Selective Oxidation of 5-Hydroxymethylfurfural to 5-Hydroxymethyl-2

Subscriber access provided by Iowa State University | Library

Article

Selective oxidation of 5-hydroxymethylfurfural to 5hydroxymethyl-2-furancarboxylic acid using Gluconobacter oxydans Mahmoud Sayed, Sang-Hyun Pyo, Nicola Rehnberg, and Rajni Hatti-Kaul ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.8b06327 • Publication Date (Web): 25 Jan 2019 Downloaded from http://pubs.acs.org on January 31, 2019

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 28 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Sustainable Chemistry & Engineering

Research Article

Selective oxidation of 5-hydroxymethylfurfural to 5-hydroxymethyl-2furancarboxylic acid using Gluconobacter oxydans

Mahmoud Sayed a, b, Sang-Hyun Pyo a,*, Nicola Rehnberg c and Rajni Hatti-Kaul a

a Biotechnology,

Department of Chemistry, Center for Chemistry and Chemical Engineering,

Lund University, Box 124, SE-22100 Lund, Sweden. b

Department of Botany, Faculty of Science, South Valley University, Qena, Egypt.

c Strategic

R&D, Bona AB, Box 210 74, 200 21 Malmö, Sweden.

* Corresponding author Tel: +46-46-222-4838; Fax: +46-46-222-4713 E-mail: [email protected] (S.-H. Pyo)

1 ACS Paragon Plus Environment

ACS Sustainable Chemistry & Engineering 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Abstract 5-Hydroxymethylfurfural (HMF), an important biobased platform chemical, can be potentially oxidized to several products that can serve as versatile building blocks for polymers. 5-Hydroxymethyl-2-furan carboxylic acid (HMFCA) is formed by incomplete oxidation of HMF, but the reaction often suffers from substrate inhibition and overoxidation to other products. In this study, resting cells of Gluconobacter oxydans DSM 50049 were shown to oxidize HMF quantitatively to HMFCA with exquisite selectivity. Complete conversion of 31.5 g L-1 crude HMF to HMFCA was achieved within 6 h under pHcontrolled conditions. Initial productivity of 10 g L-1 h-1 was reduced to 2 g L-1 h-1 towards the end of the reaction. Thereafter, additional HMF added to the reaction mixture (12 g L-1) was converted up to 94 % within 17 h, with 100 % selectivity resulting in final HMFCA concentration of 44.6 g L-1 and yield of 6.2 g g-1 cell dry weight. Recovery of HMFCA from the reaction could be achieved by adsorption to anion exchange resins Amberlite IRA-400 (Cl- form) and Ambersep® 900 (OH- form), the former showing higher binding (169 mg/g resin) and product recovery. Alternatively, liquid-liquid extraction with ethyl acetate provided a facile separation technique for the recovery of pure HMFCA.

Keywords 5-hydroxymethyl-2-furan carboxylic acid (HMFCA), 5-hydroxymethylfurfural (HMF); selective oxidation, whole cells biocatalyst, Gluconobacter oxydans DSM 50049


Page 2 of 28

Page 3 of 28 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Introduction 5-Hydroxymethylfurfural (HMF), formed by acid catalyzed dehydration of hexose sugars, and a common by-product generated during the pretreatment of biomass under relatively harsh conditions, is identified among top chemical opportunities from biorefinery carbohydrates. Thanks to its functional groups, including the furan ring, an aldehyde and an alcohol group, HMF is highly reactive making it a valuable platform chemical for a variety of molecules of interest as biofuel, solvents and building blocks for polymers.1-4 Hence extensive R&D efforts are ongoing to develop processes for production and transformation of HMF. Oxidation of HMF yields several important furan chemicals, such as 2,5-diformylfuran (DFF), 5-hydroxymethyl-2-furancarboxylic acid (HMFCA), 5-formyl-2-furancarboxylic acid (FFCA), and 2,5-furandicarboxylic acid (FDCA) (Scheme 1), through the use of chemical and biological catalysts.5, 6 Already in 2004, FDCA was ranked among the US Department of Energy list of top 10 chemicals from carbohydrates5, 7 and is being developed as a biobased alternative to the fossil based terephthalic acid (TPA) for the production of a new polymer, polyethylene furanoate (PEF) with superior thermal and barrier properties than polyethylene terephthalate (PET).8 5, 9, 10 Meanwhile, HMFCA, a less known oxidation product than FDCA, is formed by selective oxidation of the aldehyde group in HMF, and can serve as an interesting monomer for various polymers and also as a therapeutic based on its antimicrobial and antitumor activity.11,

12

The selective (incomplete) oxidation is however more

complicated due to the possibility of the overoxidation to FDCA.13 Only a few reports are available on the oxidation of HMF to HMFCA, the majority of which make use of metal catalysts such as MnO2, Pt/C and Au-Pd, often resulting in low selectivity and hence giving poor product yields.5, 13, 14 Recently, some systems with high HMF conversion and HMFCA yields have been reported. For example, total HMF conversion with 86.9% HMFCA yield was obtained using



a montmorillonite K-10 clay immobilized molybdenum acetylacetonate complex in toluene, 15

and 97.2% HMF conversion with 72.9% HMFCA yield were obtained using Cs-substituted

