Gluconic Acid Production from Potato Waste by Gluconobacter

Research Article pubs.acs.org/journal/ascecg

Gluconic Acid Production from Potato Waste by Gluconobacter oxidans Using Sequential Hydrolysis and Fermentation Yi Jiang,†,⊥ Kuimei Liu,†,§,⊥ Hongsen Zhang,‡ Yingli Wang,∥ Quanquan Yuan,† Ning Su,† Jie Bao,‡ and Xu Fang*,† †

State Key Laboratory of Microbial Technology, School of Life Sciences, Shandong University, Jinan 250100, China State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, China § Rongcheng Campus, Harbin University of Science and Technology, Weihai, 264300, China ∥ Weihai Aquatic School, Weihai, 264300, China ‡

ABSTRACT: Potato pulp, which is the industrial residue of potato processing for starch production, can be used for biochemicals production. In this study, a green, sustainable, highly productive technology for producing gluconic acid from potato pulp was developed. The cocktails of cellulases from Trichoderma reesei TX and Penicillum oxalicum JUA10−1 with commercial pectinase were prepared and used to hydrolyze hydrothermally treated potato pulp into fermentable sugars at a solids content of 25% (w/v). Eighty percent of the glucan in the potato pulp was able to be converted into glucose when 8 FPU/g of dry material (DM) of cellulase and 1000 PGU/g DM of pectinase were added. The enzymatic hydrolysates of the potato pulp were fermented into 81.4 g/L gluconic acid by Gluconobacter oxidans DSM 2003 for 20 h at 30 °C. The overall conversion yield from glucose to gluconic acid was 94.9%, and the productivity was 4.07 g/L/h, which was significantly better than that of previous studies. Finally, 546.48 g of gluconic acid was produced from per kilogram of dried potato pulp with this process. KEYWORDS: Potato pulp, Gluconic acid, Cellulase, Trichoderma reesei, Penicillum oxalicum, Gluconobacter oxidans

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INTRODUCTION Agricultural residue is an abundant, inexpensive, and renewable source for the sustainable production of biochemicals through bioprocess engineering.1 In addition, it can solve the environmental contamination that is caused by microorganisms during long storage periods and address the greenhouse gas problem. Potato pulp is an agricultural waste product that is obtained from potato starch production, and it contains cellulose, starch, and some proteins. This pulp contains a considerable amount of pectin. However, it also has a high moisture content and belongs among the easy-decay materials. Spoilage and deterioration lead to environmental pollution, but drying potato pulp is likely to have high costs and increase the burden on the enterprises that process it. Therefore, this pulp is usually buried as feed or waste disposal, resulting in soil and water pollution and a problem with low waste utilization.2 In China, a huge amount of potato pulp from starch production is produced each year.3 Potato pulp is significantly different from conventional agricultural lignocellulosic biomasses, such as corn stover, wheat straw, rice straw, and so on, because of its loose and hydrated structure as well as its low lignin content. These properties of potato pulp make it easier to hydrolyze into sugar.4 Thus, potato pulp is a suitable resource for producing biochemicals, such as gluconate, gluconic acid, lactic acid, bioethanol,5,6 and so on. © 2017 American Chemical Society

Compared to the acid-catalyzed hydrolysis method, enzymecatalyzed hydrolysis is an efficient and environmentally friendly method that usually occurs in potato waste with high viscosity, and it reduces the discharge of industrial wastewater and high energy consumption.7,8 Cellulase preparations based on mutant strains of T. reesei (also known as Hypocrea jecorina) are conventionally used to hydrolyze cellulose and hemicellulose to glucose.9 P. oxalicum has a more diverse lignocellulolytic enzyme system, particularly for cellulose binding domaincontaining proteins and hemicellulases. Further, proteomic analysis of secretomes revealed that more lignocellulolytic enzymes were produced by P. oxalicum with better performance than that from T. reesei with cellulose-wheat bran as the carbon source.10 Cellulase reportedly also contributes to starch liberation during cassava pulp hydrolysis.11 Wang et al. obtained 153.46 and 168.13 g/L glucose from high-gravity sweet potato residues with cellulase and a mixture of cellulase and pectinase, respectively. This finding suggested that pectinase can assist cellulase in liberating glucose.12 Gluconic acid, the oxidation product of glucose aldehydes, is a nontoxic chemical that is neither caustic nor corrosive; it is a Received: April 1, 2017 Revised: May 22, 2017 Published: May 23, 2017 6116

