Fed-Batch and Sequential-Batch Approaches To Enhance the

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Biotechnology and Biological Transformations

Fed-batch and Sequential-batch approaches to enhance the bioproduction of 2-phenylethanol and 2-phenethyl acetate in solid-state fermentation residue-based systems Oscar Mauricio Martínez-Avila, Antoni Sánchez, Raquel Barrena, and Xavier Font J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b00524 • Publication Date (Web): 28 Feb 2019 Downloaded from http://pubs.acs.org on March 5, 2019

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Journal of Agricultural and Food Chemistry

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Fed-batch and Sequential-batch approaches to enhance the bioproduction of 2-phenylethanol

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and 2-phenethyl acetate in solid-state fermentation residue-based systems

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Oscar Martínez-Avila†,a, Antoni Sánchez†,b, Xavier Font†,c, Raquel Barrena†,*

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†

Composting Research group, Department of Chemical, Biological and Environmental

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Engineering. Escola d’Enginyeria, Universitat Autònoma de Barcelona, Cerdanyola del

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Vallès, 08193 Barcelona, Spain.

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a [email protected],

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b [email protected],

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c [email protected],

ORCID: 0000-0001-7561-4013

ORCID: 0000-0003-4254-8528

ORCID: 0000-0003-4981-7436

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*Corresponding author, contact details:

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Tel.: +34 935814793

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Fax: +34 935812013

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E-mail address: [email protected]

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ORCID: 0000-0002-6077-7765

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1 ACS Paragon Plus Environment


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Abstract

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This study describes the use of alternative operational strategies in the solid-state fermentation

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of the agro-industrial leftover sugarcane bagasse (SCB) supplemented with L-phenylalanine,

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for bioproducing natural 2-phenylethanol (2-PE) and 2-phenethyl acetate (2-PEA) using K.

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marxianus. Here, fed-batch and sequential-batch have been assessed at two scales (1.6 and 22

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L) as tools to increase the production, as well as to enhance the sustainability of this residue-

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based process.

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While in the reference batch strategy a maximum of 17 mg 2-PE+2-PEA per gram of added

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SCB were reached at both scales, the implementation of fed-batch mode induced a production

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increase of 11.6 and 12.5%, respectively. Also, the production was increased in 16.9 and 2.4%

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compared to the batch when a sequential-batch mode was used. Furthermore, the use of these

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strategies was accompanied by lower consumption of key resources like the inoculum, air and

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time, promoting savings between 22 and 76% at both scales.

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Keywords

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Kluyveromyces marxianus, Aroma compounds, Rose-like compounds, Scale-up, Waste to

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product.

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Introduction

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Aroma compounds are typically used as additives in industries like food, fragrance and

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cosmetic to improve the organoleptic properties of the products. Among these, the 2-

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phenylethanol (2-PE) is one of the most useful due to the rose-like scent it produces1,2. This

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higher alcohol has been widely used in perfumes, cosmetics, and personal care products as

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fragrance and preservative3,4, but also in disinfectants and cleaning products due to its biocide

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capability5,6. Besides, 2-PE is used as precursor for obtaining other valuable chemicals like 2-

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phenethyl acetate (2-PEA) that produces a floral-like aroma7. These rose-like compounds are

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considered generally recognized as safe (GRAS) flavoring agents8 making them value-added

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chemicals. Although most of the 2-PE and 2-PEA production is obtained by chemical

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synthesis1,2,9, these routes use fossil-based raw materials that induce off-odors altering the

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product’s organoleptic profiles and therefore limiting their use in specific applications1.

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On the contrary, their natural counterparts are extracted from the essential oils contained in

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some flowers and plants. Nevertheless, the recovery process is expensive because of the low

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content of these compounds and the dependence on external factors10–12. Consequently, the

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exploration of alternative routes for obtaining these and other valuable aromas has turned into

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an important subject13. Thereby, microbial biosynthesis and bioconversion appear as potential

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alternatives to produce natural aroma compounds due to the ability to transform some bio-

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based raw materials into value-added products14. In this sense, different valuable aromas have

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been previously produced by microbial biosynthesis through submerged fermentation (SmF)

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systems. For instance, -decalactone starting from ricinoleic acid as precursor and using

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lipolytic enzymes15, vanillin as a product of the bioconversion of ferulic acid through specific

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bacteria16, or isoamyl acetate obtained from the bioconversion of its correspondent fusel

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alcohol using yeasts17.



