Gluconic Acid Production from Potato Waste by Gluconobacter

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Gluconic acid production from potato waste by Gluconobacter oxydans using sequential hydrolysis and fermentation Yi Jiang, Kuimei Liu, Hongsen Zhang, Yingli Wang, Quanquan Yuan, Ning Su, Jie Bao, and Xu Fang ACS Sustainable Chem. Eng., Just Accepted Manuscript • Publication Date (Web): 23 May 2017 Downloaded from http://pubs.acs.org on May 31, 2017

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ACS Sustainable Chemistry & Engineering

Gluconic acid production from potato waste by Gluconobacter oxydans using sequential hydrolysis and fermentation Yi Jiang1§, Kuimei Liu1, 3§, Hongsen Zhang2, Yingli Wang4, Quanquan Yuan1, Ning Su1, Jie Bao2, Xu Fang1,* 1 State Key Laboratory of Microbial Technology, School of Life Sciences, Shandong University, Jinan 250100, China 2 State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, China 3 Rongcheng Campus, Harbin University of Science and Technology, Weihai, 264300, China 4 Weihai Aquatic school, Weihai, 264300, China §These authors contributed equally to this work. *

Corresponding author, (*(X, F) Phone: +86-0531-88364004, Fax:

+86-0531-88364363 E-mail: [email protected])

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ACS Paragon Plus Environment


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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

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producing gluconic acid from potato pulp was developed. The cocktails

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of cellulases from Trichoderma reesei TX and Penicillum oxalicum

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JUA10-1 with commercial pectinase were prepared and used to hydrolyze

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hydrothermally treated potato pulp into fermentable sugars at the solids

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content of 25 % (w/v). Eighty percent of the glucan in the potato pulp

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was able to be converted into glucose when 8 FPU/g dry material (DM)

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of cellulase and 1,000 PGU/g DM of pectinase was added. The enzymatic

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hydrolysates of the potato pulp were fermented into 81.4 g/L gluconic

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acid by Gluconobacter oxydans DSM 2003 for 20 hours at 30 °C. The

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overall conversion yield from glucose to gluconic acid was 94.9 %, and

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the productivity was 4.07 g/L/h, which was significantly better than that

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of previous studies. Finally, 546.48 gram gluconic acid was produced

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from per kilogram of dried potato pulp with this process.

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Keywords: potato pulp, gluconic acid, cellulase, Trichoderma reesei,

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Penicillum oxalicum, Gluconobacter oxydans

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Introduction:

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Agricultural residue is an abundant, inexpensive, and renewable

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source for the sustainable production of biochemicals through bioprocess

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engineering 1. In addition, it can solve the environment contamination

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that is caused by microorganisms during long storage periods, and

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address the greenhouse gas problem.

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Potato pulp is an agricultural waste product that is obtained from

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potato starch production, and it contains cellulose, starch and some

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proteins. This pulp contains a considerable amount of pectin. However, it

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also has a high moisture content and belongs among the easy-decay

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materials. Spoilage and deterioration lead to environmental pollution, but

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drying potato pulp is likely to have high costs and increase the burden on

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the enterprises that process it. Therefore, this pulp is usually buried as

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feed or waste disposal, resulting in soil and water pollution and a problem

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with low waste utilization 2. In China, a huge amount of potato pulp from

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starch production is produced each year 3. Potato pulp is significantly

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different from conventional agricultural lignocellulosic biomasses, such

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as corn stover, wheat straw, rice straw, and so on, because of its loose and

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hydrated structure as well as its low lignin content. These properties of

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potato pulp make it easier to hydrolyze into sugar 4. Thus, potato pulp is a

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suitable resource for producing biochemicals, such as gluconate, gluconic

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acid, lactic acid, bioethanol 5-6, and so on. 3



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Compared to the acid-catalyzed hydrolysis method,

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enzyme-catalyzed hydrolysis is an efficient and environmentally friendly

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method that usually occurs in potato waste with high viscosity, and it

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reduces the discharge of industrial wastewater and high energy

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consumption 7-8. Cellulase preparations based on mutant strains of T.

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reesei (also known as Hypocrea jecorina) are conventionally used to

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hydrolyze cellulose and hemicellulose to glucose 9. P. oxalicum has a

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more diverse lignocellulolytic enzyme system, particularly for cellulose

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binding domain-containing proteins and hemicellulases. Further,

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proteomic analysis of secretomes revealed that more lignocellulolytic

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enzymes were produced by P. oxalicum with better performance than that

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form T. reesei with cellulose-wheat bran as the carbon source 10. Cellulase

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reportedly also contributes to starch liberation during cassava pulp

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hydrolysis 11. Wang et al. obtained 153.46 and 168.13 g/L glucose from

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high-gravity sweet potato residues with cellulase and a mixture of

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cellulase and pectinase, respectively. This finding suggested that

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pectinase can assist cellulase in liberating glucose 12.

