Fed-batch Fermentation Research Articles

Production of Optically Pure (S)-3-Hydroxy-γ-butyrolactone from d-Xylose Using Engineered Escherichia coli.

Biocatalysis has advantages in asymmetric synthesis due to the excellent stereoselectivity of enzymes. The present study established an efficient biosynthesis pathway for optically pure (S)-3-hydroxy-γ-butyrolactone [(S)-3HγBL] production using engineered Escherichia coli. We mimicked the 1,2,4-butanetriol biosynthesis route and constructed a five-step pathway consisting of d-xylose dehydrogenase, d-xylonolactonase, d-xylonate dehydratase, 2-keto acid decarboxylase, and aldehyde dehydrogenase. The engineered strain harboring the five enzymes could convert d-xylose to 3HγBL with glycerol as the carbon source. Stereochemical analysis by chiral GC proved that the microbially synthesized product was a single isomer, and the enantiomeric excess (ee) value reached 99.3%. (S)-3HγBL production was further enhanced by disrupting the branched pathways responsible for d-xylose uptake and intermediate reduction. Fed-batch fermentation of the best engineered strain showed the highest (S)-3HγBL titer of 3.5 g/L. The volumetric productivity and molar yield of (S)-3HγBL on d-xylose reached 50.6 mg/(L·h) and 52.1%, respectively. The final fermentation product was extracted, purified, and confirmed by NMR. This process utilized renewable d-xylose as the feedstock and offered an alternative approach for the production of the valuable chemical.

Enhanced astaxanthin production by Aurantiochytrium sp. CJ6 using sorghum distillery residue (SDR)-based growth medium and SDR-derived biochar carrier

Sorghum distillery residue (SDR) is a byproduct of Chinese liquor (baijiu) manufacturing, known for its high carbohydrate and nutrient content. This study utilized SDR as a growth medium and carrier to cultivate Aurantiochytrium sp. CJ6 for astaxanthin production. Through an isothermal torrefaction process, SDR was transformed into biochar (SDR-biochar), characterized by its rough surface and large cavities. Using SDR-biochar as a carrier for CJ6 cultivation effectively boosted cell growth and reduced CJ6 cultivation time. Continuous-feeding fed-batch (CF-FB) fermentation was employed to enhance astaxanthin synthesis. Without any carrier, a maximum astaxanthin concentration of 0.582 mg/L was achieved after 22 days of fermentation. However, when SDR-biochar was added as the carrier for CJ6 growth, a maximum astaxanthin concentration of 0.986 mg/L was reached within just 14 days. By combining CF-FB fermentation with the addition of SDR-biochar as the growth carrier, the astaxanthin productivity of CJ6 increased from 0.0264 mg/L/d to 0.070 mg/L/d, marking a remarkable 1.69-fold improvement.

Revealing the Mechanisms of Enhanced β-Farnesene Production in Yarrowia lipolytica through Metabolomics Analysis.

β-Farnesene is an advanced molecule with promising applications in agriculture, the cosmetics industry, pharmaceuticals, and bioenergy. To supplement the shortcomings of rational design in the development of high-producing β-farnesene strains, a Metabolic Pathway Design-Fermentation Test-Metabolomic Analysis-Target Mining experimental cycle was designed. In this study, by over-adding 20 different amino acids/nucleobases to induce fluctuations in the production of β-farnesene, the changes in intracellular metabolites in the β-farnesene titer-increased group were analyzed using non-targeted metabolomics. Differential metabolites that were detected in each experimental group were selected, and their metabolic pathways were located. Based on these differential metabolites, targeted strain gene editing and culture medium optimization were performed. The overexpression of the coenzyme A synthesis-related gene pantothenate kinase (PanK) and the addition of four mixed water-soluble vitamins in the culture medium increased the β-farnesene titer in the shake flask to 1054.8 mg/L, a 48.5% increase from the initial strain. In the subsequent fed-batch fermentation, the β-farnesene titer further reached 24.6 g/L. This work demonstrates the tremendous application value of metabolomics analysis for the development of industrial recombinant strains and the optimization of fermentation conditions.

