the volume of the cell-polymer suspension, the COD level significantly decreased. According to the Fig. 1 (c), the alginate polymer as an activated sludge carrier had a higher COD removal than the hybrid alginate-polyvinyl alcohol matrix, although based on the results of the analysis of variance, the difference was not significant. Leenen et al. ( 1996 ) compared several natural and synthetic polymers in their survey, and according to the results, cell immobilization and growth occurred well in the natural matrices like alginate and carrageenan. In addition, the effective diffusion coefficients of substrate in the presence of these polymers were close to the diffusion coefficients in water. However, the solubility and degradation of these polymers were reported as the disadvantages especially in their long-terms usage. In contrast, for the synthetic carriers, higher mechanical properties and lower substrate diffusion coefficients were reported (Leenen et al., 1996 ). Examination of interactive effect of variables on COD removal The interactive effect of voltage and volume of cell-polymer suspension on the COD reduction is illustrated in Fig. 2 (a). As can be seen in the Fig. 2 (a), at low cell-polymer suspension volume, with increasing voltage (decreasing bead size), COD has significantly decreased. While at high cell-polymer suspension volume, with increasing voltage, COD has significantly increased. Therefore, it can be concluded that when the amount of immobilized microbial cells in the culture was low, more mass transfer problems were observed in the system and mass transfer limitation was overcome by decreasing bead size. In the other words, it can be concluded that reducing the microbeads size does not necessarily have a positive effect on biodegradation rate and their efficiency is a function of the immobilized cell concentration in the culture. According to the optimization results as shown in Table 3 , the maximum biodegradation rate and COD removal of 74% was observed in alginate-immobilized cell with voltage and polymer-cell solution volume of 3 KV and 2.5 mL, respectively. Table 3 Optimization results of cellulose wastewater treatment using immobilized cell system. Voltage (KV) Cell-Polymer suspension volume (ml) Type of Polymer COD (mg O 2 /Lit) 3 2.5 Alginate (1%) 1736.815 The contour plot for optimizing wastewater treatment by two polymer carriers including alginate and hybrid of alginate-polyvinyl alcohol, is shown in Fig. 3 . As can be seen, the performance of the hybrid matrix was not very successful, while positive performance has been observed for alginate. Although, Cassidy et al have reported it seems that alginate polymer is not a suitable matrix in microbial consortium due to its possibility consumption by fungi (Cassidy et al., 1996 ). In this study, immobilized activated sludge in alginate shows high performance in wastewater treatment. As shown in the Fig. 3 , maximum COD removal was observed in alginate polymer at low voltage and high volume of cell polymer suspension as well as high voltage and low volume of cell polymer suspension. Conclusions Generally, molecular diffusion is the effective process in mass transfer into the gel matrix and usually is a fast process, but the volume fraction and the available space inside the matrix are very small. Therefore, in the discussion of cell immobilization with the entrapment technique, the mass transfer limitation is of particular importance. In this study, cellulose wastewater treatment by immobilized cell fabricated using electro-spraying technique was investigated. According to the obtained results, among the three studied factors, cell-polymer suspension volume had an effective role on the COD reduction. Moreover, the study of interaction effects of the variables showed that only voltage and cell-polymer suspension volume had a mutual effect on each other and as a result affected the COD removal. Besides, it can be concluded that when the amount of activated sludge in the culture was low, mass transfer limitation was observed and it was overcome by reducing the particle size. Based on the findings of this survey, it can be stated that reducing the micro-bead size was effective in the biological treatment process when the volume of used polymer was small. Moreover, in cases which is not possible to produce small-sized beads, a larger volume of polymer-cell can be applied. Declarations Conflicts of Interest: The authors declare no conflict of interest. Funding: This research received no external funding. Author Contribution F.G.A.: methodology, data curation, formal analysis, investigation, resources O.R. and A.P.: conceptualization, supervision, project administration, software, writing—review and editing. All authors have read and agreed to the published version of the manuscript. 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Siripattanakul, S. & Khan, E. Fundamentals and Applications of Entrapped Cell Bioaugmentation for Contaminant Removal. In (ed Shah, V.) Emerging Environmental Technologies, Volume II (147–169). Dordrecht: Springer Netherlands. (2010). Suzuki, T., Yamaguchi, T. & Ishida, M. Immobilization of Prototheca zopfü in calcium-alginate beads for the degradation of hydrocarbons. Process. Biochem. 33 , 541–546. https://doi.org/10.1016/S0032-9592(98)00022-3 (1998). Tiku, D. K. et al. Holistic bioremediation of pulp mill effluents using autochthonous bacteria. Int. Biodeterior. Biodegrad . 64 , 173–183. https://doi.org/10.1016/j.ibiod.2010.01.001 (2010). Tyagi, S. et al. Bioremediation of Pulp and Paper mill Effluent by Dominant Aboriginal Microbes and Their Consortium. I nt . J. Environ. Res. 8 , 561–568. https://doi.org/10.22059/ijer.2014.750 (2014). Zhang, L., Wu, W. & Wang, J. Immobilization of activated sludge using improved polyvinyl alcohol (PVA) gel. J. Environ. Sci. 19 , 1293–1297. https://doi.org/10.1016/S1001-0742(07)60211-3 (2007). Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 01 Sep, 2025 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 01 Aug, 2025 Reviews received at journal 22 Jul, 2025 Reviews received at journal 06 Jul, 2025 Reviewers agreed at journal 14 Jun, 2025 Reviewers agreed at journal 14 Jun, 2025 Reviewers invited by journal 12 Jun, 2025 Editor assigned by journal 12 Jun, 2025 Editor invited by journal 12 Jun, 2025 Submission checks completed at journal 12 Jun, 2025 First submitted to journal 11 Jun, 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. 