tungstophosphate-supported ruthenium nanoparticles.16 Also, selective photocatalytic oxidation of HMF to HMFCA at 90–95% yields under ultraviolet and visible light in aqueous Na2CO3 solution by Au/TiO2 has been achieved.17 However, the problems associated with the use of chemical catalysts such as the low selectivity, toxicity of the catalysts, as well as the harsh conditions required for both upstream and downstream processes, are major challenges. Biocatalytic oxidation offers a more selective and mild alternative to chemical processes, and has lately received some attention.12 Lipase-mediated Baeyer-Villiger oxidation of HMF to HMFCA using H2O2 as the oxidant for in situ generation of peracid from ethyl acetate or ethyl butyrate gave HMFCA with a yield of approximately 80%.18 Also, the oxidation of HMF using xanthine oxidase from Escherichia coli produced HMFCA with a yield of 94% and a selectivity of >99% after 7 h.19 Since whole cells provide a more protective environment for the enzymes, allow regeneration of cofactors, and are also less expensive, the use of whole cell oxidation is generally more preferable to pure enzymes.20, 21 The main challenges could however be the inhibitory effect of HMF on the microorganisms as for the enzymes, and the overoxidation of HMF by other oxidative enzymes present in the cells. Only one report is found on the selective oxidation of HMF to HMFCA using Comamonas testosteroni SC1588; product yield of 98% was obtained from 20 g/L substrate in the presence of histidine as an additional enhancer.12 However, the cell viability, HMF conversion, and HMFCA yield were negatively affected by just a slight increase in HMF concentration. Also, C. testosteroni SC1588 produced bis(hydroxymethyl)furan (BHMF) as a coproduct with HMFCA especially at higher HMF concentration.12


Page 4 of 28

Page 5 of 28 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Many acetic acid bacteria are known to oxidize their substrates incompletely even in the presence of excess oxygen. Among them is Gluconobacter oxydans, an obligately aerobic, acidophilic bacteria belonging to the family Acetobacteriaceae, which has been used for incomplete oxidation of a variety of sugars, alcohols and related compounds.22, 23 Some wellknown examples include the oxidation of sorbitol to L-sorbose, D-glucose to D-gluconic acid, 5-keto- and 2-keto gluconic acids, and glycerol to dihydroxyacetone.23, 24 The processes are characterized by high oxidation rates at low biomass growth and often complete secretion of the products into the medium. In our laboratory, G. oxydans has been used for regioselective

oxidation

of

1,3-propanediol

and

2-methyl-1,3-propanediol

to

3-

hydroxypropionic acid and 3-hydroxy-2-methylpropionic acid, respectively, providing a green process for the production of important building blocks for polymers.23,

25-30

The

genome sequence of G. oxydans has recently been reported and a number of membrane bound dehydrogenases and soluble oxidoreductases have been identified.31, 32 The present study reports the use of Gluconobacter oxydans for oxidation of HMF to HMFCA. Two strains of G. oxydans were screened for their ability to catalyse the oxidation reaction in aqueous medium. The most promising candidate G. oxydans DSM 50049 was used for the oxidation of HMF produced in house by fructose dehydration. The reaction was evaluated in lab scale bioreactor at increased HMF concentration, and recovery of HMFCA from the reaction solution was investigated.

2. Materials and methods 2.1. Materials The bacterial strains, Gluconobacter oxydans DSM 50049, and G. oxydans DSM 2343 were obtained from Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (Braunschweig, Germany). 5-HMF (99% purity) and HMFCA (99%) for use as standards,



Amberlite IRA-400 (Cl form) and Ambersep® 900 (OH- form) were purchased from Sigma Aldrich. Yeast extract, glycerol, sodium dihydrogen phosphate and disodium hydrogen phosphate were from Merck. All chemicals were of analytical grade.

2.2. Preparation of HMF from fructose One liter of 30% w.w-1 fructose solution in DMSO was placed in a 2 L flask, followed by addition of 60 g ion exchange resin (0.2 (w.w-1) equivalent to fructose), and shaking in an oil bath at 110 C for 3 hours. 5-HMF (75% w.w-1) was obtained from the reaction mixture by liquid/liquid extraction and concentration by rotary evaporation. The partially purified 5HMF was used for the oxidation experiments.

2.3. Cultivation of G. oxydans strains Lyophilized cells of G. oxydans DSM 50049 and DSM 2343 were inoculated into 50 mL Gluconobacter broth medium in 250 mL flasks, containing (per liter): 100 g glucose, 10 g yeast extract at pH 6.8. All the flasks were incubated in a shaker incubator (Ecotron, Infors HT, UK) at 30 C and 200 rpm for 24 h. Then 20 % glycerol stocks of the bacterial cultures were prepared by mixing 500 L culture broth with 500 L of 40 % glycerol solution, and stored at -20 C for further use. For preparing the cells for transformation of HMF, 100 µL glycerol stocks of the microorganisms were inoculated individually into 50 mL medium in 250 mL flasks, containing per liter: 25 g glycerol, 10 g yeast extract with pH adjusted to 5 respectively, and the flasks were incubated as described above. Thereafter, the culture broths were centrifuged at 4700xg for 15 min (Sorvall LYNX 4000, Thermo Scientific, Germany), the cell pellets were separated and washed twice using 0.1 M sodium phosphate buffer pH 7 prior to use in the oxidation reactions.