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Regarding the 2-PE and 2-PEA bioproduction, the L-phenylalanine (L-phe) bioconversion via

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the Ehrlich pathway represent the most widespread approach due to its conversion

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efficiency1,9, but, the use of less efficient de novo synthesis starting from simple sugars

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(Shikimate pathway) have also been reported3. Although these bioconversions are performed

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by bacteria and fungi18,19, yeasts are the main microorganisms used for this goal7,20. Among

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these, Kluyveromyces marxianus has proved to be one of the most effective 2-PE and 2-PEA

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producers7,21. Evidently, the use of L-phe as building block has the inherent limitation of the

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cost associated with this precursor (US$ 9-10 per kg at 98%9). However, the current price of

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natural 2-PE and 2-PEA (Approx. US$ 220 and 330 per kg at 99% respectively9) provides a

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margin to use the L-phe in these bioprocesses. In general, most of the bioprocesses developed

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for producing 2-PE and 2-PEA are based on the use of SmF systems3,12,18,22. Still, most of

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these technologies use sterilized synthetic media as substrates and comprise complex reaction

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systems that consume significant amounts of key resources to reach high titers9. Additionally,

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the SmF systems that use waste streams as substrates are not such efficient to use these raw

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materials in the 2-PE and 2-PEA biotransformation, making them less attractive from the

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sustainability point of view9.

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Thus, an alternative approach in line with the use of renewable sources, more energy efficient

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processes, limited hazardous materials, innocuous solvents, and waste prevention is the solid-

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state fermentation (SSF)23,24. Commonly, SSF presents high production rates and yields with

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relatively low energy requirements25, and it can be run with solid organic wastes as

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substrates24,26, providing significant potential for the development of sustainable and

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economically feasible bioprocesses. SSF has been previously used for the production of

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valuable aroma compounds such as fruity aromas27, vanillin28 or 6-pentyl-α-pyrone (coconut-

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like aroma)29 starting with agro-industrial residues of diverse origin as raw materials.


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Nevertheless, constraints such as the residues’ sterilization or the small knowledge of the

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operating modes effects on the process efficiency, limit the sustainability, and in general, the

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development of these SSF technologies26. In general, SSF systems are batch processes where

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the substrates are loaded entirely at time zero and remaining static through the fermentation.

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This approach results effective at lab scale, but the scale-up of a SSF process induce

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significant changes due to the complex mass and heat transfer phenomena existing in the

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solid-liquid-gas interphases30 hindering the progress of the SSF at higher scales.

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Consequently, the application of suitable strategies capable of limiting these adverse effects is

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crucial to the development of any SSF-based bioprocess.

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Thereby, the use of alternative operational strategies (compare with the batch mode) has been

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assessed to enhance the SSF performance. For instance, fed-batch operations (FB) have been

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used to promote a better microbial growth by fractioning the nutrients availability by using

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partial feedings31,32, or sequential-batch (SB) as a reliable way to operate SSF processes in

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semi-continuous regime33,34. Consequently, the integration of organic residues as no-cost

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feedstock with more efficient operational strategies contributes to improving the bioprocess

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performance and sustainability, as well as in the industrial advance of the technology in the

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framework of the green chemistry and circular economy35,36.

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This study aimed to evaluate the use of fed-batch and sequential-batch strategies to improve

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the combined 2-PE, and 2-PEA bioproduction in a SSF residue-based system using the agro-

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industrial leftover sugarcane bagasse (SCB) supplemented with L-phenylalanine as substrate

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and K. marxianus as inoculum. In addition, it was examined how the efficiency of these

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strategies was affected due to an increase in the scale. With this target, the above strategies

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were evaluated using the non-sterilized substrate at 1.6 L and 22 L implying an increase of

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eightfold the mass used at 1.6 L scale. In both cases, reactors were neither isolated nor

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temperature-controlled. Therefore, this is one of the few reports dealing with the use of



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alternative operational modes in SSF systems, as well as one of the few SSF bioprocess (using

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specific inoculum) that works at bench-scale and under not-sterile conditions. Thus, the

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proposed strategies constitute one further step towards the development of more sustainable

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and efficient bioprocesses for producing 2-PE and 2-PEA by using residue-based systems.

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Material and methods Inoculum

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Kluyveromyces marxianus (ATCC 10022) was obtained from Colección Española de Cultivos

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Tipo (CECT, Valencia, Spain). K. marxianus was grown at 30°C for 20 h on agar slants

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comprising: glucose (40 g L-1), yeast extract (5 g L-1), soy peptone (5 g L-1) and agar (20 g L-

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1)

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during 30 min). K. marxianus was preserved at -80°C in cryovials containing saturated pearls

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with the strain. Inoculum preparation consisted of putting one pearl into a 250 mL Erlenmeyer

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flask with 100 mL of a media consisted of glucose (40 g L-1), yeast extract (5 g L-1), soy

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peptone (5 g L-1). This culture was incubated at 30°C and 180 rpm during 20 h in a rotary

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shaker. Once grown, it was used to inoculate the non-sterilized prepared substrate in the

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different performed experiments.

