Assessing the Role of Molecular Sieves in Continuous Enzymatic Synthesis of Geranyl Acetate

Assessing the Role of Molecular Sieves in Continuous Enzymatic Synthesis of Geranyl Acetate | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Assessing the Role of Molecular Sieves in Continuous Enzymatic Synthesis of Geranyl Acetate Daniele Gonçalves de Oliveira, Daniela Remonatto, Ila Maranhão de Oliveira, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8329923/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Geranyl acetate is a geraniol ester widely used as a flavoring agent and preservative in food and cosmetic products due to its pleasant aroma, antimicrobial properties, and low toxicity. This study investigated the production of geranyl acetate by esterification of geraniol with acetic acid at a 3:1 molar ratio in a solvent-free packed bed reactor using Novozym 435 as biocatalyst. Additionally, the influence of molecular sieves on process performance was evaluated. The results showed that geranyl acetate conversions reached approximately 100% (w/w) at flow rates of 0.1 and 0.2 mL/min, both in the presence and absence of molecular sieves. Thus, the addition of these adsorbents did not provide any benefit to the reaction. Consequently, the preferred approach for continuous synthesis of geranyl acetate in a packed bed reactor is to use Novozym 435 without molecular sieves. This strategy simplifies the process, reduces costs, and maintains conversion efficiency over time, offering valuable insights for the industrial development of direct esterification processes using biocatalysts in continuous systems. Esterification Geranyl acetate Molecular sieves Packed bed reactor Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Consumers are increasingly seeking natural ingredients as alternatives to synthetic additives. This preference stems from both the pursuit of a healthier lifestyle and the growing concern for environmental preservation. Some of the natural products that most attract consumer attention are essential oils and other plant-derived compounds. Geraniol (3,7-dimethylocta- trans -2,6-dien-1-ol) is an acyclic monoterpene alcohol found in the essential oils of plants such as geranium ( Geranium sp. L.), citronella ( Cymbopogon winterianus Jowitt), lemongrass ( Cymbopogon citratus (DC.) Stapf), and rose ( Rosa × damascena Mill.) [ 1 – 3 ]. This terpene alcohol, along with its isomer nerol, exhibits notable biological activity and is frequently noted in the literature for its antimicrobial, acaricidal, pesticidal, anti-inflammatory, and chemotherapeutic properties [ 1 – 6 ]. However, its use in pharmaceutical and cosmetic products is limited by a certain level of toxicity, attributed to the presence of an aldehyde group that can react with amino residues in proteins and trigger undesirable effects [ 2 , 7 ]. A viable alternative to using this monoterpene alcohol is to convert it into derivatives, such as geranyl acetate (3,7-dimethylocta-2,6-dien-1-yl ethanoate). This geraniol ester is characterized by low toxicity and advantageous organoleptic properties, including a pleasant flavor and aroma, which make it a valuable ingredient in food, fragrance, and cosmetic products [ 6 , 8 ]. It is noteworthy that geranyl acetate has been approved for use in food products by the U.S. Food and Drug Administration (FDA) and is recognized as safe by the Flavor and Extract Manufacturers Association (FEMA) [ 9 ]. Geranyl acetate is commonly obtained by fractionating essential oils. However, this process requires large quantities of raw materials, representing an impractical and expensive route [ 10 ]. Another widely used method is conventional chemical synthesis, which also has several disadvantages, including harsh reaction conditions (e.g., high pH and temperature) and high costs associated with product purification [ 8 ]. A third route to ester production is biocatalysis. This alternative method offers several advantages over chemical methods, including greater chemoselectivity, enantioselectivity, and environmental friendliness [ 11 , 12 ]. Enzymatic catalysis using lipases (triacylglycerol acyl hydrolases, EC 3.1.1.3) is a promising alternative for the synthesis of geraniol esters. Although these enzymes can efficiently catalyze esterification, transesterification, amination, acidolysis, and hydrolysis reactions [ 4 , 13 – 15 ], some drawbacks impair their use in their free form, such as low stability, loss of activity during storage, and difficulty in reuse [ 16 ]. These limitations can be circumvented by immobilizing lipases. Accordingly, immobilized lipases represent an attractive strategy to reduce process costs, as immobilization enhances enzyme stability and facilitates biocatalyst reuse [ 13 , 17 ]. The main reactions that use immobilized lipases to produce geraniol esters are esterification (between an alcohol and a carboxylic acid) and transesterification (between an ester and an alcohol) [ 4 , 6 , 10 , 18 – 21 ]. In the current study, geranyl acetate was synthesized via direct esterification catalyzed by the immobilized enzyme Novozym 435. Enzymatic esterification is a thermodynamically controlled process; that is, it is governed by chemical equilibrium [ 22 ]. In these processes, biocatalysts are often susceptible to inactivation due to the high concentration of acid in the reaction medium, a problem typically mitigated by using an organic solvent or an excess of alcohol [ 4 , 22 , 23 ] Another important aspect of enzymatic esterification is that, at the molecular level, a minimal amount of water is essential to maintain lipase activity. On the other hand, excess water can significantly reduce enzyme effectiveness by promoting unwanted side reactions, such as hydrolysis, thereby reducing esterification yield [ 24 ]. One strategy to reduce this problem is to use molecular sieves, which remove the water produced during the reaction [ 4 , 18 , 25 – 27 ]. Kanwar et al. [ 18 ] reported that, during the esterification of geraniol with butyric acid using hydrogel-immobilized lipase, the addition of 3 Å molecular sieves resulted in geranyl butyrate yields close to 100%. Trusek-Holownia et al. [ 28 ], in studying the esterification of geraniol with acetic acid in a membrane bioreactor, achieved 80% conversion to geranyl acetate by using a column packed with molecular sieves for water removal. The esterification process can be carried out either in the presence or absence of solvents [ 8 , 22 , 26 , 29 – 32 ]. The use of solvents can increase substrate solubility, reduce enzyme inhibition, and control water activity by preventing ester hydrolysis. However, it also increases process costs and requires an additional removal step at the end of the reaction [ 26 , 30 , 33 ]. Solvent-free esterification offers several economic and environmental advantages. The absence of solvents reduces the need for purification steps and minimizes waste generation, rendering the process more sustainable and cost-effective [ 4 , 20 ]. Nevertheless, solvent-free methods also present challenges, given the effect of the reagent molar ratio on lipase activity and its influence on the reaction equilibrium [ 15 ]. A useful method for mitigating these problems is the use of an excess of one of the substrates. Remonatto et al. [ 4 ] observed that using geraniol in excess resulted in high conversion to geranyl acetate by shifting the equilibrium toward product formation and preventing biocatalyst denaturation caused by the presence of acid. Nascimento et al. [ 23 ] likewise used excess geraniol to produce geranyl cinnamate in a solvent-free system, achieving optimal conversion with a molar ratio of 1:5.68 (cinnamic acid/geraniol). The synthesis of geraniol esters is commonly carried out in two types of bioreactors: packed bed reactors (PBRs) and stirred tank reactors (STRs) [ 4 , 10 , 34 – 36 ]. The choice between these systems depends on the nature of the process [ 37 ]. PBRs are predominantly used in continuous operations [ 38 – 41 ]. By contrast, STRs play a central role in batch processes, given their ease of control and compatibility with a wide range of operating conditions [ 28 , 42 ]. It should be noted that STRs also have significant disadvantages. These include the potential for high shear stress under reaction conditions, the need for dedicated cleaning time after operation, and substantial energy consumption [ 43 ]. On the other hand, PBRs are widely used for syntheses catalyzed by immobilized enzymes due to their operational simplicity, continuous production, high efficiency, ease of product separation, and effective enzyme recovery at the end of the process [ 44 , 45 ]. These characteristics play a fundamental role in reducing the overall costs of the process. In light of these considerations, this study aimed to synthesize geranyl acetate through solvent-free direct esterification of geraniol with acetic acid catalyzed by the immobilized enzyme Novozym 435 in a continuous PBR system. A secondary aim was to assess the influence of incorporating molecular sieves for water removal on process performance. Material and methods Materials The substrates used in esterification reactions were acetic acid (99.8%, Neon, Susano, SP, Brazil) and geraniol (97%, Sigma–Aldrich, St. Louis, MO, USA). Before use, both reagents were dried overnight in the presence of an excess of molecular sieves (250 g/L). Immobilized Candida antarctica lipase B (Novozym 435) was kindly provided by Novozymes® (Araucária, PR, Brazil). The molecular sieves used were 4 Å (8–12 mesh, Sigma–Aldrich, St. Louis, USA). Oleic acid (Synth, Diadema, SP, Brazil) and ethanol (99.5%, Synth, Diadema, SP, Brazil) were used to determine esterification activity. Methods Hydrodynamic characterization of the PBR Tracer pulse experiments were conducted to characterize the hydrodynamics of the PBR. The residence time distribution was determined experimentally by injecting a geraniol solution containing a purple dye (Roxo Saramanil®, São Paulo, Brazil, purchased locally) into the reactor and measuring the tracer concentration at the outlet over time using a UV-Vis spectrophotometer (Genesys 10S, Thermo Fisher Scientific, San Jose, CA, USA) at 530 nm. The residence time distribution function, E ( t ), was