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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-6870463","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":471212208,"identity":"edc34cae-11da-473d-bc1a-06f3f9f938ce","order_by":0,"name":"Farshad Ghafari-Arsoon","email":"","orcid":"","institution":"Shahid Beheshti University","correspondingAuthor":false,"prefix":"","firstName":"Farshad","middleName":"","lastName":"Ghafari-Arsoon","suffix":""},{"id":471212209,"identity":"81c6dc5b-9578-4f99-bffa-a90bf4b7f307","order_by":1,"name":"Omid Ramezani","email":"","orcid":"","institution":"Shahid Beheshti University","correspondingAuthor":false,"prefix":"","firstName":"Omid","middleName":"","lastName":"Ramezani","suffix":""},{"id":471212213,"identity":"f5b161ab-9cc9-4a3b-a2f4-abb795b182a8","order_by":2,"name":"Ali Partovinia","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA1ElEQVRIiWNgGAWjYDADNvYGNgiLmSj1CUAtPAdI1cIgkcBGnHv4G3gPfq78YZPYJ/n82WMeBjt5BnbeB3i1SBzgS5Y8k5CW2CadY27Mw5Bs2MDMboDfmgM8BpINCYeN2aRz2KR5GJgTGJgJOFD+AI/xT7AWyePPgFrqCWsxOMBjBrJFjk2CwQyo5TBhLYYH+NIsG9LS5Nh4cswN5xgcN2wjpEXuAO/hmw02Njzy7cefPXhTUS3Pz38MvxYG+Tco7gTGKQENQMBDWMkoGAWjYBSMcAAAtycyouvMzfEAAAAASUVORK5CYII=","orcid":"","institution":"Shahid Beheshti University","correspondingAuthor":true,"prefix":"","firstName":"Ali","middleName":"","lastName":"Partovinia","suffix":""}],"badges":[],"createdAt":"2025-06-11 09:53:36","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6870463/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6870463/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-025-16985-4","type":"published","date":"2025-09-01T15:57:07+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":84705390,"identity":"94027cc2-960a-43cc-a9bf-79f63c33e061","added_by":"auto","created_at":"2025-06-16 12:17:50","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":399397,"visible":true,"origin":"","legend":"\u003cp\u003eThe effect of main variables: (\u003cstrong\u003ea\u003c/strong\u003e) Voltage (\u003cstrong\u003eb\u003c/strong\u003e) Cell-polymer suspension volume (\u003cstrong\u003ec\u003c/strong\u003e) Type of polymer used in the immobilization process on the effluent COD reduction.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6870463/v1/715b30ae0f0c37bf27322e96.jpeg"},{"id":84705389,"identity":"320e0001-ff9c-494a-8550-72cd765aa5f7","added_by":"auto","created_at":"2025-06-16 12:17:50","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":477297,"visible":true,"origin":"","legend":"\u003cp\u003eInteraction plot showing the mutual effects on COD removal: (\u003cstrong\u003ea\u003c/strong\u003e) Interactive effect of voltage and cell-polymer suspension volume; (\u003cstrong\u003eb\u003c/strong\u003e) Interactive effect of voltage and polymer type (\u003cstrong\u003ec\u003c/strong\u003e) Interactive effect of polymer type and cell-polymer suspension volume.\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6870463/v1/06a9822e1b5f8bd56e0bcdcd.jpeg"},{"id":84705395,"identity":"95a40c29-4f7c-49b5-811c-6e60246df7aa","added_by":"auto","created_at":"2025-06-16 12:17:50","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":213240,"visible":true,"origin":"","legend":"\u003cp\u003eContour plot\u003cstrong\u003e \u003c/strong\u003efor optimized point in biological wastewater treatment by immobilized cells in: (a) alginate (b) PVA/Alginate hybrid.\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6870463/v1/3839028c35db84ac1f21f795.jpeg"},{"id":90827959,"identity":"229d9469-e92b-4393-b1eb-b31988f63f4f","added_by":"auto","created_at":"2025-09-08 16:04:08","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2067592,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6870463/v1/1dbc53f6-0087-4387-bced-b7c546227180.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Optimization of immobilized activated sludge performance in electro-sprayed matrices for treatment of cellulose industry wastewater","fulltext":[{"header":"Introduction","content":"\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003ePulp and paper production is among the important industries that lead to the creation of large amounts of wastewater (Kumar et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The vast consumption of chemicals in the various processes of this industry has led to the production of highly polluted wastewater (Haq et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Pazira, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Therefore, the use of efficient methods for the treatment of these polluted wastewaters is essential and inevitable. There are various techniques including chemical and electro-coagulation, physical adsorption and biological treatment for removing pollutants from industrial wastewater. These methods can be used separately or in combination (Azimi et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Boguniewicz-Zablocka et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Haq et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Kumar et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eBiological treatment is known as an effective method for removing organic matter with low molecular weight (Leivisk\u0026auml; et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). However, the high performance and cost-effectiveness of wastewater treatment was observed by microbial cells system, there is a possibility of microbial cells inactivation in direct contact with wastewater due to the presence of various inhibitors and toxicants (Partovinia and Rasekh, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). In recent studied reported that to prevent cell disruption as well as cell activity decrement, it is necessary to immobilized microbial cells to overcome these difficulties (Bouabidi et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Khanpour-Alikelayeh et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Paliwal et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Sergio and Bustos, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2009\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eImmobilization technique is a process in which microbial cells attached or entrapped into a support while maintaining its activity. In general, carriers employed in biodegradation processes include natural and synthetic polymers (Bettmann and Rehm, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e1984\u003c/span\u003e; Je\u0026ugrave;rabkova et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e1997\u003c/span\u003e; Leenen et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e1996\u003c/span\u003e; Paje et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e1998\u003c/span\u003e; Suzuki et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e1998\u003c/span\u003e; Zhang et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). The natural polymers used mainly include alginate, carrageenan, chitosan, and agar, and the synthetic polymers include polyurethane and polyvinyl alcohol (Doria-Serrano et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Je\u0026ugrave;rabkova et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e1997\u003c/span\u003e; Partovinia and Rasekh, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Siripattanakul