DOI: 10.1021/acssuschemeng.7b00992 ACS Sustainable Chem. Eng. 2017, 5, 6116−6123

Research Article

ACS Sustainable Chemistry & Engineering

determined by heating 1 g of the sample at 600 °C in an oven for 5 h. The lignin was measured according to a previous report.25 The starch content was measured by allowing the samples to react for 2 h at 90 °C after adding α-amylase and then adding α-glucosidase and allowing them to react for 8 h at 60 °C. After the termination of this reaction, the glucose concentration was detected by HPLC (Hitachi, Tokyo, Japan). The cellulose content was calculated by isolating the starch content from the glucan content. To measure the pectin content, 20 mg of dry potato pulp was mixed with 8 mL of 0.1 M NaOH (pH 11.5) for 1 h for sufficient saponification. Hydrochloric acid was added to adjust the pH to 4.5, and then 100 mg of pectinase was added. The mixture was then allowed to react at 55 °C for 20 h. The pectin content was calculated for the released galacturonic acid.

readily biodegradable organic acid of great interest for many applications.13 Gluconic acid and its derivative bulk chemicals can be used in food, medicine, the cement industry, and other sectors.14 Numerous manufacturing processes are developed to produce gluconic acid from glucose, primarily including chemical and electrochemical catalysis,15 enzymatic biocatalysis reactions,16 and continuous fermentation processes by using various microorganisms, including bacteria and fungi.17 It is worth noting that the development of enzymatic biocatalysis processes offers numerous advantages over the traditional chemical catalysis process in terms of environmental protection. In the present work, the feasibility of converting potato pulp into gluconic acid was studied. The differences in glucan conversion by potato pulp that was treated or not treated by hydrothermal methods were compared. Different enzyme systems and their mixtures were used to achieve a higher hydrolysis efficiency with the potato pulp, and then the hydrolysates were fermented by G. oxidans to produce gluconic acid. To our knowledge, this is the first report to show that gluconic acid was produced from potato pulp.

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The content of pectin (%) =

[Gal]1 × V1 × 150.1 × 100% M1 × 194.1

(1)

where [Gal]1 is the obtained galacturonic acid concentration following enzymatic hydrolysis (g/L), V1 is the volume of enzymatically hydrolyzed potato pulp (L), and M1 is the amount of dry potato pulp (g). Two independent replicates were performed. The glucan conversion was calculated according to the following equation:

MATERIALS AND METHODS Glucan conversion (%) =

Materials, Enzymes, and Reagents. Potato pulp (PP) was generously provided by Shandong Bio Sunkeen Co., Ltd., Jining, Shandong, China. After being dried at 80 °C, the potato pulp was ground by a milling cutter and then subjected to hydrothermal treatment at 121 °C for 30 min.18 These samples were further hydrolyzed with enzymes after cooling to room temperature without pH adjustment. The cellulase preparation was produced by fermenting P. oxalicum JUA10−1 and T. reesei TX according to our previous method.12,19 G. oxidans DSM 2003 was maintained according to a previous report.20 The commercial pectinase solutions were kindly supplied by Qingdao Vland Biotech (Qingdao, Shandong, China). The α-amylase and α-glucosidase produced by Novozymes (China) Biotechnology Co., Ltd. and Shandong Longda Bioproducts Co., Ltd., respectively, were generously provided by Jiangsu Lianhai Biological Technology Co., Ltd. Galacturonic acid, galactose, citric pectin, and soluble starch were purchased from Sigma-Aldrich (St, Louis, MO, USA). Whatman No. 1 filter paper was purchased from Hangzhou Whatman-Xinhua Filter Paper, Hangzhou, Zhejiang, China. All the other chemicals were purchased from Sinopharm Chemical Reagent (Shanghai, China). Analytical Methods. The FPase activity was measured in accordance with the method of Ghose.21 The pectinase activity was measured in accordance with previous reports, with modifications.22 One milliliter of the diluted enzyme was added to 1 mL of 1% citric pectin in 0.05 M acetate buffer (pH 5.5) and allowed to react for 10 min at 45 °C. The reaction was terminated by adding 1 mL of DNS, followed by boiling for 5 min. The activities of α-amylase and αglucosidase were assayed according to previous methods.23 The protein concentration was measured using the Lowry method.24 The amounts of glucose, xylose, and galactose released from the potato pulp were analyzed according to the National Renewable Energy Laboratory protocols (http://www.nrel.gov/docs/gen/fy13/42618. pdf). The glucose concentration was detected using an Aminex HPX-87H Column (300 × 7.8 mm) purchased from Bio-Rad Laboratories, Inc. (Hercules, California, USA). The xylose and galactose contents were determined with a Sugar-D (4.6 ID × 250 mm) COSMOSIL Packed Column purchased from NACALAI TESQUE, INC (Nijo Karasuma, Nakagyo, Kyoto). The mobile phase was 1 mM sulfuric acid. The flow rate was 0.5 mL/min at 45 °C, and an RI detector (Model L-2490, Hitachi, Tokyo, Japan) was used. Gluconic acid was determined at 200 nm with a diode array detector (DAD L-2455, Hitachi, Tokyo, Japan). The Inert SustainOR C18 (5 μm, 4.6 × 250 mm) that was purchased from GL Science Inc. (Nishishinjuku 6-chome Shinjuku, Tokyo), and the mobile phase was 1 mM sulfuric acid at 0.4 mL/min at 55 °C. The ash content was

