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Novel intensified back extraction process for Itaconic Acid – towards in-situ product recovery for Itaconic acid fermentation Guneet Kaur, Miranda Maesen, Linsey Garcia-Gonzalez, Heleen De Wever, and Kathy Elst ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.7b04874 • Publication Date (Web): 26 Mar 2018 Downloaded from http://pubs.acs.org on March 26, 2018

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ACS Sustainable Chemistry & Engineering

Novel intensified back extraction process for Itaconic Acid – towards in-situ product recovery for Itaconic acid fermentation

Guneet Kaur1*, Miranda Maesen2, Linsey Garcia-Gonzalez2, Heleen De Wever2, Kathy Elst2* 1

Sino-Forest Applied Research Centre for Pearl River Delta Environment & Department of Biology,

Hong Kong Baptist University, Kowloon Tong, Kowloon, Hong Kong 2

Flemish Institute for Technological Research (VITO), Business Unit Separation and Conversion

Technology, Boeretang 200, 2400 Mol, Belgium *Corresponding author Email: [email protected], [email protected]; Tel: +852 54459953 (Guneet Kaur) Email: [email protected]; Tel.: +32 14335617 (Kathy Elst)

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Abstract

Itaconic acid (IA), an unsaturated dicarboxylic acid produced by fermentation, is a promising alternative to petrochemical-based monomers as building block for plastics, resins and synthetic fibers. Efficient recovery of IA from aqueous fermentation broth was previously achieved by aminebased reactive extraction (RE) systems. In the present work, several back extraction methods were tested in order to recover IA from four different RE solutions, three based on trioctylamine and the diluents methyloctanoate, pentylacetate and 1-octanol, and one based on N-methyldioctylamine and the diluent 1-octanol. Conventional back extraction methods using a temperature swing, NaOH or tertiary volatile amines were applied and tested at different conditions. Especially with tertiary volatile amines good back extraction efficiencies were achieved. As an intensified approach, in addition a novel back extraction-conversion method was developed to recover the itaconic acid in the form of methyl-esters. This approach was based on non-catalyzed in-situ esterification with high temperature-pressure methanol (HTPM) allowing a continuous processing. Reaction temperature, residence time, pressure and methanol excess were investigated. At 200-250 oC and residence time 10-20 min, with methanol dosed at a similar weight as the RE-layer, ester formation of >80 mole% could be obtained with a continuous esterification process. This latter method can be a suitable alternative technique for standard back extraction procedures, aiming at an easy recovery of the IA ester through distillation, followed by a direct polymerization to bioplastics.

Keywords: In-situ esterification, reactive extraction, solvent toxicity, trioctylamine, industrial biotechnology


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Introduction Itaconic acid (IA) is a versatile organic compound which has been identified by the US Department of Energy as one of the 12 promising bio-based chemicals or building blocks that can be produced by biocatalysis and be converted into a multitude of high value-added derivatives.1 IA is particularly attractive as a substitute for petroleum-based compounds such as acrylic acid and methacrylic acid, because it is equally monosaturated and therefore can be easily polymerized by addition to be used for synthesis of polymers and chemicals.2 IA finds other myriad applications such as in paints, detergents, films, thickeners, and bioactive compounds. The current market for IA is estimated to be 80 kton/year with further potential to increase.3 Thus, IA is an industrially important product. The biotechnological production method can produce up to 80 g/L IA but severe product inhibition has been observed at IA concentrations as low as 20 g/L.4 To be competitive with petro-based processes, improvement in terms of yield, titer and productivity needs to be achieved for bio-based processes. The state-of-the-art downstream processing for IA often leads to problems with purity or yield of IA due to its high polarity. Additionally, the use of crystallization as the recovery method is unfavorable because it is a cost and energy intensive process. Considering that downstream processing accounts for almost 40% of total production costs of organic acids such as IA, the development of efficient recovery methods remains essential.5 While new and innovative downstream concepts are constantly being developed, they are usually sub-optimally implemented since their interactions with upstream process are neglected at an early stage of process design, wherein critical decisions are being made. Thus, an integrated approach for process development in which both upstream and downstream are simultaneously considered, has enormous potential for process intensification. Such a provision is made by In-situ Product Recovery (ISPR) which can bring additional benefits such as alleviation of product inhibition, enhanced IA productivity while allowing efficient IA recovery and improving overall process economics.6,7 In our previous paper, we identified reactive extraction (RE) systems for purification of IA from complex aqueous solutions, as the first step towards development of ISPR for IA fermentation.8 Nine RE systems were found to be most suitable for IA recovery from aqueous broths, which included 3 ACS Paragon Plus Environment


