Co-production of HMF and gluconic acid from sucrose by chemo-enzymatic method

Accepted Manuscript Co-production of HMF and Gluconic Acid from Sucrose by Chemo-enzymatic Method Hongli Wu, Ting Huang, Fei Cao, Qiaogen Zou, Ping Wei, Pingkai Ouyang PII: DOI: Reference:

S1385-8947(17)31058-6 http://dx.doi.org/10.1016/j.cej.2017.06.107 CEJ 17188

To appear in:

Chemical Engineering Journal

Received Date: Revised Date: Accepted Date:

26 April 2017 12 June 2017 20 June 2017

Please cite this article as: H. Wu, T. Huang, F. Cao, Q. Zou, P. Wei, P. Ouyang, Co-production of HMF and Gluconic Acid from Sucrose by Chemo-enzymatic Method, Chemical Engineering Journal (2017), doi: http://dx.doi.org/ 10.1016/j.cej.2017.06.107

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Co-production of HMF and Gluconic Acid from Sucrose by Chemo-enzymatic Method +

+

Hongli Wu , Ting Huang , Fei Cao*, Qiaogen Zou, Ping Wei, Pingkai Ouyang College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University 30 South Puzhu Road, Nanjing, 211816 (P. R. China) *Corresponding author Email: [email protected] Abstract: Co-production of multi-products is one of the core principles of chemical industry, and it is also an important way to improve the atom economy. Herein, we proposed an approach to co-producing two valuable platform compounds, gluconic acid (GA) and 5-hydroxymethyl furfural (HMF), from sucrose by successive hydrolysis, oxidation and dehydration. In the enzymatic oxidation step, only glucose was oxidized to GA, meanwhile fructose was 100% retained. In the further biphasic dehydration system, only fructose was converted into HMF, whereas GA was maintained with over 95% recovery. After three reactions, the yields of HMF and GA were respectively 42.5% and 48% when the initial feedstock of sucrose was 200g/L. Two products were easily separated because GA was completely existed in aqueous phase, and HMF was mainly in organic phase. In the whole process, only commercial enzymes and mineral acid were used instead of self-made catalysts. Keywords: Co-production • HMF • Gluconic acid • Chemo-enzymatic method • Sucrose Introduction HMF, one of the most important bio-based platform compounds, is used to produce a variety of biopolymers, biofuels, commodity chemicals, pharmaceuticals, and can be synthesized from all types of monomeric or polymeric glucose and fructose, such as glucose, fructose, sucrose, starch, inulin, cellulose and so on[1-4]. Fructose is the best raw material for HMF preparation because of its natural furanose structure which is beneficial to the formation of HMF. However, its price is too high to be applied in the large-scale HMF production. This conclusion was also demonstrated by Torres[5] and Kabir Kazi[6] in their studies about the techno-economic evaluation of HMF production. Hence, lots of cheaper materials like glucose, sucrose and cellulose were chosen to manufacture HMF. Compared to fructose, glucose is a more abundant and less expensive hexose (its price was shown in Table S1) and can be obtained from cheaper raw materials like cellulose or starch. However, due to the stable nature of its pyranose ring structure, harsh reaction conditions are required to offset side-reactions and increase HMF yield. Based on this, lots of progresses have recently been made in integrating the glucose isomerization (by solid bases[7-9], enzymes[10, 11], or Lewis acids[12-16]) and fructose dehydration to produce HMF in a “one-pot” configuration. Nonetheless, several limitations were revealed in recent publications[17, 18]. For example, high HMF yields (70-90%) are reported from glucose in ionic liquid with the Lewis acid catalyst, but ionic liquids are not yet suitable for large scale applications due to the difficulty of HMF’s separation, deactivation by small amounts of water and their high cost[19]. Another choice is separating fructose after the isomerization

of

glucose.