calculated using Eq. ( 1 ) [ 46 ]. $$\:\text{E}\text{(}\text{t}\text{):=}\frac{\text{C}\text{(}\text{t}\text{)}}{{\int\:}_{\text{0}}^{\text{∞}}\text{C}\text{(}\text{t}\text{)d}\text{t}}$$ 1 where C ( t ) is the tracer concentration at time ( t ). Based on the residence time distribution function, the mean residence time ( t m ) was calculated according to Eq. ( 2 ) [ 46 ]: $$\:{\text{t}}_{\text{m}}\text{=}{\int\:}_{\text{0}}^{\text{∞}}\text{tE}\text{(}\text{t}\text{)d}\text{t}$$ 2 The integration in Eq. ( 2 ) was performed using Origin software version 8.0 (Origin Lab Corporation, Washington, USA). Geranyl acetate production Geranyl acetate was synthesized via Novozym 435-catalyzed esterification of geraniol with acetic acid at a 3:1 molar ratio in a column PBR (5 cm height × 2 cm diameter; Fig. 1 ). The reactor was packed with approximately 1.51 g of catalyst and operated in continuous mode. Substrates were pumped at a flow rate of 1 mL/min, corresponding to a space time of 1.85 h, as calculated using Eq. ( 3 ). A water bath (Marconi, model MA 184/6, Piracicaba, SP, Brazil) was connected to the feed circulation system to maintain the reaction mixture at 60°C (Fig. 2 ). In a previous study, Remonatto et al. [ 4 ] determined the optimal synthesis conditions (molar ratio and temperature) for batch STRs. $$\:\text{τ}\text{}\text{=}\frac{{V}_{\text{u}}}{{Q}_{\text{t}}}$$ 3 where \(\:{Q}_{\text{t}}\) is the flow rate of the tracer, \(\:{V}_{\text{u}}\) is the useful volume of the reactor, and \(\:\text{τ}\) is the space time. Figure 1 near here Figure 2 near here Geranyl acetate production in a PBR with molecular sieves Molecular sieves were used to remove the water generated during esterification. For this purpose, the reactor was packed with four alternating layers separated by nylon mesh: the first and third layers contained 0.75 g of immobilized lipase (Novozym 435) each, whereas the second and fourth layers contained 0.75 g of molecular sieves (Fig. 3 ). The same proportion was used by Freitas et. al [ 25 ] in a similar system and by Vadgama et al. [ 47 ] in a series of PBRs, where the first and third reactors contained 10 g of enzyme and the second reactor contained 20 g of molecular sieves. The synthesis was carried out following the parameters described in the previous section. Figure 3 near here Purification of geranyl acetate Crude geranyl acetate was purified by column chromatography (50 × 1 cm). Silica gel (Neon, Suzano, SP, Brazil) was used as stationary phase. The mobile phase consisted of ethyl acetate and cyclohexane (1:14 v/v) acidified with 2% (v/v) acetic acid. Initially, 1 mL of sample (reaction mixture after esterification) was applied to the top of the column, which was then eluted at a constant rate to separate the components. Aliquots were collected at the column outlet, transferred into 2 mL vials, and analyzed using thin-layer chromatography (Sigma–Aldrich, Brazil). Geranyl acetate was quantified by comparison with a standard solution. Aliquots showing high conversion were further analyzed for purity using gas chromatography. Quantification of geranyl acetate by gas chromatography Initially, samples were neutralized using a hydroalcoholic solution (30% v/v ethanol) in 0.8 M potassium hydroxide [ 48 ]. Geranyl acetate was quantified by gas chromatography on a Shimadzu GC-2010 chromatograph equipped with an auto-injector (AOC-20i), a fused silica capillary column (SH-Stabilwax-DA, 30 m length × 0.25 mm internal diameter × 0.25 µm film thickness), and a flame ionization detector. The temperature program was as follows: 60–210°C at 10°C/min, 210–250°C at 20°C/min, and 250°C for 1 min. Injector and detector temperatures were maintained at 250°C. Samples were diluted in hexane to a final volume of 1 µL. Injection was performed in split mode (1:100 ratio). Nitrogen (N 2 ) was used as carrier gas at 56 kPa. Geranyl acetate conversion was determined by constructing an analytical curve (Eq. 4) using purified geranyl acetate (item 3.2.2) and methyl nonadecanoate (C19) (Sigma–Aldrich) as internal standard [ 23 ]. \(\:y=1.6428x+0.0066\) ( \(\:{R}^{2}=0.998\) ) (4) where \(\:y\) is the geranyl acetate conversion (w/w) and \(\:x\) is the ratio of the geranyl acetate peak area at the end of the reaction to the area of the internal standard C19. Esterification activity The esterification activity (U/g) of the biocatalyst before and after the reactions was determined as described elsewhere [ 49 ]. For this, oleic acid was esterified with ethanol at a molar ratio of 1:1 in the presence of 0.2 g of Novozym 435 at 40°C and 150 rpm. Aliquots (300 µL) were collected at 0, 10, 20, 30, 40, and 60 min of reaction. The consumption of oleic acid was determined by titration with 0.05 M sodium hydroxide. One unit of enzyme activity (U) is defined as the amount of enzyme required to consume 1 µmol of oleic acid per minute. Results and discussion Hydrodynamic characterization of the PBR Hydrodynamic analysis of PBRs is essential for identifying preferential paths, which are regions where the fluid flows predominantly due to irregularities in bed packing or local variations in fluidity, leading to nonuniform flow [ 50 ]. Such paths may result from uneven particle distribution, inadequate bed compaction, accumulation of fine particles, or operational issues. Although preferential paths may form anywhere within the bed, they are most common near the reactor walls, in low-resistance zones, and around fluid inlet and outlet ports. These deviations decrease reaction efficiency and compromise overall reactor performance [ 51 ]. Therefore, understanding and controlling preferential paths is critical for the efficient and safe operation of PBRs. An effective approach for analyzing fluid flow patterns in PBRs is the study of mean residence time. This parameter describes the flow behavior within the reactor and provides valuable information on bed performance, including the presence of stagnation zones [ 52 ]. Given that the flow regime directly influences reactor efficiency, its evaluation offers opportunities to improve process control [ 46 , 53 ]. Consequently, mean residence time is one of the most important parameters for achieving high product conversion in PBRs. In this study, a tracer pulse assay was conducted to experimentally determine the mean residence time of reactants and characterize the behavior of the reaction mixture within the bed. The mean residence time of reagents in the PBR was determined experimentally using a purple dye as tracer. The calibration curve was constructed for quantification ( A = 0.0396 C , where C is the concentration of the tracer). The coefficient of determination ( R 2 = 0.9998) indicated a good fit of the model to experimental data. The results were used to construct the distribution curve of residence time ( E ) as a function of time ( t ), according to Eq. ( 2 ) (Fig. 4 ). Figure 4 near here For a biocatalyst volume of 1.81 mL, a useful volume of 9.5 mL, and a flow rate of 0.1 mL/min, the space time was 110.95 min, according to Eq. ( 3 ). The average residence time, as calculated by Eq. ( 2 ), was 167.61 min. This difference between theoretical and experimental values suggests the existence of preferential paths in the bed. According to Fogler [ 46 ], when the residence time is greater than the space time, there are dead volumes in the reactor, that is, areas with fluid stagnation. Cozentino et al. [ 54 ] studied the synthesis of MLM-type triacylglycerols from grape seed oil in associated PBRs. The mean experimental residence time obtained was 335.17 min, whereas the theoretical space time was 415.58 min. This 19.35% difference between theoretical and experimental values was considered small, indicating that the reactor had a satisfactory performance, without significant deviations in flow and with good distribution of biocatalyst particles. De Paula et al. [ 55 ] evaluated the enzymatic interesterification of milk fat with soybean oil in a PBR using immobilized lipase from Rhizopus oryzae . The mean residence time was 135 min, which was 8.74% higher than the theoretical space time. This difference was considered acceptable, indicating that the reactor operated effectively, lacking significant dead zones or preferential paths in the bed. These findings are reflective of a good packing quality and system performance. Influence of flow rate The behavior of continuous processes can vary according to flow rate and composition (feed substrates). Depending on the flow rate, there may be an increase in fluid shear stress, causing changes in the structure of the biocatalyst support and resulting in reduced ester conversion [ 21 ]. Thus, this study conducted a preliminary analysis of the feed flow in the synthesis of geranyl acetate via the esterification of geraniol with acetic acid, catalyzed by Novozym 435 in a PBR operating in continuous mode. To date, there are no reports in the literature on the esterification of geranyl acetate using acetic acid and geraniol in a solvent-free medium within a PBR. Syntheses were performed at flow rates of 0.1 and 0.2 mL/min, with space times of 110.95 min and 59.54 min, respectively. Figure 5 shows the geranyl acetate conversions obtained under both conditions. Figure 5 near here It is known that the lower the flow rate, the longer the residence time, which increases the contact time between substrates and enzymes. As a result, the conversion of the product of interest is enhanced. However, as shown in Fig. 5 under both flow rate conditions, a steady state was reached in less than 1 h of reaction, nearly 100% of geranyl acetate production. The yields ranged from 90% to 100% during 24 h of reaction. Therefore, it was found that increasing the flow rate two-fold to 0.2 mL/min did not result in relevant changes to geranyl acetate production. Remonatto et al. [ 4 ] esterified geranyl acetate under the same conditions as in the present study, but used STRs in batch mode. The time required to achieve 99% geranyl acetate conversion was 1 h, using an enzyme load of 5% (w/w) relative to substrates. In the preliminary assay, Novozym 435 achieved esterification activities of 2786.63 ± 150.37 U/g. Enzymes recovered after 24 h of reaction exhibited relative activities of 96.42% and 96.26% under flow