1Production of levulinic acid from corn cob residue in a fed-batch

2acid hydrolysis process

3Chen Lianga, Yangdong Hua*, Yan Wangb, Lianying Wua, and Weitao Zhanga

4a. College of Chemistry and Chemical Engineering, Ocean University of China, 238

5Songling Road, Qingdao , PR China

6b. College of Applied Technology, Qingdao University, 93 Songling Road, Qingdao,

7PR China

8
9*Correspondence to: Yangdong Hu, Ocean University of China, 238 Songling Road,
10Qingdao, 266100 PR China
11Tel: +86 0532-66782141
12E-mail: ydhuhd@ouc.edu.cn
13

14ABSTRACT:

15Levulinic acid (LA) is an important platform chemical, the production of which by

16using biomass resources such as corncob is of great significance to the sustainable

17development. Traditional hydrolysis processes yield low concentrations of levulinic

18acid with large amounts of acid being consumed. In this paper, a new fed-batch

19process for hydrolyzing corncob residues with sulfuric acid being utilized as the

20catalysis to produce levulinic acid at high concentrations is proposed. The mass

21concentration of the levulinic acid increased with growing the times of feeding and a

22107.93 g/L of the levulinic acid can be reached at the 7 th hydrolysis. However, the

23yield of levulinic acid reduces gradually during the fed-batch process. To explore the

1
24phenomenon, the reaction mechanism of cellulose acid hydrolysis was experimentally

25studied. It can be deduced that the reduction of the levulinic acid yield was caused by

26the polymerization of the soluble humin analogues and the 5-hydroxymethyl furfural

27(5-HMF) as well as the glucose.

28Keywords: Fed-batch acid hydrolysis; Levulinic acid; Corncob; Mechanism of

29humins formation.

301. INTRODUCTION

31 Biomass resources are known as low-cost, renewable, extensive and

32environment-friendly, and the utilization of which to produce correlative chemicals is

33of great significance to the sustainable development [1]. For example, an important

34platform chemical, the levulinic acid, which is predominantly adopted to produce

35various bio-chemicals such as solvents, resins, plasticisers, polymers, herbicides,

36pharmaceuticals, flavoring agents and biofuels via esterification, substitution and

37redox etc., can be obtained from cellulose-rich biomass resources [2-4].

38 At the end of the last century, Biofine company used a two-step hydrolysis

39method with dilute acids and carbohydrate-containing materials, e.g., the slurry of

40paper material, to produce the levulinic acid [5]. With over 70 % yield of the levulinic

41acid, they started a small-scale industrial production. In addition to paper slurry,

42levulinic acid can be obtained by the hydrolysis of other biomass materials such as

43sorghum [6], water hyacinth [7], bagasse [8], rice husks [9], wheat straw [10] and

44corn stalk [11], etc. Among various bioresource, the corncob, with an annual output of

45approx. 50 million tons in China [12], has been widely used in producing furfural [13,

2
4614], xylose [15] and xylitol [16]. The corncob residue contains large amounts of

47cellulose, which can be recycled to acquire downstream chemicals, such as the

48levulinic acid.

49 In the process of producing the levulinic acid from biomass resources, the acid

50catalyst is required during the hydrolysis reaction [17]. Acid catalysts, such as solid

51acid [18], inorganic acid [19, 20], organic acid [21], and acidic functionalized ionic

52liquids [22], are usually considered. Among them, inorganic acids are the most

53commonly used catalyst in producing the levulinic acid since they are cost effective.

54However, if large amounts of acid were utilized in hydrolysis processes, the recycling

55of acid residuals after reaction would be difficult, resulting in significant waste of

56resources and environmental pollution. In addition, the levulinic acid, after being

57produced, has to be concentrated for the subsequent separation and purification

58processes. The more water contents exist in the hydrolysate, the more energy has to be

59consumed in the concentration process. It is not conducive with respect to energy

60saving. Aiming at this problem, Guo and co-workers [23] proposed an improved

61hydrolysis technology, in which the hemicellulose in corncob was hydrolyzed to

62produce high concentrations of the xylose production. However, technology refers to

63the hydrolysis of the cellulose to produce high concentrations of the levulinic acid has

64rarely been reported. Therefore, it is of great significance to develop new cellulose

65hydrolysis processes by consuming small quantity of acid to produce high

66concentrations of the levulinic acid.