Alipour[20]

recently

explored

a

novel

enzyme-based

Simultaneous-Isomerization-and-Reactive-Extraction (SIRE) process to extract fructose into a water-immiscible organic phase (octanol). Then the fructose transferred from the organic phase by Back-Extraction (BE) into an acidic IL ([EMIM]HSO4) reaction medium was dehydrated into HMF. Finally, HMF was re-extracted into the low boiling point organic solvent. The whole process has undergone three extractions, which consumed amounts of

solvent and was difficult to be operated. Sucrose, another one of the most widely-traded commodities in the world with a low price (see also Table S1), is easily hydrolyzed into one molecular glucose and fructose. For the adequate use of these two carbohydrates, the Brφnsted-Lewis composite catalytic system was commonly applied to produce HMF. However, those approaches have the same limitations as the process of HMF preparation from glucose. Inedible cellulose has been recognized as most promising renewable resource for the production of HMF. Commonly, the synthesis of HMF from cellulose is also considered through the pathway of glucose isomerization and will face the same difficulty like glucose as raw material. We had explored a different route from cellulose to HMF in polar aprotic solvents and its yield reached 44%, however, its reaction conditions are very harsh[21]. Therefore, we need a new process of HMF to change this status. Co-production of multi-products is an important method for chemical engineering processes and the revenue of the co-products will reduce the overall cost. Gluconic acid (GA), an oxidation production of glucose, is a valuable platform chemical and widely used in the pharmaceutical, food, detergent, textile, leather, and concrete industries with its salts[22]. Different from other glucose oxidation methods including catalytic oxidation[23-26], electrochemical oxidation[27], fermentation[28, 29] , enzymatic oxidation, especially glucose oxidase (GOD, β-D-glucose: oxygen 1-oxidoreductase, E. C.: 1.1.3.4),

merely oxidized glucose into GA

without affecting other carbohydrates because of its high substrate specificity [30]. Based on this character, GOD was often applied to remove the residue glucose and improve the purity of the other sugars or polysaccharides[31, 32]. GA production form sucrose through the multi-enzyme reaction has also been reported in the literatures[33-35]. In these processes, a mixture solution of GA and fructose was formed. Consequently, HMF will be co-produced with GA from GA and fructose mixtures if GA is stable, does not affect the dehydration of fructose under acidic reaction condition and is easily separated in the subsequent separation process. Meanwhile, the overall revenues of HMF preparation will be greatly improved and the restrictions of raw materials and catalysts will be reduced. Herein, we reported an approach to co-producing GA and HMF from sucrose by a chemo-enzymatic method (Scheme 1). A glucose and fructose mixture was obtained from sucrose hydrolysis, and then was converted into GA and fructose using GOD and catalase (CAT, E. C.:1.11.1.6). This mixture solution was further converted into HMF by dilute sulfuric acid in a water/2-methyl tetrahydrofuran biphasic system (H2O/2-MeTHF). The presence of GA did not affect the fructose dehydration and GA was reserved in the aqueous phase. While the HMF went into the organic phase and therefore it was easy to separate from the GA. Base on the conditions of batch reaction, a continuous co-production of GA and HMF in a micro-flow reactor was also explored.

Scheme 1. The co-production of HMF and GA from sucrose.

Experimental

Chemicals. Sucrose, sodium gluconate, glucono- δ -lactone, 2-methyltetrahydro-furan, sulphuric acid were purchased from Sigma–Aldrich. Invertase (INV, E. C.: 3.2.1.26), glucose oxidase and catalase were supplied by Shanghai Baoman Biotech. Co. Other regents were provided from Aladdin.

Sucrose enzymatic hydrolysis. Solution of sucrose (200 g/L) were prepared in water (pH was adjusted to 4.5) and putted into a 50 mL conical flask. Immobilized invertase (Invertase was immobilized by 4% carrageenan, 2000IU) was added into solution. The reaction mixture was shaken in a shaker at 200 rpm and 50 °C.