rates of 0.1 and 0.2 mL/min, respectively. Thus, there was no considerable loss of immobilized lipase activity following esterification reactions in the PBR. It is expected that with the increase in flow rate, product conversion will decrease. However, given that the evaluated flow rates were low, this effect was not observed. The following experiments were conducted at the lowest flow rate (0.1 mL/min) to minimize reagent use. Another potential strategy for reducing costs would be to decrease the size of the biocatalytic bed, as the amount of enzyme used provided good yields at both flow rates. Geranyl acetate production The operational stability of the biocatalytic bed of PBR in the synthesis of geranyl acetate was analyzed. For this, a 10-day reaction was conducted at a flow rate of 0.1 mL/min. Novozym 435 provided high ester conversions (> 85%) over the 10 days of reaction in the PBR (Fig. 6 ). Despite the acidity of the reaction medium, which could have impaired biocatalyst activity, ester production was high (~ 100%) over the 10 days of esterification. Figure 6 near here Remonatto et al. [ 4 ] investigated the synthesis of geranyl acetate in batch STRs catalyzed by Novozym 435 under the same temperature and molar flow conditions. The authors reported yields higher than 77% over 10 reuse cycles. The high and stable conversions observed in the present study over 10 days of reaction can be attributed to the minimal loss of biocatalyst activity in the PBR, resulting from the laminar flow through the column. This flow regime reduced shear stress on the immobilized enzyme. By contrast, the magnetic agitation inherent to STRs can promote shear-related deactivation, leading to a more rapid decline in enzyme activity. Guajardo et al. [ 56 ] observed an increase in operational stability with the use of a PBR as compared with an STR [ 57 ] in the esterification of glycerol and benzoic acid catalyzed by Novozym 435. Salvi et al. [ 58 ] synthesized geranyl propionate using Novozym 435 and n -heptane as solvent in both PBRs and STRs. A conversion of 94% was achieved in 6 h in the STR, whereas the PBR reached 88% conversion in only 15 min. Here, the results revealed that, despite achieving a slightly lower conversion, PBR outperformed the STR in terms of productivity. This superior performance can be attributed to the greater enzyme stability within PBRs, the absence of shear stress generated by agitation in STRs, and the reduced mass transfer resistance provided by the laminar flow of substrates in the continuous PBR system. The observed phenomena are directly related to the intrinsic characteristics of the reactors rather than to the reactions themselves. In the PBR, laminar flow minimizes shear stress on the biocatalyst, preserving its activity for longer and sustaining high conversion throughout the process. By contrast, in the STR, mechanical agitation can accelerate enzyme deactivation due to the more intense shear forces. Collectively, these differences highlight the potential of PBRs to provide greater biocatalyst stability and improved efficiency in long-term operations. The accumulation of water formed during esterification can reduce lipase activity and consequently decrease ester formation, as excess water favors the reverse reaction—hydrolysis [ 26 ]. To prevent reduced productivity and improve the yield of the desired product, it is essential to maintain water at a minimum concentration. An effective strategy for removing excess water from the system is to use molecular sieves [ 27 , 28 , 59 ]. Molecular sieves have been used by several authors as one of many different approaches for water removal during geraniol ester production, resulting in good product yields. Kanwar et al. [ 18 ] esterified geraniol with butyric acid using hydrogel-immobilized lipase. They achieved a geranyl butyrate yield close to 100% with the addition of 3 Å molecular sieves. For comparison, the reaction was performed without molecular sieves, reaching 98.8% yield after 15 h of reaction at 65°C. Molecular sieves (100 mg/mL) absorbed the water generated during esterification, allowing to achieve 100% conversion in just 12 h of reaction. Trusek-Holownia et al. [ 28 ] also applied molecular sieves in the esterification of geraniol and acetic acid in a membrane bioreactor, resulting in 80% conversion. Here, the efficiency of molecular sieves was assessed in a PBR with alternating layers of Novozym 435 and molecular sieves for geranyl acetate production over 10 days. Both reactions (with and without sieves) achieved similar conversions (about 100% w/w) after 10 days of continuous operation in PBRs. However, the reaction with molecular sieves exhibited a greater variation in conversion over time. This result is likely related to the saturation of molecular sieves and the accumulation of water in their vicinity, which can restrict the passage of substrates, hinder mass transfer, and reduce contact between substrates and enzymes, compromising conversion (Fig. 7 ). Figure 7 near here Figure 7 a shows the PBR system with molecular sieves on the first day of reaction. The nylon support did not have sufficient mechanical strength to sustain the material, which led to the mixing of sieves with enzyme layers. On the fifth day of operation, it was observed liquid accumulation, possibly water, near the sieves (Fig. 7 b), which intensified until the tenth day, causing the rupture of the catalytic bed (Fig. 7 c). To avoid this type of failure, it is recommended to adopt a series reactor system, in which one of the reactors is dedicated exclusively to housing molecular sieves. Overall, the use of sieves was not beneficial in a continuous PBR system for the production of geranyl acetate. Novozym 435 showed an initial esterification activity of 2786.63 ± 150.37 U/g in the system without sieves, reducing to 1928.64 ± 78.90 U/g after 240 h (10 days) of reaction. In other words, enzyme activity decreased by 30.79%. In the PBR system containing molecular sieves, the final enzyme activity was 1793.64 U/g, representing a reduction of 35.64% compared with the initial activity. It is possible that some of the water accumulated near sieves (Fig. 5 ) was absorbed by the enzyme support, resulting in a decrease in esterification activity [ 4 , 45 ]. According to Sose et al. [ 24 ], a minimum quantity of water is essential for maintaining lipase activity. However, excessive water can significantly reduce the effectiveness of the enzyme, particularly in esterification reactions. In such cases, the surplus water shifts the equilibrium toward the reverse reaction (hydrolysis), ultimately impairing the yield of the desired ester. Molecular sieves are commonly used in esterification processes to remove excess water, thereby prolonging the operational stability of biocatalysts, preventing water absorption by the immobilized lipase support, and increasing conversion in direct esterification reactions, as water removal shifts the reaction equilibrium toward ester formation [ 22 ]. In the present study, however, the use of molecular sieves did not provide any measurable benefit, as both systems (with and without sieves) achieved similarly high conversions of geranyl acetate (~ 100%). Therefore, the continuous PBR system without molecular sieves was selected for the synthesis of geranyl acetate catalyzed by Novozym 435. Remonatto et al. [ 4 ], in studying the production of geranyl acetate in a batch STR with and without molecular sieves, observed no significant differences in geranyl acetate conversion between systems. The authors argued that the reaction conditions and the fact that substrates were previously dried with molecular sieves contributed to the low water levels. Another observation was that the addition of molecular sieves might have limited mass transfer, reducing the contact between enzymes and substrates. This limitation occurs because molecular sieves can restrict the movement of reactants in the medium, which ultimately minimizes ester conversion. Therefore, although molecular sieves are useful to adsorb water and promote esterification balance, their use can increase process costs. Similar results were obtained by Sbardelotto et al. [ 35 ], who used molecular sieves to remove water during the synthesis of geranyl butanoate. However, they observed that, under the specific conditions of their experiment, the presence or absence of molecular sieves did not have a significant impact on the results. Other parameters seemed to have a predominant influence, such as the molar ratio of reagents. Reagent excess favored the displacement of the chemical equilibrium toward geranyl butanoate formation. Here, excess geraniol was used to shift the equilibrium toward geranyl acetate production. Several studies have shown that the partial removal of water produced during esterification leads to a significant increase in conversion [ 25 , 26 , 47 , 60 – 62 ]. Most of these investigations used reactors arranged in series, typically with the first and third reactors packed with immobilized enzyme and the second filled with molecular sieves, allowing for a greater quantity of adsorbent to be employed. In the present system, however, the inclusion of molecular sieves did not provide any observable advantage. Therefore, the most suitable configuration for the continuous synthesis of geranyl acetate in a PBR is one that does not require molecular sieves. Eliminating the use of this material can offer an economic benefit, reducing operational costs. Conclusions Geranyl acetate was successfully produced in a PBR. A near 100% conversion was achieved under the two flow rates tested (0.1 and 0.2 mL/min). Likewise, systems operated with and without molecular sieves achieved conversions close to 100%. Thus, the presence of molecular sieves did not benefit the reaction. On the basis of the findings, the preferred approach for the continuous synthesis of geranyl acetate catalyzed by Novozym 435 in a PBR is to apply a flow rate of 0.1 mL/min and not use molecular sieves. This configuration not only simplifies the process and reduces costs associated with adsorbent materials but also maintains high conversion throughout the reaction by preserving enzyme activity. These results have important implications for the development of industrial direct esterification processes employing biocatalysts in continuous systems. Abbreviations PBR Packed bed reactor STR Stirred tank reactor Declarations Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Ethical Approval This article does not contain any studies with human participants or animals. Consent to Participate All authors agree mutually with the participation and publication of this work and declare that this is original research. Consent to Publish Not applicable. Funding This study was funded by the São Paulo Research Foundation (FAPESP, grants Nos. 2020/09592-1, 2024/07997-5 and 2025/00723-0) and the Brazilian National Council for Scientific and Technological Development (CNPq) for the financial support (grant No. 304399/2022-1). Author Contribution **D. G. O.:** Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Visualization, Writing – original draft. **D. R.:** Formal analysis, Methodology, Visualization, Validation, Writing – original draft. **I. M. O.:** Methodology, Data curation, Formal analysis, Writing – original draft. **B. W. P.:** Methodology, Data curation, Formal analysis, Writing – original draft**. J. V. O.:** Investigation, Visualization, Funding acquisition, Writing – review & editing **P. V. A**.