Integrated biorefinery strategy for poly(3-hydroxybutyrate) accumulation in Cupriavidus necator DSM 545 using a sugar rich syrup from cereal waste and acetate from gas fermentation

Poly(3-hydroxybutyrate) (PHB) is one of the most well-known biodegradable and biocompatible biopolymers produced by prokaryotic microorganisms. It belongs to the family of polyhydroxyalkanoates (PHAs), and it has gained significant attention in recent years due to its potential as a sustainable alternative to traditional petroleum-based plastics. Cupriavidus necator has been identified as a potential producer of PHB for industrial applications due to its ability to produce high amounts of the polymer under controlled conditions, using a wide range of waste substrates. In this study, the ability of Cupriavidus necator DSM 545 strain to produce PHB was tested in a fed-batch strategy providing two different organic substrates. The first is a sugar-based syrup (SBS), derived from cereal waste. The second is an acetate-rich medium obtained through CO2 -H2 fermentation by the acetogenic bacterium Acetobacterium woodii. The carbon sources were tested to improve the accumulation of PHB in the strain. C. necator DSM 545 proved to be able to grow and to perform high accumulation of biopolymer on waste substrates containing glucose, fructose, and acetate, reaching about 10 g/L of PHB, 83% of biopolymer accumulation in cell dry mass, in 48 h of fed-batch fermentation in 0.6 L working volume in a bioreactor. Moreover, a Life Cycle Assessment analysis was performed to evaluate the environmental impact of the process converting the sugar syrup alone and the integrated one. It demonstrated that the integrated process is more sustainable and that the most impactful step is the PHB production, followed by the polymer extraction.

Metabolic engineering of Escherichia coli for efficient production of 1,4-butanediol from crude glycerol

1,4-butanediol (1,4-BDO) is a significant non-natural chemical with diverse applications in various fields, particularly in the production of biodegradable plastic products as an essential monomer. The conversion of starch-based sugars using microbial cell factories provides a green alternative for the production of 1,4-BDO, but was limited by the low efficiency and high production cost. In this work, the classic CoA-dependent 1,4-BDO synthesis pathway was exogenously introduced into Escherichia coli, but the titer of 1,4-BDO was only 0.72 g/L using glucose as the substrate. The poorly expressed enzymes and the ineffective distribution of central carbon flux were considered the main limiting factors. Combined genetic manipulation of the synthesis pathway and bioprocess engineering, glycerol was proven to be a more favorable feedstock for 1,4-BDO synthesis, and a novel pathway functioned by carboxylic acid reductase could achieve one-step catalysis from 4-hydroxybutyric acid to 4-hydroxybutyraldehyde, which avoided the rate-limiting steps involved by hydroxybutyrate CoA transferase (Cat2) and alcohol dehydrogenase (Bld). The titer of 1,4-BDO reached 1.31 g/L and 1.07 g/L in the novel pathway using pure glycerol and the industrial by-product crude glycerol as the substrate, respectively. The fed-batch fermentation was finally conducted, and the titer of 1,4-BDO was increased to 5.75 g/L with a yield of 143.75 mg/g glycerol. Our work removed the bottleneck of the 1,4-BDO synthesis pathway by introducing carboxylic acid reductase and effectively converted crude glycerol to 1,4-BDO, which enabled us to build a sustainable microbial cell factory to cost-efficiently produce 1,4-BDO as well as other biochemicals from crude glycerol.

Simultaneous Lipid and Carotenoid Production via Rhodotorula paludigena CM33 Using Crude Glycerol as the Main Substrate: Pilot-Scale Experiments.

Rhodotorula paludigena CM33 is an oleaginous yeast that has been demonstrated to accumulate substantial quantities of intracellular lipids and carotenoids. In this study, crude glycerol, a by-product of biodiesel production, was used as a carbon source to enhance the accumulation of lipids and carotenoids in the cells. The culture conditions were first optimized using response surface methodology, which revealed that the carotenoid concentration and lipid content improved when the concentration of crude glycerol was 40 g/L. Different fermentation conditions were also investigated: batch, repeated-batch, and fed-batch conditions in a 500 L fermenter. For fed-batch fermentation, the maximum concentrations of biomass, lipids, and carotenoids obtained were 46.32 g/L, 37.65%, and 713.80 mg/L, respectively. A chemical-free carotenoid extraction method was also optimized using high-pressure homogenization and a microfluidizer device. The carotenoids were found to be mostly beta-carotene, which was confirmed by HPLC (high pressure liquid chromatography), LC-MS (liquid chromatography-mass spectrometry), and NMR (nuclear magnetic resonance). The results of this study indicate that crude glycerol can be used as a substrate to produce carotenoids, resulting in enhanced value of this biodiesel by-product.

Hypersecretory production of glucose oxidase in Pichia pastoris through combinatorial engineering of protein properties, synthesis, and secretion.

