Optimization of limonene biotransformation to limonene-1,2-diol by Colletotrichum nymphaeae CBMAI 0864

Process Biochemistry 86 (2019) 25–31

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Optimization of limonene biotransformation to limonene-1,2-diol by Colletotrichum nymphaeae CBMAI 0864

T

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Adones Sales , Glaucia Maria Pastore, Juliano Lemos Bicas University of Campinas, Faculty of Food Engineering, Department of Food Science, Monteiro Lobato Street, 80, 13083-862 Campinas, São Paulo, Brazil

A R T I C LE I N FO

A B S T R A C T

Keywords: Bioaroma Bioreactor By-product Citrus terpene Dissolved oxygen Volatilization

This study aimed to optimize the conditions of the biotransformation of limonene to limonene-1,2-diol by C. nymphaeae. In the initial modeling, in shake flasks, the use of 13.2 g.L−1 biomass, 27 °C, 250 rpm, and pH of 6.0 could maximize the production of limonene-1,2-diol (6.75 g.L−1). Subsequently, optimal conditions were transposed to a bioreactor, where agitation and aeration were also evaluated. A minimum 60% of dissolved oxygen was defined for this process. The limonene volatilization could be reduced almost three times when a larger condenser is used. Finally, when the bioreactor was operated at 27 °C, 300 rpm, 1 vvm, and with 13.2 g.L−1 biomass, limonene-1,2-diol production reached 7.1, 7.8, and 5.6 g.L−1 after 72 h when using 20 g.L−1 of R-(+)-, S-(−)-limonene or citrus terpene as substrate, respectively. This is approximately the same maximum product concentration found for the shake flasks, but it represents an almost three times higher productivity.

1. Introduction Terpenes have a strong impact on the global market, considering that such compounds are used in the food, perfumery, cosmetic, pharmaceutical, and fuel industries. Natural and sustainable appeals increasingly drive the search for these compounds and, in this context, terpenes with biotechnological origin may have a higher added value [1]. Therefore, biotransformation of natural terpene substrates is an interesting approach for producing biotech terpene compounds. However, this strategy poses some challenges, such as: (i) chemical instability, (ii) low water solubility, (iii) high volatility, and (iv) high toxicity of both substrate and product; besides the (v) low yields obtained and (vi) high costs related to fermentations [2,3]. Thus, optimization of process variables may be highly relevant to overcome these problems. Authors of studies on optimization have indicated that substantial increase in products’ concentration is feasible, and that temperature, pH, and agitation are relevant parameters in the biotransformation of terpenes [4,5]. Moreover, the use of bioreactors and scale-up studies are necessary for the application and recovery of the product in larger volumes (i.e., industrial scale) [6]. In this context, parameters, such as dissolved oxygen, primordial in aerobic processes, might consist in limiting factors to reach a desirable yield [7]. Researchers of previous studies have shown that Colletotrichum nymphaeae could biotransform limonene to limonene-1,2-diol reaching

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concentrations up to 4 g.L−1 after 192 h [8]. The culture conditions for the aforementioned bioconversion were also defined, establishing the aerobic process and using non-inductive 72 h-old biomass, 15 g.L−1 limonene substrate, and simple aqueous medium for bioconversion [9]. The limonene-1,2-diol has been associated with a significant inhibitory effect on the pro-inflammatory activities of CD4+ and CD8 + T lymphocytes [10], a potential anticancer activity [11,12], insect-attractant properties [13], besides being possibly used as flavoring for beverages, chewing gum, gelatins, and puddings [14]. Regarding the used substrate, R-(+)-limonene is the major terpene in citrus oils and is largely available in nature or as a by-product of the juice industry [15]; its biotransformation is deemed a good choice for increasing its added value [16]. Therefore, we aimed to optimize conditions of the biotransformation of limonene to limonene-1,2-diol by C. nymphaeae CBMAI 0864 in shake flasks and to further transpose this process to a laboratory-scale bioreactor. 2. Materials and methods 2.1. Microorganisms and chemicals The strain C. nymphaeae CBMAI 0864 was a courtesy of the Brazilian Collection of Environmental and Industrial Microorganisms (CBMAI) of the Multidisciplinary Center of Chemical, Biological and Agricultural Research (CPQBA, Paulínia, SP, Brazil). R-(+)-limonene (purity 99%),

Corresponding author. E-mail addresses: [email protected] (A. Sales), [email protected] (G.M. Pastore), [email protected] (J.L. Bicas).

https://doi.org/10.1016/j.procbio.2019.07.022 Received 27 May 2019; Received in revised form 12 July 2019; Accepted 31 July 2019 Available online 01 August 2019 1359-5113/ © 2019 Elsevier Ltd. All rights reserved.

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S-(−)-limonene (purity ≥95%), and limonene-1,2-diol (purity ≥97%) were purchased from Sigma–Aldrich (St. Louis, MO, USA). All other chemicals and solvents were of the best grade available. Citrus terpene (produced from orange peel by distillation process, 97% limonene in GC, data not shown), gently supplied by Cocamar® (Paranavaí, PR, Brazil), was also used in this study as a convenient source of R(+)-limonene.

Table 2 Central composite rotatable design matrix and the resulting limonene-1,2-diol concentration (g.L−1) after 192 h biotransformation and Yp/x (%, g. g−1) after 192 h-biotransformation for each assay*.