and Khan, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Overall, among the natural polymers, alginate is used more often in the entrapment technique due to the mild fabrication condition (Bettmann and Rehm, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e1984\u003c/span\u003e; Leenen et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e1996\u003c/span\u003e; Sergio and Bustos, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Moreover, alginate due to the cost-effectiveness, biocompatibility and biodegradability introduced as an appropriate choice for industrial applications (Partovinia and Rasekh, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Siripattanakul and Khan, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). An and Lo (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2001\u003c/span\u003e) successfully employed the PVA-alginate for the activated sludge immobilization. According to their results, 10-12.5% of PVA was determined as the optimum polymer concentration for sludge immobilization. They also suggested that the presence of 1% alginate in the microbeads prevent their aggregation (An and Lo, \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2001\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn the previous researches, various approaches such as emulsification technique using a homogenizer and ultrasonic method was employed to reduce the microbeads size which helps to overcome to mass transfer limitation (Ausheva et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Recently, to produce smaller-sized beads, the electrospray method has been used to produce beads with a size of 100\u0026ndash;200 \u0026micro;m (Barron and He, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Partovinia and Vatankhah, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2019\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003ePrevious studies demonstrate the ability of activated sludge in pulp and paper effluent treatment (Ince et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Paris and Blondeau, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e1999\u003c/span\u003e; Sharma et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Partovinia and Vatankhah (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) applied immobilized activated sludge to investigate the effect of beads particle size on the biodegradation efficiency of phenol and microbial growth rate. They used electrospray technique to immobilize activated sludge in a hybrid matrix of alginate and polyvinyl alcohol. According to their results, free cell and immobilized cell systems performed similarly at low pollutant concentrations, while the immobilized cell system showed superior biodegradation at higher concentrations like 2000 mg/L. Furthermore, immobilized cells in smaller beads was shown the higher degradation capacity (Partovinia and Vatankhah, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAccording to best of our knowledge, until now the effect of alginate and PVA beads as well as their hybrid in the biological treatment of cellulose industry wastewater has not been investigated. It is noteworthy that alginate beads have not always been completely successful in the biodegradation studies (Je\u0026ugrave;rabkova et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e1997\u003c/span\u003e; Paje et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e1998\u003c/span\u003e; Suzuki et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e1998\u003c/span\u003e). Therefore, the stability and activity of microbeads in cellulose industry wastewater, which includes various chemical compounds and toxicants, can be one of the main topics in this research. On the other hand, the immobilized bead size is very crucial in its performance and the bead size reduction lead to increase of mass transfer and biodegradation capacity (Ogbonna et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e1991\u003c/span\u003e). Besides, the effect of the immobilized activated sludge content (volume of polymer-cell suspension), the size of the produced beads and the type of polymer (alginate and hybrid alginate-PVA) in the biological treatment of cellulose industry wastewater and COD reduction will be evaluated. Finally, the performance of free and immobilized activated sludge in wastewater treatment will be compared.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eCellulose effluent and materials\u003c/p\u003e \u003cp\u003eIn this survey, the effluent of the activated sludge pond was selected from the Pouya Ayesh Mazand (Cellulose Group, Mazandaran, Iran). Concentrated sulfuric acid (98%), potassium dichromate, mercury sulfate, silver sulfate, and potassium hydrogen phthalate (KHP) were obtained from Merck, Germany. In this study, a thermal reactor (CR2200 model, Germany) and spectrophotometer (Jenway model 6300, England) was used for the digestion process and COD measurement, respectively.\u003c/p\u003e \u003cp\u003eChemical Oxygen Demand (COD) Analysis Method\u003c/p\u003e \u003cp\u003eChemical oxygen demand is one of the most important indicators for measuring wastewater pollution. To measure the COD, digestion solution, catalyst solution, and standard solution are required. To prepare the digestion solution, 1.5 g of potassium dichromate, 84 mL of concentrated sulfuric acid, and 16.7 g of mercury sulfate were added to 250 mL of deionized distilled water and the volume was adjusted to 500 mL after cooling. Catalyst solution was prepared by adding 2.2 g of silver sulfate into 0.409 kg of concentrated sulfuric acid and placed on a stirrer until completely mixed. Potassium hydrogen phthalate (KHP) as the standard solution was prepared by dissolving 0.425 g in 500 mL of double-distilled water, and 1 mL of this solution contains 1 mg COD (1 mg/1 mL). In this research, to measure the COD quantity, 2.5 mL of sample (e.g. wastewater, deionized water as a control sample or standard solution), 1.5 mL of digestion solution, and 3.5 mL of catalyst solution were added to Pyrex glass vials and stirred well for sufficient mixing. To perform the digestion process, the vials were placed in a thermo-reactor at 150\u0026deg;C for 120 minutes. At the end of this stage, the vials were cooled and the optical density was measured at a wavelength of 600 nm. Finally, the COD level of the samples was calculated using the standard curve (Boyles, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e1997\u003c/span\u003e; Partovinia et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Rice et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2012\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eActivated sludge immobilization in alginate and alginate-PVA hybrid beads\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePolymer preparation and microbial cell immobilization in