Recovery of Natural Î±-Ionone from Fermentation Broth - American

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Chemistry and Biology of Aroma and Taste

Recovery of natural #-ionone from fermentation broth Ilya Lukin, Guido Jach, Isabell Wingartz, Peter Welters, and Gerhard Schembecker J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b07270 • Publication Date (Web): 13 Mar 2019 Downloaded from http://pubs.acs.org on March 20, 2019

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Journal of Agricultural and Food Chemistry

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Recovery of natural α-ionone from fermentation broth

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Ilya Lukin1, Guido Jach2, Isabell Wingartz1, Peter Welters², Gerhard Schembecker1*

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1Laboratory

of Plant and Process Design, Department of Biochemical and Chemical Engineering, TU Dortmund University, Emil-Figge-Strasse 70, D-44227 Dortmund, Germany

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2Phytowelt

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*Corresponding author: E-mail address: [email protected] Tel.: +49 (0)231 755 2338; Fax: +49 (0)231 755 2341

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Keywords

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natural aromas, ionone, metabolic engineering, downstream process, process development

Green Technologies GmbH, Kölsumer Weg 33, D-41334 Nettetal, Germany

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Abstract

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Recently, the market value of aromas has constantly been rising. As the supply from natural feedstock is

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limited, the biotechnological production receives more interest. So far, only a few attempts have been

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made to produce α-ionone, a valued essential aroma of raspberry, biotechnologically. This study reports

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a production process for enantiopure (R)-α-ionone from lab scale (2 L – 150 L) with typical titer of 285

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mg/L-broth to industrial scale (up to 10,000 L) with a titer up to 400 mg/L-broth focusing on the

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development of a downstream process with maximized yield at minimized effort. The developed recovery

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consists of solid-liquid extraction from the biomass at φ = 0.4 g-n-hexane/g-biomass, for 90 minutes at

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ambient temperature and of adsorption from the aqueous supernatant at Φ = 0.5 g-DiaionHP20/mg-

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α-ionone followed by the desorption at Ψ = 30 g-n-hexane/g-DiaionHP20. Altogether, natural α-ionone

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could be gained in substantial quantity and purity of > 95 %.

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1

Introduction

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Over the past few decades, flavors and fragrances (F&F) or aromas have become profitable fine chemicals

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with a continually growing market value due to their increasing use in goods like foods, feeds and

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beverages, household products, cosmetics and toiletries, perfumery and even pharmaceuticals1.

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Moreover, there is a strongly growing consumer-driven demand of the aroma industry to provide and use

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natural compounds in their formulations2–4. Unfortunately, the availability of such aromas in natural

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sources is limited, and their recovery is expensive. One way to increase the availability and by that to

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decrease the costs of natural aromas is the application of plant biotechnology tools. Streamlining the

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metabolite flows towards the desired path can immensely increase the aroma production in the plants.

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The more profound understanding and control of regulatory mechanisms of secondary metabolism can

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reduce seasonal and regional variations of aroma in the natural feedstock. Introduction of additional

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resistances against environmental changes can lead to less crop loss. Although potent, plant biotechnology

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is still limited by long generation times, large space needs, broad byproduct spectrum, difficult product

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recovery, and general customer skepticism. Especially the recent developments in the modern analytics

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and genetic engineering opened a way for biotechnological aroma production as a serious long-term

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alternative to natural extraction5. On the one hand, a lot of bacteria and fungi are known to produce

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commercially valuable aromas naturally4. On the other hand, the “working horses” of white biotechnology

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like E. coli or S. cerevisae can be easily modified to produce the desired aroma compound in high quality

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and purity. Biotechnological aroma production is rapid, highly reproducible, flexibly scalable, can be

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carried out at mild conditions, and uses renewable substrates like sugar or even agricultural residual

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streams. It yields an enantiopure product and does not produce any toxic waste.6,7 Besides other

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advantages, biotechnologically derived aromas can be labeled as natural8 leading to a strong market

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advantage for F&F companies.

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Among various F&F compounds, the class of norisoprenoids, which are derived from the enzymatic

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cleavage of carotenoids, is in the focus of current research. In particular, α-ionone, a key flavor of ACS Paragon Plus Environment

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raspberry9–11 and blackberry12, is an important aromatic compound with a worldwide use on a scale of 100

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to 1000 tons per year13. Having an extremely low odor threshold of 0.4 ppb14 - 3.2 ppb15 and warm, flowery

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scents with a note of violet16, α-ionone is valued in cosmetics and perfumery13. As the most natural aromas,

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α-ionone is a secondary metabolite of numerous plants, and few fungi or bacteria. Naturally, it results

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from oxidative cleavage of precursors α-carotene or ε-carotene17. Although α-carotene is almost

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ubiquitous, ε-carotene is present in few plants only18. In natural sources, α-ionone is found in minor

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quantities, with concentrations ranging from some ppb to few ppm, and in combination with the

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dominating ß-ionone, which makes its extraction from natural stock tedious and expensive9,19. Combined

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with the concerns on supply security and regional as well as seasonal stock variations, this has led to the

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fact, that most of α-ionone on the market is produced chemically nowadays6,7. However, chemical

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synthesis is non-sustainable, consumes much energy, cannot be labeled natural and results in racemic

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mixtures7. The latter, in particular, is problematic as the (S)-enantiomer of α-ionone possesses undesirable

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scent properties making the resulting product less worth. Hence, there is a strong industry demand for

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natural, enantiomeric pure (R)-α-ionone.