Page 6 of 28

Page 7 of 28 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


2.4. Whole cell catalysed oxidation of HMF The cell pellets of G. oxydans DSM 50049 and DSM 2343 collected from 4 mL cultivation medium, were suspended in 1 mL of 100 mM sodium phosphate buffer (pH 7) supplemented with 5 g L-1 HMF (99%) in 4 mL vials. All vials were incubated in a thermomixer (MKR 13, HLC Biotech, Germany) at 30 C and 700 rpm for 72 h. Twenty microliter samples were collected after 3, 6, 12, 24 and 72 h of reaction for analyzing substrate and product concentrations. The effect of different reaction parameters on HMF oxidation was studied using resting cells of G. oxydans DSM 50049 in 1 mL reaction medium containing 0.1 M sodium phosphate buffer supplemented with 16 g L-1 of partially purified HMF (75% purity) in 4 mL vials. The effect of pH was studied by performing the reaction in the buffer adjusted to 5, 6, 7 and 8, respectively. To study the effect of cell concentration, 4, 8 and 12 mg cell dry weight, respectively, were added to 1 mL reaction at pH 7. All vials were incubated at 30 C and 700 rpm, and 20 µL samples were withdrawn at 0, 3, 6, 12 and 24 h for analyses. To identify the location of the oxidative enzyme, G. oxydans DSM 50049 cells grown in the glycerol containing medium were harvested from 50 mM cell suspension by centrifugation at 4700 × g for 15 min and washed twice with 100 mM sodium phosphate buffer of pH 7. The cell pellet was resuspended in the same buffer and sonicated on ice (3 × 60 s, cycle 0·75) with UP400S sonicator (Dr. Hielscher GmbH, Stahnsdorf, Germany), centrifuged at 13 400 rpm for 30 min in an eppendorf centrifuge, and the cell debris separated from the supernatant. The two fractions were analysed for HMF oxidizing activity in 0.1 M sodium phosphate buffer pH 7.

2.5. Scaling up the reaction Reactions in a larger volume were performed under optimal conditions using the partially purified HMF and the resting cells of G. oxydans DSM 50049. In 50 mL scale, the cell pellet 7 ACS Paragon Plus Environment


collected from 200 mL reaction medium was washed and re-suspended in 50 mL sodium phosphate buffer pH 7 supplemented with 24 g L-1 HMF in 250 mL shake flask, incubated at 30 C and 200 rpm, and 1mL samples were collected after 0, 3, 6, 9, 12, 24 and 33 h for analyzing the concentration of HMF and HMFCA. After every sampling, pH of the reaction was adjusted at 7 using 5 N NaOH. The reaction using a higher HMF concentration (31.5 g L1)

was then performed in 250 mL reaction volume in 0.5 L vessel under controlled conditions

at pH 7 and 1vvm of air flow, which were controlled and monitored by ez-control unit (Applikon, The Netherlands), and the temperature was controlled at 30 C using LAUDR E100 water circulation system (LabX, Canada), and stirring at 500 rpm using RCT basic magnetic stirrer (IKA®-Werke GmbH, KG, Germany). The reaction was initiated by the cells obtained from 2 L growth medium after 24 h cultivation. The pH was controlled by titration with 5 N NaOH. Seven milliliter samples were withdrawn at 3, 6, 9 and 11 h of the reaction, the initial 5 mL was discarded and 2 mL was used for analysis. Once the HMF was completely converted, more HMF was fed to the reactor to bring up the concentration to 12 g L-1, and samples were collected for monitoring its conversion. Once the reaction was complete, the reaction mixture was centrifuged and the cell-free solution was used for HMFCA recovery and purification experiments.

2.6. HMFCA recovery, purification and structure elucidation Two anion exchange resins, Amberlite IRA-400 (Cl form) and Ambersep® 900 (OH- form), were evaluated for the recovery of HMFCA from the reaction solution obtained from oxidation performed at 250 mL scale. One hundred milligram of each resin was washed with 1mL deionized water in 4 mL vials, followed by swelling the resin in 1 mL deionized water for 30 min. Then 1 mL of the reaction solution containing 44.6 g L-1 HMFCA was added to the swollen resins and the vials were incubated at 30 C with shaking for 30 min on a 8 ACS Paragon Plus Environment

Page 8 of 28

Page 9 of 28 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


thermomixer. Fifty microliter samples were collected at 0, 10 and 30 min for analyzing the concentration of unbound HMFCA. The two resins were washed twice with 1 mL water for 30 min, followed by 3x elution with 1 mL 2 N HCl each. Alternatively, HMFCA was purified from the reaction solution obtained from the 0.5 L reactor by acidifying to pH 1.5 using concentrated HCl, followed by liquid-liquid extraction using ethyl acetate at a ratio of 1:1. The extraction was repeated three times. Then the solvent was removed using a rotary evaporator and HMFCA was recovered in a concentrated form, and its identity confirmed by HPLC using commercial HMFCA as standard, and the structure was elucidated by 1H-NMR and 13C-NMR.

2.7. Analytical procedures Cell density was determined by measuring the optical density of cell broth at 620 nm using UV/Vis Spectrophotometer (Ultrospec 1000, Pharmacia Biotech, Sweden). The cell dry weight (CDW) was determined by collecting cells from 1 mL fermentation broth at 4700 ×g for 10 min in a dried pre-weighed 1.5 mL eppendorf tube. The collected cell pellet was dried overnight at 105 °C. The increase in weight of the tube equals the CDW per milliliter. The OD620nm was correlated to CDW by the following equation: CDW (g L-1) = OD620 x 0.4

(Eq. 1)