Glucose oxidase (EC 1.1.3.4, GOD) is a widely used industrial enzyme. To construct a GOD-hyperproducing Pichia pastoris strain, combinatorial strategies have been applied to improve GOD activity, synthesis, and secretion. First, wild-type GOD was subjected to saturation mutagenesis to obtain an improved variant, MGOD1 (V20W/T30S), with 1.7-fold higher kcat /KM . Subsequently, efficient signal peptides were screened, and the copy number of MGOD1 was optimized to generate a high-producing strain, 8GM1, containing eight copies of AOX1 promoter-GAS1 signal peptide-MGOD1 expression cassette. Finally, the vesicle trafficking of 8GM1 was engineered to obtain the hyperproducing strain G1EeSe co-expressing the trafficking components EES and SEC. 22, and the EES gene (PAS_chr3_0685) was found to facilitate both protein secretion and production for the first time. Using these strategies, GOD secretion was enhanced 65.2-fold. In the 5-L bioreactor, conventional fed-batch fermentation without any process optimization resulted in up to 7223.0 U/mL extracellular GOD activity (3.3-fold higher than the highest level reported to date), with almost only GOD in the fermentation supernatant at a protein concentration of 30.7 g/L. Therefore, a GOD hyperproducing strain for industrial applications was developed, and this successful case can provide a valuable reference for the construction of high-producing strains for other industrial enzymes.

Engineering the reductive tricarboxylic acid pathway in Aureobasidium pullulans for enhanced biosynthesis of poly-L-malic acid

Aureobasidium pullulans produced poly-L-malic acid (PMA) as the main metabolite in fermentation but with relatively low productivity and yield limiting its industrial application. In this study, A. pullulans ZX-10 was engineered to overexpress cytosolic malate dehydrogenase (MDH) and pyruvate carboxylase (PYC) and PMA synthetase (PMS) using a high-copy yeast episomal plasmid with the gpdA promoter from Aspergillus nidulans. Overexpressing endogenous PMS and heterologous MDH and PYC from Aspergillus oryzae respectively increased PMA production by 19 % – 37 % (0.64 – 0.74 g/g vs. 0.54 g/g for wild type) in shake-flask fermentations, demonstrating the importance of the reductive tricarboxylic acid (rTCA) pathway in PMA biosynthesis. A. pullulans co-expressing MDH and PYC produced 96.7 g/L PMA at 0.90 g/L∙h and 0.68 g/g glucose in fed-batch fermentation, which were among the highest yield and productivity reported. The engineered A. pullulans with enhanced rTCA pathway is advantageous and promising for PMA production.

Engineering Escherichia coli for cost-effective production of medium-chain fatty acids from soy whey using an optimized galactose-based autoinduction system

Medium-chain fatty acids (MCFAs) are essential chemical feedstocks. Microbial production of MCFAs offers an attractive alternative to conventional methods, but the costly media and external inducers limit its practical application. To address this issue and make MCFA production more cost-effective, an E.coli platform was developed using soy whey as a medium and galactose as an autoinducer. We first designed an efficient, stringent, homogeneous, and robust galactose-based autoinduction system for the expression of pathway enzymes by rationally engineering the promoter of the galactose-proton symporter (GalP). Subsequently, the intracellular acetyl-CoA availability and NADH regeneration were enhanced to improve the reversal of the β-oxidation cycle. The resulting strain yielded 8.20 g/L and 16.42 g/L MCFA in pH-controlled batch fermentation and fed-batch fermentation with glucose added using soy whey as medium, respectively. This study provided a cost-effective and promising platform for MCFA production, as well as future strain development for other value-added chemicals production.

High-level phenol bioproduction by engineered Pichia pastoris in glycerol fed-batch fermentation using an efficient pertraction system

This study aimed to establish a high-level phenol bioproduction system from glycerol through metabolic engineering of the yeast Pichia pastoris (Komagataella phaffii). Introducing tyrosine phenol-lyase to P. pastoris led to a production of 59 mg/L of phenol in flask culture. By employing a strain of P. pastoris that overproduces tyrosine—a precursor to phenol—we achieved a phenol production of 1052 mg/L in glycerol fed-batch fermentation. However, phenol concentrations exceeding 1000 mg/L inhibited P. pastoris growth. A phenol pertraction system utilizing a hollow fiber membrane contactor and tributyrin as the organic solvent was developed to reduce phenol concentration in the culture medium. Integrating this system with glycerol fed-batch fermentation resulted in a 214 % increase in phenol titer (3304 mg/L) compared to glycerol fed-batch fermentation alone. These approaches offer a significant framework for the microbial production of chemicals and materials that are highly toxic to microorganisms.