67 As an important topic in the hydrolysis reaction, the mechanism of acid

3
68hydrolysis of the cellulose in producing the levulinic acid has been extensively

69studied. In the hydrolysis reaction, the cellulose is firstly depolymerized to the

70glucose, then the glucose is converted to the 5-HMF. The 5-HMF can be further

71transformed into equimolar amounts of the levulinic acid and the formic acid [19, 24].

72However, for the by-product humins, the exact transformation mechanism has not

73been fully understood. Different opinions on the mechanism of humins transformation

74during the cellulose hydrolysis are still existing. Girusuta and co-workers [19]

75believed that humins can be transformed from cellulose, glucose, and 5-HMF in their

76kinetic study, respectively. Besides, majority of previous kinetics [17, 20, 25] assumed

77that humins may originate from self-polymerization of the glucose and self-

78polymerization of the 5-HMF, respectively. Kupiainen and coworkers [26] proposed

79that glucose would first be converted into an intermediate and followed by the 5-

80HMF, and the humins mainly came from the intermediate and the 5-HMF. Girisuta et

81al. and Chang et al. assumed that humins were produced directly by the conversion of

82the glucose [8, 27]. Yan et al. [28] believed that humins were derived from the 5-

83HMF, while the glucose would produce other undesired products instead of humins.

84In our previous work [29], a kinetic study was conducted for the hydrolysis of the

85corncob, in which we assumed that the humins were mainly derived from the 5-HMF,

86and a good fitting effect has been achieved. However, direct evidences are still needed

87to support these assumptions with respect to humins transformation in all above

88kinetics. In addition to the afore-mentioned kinetic studies, a few research groups

89have also developed mechanisms for the formation of humins by-products. A typical

4
 90mechanism was proposed by Sumerskii et al. [30], which involves a polycondensation

91pathway of humins, leading to a network of furan rings that is linked by ether or

92acetal bonds. Patil et al.[31, 32] put forward a pathway for the humins formation from

93the 5-HMF and the cellulose. They proposed that humins were mainly derived from

94the aldol addition/condensation of the 2,5-dioxo-6-hydroxyhexanal, which was

95converted from the 5-HMF with aldehydes and ketones. However, direct conversion

96of the cellulose and the glucose to humins was not obvious. Van and co-workers [33]

97revealed that humins were formed by a dehydration pathway, in which the furanic

98structure reacted with alcohol, acid, ketone, and aldehyde functional groups. As a

99result, they believe that humins were mainly derived from the 5-HMF. Mechanisms

100proposed by the afore-mentioned studies were predominately based on

101characterization analysis of the humins structure via instruments such as infrared,

102SEM, NMR spectroscopy and pyrolysis/GC-MS etc., rather than direct experimental

103analysis which is based on the concentration variation of each component throughout

104the experiment and/or the experimental phenomena in different hydrolysis

105experiments. The latter is considered to be a supplementary evidence which helps to

106better understand the hydrolysis reaction mechanism from a new perspective.

107 For the comprehensive utilization of the corncob, in the present work, the

108corncob, in which the hemicellulose have been removed, is adopted as the raw

109material to study the proposed new fed-batch process in producing the levulinic acid

110at high concentration. In addition, the effects of other impurities in the corncob on the

111levulinic acid yield are investigated. Furthermore, reaction mechanisms of the overall

5
112hydrolysis process are studied via direct experimental analysis. Combined with the

113mechanisms, the reasons that affect the yield of levulinic acid in the proposed

114technology are discussed.

115

1162. MATERIAL AND METHODS 

1172.1 Materials

118 The following materials were selected for this study: corncobs (30-mesh, Local

119farm, Qingdao, China), glucose (Sinopharm chemical, Shanghai, China), levulinic

120acid (Aladdin, Shanghai, China), sulfuric acid (98 wt.%, Sinopharm chemical,

121Shanghai, China), deionized water, microcrystalline cellulose (Macklin, Shanghai,

122China), and 5-HMF (Aladdin, Shanghai, China).

