Biochemical features and early adhesion of marine Candida parapsilosis strains on high-density polyethylene.

AIMS: Plastic debris are constantly released into oceans where, due to weathering processes, they suffer fragmentation into micro- and nanoplastics. Diverse microbes often colonize these persisting fragments, contributing to their degradation. However, there are scarce reports regarding the biofilm formation of eukaryotic decomposing microorganisms on plastics. Here, we evaluated five yeast isolates from deep-sea sediment for catabolic properties and early adhesion ability on high-density polyethylene (HDPE). METHODS AND RESULTS: We assessed yeast catabolic features and adhesion ability on HDPE fragments subjected to abiotic weathering. Adhered cells were evaluated through Crystal Violet Assay, Scanning Electron Microscopy, Atomic Force Microscopy and Infrared Spectroscopy. Isolates were identified as Candida parapsilosis and exhibited wide catabolic capacity. Two isolates showed high adhesion ability on HDPE, consistently higher than the reference C. parapsilosis strain, despite an increase in fragment roughness due to weathering. Isolate Y5 displayed the most efficient colonization, with production of polysaccharides and lipids after 48 h of incubation. CONCLUSION: This work provides insights on catabolic metabolism and initial yeast-HDPE interactions of marine C. parapsilosis strains. SIGNIFICANCE AND IMPACT OF THE STUDY: Our findings represent an essential contribution to the characterization of early interactions between deep-sea undescribed yeast strains and plastic pollutants found in oceans.

Post-Consumer Poly(ethylene terephthalate) (PET) Depolymerization by Yarrowia lipolytica: A Comparison between Hydrolysis Using Cell-Free Enzymatic Extracts and Microbial Submerged Cultivation.

Several microorganisms have been reported as capable of acting on poly(ethylene terephthalate) (PET) to some extent, such as Yarrowia lipolytica, which is a yeast known to produce various hydrolases of industrial interest. The present work aims to evaluate PET depolymerization by Y. lipolytica using two different strategies. In the first one, biocatalysts were produced during solid-state fermentation (SSF-YL), extracted and subsequently used for the hydrolysis of PET and bis(2-hydroxyethyl terephthalate) (BHET), a key intermediate in PET hydrolysis. Biocatalysts were able to act on BHET, yielding terephthalic acid (TPA) (131.31 µmol L-1), and on PET, leading to a TPA concentration of 42.80 µmol L-1 after 168 h. In the second strategy, PET depolymerization was evaluated during submerged cultivations of Y. lipolytica using four different culture media, and the use of YT medium ((w/v) yeast extract 1%, tryptone 2%) yielded the highest TPA concentration after 96 h (65.40 µmol L-1). A final TPA concentration of 94.3 µmol L-1 was obtained on a scale-up in benchtop bioreactors using YT medium. The conversion obtained in bioreactors was 121% higher than in systems with SSF-YL. The results of the present work suggest a relevant role of Y. lipolytica cells in the depolymerization process.

Biofilms of Pseudomonas and Lysinibacillus Marine Strains on High-Density Polyethylene.

Environmental pollution by plastic debris is estimated on a scale of 100 million metric tons, a portion of which is fragmented into micro- and nanoplastics. These fragments are often colonized by bacterial species in marine environments, possibly contributing to the biodegradation of such materials. However, further investigations are necessary to determine the impact of abiotic polymer weathering on biofilm adhesion, as well as the specific biofilm formation strategies employed by marine isolates. Here, we evaluate deep-sea sediment bacterial isolates for biofilm adhesion, extracellular matrix production, and polymer degradation ability. Our study focuses on high-density polyethylene (HDPE) fragments for their high durability and environmental persistence, subjecting fragments to abiotic weathering prior to bacterial colonization. Marine isolates identified as Pseudomonas sp. and Lysinibacillus sp. exhibited decreasing biofilm formation on weathered HDPE, especially over the first 24 h of incubation. This effect was countered by increased extracellular matrix production, likely improving cell adhesion to surfaces roughened by abiotic degradation. These adhesion strategies were contrasted with a reference Pseudomonas aeruginosa strain, which displayed high levels of biofilm formation on non-weathered HDPE and lower extracellular matrix production over the first 24 h of incubation. Furthermore, our results suggest that an increase in biofilm biomass correlated with changes to HDPE structure, indicating that these strains have a potential for biodegradation of plastic fragments. Therefore, this work provides a detailed account of biofilm formation strategies and bacteria-plastic interactions that represent crucial steps in the biodegradation of plastic fragments in marine environments.