Efficient production of single cell protein from biogas slurry using screened alkali-salt-tolerant Debaryomyces hansenii

Production of single cell protein (SCP) by recovering ammonia nitrogen from biogas slurry shows great potential against protein scarcity and unsustainable production of plant and animal proteins. Herein, a high-alkali-salt-tolerant yeast strain, Debaryomyces hansenii JL8-0, was isolated and demonstrated for high-efficient SCP production. This strain grew optimally at pH 8.50 and 2500 mg/L NH4+-N, and it could efficiently utilize acetate as the additional carbon source. Under optimal conditions, SCP biomass of 32.21 g/L and productivity of 0.32 g/L·h−1 were obtained in fed-batch fermentation. Remarkably, nearly complete (97.40 %) ammonia nitrogen from biogas slurry was recovered, probably due to its high affinity for NH4+-N. Altogether, this strain showed advantages in terms of cell biomass titer, productivity, and yield. A cultivation strategy was proposed by co-culturing D. hansenii with other compatible yeast strains to achieve high-efficient SCP production from biogas slurry, which could be a promising alternative technology for biogas slurry treatment.

Beet red food colourant can be produced more sustainably with engineered Yarrowia lipolytica

Synthetic food colourants are widely used in the food industry, but consumer concerns about safety and sustainability are driving a need for natural food-colour alternatives. Betanin, which is extracted from red beetroots, is a commonly used natural red food colour. However, the betanin content of beetroot is very low (~0.2% wet weight), which means that the extraction of betanin is incredibly wasteful in terms of land use, processing costs and vegetable waste. Here we developed a sustainability-driven biotechnological process for producing red beet betalains, namely, betanin and its isomer isobetanin, by engineering the oleaginous yeast Yarrowia lipolytica. Metabolic engineering and fermentation optimization enabled production of 1,271 ± 141 mg l−1 betanin and 55 ± 7 mg l−1 isobetanin in 51 h using glucose as carbon source in controlled fed-batch fermentations. According to a life cycle assessment, at industrial scale (550 t yr−1), our fermentation process would require significantly less land, energy and resources compared with the traditional extraction of betanin from beetroot crops. Finally, we apply techno-economic assessment to show that betanin production by fermentation could be economically feasible in the existing market conditions.

Comprehensive evaluation of recombinant lactate dehydrogenase production from inclusion bodies

A broad application spectrum ranging from clinical diagnostics to biosensors in a variety of sectors, makes the enzyme Lactate dehydrogenase (LDH) highly interesting for recombinant protein production. Expression of recombinant LDH is currently mainly carried out in uncontrolled shake-flask cultivations leading to protein that is mostly produced in its soluble form, however in rather low yields. Inclusion body (IB) processes have gathered a lot of attention due to several benefits like increased space-time yields and high purity of the target product. Thus, to investigate the suitability of this processing strategy for ldhL1 production, a fed-batch fermentation steering the production of IBs rather than soluble product formation was developed. It was shown that the space-time-yield of the fermentation could be increased almost 3-fold by increasing qs to 0.25 g g−1 h−1 which corresponds to 21% of qs,max, and keeping the temperature at 37°C after induction. Solubilization and refolding unit operations were developed to regain full bioactivity of the ldhL1. The systematic approach in screening for solubilization and refolding conditions revealed buffer compositions and processing strategies that ultimately resulted in 50% product recovery in the refolding step, revealing major optimization potential in the downstream processing chain. The recovered ldhL1 showed an optimal activity at pH 5.5 and 30∘C with a high catalytic activity and KM values of 0.46 mM and 0.18 mM for pyruvate and NADH, respectively. These features, show that the here produced LDH is a valuable source for various commercial applications, especially considering low pH-environments.