The concentrations of HMF, HMFCA and other byproducts (BHMF) were determined using HPLC (JASCO, Tokyo, Japan) equipped with Aminex HPX-87H chromatographic column connected to a guard column (Biorad, Richmond, CA, USA), refractive index detector (ERC, Kawaguchi, Japan), a JASCO UV detector operating at 215 nm, and a JASCO intelligent autosampler. The column temperature was maintained at 65 C in a chromatographic oven (Shimadzu, Tokyo, Japan). Samples were diluted with Milli-Q quality water and mixed with 20 % v.v-1 H2SO4 (20 µL.mL-1 sample) and then filtered. A 40 µL 9 ACS Paragon Plus Environment


aliquot was injected in 0.5 mM H2SO4 mobile phase flowing at a rate of 0.4 mL min-1. The peaks for the different compounds were confirmed and quantified using external standards of HMF and HMFCA. The reaction productivity (Qp) and yield with respect to substrate (Yp/s) and biocatalyst (Yp/x) were calculated using the following equations: Qp (g/L. h) = dP (g L-1)/dt (h)

(Eq. 2)

Yp/s (%) = [dP (mole)/dS (mole)]* 100

(Eq. 3)

Yp/x = [P (g L-1)/X (g L-1)]

(Eq. 4)

where (P) is the product, (t) is the time in hours, (S) is the substrate and (X) is the cell dry weight.

3. Results and discussion 3.1. Oxidation of HMF using G. oxydans cells G. oxydans DSM 50049 and DSM 2343 were grown using glycerol as carbon source, and after harvesting, the cells (4 mg mL-1 CDW) were screened for their ability to oxidize HMF (5 g L-1) in aqueous medium at 30 C and product profiles were monitored (Fig. 1 A-B). G. oxydans DSM 2343 showed only 22% conversion of HMF and accumulation of 12% of HMFCA and 10% BHMF (Figure 1A), indicating the presence of both oxidative and reductive activities. On the other hand, G. oxydans DSM 50049 converted the HMF completely to HMFCA within 3 h (Fig. 1B). No intermediate product was observed, indicating that HMF could be directly oxidized to HMFCA. A preliminary experiment to determine the location of the enzyme activity was performed by sonicating G. oxydans DSM 50049 cells, and separating the soluble and insoluble fractions that were tested individually for reactions with HMF. Only the insoluble fraction was shown to display the HMF oxidizing activity, suggesting the involvement of a membrane bound enzyme. G. oxydans cells are 10 ACS Paragon Plus Environment

Page 10 of 28

Page 11 of 28 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


known to have a large number of dehydrogenases (including aldehyde dehydrogenase), oxidoreductases as well as oxidases.31,32 While the genome sequence of the strain that did not show HMF oxidation (DSM 2343) is available,31 it is plausible that G. oxydans 50049 that catalysed the selective oxidation of HMF has a unique set of genes encoding oxidative enzymes. Based on the above results, G. oxydans DSM 50049 was selected as the biocatalyst for further investigations. Taking advantage of the selectivity of the biocatalyst, subsequent reactions were performed using significantly higher concentrations of partially purified HMF produced in the laboratory, which would not only lower the total production cost but also the environmental impact due to less chemicals and energy needed for HMF purification. Figure 2A indicates the effect of pH 5-8 on the oxidation of 16 g L-1 HMF at 30 C. The conversion of HMF and yield of HMFCA clearly depended on the initial pH as in the previous report using C. testosterone

12.

The highest conversions to HMFCA, 95.6% and 100% were

observed at pH 7 and pH 8, respectively, while significantly lower conversions were achieved at lower pH values (Fig. 2A). In spite of the higher conversion at pH 8, the initial reaction productivity was lower, 2.39 g L-1h-1 compared to 2.7 g L-1h-1 at pH 7 (Figure 2B). Irrespective of the initial reaction pH, the pH at the end of the reaction had dropped to 2.6 in all cases because of the accumulation of HMFCA, which affects the activity of the cells. This would explain the low conversion obtained at lower pH values of 5 and 6 in which the pH drop was faster. Controlling the reaction pH would thus be crucial for improving the conversion rate and product yield. The effect of cell amount was investigated by using 4, 8 and 12 mgcdw mL-1 of cells for the oxidation of 16 g L-1 HMF at pH 7 and 30 C. Figure 3 shows that increase in cell amount has a minor influence on the initial oxidation rate, 90 % conversion of HMF being achieved at 12 mgcdw/mL of cells, compared to about 83% conversion by 4 and 8 mgcdw mL-1 after 6 h 11 ACS Paragon Plus Environment


reaction (Figure 3A). This could be related to the decreased initial specific productivity from 0.84 to 0.30 g/g dry cells per hour with increase in cell concentration from 4 to 12 mg/mL, which was attributed to limited oxygen level in the closed space of the reaction vial as a result of which the available biocatalytic capacity in the cells was not fully utilized. The initial productivities of the reactions were slightly higher, 3.64 ± 0.01 and 3.52 ± 0.1 g L-1h-1 for 12 and 8 mgcdw mL-1, respectively, compared to 3.36 ± 0.2 g L-1h-1 with 4 mgcdw mL-1 (Figure 3B). The higher cell concentrations yielded 100 % conversion of HMF with 100 % yield and selectivity of HMFCA (8 and 12 mgcdw mL-1 of cell) after 12 h reaction, while 4 mgcdw mL-1 cells gave 98 % conversion of HMF with 98 % yield HMFCA after 12 h, and 100 % conversion and yield on further incubation (Fig. 3A). In all cases selectivity of HMFCA formation was 100 %. The results indicate that increasing the amount of G. oxydans DSM 50049 cells did not significantly increase the cell activity under the reaction conditions. However, the higher cell density has a protective effect against the inhibitory effect of HMFCA over a longer time period.29

3.2. Scaling up of HMFCA production HMFCA production using whole cells of G. oxydans DSM 50049 was scaled up to evaluate the effect of controlling the pH and also the performance at even higher substrate concentration, considering that even the substrate HMF has an inhibitory effect on microbial cells

33,12.