Improved production of biocatalysts by Yarrowia lipolytica using natural sources of the biopolyesters cutin and suberin, and their application in hydrolysis of poly (ethylene terephthalate) (PET).

Since plastic pollution emerged as an urgent environmental problem, different biocatalysts have been tested for poly(ethylene terephthalate) (PET) hydrolysis. This work evaluated three different possible inducers for lipases and/or esterases, two natural sources of biopolymers (apple peels and commercial cork) and PET, as supplements in the solid-state fermentation of soybean bran by Yarrowia lipolytica. The obtained enzymatic extracts displaying different levels of lipase and esterase activities were then tested for PET depolymerization. Supplementation with 5 or 20 wt% of commercial cork led to an increase of 16% in lipase activity and to an increase of 131% in esterase activity, respectively. PET supplementation also led to an increase in the esterase activity of the enzymatic extracts (up to 69%). Enzymes produced in the screening step were able to act as biocatalysts in PET hydrolysis. Enzymatic extracts obtained in fermentation samples supplemented with 20 wt% PET and 20 wt% apple peels led to the highest terephthalic acid concentration (21.2 µmol L-1) in 7 days, whereas enzymes produced in commercial cork media were more efficient for bis(2-hydroxyethyl) terephthalate (BHET) hydrolysis, one of the key-PET hydrolysis intermediates. Results suggest a good potential of the biocatalysts produced by Y. lipolytica IMUFRJ 50,682 in a low-cost media for subsequent utilization in PET depolymerization reactions. This is one of the few reports on the use of a yeast for this application.

Process strategies to improve biocatalytic depolymerization of post-consumer PET packages in bioreactors, and investigation on consumables cost reduction.

Massive plastics production has raised concerns about low recycling rates and disposal of these materials in nature, causing environmental and economic impacts. Poly(ethylene terephthalate) (PET) is one of main polymers used for manufacture of plastic packaging (e.g. bottles, trays). Enzymatic recycling of PET has been a route of increasing study aiming at to recover its monomers (terephthalic acid and ethylene glycol), resulting in a circular production chain. In this study, investigation of pH control and fractionation of enzyme feeding were explored in post-consumed PET (PC-PET) hydrolysis reactions catalyzed by Humicola insolens cutinase (HiC) in stirred reactors. It was found that the unbuffered reaction provided of pH control by 0.5 M NaOH addition showed 2.39-fold improvement in the released monomers (to a total of 26.3 mM), comparatively to the Tris-HCl-buffered reaction. In addition, it was observed a possibility of reducing the enzyme loading used in the process by half, leading to an increase of 2.41-fold in the specific terephthalic acid concentration released per protein amount, whilst maintaining a high products concentration (97 mM). A simplified cost analysis of reaction consumables was performed, and the data reported here demonstrates that these alternative process strategies contribute to costs reduction on the enzymatic depolymerization reactions of PET.

A critical view on the technology readiness level (TRL) of microbial plastics biodegradation.

Accumulation of plastic wastes and their effects on the ecosystem have triggered an alarm regarding environmental damage, which explains the massive investigations over the past few years, aiming technological alternatives for their proper destination and valorization. In this context, biological degradation emerges as a green route for plastic processing and recycling in a circular economy approach. Some of the main polymers produced worldwide are poly(ethylene terephthalate) (PET), polyethylene (PE) and polypropylene (PP), which are among the most recalcitrant materials in the environment. In comparison to other polymers, PET biodegradation has advanced dramatically in recent years concerning microbial and enzymatic mechanisms, being positioned in a higher technology readiness level (TRL). Even more challenging, polyolefins (PE and PP) biodegradation is hindered by their high recalcitrance, which is mainly related to stable carbon-carbon bonds. Potential microbial biocatalysts for this process have been evaluated, but the related mechanisms are still not fully elucidated. This review aims to discuss the latest developments on key microbial biocatalysts for degradation of these polymers, addressing biodegradation monitoring, intellectual property, and TRL analysis of the bioprocessing strategies using biodegradation performance, process time and scale as parameters for the evaluation.

2,3-Butanediol production by the non-pathogenic bacterium Paenibacillus brasilensis.