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So far, there have been only a few attempts on the biochemical production of enantiopure (R)-α-ionone

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and only one supplier of naturally labeled α-ionone could be found20. The company Phytowelt developed

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2015 the first patent explicitly pending fermentative production system for pure (R)-α-ionone, which

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already has been brought to technical scale21. The first ever patent, disclosing the possibility of

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biotechnological production of carotenoids and their derivatives, as e.g. α-ionone, via fermentation of

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genetically modified Y. lipolytica or E. coli, also dates 201522. The most recent breakthrough in the

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biotechnological production of α-ionone has been reported 2018 by Zhang et al.23. The research group

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designed an E. coli based “plug-and-play” system for the production of α-ionone, β-ionone, psi-ionone,

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retinal and retinol. In small-scale (250 mL) fed-batch biphasic fermentations with isopropyl myristate as



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the second organic phase for an in-situ product removal a titer of 480 mg/L α-ionone has been reported.

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Unfortunately, substantial amounts of β-ionone and psi-ionone were found in the reported broth,

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lowering the purity of the final product. Besides, as the most used route for the chemical synthesis of

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α-ionone is the well-known acid catalyzed cyclisation of psi- or pseudoionone24, its presence makes the

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final natural product indistinguishable from the chemically synthesized.

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Although the fermentation of aroma compounds is quite advantageous, its recovery from crude

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biochemical broth can be challenging. As we previously described, there is a lot of information available

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about the lab-scale analytical aroma recovery from the fermentation broth as well as about the industrial

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aroma recovery from plant sources. For instants, solid adsorbents were used on a lab-scale for the

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recovery, concentration, and fractionation of aromas for several decades25,26. In the form of solid-phase

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microextraction (SPME) adsorbents are currently still the most used lab-scale technique prior to the

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chromatographic analytics of aromas27. In the case of industrial scale application, hydro-distillation and

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solvent extraction are widely used for the recovery of aromas from plant material28. However, there is a

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gap in transferring the lab-scale techniques such as adsorption to an industrial aroma recovery from the

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fermentation broth as not all traditional aroma recovery techniques are suitable for the processing of

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highly diluted, solid containing fermentation broth. Product deterioration, high energy consumption, or

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high costs are only some challenges to name.29 In food and beverage processing, supercritical extraction

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and membrane technology became state-of-the-art techniques for the recovery of volatile compounds like

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aromas30,31. Dealing with biotechnologically produced aromas the recovery task strongly depends on the

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product location and the phase distribution determined by the product’s physical properties like

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hydrophobicity and volatility. The systematic development of a suitable recovery technique for the

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biochemically produced aroma compounds can help to bridge the gap from lab-scale research to economic

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industrial scale production.


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In this work, enantiopure (R)-α-ionone was produced by fermentation of metabolically engineered E. coli

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using patented technology21 comprising the use of plant-based heterologous enzyme cascade for

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ε-carotene production and subsequent α-ionone release via carotenoid-cleavage dioxygenase activity. A

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simple one-phase fed-batch fermentation was improved beforehand to generate a sufficient amount of

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product containing biomass. Besides, a robust downstream process for the recovery and purification of

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α-ionone from the fermentation broth was systematically developed with the aim to maximize the product

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yield at the minimized effort. This research reports for the first time the combination of the optimized

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enantiopure production and yield maximized recovery leading to an overall technical scale process for

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sustainable production of natural (R)-α-ionone in sufficient quantity and market suitable purity.

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2

Materials and Methods

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2.1 Strains and fermentation

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Phytowelt´s proprietary (R)-α-ionone producing E. coli strain EcPHY-G81 was used throughout the process

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development to gain ionone containing biomass samples. This plasmid-free strain harbors the full enzyme

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cascade required for ionone production in its genome and does not require the use of antibiotics or

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inducers during the fermentation procedure. The strain expresses the genes crtB, crtE and crtI from

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E. herbicola under the control of the proprietary promoter and terminator sequences to implement

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required enzyme activities (geranylgeranyl-pyrophosphate synthase, phytoene synthase, and phytoene

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desaturase) for the formation of intermediate lycopene. Additional expression of a mutated plant

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lycopene epsilon-cyclase as well as a mutated plant carotenoid cleavage dioxygenase leads to quantitative

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conversion of lycopene to the intermediate epsilon-carotene and then to the final product (R)-α-ionone.

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Fed-batch fermentations were conducted at 26 °C over 120 h at 2 L – 150 L scale in different experiments

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using the media and protocol described by Riesenberg et al.32. Feeding rates were adapted to achieve a

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reduced growth rate and elongated fermentation times. Finally reached biomass concentration was in the

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range of 60 – 70 g-CDW/L.

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2.2 Phase separation

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The phase separation of the fermentation broth was done in a laboratory centrifuge (Centrifuge 5804 R,

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Eppendorf, Germany ) at 4500 rpm (3622 rcf) at 25 °C using Falcon™ tubes.