123

1242.2 Experimental procedure

125 Corncob pretreatment: The corncobs were processed by a 3 wt. % sulfuric acid

126solution at a temperature of 373.15 K and a solid-to-liquid ratio of 1:10 for 3 h. The

127purpose of this pretreatment procedure was to simulate the operating conditions of the

128xylose industries, and to remove the hemicellulose from corncobs. Later, the

129hydrolysate was filtered, and the corncob residue was washed for three times with

130sufficient deionized water. Finally, the corncob residue was dried in a vacuum oven

131(DZF-6050, Hecheng Instrument Manufacturing Inc., Shanghai, China).

132 The fed-batch acid hydrolysis experiment: The experiment was carried out in

133a 500 ml reaction vessel (as shown in Fig. 1). The inner wall of the reactor which

6
134contacted with raw materials was made by Hastelloy (C276) for preventing corrosion

135under acidic conditions. Corncob residue of 30g (or the microcrystalline cellulose that

136containing an identical amount of the cellulose in corncob residue) and 300ml

1370.5mol/L sulfuric acid solution were put into the reaction vessel. The reaction was

138maintained at 453.15 K for 50 min and the kettle impeller speed was set to 400 rpm.

139Reaction under above conditions could ensure the cellulose to be fully converted, as

140shown in Fig. 2. The yield of glucose was nearly zero when the reaction went to 45

141minutes, indicating that the cellulose could not be converted to the glucose any more.

142After the reaction, the filter residue was filtered off and the volume of the filtrate was

143measured. The same volume of 0.5 mol/L sulfuric acid solution as the hydrolysate

144adsorbed by the residue was added for washing. Later, the washing solution was

145added into the filtrate to recover the reaction solution to 300 ml for the next

146hydrolysis reaction. Continue to hydrolyze new corncob residue (or microcrystalline

147cellulose) using this hydrolysate while maintaining the solid-liquid ratio at 1:10.

148Reaction conditions were kept the same as those for the first time to ensure the

149cellulose to be completely converted. This process was referred to as the second batch

150hydrolysis. Based on this method, the hydrolysate was continuous used to hydrolyze

151the corncob residue. The concentration of the levulinic acid after each hydrolysis was

152determined by chromatographic systems.

153 To evaluate the mechanism of humins formation in the fed-batch hydrolysis

154process, a number of verification experiments were performed.

155 Verification experiment 1: Hydrolysate filtered by a 0.45 μm filter (Jinteng,

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156Tianjin, China) of 500 ml containing 63.18 g/L levulinic acid and a corresponding

157amount of soluble humin analogues (soluble humin analogues are complex mixtures,

158the contents of which are difficult to measure and quantify. In this paper, the amount

159of soluble humin analogues in the hydrolysate was determined corresponding to the

160amount of levulinic acid and the times of hydrolysis.) was divided into two equal

161parts: Solution 1 was utilized for the next reaction immediately without treatment;

162Solution 2 was placed at room temperature for 15 days and filtered by a 0.45 μm filter

163thereafter. Two copies of 15 g celluloses were added into two solutions for the

164hydrolysis reaction, respectively. The reaction was maintained at 453.15 K for 50

165minutes and the speed of kettle impeller was set to 400 rpm. After the reaction, the

166hydrolysates were filtered again, and the components in the hydrolysate were

167determined by chromatographic systems. Took another 500 ml hydrolysate containing

16861.58 g/L levulinic acid and a corresponding amount of soluble humin analogues and

169divided the solution into two parts, i.e., solution 3 and solution 4, and repeated the

170above experimental step.

171 Verification experiment 2: Two hydrolysates containing 38.76 g/L (hydrolysate

1721) and 127.43 g/L (hydrolysate 2) of the levulinic acid, and the corresponding

173amounts of soluble humin analogues were heated up to 453.15 K, respectively. This

174temperature was maintained for 100 min and the mixing speed was kept at 400 rpm.

175The concentrations of the levulinic acid in hydrolysate 1 and 2 were measured again

176after the heating reaction.