2.2. Optimization experiments 2.2.1. Inoculum preparation A piece of agar (˜1.5 cm2) with the pre-grown culture (30 °C for 72 h) in yeast and malt (YM) agar (in g.L−1: glucose = 10; peptone = 5; yeast extract = 3; malt extract = 3; agar = 20, pH ˜6.7) was transferred to a 125 mL conical flask filled with 50 mL of YM Broth (the same composition aforementioned, without the agar). The material was homogenized under sterile conditions with an Ultra-Turrax® T18 (Ika, Wilmington, NC, USA) until complete disruption of the solid matter. After incubation at 30 °C and 150 rpm for 48 h, biomass was recovered by vacuum filtration using a Buchner funnel and paper filter Whatman #1 [17]. 2.2.2. Biotransformation procedure The resulting biomass was resuspended in 50 mL phosphate buffer 20 μmol.L−1 supplemented with 20 g.L−1 of R-(+)-limonene. Flasks were incubated at a given condition (see Section 2.2.3), and samples were collected every 48 h to monitor the produced amount of limonene1,2-diol. 2.2.3. Experimental design A central composite rotatable design with 24 experiments and 4 center points was considered to model the effect of agitation, temperature, pH, and inoculum concentration (Tables 1 and 2) on the yield (Yp/x; %, g. g−1) and concentration (g.L−1) of limonene-1,2-diol [18]. The variables considered in this study (agitation, temperature, pH, and inoculum concentration) and their levels were defined based on previous experiments [8,9,17,19]. Results were analyzed by the Protimiza® software (http:// experimental-design.protimiza.com.br). A 10% significance level was used for the central composite rotatable design.

Assay

A

T

pH

I

[diol]

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28

115 245 115 245 115 245 115 245 115 245 115 245 115 245 115 245 50 310 180 180 180 180 180 180 180 180 180 180

24 24 32 32 24 24 32 32 24 24 32 32 24 24 32 32 28 28 20 36 28 28 28 28 28 28 28 28

6.0 6.0 6.0 6.0 7.0 7.0 7.0 7.0 6.0 6.0 6.0 6.0 7.0 7.0 7.0 7.0 6.5 6.5 6.5 6.5 5.5 7.5 6.5 6.5 6.5 6.5 6.5 6.5

4.95 4.95 4.95 4.95 4.95 4.95 4.95 4.95 8.25 8.25 8.25 8.25 8.25 8.25 8.25 8.25 6.60 6.60 6.60 6.60 6.60 6.60 3.30 9.90 6.60 6.60 6.60 6.60

1.99 3.12 1.37 2.77 2.69 2.89 2.06 1.94 3.21 4.01 2.98 4.20 3.41 0.74 3.06 3.44 0.44 3.81 2.81 0.03 4.78 3.19 2.04 4.62 4.91 4.11 4.40 3.96

Yp/x 40.12 63.11 27.58 55.96 54.44 58.38 41.58 39.24 38.85 48.58 36.12 50.86 41.29 40.14 37.15 41.66 6.74 57.69 42.52 0.43 53.91 48.31 61.83 46.72 74.47 62.22 66.66 59.96

* A: agitation (rpm), T: temperature (°C), I: inoculum concentration (g.L−1), [diol]: limonene-1,2-diol concentration (g.L−1) after 192 h of biotransformation, Yp/x: yield coefficient, expressed as limonene-1,2-diol formed per unit of biomass dry weight after 192 h of biotransformation (%, g.g−1).

phosphate buffer 20 μmol.L−1 in a 2.5 L vessel coupled to the same system previously described (Section 2.3.1). The optimal values of pH, temperature, and inoculum concentration obtained from the shaker optimization were applied to the bioreactor. Different levels of agitation and airflow were evaluated using 20 g.L−1 of R-(+)-limonene as substrate. After defining agitation and airflow levels, S-(−)-limonene and citrus terpene were also tested as substrate.

2.3. Bioreactor application 2.3.3. Loss of substrate by volatilization An abiotic control was established to evaluate the loss of substrate by volatilization. In this case, after biomass resuspension (as defined in Section 2.3.2), the whole vessel was autoclaved. Subsequently, 20 g.L−1 of R-(+)-limonene was added to the vessel for operation under optimum biotransformation conditions. A silicone tubing connected the condenser outlet to a bottle containing 100 mL of hexadecane, which was used to trap the limonene from the gas stream. The limonene concentration retained in hexadecane was then quantified at different airflow rates (0.5, 1, 1.5, and 2 vvm). We also evaluated the loss of substrate using two different exhaust condensers with continuous flow of water at room temperature: 18 x 2 cm (length x internal diameter) metallic and 60 x 2 cm glass condenser.

2.3.1. Inoculum Two agar plates (˜180 cm2) with the pre-grown culture were homogenized (see Section 2.2.1.) in 6 L of YM Broth and transferred to a vessel of 7.5 L (nominal volume) controlled by a BioFlo®/CelliGen® 310 Bioreactor (www.eppendorf.com) equipped with pH and dissolved oxygen (DO) probes (www.mt.com/us/en/home.html). After incubation at 30 °C and 300 rpm (marine blade impeller) for 48 h, the biomass was recovered by vacuum filtration using a Buchner funnel and Whatman #1 filter paper. 2.3.2. Biotransformation The resulting biomass (Section 2.3.1) was resuspended with 1 L of Table 1 Variables and levels evaluated in the central composite rotatable design. Variables

Agitation (rpm) Temperature (°C) pH Inoculum concentration (g.L−1)