The reaction in 50 mL volume in 250 mL flask was carried out using 4 mg CDW

mL-1 with 24 g L-1 HMF in 100 mM sodium phosphate buffer, and pH was adjusted at 7 every 3 hours. About 91% of HMF was converted to HMFCA at 100% selectivity after 33 h of the reaction (Figure 4). Also, HMFCA yield with respect to HMF and biocatalyst (CDW) was 1.13 g g-1HMF and 5.95 g g-1cdw, respectively.


Page 12 of 28

Page 13 of 28 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Subsequently, the reaction was performed in a 0.5 L bioreactor with continuous control of pH and aeration besides temperature. The crude HMF at 31.5 gL-1 concentration in 250 mL was completely converted to HMFCA at 100% yield and selectivity after 6 h reaction with 24 mg mL-1 CDW G. oxydans DSM 50049. The productivity of the reaction was as high as 10 g L-1h-1 in the initial phase of the reaction and was decreased to 2 g L-1h-1. After this time, additional HMF was added to a concentration of 12 g L-1, 94 % of which was converted to HMFCA with 100% selectivity after 17 h from the time of addition, at a productivity of 2 g L-1h-1. Hence the cells, although active, had significantly lost their enzyme activity during the reaction (Figure 4B). In comparison, the earlier study describing the use of resting cells of C. testosterone SC1588 (30 mg/mL), activated by 5 mM furfuryl alcohol, reported the oxidation of 160 mM (around 20.2 g L-1) HMF with 98 % yield of HMFCA after 36 h in a reaction in the presence of 20 mM histidine as an enhancer. BHMF, the reduced derivative of HMF was formed as an intermediate during the reaction, which was subsequently oxidized to HMFCA, indicating that the organism utilized a different route involving different redox enzymes.12 Also, at higher concentrations of HMF, 180 mM and 200 mM, the yield of HMFCA was dramatically decreased to 52 and 15%, respectively, and FDCA and BHMF were observed as co-products. Although both HMF and HMFCA are inhibitory compounds, the latter is especially recognized for its antimicrobial activity against microorganisms.12, 33 G. oxydans DSM 50049 showed much higher resistance at 2 times higher concentration of HMF and HMFCA than that used for C. testosterone SC1588, and the final product contained 44.6 g L-1 HMFCA only without any intermediate (e.g. BHMF), by-product (e.g. FDCA) or residual substrate.

3.3. HMFCA recovery



As HMFCA is formed in a pure form in the present study, the main downstream processing needed for product recovery would be the removal of water after separation of the whole cell biocatalyst. However, as mentioned above, the HMF used was not completely pure, and HMFCA separation from the impurities present would be easier due to the presence of the carboxylic group. Moreover, integration of the purification step with the biocatalytic step would potentially result in higher productivity due to in situ product removal and reduced product inhibition. Several bioprocesses involving production of organic acids including lactic acid, citric acid and succinic acid, were significantly improved through the recovery of these acids using an anion exchange resin.34-36 Preliminary experiments on the recovery of HMFCA from 250 mL scale experiment at pH 7, were performed using 100 mg mL-1 of the anion exchange resins, Amberlite IRA-400 (Cl form) and Ambersep® 900 (OH- form), respectively. The binding of HMFCA to the resins was followed over time (Figure 5 A); the binding capacity after 30 min was 16.9 and 16.1 mg HMFCA per 100 mg of Amberlite IRA-400 (Cl form) and Ambersep® 900 (OH- form), respectively. The anion exchange resin (Cl form) showed higher binding efficiency, and also exhibited much less loss of the bound HMFCA during washing (1.8 mg mL-1 versus 5 mg mL-1) compared to the (OH- form) resin (Figure 5B). Moreover, 83.6% of the bound HMFCA to (Cl form) resin was recovered after 3 elution steps using 1 mL each of 2 M HCl, while only 68.3 % was recovered from (OH form) under similar conditions. The elution step may be improved further by increasing the HCl concentration; however the results suggest that a large amount of the resin (~265 g.L-1) would be needed for capturing the product and also the elution results in further dilution of the product. Alternatively, HMFCA was directly recovered from reaction medium by liquid-liquid extraction. The product solution was adjusted to around pH 1.5 using concentrated HCl, and


Page 14 of 28

Page 15 of 28 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


then subjected to liquid-liquid extraction with ethyl acetate. HMFCA with a purity of 98% was obtained in quantitative yield simply by evaporation of the low boiling point ethyl acetate (77 C) under reduced pressure at 40 C. Figure 6A-D shows the chromatograms of the samples from different points of the HMFCA production process. In particular, chromatogram 6A representing the crude HMF at 0 h of the biotransformation reaction, shows the presence of high amounts of residual DMSO, the solvent used for production of HMF and having a boiling point close to that of HMF. DMSO accompanied the product HMFCA after the reaction (Figure 6B and C), but was removed during product recovery by ion exchange adsorption and extraction in ethyl acetate (Figure 6D). If the HMF production were done in an alternative medium, e.g. a biphasic system using a low boiling solvent instead of DMSO,

1

the solvent impurity in the reaction product would be negligible.

HMFCA structure was confirmed by 1H-NMR (Figure S1).