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2.3 Product recovery

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All extraction and adsorption experiments were performed on an overhead shaker (PTR-60, Grant-bio, UK)

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at 50 rpm and ambient temperature using 15 mL Falcon™ tubes. The recovery of α-ionone during the

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fermentation was made via extraction with ethyl acetate. The solvent was added to the fermentation

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slurry at a phase ratio of 0.2 g-solvent/g-CWW. The two-stage extraction was carried out for 30 minutes

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per stage. Fresh solvent was used in every extraction stage. After the phase separation, the solvents of

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both stages were pooled and analyzed for α-ionone concentration using HPLC. During the optimization of

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the preparative α-ionone extraction different solvents, phase ratios, temperatures, extraction times and

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a number of stages were varied. Therefore, the fermentation broth was separated into the solid and

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aqueous phases, and the solvent was added to the wet biomass at a given phase ratio, and the extraction

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was carried out at defined conditions. For all experiments, the extraction was performed for 90 minutes

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at ambient temperature. For the screening of the extraction kinetics, the extraction time was varied from

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10 minutes to 120 minutes. For the investigations of the temperature effect, the overhead shaker was

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placed in a temperature controlled incubator. After the extraction, the samples were centrifuged at

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4500 rpm (3622 rcf) for 10 minutes, the phases were separated, and the solvent was analyzed for α-ionone

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concentration using HPLC.

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α-ionone was recovered from the aqueous supernatant by adsorption on solid resins. Different types of

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adsorbents were added to the cell-free aqueous supernatant at a defined ratio and mixed at ambient

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temperature overnight. Afterward, the samples were centrifuged at 4500 rpm (3622 rcf) for 10 minutes,

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and the adsorbent was collected. α-ionone was desorbed by adding a solvent at a defined ratio to the

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resin. The samples were mixed overnight. After the phase separation (4500 rpm (3622 rcf) for 10 minutes)

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the solvent phase was collected for the α-ionone analysis using HPLC.



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2.4 Analytics

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α-ionone concentration was determined by HPLC analysis (Knauer, Germany) using a

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NUCLEODUR™ 250x4 C8ec column (Macherey-Nagel, Germany). Acetonitrile (HiPerSolv

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CHROMANORM®, VWR International, USA) and Millipore Water were used for elution. The

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solvents were mixed with 1 % v/v acetic acid (GPR Rectapur 99 – 100 %, VWR Chemicals, USA)

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and stored separately. The oven temperature was 40 °C and the following gradient was applied:

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starting at 60 % ACN isocratic for 4 min to 100 % ACN gradient for 4 min to 100 % ACN isocratic

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for 1 min and finally to 60 % ACN equilibration step for 6 min. The approximate total retention

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time of α-ionone was 12.2 min. The concentration was measured by a DAD detector at 245 nm

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(DAD Knauer, Germany) according to prior calibration with analytical standard α-ionone

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(Sigma-Aldrich/Merck, Germany).


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3

Results and discussion

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The production strain has been consecutively optimized for the biosynthesis of the desired target product.

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The strain used in this paper has been chosen because it proved to be remarkably robust through all stages

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of up-scaling with highly reproducible results in all fermentation experiments. The main parameters of the

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fermentation process like media components, feeding strategy, temperature, and duration, are commonly

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used measures to achieve the highest product yields and economically feasible processes. These issues

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have already been addressed beforehand and lead to the fermentative process being used in this study to

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generate representative samples for downstream process development and evaluation.

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3.1 Fermentative α-ionone production

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A typical result of the fermentation of the used strain PHY-G81 at 2 – 150 L scale is given in figure 1. Cells

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show steady growth over 123 h of fermentation time, with a somewhat slower growth after about 72 h.

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The yield of α-ionone also increased with the time, but at a steeper slope, indicating increasing specific

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productivity of the cells over time. Highest product titer was 282 mg/L (123 h). Further elongation of the

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fermentation is likely to give even high titers. However, reaching the maximum volume of broth possible

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for the given fermentation vessel limits the possible fermentation times. Scale up trials were successfully

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accomplished and titers of 400 mg/L were reached at 10.000 L scale within 120 h fermentation time using

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this process.

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Carotenoid cleavage enzymes frequently used to release ionones from carotenoid precursor are often

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promiscuous and accept a range of different carotenoids as substrates, which leads to the formation of

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side-products such as pseudoionone (via cleavage of lycopene). The presence of pseudoionone in high

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concentrations causes, in fact, severe problems, as it cannot be separated from the desired α-ionone at

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reasonable costs and labor due to its quite similar chemical properties. Thus, the production of α-ionone ACS Paragon Plus Environment


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to the desired purity (> 95%) can be severely hampered. In our process, pseudoionone concentrations

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remained typically around 1-2 % and represented no obstacle for the downstream process.

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Thus, the upstream part already works satisfactorily at production scale. With respect to the economics of

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the whole production process, however, extraction and purification of the desired product are of equal

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importance to gain yield maximized production/recovery of natural α-ionone at lowest costs, which is the

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focus of the work described here.

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3.2 Determination of product phase distribution

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In order to develop an industrially suitable α-ionone downstream process, knowledge about the product’s

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phase distribution is crucial. We previously reviewed the choice of potential techniques for the recovery

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and purification of volatile aroma compounds depending on their phase partitioning and physical

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properties29. As a semivolatile (Hpc = 4.5x10-5 m³atm/mol33) but rather hydrophobic (logKow = 3.8534)

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compound, α-ionone can partition between all phases of a fermentation broth. The molecule can stick to

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the hydrophobic biomass, be diluted into the aqueous supernatant up to its saturation and can evaporate

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into the headspace of the bioreactor. The maximum product content in the fermentation broth was

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determined by leaching extraction of the slurry at high phase ratio. By performing the extraction of the

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separated phases at the same conditions, the products partitioning between biomass and aqueous

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supernatant was evaluated. Because of the limited aqueous solubility of α-ionone (csat = 0.1 g/L35), 85 –

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92 % w/w of the molecule is found in or on the biomass leading to the higher phase specific yield (figure

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2) while the remainder is solved in the fermentation supernatant. At fermentation conditions

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(1 vvm, T = 26 °C) α-ionone has also been qualitatively detected in the reactor’s off-gas at around 2 – 5 %.