2.4. Extraction, identification, and quantification of biotransformation products

Levels −2

−1

0

+1

+2

50 20 5.5 3.30

115 24 6.0 4.95

180 28 6.5 6.60

245 32 7.0 8.25

310 36 7.5 9.90

Samples were extracted (40 s in vortex) using the same volume of ethyl acetate. After phase separation, the organic fraction was dried over sodium sulfate, and 1 μL of this phase was injected into a gas chromatograph with a flame ionization detector (GC–FID) HP-7890 (Agilent Technologies, Santa Clara, CA,) coupled to an HP-5 column (30-m length × 0.25-mm i.d. × 0.25-μm film thickness) operating at 26

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and the influence on the catalytic activity of the microorganism [21,22]. Therefore, intermediate temperatures (26–30 °C) are usually indicated for terpene biotransformations [17,23,24]. In addition, terpene biotransformations are usually affected by pH. Although this parameter did not present significant effect on the biotransformation limonene to α-terpineol (in the range of 5.2–8.2) [17], the mechanism of action of epoxy hydrolases, such as the enzyme supposedly required for this particular biotransformation [8], involves a nucleophilic attack of aspartate residue to open the epoxide ring [25–27]. Therefore, very acidic conditions would protonate the aspartate residue and disfavor this reaction. Indeed, biotransformation of limonene to limonene-1,2diol is usually carried out under near-neutral conditions [19,28]. Regarding agitation, this is an aerobic process and requires cofactors [9]; therefore, oxygen supply is essential. In shake flasks, aeration depends on the value of rotations per minute [17,29,30], and this is suggested to explain the positive effect of this agitation on the limonene-1,2-diol production. As for the effect of inoculum, as expected, the best inoculum loads to maximize limonene-1,2-diol concentrations differs from the condition to reach an ideal yield: in case of limonene-1,2-diol concentration, 7.5 to 10 g.L−1 inoculum load is ideal, whereas 4.5 g.L−1 or less is indicated to get an optimum Yp/x. The central point for biomass concentration (6.6 g.L−1) remains in an intermediate position, in which both limonene-1-2-diol concentration and yield are at c.a. 90% of the maximum values (e.g., at 9 g.L−1 or 3.5 g.L−1, respectively). Therefore, 6.6 g.L−1 of biomass concentration was chosen for further trials. To evaluate if higher inoculum loads would result in a further increase in limonene-1,2-diol concentration (as suggested in Fig. 1C and E), a new biotransformation was carried out at the optimized conditions (250 rpm, 27 °C, and pH 6) using different biomass concentrations (6.6, 13.2, and 26.4 g.L−1). In Fig. 3 we show the results. In addition, a ˜20% increase in limonene-1,2-diol concentration is achieved when biomass concentration is doubled (from 6.6–13.2 g.L−1), but there is no gain in limonene concentrations for biomass load higher than that (no statistical differences are observed between 13.2 and 26.4 g.L−1). At 192 h, the limonene-1,2-diol concentration (g.L−1) and yield (Yp/x) obtained for 6.6 g.L−1 of biomass (5.3 g.L−1 and 81%, respectively) is 19% and 23% higher than the responses predicted by the models (Eqs. (1) and (2)). Considering that our main goal was to achieve the highest limonene-1,2-diol concentration, we opted to conduct the biotransformation in bioreactor at 13.2 g.L−1 of biomass concentration, knowing that the yield of diol in terms of biomass would be dramatically lower. The range obtained from limonene-1,2-diol concentration is among the highest ones reported. Bier et al. [23], in a study on endophytic fungus isolated from Pinus taeda and identified as Phomopsis sp., produced 2.08 g.L−1 limonene-1,2-diol from R-(+)-limonene. Other endophytic fungi isolated from Eupatorium buniifolium, i.e., Alternaria alternata and Neofusicoccum sp., were also able to produce 1.75 and 2.23 g.L−1 of limonene-1,2-diol from the R-(+)-limonene, respectively [28]. Molina et al. [19] reached 3.7 g.L-1 using Fusarium oxysporum. Abraham et al. [24], using a reactor with 70 liters of medium and the fungus Corynespora cassiicola, converted 1.3 kg R-(+)-limonene to 900 g limonene-1,2-diol (12.9 g.L−1). In Table 3 we summarize the production parameters of our study and compare them with previously published research on the biotransformation of limonene to limonene1,2-diol.