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Gluconic acid, the oxidation product of glucose aldehydes, is a

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non-toxic chemical that is neither caustic nor corrosive; it is a readily

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biodegradable organic acid of great interest for many applications 13.

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Gluconic acid and its derivative bulk chemicals can be used in food,

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medicine, the cement industry, and other sectors 14. Numerous 4


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manufacturing processes are developed to produce gluconic acid from

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glucose, primarily including chemical and electrochemical catalysis 15,

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enzymatic biocatalysis reactions 16 and continuous fermentation processes

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by using various microorganisms, including bacteria and fungi 17. It is

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worth noting that the development of enzymatic biocatalysis processes

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offers numerous advantages over the traditional chemical catalysis

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process in terms of environmental protection.

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In the present work, the feasibility of converting potato pulp into

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gluconic acid was studied. The differences in glucan conversion by potato

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pulp that was treated or not treated by hydrothermal method were

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compared. Different enzyme systems and their mixtures were used to

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achieve a higher hydrolysis efficiency with the potato pulp, and then the

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hydrolysates were fermented by G. oxydans to produce gluconic acid. To

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our knowledge, this is the first report to show that gluconic acid was

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produced from potato pulp.

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MATERIALS AND METHODS

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Materials, enzymes and reagents

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Potato pulp (PP) was generously provided by Shandong Bio

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Sunkeen Co., Ltd, Jining, Shandong, China. After being dried at 80 oC,

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the potato pulp was ground by a milling cutter and then subjected to

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hydrothermal treatment at 121 °C for 30 minutes 18. These samples were

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further hydrolyzed with enzymes after cooling to room temperature 5



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without pH adjustment. The cellulase preparation was produced by

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fermenting P. oxalicum JUA10-1 and T. reesei TX according to our

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previous method 12,19. G. oxydans DSM 2003 was maintained according

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to a previous report 20. The commercial pectinase solutions were kindly

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supplied by Qingdao Vland Biotech (Qingdao, Shandong, China). The

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α-amylase and α-glucosidase produced by Novozymes (China)

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Biotechnology Co., Ltd and Shandong Longda Bioproducts Co., Ltd,

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respectively, were generously provided by Jiangsu Lianhai Biological

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Technology Co., Ltd. Galacturonic acid, galactose, citric pectin and

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soluble starch were purchased from Sigma-Aldrich (St, Louis, MO, USA).

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Whatman No. 1 filter paper was purchased from Hangzhou

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Whatman-Xinhua Filter Paper, Hangzhou, Zhejiang, China. All the other

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chemicals were purchased from Sinopharm Chemical Reagent (Shanghai,

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China).

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Analytical methods

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The FPase activity was measured in accordance with the method of

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Ghose 21. The pectinase activity was measured in accordance with

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previous reports, with modifications 22. One milliliter of the diluted

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enzyme was added to 1 mL of 1 % citric pectin in 0.05 M acetate buffer

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(pH 5.5) and allowed to react for 10 minutes at 45 °C. The reaction was

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terminated by adding 1 mL of DNS, followed by boiling for 5 minutes.

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The activities of α-amylase and α-glucosidase were assayed according to 6


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previous methods 23. The protein concentration was measured using the

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Lowry method 24. The amounts of glucose, xylose and galactose released

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from the potato pulp were analyzed according to the National Renewable

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Energy Laboratory protocols

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(http://www.nrel.gov/docs/gen/fy13/42618.pdf). The glucose

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concentration was detected using an Aminex HPX-87H Column (300×7.8

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mm) purchased from Bio-Rad Laboratories, Inc. (Hercules, California,

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USA). The xylose and galactose contents were determined with a

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Sugar-D (4.6 ID×250 mm) COSMOSIL Packed Column purchased from

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NACALAI TESQUE, INC (Nijo Karasuma, Nakagyo, Kyoto). The

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mobile phase was 1 mM sulfuric acid, the flow rate was 0.5mL/min at

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45 °C and an RI detector (Model L-2490, Hitachi, Tokyo, Japan) was

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used. Gluconic acid was determined at 200 nm with diode array detector

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(DAD L-2455, Hitachi, Tokyo, Japan). The Inert SustainOR C18 (5 µm,

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4.6×250 mm) that was purchased from GL Science Inc. (Nishishinjuku

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6-chome Shinjuku, Tokyo) and the mobile phase was 1 mM sulfuric acid

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at 0.4 mL/min at 55 °C. The ash content was determined by heating one

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gram of the sample at 600 °C in an oven for 5 hours. The lignin was

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measured according to a previous report 25. The starch content was

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measured by allowing the samples to react for 2 hours at 90 °C after

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adding α-amylase and then adding α-glucosidase and allowing them to

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react for 8 hours at 60 °C. After the termination of this reaction, the 7



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glucose concentration was detected by HPLC (Hitachi, Tokyo, Japan).