2,3-Butanediol (2,3-BDO) is of considerable importance in the chemical, plastic, pharmaceutical, cosmetic, and food industries. The main bacterial species producing this compound are considered pathogenic, hindering large-scale productivity. The species Paenibacillus brasilensis is generally recognized as safe (GRAS) and is phylogenetically similar to P. polymyxa, a species widely used for 2,3-BDO production. Here, we demonstrate, for the first time, that P. brasilensis strains produce 2,3-BDO. Total 2,3-BDO concentrations for 15 P. brasilensis strains varied from 5.5 to 7.6 g/l after 8 h incubation at 32 °C in modified YEPD medium containing 20 g/l glucose. Strain PB24 produced 8.2 g/l of 2,3-BDO within a 12-h growth period, representing a yield of 0.43 g/g and a productivity of 0.68 g/l/h. An increase in 2,3-BDO production by strain PB24 was observed using higher concentrations of glucose, reaching 27 g/l of total 2,3-BDO in YEPD containing about 80 g/l glucose within a 72-h growth period. We sequenced the genome of P. brasilensis PB24 and uncovered at least six genes related to the 2,3-BDO pathway at four distinct loci. We also compared gene sequences related to the 2,3-BDO pathway in P. brasilensis PB24 with those of other spore-forming bacteria, and found strong similarity to P. polymyxa, P. terrae, and P. peoriae 2,3-BDO-related genes. Regulatory regions upstream of these genes indicated that they are probably co-regulated. Finally, we propose a production pathway from glucose to 2,3-BDO in P. brasilensis PB24. Although the gene encoding S-2,3-butanediol dehydrogenase (butA) was found in the genome of P. brasilensis PB24, only R,R-2,3- and meso-2,3-butanediol were detected by gas chromatography under the growth conditions tested here. Our findings can serve as a basis for further improvements to the metabolic capabilities of this little-studied Paenibacillus species in relation to production of the high-value chemical 2,3-butanediol.

Screening of commercial enzymes for poly(ethylene terephthalate) (PET) hydrolysis and synergy studies on different substrate sources.

Poly(ethylene terephthalate) (PET) is one of the most consumed plastics in the world. The development of efficient technologies for its depolymerization for monomers reuse is highly encouraged, since current recycling rates are still very low. In this study, 16 commercial lipases and cutinases were evaluated for their abilities to catalyze the hydrolysis of two PET samples. Humicola insolens cutinase showed the best performance and was then used in reactions on other PET sources, solely or in combination with the efficient mono(hydroxyethyl terephthalate)-converting lipase from Candida antarctica. Synergy degrees of the final titers of up to 2.2 (i.e., more than double of the concentration when both enzymes were used, as compared to their use alone) were found, with increased terephthalic acid formation rates, reaching a maximum of 59,989 µmol/L (9.36 g/L). These findings open up new possibilities for the conversion of post-consumer PET packages into their minimal monomers, which can be used as drop in at existing industrial facilities.

Enzymatic post-consumer poly(ethylene terephthalate) (PET) depolymerization using commercial enzymes.

Poly(ethylene terephthalate) (PET) is a synthetic polymer widely used globally. The high PET resistance to biotic degradation and its improper destination result in the accumulation of this plastic in the environment, largely affecting terrestrial and aquatic animals. This work investigated post-consumer PET (PC-PET) degradation using five commercial hydrolase enzymes (Novozym 51032, CalB, Palatase, Eversa, Lipozyme TL). Humicola insolens cutinase (HiC, Novozym 51032) was the most active among the enzymes studied. Several important reaction parameters (enzyme type, dual enzyme system, enzyme concentration, temperature, ultrasound treatment) were evaluated in PC-PET hydrolysis using HiC. The concentration and the proportion (molar ratio) of hydrolysis products, terephthalic acid (TPA), mono(2-hydroxyethyl) terephthalate (MHET), and bis(2-hydroxyethyl) terephthalate (BHET), were significantly changed depending on the reaction temperature. The TPA released at 70 °C was 3.65-fold higher than at 50 °C. At higher temperatures, the conversion of MHET into TPA was favored. The enzymatic PET hydrolysis by HiC was very sensitive to the enzyme concentration, indicating that it strongly adsorbs on the polymer surface. The concentration of TPA, MHET, and BHET increased as the enzyme concentration increased, and a maximum was achieved using 40-50 vol % of HiC. The presented results add relevant data to optimizing enzyme-based PET recycling technologies.

Fungal Screening for Potential PET Depolymerization.