Glucose enzymatic oxidation. Glucose and fructose (200g, respectively) were dissolved in 2 L water and put into a 5 L Bioflo 100 fermentor. After purged with air, glucose oxidase (10000IU) and catalase (40000IU) was added into solution. The reaction mixture was stirred at 45 °C. During whole reaction, pH was maintained 6.5-7.5 by continuously adding 10 M NaOH solution. Before this mixture was used in furthermore dehydration, 21mL sulfuric acid (98%) was add into product solution, and the protein precipitations were removed by centrifugation (10000 rpm).

GA’s decomposition. The 0.61g sodium gluconate and 85uL sulfuric acid (98%) was dissolved in 5mL water. The solution was carried out in 15 mL sealed ACE 8648 glass tube, and purged with nitrogen before sealing. The reaction mixture was stirred for 2 min at room temperature and then, heated at 130, 150, 170, and 190 °C for 0.5 h or 1 h. After rapidly being quenched in ice-water, samples were analyzed by HPLC. Although a certain amount of glucono-δ-lactone was formed from GA in acidic condition, it can be converted back into GA after adding 0.1M NaOH solution. Therefore, all samples were adjusted to pH=9.0 before HPLC analysis.

Dehydration with Microwave irritation. Fructose and sodium gluconate (typically 100 mg and 121 mg) were weighed out into a 10 mL microwave tube with 1 mL water (or salt water). After adding 18.1uL H2SO4 (15.4 uL for acidizing sodium gluconate and 2.7uL for catalyst) and 5 mL 2-MeTHF, the tube was then sealed using the microwave tube lid. These samples were placed in a CEM Discovery SP microwave apparatus, operating at a frequency of 2.45 GHz with continuous radiation power from 0 to 300 W. The reactor was equipped with a magnetic stirrer as well as with pressure, temperature and power controls. The mixtures were reacted under 140–170 °C for 2-10 min. After microwave treatment, the aqueous layer and the organic solvent layer were separated and filtered with a 0.2 μm syringe filter. Each sample was diluted with water prior to analysis.

Continuous micro-flow reaction. Continuous micro-Flow reactions were performed with Vapourtec ® R2-C/R4 instruments. All flow reactions reported were run in polymer tubing (PFA) with an outer diameter of 1/16 inch and 1 mm inner diameter. A solution of 2-MeTHF and an aqueous solution including 100 g/L fructose, 120 g/L sodium gluconate (before acidizing) and 50mM H2SO4 were fed to the reactor. The flow rates of 2-MeTHF and aqueous solution were 0.48mL/min and 0.12 mL/min, respectively. The reaction mixture passed through the reactor under 150 °C for 10 min and was subsequently cooled in an ice bath to stop the reaction. The pressure in the system was kept at 10 bars by a back pressure regulator (BPR Assembly P-791, Upchurch Scientific, USA) to avoid solvent vaporization. Crude HMF was obtained after the organic phase was separated and condensed.

Analysis. Reaction product samples were analyzed by high performance liquid chromatography (Agilent 1260 Infinity). Carbohydrates (except glucose) were detected with a RI detector (1260 RID). HMF and gluconic acid were detected with a UV-Vis detector (1260 VWD VL) at wavelengths 210 nm. The column used was a Biorad© Aminex HPX-87H sugar column. The mobile phase was 0.005 M H2SO4 flowing at a rate of 0.6 mL/min. The column oven was set to 30 °C. The concentration of glucose in the glucose oxidation reaction was analyzed using a biosensor equipped with glucose oxidase electrode (SBA-40C, Shandong Academy of Sciences, China), because the glucose’s peak was coincided with the GA’s peak on HPX-87H sugar column. GA’s yield in the enzymatic oxidation and HMF’s yield in the dehydration were calculated as follows:

Fructose’s recovery in the enzymatic oxidation and GA’s recovery in the dehydration were calculated as follow:

Result and discussion Enzymatic hydrolysis and oxidation The glucose-fructose mixture solution can be prepared from the hydrolysis of sucrose, the isomerization of glucose, and even directly come from the high fructose syrup. Herein, we used INV as catalyst to hydrolyze sucrose. The immobilized INV was effective (sucrose was converted completely in 10min) and can be used over 10 batches (shown in Fig. S1). In glucose-fructose mixture, only glucose was converted into sodium gluconate (NaGlc, sodium ion come from NaOH solution which was added to control the reaction pH value) after 8h and fructose was hardly consumed (shown in Fig. 1). The byproduct of enzymatic glucose oxidation, hydrogen peroxide, is decomposed into oxygen and water by CAT. In order to reduce the effect of glucose on the subsequent dehydration reaction, we extended the oxidation reaction time to 22h and ensured that no glucose was detected. These solutions can be directly used to co-produce HMF and GA.

Fig. 1. Enzymatic oxidation with the co-catalysis of glucose oxidase and catalase in 5L Bioflo 100 fermentor: (■) glucose, (▲) fructose, (●) NaGlc. Reaction condition: Glucose and fructose loading (2 L) were all 100g/L, the activity glucose oxidase and catalase were 10000IU and 40000IU, respectively. T=45 ºC, The solution was controlled pH=6.5-7.5 using 10M NaOH solution, and air rate was 4 L/min. GA’s stability under dehydration condition In order to realize our proposed co-production with HMF, GA must be stable under the following dehydration conditions. The stability of GA complexation with Nd(III) and Ln(III) in acidic solutions at 25 ºC have been studied by Zhang[36] and Panda[37, 38]. However, the stability of GA in acidic solution at higher temperature conditions has not reported. Fig 2 shows the stability of the GA with 50mM sulfuric acid at 130 ºC, 150 ºC, 170 ºC, and 190 ºC. The recoveries of GA were all over 95% after 0.5 h at the tested temperature range from 150 ºC to 190 ºC. The higher was the reaction temperature, the more was the decomposition of GA. Only 86% GA was detected at 190 ºC after 1 h. However, the recoveries of GA were still over 96% at 130 ºC after 3 h and 92% at 150 ºC after 3 h, respectively (data was shown in Fig. S2, and the MS spectrum of GA was shown in Fig. S3). These results indicated that the loss of GA can be reduced at lower dehydration temperature.

Fig. 2 The stability of GA under acidic condition. The white bars are GA’s recovery at 0.5h, and the stripped bars are GA’s recovery at 1h. Reaction condition: 5 mL 100g/L sodium gluconate solution was sealed in a 20 mL ACE 8648 glass tube, and put in oil bath at 130 ºC, 150 ºC, 170 ºC, and 190 ºC. The sulfuric acid concentration was 50 mM.