alginate\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eAlginate microbeads were prepared by dissolving sodium alginate powder in distilled water (1% w/w) on a stirrer for 30 min at 60 \u0026ordm;C. In order to immobilize the cells, at first 40 mL of activated sludge solution (MLSS\u0026thinsp;=\u0026thinsp;15000 mg/Lit) was centrifuged at 3000 rpm for 3 min. Then, the aqueous phase was decanted and microbial cells were added to 10 mL of alginate polymer solution and gently mixed for 1 hour to cells were uniformly dispersed in the solution for the electrospray process. Then, 1 ml of the polymer-cell solution was transferred to a 1 mL syringe with a steel needle of 19 G. An electric field with a voltage of 0\u0026ndash;12 KV was employed and the solution transferred dropwise with the rate of 2.5 mL/h by a syringe pump into the calcium chloride solution (crosslinking agent). The fabricated beads were left 2 h in the crosslinking solution for hardening process and then washed with distilled water to remove un-immobilized cells on the beads surface (Partovinia and Vatankhah, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003ePolymer preparation and microbial cell immobilization in alginate-PVA hybrid\u003c/h3\u003e\n\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eTo fabrication of hybrid alginate-polyvinyl alcohol micro-carriers, first alginate 1% (w/v) and PVA 10% (w/v) solutions were prepared, mixed at a ratio of 20:80 (Alginate: PVA), then electrospray process was employed.\u003c/p\u003e \u003cp\u003eBiological wastewater treatment using experimental design approach\u003c/p\u003e \u003cp\u003eThe biological experiments were carried out in 150 mL Erlenmeyer flasks with a working volume of 30 mL in shaker incubator with 150 rpm and 30 \u0026ordm;C for 4 days. Statistical optimization and modelling were carried out by response surface methodology (RSM) based on central composite design (CCD) using three variables. These factors and ​​related ranges are given in the Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eMain factors and their coded levels used in the experimental design\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eFactors\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"5\" nameend=\"c6\" namest=\"c2\"\u003e \u003cp\u003eRange and levels (coded)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-2\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-1\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eA: Voltage (KV)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eB: Cell-polymer suspension volume (mL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eC: Carrier type\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"5\" nameend=\"c6\" namest=\"c2\"\u003e \u003cp\u003eAlginate\u003c/p\u003e \u003cp\u003eHybrid Alginate-PVA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe total number of trials in the CCD method is calculated as 2\u003csup\u003ek\u003c/sup\u003e\u0026thinsp;+\u0026thinsp;2k\u0026thinsp;+\u0026thinsp;n, where k and n are the number of main factors and central points, respectively. In this study, k and n are 2 and 5, respectively, resulting in 13 experiments for alginate and 13 experiments for hybrid matrix (Montgomery, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eStatistical analysis method\u003c/p\u003e \u003cp\u003eIn this study, Design Expert software (version 7) was used for analysis of variance and statistical optimization. Response surface methodology with central composite design was employed to statistically evaluate the main and interactive effects of selected variables and optimize biological treatment.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e\n"},{"header":"Results and Discussion","content":"\u003cdiv class=\"BlockQuote\"\u003e\n \u003cp\u003eCellulose wastewater treatment by freely cell suspended system\u003c/p\u003e\n \u003cp\u003eThe initial COD for the selected cellulose industry effluent was measured 6715 mg/L. According to the results, measured COD of control system without inoculation and treatment by freely cell suspension of activated sludge was 6560 and 2320 mg/L after 96 h, respectively. Therefore, it can be concluded that the activated sludge with 65% COD removal after 4 days had a significant role in reducing COD and the organic pollution load of the wastewater. Similarly, Tyagi et al. (\u003cspan class=\"CitationRef\"\u003e2014\u003c/span\u003e) employed combination of two bacterial species \u003cem\u003eBacillus subtilis\u003c/em\u003e and \u003cem\u003eMicrococcus luteus\u003c/em\u003e and a fungus \u003cem\u003ePhanerochaete crysosporium\u003c/em\u003e to treat paper mill wastewater. Their results showed that microbial combination was able to reduce 94.7% COD after 9 days (Tyagi et al., \u003cspan class=\"CitationRef\"\u003e2014\u003c/span\u003e). Haq et al. (\u003cspan class=\"CitationRef\"\u003e2016\u003c/span\u003e) showed that the strain \u003cem\u003eSerratia liquefaciens\u003c/em\u003e LD-5 successfully reduced 85% COD of pulp and paper effluent after 144 h of treatment at 30\u0026deg;C, pH 7.6 and 120 rpm (Haq et al., \u003cspan class=\"CitationRef\"\u003e2016\u003c/span\u003e). Tiku et al. (\u003cspan class=\"CitationRef\"\u003e2010\u003c/span\u003e) used three bacteria as the consortium exhibited high efficiency to reduce the COD of the effluent, about 76% reduction in COD within 10 h (Tiku et al., \u003cspan class=\"CitationRef\"\u003e2010\u003c/span\u003e). Gholami et al. (\u003cspan class=\"CitationRef\"\u003e2022\u003c/span\u003e) showed that the maximum COD removal in recycled paper and cardboard mill wastewater treatment, by \u003cem\u003eCitrobacter freundii\u003c/em\u003e AT-4 was 79.54% after 10 days of incubation. In the case of \u003cem\u003eBacillus subtilis\u003c/em\u003e AT-5 and \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e AT-1, the maximum COD removal were 70.08% and 71.26%, respectively (Gholami et al., \u003cspan class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e\n \u003cp\u003eCellulose wastewater treatment by immobilized activated sludge\u003c/p\u003e\n \u003cp\u003eThe results of biological wastewater treatment using the immobilized activated sludge after 96 hours are given in the Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e. As can be shown in Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e, the highest COD removal observed in experiment, in which the activated sludge was immobilized in alginate polymer and the voltage and polymer-cell solution volume were 6 KV and 2 mL, respectively. In this case, the highest COD removal was achieved almost 78%, which is higher than the 65% COD removal by free cells. On the other hand, according to the results, the lowest COD removal equal to 40% was observed for immobilized cells in hybrid alginate-polyvinyl alcohol polymer at voltage and polymer-cell solution