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The cellulose content was calculated by isolating the starch content from

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the glucan content. To measure the pectin content, 20 mg of dry potato

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pulp was mixed with 8 mL of 0.1 M NaOH (pH 11.5) for 1 hour for

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sufficient saponification; hydrochloric acid was added to adjust the pH to

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4.5 and then 100 mg of pectinase was added; the mixture was then

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allowed to react at 55 °C for 20 hours. The pectin content was calculated

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for the released galacturonic acid.

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The content of pectin (%) =

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Where [Gal]1 is the obtained galacturonic acid concentration following

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enzymatic hydrolysis (g/L); V1 is the volume of enzymatically hydrolyzed

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potato pulp (L); and M1 is the amount of dry potato pulp (g). Two

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independent replicates were performed.

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The glucan conversion was calculated according to the following

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equation:

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Glucan conversion (%) =

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Where [Glu ]2 is the glucose concentration resulting from the enzymatic

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hydrolysis (g/L); V2 is the volume of enzymatically hydrolyzed potato

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pulp (L); M2 is the amount of dry potato pulp (g); and W is the percentage

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of glucan in the dry potato pulp (%). Two independent replicates were

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performed.

[Gal ]1  V1  150.1  100% M 1  194.1

[Glu ]2  V2  100% M 2 W

(1)

(2)

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153 154


Degradation of Potato Pulp by cellulytic enzymes and pectinase The potato pulp was degraded by adding 0, 2, 4, 8, 10, and 20 FPU/

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g cellulolytic enzyme from T. reesei TX or P. oxalicum JUA10-1. The

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solids loading is 25% (w/w) and then this indicates 25 gram of the dry

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substrate in 100 gram of the hydrolysates. The commercial pectinase was

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added at 0, 500, 1000, 2000, 4000, and 6000 PGU per gram of dry potato

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pulp. The reaction was sustained at 200 rpm and 45 °C for 8 hours. After

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that, the hydrolysates were further enzymatically degraded by adding 53

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U α-amylase and allowing them to react for 2 hours at 90 °C. After

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α-amylase was added, 53 U α-glucosidase was added and the samples

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were allowed to react for 8 hours at 60 °C. After the termination of this

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reaction, the concentrations of glucose and galacturonic acid were

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detected by HPLC.

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Gluconic acid production after enzymatic hydrolysis

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After enzymatic hydrolysis by a cellulase preparation from T. reesei

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TX or P. oxalicum JU A10-1, the hydrolysates were further fermented by

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G. oxydans DSM 2003. The fermentation conditions were as follows: 100

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mL of the G. oxydans culture was inoculated into 900 mL sterilizated

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hydrolysates in 3 L fermenterwith the loading amount of 1 L, the

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incubation amount was 10 %, the amount of enzymatic hydrolysate was

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900 mL, and the fermentations were incubated at 30 °C, 2.5 vvm

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ventilation with a rotating speed of 220 rpm. The concentration of 9



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galacturonic acid was detected by HPLC. The gluconic acid yield based on glucan conversion were calculated

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according to the following equation:

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Gluconic acid yield (%) =

[GA] V  [GA]0 V 0 100% M 3 W 1.1111.089

(3)

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Where [GA]0 and [GA], the initial and final gluconic acid

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concentrations (g/L); V0 and V, the initial and final volumes of

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fermentation broth (L); M3 is the amount of dry potato pulp (g); and W is

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the percentage of glucan in dry potato pulp (%); 1.111 is the conversion

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factor for glucan to equivalent glucose; 1.089 is the conversion factor for

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glucose to equivalent gluconic acid based on the stoichiometric balance..

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Two independent replicates were performed.

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RESULTS

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Chemical composition of potato pulp

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Potato pulp is the waste that remains after starch is extracted from

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potatoes during starch production. The chemical composition of potato

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pulp was investigated to evaluate the potential gluconic acid production

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from the potato pulp. The major components of the potato pulp were

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starch, pectin and cellulose, which contributed 40.99 %, 40.62 % and

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11.44 % (w/w) of the dry potato pulp, respectively. The lignin content

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(2.12 %) was very low. The potato pulp was degraded by enzymatic

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hydrolysis according to Method (5), and then it was found that the

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hydrolysate primarily consisted of glucose, galacturonic acid and 10


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galactose. Furthermore, it was found that 529.4 ±11.0 g glucose, 406.7 ±

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17.0 g galacturonic acid, and 64.3 ±1.60 g galactose were released from

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each kilogram of dry potato pulp according to the National Renewable

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Energy Laboratory protocols (see “Methods” section), but there is no

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xylose released from the potato pulp. Therefore, a scheme workflow was

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designed to illustrate the process for producing gluconic acid from potato

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pulp (Figure 1).