Approximately 400 billion PET bottles are produced annually in the world, of which from 8 to 9 million tons are discarded in oceans. This requires developing strategies to urgently recycle them. PET recycling can be carried out using the microbial hydrolysis of polymers when monomers and oligomers are released. Exploring the metabolic activity of fungi is an environmentally friendly way to treat harmful polymeric waste and obtain the production of monomers. The present study addressed: (i) the investigation of potential of strains with the potential for the depolymerization of PET bottles from different manufacturers (crystallinity of 35.5 and 10.4%); (ii) the search for a culture medium that favors the depolymerization process; and (iii) gaining more knowledge on fungal enzymes that can be applied to PET recycling. Four strains (from 100 fungal strains) were found as promising for conversion into terephthalic acid from PET nanoparticles (npPET): Curvularia trifolii CBMAI 2111, Trichoderma sp. CBMAI 2071, Trichoderma atroviride CBMAI 2073, and Cladosporium cladosporioides CBMAI 2075. The fermentation assays in the presence of PET led to the release of terephthalic acid in concentrations above 12 ppm. Biodegradation was also confirmed using mass variation analyses (reducing mass), scanning electron microscopy (SEM) that showed evidence of material roughness, FTIR analysis that showed band modification, enzymatic activities detected for lipase, and esterase and cutinase, confirmed by monomers/oligomers quantification using high performance liquid chromatography (HPLC-UV). Based on the microbial strains PET depolymerization, the results are promising for the exploration of the selected microbial strain.

Design of Enzyme Loaded W/O Emulsions by Direct Membrane Emulsification for CO2 Capture.

Membrane-based gas separation is a promising unit operation in a low-carbon economy due to its simplicity, ease of operation, reduced energy consumption and portability. A methodology is proposed to immobilise enzymes in stable water-in-oil (W/O) emulsions produced by direct membrane emulsification systems and thereafter impregnated them in the pores of a membrane producing emulsion-based supported liquid membranes. The selected case-study was for biogas (CO2 and CH4) purification. Upon initial CO2 sorption studies, corn oil was chosen as a low-cost and non-toxic bulk phase (oil phase). The emulsions were prepared with Nadir® UP150 P flat-sheet polymeric membranes. The optimised emulsions consisted of 2% Tween 80 (w/w) in corn oil as the continuous phase and 0.5 g.L-1 carbonic anhydrase enzyme with 5% PEG 300 (w/w) in aqueous solution as the dispersed phase. These emulsions were impregnated onto a porous hydrophobic PVDF membrane to prepare a supported liquid membrane for gas separation. Lastly, gas permeability studies indicated that the permeability of CO2 increased by ~15% and that of CH4 decreased by ~60% when compared to the membrane without carbonic anhydrase. Thus, a proof-of-concept for enhancement of CO2 capture using emulsion-based supported liquid membrane was established.

Production of multifunctional lipases by Penicillium verrucosum and Penicillium brevicompactum under solid state fermentation of babassu cake and castor meal.

The main objective of this work was to optimize lipase production, in terms of hydrolytic and esterification activities, by Penicillium brevicompactum and Penicillium verrucosum in solid state fermentation using agroindustrial residues as raw material. Maxima hydrolytic activities of 48.6 and 87.7 U/g were achieved when P. brevicompactum was cultured in babassu cake and castor meal, respectively. Higher esterification activities (around 244 U/g) were achieved when P. brevicompactum was used as microorganism and babassu cake as raw material. Different experimental conditions led to these promising values, clearly showing that no correlation can be attributed between hydrolytic and esterification activities. In spite of the several applications of lipases which are capable of catalyze synthesis reactions, only few works in this subject are presented in the literature, especially when low cost raw materials are used.

Cellulases from Penicillium funiculosum: production, properties and application to cellulose hydrolysis.

The objective of this work is to investigate the utilization of two abundant agricultural residues in Brazil for the production and application of cellulolytic enzymes. Different materials obtained after pretreatment of sugarcane bagasse, as well as pure synthetic substrates, were considered for cellulase production by Penicillium funiculosum. The best results for FPase (354 U L(-1)) and beta-glucosidase (1,835 U L(-1)) production were observed when sugarcane bagasse partially delignified cellulignin (PDC) was used. The crude extract obtained from PDC fermentation was then partially characterized. Optimal temperatures for cellulase action ranged from 52 to 58 degrees C and pH values of around 4.9 contributed to maximum enzyme activity. At 37 degrees C, the cellulases were highly stable, losing less than 15% of their initial activity after 23 h of incubation. There was no detection of proteases in the P. funiculosum extract, but other hydrolases, such as endoxylanases, were identified (147 U L(-1)). Finally, when compared to commercial preparations, the cellulolytic complex from P. funiculosum showed more well-balanced amounts of beta-glucosidase, endo- and exoglucanase, resulting in the desired performance in the presence of a lignocellulosic material. Cellulases from this filamentous fungus had a higher glucose production rate (470 mg L(-1) h(-1)) when incubated with corn cob than with Celluclast, GC 220 and Spezyme (312, 454 and 400 mg L(-1) h(-1), respectively).