split mode (split ratio of 1:10). Helium was used as the carrier gas (1.0 mL.min–1), and the oven temperature was kept at 80 °C for 3 min, raised at 20 °C min–1 until 200 °C and held for 4 min. Temperatures of the injector and detector were kept at 250 °C [20]. Identification of volatile compounds was performed on a GC–MS system with an HP-7890 gas chromatograph coupled to an HP-5975C mass spectrometer (Agilent Technologies, Santa Clara, CA, USA). A column HP-5MS with 30-m length x 0.25-mm i.d. × 0.25-μm film thickness was used to separate volatile components. Helium was used as carrier gas at constant flow rate of 1.0 mL.min−1. Temperature of the gas chromatography oven was the same as aforementioned. The mass spectrometer transfer line was set at 250 °C, impact energy of 70 +eV, and mass range of 35–500 m/z. Compounds were identified by comparing the spectra with NIST library and with the commercial standard. Concentrations of limonene and limonene-1,2-diol (g.L−1) were determined in GC-FID (as previously mentioned) based on the calibration curve created by adding known concentrations of limonene (0.1, 0.5, 1.0, 5.0, 10.0, 15.0, and 20.0 g.L−1) and limonene-1,2-diol (0.05, 0.1, 0.5, 1.0, 3.0, 7.0, and 10.0 g.L−1) in heat-inactivated (autoclaved) biomass resuspended in phosphate buffer (6.6 g.L−1 dry weight), in order to consider the matrix effect, and fixed concentration of n-decane (1.0 g.L−1) as internal standard. The substrate or product’s concentration were determined by substituting the relative area (area of the analyte peak divided by the area of n-decane peak) in the calibration curve (Limonene relative area = /[Limonene] x 1.1522, R2 = 0.9996; Limonene-1,2-diol relative area = [Limonene-1,2-diol] x 1.0865, R2 = 0.9957). The yield (Yp/x) was calculated by dividing the amount of limonene-1,2-diol (g.L−1) produced by the biomass concentration (g.L−1) used in the biotransformation. 3. Results and discussion 3.1. Optimization in flasks Overall, regardless of the tested condition, the highest product concentration was found after 192 h of biotransformation (Supplementary Fig. 1). Therefore, this biotransformation time was chosen to model the influence of the parameters in this bioprocess. Data for 192 h-biotransformation were treated by the Protimiza® software, and regression coefficients for each of the analyzed parameters are shown in Supplementary Table 1. The coded model, including only the significant parameters (p lower than approximately 0.1), is presented in Eqs. (1) (for limonene-1,2-diol concentration) and (2) (for Yp/x). Lim-1,2-diol (g.L−1) = 4.34 + 0.49 A - 0.50 A² - 0.35 T - 0.67 T² - 0.19 pH² + 0.58 I - 0.20 I² - 0.26 A pH (1) Yp/x (%.g. g−1) = 65.83 + 7.61 A - 7.07 A² - 5.79 T - 9.75 T² - 2.34 pH² 3.17 I - 4.43 A pH + 3.04 T I (2) where A, T, and I are agitation, temperature, and inoculum concentration, respectively. To verify the validity of these models, we performed an analysis of variance (ANOVA) (Supplementary Table 2). Results indicate that the models are statistically significant: F value is 6 to 7 times greater (regression/residues) or approximately 3 times lower (lack of fit/pure error) than the respective F values at p of 0.90. Moreover, R2 values varied from 0.84 to 0.86, which are satisfactory for biological systems [17,18]. In Figs. 1 and 2 we graphically present the Eqs. (1) and (2). In both figures, we may perceive that a maximum plateau is observed for temperature, agitation, and pH values from 25 to 29 °C, 200 to 250 rpm, and 6.0 to 7.0, respectively, regardless of the considered response. This behavior was expected, based on literature and on mechanistic data expected for this biotransformation, as discussed next. Temperature is known to have a significant effect on the biotransformation of terpenes due to the high volatility of such compounds

3.2. Biotransformation in bioreactor After 48 h of growth of C. nymphaeae in bioreactor, glucose was completely consumed, pH increased, and the oxygen consumption decreased, indicating the end of log phase (Supplementary Fig. 2). At this time the process was interrupted, and the resulting biomass was used in the biotransformation process.

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Fig. 1. Contour plots for limonene-1,2-diol concentration after 192 h-biotransformation in relation to: agitation and temperature (A); agitation and pH (B); agitation and inoculum (C); temperature and pH (D); temperature and inoculum (E); pH and inoculum (F). For each figure, unmentioned variables were fixed at their central points.

Fig. 2. Contour plots of the yield of limonene-1,2-diol by biomass (Yp/x) after 192 h-biotransformation in relation to: agitation and temperature (A); agitation and pH (B); agitation and inoculum (C); temperature and pH (D); temperature and inoculum (E); pH and inoculum (F). For each figure, unmentioned variables were fixed at their central points. 28

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terpene biotransformation processes. Aroma compounds usually have high volatility, which may reinforce the volatilization rates by temperature, agitation, and aeration [34]. Therefore, in order to monitor limonene loss in the bioreactor, different agitation speeds and condenser systems were applied to an abiotic system containing limonene, whose concentration was monitored over time. In Fig. 5 we show that the substrate loss linearly increased with the aeration rate: for the metal 18 x 2 cm condenser, loss increased at a rate of 141 mg.L−1. h−1.vvm−1, which was ˜2.8 times higher than the glass 60 x 2 cm condenser (51 mg.L−1. h−1.vvm−1). Combining the results of Figs. 3 and 4, we considered that the ideal conditions for minimizing substrate loss while keeping DO > 60% would be 300 rpm and 1vvm. Authors of former studies also addressed the concern with substrate loss in biotransformations. In the biotransformation of citronellol by Cystoderma carcharias, the total loss of the volatile substrate via exhaust air was 4.5% [33]. The use of adsorption of volatilized compounds in organic solvent for future quantification was used for terpene biotransformations in solid medium by Penicillium digitatum [35]. In order to avoid substrate loss by volatilization in biotransformation studies, different approaches have been reported. The use of co-substrates (e.g., ethanol) [36] or biphasic systems [37] are some examples. Moreover, the use of bioreactors with a particular design (closed loop bioreactors) is another interesting strategy [38].

Fig. 3. Kinetics of limonene-1,2-diol production at optimal conditions (250 rpm, 27 °C, pH 6) using 6.6 (○), 13.2 (△), and 26.4 (□) g.L−1 biomass concentrations.