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The product losses through the off-gas were, as expected, rather low ranging around 2 – 5 %. Following

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the guidelines proposed by the WHO36 based on the boiling temperature, α-Ionone, with the boiling point

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of 256 °C37 under standard conditions, can be classified as semi volatile organic compound with a rather


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slow volatilization from aqueous media. The Henry’s law volatility coefficient of α-ionone is three orders

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of magnitude lower than e.g. of limonene (Hpc = 1.4 – 5.8x10-2 m³atm/mol33), a common highly volatile

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perfumes top note aroma. For that reason, the process development is focused on the product recovery

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from the biomass. In order to maximize the yield, the possibilities of the product recovery from the

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fermentation supernatant are explored.

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3.3 Development of α-ionone recovery process

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With the majority of the product sticking to the biomass (85 – 92 % w/w), a solid-liquid solvent extraction

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seems to be the most suitable recovery technique. In general, product extraction could be done either

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way, after the phase separation from the solid phase only, or via simultaneous extraction of both phases

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through the application of a water immiscible solvent. Because of the biomass concentration in the

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fermentation slurry of around cx = 60 – 70 g-CDW/L and the limited aqueous solubility of α-ionone, the

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specific product yield of the slurry 𝑌𝑠𝑙𝑢𝑟𝑟𝑦 = 0.22 ± 0.01 mg/g-slurry is much lower than that of a solid phase 𝑝

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𝑌𝑠𝑜𝑙𝑖𝑑 = 2.28 ± 0.1 mg/g-solids. In this case, the solvent consumption and the specific recovery costs of the 𝑝

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slurry extraction are much higher than that of the biomass extraction. Thus, a solid-liquid phase separation

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is applied to the fermentation broth prior to the extraction step, and α-ionone is extracted from the

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biomass only using a suitable solvent.

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3.3.1

Product recovery from the solid phase

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The success of an extraction largely depends on solvent selection. High affinity towards and high capacity

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for the product, broad availability and low costs, low viscosity and evaporation enthalpy are the common

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criteria for the solvent screening38. In addition, in the case of a natural product, the solvent should meet

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the regulations. A way to lower the experimental effort screening the organic solvents for an extraction

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task is the use of the solvent pre-selection tool MOPASOOL© based on a systematic procedure described ACS Paragon Plus Environment


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by Bergs et al.39,40. The tool ranks the possible performance of the solvents from a database according to

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the calculated hydrophobicity similarities between the desired target compound and the solvent following

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the principle “like dissolves in like”. Restrictions to specific application areas, like for green or foodstuff

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solvents, further reduce the number of potential solvents to screen. The tool supports the selection

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process narrowing the range of possibilities to search for the best performing solvent, however, does not

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predict any recoveries. The results of the solvent ranking for α-ionone extraction as well as their

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experimental performance can be seen in figure 3. It is evident that solvent performance is estimated to

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increase from hydrophilic to hydrophobic solvents. As the MOPASOOL© tool is limited to organic solvents

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only, some hydrophobic plant oils and alcohols were added to the screening.

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In good agreement with the theoretical preselection, the most non-polar solvent n-hexane showed one of

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the highest recoveries. The medium polar acetone, however, performed surprisingly well with the highest

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yield on average. As n-hexane is a non-polar solvent, it does not extract hydrophilic components (all above

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residual water) from the biomass leading to high purity of the extract and is therefore chosen for α-ionone

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extraction from the biomass. Although the plant-derived solvents are ecologically friendly, sustainable,

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and are considered to be food grade, their low extraction performance will not lead to an economic

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α-ionone production process. Especially the high viscosity of plant oils may lead to diffusion limitations of

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the product during the extraction limiting the yield. Besides, they tend to oxidize fast generating

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unpleasant off-flavors41 and may be difficult to separate from the product.

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In order to further maximize the product yield and to minimize the recovery costs, the phase ratio and the

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number of the extraction stages were varied on a narrow scale each parameter at a time as the interactions

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between solvent phase ratio and stage number in a cross-flow extraction is less important as e.g. in

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countercurrent operation. Cross-flow leaching experiments indicate that the most α-ionone is extracted ACS Paragon Plus Environment

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after two stages already at ϕ = 0.5 g-solvent/g-sample. Further reduction of the solvent phase ratio has

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led to the minimum ϕ = 0.4 g-solvent/g-sample. Up to 82 % w/w of the product could be extracted at the

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first stage and the accumulated yield of the first two stages was 96 – 98 % w/w (figure 4). The product’s

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purity was independent of the solvent phase ratio and the number of stages by 98 % peak purity (figure 5).

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The only detected byproduct was pseudoionone, the non-cyclic equivalent of α-ionone derived from the

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cleavage of ε-carotene precursor lycopene. Pseudoionone has very similar physical and chemical

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properties to α-ionone which leads to equal partitioning between the biomass and the extraction solvent.