177 Verification experiment 3: A 400 ml 0.5 mol/L sulfuric acid solution, and a 400

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178ml hydrolysates solution with 73 g/L levulinic acid together with the corresponding

179amounts of soluble humin analogues were chosen for the hydrolysis reaction. Two

180copies of 24 g microcrystalline cellulose were added into above two solutions,

181respectively. The temperature of the reactor was rapidly raised to 423.15 K, allowing

182the reaction to be processed. The time dependent concentration of the glucose can be

183observed by sampling at each time point. Samples at each reaction time point were

184taken out through a sampling tube. To clean the sampling tube before and after

185sampling, high-pressure nitrogen gas was injected into the reactor through the

186sampling tube. Then, samples were filtered through the 0.45 μm syringe filter. After

187being diluted, the components in the samples were determined by chromatographic

188systems.

189 Verification experiment 4: A 450 ml 0.5 mol/L pure sulfuric acid solution, a

190450 ml levulinic acid solution (containing 50 g/L LA and 0.5 mol/L sulfuric acid) and

191a 450 ml hydrolysate solution (containing 47 g/L LA, 0.5 mol/L sulfuric acid and a

192corresponding amount of soluble humin analogues) were chosen, whilst three copies

193of 36 g dextrose monohydrate were added into above three solutions, respectively.

194The temperature of the reactor was rapidly raised to 423.15 K to ensure the reaction to

195be processed. The concentration of the component in each sample at each time point

196was measured afterwards.

197 Conditions of four verification experiments were summarized, as shown in Table

1981.

199
2002.3 Analytical method

9
201 The analysis of the corncob was based on the Laboratory Analytical Procedure

202developed by the National Renewable Energy Laboratory [34-36]. The components in

203corncob (before after pretreatment) are given in Table 2.

204 The glucose, the 5-HMF, and the LA contents in solution were analyzed by

205HPLC (L-2000, Hitachi Limited, Tokyo, Japan) with a refractive index detector and a

206Aminex HPX-87H column (300 mm × 7.8 mm, Bio-Rad, Hercules, USA). The

207mobile phase was aqueous solution of the sulfuric acid (0.005 mol/L), and the flow

208rate was 0.55 ml/min. The temperature of the column was set at 333.15 K, and the

209temperature of the refractive index detector was set at 311.15 K. A standard curve was

210plotted by using the chromatographic pure chemical, and the concentration of each

211component in the samples was measured thereafter by standard curves.

212

2133. RESULT AND DISCUSSION

214
2153.1 Study of the LA production in the fed-batch acid hydrolysis process

216 Sulfuric acid aqueous solution of 0.5 mol/L was used to hydrolyze the corncob

217residue at 453.15 K. The concentration of the levulinic acid in solution after the

218proposed fed-batch hydrolysis is listed in Table 3. The concentration of the levulinic

219acid increased with the times of the corncob residue hydrolysate usage. Meanwhile,

220total amount of the levulinic acid increased correspondingly, resulting in

221accumulations of the levulinic acid in the hydrolysate. After the first hydrolysis

222reaction, total amount of the levulinic acid in solution was 7.09 g. The yield of the

223levulinic acid Y (%) during the fed-batch hydrolysis can be calculated by using Eq.

10
224(1):

225Y =¿ (1)

226where mi denotes the increment of the levulinic acid at the ith feeding, with i the times

227of feeding; and n is the theoretical yield of the levulinic acid (Per molar of cellulose

228monomer could transform into equimolar amounts of levulinic acid and formic acid.

229Assuming that there is no side reaction in this process, all cellulose was converted to

230levulinic acid and formic acid, and no humins was formed), with n = 30 g × 0.607 ×

231(116 / 162) = 13.04 g in this case.

232 As can be seen from Table 3 and Fig. 3, the concentration of the levulinic acid

233reached to 107.93 g/L after the 7th feeding, which effectively reduces the amount of

234acid used in the reaction. As a result, it can effectively reduce the energy consumption

235for the subsequent separation and purification processes in industrial production. In

236addition, the remaining lignin and solid humins residue can be adopted for yielding

237high-value products such as activated carbon, thus achieving comprehensive

238utilization of the corncob.