3.2.1. Impact of dissolved oxygen on the biotransformation of limonene to limonene-1,2-diol by C. nymphaeae To analyze the impact of the dissolved oxygen (DO) on the biotransformation of limonene to limonene-1,2-diol by C. nymphaeae, and to define its ideal range, this bioprocess was conducted in a bioreactor with varied combinations of agitation and aeration overtime. As expected, such parameters directly influenced DO in the bioconversion medium, in such a way that limonene-1,2-diol production rate greatly decreased when DO was below ˜60% (Fig. 4). Therefore, this was considered the minimum DO to support the production of limonene1,2-diol by C. nymphaeae in bioreactor. Using an integrated array of microbioreactors, Lee et al. [31] kept an Escherichia coli fermentation at a minimum DO 40% even without any aeration. Gomes et al. [32] observed that using 300 rpm and 0.3 vvm, a complete depletion of DO occurred in the medium of the biotransformation of methyl ricinoleate into γ-decalactone by Yarrowia lipolytica. On the other hand, using 600 rpm and 0.9 vvm, DO was above 70% during the whole experiment, maintaining maximum production. Similarly, we showed that different combinations of agitation and aeration resulting in a DO > 60% was necessary to maintain the microbial biotransformation. Considering that aeration rate may greatly influence the loss of volatiles from the culture medium [33], we decided to verify the loss of substrate at different aeration conditions in order to identify the ideal conditions for this biotransformation.

3.2.3. Biotransformation using different substrate sources Parameters obtained from shaker optimization (pH, temperature, and biomass concentration) and bioreactor studies (agitation and aeration) were used for the biotransformation of R-(+)-, S-(‒)-limonene, and citrus terpene (Fig. 6). These substrates yielded a maximum of 7.1, 7.8, and 5.6 g limonene-1,2-diol L−1, respectively. In the case of R-(+)-limonene, when compared with the biotransformation in shake flasks, this maximum limonene-1,2-diol concentration slightly increased only (˜5.5%); however, productivity was much higher (2.7 times), since the biotransformation time required to reach the maximum concentration reduced from 192 to 72 h (Figs. 3 and 6, Table 3). This was considered quite satisfactory, since the scale-up procedure reached at least the same concentration of the bench scale [39]. The production kinetics of limonene-1,2-diol in bioreactor were very similar regardless of the used limonene enantiomer (Fig. 6). The less common enantiomer of this substrate, S-(‒)-limonene, is found in Mentha species and conifer oils, and it has already been reported by other authors [19,40,41] as a biotechnological precursor of limonene1,2-diol. However, the concentrations obtained in such studies were

3.2.2. Effect of aeration on the substrate loss Loss of substrate by volatilization is one of the main problems in

Table 3 Comparison of parameters considered in the present study with previously reported bioprocesses for the production of limonene-1,2-diol via biotransformation of limonene. Microorganism

System

TVa

Sb

SCc

BLd

MCe

TMf

YP/Sg

YP/Xh

VPi

SPj

Ref.k

Colletotrichum nymphaeae C. nymphaeae C. nymphaeae C. nymphaeae Corynespora cassiicola Fusarium oxysporum Phomopsis sp. Phomopsis sp. Alternaria alternata Neofusicoccum sp. C. nymphaeae Coletotrichum acutatum C. nymphaeae

Flasks Bioreactor Bioreactor Bioreactor Bioreactor Flasks Flasks Flasks Flasks Flasks Flasks Flasks Flasks

50 mL 1L 1L 1L 70 L 200 mL 40 mL 40 mL 20 mL 20 mL 50 mL 50 mL 50 mL

R R S ct R S R ct R R R R R

20 20 20 20 20 5 10 5 2.5 2.5 15 15 15

13.2 13.2 13.2 13.2 n.a.* 50** 3.0 13.0 100** 100** 6.4 6.2 6.6

6.75 7.11 7.84 5.55 12.9 3.7 2.08 2.1 1.75 2.23 4.06 2.08 4.19

192 72 72 72 96 72 120 144 72 72 192 192 192

33.75 35.55 39.2 27.75 65.4 74.0 20.8 42.0 70.0 89.2 27.07 13.87 27.93

51.14 53.86 59.39 42.05 n.a.* 7.4 69.33 16.15 1.75 2.23 63.44 33.55 63.48

35.16 98.75 108.9 77.08 134.4 51.39 17.33 14.58 24.31 30.97 21.15 10.83 21.82

0.27 0.75 0.83 0.58 n.a.* 0.1 0.58 0.11 0.02 0.03 0.33 0.18 0.33

1 1 1 1 2 3 4 4 5 5 6 6 7

TV Total volume of biotransformation medium; b S Substrate: R: R-(+)-limonene; S: S-(–)-limonene, ct: citrus terpene; c SC Initial substrate concentration (g.L−1); d BL Biomass load (g.L−1 dry weight); e MC Maximal concentration (g.L−1); f TM Duration for achieving the maximal limonene-1,2-diol concentration (h); g Yield coefficient, expressed as product formed per unit of substrate added (%, g. g−1), since no information was available regarding consumed substrate; h Yield coefficient, expressed as product formed per unit of biomass dry weight (%, g. g−1); i VP Volumetric productivity (mg.L.−1. h−1); j SP Specific productivity (%, g. g−1. h−1); k References: 1. Our study; 2. Abraham et al. (1985) [24]; 3. Molina et al. (2015) [19]; 4. Bier at al. (2017) [23]; 5. Cecati et al. (2018) [28]; 6. Sales et al. (2018) [8]; 7. Sales et al. (2019) [9]. *n.a.: not available; ** Fresh weight biomass. a