Dehydration of fructose into HMF from GA and fructose mixture solution Based on the stability of GA under high temperature and acidic condition, the mixture solution of GA and fructose was chosen as the raw materials to prepare HMF. The water–solvent biphasic systems for HMF production which have been reported to decrease the rate of HMF decomposition thereby improving the HMF yield was used, and were easy to separate the two compounds of GA and HMF (The distributions of fructose, GA and HMF in the two phases were shown in Table S2. Fructose and GA were not soluble in organic solvents, and HMF mainly existed in the organic phase). Herein, we selected a green solvent of 2-Methyltetrahydrofuran (2-MeTHF, b.p. 78 ºC) which is from biomass[39] and has an acceptable extraction effect with unsalted water[40]. Assisted by microwave irradiation heating, the dehydration time of fructose can be reduced to less than 10min (HMF’s yield reached 78.3% in Table S3), which further reduces the loss of GA. The effect of concentration and ratio of NaGlc and fructose for fructose dehydration in H 2O/2-MeTHF biphasic system with microwave-assistant irradiation is shown in Table 1(Entry 1-7). Whatever any concentration or ratio, the recoveries of GA in aqueous phase were all over 95%. As the fructose concentration increased, the yield of HMF decreased from 76% to 67% with over 99% fructose’s conversion. The ratio of NaGlc and fructose (from 0.5 to 2, entry 2, 3, 5) in the feed did not have a major effect on the HMF’s yield (from 76% to 71%). It is important to note that HMF’s yield has visibly deteriorated due to increasing the acid concentration, despite a minimal effect on GA’s recovery (Entry 4). Compared to the preparation of HMF from fructose alone, the HMF's yield has a small reduction (Entry 1 and 2, Table S3). Furthermore, the dehydration of fructose was not affected when GA was replaced by glucono-δ-lactone (Entry 8). The effect of reaction temperature and time on the dehydration of fructose under the co-existence of NaGlc is shown in Table 1(Entry 9-14). From 140 to 170 ºC, the recoveries of GA in aqueous phase were never below 98%. However, the HMF yield of HMF changed with temperature and time because of the incomplete fructose conversion. At 140 ºC, the conversion of fructose was only 68.6% after 10 min, and the HMF yield was 53.3% (Entry 9). At a temperature of 150 ºC, 98% of fructose was converted in 10min, and the HMF yield increased to 74.5% (Entry 6). Further increasing the temperature shortened the complete conversion time to 5 min, and the yields of HMF were 73 and 71% at 160 ºC and 170 ºC, respectively. Using the actual enzymatic oxidation solution as raw material, the recovery of GA in aqueous phase was 98.9% and the yield of HMF was 73.8% with over 99% fructose’s conversion (Entry 15). Except of 2-MeTHF, we also chose other solvents that commonly used in the literatures, such as MIBK, 2-butanone, 2-butanol to be as extraction phase and tested the effect of solvents for the co-production of GA and HMF (shown in Table 1, entry 16-18). From these results, MIBK and 2-butanone were presented the good yields of HMF, whereas the recoveries of GA were unacceptable. On the contrast, 2-butanol has the better recovery of GA than MIBK and 2-butanone; however the yield of HMF was low. Here, in particularly, water without adding salt was used to form the biphasic system with those organic solvents. Table 1. The GA and HMF co-production in biphasic systema. Concentratio Entry

n of NaGlc and fructose

Sulfuric Solvents

(g/L)

Temperatur

Time

acid

e

(min)

concentra

(ºC)

GA’s recovery (%)

tion (mM)

Fructose’ s

HMF’s

conversio

yield (%)

n (%)

1

0/50

2-MeTHF

50

150

10

0

99.8

75.0

2

30/50

2-MeTHF

50

150

10

99.0

99.7

75.5

3

60/50

2-MeTHF

50

150

10

98.6

99.8

76.1

4

60/50

2-MeTHF

100

150

10

99.2

99.8

63.3

5

121/50

2-MeTHF

50

150

10

98.1

99.8

71.5

6

121/100

2-MeTHF

50

150

10

99.9

99.5

74.5

7

242/200

2-MeTHF

50

150

10

95.0

99.5

67.7

8

99b/100

2-MeTHF

50

150

10

99.0

99.5

72.6

9

121/100

2-MeTHF

50

140

10

99.0

68.6

53.3

10

121/100

2-MeTHF

50

150

2

101.2

59.9

43.0

11

121/100

2-MeTHF

50

150

5

100.2

70.3

54.5

12

121/100

2-MeTHF

50

160

2

100.0

84.0

63.2

13

121/100

2-MeTHF

50

160

5

98.4

97.0

73.5

14

121/100

2-MeTHF

50

170

5

98.9

99.9

71.7

15c

129/106

2-MeTHF

50

150

10

98.9

99.8

73.8

16

121/100

MIBK

50

150

10

76.7

99

63.0

17

121/100

2-butanol

50

150

10

96.7

99

52.7

18

121/100

50

150

10

90.0

99

60.2

2-butanon e

a:

Reaction condition: Vorg./Vaq. = 5, MWI=150w.

b:

GA was here replaced by glucono-δ-lactone.

c:

The mixture solution is from actual enzymatic oxidation in this entry.