4. Conclusions The whole cells of G. oxydans DSM 50049 offer a much superior catalytic system for selective oxidation of HMF to HMFCA than those previously reported, and the process meets several of the green chemistry principles, including use of renewable resources, high atom economy, prevention of waste, less hazardous chemical synthesis, safer solvents and auxiliaries, high energy efficiency, safer solvents, etc. HMF, produced without extensive purification, was quantitatively oxidized to HMFCA, which was easily recovered in a pure form. The process can be further improved by fed-batch or continuous transformation using a high cell density reactor integrated with in situ removal of product. These approaches are currently being investigated. It is also of interest to identify the enzyme system in G. oxydans DSM 50049 responsible for the selective oxidation of HMF.



Conflicts of interest The authors declare that they have no competing interests.

Acknowledgement This work was performed within the research program, Sustainable Plastics and Transition Pathways (STEPS) at Lund University supported by the Swedish Foundation for Strategic Environmental Research (Mistra) (grant no. 2016/1489). The authors thank Niklas Warlin at Center for Analysis and Synthesis for help with NMR of the product.

Supporting Information The Supporting information is available. 1H-NMR

of 5-hydroxymethyl-2-furan carboxylic acid (HMFCA) in DMSO-d6 (PDF)


Page 16 of 28

Page 17 of 28 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


6. References 1. Román-Leshkov, Y.; Chheda, J. N.; Dumesic, J. A. Phase modifiers promote efficient production of hydroxymethylfurfural from fructose. Science 2006, 312, 1933-1937, DOI: 10.1126/science.1126337. 2. Saha, B.; Abu-Omar, M. M. Advances in 5-hydroxymethylfurfural production from biomass in biphasic solvents. Green Chem. 2014, 16, 24-38, DOI: 10.1039/C3GC41324A. 3. Bozell, J. J.; Petersen, G. R. Technology development for the production of biobased products from biorefinery carbohydrates—the US Department of Energy’s “Top 10” revisited. Green Chem. 2010, 12, 539-554, DOI: 10.1039/B922014C. 4. Ordomsky, V. V.; van der Schaaf, J.; Schouten, J. C.; Nijhuis, T. A. Fructose dehydration to 5‐hydroxymethylfurfural over solid acid catalysts in a biphasic system. ChemSusChem 2012, 5, 1812-1819, https: //doi.org/10.1002/cssc.201200072. 5. Hayashi, E.; Komanoya, T.; Kamata, K.; Hara, M. Heterogeneously‐catalyzed aerobic oxidation of 5‐hydroxymethylfurfural to 2, 5‐furandicarboxylic acid with MnO2. ChemSusChem 2017, 10, 654-658, https: //doi.org/10.1002/cssc.201601443. 6. McKenna, S.; Mines, P.; Law, P.; Kovacs-Schreiner, K.; Birmingham, W.; Turner, N.; Leimkühler, S.; Carnell, A. The continuous oxidation of HMF to FDCA and the immobilisation and stabilisation of periplasmic aldehyde oxidase (PaoABC). Green Chem. 2017, 19, 4660-4665, DOI: 10.1039/C7GC01696D. 7. Werpy, T.; Petersen, G.; Aden, A.; Bozell, J.; Holladay, J.; White, J.; Manheim, A.; Eliot, D.; Lasure, L.; Jones, S. Top value added chemicals from biomass. Volume 1-Results of screening for potential candidates from sugars and synthesis gas; Department of Energy Washington DC: 2004. 8. Neaţu, F.; Petrea, N.; Petre, R.; Somoghi, V.; Florea, M.; Parvulescu, V. Oxidation of 5hydroxymethyl furfural to 2, 5-diformylfuran in aqueous media over heterogeneous manganese based catalysts. Catal. Tod. 2016, 278, 66-73, https: //doi.org/10.1016/j.cattod.2016.03.031. 9. Burgess, S. K.; Leisen, J. E.; Kraftschik, B. E.; Mubarak, C. R.; Kriegel, R. M.; Koros, W. J. Chain mobility, thermal, and mechanical properties of poly (ethylene furanoate) compared to poly (ethylene terephthalate). Macromolecules 2014, 47, 1383-1391, DOI: 10.1021/ma5000199. 10. Papageorgiou, G. Z.; Papageorgiou, D. G.; Terzopoulou, Z.; Bikiaris, D. N. Production of bio-based 2,5-furan dicarboxylate polyesters: Recent progress and critical aspects in their synthesis and thermal properties. Eur. Polym. J. 2016, 83, 202-229, https: //doi.org/10.1016/j.eurpolymj.2016.08.004. 11. Munekata, M.; Tamura, G. Antitumor activity of 5-hydroxy-methyl-2-furoic Acid. Agric. Biol. Chem. 1981, 45, 2149-2150, DOI: 10.1080/00021369.1981.10864851. 12. Zhang, X.-Y.; Zong, M.-H.; Li, N. Whole-cell biocatalytic selective oxidation of 5hydroxymethylfurfural to 5-hydroxymethyl-2-furancarboxylic acid. Green Chem. 2017, 19, 4544-4551, DOI: 10.1039/C7GC01751K.