239 A comparison of LA concentration in this work was made with other references,

240as shown in Table 4. Clearly, the proposed fed-batch process in this work exhibits

241much higher concentration of the levulinic acid than others. However, as the times of

242reactions increased, the yield of the levulinic acid presented a decreasing tendency. As

243shown in Table 3, the yield of the levulinic acid at the 5 th hydrolysis was 26.84%,

244nearly half of the levulinic acid yield comparing to the 1 st hydrolysis. When the

245reaction progressed to the 7th, the yield was only 19.71%. More times of the

11
246hydrolysis reaction were considered to be meaningless since the accumulation of the

247levulinic acid was not significant at the 7 th feeding. Therefore, the reaction was

248stopped at the 7th feeding and we expected this as an adequate treatment cycle for the

249fed-batch acid hydrolysis process. The yield at the 4th feeding was 70.68% of the 1st,

250while the yield at the 5th feeding was only 49.36% of the 1st. The yield decreased

251significantly between 4th and 5th feeding process. With respect to the yield of levulinic

252acid, a four times feeding is considered to be reasonable, as it not only obtains

253relatively high concentrations of the levulinic acid, but also effectively reduces the

254operating costs.

2553.2 Effect of other impurities in corncob residue on the yield of levulinic acid

256 The corncob residue after producing the xylose or the furfural contains much

257lignin, a small amount of inorganic salts and some ashes. The microcrystalline

258cellulose containing an equal amount of cellulose in the corncob residue was used to

259explore the effect of other impurities in the corncob residue on the yield of the

260levulinic acid in the hydrolysis reaction. As shown in Fig. 4, by using the

261microcrystalline cellulose and the corncob residue as raw materials respectively, the

262increment of the levulinic acid concentration was roughly consistent during the fed-

263batch hydrolysis process. It indicates that lignin, trace inorganic salts and ashes in

264corncob residue have almost no effect on the yield of the levulinic acid. Also, it

265implies that high molecular cellulose was depolymerized into low molecular

266cellulose, and low molecular cellulose was disaggregated into monosaccharides in the

267process of hydrolysis. At a sufficiently low solid-liquid ratio, the chain length of

12
268cellulose may have little effect on the yield of the levulinic acid. The small difference

269between microcrystalline cellulose and corncob residue shown in Fig. 4 could be

270attributed to the concentration deviations of LA caused by the experimental error, as

271can been seen that the standard deviations are partially or even completely

272overlapped. Meanwhile, the maximum difference of LA mean concentration (appears

273at the 4th feeding) is 4.8 %, which is considered to be an acceptable experimental

274error.

275

2763.3 Mechanisms of the humins formation and the yield of LA in the fed-batch
277acid hydrolysis process
278 After hydrolysis of cellulose in corncob residue, a red-brown solution and a

279hydrolyzed residue were obtained. The sum of the solid humin yield and products

280detected by HPLC analysis did not follow the mass balance in each feeding reaction,

281indicating that water-soluble oligomers were generated. The existence of water-

282soluble oligomers in solution was further confirmed by a set of experiments (detailed

283experimental verification can refer to Supplementary Material S1). The red-brown

284solution mainly contains the levulinic acid, the formic acid and water-soluble

285oligomers. The water-soluble oligomers are named as soluble humin analogues in this

286paper. As increasing the times of the hydrolysis reaction, the amount of soluble humin

287analogues in the hydrolysate increased gradually. As can be seen from Table 3, the

288hydrolysis residue increases successively with times of the hydrolysis reaction,

289indicating that the amount of solid humins has increased gradually in hydrolysis

290residue and more ingredients involve in the production of the solid humins. During

13
291the verification experiment 1, it was found that when hydrolysate was filtered through

292a 0.45μm filter and was placed at room temperature for 15 days, the clear solution

293became turbid and a small amount of precipitation was formed. Later, the

294precipitation was filtered and detected by infrared spectroscopy. As shown in Fig. 5.

295the characteristic peaks of the infrared spectrum lines of the precipitation and the solid

296humins residue derived from the cellulose acid-hydrolysis are nearly identical,

297implying that the precipitation and solid humins are the same substance. However, the

298concentrations of the levulinic acid and the formic acid were unchanged. The soluble

299humin analogues may self-polymerize or flocculate to precipitate a small amount of

300solid humins. Besides, the soluble humin analogues may contain some reactive sites

301which participate in self-polymerization or other reactions. As a result, the reduced

302yield of the levulinic acid may also be related to the existing soluble humin analogues

303in solution.