Cost-effective production of ergothioneine using Rhodotorula mucilaginosa DL-X01 from molasses and fish bone meal enzymatic hydrolysate

Ergothioneine (EGT) is a high-value natural antioxidant that cannot be synthesized by the human body. This study showed that Rhodotorula mucilaginosa DL-X01 can use untreated molasses and fish bone meal enzymatic hydrolysate as the substrates to synthesize EGT. By optimizing the growth conditions, the EGT yield reached 29.39 mg/L when molasses and fish bone meal (FBM) were added at 60 g/L and 400 g/L respectively. Finally, the EGT yield was increased to 216.25 mg/L by fed-batch fermentation in a 5 L bioreactor. Compared with the fermentation by yeast extract peptone dextrose medium, the feedstock cost of EGT production was reduced by 330.91 % by using molasses and FBM as substrates. These results showed that R. mucilaginosa DL-X01 can produce high-value EGT using two cheap processing by-products, molasses and FBM, which is of great significance for environmental protection and sustainable development.

Production of single cell protein rich in potassium by Nectaromyces rattus using biogas slurry and molasses

Single-cell protein (SCP) is a vital supplement for animal protein feed. This study utilized biogas slurry and sugarcane molasses to ferment Nectaromyces rattus for the production of SCP. The optimal batch fermentation conditions were obtained in a 5L jar with a tank pressure of 0.1 MPa, an initial speed of 300 rpm, and an inoculum volume of 30%. The highest cell dry weight concentrations of the fed-batch fermentation without reflux and the fed-batch fermentation with reflux were 46.33 g/L and 29.71 g/L, respectively. The nitrogen conversion rates (47.05% and 44.12%) and the cell yields of total organic carbon (1 g/g and 1.17 g/g) of both fermentation modes were compared. The SCP contained 42.32% amino acids. Its high concentrations of potassium (19859.96 mg/kg) and phosphorus (7310.44 mg/kg) present a novel approach for the extraction of these essential nutrients from biogas slurry. The enrichment of K was related to the H+ efflux and sugar transport.

Overproduction of Xanthophyll Pigment in Flavobacterium sp. JSWR-1 under Optimized Culture Conditions.

Flavobacterium can synthesize xanthophyll, particularly the pigment zeaxanthin, which has significant economic value in nutrition and pharmaceuticals. Recently, the use of carotenoid biosynthesis by bacteria and yeast fermentation technology has shown to be very efficient and offers significant advantages in large-scale production, cost-effectiveness, and safety. In the present study, JSWR-1 strain capable of producing xanthophyll pigment was isolated from a freshwater reservoir in Wanju-gun, Republic of Korea. Based on the morphological, physiological, and molecular characteristics, JSWR-1 classified as belonging to the Flavobacterium species. The bacterium is strictly aerobic, Gram-negative, rod-shaped, and psychrophilic. The completed genome sequence of the strain Flavobacterium sp. JSWR-1 is predicted to be a single circular 3,425,829-bp chromosome with a G+C content of 35.2% and 2,941 protein-coding genes. The optimization of carotenoid production was achieved by small-scale cultivation, resulting in zeaxanthin being identified as the predominant carotenoid pigment. The enhancement of zeaxanthin biosynthesis by applying different light-irradiation, variations in pH and temperature, and adding carbon and nitrogen supplies to the growth medium. A significant increase in intracellular zeaxanthin concentrations was also recorded during fed-batch fermentation achieving a maximum of 16.69 ± 0.71 mg/l, corresponding to a product yield of 4.05 ± 0.15 mg zeaxanthin per gram cell dry weight. Batch and fed-batch culture extracts exhibit significant antioxidant activity. The results demonstrated that the JSWR-1 strain can potentially serve as a source for zeaxanthin biosynthesis.

Iterative gene integration mediated by 26S rDNA and non-homologous end joining for the efficient production of lycopene in Yarrowia lipolytica

Because of its potent antioxidant effects, lycopene has been used in various industries including, but not limited to, food, medical, and cosmetic industries. Yarrowia lipolytica, a non-conventional yeast species, is a promising chassis due to its natural mevalonate (MVA) pathway, abundant precursor acetyl coenzyme A content, and oleaginous properties. Several gene editing tools have been developed for Y. lipolytica along with engineering strategies for tetraterpenoid production. In this study, we engineered Y. lipolytica following multi-level strategies for efficient lycopene accumulation. We first evaluated the performance of the key lycopene biosynthetic genes crtE, crtB, and crtI, expressed via ribosomal DNA (rDNA) mediated multicopy random integration in the HMG1- and GGS1-overexpressing background strain. Further improvement in lycopene production was achieved by overexpressing the key genes for MVA synthesis via non-homologous end joining (NHEJ) mediated multi-round iterative transformation. Efficient strategies in the MVA and lipid synthesis pathways were combined to improve lycopene production with a yield of 430.5 mg/L. This strain produced 121 mg/g dry cell weight of lycopene in a 5-L fed-batch fermentation system. Our findings demonstrated iterative gene integration mediated by 26S rDNA and NHEJ for the efficient production of lycopene in Y. lipolytica. These strategies can be applied to induce Y. lipolytica to produce other tetraterpenoids.Graphical