[Glu]2 × V2 × 100% M2 × W

(2)

where [Glu]2 is the glucose concentration resulting from the enzymatic hydrolysis (g/L), V2 is the volume of enzymatically hydrolyzed potato pulp (L), M2 is the amount of dry potato pulp (g), and W is the percentage of glucan in the dry potato pulp (%). Two independent replicates were performed. Degradation of Potato Pulp by Cellulytic Enzymes and Pectinase. The potato pulp was degraded by adding 0, 2, 4, 8, 10, and 20 FPU/g of cellulolytic enzyme from T. reesei TX or P. oxalicum JUA10−1. The solids loading is 25% (w/w), and this indicates 25 g of the dry substrate in 100 g of the hydrolysates. The commercial pectinase was added at 0, 500, 1000, 2000, 4000, and 6000 PGU per gram of dry potato pulp. The reaction was sustained at 200 rpm and 45 °C for 8 h. After that, the hydrolysates were further enzymatically degraded by adding 53 U α-amylase and allowing them to react for 2 h at 90 °C. After α-amylase was added, 53 U α-glucosidase was added, and the samples were allowed to react for 8 h at 60 °C. After the termination of this reaction, the concentrations of glucose and galacturonic acid were detected by HPLC. Gluconic Acid Production after Enzymatic Hydrolysis. After enzymatic hydrolysis by a cellulase preparation from T. reesei TX or P. oxalicum JU A10−1, the hydrolysates were further fermented by G. oxidans DSM 2003. The fermentation conditions were as follows: A total of 100 mL of the G. oxidans culture was inoculated into 900 mL of sterilizated hydrolysates in a 3 L fermenter with the loading amount of 1 L. The incubation amount was 10%, the amount of enzymatic hydrolysate was 900 mL, and the fermentations were incubated at 30 °C and 2.5 vvm ventilation with a rotating speed of 220 rpm. The concentration of galacturonic acid was detected by HPLC. The gluconic acid yield based on glucan conversion was calculated according to the following equation: Gluconic acid yield (%) =

[GA] × V − [GA]0 × V0 × 100% M3 × W × 1.111 × 1.089 (3)

where [GA]0 and [GA] are the initial and final gluconic acid concentrations (g/L), V0 and V are the initial and final volumes of fermentation broth (L), M3 is the amount of dry potato pulp (g), and W is the percentage of glucan in dry potato pulp (%); 1.111 is the conversion factor for glucan to equivalent glucose, and 1.089 is the conversion factor for glucose to equivalent gluconic acid based on the stoichiometric balance. Two independent replicates were performed. 6117


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Figure 1. A scheme of the overall process for producing gluconic acid from potato pulp.