304 A series of experiments were designed to verify the above assumptions

305(verification experiment 1). From Table 5, the increase of the levulinic acid

306concentration in solutions 1, 2, 3 and 4 were 14.36 g/L, 16.41 g/L, 14.98 g/L and

30717.03 g/L, respectively. After treatment, the soluble humin analogues were self-

308polymerized or flocculated so that small amounts of solid humins were formed, and

309the polymerization of soluble humin analogues with other substances in solution was

310reduced. As a result, the yields of the levulinic acid after treatment are higher than that

311of the levulinic acid without treatment.

312 The above experimental results showed that the soluble humin analogues reacted

14
313with other substances in the solution, thus affecting the yield of the levulinic acid.

314There are two possibilities for this situation: one is that soluble humin analogues

315directly reacted with levulinic acid, causing an reduction of the levulinic acid yield

316when the hydrolysate was reused. Relevant experiments (Verification experiment 2)

317were designed to verify the above hypothesis, as shown in Table 6. After the heating

318reaction, the concentrations of the levulinic acid in two hydrolysates were 37.87 g/L

319and 126.66 g/L, respectively, only a bit lower than that of the levulinic acid (38.76 g/L

320and 127.43 g/L) before heating. The decrease of levulinic acid in two hydrolysates

321was not obvious. Therefore, it can be concluded that the main reason for the decrease

322of the levulinic acid yield in the fed-batch process was not caused by the reaction

323between levulinic acid and soluble humin analogues.

324 Another possibility is that the soluble humin analogues were further polymerized

325with the glucose and the intermediate 5-HMF to generate new humins, thus reducing

326the yield of levulinic acid when the hydrolyzate was reused. Levulinic acid was

327obtained by the 5-HMF, while 5-HMF was derived from the glucose. If the glucose

328and the 5-HMF polymerize with soluble humin analogues to form new humins in the

329reaction, the yield of the levulinic acid will eventually be affected.

330 In previous studies [30-33], the furan ring structure was detected in humins that

331formed from cellulose, indicating that the 5-HMF, which is characterized by a furan

332ring, participated in the humins forming reaction. However, for the glucose, there is

333no direct evidence that glucose could be involved in humins production. Hence, the

334reaction of soluble humin analogues with the glucose will be further studied in this

15
335paper. Experimental process is shown in verification experiment 3 and the result is

336shown in Fig. 6. At the same time point, the concentration of the glucose in sulfuric

337acid solution was higher than that of hydrolysates, indicating that the glucose in

338hydrolysates was consumed faster than the pure sulfuric acid solution (The catalysis

339of organic acids in hydrolysates is very limited at high concentration of the sulfuric

340acid solution, thus the organic acids catalytic action can be ignored). Meanwhile, as

341shown in Fig. 7, the increment of levulinic acid in sulfuric acid solution was higher

342than that of hydrolysate solution at the same time point. Combining Figs. 6 and 7, it

343indicates that glucose was involved in other reaction apart from the reaction of

344glucose degradation into 5-HMF. An adequate explanation is that soluble humin

345analogues would polymerize with some glucose in solution, the higher the

346concentration of soluble humin analogues in solution, the easier the soluble humin

347analogues polymerized with the glucose. With increasing the times of the hydrolysis

348reaction, the concentration of soluble humin analogues in solution increased

349continuously, enhancing the possibility of the polymerization of soluble humin

350analogues with the glucose. However, the selectivity of the conversion of glucose to

3515-hydroxymethylfurfural decreased, giving rise to a reduced yield of the levulinic

352acid. Our previous kinetic study [29] suggested that humins were only derived from

353the 5-hydroxymethylfurfural, while good fitting results have been achieved. The

354reason may be that when the solid-liquid ratio was low enough, the concentration of

355soluble humin analogues in solution can be neglected. Humins mainly came from

356mutual polymerization of the 5-hydroxymethylfurfural, and a very small amount of

16
357humins were less likely to react with the glucose. Therefore, based on our previous

358model, good fitting effect can be obtained. However, when the concentration of

359soluble humin analogues in solution increased with feeding times, the model was no

360longer applicable. Since all previous kinetic studies cannot explain this situation, a

361new model is therefore required.