**:** Methodology, Data curation, Formal analysis, Writing – original draft. **M. O. C.:** Investigation, Visualization, Funding acquisition, Writing – review & editing. **A. V. P.:** Conceptualization, Funding acquisition, Project administration, Supervision, Writing – review & editing. Acknowledgement We thank the Department of Bioprocess Engineering and Biotechnology of the School of Pharmaceutical Sciences, São Paulo State University, Brazil. We are grateful for the financial support provided by the Brazilian National Council for Scientific and Technological Development (CNPq, grant No. 304399/2022-1), the São Paulo State Research Foundation (FAPESP, grants Nos. 2020/09592-1, 2024/07997-5 and 2025/00723-0), and the Brazilian Federal Agency for Support and Evaluation of Graduate Education (CAPES). Data Availability Data that support the findings of this study are available from the corresponding author upon request. References Mączka W, Wińska K, Grabarczyk M (2020) One Hundred Faces of Geraniol. Molecules 25:3303. https://doi.org/10.3390/molecules25143303 dos Silva G S e, Marques JN, de Linhares J et al (2022) EPM, Review of anticancer activity of monoterpenoids: Geraniol, nerol, geranial and neral. 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06:44:56","extension":"png","order_by":23,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":33424,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineImagem7png.png","url":"https://assets-eu.researchsquare.com/files/rs-8329923/v1/2e9e8cfd9ee8472a9a7f47af.png"},{"id":99341958,"identity":"850b1f87-019b-40be-8246-1a095e9bc00b","added_by":"auto","created_at":"2026-01-01 06:44:56","extension":"xml","order_by":24,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":141705,"visible":true,"origin":"","legend":"","description":"","filename":"5637eed077f0403d92c47df16c540e4b1structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-8329923/v1/06e0987e69243eb93fe47da0.xml"},{"id":99341955,"identity":"dce0f410-079e-4100-9354-51a480af3b31","added_by":"auto","created_at":"2026-01-01 06:44:56","extension":"html","order_by":25,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":155335,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8329923/v1/a9837705e9b4113d558c4970.html"},{"id":99341926,"identity":"1e4a67cc-2be4-47a9-9416-bf7160f5b425","added_by":"auto","created_at":"2026-01-01 06:44:55","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2460751,"visible":true,"origin":"","legend":"\u003cp\u003ePacked bed column reactor in (A) front view and (B) top view\u003c/p\u003e","description":"","filename":"Imagem1.png","url":"https://assets-eu.researchsquare.com/files/rs-8329923/v1/67e6750dd6a974bdead71c88.png"},{"id":99341929,"identity":"3d7189a5-eb0c-4dd0-9080-0b4d9fe5a168","added_by":"auto","created_at":"2026-01-01 06:44:55","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2027162,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic of the experimental setup: (1) substrate reservoir, (2) magnetic stirrer, (3) peristaltic pump, (4) packed bed reactors, (5) product reservoir, and (6) thermostatic bath\u003c/p\u003e","description":"","filename":"Imagem2.png","url":"https://assets-eu.researchsquare.com/files/rs-8329923/v1/99139da720fc20215e17b8d4.png"},{"id":99789004,"identity":"8347dbad-bd9d-49d2-a4ab-1eb2bf6b9310","added_by":"auto","created_at":"2026-01-08 12:48:31","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1654415,"visible":true,"origin":"","legend":"\u003cp\u003ePacked bed column reactor in cutaway front view\u003c/p\u003e","description":"","filename":"Imagem3.png","url":"https://assets-eu.researchsquare.com/files/rs-8329923/v1/2061aac4e1cbd864e388114e.png"},{"id":99341927,"identity":"15678a90-f3b2-4d61-b09b-037cc24f0645","added_by":"auto","created_at":"2026-01-01 06:44:55","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":67651,"visible":true,"origin":"","legend":"\u003cp\u003eDistribution of residence time (\u003cem\u003et\u003c/em\u003e\u003csub\u003eE(\u003c/sub\u003e\u003csub\u003e\u003cem\u003et\u003c/em\u003e\u003c/sub\u003e\u003csub\u003e)\u003c/sub\u003e) as a function of time for a tracer test performed in a fixed bed reactor using the commercial immobilized lipase Novozym 435\u003c/p\u003e","description":"","filename":"Imagem4.png","url":"https://assets-eu.researchsquare.com/files/rs-8329923/v1/e024ff3d5e3a6a7b1c25b831.png"},{"id":99341931,"identity":"7fa421b6-e6b8-4adc-9be2-869dae185469","added_by":"auto","created_at":"2026-01-01 06:44:55","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1321980,"visible":true,"origin":"","legend":"\u003cp\u003eGeranyl acetate conversions* in a packed bed reactor (PBR) operated in continuous mode at flow rates of 0.1 and 0.2 mL/min using Novozym 435 lipase and geraniol/acetic acid as substrate at a 3:1 molar ratio and 60 °C for 24 h\u003c/p\u003e\n\u003cp\u003e*The responses represent the maximum ester conversion (% w/w) under assay conditions (33.4%), given the limitations imposed by the 3:1 geraniol/acetic acid molar ratio\u003c/p\u003e","description":"","filename":"Imagem5.png","url":"https://assets-eu.researchsquare.com/files/rs-8329923/v1/74d5a6a58933d47c44059ef3.png"},{"id":99788925,"identity":"6b49216e-2266-4c11-8f01-144fe42f6013","added_by":"auto","created_at":"2026-01-08 12:48:19","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1464300,"visible":true,"origin":"","legend":"\u003cp\u003eGeranyl acetate conversions in a packed bed reactor (PBR) operated in continuous mode with and without molecular sieves using Novozym 435 lipase and geraniol/acetic acid as substrate at a 3:1 molar ratio and 60 °C for 10 days\u003c/p\u003e","description":"","filename":"Imagem6.png","url":"https://assets-eu.researchsquare.com/files/rs-8329923/v1/472ca9296300b1cf6081b24b.png"},{"id":99341934,"identity":"1379bb30-698c-4d9c-a6ee-27a214d44e46","added_by":"auto","created_at":"2026-01-01 06:44:55","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1110750,"visible":true,"origin":"","legend":"\u003cp\u003eReaction system used for geranyl acetate production in a packed bed reactor (PBR) operated in continuous mode with and without molecular sieves after (A) 1, (B) 5, and (c) 10 days\u003c/p\u003e","description":"","filename":"Imagem7png.png","url":"https://assets-eu.researchsquare.com/files/rs-8329923/v1/79b343ea343e68f0952f51df.png"},{"id":100621776,"identity":"345240a5-eae3-4512-aa5d-baddbc3f5b8e","added_by":"auto","created_at":"2026-01-19 18:35:10","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":10230048,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8329923/v1/0b898023-a0e8-4417-b7a7-af76a6868caa.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Assessing the Role of Molecular Sieves in Continuous Enzymatic Synthesis of Geranyl Acetate","fulltext":[{"header":"Introduction","content":"\u003cp\u003eConsumers are increasingly seeking natural ingredients as alternatives to synthetic additives. This preference stems from both the pursuit of a healthier lifestyle and the growing concern for environmental preservation. Some of the natural products that most attract consumer attention are essential oils and other plant-derived compounds. Geraniol (3,7-dimethylocta-\u003cem\u003etrans\u003c/em\u003e-2,6-dien-1-ol) is an acyclic monoterpene alcohol found in the essential oils of plants such as geranium (\u003cem\u003eGeranium\u003c/em\u003e sp. L.), citronella (\u003cem\u003eCymbopogon winterianus\u003c/em\u003e Jowitt), lemongrass (\u003cem\u003eCymbopogon citratus\u003c/em\u003e (DC.) Stapf), and rose (\u003cem\u003eRosa \u0026times; damascena\u003c/em\u003e Mill.) [\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. This terpene alcohol, along with its isomer nerol, exhibits notable biological activity and is frequently noted in the literature for its antimicrobial, acaricidal, pesticidal, anti-inflammatory, and chemotherapeutic properties [\u003cspan additionalcitationids=\"CR2 CR3 CR4 CR5\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. However, its use in pharmaceutical and cosmetic products is limited by a certain level of toxicity, attributed to the presence of an aldehyde group that can react with amino residues in proteins and trigger undesirable effects [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eA viable alternative to using this monoterpene alcohol is to convert it into derivatives, such as geranyl acetate (3,7-dimethylocta-2,6-dien-1-yl ethanoate). This geraniol ester is characterized by low toxicity and advantageous organoleptic properties, including a pleasant flavor and aroma, which make it a valuable ingredient in food, fragrance, and cosmetic products [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. It is noteworthy that geranyl acetate has been approved for use in food products by the U.S. Food and Drug Administration (FDA) and is recognized as safe by the Flavor and Extract Manufacturers Association (FEMA) [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eGeranyl acetate is commonly obtained by fractionating