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Enzymatic hydrolysis of potato pulp with the commercial pectinase

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or cellulolytic enzymes from T. reesei TX or P. oxalicum JU A10-1

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First, the glucan conversion of potato pulp by the cellulolytic

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enzyme from T. reesei TX or P. oxalicum JU A10-1 was investigated. As

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shown in Figure 2a, when the potato pulp was degraded only with added

209

α-amylase and α-glucosidase, the glucose yield was only approximately

210

18 %. The glucan conversion was significantly increased from 51 % to

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61 % as the units of Fpase activity in P. oxalicum JU A10-1 increased

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from 2 to 8 FPU per gram of dry potato pulp. However, there was a slight

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change in the glucan conversion when the loading amount of T. reesei

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was increased from 2 to 20 FPU per gram of dry potato pulp. In addition,

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it was also found that the hydrolyzing efficiency of the cellulolytic

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enzyme from P. oxalicum JUA10-1 was higher than that of T. reesei TX.

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When the loading amount of cellulolytic enzyme from JUA10-1 was 8

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FPU per gram of dry potato pulp, the glucan conversion (63 %) was 11



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2-fold higher than that of T. reesei TX (Figure 2a). To gain higher glucan conversion using the potato pulp, we tried to

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treat potato pulp with a hydrothermal method (121 °C, 30 min) 26. As

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shown in Figure 2b, the glucan conversion was increased in both T. reesei

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TX and P. oxalicum JU A10-1 as the Fpase activity increased. For P.

224

oxalicum JU A10-1, the highest conversion reached 73 % when the

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amount of P. oxalicum enzyme was 20 FPU/g.

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Furthermore, we tried to hydrolyze the potato pulp by adding

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commercial pectinase. Figure 3 showed that the glucan conversion

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improved as the pectinase loading amount was increased, when the

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loading amount of pectinase was no more than 2,000 PGU/g. When the

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pectinase loading amount ranged from 2,000 to 6,000 PGU/g, the glucan

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conversion remained at approximately 40 %. Pectinase can reportedly

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increase the accessibility of cellulase to cellulose by cleaving pectin 12.

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Therefore, we tried to mix cellulolytic enzyme from the T. reesei/P.

234

oxalicum system with pectinase to improve the glucan conversion.

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Enzymatic hydrolysis of potato pulp with enzyme cocktail

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Figure 4 shows the hydrolysis efficiency for T. reesei or P. oxalicum

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enzyme mixed with 2,000 PGU/g pectinase. The glucan conversion kept

238

increasing when the loading amount of the T. reesei enzyme ranged from

239

0 to 4 FPU/g; however, there was no significant difference when the

240

loading amount was higher than 4 FPU/g. For the enzyme cocktail of P. 12


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oxalicum enzyme and pectinase, the glucan conversion kept increasing

242

when the loading amount was no more than 10 FPU/g. When the

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additional amount of P. oxalicum was 10 FPU/g, the highest glucan

244

conversion reached 78 %. The glucan conversion reached 64.69% or

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73.65% when 8 FPU/g of T. reesei or P. oxalicum enzyme mixed the

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pectinase (Figure 4). These conversions were higher than those without

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addition of pectinase, of which the glucan conversion was 53.08% and

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59.50%, respectively (Figure 2b).

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These results suggested that the high glucan conversion of potato

250

pulp treated by hydrothermal method was obtained with cellulolytic

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enzyme from T. reesei or P. oxalicum mixed with commercial pectinase

252

(Figure 4).

253

Furthermore, we investigated the change of glucan conversion when

254

different loading amount of pectinase was added into the cocktail enzyme.

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As shown in Figure 5, the conversion increased when the loading amount

256

of commercial pectinase mixed with T. reesei enzyme was increased from

257

0 to 2,000 PGU/g, while the conversion began to decrease when the

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loading amount ranged from 2,000 to 6,000 PGU/g. For the T. reesei

259

enzyme, the glucan conversion increased when the loading amount of

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commercial pectinase mixed with P. oxalicum enzyme was sharply

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increased from 0 to 500 PGU/g and remained roughly unchanged from

262

500 to 4,000 PGU/g. However, when the pectin loading amount was 13



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Page 14 of 50

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greater than 4,000 PGU/g, the conversion was also decreased. Moreover,

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it is obvious that the hydrolysis efficiency of P. oxalicum enzyme mixed

265

with commercial pectinase was higher than that of T. reesei.

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Gluconic acid production using potato pulp hydrolysate by G.