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For an increased space-time-yield of the process, the extraction time should be minimized as well. The

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measurement of the extraction kinetics showed that the minimal extraction time of α-ionone from the

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biomass should be 90 minutes (figure 6). Although α-ionone has a high affinity towards hydrophobic

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n-hexane, the extraction kinetics seem to be rather slow. The main reason lies presumably in the diffusion

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limitations of α-ionone from the cells to the solvent phase. It is assumed, that α-ionone molecules are

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incorporated into the hydrophobic cell membrane which presents an additional diffusion resistance.

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Moreover, the biomass coming from the solid-liquid separation has a high content of the residual

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moisture. Because n-hexane is immiscible with water, α-Ionone needs to diffuse through a hydrate shell

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of the cells to the bulk solvent phase. In addition, in the presence of the hydrophobic solvent, wet cells

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tend to form agglomerates minimizing the mass transfer area and slowing the extraction kinetics. Vigorous

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mixing using an impeller should help to overcome such limitations.

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Usually, elevated extraction temperatures help to increase product’s diffusion towards solvent bulk phase.

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It is often expected, that effect of temperature increase has a positive interaction with the solvent phase

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ratio. The thermodynamic portioning coefficient of the target compound should increase at elevated

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temperature helping to further reduce the solvent consumption. In order to evaluate the single factor ACS Paragon Plus Environment


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effect, the temperature was varied at three levels while all other parameters previously optimized were

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held constant (figure 7). Surprisingly, here the temperature had no visible effect neither on the α-ionone

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yield nor on extract purity. The extraction equilibrium seems to be strongly shifted towards extract by the

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use of highly hydrophobic solvent n-hexane. As the solvent loss increases with increased extraction

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temperature, it is recommended to perform the extraction at the ambient temperature.

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In summary, the optimal conditions for α-ionone extraction from the biomass at which maximum yield

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could be achieved with minimum solvent consumption, minimum temperature control and in lowest

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process time are estimated as follows: n-hexane at ϕ = 0.4 g-solvent/g-biomass for 90 minutes at ambient

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temperature. The product gained had a market-ready purity of > 95% (figure 5). As the applied solvent n-

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hexane is considered food grade under both the EU and the US legislation with the maximum solvent

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residue in the final product less than 1 – 30 ppm (EU)42 and less than 5 – 25 ppm (US)43, the obtained

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α-ionone can be labeled natural food grade. In order to increase the overall α-ionone yield of the

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downstream process, additional 8 – 15 % w/w of the product should be recovered from the aqueous phase

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too.

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3.3.2 Product recovery from the aqueous phase

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For the recovery of α-ionone from the aqueous supernatant of the fermentation broth, liquid-liquid

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extraction, adsorption on synthetic resins, distillation, pervaporation, and even strip-absorption could be

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applied. As α-ionone is a high-boiling compound (Tb = 256 °C 44) at a low concentration in an aqueous

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matrix, large amounts of low-boiling water will be evaporated during distillation or pervaporation making

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the recovery process expensive. Stripping of a semivolatile product is indeed possible but will be tedious

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and time-consuming as the volatilization will be slow. Liquid-liquid extraction is possible and a water-

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immiscible high hydrophobic solvent as n-hexane could be applied. Taking into account that the aqueous ACS Paragon Plus Environment

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phase takes 95 % w/w of the fermentation broth but contains maximum 15 % w/w of the product, the

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solvent consumption and by that the specific costs of the liquid-liquid extraction will be high. The specific

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yield of the aqueous supernatant is with 𝑌𝑎𝑞𝑢𝑒𝑜𝑢𝑠 = 0.03 ± 0.0 mg/g-phase extremely low. Adsorption of α𝑝

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ionone on synthetic resins may help to reduce the volume of the product containing stream and by that

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the solvent consumption and the costs of the recovery. As it is often the case, at higher concentrations

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aromas may have an inhibitory effect on the producing microorganism. By continuously removing the

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product from the liquid phase in-line, the equilibrium can be shifted towards production increasing the

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overall productivity of the process. Figure 8 shows the results of the screening of selected adsorbents for

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the batch recovery of α-ionone from the aqueous supernatant of a fermentation broth. As the adsorption

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is driven by the available binding surface, adsorbents were added at varied mass providing equal surface

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area. Commonly available non-polar XAD resins are expected to deliver the best performance adsorbing a

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rather hydrophobic target. A polar Diaion HP20, as well as universal activated carbon adsorbent, were

313

added to prove the hypothesis.

314 315

The analysis of the liquid phase after desorption showed that at ψ = 5 m²/ml-broth the fermentation broth

316

had been completely depleted of α-ionone. It is entirely adsorbed by all resins provided. However, the

317

amount of the product that could be recovered depended on the adsorbents used, with the recovery been

318

limited by the desorption step. Stronger hydrophobic resins like XAD showed a lower amount of recovered

319

product. Even when hydrophobic n-hexane is used as a desorption solvent, the strong binding of α-ionone

320

to the resin cannot be overcome. The best desorption rates could be achieved with a least hydrophobic

321

resin Diaion HP20. It was necessary to immensely increase the phase ratio of n-hexane up to

322

Φ = 30 g-solvent/g-resin (figure 9) to reach full desorption.