volume of 3 KV and 1.5 mL, respectively. In most previous studies, it has also been shown that immobilized microbial cells generally have a higher biodegradation rate than free cells (Paliwal et al., \u003cspan class=\"CitationRef\"\u003e2015\u003c/span\u003e; Paliwal et al., \u003cspan class=\"CitationRef\"\u003e2016\u003c/span\u003e; Partovinia and Rasekh, \u003cspan class=\"CitationRef\"\u003e2018\u003c/span\u003e). The reason for this can be attributed to various factors such as the protective role of the carrier material against toxic compounds, the quorum sensing and cooperative effect of microorganisms which live together, the reduction of the inhibitory effect of toxicants, and controlled mass transfer. Paliwal et al. (\u003cspan class=\"CitationRef\"\u003e2015\u003c/span\u003e) reported that co-immobilization of bacterial consortium in corncobs as the agro-industrial residue enhanced the decolorization efficacy of these consortium by retaining the ligninolytic enzymes production (Paliwal et al., \u003cspan class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e\n\u003c/div\u003e\n\u003ctable id=\"Tab2\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eResults of COD content in treated wastewater using central composite experimental design matrix for actual variables.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eRun\u003c/p\u003e\n \u003cp\u003eno.\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eFactor 1\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eFactor 2\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eFactor 3\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eResponse\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eA:Voltage\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eB:Cell-Polymer suspension volume\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eC:Type of Polymer\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eChemical Oxygen Demand\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eKV\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003emL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003emg O\u003csub\u003e2\u003c/sub\u003e/L\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAlginate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3140\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAlginate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1521\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAlginate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2318\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAlginate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2492\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAlginate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1719\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAlginate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1521\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAlginate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3792\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAlginate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1903\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAlginate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2270\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAlginate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1458\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAlginate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3164\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAlginate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3130\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAlginate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2396\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePVA:ALG\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4043\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePVA:ALG\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2140\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePVA:ALG\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2260\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePVA:ALG\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2502\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePVA:ALG\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2101\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePVA:ALG\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2473\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePVA:ALG\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3705\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePVA:ALG\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3797\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePVA:ALG\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2700\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e23\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePVA:ALG\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1956\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePVA:ALG\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2656\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePVA:ALG\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2656\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e26\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePVA:ALG\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2072\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003ch3\u003e\u003cbr\u003e\u003c/h3\u003e\n\u003ch3\u003eThe analysis of variance for biological wastewater treatment\u003c/h3\u003e\n\u003cdiv class=\"BlockQuote\"\u003e\n \u003cp\u003eThe ANOVA results of wastewater treatment by the immobilized activated sludge are presented in the Table \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e. The obtained p-values for voltage, cell-polymer suspension volume, and polymer type were 0.3321, 0.0962, and 0.1589, respectively, which indicates that among the three selected variables, only the cell-polymer suspension volume, possibly have an effective role on COD reduction. Also, the values obtained for P\u003csub\u003eAB\u003c/sub\u003e, P\u003csub\u003eAC\u003c/sub\u003e, and P\u003csub\u003eBC\u003c/sub\u003e (0.0245, 0.7421, and 0.3987, respectively) revealed that just interaction of voltage and cell-polymer suspension volume has a significant effect on biological wastewater treatment and COD reduction.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"gridtable\"\u003e\n \u003ctable id=\"Tab3\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eANOVA results of the quadratic model for cellulose wastewater treatment using immobilized cell system.