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under sterile conditions (media and materials were sterilized by autoclaving at 120°C

Preparation of the solid substrate

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Sugarcane bagasse (SCB) (supplied by the factory Ingenio Ntra. Sra. del Carmen (Málaga,

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Spain)) was first dried at 60°C overnight in an air oven. The dried SCB was ground using a

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granulator mill obtaining a particle size distribution between 0.5-32 mm. Then, it was stored

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at -20°C until used. Moisture, pH and L-phenylalanine content of the solid media were

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adjusted to the initial conditions required in each experiment (see result and discussion

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section) by using a 1:1 (v:v) mixture of a phosphate buffer pH 7 (0.1 M) and a nutrient

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solution including 1.5 g Fe(NO3)3.9H2O L-1, 0.8 g ZnSO4.7H2O L-1, 0.4 g MnSO4.4H20 L-1

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and 3.0 g MgSO4.7H2O L-1. L-phenylalanine was added to this solution, and once it was


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dissolved, the solution was mixed with the dried SCB. Once obtained the prepared substrate

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(Figure S1, Supporting Information), it was inoculated using 108 colony forming units of K.

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marxianus (CFU) per gram of total solids content (TS) of substrate (g TS).

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SSF experiments

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Table 1 contains the main description of the evaluated operational strategies.

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Table 1.

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Bench-scale 1.6 L reactors

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Reaction system consisted of three polyvinyl chloride cylindrical bioreactors of 1.6 L working

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volume. Reactors were provided with a metallic net in the bottom, so the solid substrate was

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held on it. Air entered at the bottom, and it was conducted through the solid bed until it

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reached the top. The system monitored the temperature of the solid media at the midpoint of

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the bed (Pt-100 sensors, Sensotrans), and the exhausted gases were conducted to an oxygen

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sensor (αLphase Ltd.) connected to a self-made data acquisition system (Arduino®-based)

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that recorded oxygen concentrations and temperature each minute (Figure S2, Supporting

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Information). The respirometric analysis consisted of computing the oxygen uptake rate

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(OUR) and the cumulative oxygen consumption (COC) as stated by Ponsá et al.37. For fed-

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batch tests, the addition of fresh material was done by emptying the reactor, and manually,

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mixing the content with the fresh material into 5 L plastic trays. Then, the mixture was

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quantitatively loaded back into the reactor. For sequential-batch tests, substrate replacement

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was performed by putting the withdrawn content into 5 L plastic trays, and then, manually

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mixing the predefined fractions of fermented material and the fresh substrate. Then, the mixed

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substrate was loaded back into the reactor.

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Bench-scale 22 L reactor

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Experiments were carried out in a cylindrical 22 L stainless steel reactor with an automatic

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helical ribbon mixer, and a removable inner basket where the prepared substrate was placed as



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detailed by Martínez et al.26. The system maintained the same geometry and height to

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diameter ratio (h:) to the 1.6 L system (h: of 2). The reactor was filled up to 90% of his

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capacity, and it was monitored similarly to the 1.6 L reactors. For fed-batch tests, the fresh

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material was added directly into the reactor basket, and the content was mixed at 14 rpm for 5

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min. For sequential-batch tests, replacement of the reactor content was performed by taking

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out the removable basket and leaving all the withdrawn content into a 25 L tray. Then, after

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repositioning the removable basket into the reactor, the predefined fractions of fermented

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material and fresh substrate were loaded back into the reactor´s basket. Once the reactor was

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loaded, the content was mixed at 14 rpm for 5 min.

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Analytical methods 2-PE and 2-PEA content in the gas phase

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The 2-PE and 2-PEA content in the exhaust gases of the systems were determined by thermal

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desorption gas chromatography mass spectrometry (TD-GC-MS) as described by Martínez et

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al.24 (Supporting Information).

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L-phe, 2-PE, and 2-PEA content in the solid phase

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L-phe, 2-PE, and 2-PEA concentrations in the solid phase were quantified by HPLC (high-

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performance liquid chromatography) (Ultimate 3000, ThermoFisher) using a reverse phase

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Supelcosil LC-18 column (250 mm length, 4.6 mm diameter, 5µm particle size) as detailed by

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Martínez et al.24 after a solid-liquid extraction (Supporting Information).

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Sugar content

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Reducing sugars of the solid substrate were estimated using the DNS method38 on the

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supernatant obtained after a solid-liquid extraction of the fermented substrate using distilled

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water in a 1:7 (w/v) ratio at 50°C for 30 min. The supernatant was filtered through a 0.45 µm

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membrane and adequately diluted before its analysis. Concentrations were computed based on

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calibration curves using glucose as reference standard in the range 0.2-2.0 g glucose L-1.


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K. marxianus population

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K. marxianus population was determined as described by Martínez et al.24 after the solid-

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liquid extraction of 10 g of solid sample with 100 mL of a 9 g NaCl L-1 solution (Supporting

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Information).

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pH and moisture content

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Moisture content (MC), total solids (TS), volatile solids (VS) and pH have been measured

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according to the standard procedures39.

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Statistical analysis

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Statistical differences of the assessed strategies were analyzed using a one-way ANOVA

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(p

Fed-Batch and Sequential-Batch Approaches To Enhance the

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