323

Desorption might be additionally limited by the residual moisture from the adsorption step. The hydrate

324

shell around the resins might present an additional diffusion resistance for the mass transfer of α-ionone ACS Paragon Plus Environment


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from the resin to the bulk solvent phase. For an industrial application, product desorption might be done

326

using a gradient elution starting with a semi-polar solvents like alcohol and switching to n-hexane for full

327

desorption.

328 329

In summary, α-ionone can be adsorbed from the aqueous supernatant of the fermentation broth with

330

Diaion HP20 resin. In a batch experiment, it was necessary to provide at least ψ = 5 m²/ml-broth of binding

331

surface or ψ* = 0.5 g-resin/mg-α-ionone for the maximized yield. In order to reach a full recovery of bound

332

α-ionone from the resin at least Φ = 30 g-solvent/g-resin n-hexane was needed. Due to the high

333

hydrophobicity of n-hexane the obtained product contained only α-ionone and pseudoionone at the same

334

ratio as in the aqueous supernatant of the fermentation broth.

335

336

4

Discussion

337

Besides the usual optimization of biosynthetic capacities of the production strain and the fermentation

338

process parameters the establishment of best-suited procedures for the downstream processing of the

339

desired product (R)-α-ionone is crucial to gain a complete yield maximized production process.

340

Upregulation of lycopene cyclase leads to the faster formation of ε-carotene reducing the amount of

341

residual lycopene and with that of the unwanted byproduct pseudoionone. The developed process

342

presents a way for sustainable production of sufficient quantity of natural enantiopure (R)-α-ionone in

343

market suitable purity. The market price of chemical α-ionone (> 90 %) is with around 100 €/kg rather

344

low45. However, when coming to a natural α-ionone, the price for even low purity product (> 86 %) is with

345

1,390 €/kg more than an order of magnitude higher46. So far, only one bulk supplier offers α-ionone

346

(> 90 %) which is labeled natural under US regulations for around 600 $/kg47. Unfortunately, no explicit

347

information could be found on the source of origin and on the enantiopurity of the product. In order to ACS Paragon Plus Environment

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348

achieve economic competitiveness, the biochemical α-ionone production should be further optimized.

349

One of the bottlenecks of the process developed remains a cell density of up to 70 g-CDW/L which leads

350

to a product titer lower than elsewhere reported. As the most product was found on or in the biomass,

351

the most effort should be put into a further increase in cell density which will lead to lower specific

352

fermentation costs. The highest reported cell density of E. coli fed-batch fermentation was 148 g-CDW/L48.

353

Shiloach and Fass reviewed in their article several methods on the way to high cell density E. coli

354

fermentation. The proposed strategies were alongside the proper choice of growth medium, the intense

355

oxygen supply and the suppression of acetate accumulation during the fermentation.49

356 357

Further process optimization can be done at the interface of the fermentation and downstream process.

358

As the high concentration of α-ionone is known to be inhibitory for E. coli growth23, an in-situ product

359

recovery is an often reported approach to increase the process productivity. As we showed, most of

360

α-ionone is located in or on the cells making an ISPR quite challenging. Recovering the product from the

361

aqueous phase should indeed lead to the partitioning of more product from the biomass through the

362

fermentation supernatant to the ISPR phase. However, as the aqueous solubility of α-ionone is limited,

363

most of the product will still stick to the cells. Nevertheless, successful product removal during the

364

fermentation should immensely increase the overall process productivity. However, economics also have

365

to be taken into account. Often used two-phase fermentation requires sometimes expensive second

366

phases, e.g. n-dodecane. For n-dodecane regeneration, there will be a need for a backextraction of the

367

product with e.g. n-hexane. If the second phase is highly viscose not only the emulsification of such might

368

be challenging on an industrial scale, but also the oxygen transfer into the medium might be limited

369

decreasing the production rate. As we mentioned above, adsorption of α-ionone on a synthetic resin can

370

be easily done in-situ, or more precisely in-line. During the fermentation, a fraction of the filtered cell-free

371

supernatant can be lead through an adsorption column packed with Diaion HP20 resin. The depleted



372

supernatant will be fed back to the reactor. Once the adsorption column has reached its loading capacity,

373

the desorption and equilibration can be done offline. An elegant way to remove α-ionone from both the

374

solid and the liquid phase simultaneously during the production step is an in-situ stripping. Increased

375

aeration and slightly increased fermentation temperature will increase the volatility of the product leading

376

to increased partitioning to the gaseous phase. Besides, such conditions are beneficial for an increased cell

377

grows, as stated previously. After the stripping, the product can be recovered from the reactor’s off-gas

378

using absorption. Especially for an industrial scale production absorption seems to be superior compared

379

to condensation with liquid nitrogen often used on lab scale.

380 381

Increasing supplementation of products with aromas combined with the rising customer's demand for all-

382

natural formulations has led to an increased interest for the biotechnological production of aroma

383

compounds. In particular, many norisoprenoids like α-ionone are in the focus of current research. This

384

work addresses the downstream processing for a given fermentative α-ionone production process.

385

Altogether, the simple and robust overall process for the production, recovery, and purification of natural

386

enantiopure (R)-α-ionone may help to bridge the gap from the lab-scale research to the industrial scale

387

commercialization. The process developed has the potential to be transferred to the production of other

388

natural aroma compounds.

389

Acknowledgment

390

This research was supported by the Federal Ministry for Economic Affairs and Energy (BMWi) of the

391

Federal Republic of Germany (grant No. 16KN065221).