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trioctylamine (TOA), dioctylammine and N-methyldioctylamine as amine extractants dissolved in 1octanol, pentylacetate and methyloctanoate. All these amines showed a high extraction efficiency ranging between 97 and 99% along with high partition coefficient (K values) and an overall loading factor Z of 0.50. A reasonably low amine concentration of 0.5 M was found to be sufficiently good for achieving maximum extraction of IA from aqueous solutions containing high (65 g/L or 0.50 M) IA concentrations and thus provided an efficient method for extraction of IA. Following our previous findings, more investigations on primary extraction of IA from aqueous broths have been performed by other groups. The use of various amines and solvents for RE systems and adsorption via ionexchange resins has been explored for IA extraction.9-11 Even though satisfactory results have been obtained on the RE of IA from the aqueous fermentation broth, the back extraction from the loaded organic phase is still needed to recover the IA required for further processing. However, to the best of our knowledge, practically no effort has been made to study the back extraction of the extractant-free IA from the organic phase. Therefore, it was the aim of the present study to develop suitable back extraction protocols for separation of IA from the loaded reactive extractant organic phase. The regeneration of the acid from the organic phase requires the reversal of reaction, i.e. to recover acid as product phase and acid-free extractant for recycle. The acid can be back extracted from the organic phase using various regeneration techniques. Among these, the use of a mineral base e.g. NaOH, a volatile organic base e.g. a low molecular tertiary amine, temperature and diluent swing has been reported for organic acids. Back extraction with NaOH or volatile low molecular tertiary amines is based on the formation of carboxylate ions or salts having a higher solubility in the aqueous phase compared to the organic phase. Yabannavar and Wang (1991) performed the back extraction of lactic acid by contacting loaded organic phase of lactic acid, Alamine 336 and oleyl alcohol with NaOH solution.12 In order to avoid creation of a salt by-product, as is the case with the use of NaOH, back extraction with volatile amine bases e.g. ammonia and short chain organic amines such as trimethylamine (TMA) have also been used.13 In this method, the acid was back-extracted from the loaded organic phase into an aqueous TMA solution. The aqueous solution of the trimethylammonium salt was then heated to remove water and TMA, leaving the acid in crystalline form. In the next step, 4 ACS Paragon Plus Environment

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TMA could be recycled, thereby avoiding costs for make-up chemicals and disposal of by-products. Using this method, lactic acid extracted in organic phase was back extracted with an aqueous TMAsolution by Wasewar et al. (2004).14 The temperature and diluent swing methods take advantage of the change in the equilibrium distribution ratio that results with changes in temperature and/or diluent composition, to back-extract the acid into an aqueous phase. The temperature swing method operates on the fact that the values of the equilibrium constants decrease with an increase in temperature. Therefore, in this back extraction method, the first extraction is carried out at ambient temperature and back extraction into water is performed at a higher temperature. The end-product is a somewhat more concentrated aqueous solution of the acid which can be sent to an evaporative crystallizer.15 Since the amount of change in the extraction equilibrium between temperatures determines the concentration of the acid achievable in the aqueous stream, the latter can be higher than that in the original aqueous feed stream. None of these methods have thus far been investigated for potential recovery of IA from organic phase. Furthermore, since the potential toxicity of extractants to fermenting microorganism is a concern in integrated systems, the biocompatibility of RE systems with IA fermentation is also a point of consideration.16,17 The above back extraction methods were applied in the present work for the back extraction of IA from previously identified RE systems, while also testing the toxicity of the used extractant systems. As a further development, a novel approach for the recovery of IA from the RE system was then studied in this work. In contrast to the conventional back extraction methods that aim to recover pure IA, the new process targets the recovery of the IA-ester. The process is based on the non-catalyzed high pressure methanol process (HTPM) previously developed for lipids (trans-)esterification,18 and aims to achieve the in-situ esterification of the acid within its amine-complex obtained as a result of RE. The main idea was that upon esterification, the acid can be released in ester form from the amine complex. The resulting ester, being more volatile, allows a potentially easier recovery of IA, for instance by distillation. Additionally, the ester is also a suitable starting material for subsequent polymerization to bioplastics, which is one of the major industrial applications of IA. As in this process, the IA is not only back-extracted but also converted to an ester at the same time, it is further