13. Kucherov, F. A.; Romashov, L. V.; Galkin, K. I.; Ananikov, V. P. Chemical transformations of biomass-derived c6-furanic platform chemicals for sustainable energy research, materials science, and synthetic building blocks. ACS Sustain. Chem. Eng. 2018, 6(7), 8064-8092, DOI: 10.1021/acssuschemeng.8b00971. 14. Antonyraj, C. A.; Huynh, N. T. T.; Park, S.-K.; Shin, S.; Kim, Y. J.; Kim, S.; Lee, K.-Y.; Cho, J. K. Basic anion-exchange resin (AER)-supported Au-Pd alloy nanoparticles for the oxidation of 5-hydroxymethyl-2-furfural (HMF) into 2,5-furan dicarboxylic acid (FDCA). Appl. Catal. A: General. 2017, 547, 230-236, https: //doi.org/10.1016/j.apcata.2017.09.012. 15. Zhang, Z.; Liu, B.; Lv, K.; Sun, J.; Deng, K. Aerobic oxidation of biomass derived 5hydroxymethylfurfural into 5-hydroxymethyl-2-furancarboxylic acid catalyzed by a montmorillonite K-10 clay immobilized molybdenum acetylacetonate complex. Green Chem. 2014, 16, 2762-2770, DOI: 10.1039/C4GC00062E. 16. Wang, F.; Zhang, Z. Cs-substituted tungstophosphate-supported ruthenium nanoparticles: An effective catalyst for the aerobic oxidation of 5-hydroxymethylfurfural into 5-hydroxymethyl-2furancarboxylic acid. J. Taiwan Inst. Chem. Eng. 2017, 70, 1-6, https: //doi.org/10.1016/j.jtice.2016.10.003. 17. Zhou, B.; Song, J.; Zhang, Z.; Jiang, Z.; Zhang, P.; Han, B. Highly selective photocatalytic oxidation of biomass-derived chemicals to carboxyl compounds over Au/TiO 2. Green Chem. 2017, 19, 1075-1081, DOI: 10.1039/C6GC03022J. 18. Krystof, M.; Pérez‐Sánchez, M.; Domínguez de María, P. Lipase‐mediated selective oxidation of furfural and 5‐hydroxymethylfurfural. ChemSusChem 2013, 6, 826-830, https: //doi.org/10.1002/cssc.201200954. 19. Qin, Y.-Z.; Li, Y.-M.; Zong, M.-H.; Wu, H.; Li, N. Enzyme-catalyzed selective oxidation of 5-hydroxymethylfurfural (HMF) and separation of HMF and 2,5-diformylfuran using deep eutectic solvents. Green Chem. 2015, 17, 3718-3722, DOI: 10.1039/C5GC00788G. 20. de Carvalho, C. C. Whole cell biocatalysts: essential workers from Nature to the industry. Microb. Biotechnol. 2017, 10, 250-263, https: //doi.org/10.1111/1751-7915.12363. 21. Wachtmeister, J.; Rother, D. Recent advances in whole cell biocatalysis techniques bridging from investigative to industrial scale. Curr. Opin. Biotechnol. 2016, 42, 169-177, https: //doi.org/10.1016/j.copbio.2016.05.005. 22. Švitel, J.; Kutnik, P. Potential of acetic acid bacteria for oxidation of low‐molecular monoalcohols. Lett. Appl. Microbiol. 1995, 20, 365-368, https: //doi.org/10.1111/j.1472765X.1995.tb01322.x. 23. Gupta, A.; Singh, V. K.; Qazi, G.; Kumar, A. Gluconobacter oxydans: its biotechnological applications. J. Mol. Microbiol. Biotechnol. 2001, 3, 445-456. 24. Bories, A.; Claret, C.; Soucaille, P. Kinetic study and optimisation of the production of dihydroxyacetone from glycerol using Gluconobacter oxydans. Process Biochem. 1991, 26, 243248, https: //doi.org/10.1016/0032-9592(91)85006-A.


Page 18 of 28

Page 19 of 28 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


25. Molinari, F.; Aragozzini, F.; Cabral, J.; Prazeres, D. Continuous production of isovaleraldehyde through extractive bioconversion in a hollow-fiber membrane bioreactor. Enzyme Microb Technol. 1997, 20, 604-611, https: //doi.org/10.1016/S0141-0229(96)00205-0. 26. Sayed, M.; Dishisha, T.; Sayed, W. F.; Salem, W. M.; Temerk, H. A.; Pyo, S.-H. Selective oxidation of trimethylolpropane to 2, 2-bis (hydroxymethyl) butyric acid using growing cells of Corynebacterium sp. ATCC 21245. J. Biotechnol. 2016, 221, 62-69, https: //doi.org/10.1016/j.jbiotec.2016.01.022. 27. Dishisha, T.; Pyo, S.-H.; Hatti-Kaul, R. Bio-based 3-hydroxypropionic-and acrylic acid production from biodiesel glycerol via integrated microbial and chemical catalysis. Microb. Cell Fact. 2015, 14, 200, https: //doi.org/10.1186/s12934-015-0388-0. 28. Pyo, S.-H.; Dishisha, T.; Dayankac, S.; Gerelsaikhan, J.; Lundmark, S.; Rehnberg, N.; Hatti-Kaul, R. A new route for the synthesis of methacrylic acid from 2-methyl-1, 3-propanediol by integrating biotransformation and catalytic dehydration. Green Chem. 2012, 14, 1942-1948, DOI: 10.1039/C2GC35214A. 29. Sayed, M.; Dishisha, T.; Sayed, W. F.; Salem, W. M.; Temerk, H. M.; Pyo, S.-H. Enhanced selective oxidation of trimethylolpropane to 2,2-bis (hydroxymethyl) butyric acid using Corynebacterium sp. ATCC 21245. Process Biochem. 2017, 63, 1-7, https: //doi.org/10.1016/j.procbio.2017.08.007. 30. Eggeling, L.; Sahm, H. The formaldehyde dehydrogenase of Rhodococcus erythropolis, a trimeric enzyme requiring a cofactor and active with alcohols. Eur. J. Biochem.1985, 150, 129134, https: //doi.org/10.1111/j.1432-1033.1985.tb08997.x. 31. Prust, C.; Hoffmeister, M.; Liesegang, H.; Wiezer, A.; Fricke, W. F.; Ehrenreich, A.; Gottschalk, G.; Deppenmeier, U. Complete genome sequence of the acetic acid bacterium Gluconobacter oxydans. Nature Biotechnol. 2005, 23, 195-200, DOI:10.1038/nbt1062. 32, Wei, L. J; Zhou, J. L.; Zhu, D. N.; Cai, B. Y.; Lin, J. P.; Hua, Q.; & Wei, D. Z. Functions of membrane-bound alcohol dehydrogenase and aldehyde dehydrogenase in the bio-oxidation of alcohols in Gluconobacter oxydans dsm 2003. Biotechnol. Bioprocess Eng.2012, 17, 1156-1164, DOI: 10.1007/s12257-012-0339-0. 33. Mussatto, S. I.; Roberto, I. C. Alternatives for detoxification of diluted-acid lignocellulosic hydrolyzates for use in fermentative processes: a review. Bioresource Technol. 2004, 93, 1-10, https: //doi.org/10.1016/j.biortech.2003.10.005. 34. Kurzrock, T.; Weuster-Botz, D. Recovery of succinic acid from fermentation broth. Biotechnol. Lett. 2010, 32, 331-339, DOI: 10.1007/s10529-009-0163-6. 35. López-Garzón, C. S.; Straathof, A. J. J. Recovery of carboxylic acids produced by fermentation. Biotechnol. Adv. 2014, 32, 873-904, https: //doi.org/10.1016/j.biotechadv.2014.04.002. 36. Cao, X.; Yun, H. S.; Koo, Y.-M. Recovery of L-(+)-lactic acid by anion exchange resin Amberlite IRA-400. Biochem. Eng. J. 2002, 11, 189-196, https: //doi.org/10.1016/S1369703X(02)00024-4.