392 393


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Nomenclature

394

𝑐𝑠𝑎𝑡

saturation aqueous solubility

[g/L]

𝑐𝑥

biomass concentration

[g-CDW/L]

𝐻𝑝𝑐

Henry’s law constant

[atm*m³/mol]

𝑙𝑜𝑔𝐾𝑂𝑊

octanol-Water partitioning coefficient

[-]

n

number of replications

[-]

𝑡

time

[min]

𝑇𝑏

boiling point

[°C]

𝑌𝑎𝑞𝑢𝑒𝑜𝑢𝑠 𝑝

specific product yield of aqueous phase

[mg/g-phase]

𝑌𝑠𝑜𝑙𝑖𝑑 𝑝

specific product yield of solid phase

[mg/g-phase]

𝑌𝑠𝑙𝑢𝑟𝑟𝑦 𝑝

specific product yield of slurry

[mg/g-phase]

ϕ

solvent phase ratio

[g-solvent/g-CWW]

Φ

desorbent phase ratio

[g-solvent/g-resin]

ψ

specific adsorbent surface ratio

[m²/ml-broth]

Ψ*

specific adsorbent surface ratio

[m²/mg-α-ionone]

395

396

Abbreviations ACN

Acetonitrile

CCD

Carotine Cleavage Dioxygenase

CDW

Cell Dry Weight

CWW

Cell Wet Weight

DAD

Diode Array Detector

F&F

Flavors and Fragrances

HPLC

High Pressure Liquid Chromatography

ISPR

In-Situ Product Recovery

ppb

Parts Per Billion

rpm rcf

Rotations Per Minute Relative Centrifugal Force

vvm

Volume per Volume per Minute

397



398 399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442

5

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20. Vigon. Ionone Alpha Natural. [Pricing & Purchase]. 2018. Available at: https://www.vigon.com/product/ionone-alpha-natural/. Accessed September 28, 2018. 21. Jach G, Azdouffal S, Schullehner K, Welters P, Natanek A, inventors; Phytowelt Green Technologies GmbH. Method of fermentative α-Ionone production. WO2017036495A1. March 9, 2017. 22. Wang Y, inventor; ACH INNOTEK, LLC. COMPOSITIONS AND METHODS O F BIOSYNTHESIZING CAROTENOIDS AND Compositions and methods of biosynthesizing carotenoids and their derivatives. US WO 2016/154314 A1. 23. Zhang C, Chen X, Lindley ND, Too H-P. A "plug-n-play" modular metabolic system for the production of apocarotenoids. Biotechnology and bioengineering. 2018;115(1):174-183. 24. Royals EE. Cyclization of Pseudoionone by acidic reagents. Industrial & Engineering Chemistry. 1946;38(5):546-548. 25. Parliment TH. Concentration and Fractionation of Aromas on Reverse-Phase Adsorbents. Journal of Agricultural and Food Chemistry. 1981;29(4):836-841. 26. Harper M. Sorbent trapping of volatile organic compounds from air. Journal of Chromatography A. 2000;885(1-2):129-151. 27. Wardencki W, Michulec M, Curylo J. A review of theoretical and practical aspects of solid-phase microextraction in food analysis. International Journal of Food Science and Technology. 2004;39(7):703-717. 28. Reineccius G. 18 Flavour-Isolation Techniques. In: Berger RG, ed. Flavours and Fragrances. Chemistry, Bioprocessing and Sustainability: 13 Chemical Conversions of Natural Precursors. Berlin Heidelberg: Springer; 2007. 29. Lukin I, Merz J, Schembecker G. Techniques for the recovery of volatile aroma compounds from biochemical broth: A review. Flavour and Fragrance Journal. 2018;33(3):203-216. 30. Kazazi H, Rezaei K, Ghotbisharif S, Emamdjomeh Z, Yamini Y. Supercriticial fluid extraction of flavors and fragrances from Hyssopus officinalis L. cultivated in Iran. Food Chemistry. 2007;105(2):805-811. 31. Bundschuh E, Tylla M, Baumann G, Griescher K. Gewinnung von natürlichen Aromen aus Reststoffen der Lebensmittelproduktion mit Hilfe der CO2-hochdruckextraktion. Lebensmittel-Wissenschaft und Technologie;1986(19):493-496. 32. Riesenberg D, Schulz V, Knorre WA, et al. High cell density cultivation of Escherichia coli at controlled specific growth rate. Journal of Biotechnology. 1991;20(1):17-27. 33. Sander R. Compilation of Henry’s law constants (version 4.0) for water as solvent. Atmospheric Chemistry and Physics. 2015;15(8):4399-4981. 34. Griffin S, Wyllie S, Markham J. Determination of octanol–water partition coefficient for terpenoids using reversed-phase high-performance liquid chromatography. Journal of Chromatography A. 1999;864(2):221-228. 35. Etzweiler F, Senn E, Schmidt HWH. Method for measuring aqueous solubilities of organic compounds. Analytical Chemistry. 1995;67(3):655-658. 36. World Health Organization. Indoor air quality: organic pollutants: Report on a WHO Meeting Berlin 23-27 August 1987. Copenhagen: World Health Organization Regional Office for Europe; 1989; EURO SReports and Sutdies 111. 37. Sell CS. Terpenoids. In: Inc JW&S, ed. Kirk-Othmer Encyclopedia of Chemical Technology. Hoboken, NJ, USA: John Wiley & Sons, Inc; 2000. 38. Sattler KD. Thermische Trennverfahren: Grundlagen, Auslegung, Apparate. 3rd ed. Weinheim: WileyVCH; 2001.