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addressed as back extraction-conversion. The workflow for IA process intensification is presented in Figure 1. Experimental Section TOA, methyloctanoate, IA and TMA were procured from Merck (Darmstadt, Germany) while Nmethyldioctylamine, pentylacetate, methanol 1-octanol and sodium hydroxide were supplied by Sigma Aldrich (Saint Louis, USA). TMA was bought as a 7.3 M solution in water from Acros Organics (Geel, Belgium). IA and the medium components for the mineral medium were purchased from Merck, Germany and used as aqueous solutions in demineralized water. All chemicals were used without purification. Back extraction was performed in 1.5 mL Eppendorf centrifuge tubes. Toxicity of RE systems on IA fermentation The influence of the RE solvents on the growth and IA production of Aspergillus terreus NRRL 1960 was investigated in shake flask experiments. To assess the toxicity, the fermentation medium19 was saturated with the RE solvents as described in the Supplementary information. The solvents included both diluents (used alone) and diluents and amine (used together) which were selected as the best RE systems for IA recovery from the aqueous broth. The cultivation was followed for 7 days and culture samples were taken at days 3 and 7 for the determination of biomass, glucose and IA concentration (see Supplementary information). Conventional back extraction methods In the first phase of back extraction studies, the more conventional approaches to recover an acid from an organic RE mixture were investigated for IA. The IA back extraction systems included the use of mineral base, volatile base and temperature swing. To this, the four most best performing RE systems were selected from our previous study,8 i.e. TOA in 1-octanol (TOC), TOA in methyloctanoate (TMO), TOA in pentylacetate (TPA) and N-methyldioctylamine in octanol (MOC). These RE-systems were back extracted with the three different back extraction procedures at standardized conditions. Back extraction with the volatile amine TMA was found to be the most performant for all RE systems tested and was selected for more detailed assessment. Moreover, the RE systems were limited to TOC and TPA as they provided the highest back extraction efficiency as well as the lowest toxicity towards the fermentation. The back extraction of these two RE systems 6 ACS Paragon Plus Environment

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were then analyzed in more detail on the efficiency in the function of IA concentration as well as the details of extraction and back extraction. For screening experiments, an IA-solution representative for the end of the fermentation (i.e., 65 g/L or 0.50 M) was first extracted with the four different RE systems at 1 M amine. The organic layer, containing the IA, was recovered and its IA concentration was determined as in previous work.8 The extraction yield ranged between 96-99 mole% providing 4 different RE systems with similar starting IA concentrations. The exact IA concentration was taken as a starting point for the assessment of the performance of the subsequent back extraction which was applied as detailed below. In general terms, each artificial RE mixture was contacted with each of the various back extractants in equimolar volumes in 1.5 mL Eppendorf centrifuge tubes. The tubes were shaken during a specified time at a specific temperature. This was followed by centrifugation at 3500 xg for 10 min to facilitate the separation of the two liquids. The aqueous (back extraction) layer was separated from the organic RE layer, and subjected to analysis as described in Analytical methods section. Experiments for the first back extraction method using a mineral base were performed on the four RE systems, as mentioned above. These were contacted with varying concentrations of NaOH aqueous solutions between 0.5 M and 2.5 M at room temperature (25 oC). The contact time was varied from 5 up to 60 min and the efficiency of the system for removing IA from organic phase was determined. The second conventional back extraction method involved the use of a temperature swing. The experiments were conducted by contacting the four RE systems with an equivolume of demineralized water at 80 oC for different contact times of 5, 30 and 60 min. As indicated above, immediately after the reaction time, the aqueous solution was separated from the organic layer by centrifugation and analyzed for its IA concentration to assess the back extraction efficiency. In the third method, the back extraction was performed using a volatile base. Two volatile bases including TMA and triethylamine (TEA) were employed to investigate the recovery of IA from the four RE phases. The amine concentration used in this experiment was 0.5 M and 1.1 M in water. Both extractions were performed at room temperature. 7 ACS Paragon Plus Environment