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Fig. 4. Dissolved oxygen (DO, continuous line) and limonene-1,2-diol production (dashed line) from R-(+)-limonene biotransformation by C. nymphaeae (13.2 g.L−1) in a 2.5 L bioreactor operating at different levels of agitation (rpm) and airflow (vvm) at 27 °C and pH 6.0.

reaching viable large-scale processes. We showed that the use of 13.2 g.L−1 biomass as inoculums, 27 °C, 250 rpm, and pH 6.0 could maximize the production of limonene-1,2-diol via biotransformation of limonene by C. nymphaeae. The use of DO as a parameter for defining agitation and airflow in bioreactor was very practical. Agitation at 300 rpm and airflow at 1 vvm were sufficient to maintain DO > 60%, the minimum required for this process. We also identified that the substrate volatilization in bioreactor could be significantly reduced using a 60 cm condenser and lower agitation rates. Therefore, when the bioreactor was operated at 27 °C, 300 rpm, 1 vvm, and containing 13.2 g.L−1 of C. nymphaeae biomass, the limonene-1,2-diol production reached 7.1, 7.8 and 5.6 g.L−1 after 72 h when using 20 g.L−1 R-(+)-, S-(−)-limonene, or citrus terpene as substrate, respectively. This is approximately the same product concentration found for the shake flasks, but in the bioreactor the productivity was almost three times higher. We consider that the strategies adopted in this study could be useful for researchers studying other bioprocesses as well.

Fig. 5. Loss of R-(+)-limonene by volatilization using (•) metal 18 x 2 cm and (◼) glass 60 x 2 cm condensers in a 2.5 L bioreactor containing autoclaved biomass (13.2 g.L−1) and operating at 300 rpm, 27 °C, pH 6, and different aeration rates.

Funding Funding sources had no involvement in the study design; collection, analysis and interpretation of data; writing of the report; and decision to submit the article for publication. Declaration of Competing Interest None. Acknowledgements Fig. 6. Production of limonene-1,2-diol by C. nymphaeae using R-(+)- (□), S(‒)-limonene (○), and citrus terpene (△) as substrates in a 2.5 L bioreactor operating with 13.2 g.L−1 biomass at 300 rpm, 27 °C, 1 vvm, and pH 6.0.

The authors acknowledge the Brazilian Collection of Environmental and Industrial Microorganisms (CBMAI-CPQBA) for the strain and Espaço da Escrita – Coordenadoria Geral da Universidade (UNICAMP) for the language services provided. This study was funded by the National Council of Technological and Scientific Development (CNPq) (grant no. 400411/2016-4); São Paulo Research Foundation (FAPESP) (grant no. 2016/21619-7), and Coordination for the Improvement of Higher Education Personnel (CAPES) (scholarship for A. Sales and process no. 23038.000795/2018-61, and finance code 001).

lower than those we found (7.8 g.L−1). Citrus terpene, a by-product of orange juice industry, with estimated global production of 30,000 tons per year [15] is an inexpensive source of R-(+)-limonene. We showed that this by-product could replace the standard R-(+)-limonene, resulting in a similar production profile, although resulting in a 21.1% lower product concentration at the same biotransformation time (72 h). Other authors have already reported the use of citrus by-products to replace limonene in biotransformation, but none with a product concentration as high as the one described in this study. Bier et al. [23], for instance, reported the biotransformation in a natural orange extract medium by Phomopsis sp. resulting in a production of 2.1 g limonene1,2-diol L−1. Marostica et al. [42] used orange peel oil in a biotransformation process with Fusarium oxysporum, and obtained c.a. 400 mg.L−1 of α-terpineol.

Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.procbio.2019.07.022. References [1] L. de O. Felipe, A.M. de Oliveira, J.L. Bicas, Bioaromas – Perspectives for sustainable development, Trends Food Sci. Technol. 62 (2017) 141–153, https://doi.org/ 10.1016/j.tifs.2017.02.005. [2] G. Molina, J.L. Bicas, E.A. Moraes, M.R. Marostica Jr, G.M. Pastore, Recent developments and industrial perspectives in the microbial production of bioflavors, Appl.

4. Conclusions Knowing the optimal conditions of shaker bioprocess is essential for 30

Process Biochemistry 86 (2019) 25–31

A. Sales, et al.

[3]

[4]

[5]

[6]

[7]

[8]

[9]

[10]

[11]

[12]

[13]

[14] [15]

[16]

[17]

[18] [19]

[20]

[21]

[22]

[23]