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39. Bergs D, Merz J, Delp A, Joehnck M, Martin G, Schembecker G. A Standard Procedure for the Selection of Solvents for Natural Plant Extraction in the Early Stages of Process Development. Chemical Engineering & Technology. 2013;36(10):1739-1748. 40. Bergs D. A contribution to chromatographic purification of natural products. 1. Aufl. München: Verl. Dr. Hut; 2013. Schriftenreihe Anlagen- und Prozesstechnik; 7. 41. Choe E, Min DB. Mechanisms and factors for edible oil oxidation. Comprehensive Reviews in Food Science and Food Safety. 2006;5(4):169-186. 42. The European parliament and the council of the European Union. DIRECTIVE 2009/32/EC OF THE EUROPEAN PARLIAMENT AND OF THE COUNCIL of 23 April 2009 on the approximation of the laws of the Member States on extraction solvents used in the production of foodstuffs and food ingredients. 2010. Available at: https://eur-lex.europa.eu/legal-content/EN/TXT/?uri=CELEX:02009L003220100916. Accessed December 10, 2018. 43. U.S. Food & Drug Administration. Food Additive Status List. 2018. Available at: https://www.fda.gov/Food/IngredientsPackagingLabeling/FoodAdditivesIngredients/ucm091048.ht m#ftnH. Accessed December 10, 2018. 44. Sell CS. Terpenoids. In: John Wiley & Sons Inc, ed. Kirk-Othmer Encyclopedia of Chemical Technology. Hoboken, NJ, USA: John Wiley & Sons, Inc; 2000. 45. Merck KgaA. α-Ionone: ≥90%, stabilized. 2019. Available at: https://www.sigmaaldrich.com/catalog/product/aldrich/w259403?lang=de®ion=DE&cm_sp=Insit e-_-prodRecCold_xviews-_-prodRecCold5-3. Accessed 19.02.19. 46. Merck KgaA. α-Ionone: natural, ≥86%. Available at: https://www.sigmaaldrich.com/catalog/product/aldrich/w259411?lang=de®ion=DE&cm_sp=Insit e-_-prodRecCold_xviews-_-prodRecCold5-2. Accessed 19.02.19. 47. Vigon. Ionone Alpha Natural: Pricing & Purchase. 2018. Available at: https://www.vigon.com/product/ionone-alpha-natural/. Accessed September 28, 2018. 48. Korz DJ, Rinas U, Hellmuth K, Sanders EA, Deckwer W-D. Simple fed-batch technique for high cell density cultivation of Escherichia coli. Journal of Biotechnology. 1995;39(1):59-65. 49. Shiloach J, Fass R. Growing E. coli to high cell density - a historical perspective on method development. Biotechnology advances. 2005;23(5):345-357.


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518 519


Figure Captions

520 521

Figure 1: Typical results for the fermentation using the patent pending α-ionone process delivering samples for this study.

522 523

Figure 2: Distribution of α-ionone between different phases of a fermentation broth. Mass of α-ionone extracted from separated phases and from native fermentation slurry (n-hexane,

524

ϕ = 1.0 g-solvent/g-sample, 90 minutes, one stage; n = 2).

525 526 527

Figure 3: Solvent screening for the extraction of α-ionone from the biomass (ϕ = 1.0 g-solvent/g-sample, 90 minutes; n = 2). Inlay shows the solvent ranking according to MOPASOOL© based on hydrophobicity calculations.

528 529

Figure 4: Solvent phase ratio screening. Yield of α-ionone for the extraction of wet cells with n-hexane at different solvent phase ratios for up to four stages (t = 90 min; n = 2).

530

Figure 5: HPLC chromatogram (RP C8ec 250x7 mm; H2O:ACN gradient elution) for the solvent extraction

531 532

of the biomass (n-hexane, 90 minutes, ϕ = 0.4 g-solvent/g-biomass, ambient temperature). The inlay shows an absorption spectrum scan of the peak 2.

533 534

Figure 6: Kinetics of α-ionone solvent extraction from the biomass (n-hexane, ϕ = 0.4 g-solvent/g-sample; n = 2).

535

Figure 7: Screening of the extraction temperature. Yield and purity of α-ionone for the extraction from

536

the biomass at different temperatures (n-hexane, ϕ = 0.4 g-solvent/g-biomass, 90 minutes; n = 2).

537 538

Figure 8: Adsorbent screening for the recovery of α-ionone from the aqueous supernatant of a fermentation broth (adsorption: 5 ml sample, overnight (t > 8 h); desorption: n-hexane,

539

Φ = 10 g-solvent/g-adsorber, overnight (t > 8 h); n = 2).

540

Figure 9: Screening of the desorbent phase ratio for the recovery of α-ionone from the aqueous

541 542

supernatant of a fermentation broth (adsorption: Diaion HP20, ψ = 5 m²/ml-broth, overnight (t > 8 h); desorption: n-hexane, overnight (t > 8 h) per step; n = 2).

543

TOC figure: Production and recovery of natural enantiopure (R)-α-ionone from fermentation broth.



figure1

figure 2


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figure 3

figure 4



figure 5

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figure 7

figure 8



figure 9

TOC figure


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Recovery of Natural Î±-Ionone from Fermentation Broth - American

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