Page 20 of 28

Scheme. 1 5-Hydroxymethylfurfural (HMF) transformation to value added building blocks. 5-Hydroxymethylfuran-2-carboxylic

acid

(HMFCA),

5-formyl-2-furancarboxylic

acid

(FFCA), 2,5-furan dicarboxylic acid (FDCA), and 2,5-bis(hydroxymethyl)furan (BHMF). The scheme shows mainly the oxidation [O] reactions.

O

O

HO

H

BHMF

C6-sugars (biomass)

O HO

H

[O]

Reduction Dehydration

O

O

OH

DFF

O H

[O]

5-Hydroxymethylfurfural (HMF)

[O] O

O HO

O

[O]

OH

HMFCA

[O]

[O] O

O

HO

OH

FDCA


O

O

H

O OH

FFCA

Page 21 of 28 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure legends Fig. 1. Screening of (A) G. oxydans DSM 2343 and (B) G. oxydans DSM 50049 resting cells for oxidation of 5 g L-1 HMF. Symbols: HMF (), HMFCA (▲) and BHMF (●). Experimental details are provided in the text. Fig. 2. Effect of pH 5 (■), 6 (▲), 7 (●) and 8 () on the selective oxidation of the crude 5HMF (16 g.L-1) to HMFCA using resting cells of G. oxydans DSM 50049 in 100 mM phosphate buffer. (A) Content (%) of HMFCA, and (B) initial productivity (g.L-1h-1) calculated after the first 3 h of the reaction at different pH conditions. Fig. 3. Effect of different amounts of G. oxydans DSM 50049 cells, 4 (■), 8 () and 12 mg.ml-1 (●) CDW on the selective oxidation of 16 g.L-1 HMF (empty symbols) to HMFCA. (A) Conversion (%) of HMF and content (%) of HMFCA, and (B) initial productivity (g.L-1h-1) calculated after 3 h reaction. Fig. 4. Profiles of selective oxidation of HMF (●) to HMFCA (■) using G. oxydans DSM 50049 cells on scaling up the reaction. Reactions with (A) 24 g L-1 HMF in 50 mL scale with intermittent pH control, and (B) with 43.5 g L-1 HMF in 250 mL solution in a bioreactor with continuous pH control. HMFCA productivity (▲) was calculated at different time points to evaluate the performance of the reaction. Fig. 5. Binding profile of HMFCA from 1 mL reaction solution to 100 mg Amberlite IRA400 (Cl form) (red) and Ambersep® 900 (OH- form) (blue). (A) Concentration of unbound HMFCA during adsorption step, and (B) concentration of HMFCA desorbed during washing (W) and elution (E) steps. Fig. 6. HPLC chromatograms of samples of (A) initial HMF solution used for oxidation by G. oxydans DSM 50049; (B) end product of the oxidation reaction (C); aqueous phase after liquid-liquid extraction with ethyl acetate (EA); and (D) concentrated HMFCA from ethyl acetate phase.



Fig. 1.

A

B


Page 22 of 28

Page 23 of 28 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Fig. 2

A

B



Fig. 3

A

B


Page 24 of 28

Page 25 of 28 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Fig. 4

A

B



Fig. 5

A

B


Page 26 of 28

Page 27 of 28 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Fig. 6



For Table of Contents Use Only Synopsis Gluconobacter oxydans DSM 50049 cells oxidize high concentration of HMF totally to HMFCA with exquisite selectivity.


Page 28 of 28

Selective Oxidation of 5-Hydroxymethylfurfural to 5-Hydroxymethyl-2

Recommend Documents