essential oils. However, this process requires large quantities of raw materials, representing an impractical and expensive route [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Another widely used method is conventional chemical synthesis, which also has several disadvantages, including harsh reaction conditions (e.g., high pH and temperature) and high costs associated with product purification [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. A third route to ester production is biocatalysis. This alternative method offers several advantages over chemical methods, including greater chemoselectivity, enantioselectivity, and environmental friendliness [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Enzymatic catalysis using lipases (triacylglycerol acyl hydrolases, EC 3.1.1.3) is a promising alternative for the synthesis of geraniol esters. Although these enzymes can efficiently catalyze esterification, transesterification, amination, acidolysis, and hydrolysis reactions [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan additionalcitationids=\"CR14\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], some drawbacks impair their use in their free form, such as low stability, loss of activity during storage, and difficulty in reuse [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. These limitations can be circumvented by immobilizing lipases. Accordingly, immobilized lipases represent an attractive strategy to reduce process costs, as immobilization enhances enzyme stability and facilitates biocatalyst reuse [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe main reactions that use immobilized lipases to produce geraniol esters are esterification (between an alcohol and a carboxylic acid) and transesterification (between an ester and an alcohol) [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan additionalcitationids=\"CR19 CR20\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. In the current study, geranyl acetate was synthesized via direct esterification catalyzed by the immobilized enzyme Novozym 435. Enzymatic esterification is a thermodynamically controlled process; that is, it is governed by chemical equilibrium [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. In these processes, biocatalysts are often susceptible to inactivation due to the high concentration of acid in the reaction medium, a problem typically mitigated by using an organic solvent or an excess of alcohol [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/p\u003e \u003cp\u003eAnother important aspect of enzymatic esterification is that, at the molecular level, a minimal amount of water is essential to maintain lipase activity. On the other hand, excess water can significantly reduce enzyme effectiveness by promoting unwanted side reactions, such as hydrolysis, thereby reducing esterification yield [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. One strategy to reduce this problem is to use molecular sieves, which remove the water produced during the reaction [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan additionalcitationids=\"CR26\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Kanwar et al. [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e] reported that, during the esterification of geraniol with butyric acid using hydrogel-immobilized lipase, the addition of 3 \u0026Aring; molecular sieves resulted in geranyl butyrate yields close to 100%. Trusek-Holownia et al. [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], in studying the esterification of geraniol with acetic acid in a membrane bioreactor, achieved 80% conversion to geranyl acetate by using a column packed with molecular sieves for water removal.\u003c/p\u003e \u003cp\u003eThe esterification process can be carried out either in the presence or absence of solvents [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan additionalcitationids=\"CR30 CR31\" citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. The use of solvents can increase substrate solubility, reduce enzyme inhibition, and control water activity by preventing ester hydrolysis. However, it also increases process costs and requires an additional removal step at the end of the reaction [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Solvent-free esterification offers several economic and environmental advantages. The absence of solvents reduces the need for purification steps and minimizes waste generation, rendering the process more sustainable and cost-effective [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Nevertheless, solvent-free methods also present challenges, given the effect of the reagent molar ratio on lipase activity and its influence on the reaction equilibrium [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. A useful method for mitigating these problems is the use of an excess of one of the substrates. Remonatto et al. [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e] observed that using geraniol in excess resulted in high conversion to geranyl acetate by shifting the equilibrium toward product formation and preventing biocatalyst denaturation caused by the presence of acid. Nascimento et al. [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e] likewise used excess geraniol to produce geranyl cinnamate in a solvent-free system, achieving optimal conversion with a molar ratio of 1:5.68 (cinnamic acid/geraniol).\u003c/p\u003e \u003cp\u003eThe synthesis of geraniol esters is commonly carried out in two types of bioreactors: packed bed reactors (PBRs) and stirred tank reactors (STRs) [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan additionalcitationids=\"CR35\" citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. The choice between these systems depends on the nature of the process [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. PBRs are predominantly used in continuous operations [\u003cspan additionalcitationids=\"CR39 CR40\" citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. By contrast, STRs play a central role in batch processes, given their ease of control and compatibility with a wide range of operating conditions [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. It should be noted that STRs also have significant disadvantages. These include the potential for high shear stress under reaction conditions, the need for dedicated cleaning time after operation, and substantial energy consumption [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. On the other hand, PBRs are widely used for syntheses catalyzed by immobilized enzymes due to their operational simplicity, continuous production, high efficiency, ease of product separation, and effective enzyme recovery at the end of the process [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. These characteristics play a fundamental role in reducing the overall costs of the process.\u003c/p\u003e \u003cp\u003eIn light of these considerations, this study aimed to synthesize geranyl acetate through solvent-free direct esterification of geraniol with acetic acid catalyzed by the immobilized enzyme Novozym 435 in a continuous PBR system. A secondary aim was to assess the influence of incorporating molecular sieves for water removal on process performance.\u003c/p\u003e"},{"header":"Material and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eMaterials\u003c/h2\u003e \u003cp\u003eThe substrates used in esterification reactions were acetic acid (99.8%, Neon, Susano, SP, Brazil) and geraniol (97%, Sigma\u0026ndash;Aldrich, St. Louis, MO, USA). Before use, both reagents were dried overnight in the presence of an excess of molecular sieves (250 g/L). Immobilized \u003cem\u003eCandida antarctica\u003c/em\u003e lipase B (Novozym 435) was kindly provided by Novozymes\u0026reg; (Arauc\u0026aacute;ria, PR, Brazil). The molecular sieves used were 4 \u0026Aring; (8\u0026ndash;12 mesh, Sigma\u0026ndash;Aldrich, St. Louis, USA). Oleic acid (Synth, Diadema, SP, Brazil) and ethanol (99.5%, Synth, Diadema, SP, Brazil) were used to determine esterification activity.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eMethods\u003c/h3\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eHydrodynamic characterization of the PBR\u003c/h2\u003e \u003cp\u003eTracer pulse experiments were conducted to characterize the hydrodynamics of the PBR. The residence time distribution was determined experimentally by injecting a geraniol solution containing a purple dye (Roxo Saramanil\u0026reg;, S\u0026atilde;o Paulo, Brazil, purchased locally) into the reactor and measuring the tracer concentration at the outlet over time using a UV-Vis spectrophotometer (Genesys 10S, Thermo Fisher Scientific, San Jose, CA, USA) at 530 nm. The residence time distribution function, \u003cem\u003eE\u003c/em\u003e(\u003cem\u003et\u003c/em\u003e), was calculated using Eq.\u0026nbsp;(\u003cspan refid=\"Equ1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e].\u003cdiv id=\"Equ1\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ1\" name=\"EquationSource\"\u003e\n$$\\:\\text{E}\\text{(}\\text{t}\\text{):=}\\frac{\\text{C}\\text{(}\\text{t}\\text{)}}{{\\int\\:}_{\\text{0}}^{\\text{\u0026infin;}}\\text{C}\\text{(}\\text{t}\\text{)d}\\text{t}}$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e1\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003ewhere \u003cem\u003eC\u003c/em\u003e(\u003cem\u003et\u003c/em\u003e) is the tracer concentration at time (\u003cem\u003et\u003c/em\u003e).\u003c/p\u003e \u003cp\u003eBased on the residence time distribution function, the mean residence time (\u003cem\u003et\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e) was calculated according to Eq.\u0026nbsp;(\u003cspan refid=\"Equ2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]:\u003cdiv id=\"Equ2\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ2\" name=\"EquationSource\"\u003e\n$$\\:{\\text{t}}_{\\text{m}}\\text{=}{\\int\\:}_{\\text{0}}^{\\text{\u0026infin;}}\\text{tE}\\text{(}\\text{t}\\text{)d}\\text{t}$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e2\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003eThe integration in Eq.\u0026nbsp;(\u003cspan refid=\"Equ2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) was performed using Origin software version 8.0 (Origin Lab Corporation, Washington, USA).