Cytoplasmic production of Fabs in chemically defined media in fed-batch fermentation

Fragment of antigen-binding region (Fab) of antibodies are important biomolecules, with a broad spectrum of functionality in the biomedical field. While full length antibodies are usually produced in mammalian cells, the smaller size, lack of N-glycosylation and less complex structure of Fabs make production in microbial cell factories feasible. Since Fabs contain disulfide bonds, such production is often done in the periplasm, but there the formation of the inter-molecular disulfide bond between light and heavy chains can be problematic. Here we studied the use of the CyDisCo system (cytoplasmic disulfide bond formation in E. coli) to express two Fabs (Herceptin and Maa48) in the cytoplasm of E. coli in fed-batch fermentation using a generic chemically defined media. We were able to solubly express both Fabs with purified yields of 565 mg/L (Maa48) and 660 mg/L (Herceptin) from low density fermentation. Both proteins exhibited CD spectra consistent with natively folded protein and both were biologically active. To our knowledge this is the first demonstration of high-level production of biological active Fabs in the cytoplasm of E. coli in industrially relevant fermentation conditions.

Expression of ATP-binding cassette transporter proteins AatA or MdlB facilitates butyric acid production in Clostridium tyrobutyricum

ATP-binding cassette (ABC) transporters, one of the largest super families of membrane-associated microbial proteins involved in the transport of a wide range of substrates, play a vital role in bacterial physiology. In this study, we investigated the utility of heterologous expression of the ABC transporter AatA from Acetobacter pasteurianus AC2005 and homologous overexpression of the ABC transporter protein MdlB to improve butyric acid production in Clostridium tyrobutyricum. Compared to the starting wild-type strain C. tyrobutyricum ATCC 25755, recombinant strains of C. tyrobutyricum/aatA and C. tyrobutyricum/mdlB showed increased butyrate yield, productivity, selectivity, and titer during fed-batch fermentation. qRT-PCR revealed that AatA and MdlB upregulated the expression levels of ack, the gene encoding a key enzyme in the acetate synthesis pathway. This result was consistent with the observed increase in acetate titer in the recombinant strains. Moreover, overexpression of AatA or MdlB promoted the synthesis and secretion of acetic acid. An assay of cellular ATPase activities established that the two recombinant strains demonstrated two-folder higher ATPase activities compared to the wild-type strain. This increase in ATPase activity is proposed to accelerate sugar utilization, induce extracellular secretion of acetate for butyrate production, and shorten fermentation periods to improve butyrate productivity.

Paper sludge saccharification for batch and fed-batch production of bacterial cellulose decorated with magnetite for dye decolorization by experimental design

Cellulosic wastes represent a great environmental challenge, with potential conversion to product-added value through microbial fermentation. Currently, bacterial cellulose (BC) is considered a promising natural polymer for multiple applications. However, the high production cost challenges its wide application. Hence, the current study evaluated the applicability of paper sludge as a cost-effective medium for both cellulases and BC production. The local isolate Streptomyces rochei revealed the highest cellulase production titer (about 3 U/mL) at optimized conditions. For BC production, batch and fed-batch fermentation strategies were evaluated using enzymatically hydrolyzed paper sludge. The results asserted the advantage of fed-batch fermentation for advanced BC production (3.10 g/L) over batch fermentation (1.06 g/L) under the same cultivation conditions. The developed BC membranes were characterized through different instrumental analyses, which revealed an increase in fiber diameters and crystallinity under fed-batch fermentation. Furthermore, BC/magnetite (BC/Fe3O4) nanocomposite was developed by an in-situ approach. The newly developed composite was evaluated for dye removal applications, using methyl orange (MO) as a model. The dye removal conditions were optimized through Box Behnken design (BBD), which indicated maximal MO removal (83.5%) at pH 3.0 and BC/Fe3O4 concentration of 0.1 mg/dL after 60 min. Therefore, the current study asserts the good applicability of enzymatically hydrolyzed paper sludge as a medium for cost-effective BC production and the high capacity of BC/magnetite nanocomposite for MO decolorization. The study paves the way for the cost-effective implementation of BC/magnetite nanocomposite for dye removal.Graphical