121 (2017) 137–144, https://doi.org/10.1016/j.funbio.2016.11.003. [24] W.R. Abraham, H.M.R. Hoffmann, K. Kieslich, G. Reng, B. Stumpf, Microbial transformations of some monoterpenoids and sesquiterpenoids, Enzym. Org. Synth. Ciba Found (1985) 146–170, https://doi.org/10.1002/9780470720929.ch11. [25] R.V.A. Orru, A. Archelas, R. Furstoss, K. Faber, Epoxide hydrolases and their synthetic applications, in: K. Faber (Ed.), Biotransformations, Springer, Berlin, 1999, pp. 145–167, , https://doi.org/10.1007/3-540-69791-8_7. [26] C. Morisseau, B.D. Hammock, EPOXIDE HYDROLASES: mechanisms, inhibitor designs, and biological roles, Annu. Rev. Pharmacol. Toxicol. 45 (2004) 311–333, https://doi.org/10.1146/annurev.pharmtox.45.120403.095920. [27] A. Steinreiber, K. Faber, Microbial epoxide hydrolases for preparative biotransformations, Curr. Opin. Biotechnol. 12 (2001) 552–558, https://doi.org/10. 1016/S0958-1669(01)00262-2. [28] F.M. Cecati, C. Magallanes-Noguera, C.E. Tonn, C.E. Ardanaz, M. Kurina-Sanz, Ecofriendly chemical diversification of Eupatorium buniifolium essential oil by endophytic fungi, Process Biochem. 64 (2018) 93–102, https://doi.org/10.1016/j. procbio.2017.09.028. [29] J.C.R. Demyttenaere, H.L. De Footer, Biotransformation of geraniol and nerol by spores of Penicillium italicum, Phytochemistry 41 (1996) 1079–1082, https://doi. org/10.1016/0031-9422(95)00797-0. [30] Z. Liu, X. Wang, Q. Qi, Q. Hua, Quantification and analysis of metabolic characteristics of aerobic succinate-producing Escherichia coli under different aeration conditions, Process Biochem. 47 (2012) 1532–1538, https://doi.org/10.1016/j. procbio.2012.01.022. [31] H.L.T. Lee, P. Boccazzi, R.J. Ram, A.J. Sinskey, Microbioreactor arrays with integrated mixers and fluid injectors for high-throughput experimentation with pH and dissolved oxygen control, Lab Chip 6 (2006) 1229, https://doi.org/10.1039/ b608014f. [32] N. Gomes, M. Aguedo, J. Teixeira, I. Belo, Oxygen mass transfer in a biphasic medium: Influence on the biotransformation of methyl ricinoleate into γ-decalactone by the yeast Yarrowia lipolytica, Biochem. Eng. J. 35 (2007) 380–386, https://doi.org/10.1016/j.bej.2007.02.002. [33] J. Onken, R.G. Berger, Biotransformation of citronellol by the basidiomycete Cystoderma carcharias in an aerated-membrane bioreactor, Appl. Microbiol. Biotechnol. 51 (1999) 158–163, https://doi.org/10.1007/s002530051376. [34] T. Chatterjee, D.K. Bhattacharyya, Biotransformation of limonene by Pseudomonas putida, Appl. Microbiol. Biotechnol. 55 (2001) 541–546, https://doi.org/10.1007/ s002530000538. [35] J. Demyttenaere, N. De Kimpe, Biotransformation of terpenes by fungi: Study of the pathways involved, J. Mol. Catal. B Enzym. 11 (2001) 265–270, https://doi.org/10. 1016/S1381-1177(00)00040-0. [36] Q. Tan, D.F. Day, K.R. Cadwallader, Bioconversion of (R)-(+)-limonene by P. digitatum (NRRL 1202), Process Biochem. 33 (1998) 29–31, https://doi.org/10. 1016/S0032-9592(97)00048-4. [37] C.C.R. de Carvalho, F. van Keulen, M. Manuela, R. da Fonseca, Production and recovery of limonene-1,2-Diol and simultaneous resolution of a diastereomeric mixture of limonene-1,2-epoxide with whole cells of Rhodococcus Erythropolis DCL14, Biocatal. Biotransformation 18 (2000) 223–235, https://doi.org/10.3109/ 10242420009015246. [38] M. Pescheck, M.A. Mirata, B. Brauer, U. Krings, R.G. Berger, J. Schrader, Improved monoterpene biotransformation with Penicillium sp. by use of a closed gas loop bioreactor, J. Ind. Microbiol. Biotechnol. 36 (2009) 827–836, https://doi.org/10. 1007/s10295-009-0558-3. [39] J.K. Kim, G.H. Han, B.R. Oh, Y.N. Chun, C.Y. Eom, S.W. Kim, Volumetric scale-up of a three stage fermentation system for food waste treatment, Bioresour. Technol. 99 (2008) 4394–4399, https://doi.org/10.1016/j.biortech.2007.08.031. [40] Y. Noma, S. Yamasaki, Y. Asakawa, Biotransformation of limonene and related compounds by Aspergillus cellulosae, Phytochemistry 31 (1992) 2725–2727, https://doi.org/10.1016/0031-9422(92)83619-A. [41] J.C.R. Demyttenaere, K. Van Belleghem, N. De Kimpe, Biotransformation of (R)(+)- and (S)-(-)-limonene by fungi and the use of solid phase microextraction for screening, Phytochemistry 57 (2001) 199–208, https://doi.org/10.1016/S00319422(00)00499-4. [42] M.R. Maróstica, G.M. Pastore, Production of R-(+)-α-terpineol by the biotransformation of limonene from orange essential oil, using cassava waste water as medium, Food Chem. 101 (2007) 345–350, https://doi.org/10.1016/j.foodchem. 2005.12.056.