Enzyme Immobilization in Covalent Organic Frameworks: Strategies and Applications in Biocatalysis.

The development of efficient catalytic systems is a fundamental aspect for the straightforward production of chemicals. During the last years, covalent organic frameworks (COFs) emerged as an exciting class of organic nanoporous materials. Due to their pre-designable structure, they can be prepared with distinct physicochemical characteristics, specific pore sizes, and tunable functional groups. Moreover, associated with their stability in different media, these materials are considered promising supports for enzyme immobilization. Herein, it is highlighted the recent literature of enzyme immobilization in COFs, the main immobilization strategies, and the catalytic applications of these composites.

Evaluation of 1,3-propanediol production by twoCitrobacter freundiistrains using crude glycerol and soybean cake hydrolysate.

Biodiesel production processes using soybean as feedstock generates soybean cake and crude glycerol as by-products. These by-product streams were used as sole feedstocks for the production of 1,3-propanediol (PDO) using two bacterial strains of Citrobacter freundii. Soybean cake has been converted into a nutrient-rich hydrolysate by crude enzymes produced via solid state fermentation. The effect of initial glycerol and free amino nitrogen concentration on bacterial growth and PDO production has been evaluated in batch bioreactor cultures showing that C. freundii VK-19 is a more efficient PDO producer than C. freundii FMCC-8. The cultivation of C. freundii VK-19 in fed-batch bioreactor cultures using crude glycerol and soybean cake hydrolysates led to PDO concentration of 47.4 g/L with yield and productivity of 0.49 g/g and 1.01 g/L/h, respectively. The effect of PDO, metabolic by-products, and sodium and potassium salts on bacterial growth was evaluated showing that potassium salts initially enhance bacterial growth, whereas sodium salts cause significant inhibition to bacterial growth. Soybean cake hydrolysate and crude glycerol could be utilized for PDO production, but the fermentation efficiency is influenced by the catalyst used during biodiesel production.

A newly isolated Enterobacter sp. strain produces 2,3-butanediol during its cultivation on low-cost carbohydrate-based substrates.

2,3-Butanediol (BDO) is an important platform chemical with a wide range of applications in various industries. In the present study, a newly isolated wild Enterobacter sp. strain (FMCC-208) was evaluated towards its ability to produce BDO on media composed of sugars derived from sucrose refinery plant. Optimum values of temperature and pH as well as substrate inhibition were determined through batch experiments. The ability of the strain to convert various monosaccharides was also investigated. Maximum BDO concentrations of 90.3 and 10 g l-1 of acetoin were obtained during a fed-batch bioreactor experiment with cane molasses and sucrose employed as substrates. A high volumetric productivity was noted in a fed-batch experiment using molasses and sucrose as carbon sources at T = 37°C, in which 73.0 g l-1 of BDO together with 12.4 g l-1 of acetoin was produced where 1.15 g l-1 h-1 of diol/acetoin was produced. In previously pasteurized media, 70.0 g l-1 of BDO and 5.0 g l-1 of acetoin were produced (yield = 0.39 g g-1). Finally, besides BDO production, growth on molasses was accompanied by non-negligible decolorization (25-35%) of the residue. Therefore, the strain is a promising candidate for the conversion of sucrose-based materials into BDO.

Biocatalytic esterification of fatty acids using a low-cost fermented solid from solid-state fermentation with Yarrowia lipolytica.