267

oxydans DSM 2003

268

After the hydrolysates were obtained, the glucose was further

269

fermented into gluconic acid by G. oxydans DSM 2003. No inhibitor such

270

as 5-hydroxymethylfurfural, acetic acid and phenolic compounds in the

271

hydrolysate of potato pulp was detected by HPLC. Figure 6 showed that

272

glucose from the enzymatic hydrolysate of T. reesei or P. oxalicum can be

273

fully consumed after a 20-hour culture, and the conversion from glucose

274

to gluconic acid reached 93.8 % or 94.9 %, respectively. Moreover, the

275

concentration of gluconic acid reached 82.9 g/L or 81.4 g/L and the

276

production rate was 4.15 or 4.07 g/L/h, respectively (Figure 5). Finally,

277

546.48 gram gluconic acid was produced from per kilogram of potato

278

pulp.

279

Discussion

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Mayer et al. reported that the major components of potato pulp were

281

starch, cellulose, pectin and hemicelluloses 2. Cellulose is a polymer

282

consisting of unbranched β-D- 1, 4-glycosidic linkage units 27. Starch is a

283

biopolymer of D-glucose units composed of amylose and amylopectin 28.

284

Pectin is a linear polysaccharide polymer containing α-(1→ 4) –linked 14


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acid as backbones, and their methyl esters 29. Moreover, it

285

D-galacturonic

286

was found that fermentable hexose (593.70 grams) and galacturonic acid

287

(406.70 grams) released from dry potato pulp (per kilogram), and then

288

there was no xylose with the acid hydrolysis method. These results

289

indicate that fermentable sugar was released from the potato pulp through

290

the breaking of the linkages in the cellulose, starch and pectin with

291

corresponding enzymes. During this process, obtaining fermentable sugar

292

from potato pulp is a key step. The method was physical, chemical or

293

based on enzymatic hydrolysis. T. reesei has always been used to produce

294

cellulase cocktails due to its high protein secretion capacity. Although T.

295

reesei shows the ability to produce high cellulase and hemicellulose

296

activities, relatively low specific activity and low β-glucosidase activity

297

restrict the application of T. reesei 30-32. However, P. oxalicum exhibits

298

higher β-glucosidase activity and pectinase activity in its enzyme system

299

than that of T. reesei 10,12,33. Altogether, cellulase from P. oxalicum is more

300

suitable for the enzymatic hydrolysis of potato pulp.

301

A scheme flow chart was provided to depict the process for

302

gluconic acid production from potato pulp in Figure 1. Generally an

303

increased rate of enzymatic hydrolysis can be achieved after pretreatment.

304

The hydrothermal method has been selected to pretreat dry potato pulp

305

due to low cellulase inhibitors 34. In comparing between the results in

306

Figure 2a and Figure 2b, it can be seen that when potato pulp was treated 15



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Page 16 of 50

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with the hydrothermal method, the glucan conversion of cellulolytic

308

enzyme from P. oxalicum JU A10-1 was more than 10 % higher than that

309

of untreated potato pulp from 53.57 % to 68.96 % when the amount of P.

310

oxalicum JU A10-1 enzyme was 20 FPU/g. For T. reesei TX enzyme,

311

there is greater improvement, and from 52.32 % to 67.59 % when the

312

amount of T. reesei TX enzyme was 20 FPU/g. This finding suggested

313

that the hydrothermal treatment of potato pulp enhanced the hydrolyzing

314

efficiency of the cellulolytic enzyme.

315

Zavareze Eda et al. reported that the physical and chemical

316

properties of starch are not always appropriate for certain types of

317

processing 35. Starch is often modified by specific methods, such as

318

physical, chemical, and enzymatic treatment. The physical modification

319

by hydrothermal method reportedly altered the physicochemical

320

properties of starch without destroying its granular structure 36. Moreover,

321

the advantage of thermochemical conversion is that it is a fast process

322

with low residence time, and it has applications to a broad range of

323

feedstocks in a continuous manner 37. This is consistent with what we

324

found in Figure 2a and Figure 2b which the glucan conversion of

325

cellulolytic enzyme from both P. oxalicum JU A10-1 and T. reesei TX

326

were more than 10 % higher than that of untreated potato pulp. Therefore,

327

it is hypothesized that the alteration of the potato pulp structure by

328

hydrothermal treatment made potato pulp more susceptible to hydrolysis 16


Page 17 of 50

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

329


by T. reesei.

330

Potato pulp is primarily composed of starch, pectin and cellulose.

331

The filter paperase (Fpase), pectinase and α-amylase activities of three

332

(the commercial pectinase and cellulolytic enzyme from T. reesei TX or P.