The effect of salt and metal cations Many articles had verified that addition of salt to aqueous solutions of fructose in biphasic systems can improve HMF yields by increasing the partitioning of HMF into the extracting phase [40, 41]. We investigated the “salt-out” effect of co-production reaction using the 3.5 wt. % NaCl solution (seawater’s salt concentration) and saturated NaCl solution. The yield of HMF increased from 75 to 80% in the 3.5 wt. % NaCl solution, but the saturated NaCl solution resulted in a decreasing of the GA recovery and HMF yield (shown in Table. 2). Table 2 Effect of NaCl concentration on the GA and HMF co-production in biphasic systems. a

a:

NaCl concentration

GA’s recovery

Fructose’s conversion

(wt. %)

(%)

(%)

0

98.6

99.9

75.0

3.5

97.4

99.9

80.1

35

94.0

99.9

71.8

HMF’s yield (%)

Reaction condition: NaGlc/fructose 60/50 g/L, 150 ºC 10min, 50mM sulfuric acid, V org./Vaq. = 5, MWI=150w

In the process of glucose oxidation reaction by GOD, various metal oxides or carbonates were used to maintain the pH value. Hence, metal ions often exist in the reaction solution after oxidation. We investigated the effects of various metal ions on the dehydration reaction (Fig. 3). The HMF’s yields were 78-79% for the monovalent cations tested. Alkaline-earth metal cations (including Mg2+ and Ca2+) caused a decrease in the GA recovery because GA with alkaline-earth metal ions formed the insoluble salt, and then precipitated from reaction solution. However, high yields of HMF were gained with the existence of alkaline-earth metal ions for fructose dehydration reaction. Other divalent or trivalent metal cations, such as Cu2+, Zn2+, Fe2+and Cr3+, decreased the GA recovery (89, 76, 72 and 52%, respectively) and HMF yield (60, 59, 59 and 58%, respectively). This decreasing was probably due to the carbohydrate isomerization which was always catalyzed by these four metal cations as Lewis acid. Hence, we cannot use these metal oxides or carbonates which included divalent or trivalent metal cations as a neutralizing agent in the process of glucose oxidation.

Fig. 3. The effect of metal cations on the GA and HMF co-production. Black filled bars are GA’s recovery, white bars are fructose conversion, and stripped bars are HMF yield. Reaction condition: metal ions’ concentrations are all 20g/L and NaGlc/fructose is 60/50 g/L, T=150 ºC, 50mM sulfuric acid, Vorg. /Vaq. = 5, MWI=150w, 10min. Continuous micro-flow reaction. In literatures, the dehydration of fructose using a biphasic system was always carried out in batch reaction. However, for the development of an industrial scale process, the production of HMF in continuous reactor is indispensable. Recently, Shimanouchi[42] and Tuercke[43] made an impressive progress of HMF production in micro-flow reactors, while the yields were 88.5% and 85%, respectively. These results indicated that the micro-flow reactor can not only continuously produce HMF, but also offer the enormous surface-to-volume ratio in the liquid-liquid biphasic system, combined with efficiency and safety increasing compared to conventional chemical reactors. Basing on the optimization of reaction conditions (Table S4-5), we have implemented the 5 batches continuous co-production of GA and HMF in a Vapourtec® R2-C/R4 micro-flow reactor. The reaction conditions are similar to the batch experiments and only replaced by the oil bath instead of the microwave heating. The results in Table 3 showed that the micro-flow system presented here could achieve a stable yield of HMF (over 85%, it is higher than batch reaction); meanwhile the average recovery of GA was also comparable with batch reaction (95.6%). It proved the excellent prospects for the continuous co-production of GA and HMF in the micro-flow reactor. Table 3. The continuous preparation of GA and HMF in a micro-flow reactor. a Crude HMFb

HMF’s

volume (mL)

(g)

purityc (%)

1

200

2.87

85

87

96

2

265

3.76

84

85

96

3

210

3.05

86

89

95

4

230

3.32

87

89

95

5

280

4.09

84

87

96

Batch

a:

GA’s

Total reaction

HMF’s yield (%)

recovery (%)

Shimanouchi[42]

88.5

Tuercke[43]

85

Reaction condition: The concentration of NaGlc and fructose is 120 and 100 g/L, respectively. T=150 ºC,

residence time =10min, Water: 2-MeTHF=4:1, 50mM sulfuric acid.

b:

Crude HMF means the masses of concentrate which were obtained from extract solvent.

c:

HMF purity was determined by GC with external standard method.