This study aimed to evaluate the use of a lyophilized fermented solid (named solid enzymatic preparation, SEP), with lipase activity, as a low-cost biocatalyst for esterification reactions of fatty acids present in acid raw materials for biodiesel synthesis. The SEP was obtained by solid-state fermentation (SSF) of soybean bran using the strain of Yarrowia lipolytica IMUFRJ 50682 and contains the lipases secreted by this yeast. The esterification reaction of ethanol and the predominant fatty acids present in different acid oil sources for biodiesel production (oleic, linoleic, stearic and palmitic acids) was investigated. Oleic acid conversion of above 85% was obtained after 24 h, using 30 wt% of SEP and ethanol/oleic acid molar ratio of 1, at 30 °C, in a reaction medium with and without solvent (n-hexane). Similar results were achieved with stearic (79%), palmitic (82%) and linoleic (90%) acids. The reusability of SEP was investigated over ten successive batches by washing it with different solvents (ethanol, water or n-hexane) between the cycles of ethyl oleate synthesis. Washing with water allowed the SEP to be reused for six cycles maintaining over 80% of the conversion reached in the first cycle. These results show the potential of this biocatalyst to reduce the content of free fatty acids in acid oils for biodiesel synthesis with a potential to be applied in a broad plethora of raw materials.

Improvement on bioprocess economics for 2,3-butanediol production from very high polarity cane sugar via optimisation of bioreactor operation.

This study focuses on the optimisation of 2,3-butanediol (BDO) production in fed-batch cultures carried out with the bacterial strain Enterobacter ludwigii using very high polarity (VHP) sugar from sugarcane mills. Various kLa values were evaluated using either complex or synthetic fermentation media demonstrating that the latter enhance BDO production efficiency with low by-product formation. The pH (6.3) and temperature (33.9 °C) employed in fed-batch bioreactor cultures has been optimised via experimental design. Fed-batch cultures carried out at the optimum temperature and pH and varying kLa values resulted in BDO concentration, yield and productivity of 86.8 g/L, 0.37 g/g and 3.95 g L-1 h-1. Using this fermentation efficiency, the minimum selling price of BDO for annual production capacities of 10,000 t and 50,000 t was estimated at $3.12/kg and $2.67/kg, respectively, for a VHP cane sugar market price of $0.4/kg.

Hydrocarbon-associated substrates reveal promising fungi for poly (ethylene terephthalate) (PET) depolymerization.

Recalcitrant characteristics and insolubility in water make the disposal of synthetic polymers a great environmental problem to be faced by modern society. Strategies towards the recycling of post-consumer polymers, like poly (ethylene terephthalate, PET) degradation/depolymerization have been studied but still need improvement. To contribute with this purpose, 100 fungal strains from hydrocarbon-associated environments were screened for lipase and esterase activities by plate assays and high-throughput screening (HTS), using short- and long-chain fluorogenic probes. Nine isolates were selected for their outstanding hydrolytic activity, comprising the genera Microsphaeropsis, Mucor, Trichoderma, Westerdykella, and Pycnidiophora. Two strains of Microsphaeropsis arundinis were able to convert 2-3% of PET nanoparticle into terephthalic acid, and when cultured with two kinds of commercial PET bottle fragments, they also promoted weight loss, surface and chemical changes, increased lipase and esterase activities, and led to PET depolymerization with release of terephthalic acid at concentrations above 20.0 ppm and other oligomers over 0.6 ppm. The results corroborate that hydrocarbon-associated areas are important source of microorganisms for application in environmental technologies, and the sources investigated revealed important strains with potential for PET depolymerization.

Fumaric acid production using renewable resources from biodiesel and cane sugar production processes.

The microbial production of fumaric acid by Rhizopus arrhizus NRRL 2582 has been evaluated using soybean cake from biodiesel production processes and very high polarity (VHP) sugar from sugarcane mills. Soybean cake was converted into a nutrient-rich hydrolysate via a two-stage bioprocess involving crude enzyme production via solid state fermentations (SSF) of either Aspergillus oryzae or R. arrhizus cultivated on soybean cake followed by enzymatic hydrolysis of soybean cake. The soybean cake hydrolysate produced using crude enzymes derived via SSF of R. arrhizus was supplemented with VHP sugar and evaluated using different initial free amino nitrogen (FAN) concentrations (100, 200, and 400 mg/L) in fed-batch cultures for fumaric acid production. The highest fumaric acid concentration (27.3 g/L) and yield (0.7 g/g of total consumed sugars) were achieved when the initial FAN concentration was 200 mg/L. The combination of VHP sugar with soybean cake hydrolysate derived from crude enzymes produced by SSF of A. oryzae at 200 mg/L initial FAN concentration led to the production of 40 g/L fumaric acid with a yield of 0.86 g/g of total consumed sugars. The utilization of sugarcane molasses led to low fumaric acid production by R. arrhizus, probably due to the presence of various minerals and phenolic compounds. The promising results achieved through the valorization of VHP sugar and soybean cake suggest that a focused study on molasses pretreatment could lead to enhanced fumaric acid production.