333

oxalicum JU A10-1) enzyme systems was investigated. The specific

334

Fpase activities of T. reesei TX, P. oxalicum JU A10-1 and commercial

335

pectinase were 0.28, 0.60 and 0.01 U/mg, for the specific pectinase they

336

were 0.81, 13.97 and 529.79 U/mg, and for the α-amylase activity they

337

were 0.01, 0.04 and 0.07 U/mg, respectively. Thus, we suggested that the

338

cellulolytic enzyme of T. reesei TX or P. oxalicum JU A10-1, commercial

339

pectinase and their mixture may have the potential to hydrolyze the

340

potato pulp efficiently. Furthermore, it was found that the Fpase,

341

pectinase or α-amylase activities of P. oxalicum enzyme were higher than

342

those of T. reesei. Thus, the glucan conversion of P. oxalicum was higher

343

than that of T. reesei for the biomass, with or without hydrothermal

344

treatment (Figure 2), because the cellulolytic enzyme of P. oxalicum was

345

suitable for hydrolyzing the potato pulp, compared with that of T. reesei,

346

which is consistence with previous report that P. oxalicum produced

347

native enzyme systems with better performance than that of T. reesei 38.

348

In Figure 4 and Figure 5, there is a synergy between commercial

349

pectinase and the cellulolytic enzyme of T. reesei TX or P. oxalicum JU

350

A10-1 for glucan conversion. It is also reported that pectinase acts as a 17



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

Page 18 of 50

351

“helper protein” in releasing glucose by clearing barriers, such as pectin.

352

Therefore, pectinase increases cellulose accessibility to cellulase, in turn

353

helping to improve glucan conversion 12, which is consistent with our

354

results. Panouilléet al. reported that by combining commercial cellulases

355

and proteases, the final extraction ratio was higher than it was when

356

treated with the chemical method 39. It is also reported that the addition of

357

pectinase improved the glucose and xylose yields when pretreated

358

corncob was hydrolyzed with enzyme complexes 40. These findings

359

suggested that the synergistic effects of different enzymes have a

360

profound impact on hydrolyzing substrates.

361

Finally, the hydrolysates were fermented by G. oxydans DSM 2003.

362

G. oxydans is a gram-negative bacteria. Over 90% glucose

363

dehydrogenase of G. oxydans which transform glucose into gluconic acid

364

were located on the cell wall membrane, instead of the intracellular

365

location. This property helped improving the conversion from glucose to

366

gluconic acid than commonly used strian A. niger 41-43. And then the

367

conversion from glucose to gluconic acid in this study reached 94.9 %.

368

The change of galacturonic acid content during the fermentation process

369

was also investigated, and there was no obvious change. This result

370

showed that G. oxydans DSM 2003 cannot utilize galacturonic acid, and

371

glucose was the major carbon source that was metabolized by G. oxydans.

372

The methods for producing gluconic acid from different substrates are 18


Page 19 of 50

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


373

compared in Table 1. Gluconic acid was produced from various materials,

374

such as Indian cane molasses, corn stover, office waste paper, figs, grape

375

must and golden syrup. To our knowledge, this is the first report to show

376

that gluconic acid was produced from potato pulp, which is an abundant,

377

inexpensive, and renewable industrial biomass. In the previous process,

378

gluconic acid was mostly produced by Aspergillus niger, and it took 2~15

379

days to take 44-46. However, the production time for this novel process is

380

less 1 day, and our productivity (4.07 g/L/h) is the highest among these

381

processes. Moreover, the conversion from glucose to gluconic acid in this

382

study reached 94.9 % from the enzymatic hydrolysate of P. oxalicum.

383

Thus, this process reduced the energy consumption and production cost.

384

Furthermore, there is no environmental pollution problem caused by this

385

process. Compared with potassium ferrocyanide and dilute acid treatment,

386

the enzymatic hydrolysis and hydrothermal treatment in this study is an

387

environmentally friendly process.

388 389

Abbreviations Used

390

Dry material (DM), Potato pulp (pp), Filter paperase (FPase), Filter

391

paperase unit (FPU); Polygalacturonase unit (PGU)

392

Acknowledgments

393

We thank Ms. Shaoli Hou for her experimental help.

394

Supporting Information description 19



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Page 20 of 50

395

This work was supported by Shandong Province Technology Innovation

396

and Transformation Program (No. 2015ZDXX0403A01), the National

397

Natural Science Foundation of China (NO.31570040), the 111 Project

398

and the State Key Laboratory of Microbial Technology Open Projects

399

Fund. The funders did not take part in any of the design of the

400

experiments, the collection and analysis of data, the preparation of the

401

manuscript, or the decision to publish.

402

References

403

1. Hasunuma, T.; Okazaki, F.; Okai, N.; Hara, K. Y.; Ishii, J.; Kondo, A.,

404

A review of enzymes and microbes for lignocellulosic biorefinery and

405

the possibility of their application to consolidated bioprocessing

406

technology. Bioresource technology 2013, 135, 513-522.