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eGeranyl acetate production\u003c/h3\u003e\n\u003cp\u003eGeranyl acetate was synthesized via Novozym 435-catalyzed esterification of geraniol with acetic acid at a 3:1 molar ratio in a column PBR (5 cm height \u0026times; 2 cm diameter; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The reactor was packed with approximately 1.51 g of catalyst and operated in continuous mode. Substrates were pumped at a flow rate of 1 mL/min, corresponding to a space time of 1.85 h, as calculated using Eq.\u0026nbsp;(\u003cspan refid=\"Equ3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). A water bath (Marconi, model MA 184/6, Piracicaba, SP, Brazil) was connected to the feed circulation system to maintain the reaction mixture at 60\u0026deg;C (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). In a previous study, Remonatto et al. [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e] determined the optimal synthesis conditions (molar ratio and temperature) for batch STRs.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv id=\"Equ3\" class=\"Equation\"\u003e \u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ3\" name=\"EquationSource\"\u003e\n$$\\:\\text{\u0026tau;}\\text{}\\text{=}\\frac{{V}_{\\text{u}}}{{Q}_{\\text{t}}}$$\u003c/div\u003e \u003cdiv class=\"EquationNumber\"\u003e3\u003c/div\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003ewhere \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{Q}_{\\text{t}}\\)\u003c/span\u003e\u003c/span\u003e is the flow rate of the tracer, \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{V}_{\\text{u}}\\)\u003c/span\u003e\u003c/span\u003e is the useful volume of the reactor, and \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\text{\u0026tau;}\\)\u003c/span\u003e\u003c/span\u003e is the space time.\u003c/p\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e near here\u003c/p\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e near here\u003c/p\u003e\n\u003ch3\u003eGeranyl acetate production in a PBR with molecular sieves\u003c/h3\u003e\n\u003cp\u003eMolecular sieves were used to remove the water generated during esterification. For this purpose, the reactor was packed with four alternating layers separated by nylon mesh: the first and third layers contained 0.75 g of immobilized lipase (Novozym 435) each, whereas the second and fourth layers contained 0.75 g of molecular sieves (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The same proportion was used by Freitas et. al [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e] in a similar system and by Vadgama et al. [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e] in a series of PBRs, where the first and third reactors contained 10 g of enzyme and the second reactor contained 20 g of molecular sieves. The synthesis was carried out following the parameters described in the previous section.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e near here\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003ePurification of geranyl acetate\u003c/h2\u003e \u003cp\u003eCrude geranyl acetate was purified by column chromatography (50 \u0026times; 1 cm). Silica gel (Neon, Suzano, SP, Brazil) was used as stationary phase. The mobile phase consisted of ethyl acetate and cyclohexane (1:14 v/v) acidified with 2% (v/v) acetic acid. Initially, 1 mL of sample (reaction mixture after esterification) was applied to the top of the column, which was then eluted at a constant rate to separate the components. Aliquots were collected at the column outlet, transferred into 2 mL vials, and analyzed using thin-layer chromatography (Sigma\u0026ndash;Aldrich, Brazil). Geranyl acetate was quantified by comparison with a standard solution. Aliquots showing high conversion were further analyzed for purity using gas chromatography.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eQuantification of geranyl acetate by gas chromatography\u003c/h3\u003e\n\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eInitially, samples were neutralized using a hydroalcoholic solution (30% v/v ethanol) in 0.8 M potassium hydroxide [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. Geranyl acetate was quantified by gas chromatography on a Shimadzu GC-2010 chromatograph equipped with an auto-injector (AOC-20i), a fused silica capillary column (SH-Stabilwax-DA, 30 m length \u0026times; 0.25 mm internal diameter \u0026times; 0.25 \u0026micro;m film thickness), and a flame ionization detector. The temperature program was as follows: 60\u0026ndash;210\u0026deg;C at 10\u0026deg;C/min, 210\u0026ndash;250\u0026deg;C at 20\u0026deg;C/min, and 250\u0026deg;C for 1 min. Injector and detector temperatures were maintained at 250\u0026deg;C. Samples were diluted in hexane to a final volume of 1 \u0026micro;L. Injection was performed in split mode (1:100 ratio). Nitrogen (N\u003csub\u003e2\u003c/sub\u003e) was used as carrier gas at 56 kPa. Geranyl acetate conversion was determined by constructing an analytical curve (Eq.\u0026nbsp;4) using purified geranyl acetate (item 3.2.2) and methyl nonadecanoate (C19) (Sigma\u0026ndash;Aldrich) as internal standard [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cspan class=\"InlineEquation\"\u003e \u003cspan class=\"mathinline\"\u003e\\(\\:y=1.6428x+0.0066\\)\u003c/span\u003e \u003c/span\u003e (\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{R}^{2}=0.998\\)\u003c/span\u003e\u003c/span\u003e) (4)\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003ewhere \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:y\\)\u003c/span\u003e\u003c/span\u003e is the geranyl acetate conversion (w/w) and \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:x\\)\u003c/span\u003e\u003c/span\u003e is the ratio of the geranyl acetate peak area at the end of the reaction to the area of the internal standard C19.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\n\u003ch3\u003eEsterification activity\u003c/h3\u003e\n\u003cp\u003eThe esterification activity (U/g) of the biocatalyst before and after the reactions was determined as described elsewhere [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. For this, oleic acid was esterified with ethanol at a molar ratio of 1:1 in the presence of 0.2 g of Novozym 435 at 40\u0026deg;C and 150 rpm. Aliquots (300 \u0026micro;L) were collected at 0, 10, 20, 30, 40, and 60 min of reaction. The consumption of oleic acid was determined by titration with 0.05 M sodium hydroxide. One unit of enzyme activity (U) is defined as the amount of enzyme required to consume 1 \u0026micro;mol of oleic acid per minute.\u003c/p\u003e"},{"header":"Results and discussion","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eHydrodynamic characterization of the PBR\u003c/h2\u003e \u003cp\u003eHydrodynamic analysis of PBRs is essential for identifying preferential paths, which are regions where the fluid flows predominantly due to irregularities in bed packing or local variations in fluidity, leading to nonuniform flow [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. Such paths may result from uneven particle distribution, inadequate bed compaction, accumulation of fine particles, or operational issues. Although preferential paths may form anywhere within the bed, they are most common near the reactor walls, in low-resistance zones, and around fluid inlet and outlet ports. These deviations decrease reaction efficiency and compromise overall reactor performance [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. Therefore, understanding and controlling preferential paths is critical for the efficient and safe operation of PBRs.\u003c/p\u003e \u003cp\u003eAn effective approach for analyzing fluid flow patterns in PBRs is the study of mean residence time. This parameter describes the flow behavior within the reactor and provides valuable information on bed performance, including the presence of stagnation zones [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. Given that the flow regime directly influences reactor efficiency, its evaluation offers opportunities to improve process control [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. Consequently, mean residence time is one of the most important parameters for achieving high product conversion in PBRs. In this study, a tracer pulse assay was conducted to experimentally determine the mean residence time of reactants and characterize the behavior of the reaction mixture within the bed.\u003c/p\u003e \u003cp\u003eThe mean residence time of reagents in the PBR was determined experimentally using a purple dye as tracer. The calibration curve was constructed for quantification (\u003cem\u003eA\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0396\u003cem\u003eC\u003c/em\u003e, where \u003cem\u003eC\u003c/em\u003e is the concentration of the tracer). The coefficient of determination (\u003cem\u003eR\u003c/em\u003e\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.9998) indicated a good fit of the model to experimental data. The results were used to construct the distribution curve of residence time (\u003cem\u003eE\u003c/em\u003e) as a function of time (\u003cem\u003et\u003c/em\u003e), according to Eq.\u0026nbsp;(\u003cspan refid=\"Equ2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e near here\u003c/p\u003e \u003cp\u003eFor a biocatalyst volume of 1.81 mL, a useful volume of 9.5 mL, and a flow rate of 0.1 mL/min, the space time was 110.95 min, according to Eq.\u0026nbsp;(\u003cspan refid=\"Equ3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The average residence time, as calculated by Eq.\u0026nbsp;(\u003cspan refid=\"Equ2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), was 167.61 min. This difference between theoretical and experimental values suggests the existence of preferential paths in the bed. According to Fogler [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e], when the residence time is greater than the space time, there are dead volumes in the reactor, that is, areas with fluid stagnation.