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For the most promising systems, additional experiments were also performed to assess back extraction performance as a function of IA concentration. An IA concentration range between 10 g/L (0.08 M, below IA inhibition) and 65 g/L (0.50 M, end of fermentation) was selected for these experiments. In order to have well-characterized starting products, the mixture obtained from the RE of the fermentation broth was mimicked by composing artificial mixtures of IA, amine and diluent. The concentration range of IA (0.08-0.50 M), type and concentration of amine (TOA, 0.25-1 M) and type of diluent (methyloctanoate and pentylacetate) were based on the results of previous work.8 These artificial mixtures were then back extracted at room temperature with an aqueous TMAsolution at varying concentrations according to the procedure outlined above. Novel in-situ esterification based back extraction-conversion of IA For the new back extraction-conversion method, a representative RE mixture composed of 0.70 M TOA, 0.35 M IA (i.e., 45 g/L) and methyloctanoate was contacted with methanol. To this purpose, the RE mixture and the methanol were pumped separately into a reactor consisting of a static mixer to ensure mixing followed a tubular reactor with a total internal volume of 17 mL. The flow rates of both the RE layer and methanol were set to achieve the targeted residence time in the reactor as well as the targeted methanol/reactive mixture ratio. The reactor was mounted into an oven heated to obtain the specified temperature. The pressure in the system was ensured by a backpressure regulator at the exit of the reactor. The cooled products were collected for analysis. The effect of various factors such as reaction temperature, residence time, pressure, methanol ratio and concentration of TOA on process performance was investigated. Since temperature and residence time were expected to be of major influence on the reaction, both were systematically studied in a coupled way. The temperature was varied from 150 to 250 oC at different residence times of 3 to 37 min and at a fixed pressure of 150 bar. As a high process pressure can be beneficial to the process but also reduce economic viability, its influence was investigated at fixed reaction conditions (200 oC, 17 min). The pressure was varied between 50 and 225 bar in steps of 25 bars. The methanol concentration was additionally varied to ascertain the optimal concentration for a high ester yield. As a starting point, methanol was dosed at approximately equal masses as compared to the RE-layer. As this corresponded to a high molar excess of methanol against IA (74:1 moles/moles based on the exact 8 ACS Paragon Plus Environment

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dosing to the reactor), two additional experiments were performed. The composition of the REextraction layer was maintained while the methanol dosing was reduced. Based on the actual masses dosed to the reactor, the two experiments were performed at a molar excess of 44:1 (low) and 24:1 (lowest) respectively. The flow rates were adjusted to maintain the requested residence time in the system. Finally, experiments were also conducted to investigate the effect of different TOA/IA ratios in the RE layer on the yields of the two IA esters i.e. monomethylitaconate and dimethylitaconate after esterification. For this study, the TOA/IA molar ratio of 1.23 as low and 3.0 as high was used and compared with normally used molar ratio of 2.0.13 The IA concentration in the RE-layer remained at 0.35 M. Analytical methods The following analytical procedures were applied. Conventional back extraction methodologies In the first phase of the screening experiments in which the back extractability of various RE systems was investigated using different conventional back-extraction methods, IA was measured photospectrometrically at 260 nm using a Tecan infinite 200 Pro plate reader (Mannedorf, Switzerland). A slightly longer wavelength was chosen as compared to the analysis with High Performance Liquid Chromatography (HPLC, see further) to avoid interference of TMA-absorption. The samples were adequately diluted in an aqueous buffer solution of potassium dihydrogen phosphate at pH 2.5 to obtain an absorbance within the linear range of the calibration curve. For every sample, 200 µL was added to a UV-transparent microwell plate and measured against a set of 6 known IA-standards and a blank. All samples, standards and blank were analyzed in triplicate. In the second phase of the work, i.e., the equilibrium studies in which the back extraction with TMA was investigated in more detail, the IA-concentration was determined with HPLC having a refractive index detector (RID) coupled to a UV-detector. The analytical protocol was based on previous work,8 with some minor modifications for analyzing IA in TMA-containing samples. The samples were first appropriately diluted with demineralized water to have a concentration suitable for HPLC analysis, after which 50 µl of HCl 37 % (w/w) was added for acidification. Separation was achieved on an Agilent Hi-PLEX H column (300 mm x 7.7 mm) using 0.005 M sulphuric acid as 9 ACS Paragon Plus Environment