407

2. Mayer, F.; Hillebrandt, J. O., Potato pulp: microbiological

408

characterization, physical modification, and application of this

409

agricultural waste product. Applied microbiology and biotechnology

410

1997, 48 (4), 435-440.

411

3. Wang,. Z. Z.; Yan, ZH ; Review: Exploitation and Utilization of

412

Potato Pulp [J]. Journal of the Chinese Cereals and Oils Association.

413

2007,2, 031.

414

4. Delgado, R.; Castro, A. J.; Vázquez, M., A kinetic assessment of the

415

enzymatic hydrolysis of potato ( Solanum tuberosum ). Food Science

416

& Technology. 2009, 42(4), 797-804. 20


Page 21 of 50

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

417


5. Lesiecki, M.; Białas, W.; Lewandowicz, G., Enzymatic hydrolysis of

418

potato pulp. Acta Scientiarum Polonorum Technologia Alimentaria.

419

2012, 11(1), 53-59.

420

6. Smerilli, M.; Neureiter, M.; Wurz, S.; Haas, C.; Fruhauf, S.; Fuchs,

421

W., Direct fermentation of potato starch and potato residues to lactic

422

acid by Geobacillus stearothermophilus under non-sterile conditions.

423

J Chem Technol Biotechnol. 2015, 90(4), 648-657.

424

7. Arapoglou, D.; Varzakas, T.; Vlyssides, A.; Israilides, C., Ethanol

425

production from potato peel waste (PPW). Waste management. 2010,

426

30(10), 1898-1902.

427

8. Izmirlioglu, G.; Demirci, A., Ethanol Production from Waste Potato

428

Mash by Using Saccharomyces Cerevisiae. Applied Sciences. 2012,

429

2(4), 738-753.

430 431 432

9.

Gusakov, A. V., Alternatives to Trichoderma reesei in biofuel production. Trends Biotechnol 2011, 29(9), 419-425.

10. Liu, G., Zhang, L., Wei, X., Zou, G., Qin, Y., Ma, L., Li J., Zheng H.,

433

Wang S., Wang C., Xun L., Zhao G., Zhou Z., Qu Y., Genomic and

434

secretomic analyses reveal unique features of the lignocellulolytic

435

enzyme system of Penicillium decumbens. PloS one, 2013, 8(2),

436

e55185.

437 438

11. Sriroth, K.; Chollakup, R.; Chotineeranat, S.; Piyachomkwan, K.; Oates, C. G., Processing of cassava waste for improved biomass 21



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

439

Page 22 of 50

utilization. Bioresource Technol. 2015, 71(1), 63-69.

440

12. Wang, F.; Jiang, Y.; Guo, W.; Niu, K.; Zhang, R.; Hou, S.; Wang, M.;

441

Yi, Y.; Zhu, C.; Jia, C.; Fang, X., An environmentally friendly and

442

productive process for bioethanol production from potato waste.

443

Biotechnology for biofuels. 2016, 9(1), 50.

444

13. Qi, P.; Chen, S.; Chen, J.; Zheng, J.; Zheng, X.; Yuan, Y., Catalysis

445

and Reactivation of Ordered Mesoporous Carbon-Supported Gold

446

Nanoparticles for the Base-Free Oxidation of Glucose to Gluconic

447

Acid. ACS Catalysis. 2015, 5(4), 2659-2670.

448

14. Cañete-Rodríguez, A. M.; Santos-Dueñas, I. M.; Jiménez-Hornero, J.

449

E.; Ehrenreich, A.; Liebl, W.; García-García, I., Gluconic acid:

450

Properties, production methods and applications—An excellent

451

opportunity for agro-industrial by-products and waste bio-valorization.

452

Process Biochemistry. 2016, 51(12), 1891-1903.

453

15. Bin, D.; Wang, H.; Li, J.; Wang, H.; Yin, Z.; Kang, J.; He, B.; Li, Z.,

454

Controllable oxidation of glucose to gluconic acid and glucaric acid

455

using an electrocatalytic reactor. Electrochimica Acta. 2014, 130,

456

170-178.

457

16. Neves, L. C. M., & Vitolo, M.., Use of glucose oxidase in a

458

membrane reactor for gluconic acid production. Applied biochemistry

459

and biotechnology. 2007, 161-170.

460

17. Anastassiadis, S.; Morgunov, I. G., Gluconic acid production. Recent 22


Page 23 of 50

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

461 462


patents on biotechnology. 2007, 1, 167-80. 18. Kurian, J. K.; Gariepy, Y.; Orsat, V.; Raghavan, G. S. V., Comparison

463

of steam-assisted versus microwave-assisted treatments for the

464

fractionation of sweet sorghum bagasse. Bioresources and

465

Bioprocessing. 2015, 2 (1), 1-16.