\u0026nbsp;\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"7\" valign=\"top\" style=\"width: 613px;\"\u003e\n \u003cp\u003eAnalysis of variance table\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 138px;\"\u003e\n \u003cp\u003eSource\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 91px;\"\u003e\n \u003cp\u003eSum of Squares\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 71px;\"\u003e\n \u003cp\u003eDegree of freedom\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 77px;\"\u003e\n \u003cp\u003eMean Square\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003eF Value\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003ep-value\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 108px;\"\u003e\n \u003cp\u003eSignificance\u003c/p\u003e\n \u003cp\u003estatus\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 138px;\"\u003e\n \u003cp\u003eModel\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 91px;\"\u003e\n \u003cp\u003e7.976E+06\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 71px;\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 77px;\"\u003e\n \u003cp\u003e9.970E+05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e3.13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003e0.0227\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 108px;\"\u003e\n \u003cp\u003eSignificant\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 138px;\"\u003e\n \u003cp\u003eA:Voltage\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 91px;\"\u003e\n \u003cp\u003e3.169E+05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 71px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 77px;\"\u003e\n \u003cp\u003e3.169E+05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e1.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003e0.3321\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 108px;\"\u003e\n \u003cp\u003eNot significant\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 138px;\"\u003e\n \u003cp\u003eB:Cell-Polymer suspension volume\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 91px;\"\u003e\n \u003cp\u003e9.866E+05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 71px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 77px;\"\u003e\n \u003cp\u003e9.866E+05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e3.10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003e0.0962\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 108px;\"\u003e\n \u003cp\u003ePossibly significant\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 138px;\"\u003e\n \u003cp\u003eC:Type of Polymer\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 91px;\"\u003e\n \u003cp\u003e6.905E+05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 71px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 77px;\"\u003e\n \u003cp\u003e6.905E+05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e2.17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003e0.1589\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 108px;\"\u003e\n \u003cp\u003eNot significant\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 138px;\"\u003e\n \u003cp\u003eAB\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 91px;\"\u003e\n \u003cp\u003e1.938E+06\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 71px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 77px;\"\u003e\n \u003cp\u003e1.938E+06\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e6.10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003e0.0245\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 108px;\"\u003e\n \u003cp\u003eSignificant\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 138px;\"\u003e\n \u003cp\u003eAC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 91px;\"\u003e\n \u003cp\u003e35574\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 71px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 77px;\"\u003e\n \u003cp\u003e35574\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e0.11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003e0.7421\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 108px;\"\u003e\n \u003cp\u003eNot significant\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 138px;\"\u003e\n \u003cp\u003eBC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 91px;\"\u003e\n \u003cp\u003e2.384E+05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 71px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 77px;\"\u003e\n \u003cp\u003e2.384E+05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e0.75\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003e0.3987\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 108px;\"\u003e\n \u003cp\u003eNot significant\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 138px;\"\u003e\n \u003cp\u003eA^2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 91px;\"\u003e\n \u003cp\u003e6.911E+05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 71px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 77px;\"\u003e\n \u003cp\u003e6.911E+05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e2.17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003e0.1587\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 108px;\"\u003e\n \u003cp\u003eNot significant\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 138px;\"\u003e\n \u003cp\u003eB^2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 91px;\"\u003e\n \u003cp\u003e2.092E+06\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 71px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 77px;\"\u003e\n \u003cp\u003e2.092E+06\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e6.58\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003e0.0201\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 108px;\"\u003e\n \u003cp\u003eSignificant\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 138px;\"\u003e\n \u003cp\u003eResidual\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 91px;\"\u003e\n \u003cp\u003e5.407E+06\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 71px;\"\u003e\n \u003cp\u003e17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 77px;\"\u003e\n \u003cp\u003e3.180E+05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 108px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 138px;\"\u003e\n \u003cp\u003eLack of Fit\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 91px;\"\u003e\n \u003cp\u003e2.895E+06\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 71px;\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 77px;\"\u003e\n \u003cp\u003e3.217E+05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e1.02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003e0.4917\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 108px;\"\u003e\n \u003cp\u003eNot significant\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 138px;\"\u003e\n \u003cp\u003ePure Error\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 91px;\"\u003e\n \u003cp\u003e2.511E+06\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 71px;\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 77px;\"\u003e\n \u003cp\u003e3.139E+05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 