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RESULTS Chemical Composition of Potato Pulp. Potato pulp is the waste that remains after starch is extracted from potatoes during starch production. The chemical composition of potato pulp was investigated to evaluate the potential gluconic acid production from the potato pulp. The major components of the potato pulp were starch, pectin, and cellulose, which contributed 40.99%, 40.62%, and 11.44% (w/w) of the dry potato pulp, respectively. The lignin content (2.12%) was very low. The potato pulp was degraded by enzymatic hydrolysis according to method 5, and then it was found that the hydrolysate primarily consisted of glucose, galacturonic acid, and galactose. Furthermore, it was found that 529.4 ± 11.0 g of glucose, 406.7 ± 17.0 g of galacturonic acid, and 64.3 ± 1.60 g of galactose were released from each kilogram of dry potato pulp according to the National Renewable Energy Laboratory protocols (see Methods section), but there is no xylose released from the potato pulp. Therefore, a scheme workflow was designed to illustrate the process for producing gluconic acid from potato pulp (Figure 1). Enzymatic Hydrolysis of Potato Pulp with the Commercial Pectinase or Cellulolytic Enzymes from T. reesei TX or P. oxalicum JU A10−1. First, the glucan conversion of potato pulp by the cellulolytic enzyme from T. reesei TX or P. oxalicum JU A10−1 was investigated. As shown in Figure 2a, when the potato pulp was degraded only with

Figure 2. Glucose released from potato pulp by cellulolytic enzyme from T. reesei TX or P. oxalicum JUA10−1. The concentration of potato pulp in the reaction system was 25% (w/v). The potato pulp was not treated at 121 °C (a) or treated at 121 °C (b) for 30 min, and cellulolytic enzyme was added at 0, 2, 4, 8, 10, and 20 FPU/g dry potato pulp. Two independent replicates were performed. Hollow bars represent T. reesei TX, and black bars represent P. oxalicum JUA10−1.

added α-amylase and α-glucosidase, the glucose yield was only approximately 18%. The glucan conversion was significantly increased from 51% to 61% as the units of Fpase activity in P. oxalicum JU A10−1 increased from 2 to 8 FPU per gram of dry potato pulp. However, there was a slight change in the glucan conversion when the loading amount of T. reesei was increased from 2 to 20 FPU per gram of dry potato pulp. In addition, it was also found that the hydrolyzing efficiency of the cellulolytic enzyme from P. oxalicum JUA10−1 was higher than that of T. 6118


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ACS Sustainable Chemistry & Engineering reesei TX. When the loading amount of cellulolytic enzyme from JUA10−1 was 8 FPU per gram of dry potato pulp, the glucan conversion (63%) was 2-fold higher than that of T. reesei TX (Figure 2a). To gain higher glucan conversion using the potato pulp, we tried to treat potato pulp with a hydrothermal method (121 °C, 30 min).26 As shown in Figure 2b, the glucan conversion was increased in both T. reesei TX and P. oxalicum JU A10−1 as the Fpase activity increased. For P. oxalicum JU A10−1, the highest conversion reached 73% when the amount of P. oxalicum enzyme was 20 FPU/g. Furthermore, we tried to hydrolyze the potato pulp by adding commercial pectinase. Figure 3 showed that the glucan

glucan conversion kept increasing when the loading amount of the T. reesei enzyme ranged from 0 to 4 FPU/g; however, there was no significant difference when the loading amount was higher than 4 FPU/g. For the enzyme cocktail of P. oxalicum enzyme and pectinase, the glucan conversion kept increasing when the loading amount was no more than 10 FPU/g. When the additional amount of P. oxalicum was 10 FPU/g, the highest glucan conversion reached 78%. The glucan conversion reached 64.69% or 73.65% when 8 FPU/g of T. reesei or P. oxalicum enzyme mixed with the pectinase (Figure 4). These conversions were higher than those without the addition of pectinase, of which the glucan conversion was 53.08% and 59.50%, respectively (Figure 2b). These results suggested that the high glucan conversion of potato pulp treated by the hydrothermal method was obtained with cellulolytic enzyme from T. reesei or P. oxalicum mixed with commercial pectinase (Figure 4). Furthermore, we investigated the change of glucan conversion when different loading amounts of pectinase were added into the cocktail enzyme. As shown in Figure 5, the

Figure 3. Glucose released from potato pulp by commercial pectinase. The concentration of potato pulp in the reaction system was 25% (w/ v). The potato pulp was treated at 121 °C for 30 min, and commercial pectinase was added at 0, 500, 1000, 2000, 4000, and 6000 PGU/g dry potato pulp. Two independent replicates were performed.