Based on our co-production process, the production of 1 ton HMF need 3.36 ton sucrose, and co-produces 1.75 ton NaGlc (The whole process was show in Fig. 4. The reaction parameter and mass balance were shown in Table S3 and S4). After the two phases’ separation, HMF is easily obtained from organic phase through evaporating 2-MeTHF (a low boiling point solvent), and NaGlc can be generated from the aqueous phase after concentration and crystallization. O2

Sucrose 3.36t INV 0.34kg Water

Glucose 1.77t Fructose 1.77t GOD 6.2kg + CAT 3.5kg Water

H2SO4

NaGlc 1.95t Fructose 1.77t Water

GA 1.75t

MeTHF, 171t

HMF 1t

Water

MeTHF

Fig. 4 Overall process block flow diagram of co-production of HMF and GA Conclusion In summary, we report a novel approach to simultaneously produce NaGlc and HMF from sucrose by chemo-enzymatic method. A glucose and fructose mixture solution was obtained from sucrose hydrolysis, and then glucose was oxidized to gluconic acid by glucose oxidase and catalase whereas enzymatic reaction did not affect fructose. In water/ 2-methyltetrahydrofuran biphasic system, fructose was dehydrated to HMF using sulphuric acid as catalyst under microwave radiation condition. HMF's yield reached 76%, and GA's recovery was over 95% in 10min 150 °C using 50mM sulphuric acid when the feedstock of NaGlc and fructose were 60.5 and 50g L-1, respectively. In the micro-flow reactor, the higher yield of HMF (over 85%) and the stable recovery of GA (over 95%) were achieved, which proved the excellent prospects for the continuous co-production of GA and HMF. The whole processes have several advantages. First, HMF can be produced with NaGlc as co-product from inexpensive sucrose. Additionally, this method also can be applied to any source of glucose and fructose mixtures, such as high fructose corn syrup or waste molasses. Secondly, the whole process only used the commercial enzymes and mineral acid instead of any self-made catalysts. Finally, two products are easily separated because they are in different phases. Furthermore, the immobilization of GOD and CAT, the screening of more suitable solvent and the extension of GA into δ-glucono-lactone or arabinose will further improve products revenues and reduce the HMF’s cost. Author contributions +The authors contributed equally to this work. Abbreviations HMF, 5-hydroxylmathyl furfural; GA, gluconic acid; NaGlc, sodium gluconate; 2-MeTHF, 2-methyl tetrahydrofuran; GOD, glucose oxidase; INV, invertase; CAT, catalase. Acknowledgements

This work was supported by the Jiangsu National Synergetic Innovation Center for Advanced Materials (SICAM). We also gratefully acknowledge the financial support provided by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD) and the Opening Foundation of State Key Laboratory of Materials-Oriented Chemical Engineering (KL16-06). The authors would like to acknowledge Wei Zhao (Shandong Fuyang Biotechnology Co. Ltd.) for the help with the industrial usages of GOD and CAT. The authors also thank Professor George W. Huber (University of Wisconsin, Madison) for his valuable comments and suggestions. References

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Co-production of HMF and Gluconic Acid from Sucrose by Chemo-enzymatic Method Hongli Wu, Ting Huang, Fei Cao, Qiaogen Zou, Ping Wei, Pingkai Ouyang

The co-production of HMF with 42.5% yield and gluconic acid with 48% yield from sucrose by chem-enzymatic method was explored.

Highlights 

Co-production of gluconic acid and HMF from sucrose was presented.



The yields of HMF and gluconic acid reached 42.5% and 48%, respectively.



Gluconic acid and HMF were easily separated because they were in different phases.