62px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 108px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 138px;\"\u003e\n \u003cp\u003eCor Total\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 91px;\"\u003e\n \u003cp\u003e1.338E+07\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 71px;\"\u003e\n \u003cp\u003e25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 77px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 108px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eBy applying multiple regression analysis, quadratic equation 1 was found to be the best fit for experimental data of COD in immobilized cell system:\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\u0026nbsp;\u003ctable id=\"Taba\" border=\"1\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCOD (mg O\u003csub\u003e2\u003c/sub\u003e/Lit)\u0026thinsp;=\u0026thinsp;2304.6\u0026ndash;114.9 A \u0026minus;\u0026thinsp;202.7 B\u0026thinsp;+\u0026thinsp;462.2 AB\u0026thinsp;+\u0026thinsp;248.5 B\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e(1)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003ewhere A and B represent actual values for voltage and cell-polymer suspension volume, respectively.\u003c/p\u003e\n\u003ch3\u003eThe effect of main variables of biodegradation and COD removal\u003c/h3\u003e\n\u003cdiv class=\"BlockQuote\"\u003e\n \u003cp\u003eFigure \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e(a) shows that with increasing the voltage and decreasing the size of the alginate beads, the COD level decreased, but in general, no significant difference was observed in the COD reduction results. Similarly, Partovinia and Vatankhah (\u003cspan class=\"CitationRef\"\u003e2023\u003c/span\u003e) showed the phenol biodegradation using the cell immobilization technique was higher in smaller beads size of alginate (Partovinia and Vatankhah, \u003cspan class=\"CitationRef\"\u003e2023\u003c/span\u003e). Figure \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e(b) shows that with increasing the volume of the cell-polymer suspension, the COD level significantly decreased. According to the Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e(c), the alginate polymer as an activated sludge carrier had a higher COD removal than the hybrid alginate-polyvinyl alcohol matrix, although based on the results of the analysis of variance, the difference was not significant. Leenen et al. (\u003cspan class=\"CitationRef\"\u003e1996\u003c/span\u003e) compared several natural and synthetic polymers in their survey, and according to the results, cell immobilization and growth occurred well in the natural matrices like alginate and carrageenan. In addition, the effective diffusion coefficients of substrate in the presence of these polymers were close to the diffusion coefficients in water. However, the solubility and degradation of these polymers were reported as the disadvantages especially in their long-terms usage. In contrast, for the synthetic carriers, higher mechanical properties and lower substrate diffusion coefficients were reported (Leenen et al., \u003cspan class=\"CitationRef\"\u003e1996\u003c/span\u003e).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n \u003ch2\u003eExamination of interactive effect of variables on COD removal\u003c/h2\u003e\n \u003cdiv class=\"BlockQuote\"\u003e\n \u003cp\u003eThe interactive effect of voltage and volume of cell-polymer suspension on the COD reduction is illustrated in Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e(a). As can be seen in the Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e(a), at low cell-polymer suspension volume, with increasing voltage (decreasing bead size), COD has significantly decreased. While at high cell-polymer suspension volume, with increasing voltage, COD has significantly increased. Therefore, it can be concluded that when the amount of immobilized microbial cells in the culture was low, more mass transfer problems were observed in the system and mass transfer limitation was overcome by decreasing bead size. In the other words, it can be concluded that reducing the microbeads size does not necessarily have a positive effect on biodegradation rate and their efficiency is a function of the immobilized cell concentration in the culture.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv class=\"BlockQuote\"\u003e\n \u003cp\u003eAccording to the optimization results as shown in Table \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e, the maximum biodegradation rate and COD removal of 74% was observed in alginate-immobilized cell with voltage and polymer-cell solution volume of 3 KV and 2.5 mL, respectively.\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003ctable id=\"Tab4\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eOptimization results of cellulose wastewater treatment using immobilized cell system.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eVoltage (KV)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eCell-Polymer suspension volume (ml)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eType of Polymer\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eCOD\u003c/p\u003e\n \u003cp\u003e(mg O\u003csub\u003e2\u003c/sub\u003e/Lit)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAlginate (1%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1736.815\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003cp\u003e\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv class=\"BlockQuote\"\u003e\n \u003cp\u003eThe contour plot for optimizing wastewater treatment by two polymer carriers including alginate and hybrid of alginate-polyvinyl alcohol, is shown in Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e. As can be seen, the performance of the hybrid matrix was not very successful, while positive performance has been observed for alginate. Although, Cassidy et al have reported it seems that alginate polymer is not a suitable matrix in microbial consortium due to its possibility consumption by fungi (Cassidy et al., \u003cspan class=\"CitationRef\"\u003e1996\u003c/span\u003e). In this study, immobilized activated sludge in alginate shows high performance in wastewater treatment. As shown in the Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e, maximum COD removal was observed in alginate polymer at low voltage and high volume of cell polymer suspension as well as high voltage and low volume of cell polymer suspension.