conversion improved as the pectinase loading amount was increased, when the loading amount of pectinase was no more than 2000 PGU/g. When the pectinase loading amount ranged from 2000 to 6000 PGU/g, the glucan conversion remained at approximately 40%. Pectinase can reportedly increase the accessibility of cellulase to cellulose by cleaving pectin.12 Therefore, we tried to mix cellulolytic enzyme from the T. reesei/P. oxalicum system with pectinase to improve the glucan conversion. Enzymatic Hydrolysis of Potato Pulp with Enzyme Cocktail. Figure 4 shows the hydrolysis efficiency for T. reesei or P. oxalicum enzyme mixed with 2000 PGU/g pectinase. The

Figure 5. Glucose released from the potato pulp by an enzyme cocktail of cellulolytic enzyme (8 FPU/g dry potato pulp) from T. reesei TX (hollow bars) or P. oxalicum JUA10−1 (black bars) mixed with commercial pectinase. The concentration of potato pulp in the reaction system was 25% (w/v). The potato pulp was treated at 121 °C for 30 min. Two independent replicates were performed.

conversion increased when the loading amount of commercial pectinase mixed with T. reesei enzyme was increased from 0 to 2000 PGU/g, while the conversion began to decrease when the loading amount ranged from 2000 to 6000 PGU/g. For the T. reesei enzyme, the glucan conversion increased when the loading amount of commercial pectinase mixed with P. oxalicum enzyme was sharply increased from 0 to 500 PGU/ g and remained roughly unchanged from 500 to 4000 PGU/g. However, when the pectin loading amount was greater than 4000 PGU/g, the conversion was also decreased. Moreover, it is obvious that the hydrolysis efficiency of P. oxalicum enzyme mixed with commercial pectinase was higher than that of T. reesei. Gluconic Acid Production Using Potato Pulp Hydrolysate by G. oxidans DSM 2003. After the hydrolysates were obtained, the glucose was further fermented into gluconic acid by G. oxidans DSM 2003. No inhibitor such as 5hydroxymethylfurfural, acetic acid, and phenolic compounds in the hydrolysate of potato pulp was detected by HPLC. Figure 6 showed that glucose from the enzymatic hydrolysate of T. reesei or P. oxalicum can be fully consumed after a 20 h culture, and the conversion from glucose to gluconic acid reached 93.8% or 94.9%, respectively. Moreover, the concen-

Figure 4. Glucose released from potato pulp by an enzyme cocktail of commercial pectinase (2000 PGU/g dry potato pulp) mixed with cellulolytic enzyme from T. reesei TX (hollow bars) or P. oxalicum JUA10−1 (black bars). The concentration of potato pulp in the reaction system was 25% (w/v). The potato pulp was treated at 121 °C for 30 min. Two independent replicates were performed. 6119


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Figure 6. Gluconic acid production by G. oxidans DSM 2003. The hydrolysate was prepared with hydrothermal treatment using T. reesei TX (a) or P. oxalicum JUA10−1 (b) mixed with commercial pectinase (2000 PGU/g dry potato pulp) and then was further fermented with G. oxidans DSM 2003 in a 3 L fermenter. The circle symbols represent glucose, and the triangle symbols represent gluconic acid.