362 The above experiments showed that soluble humin analogues did not directly

363react with the levulinic acid, whereas it would polymerize with glucose and

364intermediate 5-HMF to generate new humins. However, the problem that whether

365some humins were derived from the polymerization between levulinic acid and

366glucose has not been known yet. For this a further study was performed, the specific

367experimental process was mentioned in verification experiment 4. As shown in Fig. 8,

368real-time concentrations of the glucose in pure sulfuric acid solution and levulinic

369acid solution were almost the same, and the degradation of glucose was not

370accelerated in solution with levulinic acid. It indicates that the levulinic acid did not

371directly react with the glucose. However, the concentration of the glucose in

372hydrolysates at each time point was slightly lower than that of the prior, implying that

373the glucose was involved in other reactions aside from being converted to the 5-HMF.

374As can be seen from Fig. 9, the increase of both the concentration at each time point

375and the final yield of levulinic acid in former two solutions were nearly identical. As

376for the hydrolysate, the situations were lower than that of the former two solutions,

377which further showed that part of the glucose and the 5-HMF were involved in other

378reactions.

17
379 Based on above validation experiments and, it (shown in Fig. 10) can be

380concluded that the yield of levulinic acid in the fed-batch process reduced due to the

381accumulation of soluble humin analogues with increasing times of the hydrolysis.

382Soluble humin analogues can easily polymerize with glucose and 5-HMF, resulting in

383decreased selectivity of glucose to 5-HMF and 5-HMF to levulinic acid, and thus the

384reduced yield of the levulinic acid. Soluble humin analogues did not polymerize

385directly with the levulinic acid, while glucose did not directly react with the levulinic

386acid. It can be deduced from the reaction mechanism of the cellulose acid hydrolysis

387that by reducing the amount of soluble humin analogues in solution before each

388feeding, the yield of levulinic acid is expected to be improved.

389
3904. CONCLUSIONS
391 In this work, a new fed-batch process for producing levulinic acid by reusing

392hydrolyzate to hydrolyze corncob residue was proposed. After the first hydrolysis, the

393mass concentration of the levulinic acid in solution was 23.65 g/L. After feeding to 2,

3943, 4, 5, 6, 7 times, the mass concentration of the levulinic acid reached 42.82, 59.90,

39576.59, 88.26, 99.38, 107.93 g/L, respectively. The new process could effectively

396reduce the amount of acid used in the reaction, allowing a reduction of the energy

397consumption for the subsequent separation and purification processes.

398 The reaction mechanism of cellulose acid hydrolysis to produce levulinic acid

399and by-product humins was discussed. It was found that the decrease of levulinic acid

400yield during the fed-batch process was caused by the accumulation of soluble humin

401analogues with increasing times of the hydrolysis. The accumulated soluble humin

18
402analogues in solution can easily polymerize with glucose and 5-HMF, resulting in a

403decrease of the selectivity to the levulinic acid.

404
405ACKNOWLEDGEMENT
406
407This work was financially supported by the Nature Science Foundation of China

40821376231 and the Nature Science Foundation of China 21176264.

409

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508
509
510
511
512
513
514
515
516
517
518
519
520
521

23
522
523
524
525
526TABLES:
527
528Table 1. Comparison of the verification experiments.

Verification Solution Treatment method Purpose

experiment
Filtered
hydrolysate 1 No treatment and
1 Filtered add cellulose for To verify whether the
hydrolysate 3 hydrolysis decrease of yield is related
to soluble humin
Filtered Placed for 15 days
analogues
hydrolysate 2 and filtered,
Filtered then add cellulose
hydrolysate 4 for hydrolysis
Hydrolysate 1 To verify whether the
2 Hydrolysate 2 Heat at 453.15 k for levulinic acid could react
100 min with soluble humin
analogues
Sulfuric acid Verify that whether the
3 solution Add cellulose for glucose and 5-HMF could
Hydrolysates hydrolysis polymerize with soluble
solution humin analogues
Sulfuric acid
solution Add dextrose To verify whether the
4 Levulinic acid monohydrate for glucose and 5-HMF could
solution hydrolysis polymerize with levulinic
Hydrolysate to produce some humins.
solution
529
530Table 2. Analyses of the corncobs before and after pretreatment.