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mobile phase at a flow rate of 0.7 mL/min and 60 °C temperature. IA was measured with the UV detector at 247 nm. The samples were measured in duplicate. Novel back extraction-conversion by in-situ esterification For the analysis of samples obtained from in-situ esterification of IA in the new back extractionconversion methodology, the analytical procedure was slightly adapted to achieve a good separation between IA and its monomethyl- and dimethylester. The mobile phase was composed of 0.005 M sulphuric acid which was added at a flow rate of 0.7 mL/min. IA and monomethylitaconate were measured at a wavelength of 247 nm while dimethylitaconate was detected at 215 nm. The samples were measured in duplicate. Calculations For the conventional back extraction methods, the extraction yield was calculated from the IA concentration measured in the back extraction layer after extraction relative to the starting IA concentration of the RE-layer. The data are expressed as mole%. For the in-situ esterification, the residual IA and the IA conversion yield (expressed as mole%) were calculated from the concentrations of IA, monomethylitaconate and dimethylitaconate measured after treatment, as compared to the starting IA acid concentration corrected for the methanol added and their differences in molecular mass. The mass balance was calculated by a summation of the residual IA and the conversion yields of the formed mono- and dimethylitaconate (all in mole %). For measurement of IA from RE systems (prior to subjecting to back extraction procedure), the method as reported by Kaur and Elst (2014) was used.8 Results and Discussion Conventional back extraction methods of IA from RE systems The use of NaOH as back extraction system for IA-amine reactive extractant was investigated at different NaOH concentrations and residence times. An overview of results of back extraction with 1 M NaOH is shown in Figure 2. As shown in the figure, the application of mineral base was indeed successful in recovering IA from the loaded organic phase. The system was fast, since the equilibrium was achieved in less than 5 min for all the RE systems under investigation. However, among the 4 reactive extractants tested, the recovery efficiencies for IA varied between 75 mole% and 95 mole%. 10 ACS Paragon Plus Environment

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The effect of back extraction with NaOH concentrations greater than 1 M resulted in average extraction efficiencies of 96.5±1.3 mole% for TMO, 90.8±6.3 mole% for TPA, 81.7±4.9 mole% for TOC and 76.8±2.3 mole% for MOC (Figure 3). This can be explained on the basis of individual affinities wherein the back extraction from methyloctanoate and pentylacetate is easier to perform than from octanol. Octanol, typically classified as an active diluent, is known to stabilize the amineacid complex in the primary RE, making the back extraction less favorable. The obtained results are consistent with literature reports wherein a molar ratio of 2 and above of NaOH/organic acid was reported to result in higher extraction efficiencies in back extraction.12,16 Although satisfactorily efficient, back extraction with a non-volatile base such as NaOH generates the release of a salt rich itaconate solution requiring further downstream processing purification steps. The use of temperature swing as a simplified method of back extraction of IA was also attempted. In this procedure, the RE medium is contacted with pure water as a back-extractant, and the shifting of equilibrium at higher temperature is used to back extract the IA in the aqueous medium. If effective, this back extraction procedure provides a very simple method for the recovery of IA, since no additional chemicals are involved, and the back extraction solution can immediately be subjected to crystallization to recover the IA crystals.15 The results obtained for back extraction of IA using temperature swing in the present study indicated that an increasing extraction efficiency was noted with increasing time (results not shown). However, as shown in Figure 3, temperature swing with an aqueous solution at 80 oC was very ineffective in recovering IA from the organic phase. Maximum extraction efficiencies of ~10-15 mole% were achieved for TPA and TMO RE systems while those for TOC and MOC were even lower reaching

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