\u003c/p\u003e \u003cp\u003eCozentino et al. [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e] studied the synthesis of MLM-type triacylglycerols from grape seed oil in associated PBRs. The mean experimental residence time obtained was 335.17 min, whereas the theoretical space time was 415.58 min. This 19.35% difference between theoretical and experimental values was considered small, indicating that the reactor had a satisfactory performance, without significant deviations in flow and with good distribution of biocatalyst particles.\u003c/p\u003e \u003cp\u003eDe Paula et al. [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e] evaluated the enzymatic interesterification of milk fat with soybean oil in a PBR using immobilized lipase from \u003cem\u003eRhizopus oryzae\u003c/em\u003e. The mean residence time was 135 min, which was 8.74% higher than the theoretical space time. This difference was considered acceptable, indicating that the reactor operated effectively, lacking significant dead zones or preferential paths in the bed. These findings are reflective of a good packing quality and system performance.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eInfluence of flow rate\u003c/h2\u003e \u003cp\u003eThe behavior of continuous processes can vary according to flow rate and composition (feed substrates). Depending on the flow rate, there may be an increase in fluid shear stress, causing changes in the structure of the biocatalyst support and resulting in reduced ester conversion [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Thus, this study conducted a preliminary analysis of the feed flow in the synthesis of geranyl acetate via the esterification of geraniol with acetic acid, catalyzed by Novozym 435 in a PBR operating in continuous mode. To date, there are no reports in the literature on the esterification of geranyl acetate using acetic acid and geraniol in a solvent-free medium within a PBR. Syntheses were performed at flow rates of 0.1 and 0.2 mL/min, with space times of 110.95 min and 59.54 min, respectively. Figure\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e shows the geranyl acetate conversions obtained under both conditions.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e near here\u003c/p\u003e \u003cp\u003eIt is known that the lower the flow rate, the longer the residence time, which increases the contact time between substrates and enzymes. As a result, the conversion of the product of interest is enhanced. However, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e under both flow rate conditions, a steady state was reached in less than 1 h of reaction, nearly 100% of geranyl acetate production. The yields ranged from 90% to 100% during 24 h of reaction. Therefore, it was found that increasing the flow rate two-fold to 0.2 mL/min did not result in relevant changes to geranyl acetate production. Remonatto et al. [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e] esterified geranyl acetate under the same conditions as in the present study, but used STRs in batch mode. The time required to achieve 99% geranyl acetate conversion was 1 h, using an enzyme load of 5% (w/w) relative to substrates.\u003c/p\u003e \u003cp\u003eIn the preliminary assay, Novozym 435 achieved esterification activities of 2786.63\u0026thinsp;\u0026plusmn;\u0026thinsp;150.37 U/g. Enzymes recovered after 24 h of reaction exhibited relative activities of 96.42% and 96.26% under flow rates of 0.1 and 0.2 mL/min, respectively. Thus, there was no considerable loss of immobilized lipase activity following esterification reactions in the PBR.\u003c/p\u003e \u003cp\u003eIt is expected that with the increase in flow rate, product conversion will decrease. However, given that the evaluated flow rates were low, this effect was not observed. The following experiments were conducted at the lowest flow rate (0.1 mL/min) to minimize reagent use. Another potential strategy for reducing costs would be to decrease the size of the biocatalytic bed, as the amount of enzyme used provided good yields at both flow rates.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eGeranyl acetate production\u003c/h2\u003e \u003cp\u003eThe operational stability of the biocatalytic bed of PBR in the synthesis of geranyl acetate was analyzed. For this, a 10-day reaction was conducted at a flow rate of 0.1 mL/min. Novozym 435 provided high ester conversions (\u0026gt;\u0026thinsp;85%) over the 10 days of reaction in the PBR (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Despite the acidity of the reaction medium, which could have impaired biocatalyst activity, ester production was high (~\u0026thinsp;100%) over the 10 days of esterification.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e near here\u003c/p\u003e \u003cp\u003eRemonatto et al. [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e] investigated the synthesis of geranyl acetate in batch STRs catalyzed by Novozym 435 under the same temperature and molar flow conditions. The authors reported yields higher than 77% over 10 reuse cycles. The high and stable conversions observed in the present study over 10 days of reaction can be attributed to the minimal loss of biocatalyst activity in the PBR, resulting from the laminar flow through the column. This flow regime reduced shear stress on the immobilized enzyme. By contrast, the magnetic agitation inherent to STRs can promote shear-related deactivation, leading to a more rapid decline in enzyme activity. Guajardo et al. [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e] observed an increase in operational stability with the use of a PBR as compared with an STR [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e] in the esterification of glycerol and benzoic acid catalyzed by Novozym 435.\u003c/p\u003e \u003cp\u003eSalvi et al. [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e] synthesized geranyl propionate using Novozym 435 and \u003cem\u003en\u003c/em\u003e-heptane as solvent in both PBRs and STRs. A conversion of 94% was achieved in 6 h in the STR, whereas the PBR reached 88% conversion in only 15 min. Here, the results revealed that, despite achieving a slightly lower conversion, PBR outperformed the STR in terms of productivity. This superior performance can be attributed to the greater enzyme stability within PBRs, the absence of shear stress generated by agitation in STRs, and the reduced mass transfer resistance provided by the laminar flow of substrates in the continuous PBR system.\u003c/p\u003e \u003cp\u003eThe observed phenomena are directly related to the intrinsic characteristics of the reactors rather than to the reactions themselves. In the PBR, laminar flow minimizes shear stress on the biocatalyst, preserving its activity for longer and sustaining high conversion throughout the process. By contrast, in the STR, mechanical agitation can accelerate enzyme deactivation due to the more intense shear forces. Collectively, these differences highlight the potential of PBRs to provide greater biocatalyst stability and improved efficiency in long-term operations.\u003c/p\u003e \u003cp\u003eThe accumulation of water formed during esterification can reduce lipase activity and consequently decrease ester formation, as excess water favors the reverse reaction\u0026mdash;hydrolysis [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. To prevent reduced productivity and improve the yield of the desired product, it is essential to maintain water at a minimum concentration. An effective strategy for removing excess water from the system is to use molecular sieves [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]. Molecular sieves have been used by several authors as one of many different approaches for water removal during geraniol ester production, resulting in good product yields. Kanwar et al. [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e] esterified geraniol with butyric acid using hydrogel-immobilized lipase. They achieved a geranyl butyrate yield close to 100% with the addition of 3 \u0026Aring; molecular sieves. For comparison, the reaction was performed without molecular sieves, reaching 98.8% yield after 15 h of reaction at 65\u0026deg;C. Molecular sieves (100 mg/mL) absorbed the water generated during esterification, allowing to achieve 100% conversion in just 12 h of reaction. Trusek-Holownia et al. [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e] also applied molecular sieves in the esterification of geraniol and acetic acid in a membrane bioreactor, resulting in 80% conversion. Here, the efficiency of molecular sieves was assessed in a PBR with alternating layers of Novozym 435 and molecular sieves for geranyl acetate production over 10 days.\u003c/p\u003e \u003cp\u003eBoth reactions (with and without sieves) achieved similar conversions (about 100% w/w) after 10 days of continuous operation in PBRs. However, the reaction with molecular sieves exhibited a greater variation in conversion over time. This result is likely related to the saturation of molecular sieves and the accumulation of water in their vicinity, which can restrict the passage of substrates, hinder mass transfer, and reduce contact between substrates and enzymes, compromising conversion (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e near here\u003c/p\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea shows the PBR system with molecular sieves on the first day of reaction. The nylon support did not have sufficient mechanical strength to sustain the material, which led to the mixing of sieves with enzyme layers. On the fifth day of operation, it was observed liquid accumulation, possibly water, near the sieves (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eb), which intensified until the tenth day, causing the rupture of the catalytic bed (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ec). To avoid this type of failure, it is recommended to adopt a series reactor system, in which one of the reactors is dedicated exclusively to housing molecular sieves. Overall, the use of sieves was not beneficial in a continuous PBR system for the production of geranyl acetate.