that the hydrothermal treatment of potato pulp enhanced the hydrolyzing efficiency of the cellulolytic enzyme. Zavareze et al. reported that the physical and chemical properties of starch are not always appropriate for certain types of processing.35 Starch is often modified by specific methods, such as physical, chemical, and enzymatic treatment. The physical modification by hydrothermal method reportedly altered the physicochemical properties of starch without destroying its granular structure.36 Moreover, the advantage of thermochemical conversion is that it is a fast process with low residence time, and it has applications to a broad range of feedstocks in a continuous manner.37 This is consistent with what we found in Figure 2a and b, in which the glucan conversions of cellulolytic enzyme from both P. oxalicum JU A10−1 and T. reesei TX were more than 10% higher than that of untreated potato pulp. Therefore, it is hypothesized that the alteration of the potato pulp structure by hydrothermal treatment made potato pulp more susceptible to hydrolysis by T. reesei. Potato pulp is primarily composed of starch, pectin, and cellulose. The filter paperase (Fpase), pectinase, and α-amylase activities of three (the commercial pectinase and cellulolytic enzyme from T. reesei TX or P. oxalicum JU A10−1) enzyme systems were investigated. The specific Fpase activities of T. reesei TX, P. oxalicum JU A10−1, and commercial pectinase were 0.28, 0.60, and 0.01 U/mg. For the specific pectinase, they were 0.81, 13.97, and 529.79 U/mg, and for the α-amylase activity, they were 0.01, 0.04, and 0.07 U/mg, respectively. Thus, we suggested that the cellulolytic enzyme of T. reesei TX or P. oxalicum JU A10−1, commercial pectinase, and their mixture may have the potential to hydrolyze the potato pulp efficiently. Furthermore, it was found that the Fpase, pectinase, or α-amylase activities of P. oxalicum enzyme were higher than those of T. reesei. Thus, the glucan conversion of P. oxalicum was higher than that of T. reesei for the biomass, with or without hydrothermal treatment (Figure 2), because the cellulolytic enzyme of P. oxalicum was suitable for hydrolyzing the potato pulp, compared with that of T. reesei, which is consistent with a previous report that P. oxalicum produced native enzyme systems with better performance than that of T. reesei.38 In Figures 4 and 5, there is a synergy between commercial pectinase and the cellulolytic enzyme of T. reesei TX or P. oxalicum JU A10−1 for glucan conversion. It is also reported that pectinase acts as a “helper protein” in releasing glucose by clearing barriers, such as pectin. Therefore, pectinase increases cellulose accessibility to cellulase, in turn helping to improve

tration of gluconic acid reached 82.9 g/L or 81.4 g/L, and the production rate was 4.15 or 4.07 g/L/h, respectively (Figure 5). Finally, 546.48 g of gluconic acid was produced per kilogram of potato pulp.

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DISCUSSION Mayer et al. reported that the major components of potato pulp were starch, cellulose, pectin, and hemicelluloses.2 Cellulose is a polymer consisting of unbranched β-D-1,4-glycosidic linkage units.27 Starch is a biopolymer of D-glucose units composed of amylose and amylopectin.28 Pectin is a linear polysaccharide polymer containing α-(1→ 4)-linked D-galacturonic acid as backbones, and their methyl esters.29 Moreover, it was found that fermentable hexose (593.70 g) and galacturonic acid (406.70 g) were released from dry potato pulp (per kilogram), and then there was no xylose with the acid hydrolysis method. These results indicate that fermentable sugar was released from the potato pulp through the breaking of the linkages in the cellulose, starch, and pectin with corresponding enzymes. During this process, obtaining fermentable sugar from potato pulp is a key step. The method was physical, chemical, or based on enzymatic hydrolysis. T. reesei has always been used to produce cellulase cocktails due to its high protein secretion capacity. Although T. reesei shows the ability to produce high cellulase and hemicellulose activities, relatively low specific activity and low β-glucosidase activity restrict the application of T. reesei.30−32 However, P. oxalicum exhibits higher βglucosidase activity and pectinase activity in its enzyme system than that of T. reesei.10,12,33 Altogether, cellulase from P. oxalicum is more suitable for the enzymatic hydrolysis of potato pulp. A scheme flowchart was provided to depict the process for gluconic acid production from potato pulp in Figure 1. Generally an increased rate of enzymatic hydrolysis can be achieved after pretreatment. The hydrothermal method has been selected to pretreat dry potato pulp due to low cellulase inhibitors.34 In comparing between the results in Figure 2a and b, it can be seen that when potato pulp was treated with the hydrothermal method, the glucan conversion of cellulolytic enzyme from P. oxalicum JU A10−1 was more than 10% higher than that of untreated potato pulp from 53.57% to 68.96% when the amount of P. oxalicum JU A10−1 enzyme was 20 FPU/g. For the T. reesei TX enzyme, there is greater improvement, and from 52.32% to 67.59% when the amount of T. reesei TX enzyme was 20 FPU/g. This finding suggested 6120