Microb. Eng. (2013) 122–157, https://doi.org/10.1201/b15250-6 CRC Press, Boca Raton. J.L. Bicas, A.P. Dionísio, G.M. Pastore, Bio-oxidation of terpenes: an approach for the flavor Industry, Chem. Rev. 109 (2009) 4518–4531, https://doi.org/10.1021/ cr800190y. Q. Tan, D.F. Day, Bioconversion of limonene to a-terpineol by immobilized Penicillium digitatum, Appl. Microbiol. Biotechnol. 49 (1998) 96–101, https://doi. org/10.1007/s002530051143. A. Adams, J.C.R. Demyttenaere, N. De Kimpe, Biotransformation of (R)-(+)- and (S)-(–)-limonene to alpha-terpineol by Penicillium digitatum — investigation of the culture conditions, Food Chem. 80 (2003) 525–534. F. Garcia-Ochoa, E. Gomez, Bioreactor scale-up and oxygen transfer rate in microbial processes: an overview, Biotechnol. Adv. 27 (2009) 153–176, https://doi. org/10.1016/j.biotechadv.2008.10.006. S. Schäfer, J. Schrader, D. Sell, Oxygen uptake rate measurements to monitor the activity of terpene transforming fungi, Process Biochem. 39 (2004) 2221–2228, https://doi.org/10.1016/j.procbio.2003.11.023. A. Sales, L.F. Afonso, J.A. Americo, M. de Freitas Rebelo, G.M. Pastore, J.L. Bicas, Monoterpene biotransformation by Colletotrichum species, Biotechnol. Lett. 40 (2018) 561–567, https://doi.org/10.1007/s10529-017-2503-2. A. Sales, R.C. Moreira, G.M. Pastore, J.L. Bicas, Establishment of culture conditions for bio-transformation of R-(+)-limonene to limonene-1,2-diol by Colletotrichum nymphaeae CBMAI 0864, Process Biochem. 78 (2019) 8–14, https://doi.org/10. 1016/j.procbio.2019.01.022. C.M. Lappas, N.T. Lappas, D-Limonene modulates T lymphocyte activity and viability, Cell. Immunol. 279 (2012) 30–41, https://doi.org/10.1016/j.cellimm.2012. 09.002. T.K. Lee, H.S. Roh, J.S. Yu, J. Baek, S. Lee, M. Ra, S.Y. Kim, K.H. Baek, K.H. Kim, Pinecone of Pinus koraiensis Inducing apoptosis in human lung cancer cells by activating caspase-3 and its chemical constituents, Chem. Biodivers. 14 (2017) e1600412, , https://doi.org/10.1002/cbdv.201600412. K.N.C. Murthy, G.K. Jayaprakasha, S.M. Mantur, B.S. Patil, Citrus monoterpenes: potential source of phytochemicals for cancer prevention, ACS Symp. Ser. (2012) 545–558, https://doi.org/10.1021/bk-2012-1093.ch030. E. Bowen, Potential by-products from microbial transformation of D-limonene, Florida State Hortic. Soc. 88 (1975) 304–308 http://www.fshs.org/Proceedings/ PasswordProtected/1975Vol.88/304-308(BOWEN).pdf. G.A. Burdock, Fenaroli’s Handbook of Flavor Ingredients, 6th ed., CRC Press, Boca Raton, 2010, https://doi.org/10.1016/S0015-6264(76)80260-X. W. Schwab, C. Fuchs, F.C. Huang, Transformation of terpenes into fine chemicals, Eur. J. Lipid Sci. Technol. 115 (2013) 3–8, https://doi.org/10.1002/ejlt. 201200157. D. Mamma, P. Christakopoulos, Biotransformation of Citrus by-Products into value added products, Waste Biomass Valorization 5 (2014) 529–549, https://doi.org/10. 1007/s12649-013-9250-y. J.L. Bicas, F.F.C. Barros, R. Wagner, H.T. Godoy, G.M. Pastore, Optimization of R(+)-α-terpineol production by the biotransformation of R-(+)-limonene, J. Ind. Microbiol. Biotechnol. 35 (2008) 1061–1070, https://doi.org/10.1007/s10295008-0383-0. M.I. Rodrigues, A.F. Iemma, Experimental Design and Process Optimization, CRC Press, Boca Raton, 2014. G. Molina, M.L. Bution, J.L. Bicas, M.A.H. Dolder, G.M. Pastore, Comparative study of the bioconversion process using R-(+)- and S-(-)-limonene as substrates for Fusarium oxysporum 152B, Food Chem. 174 (2015) 606–613, https://doi.org/10. 1016/j.foodchem.2014.11.059. J.L. Bicas, P. Fontanille, G.M. Pastore, C. Larroche, A bioprocess for the production of high concentrations of R-(+)-alpha-terpineol from R-(+)-limonene, Process Biochem. 45 (2010) 481–486, https://doi.org/10.1016/j.procbio.2009.11.007. R.G. Berger, J.A.M. De Bont, G. Eggink, M.M. Da Fonseca, M. Gehrke, J.-B. Gros, F. Van Keulen, U. Krings, C. Larroche, D.J. Leak, M.J. Van Der Werf, Biotransformations in the flavour industry, Curr. Top. Flavours Fragrances (2011) 139–170, https://doi.org/10.1007/978-94-011-4022-5_8. M. Trytek, K. Jędrzejewski, J. Fiedurek, Bioconversion of α-pinene by a novel coldadapted fungus Chrysosporium pannorum, J. Ind. Microbiol. Biotechnol. 42 (2015) 181–188, https://doi.org/10.1007/s10295-014-1550-0. M.C.J. Bier, A.B.P. Medeiros, C.R. Soccol, Biotransformation of limonene by an endophytic fungus using synthetic and orange residue-based media, Fungal Biol.

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