\u003c/p\u003e \u003cp\u003eNovozym 435 showed an initial esterification activity of 2786.63\u0026thinsp;\u0026plusmn;\u0026thinsp;150.37 U/g in the system without sieves, reducing to 1928.64\u0026thinsp;\u0026plusmn;\u0026thinsp;78.90 U/g after 240 h (10 days) of reaction. In other words, enzyme activity decreased by 30.79%. In the PBR system containing molecular sieves, the final enzyme activity was 1793.64 U/g, representing a reduction of 35.64% compared with the initial activity. It is possible that some of the water accumulated near sieves (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e) was absorbed by the enzyme support, resulting in a decrease in esterification activity [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAccording to Sose et al. [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], a minimum quantity of water is essential for maintaining lipase activity. However, excessive water can significantly reduce the effectiveness of the enzyme, particularly in esterification reactions. In such cases, the surplus water shifts the equilibrium toward the reverse reaction (hydrolysis), ultimately impairing the yield of the desired ester. Molecular sieves are commonly used in esterification processes to remove excess water, thereby prolonging the operational stability of biocatalysts, preventing water absorption by the immobilized lipase support, and increasing conversion in direct esterification reactions, as water removal shifts the reaction equilibrium toward ester formation [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. In the present study, however, the use of molecular sieves did not provide any measurable benefit, as both systems (with and without sieves) achieved similarly high conversions of geranyl acetate (~\u0026thinsp;100%). Therefore, the continuous PBR system without molecular sieves was selected for the synthesis of geranyl acetate catalyzed by Novozym 435.\u003c/p\u003e \u003cp\u003eRemonatto et al. [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], in studying the production of geranyl acetate in a batch STR with and without molecular sieves, observed no significant differences in geranyl acetate conversion between systems. The authors argued that the reaction conditions and the fact that substrates were previously dried with molecular sieves contributed to the low water levels. Another observation was that the addition of molecular sieves might have limited mass transfer, reducing the contact between enzymes and substrates. This limitation occurs because molecular sieves can restrict the movement of reactants in the medium, which ultimately minimizes ester conversion. Therefore, although molecular sieves are useful to adsorb water and promote esterification balance, their use can increase process costs.\u003c/p\u003e \u003cp\u003eSimilar results were obtained by Sbardelotto et al. [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e], who used molecular sieves to remove water during the synthesis of geranyl butanoate. However, they observed that, under the specific conditions of their experiment, the presence or absence of molecular sieves did not have a significant impact on the results. Other parameters seemed to have a predominant influence, such as the molar ratio of reagents. Reagent excess favored the displacement of the chemical equilibrium toward geranyl butanoate formation. Here, excess geraniol was used to shift the equilibrium toward geranyl acetate production.\u003c/p\u003e \u003cp\u003eSeveral studies have shown that the partial removal of water produced during esterification leads to a significant increase in conversion [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e, \u003cspan additionalcitationids=\"CR61\" citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e]. Most of these investigations used reactors arranged in series, typically with the first and third reactors packed with immobilized enzyme and the second filled with molecular sieves, allowing for a greater quantity of adsorbent to be employed. In the present system, however, the inclusion of molecular sieves did not provide any observable advantage. Therefore, the most suitable configuration for the continuous synthesis of geranyl acetate in a PBR is one that does not require molecular sieves. Eliminating the use of this material can offer an economic benefit, reducing operational costs.\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusions","content":"\u003cp\u003eGeranyl acetate was successfully produced in a PBR. A near 100% conversion was achieved under the two flow rates tested (0.1 and 0.2 mL/min). Likewise, systems operated with and without molecular sieves achieved conversions close to 100%. Thus, the presence of molecular sieves did not benefit the reaction. On the basis of the findings, the preferred approach for the continuous synthesis of geranyl acetate catalyzed by Novozym 435 in a PBR is to apply a flow rate of 0.1 mL/min and not use molecular sieves. This configuration not only simplifies the process and reduces costs associated with adsorbent materials but also maintains high conversion throughout the reaction by preserving enzyme activity. These results have important implications for the development of industrial direct esterification processes employing biocatalysts in continuous systems.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003ePBR\u0026nbsp; \u0026nbsp; \u0026nbsp;Packed bed reactor\u003c/p\u003e\n\u003cp\u003eSTR \u0026nbsp; \u0026nbsp; Stirred tank reactor\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eDeclaration of competing interest\u003c/h2\u003e \u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eEthical Approval\u003c/h2\u003e \u003cp\u003eThis article does not contain any studies with human participants or animals.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eConsent to Participate\u003c/strong\u003e \u003cp\u003eAll authors agree mutually with the participation and publication of this work and declare that this is original research.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eConsent to Publish\u003c/strong\u003e \u003cp\u003eNot applicable.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis study was funded by the S\u0026atilde;o Paulo Research Foundation (FAPESP, grants Nos. 2020/09592-1, 2024/07997-5 and 2025/00723-0) and the Brazilian National Council for Scientific and Technological Development (CNPq) for the financial support (grant No. 304399/2022-1).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003e**D. G. O.:** Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Visualization, Writing \u0026ndash; original draft. **D. R.:** Formal analysis, Methodology, Visualization, Validation, Writing \u0026ndash; original draft. **I. M. O.:** Methodology, Data curation, Formal analysis, Writing \u0026ndash; original draft. **B. W. P.:** Methodology, Data curation, Formal analysis, Writing \u0026ndash; original draft**. J. V. O.:** Investigation, Visualization, Funding acquisition, Writing \u0026ndash; review \u0026amp; editing **P. V. A**.**:** Methodology, Data curation, Formal analysis, Writing \u0026ndash; original draft. **M. O. C.:** Investigation, Visualization, Funding acquisition, Writing \u0026ndash; review \u0026amp; editing. **A. V. P.:** Conceptualization, Funding acquisition, Project administration, Supervision, Writing \u0026ndash; review \u0026amp; editing.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eWe thank the Department of Bioprocess Engineering and Biotechnology of the School of Pharmaceutical Sciences, S\u0026atilde;o Paulo State University, Brazil. We are grateful for the financial support provided by the Brazilian National Council for Scientific and Technological Development (CNPq, grant No. 304399/2022-1), the S\u0026atilde;o Paulo State Research Foundation (FAPESP, grants Nos. 2020/09592-1, 2024/07997-5 and 2025/00723-0), and the Brazilian Federal Agency for Support and Evaluation of Graduate Education (CAPES).\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eData that support the findings of this study are available from the corresponding author upon request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eMączka W, Wińska K, Grabarczyk M (2020) One Hundred Faces of Geraniol. 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Biotechnol Lett 10:799\u0026ndash;804. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/BF01027576\u003c/span\u003e\u003cspan address=\"10.1007/BF01027576\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Esterification, Geranyl acetate, Molecular sieves, Packed bed reactor","lastPublishedDoi":"10.21203/rs.3.rs-8329923/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8329923/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eGeranyl acetate is a geraniol ester widely used as a flavoring agent and preservative in food and cosmetic products due to its pleasant aroma, antimicrobial properties, and low toxicity. This study investigated the production of geranyl acetate by esterification of geraniol with acetic acid at a 3:1 molar ratio in a solvent-free packed bed reactor using Novozym 435 as biocatalyst. Additionally, the influence of molecular sieves on process performance was evaluated. The results showed that geranyl acetate conversions reached approximately 100% (w/w) at flow rates of 0.1 and 0.2 mL/min, both in the presence and absence of molecular sieves. Thus, the addition of these adsorbents did not provide any benefit to the reaction. Consequently, the preferred approach for continuous synthesis of geranyl acetate in a packed bed reactor is to use Novozym 435 without molecular sieves. This strategy simplifies the process, reduces costs, and maintains conversion efficiency over time, offering valuable insights for the industrial development of direct esterification processes using biocatalysts in continuous systems.\u003c/p\u003e","manuscriptTitle":"Assessing the Role of Molecular Sieves in Continuous Enzymatic Synthesis of Geranyl Acetate","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-01 06:44:46","doi":"10.21203/rs.3.rs-8329923/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d757c692-4033-4e65-b529-b717378eeb4e","owner":[],"postedDate":"January 1st, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-01-19T18:00:51+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-01 06:44:46","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8329923","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8329923","identity":"rs-8329923","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}