Compound After pretreatment Before pretreatment

value (wt.%) value (wt.%)
Cellulose 60.70 35.40
Lignin 31.40 20.20
Hemicellulose 2.70 37.60
Salt and ash 3.00 3.80
Others 2.20 3.00

24
531
532Table 3. Concentration of the levulinic acid in solution after fed-batch hydrolysis.

Times of the Filtrate + Volume Levulinic Total Yield The

hydrolysate washing lost due to acid amount (%) amount
usage solution adsorptio concentration of of
(ml) n (g/L) levulinic residue
(ml) acid (g)
(g)
1 285+15 15 23.65 7.09 54.37 12.49
2 285+17 17 42.82 12.85 44.18 13.15
3 281+19 19 59.90 17.97 39.27 13.63
4 280+20 20 76.59 22.98 38.43 13.85
5 278+22 22 88.26 26.48 26.84 14.69
6 273+27 27 99.38 29.81 25.54 14.97
7 275+25 25 107.93 32.38 19.71 15.13
533

534Table 4. Comparison of the LA concentrations.

Biomass Cellulose Catalyst Solid LA LA References

content and to concentratio concentrati
(%) concentrat liquid n after 1st on after
ion ratio reaction feeding 7th
(mol/L) (g/L) time
(g/L)
Sugar 43.30 H2SO4 1:9 19.50 - Girisuta et
cane 0.55 al. [8]
bagasse
Wheat 40.40 H2SO4 1:16 10.40 - Chang et
straw 0.30 al. [10]
Corn 41.50 FeCl3 0.50 1:9 16.00 - Zhi et al.
stalk [11]
Corncob 60.70 H2SO4 1:10 23.70 107.90 This work
residue 0.50
535

536Table 5. The influence of soluble humin analogues on LA concentration in the

537hydrolysis reaction.

Solution LA Processing The amount LA LA

concentration method of concentration concentration
before precipitation after reaction increase

25
 reaction produced (g/L) (g/L)
(g/L) (g/L)
1 63.18 No 0 77.54 14.36
treatment
2 63.18 Placed for 0.08g 79.59 16.41
15 days
3 61.58 No 0 76.57 14.99
treatment
4 61.58 Placed for 0.07g 78.61 17.03
15 days
538
539Table 6. Variation of the levulinic acid concentration before and after heating.

Hydrolysate Heating Heating LA LA

temperature time concentration concentration
(K) (min) before heating after heating
(g/L) (g/L)
1 453.15 100 38.76 37.87
2 453.15 100 127.43 126.66
540
541
542
543
544
545
546
547
548
549
550
551
552
553
554
555
556
557
558
559
560
561
562
563
564

26
565
566
567
568
569
570
571
572
573FIGURES：
574

575

576Figure 1. Schematic diagram of the reactor.

577

578

579Figure 2. Variation of each component concentration vs. time at 453.15 K and H2SO4
580= 0.5 mol/L.
27
581
582

583
584Figure 3. Variation of the levulinic acid concentration and the yield with feeding

585times of the hydrolysis.

586

587

588Figure 4. Comparison of the LA concentration by corncob residue and the

589microcrystalline cellulose in the hydrolysis reaction.

28
590

591Figure 5. IR spectra of humin solid residue and precipitated solids in solution.

592

593Figure 6. Variation of the glucose concentration vs. reaction time in different

594solutions.
595

29
596

597Figure 7. Increment of the levulinic acid vs. reaction time in sulfuric acid solution and
598hydrolysate solution, respectively.
599

600

601Figure 8. Variation of glucose concentration vs. reaction time in three different

602solutions.
603

30
604

605Figure 9. Variation of the levulinic acid concentration vs. reaction time in three
606different solutions

607

608
609Figure 10. Path way of glucose decomposition to LA and humins
610
611

31