\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e"},{"header":"Conclusions","content":"\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eGenerally, molecular diffusion is the effective process in mass transfer into the gel matrix and usually is a fast process, but the volume fraction and the available space inside the matrix are very small. Therefore, in the discussion of cell immobilization with the entrapment technique, the mass transfer limitation is of particular importance. In this study, cellulose wastewater treatment by immobilized cell fabricated using electro-spraying technique was investigated. According to the obtained results, among the three studied factors, cell-polymer suspension volume had an effective role on the COD reduction. Moreover, the study of interaction effects of the variables showed that only voltage and cell-polymer suspension volume had a mutual effect on each other and as a result affected the COD removal. Besides, it can be concluded that when the amount of activated sludge in the culture was low, mass transfer limitation was observed and it was overcome by reducing the particle size. Based on the findings of this survey, it can be stated that reducing the micro-bead size was effective in the biological treatment process when the volume of used polymer was small. Moreover, in cases which is not possible to produce small-sized beads, a larger volume of polymer-cell can be applied.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eConflicts of Interest:\u003c/h2\u003e \u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding:\u003c/h2\u003e \u003cp\u003eThis research received no external funding.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eF.G.A.: methodology, data curation, formal analysis, investigation, resources O.R. and A.P.: conceptualization, supervision, project administration, software, writing\u0026mdash;review and editing. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThe authors gratefully acknowledge the Bio-products and Bio-systems Laboratories of Zirab Campus-Shahid Beheshti University, for providing the facilities and technical support to carry out the research work.\u003c/p\u003e\u003ch2\u003eData Availability Statement:\u003c/h2\u003e \u003cp\u003eAll data have been included in the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAn, M. \u0026amp; Lo, K. V. Activated sludge immobilization using the pva-alginate-borate method. \u003cem\u003eJ. Environ. Sci. Health A\u003c/em\u003e. \u003cb\u003e36\u003c/b\u003e, 101\u0026ndash;115. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1081/ESE-100000475\u003c/span\u003e\u003cspan address=\"10.1081/ESE-100000475\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2001).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAusheva, K. A., Goncharuk, D. A., Babusenko, E. S., Nekhaev, S. A. \u0026amp; Sultygova, Z. S. Markvichev NS Development of a bacterial preparation based on immobilized cells. \u003cem\u003eTheor. Found. Chem. 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Sci.\u003c/em\u003e \u003cb\u003e19\u003c/b\u003e, 1293\u0026ndash;1297. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/S1001-0742(07)60211-3\u003c/span\u003e\u003cspan address=\"10.1016/S1001-0742(07)60211-3\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2007).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"cellulose wastewater, chemical oxygen demand, electrospray technique, immobilized sludge, microbeads","lastPublishedDoi":"10.21203/rs.3.rs-6870463/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6870463/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eToday, the abundant use of cellulose industry products has led to an increase in production and, as a result, an increase in the volume of water consumed by this industry. On the other hand, the high volumetric flow rate of produced wastewater, chemical oxygen demand (COD), suspended solids and high turbidity of these wastewaters have caused many problems. In recent years, various methods, including physical, chemical and biological, have been used for wastewater treatment. In this study, firstly, the efficiency of the activated sludge collected from cellulose wastewater was investigated in freely suspended cell system in shake flask experiments at 150 rpm and 30 ̊C. Afterward, to investigate the performance of immobilized sludge, alginate and hybrid alginate-polyvinyl alcohol (PVA) polymers were used for microbeads production. In this survey, the electrospray technique and response surface methodology (RSM) were employed to produce microbeads and statistical optimization, respectively. In order to optimize the bioremediation process, three variables including electrospray voltage (0\u0026ndash;12 KV), the volume of cell-polymer suspension (1\u0026ndash;3 ml), and two type of carriers were selected. In order to analyze the wastewater, the results related to COD were evaluated. The optimization results showed that the maximum biodegradation and COD removal of 74% (from 6715 to 1736 mg/L) after 4 days was observed by the alginate-immobilized cells produced with the voltage and polymer-cell solution volume of 3 KV and 2.5 mL, respectively.\u003c/p\u003e","manuscriptTitle":"Optimization of immobilized activated sludge performance in electro-sprayed matrices for treatment of cellulose industry wastewater","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-16 12:17:45","doi":"10.21203/rs.3.rs-6870463/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-08-01T12:19:37+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-22T13:48:34+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-06T07:45:31+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"258341750421438709187568630201328864084","date":"2025-06-14T09:22:06+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"318431011131562993912660038815660213051","date":"2025-06-14T04:36:00+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-06-12T07:41:06+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-06-12T07:30:26+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-06-12T05:57:36+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-06-12T05:56:42+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-06-11T09:50:52+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"50ffb8c4-f59a-473a-8457-5edcc92b09ca","owner":[],"postedDate":"June 16th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":50044563,"name":"Biological sciences/Biotechnology/Environmental biotechnology"},{"id":50044564,"name":"Earth and environmental sciences/Environmental sciences/Environmental chemistry/Pollution remediation"}],"tags":[],"updatedAt":"2025-09-08T16:00:37+00:00","versionOfRecord":{"articleIdentity":"rs-6870463","link":"https://doi.org/10.1038/s41598-025-16985-4","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-09-01 15:57:07","publishedOnDateReadable":"September 1st, 2025"},"versionCreatedAt":"2025-06-16 12:17:45","video":"","vorDoi":"10.1038/s41598-025-16985-4","vorDoiUrl":"https://doi.org/10.1038/s41598-025-16985-4","workflowStages":[]},"version":"v1","identity":"rs-6870463","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6870463","identity":"rs-6870463","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}