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53

52

51

50

72.4% 144 h 69.3

0.481

86.97% 44 h 85.2

1.936

72 h 67.43

0.937

96%

0.804 10 days

15 days

72 h 80−100

80−85

0.033−0.035

95.8%

49 63%

0.685

48 60% 1.11−1.39

88 h 76.67

Aspergillus niger SIIM M276 Aspergillus niger IAM2094 Aspergillus niger ATCC 10577 Aspergillus niger ORS4.410 Aspergillus niger ATCC60363 Aspergillus niger NCIM 530 Aspergillus niger ORS4.410

0.87

94.83%

0.57 94.90% 4.07

glucan conversion,12 which is consistent with our results. Panouillé et al. reported that by combining commercial cellulases and proteases, the final extraction ratio was higher than it was when treated with the chemical method.39 It is also reported that the addition of pectinase improved the glucose and xylose yields when pretreated corncob was hydrolyzed with enzyme complexes.40 These findings suggested that the synergistic effects of different enzymes have a profound impact on hydrolyzing substrates. Finally, the hydrolysates were fermented by G. oxidans DSM 2003. G. oxidans is a Gram-negative bacteria. Over 90% glucose dehydrogenase of G. oxidans, which transforms glucose into gluconic acid, was located on the cell wall membrane, instead of the intracellular location. This property helped improve the conversion from glucose to gluconic acid compared to that with the commonly used strian A. niger.41−43 And then the conversion from glucose to gluconic acid in this study reached 94.9%. The change of galacturonic acid content during the fermentation process was also investigated, and there was no obvious change. This result showed that G. oxidans DSM 2003 cannot utilize galacturonic acid, and glucose was the major carbon source that was metabolized by G. oxidans. The methods for producing gluconic acid from different substrates are compared in Table 1. Gluconic acid was produced from various materials, such as Indian cane molasses, corn stover, office waste paper, figs, grape must, and golden syrup. To our knowledge, this is the first report to show that gluconic acid was produced from potato pulp, which is an abundant, inexpensive, and renewable industrial biomass. In the previous process, gluconic acid was mostly produced by Aspergillus niger, and it took 2−15 days to take.44−46 However, the production time for this novel process is less 1 day, and our productivity (4.07 g/L/ h) is the highest among these processes. Moreover, the conversion from glucose to gluconic acid in this study reached 94.9% from the enzymatic hydrolysate of P. oxalicum. Thus, this process reduced the energy consumption and production cost. Furthermore, there is no environmental pollution problem caused by this process. Compared with potassium ferrocyanide and dilute acid treatment, the enzymatic hydrolysis and hydrothermal treatment in this study is an environmentally friendly process.

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AUTHOR INFORMATION

Corresponding Author

enzymatic hydrolysis enzymatic hydrolysis enzymatic hydrolysis

20 h 81.40 ± 1.06 G. oxidans DSM 2003

fermentation time gluconic acid concentration (g/L) strains

*Phone: +86-0531-88364004. Fax: +86-0531-88364363. Email: [email protected].

thermal treatment

Author Contributions ⊥

These authors contributed equally to this work.

Funding

This work was supported by Shandong Province Technology Innovation and Transformation Program (No. 2015ZDXX0403A01), the National Natural Science Foundation of China (NO.31570040), the 111 Project, and the State Key Laboratory of Microbial Technology Open Projects Fund. The funders did not take part in any of the design of the experiments, the collection and analysis of data, the preparation of the manuscript, or the decision to publish.

banana must

concentrated rectified grape must golden syrup

cut without further pretreatment chopped without further pretreatment waste office paper

Jie Bao: 0000-0001-6521-3099 Xu Fang: 0000-0002-9196-5697

Rectified grape must

dilute acid pretreatment corn stover

semidried figs

hydrothermal treatment

ORCID

potato pulp

hydrolysis method pretreatment method materials

Table 1. Comparison of Gluconic Acid Production with Different Processes Using Different Substrates

productivity (g/L/h)

glucose to gluconic acid conversion (%)

gluconic acid yield (g/g, dry weight)

This study 47


Notes

The authors declare no competing financial interest. 6121


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ACKNOWLEDGMENTS We thank Ms. Shaoli Hou for her experimental help. ABBREVIATIONS USED DM, dry material pp, potato pulp FPase, filter paperase FPU, filter paperase unit PGU, polygalacturonase unit

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Gluconic Acid Production from Potato Waste by Gluconobacter

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