466

19. Wang, M.; Han, L.; Liu, S.; Zhao, X.; Yang, J.; Loh, S. K.; Sun, X.;

467

Zhang, C.; Fang, X., A Weibull statistics-based lignocellulose

468

saccharification model and a built-in parameter accurately predict

469

lignocellulose hydrolysis performance. Biotechnology journal. 2015,

470

10(9), 1424-1433.

471

20. Zhang, H.; Liu, G.; Zhang, J.; Bao, J., Fermentative production of

472

high titer gluconic and xylonic acids from corn stover feedstock by

473

Gluconobacter oxydans and techno-economic analysis. Bioresource

474

technology. 2016, 219, 123-31.

475 476 477

21. Ghose, T., Measurement of cellulase activities. Pure & Applied Chemistry. 1987, 59(2), 257-268. 22. Rubtsova, E. A.; Bushina, E. V.; Rozhkova, A. M.; Korotkova, O. G.;

478

Nemashkalov, V. A.; Koshelev, A. V.; Sinitsyn, A. P., Novel Enzyme

479

Preparations with High Pectinase and Hemicellulase Activity Based

480

on Penicillium canescens Strains. Applied biochemistry and

481

microbiology. 2015, 51 (5), 502-10.

482

23. Arellanocarbajal, F.; Olmossoto, J., Thermostable alpha-1,4- and 23



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

483

alpha-1,6-glucosidase enzymes from Bacillus sp. isolated from a

484

marine environment. World Journal of Microbiology and

485

Biotechnology 2002, 18(8), 791-795.

486

24. Lowry, O. H.; Rosebrough, N. J.; Farr, A. L.; Randall, R. J., Protein

487

measurement with the Folin phenol reagent. J Biol Chem 1951,

488

193(1), 265-275.

489

25. Lu, J.; Li, X.; Yang, R.; Zhao, J.; Qu, Y., Tween 40 pretreatment of

490

unwashed water-insoluble solids of reed straw and corn stover

491

pretreated with liquid hot water to obtain high concentrations of

492

bioethanol. Biotechnol Biofuels 2013, 6(1), 159.

493

Page 24 of 50

26. Romani, A.; Garrote, G.; Alonso, J. L.; Parajo, J. C., Bioethanol

494

production from hydrothermally pretreated Eucalyptus globulus wood.

495

Bioresource technology 2010, 101(22), 8706-8712.

496

27. Dixit, G.; Shah, A. R.; Madamwar, D.; Narra, M., High solid

497

saccharification using mild alkali-pretreated rice straw by

498

hyper-cellulolytic fungal strain. Bioresources and Bioprocessing 2015,

499

2(1), 46.

500

28. Dutta, H.; Mahanta, C. L., Effect of hydrothermal treatment varying

501

in time and pressure on the properties of parboiled rices with different

502

amylose content. Food Research International 2012, 49(2), 655-663.

503 504

29. Qi, P. X.; Wickham, E. D.; Garcia, R. A., Structural and thermal stability of beta-lactoglobulin as a result of interacting with sugar beet 24


Page 25 of 50

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


505

pectin. Journal of agricultural and food chemistry 2014, 62(30),

506

7567-76.

507

30. Adsul, M. G.; Bastawde, K. B.; Varma, A. J.; Gokhale, D. V., Strain

508

improvement of Penicillium janthinellum NCIM 1171 for increased

509

cellulase production. Bioresource technology 2007, 98(7), 1467-73.

510

31. Cheng, Y.; Song, X.; Qin, Y.; Qu, Y., Genome shuffling improves

511

production of cellulase by Penicillium decumbens JU-A10. Journal of

512

applied microbiology 2009, 107(6), 1837-46.

513

32. Chand, P.; Aruna, A.; Maqsood, A. M.; Rao, L. V., Novel mutation

514

method for increased cellulase production. Journal of applied

515

microbiology 2005, 98(2), 318-23.

516

33. Gusakov, A. V.; Sinitsyn, A. P., Cellulases from Penicillium species

517

for producing fuels from biomass. Biofuels 2012, 3(4), 463-477.

518

34. Rajan, K.; Carrier, D. J., Insights into exo-Cellulase Inhibition by the

519

Hot Water Hydrolyzates of Rice Straw. Acs Sustain Chem Eng 2016,

520

4(7), 3627-3633.

521

35. Zavareze Eda, R.; Pinto, V. Z.; Klein, B.; El Halal, S. L.; Elias, M.

522

C.; Prentice-Hernandez, C.; Dias, A. R., Development of oxidised and

523

heat-moisture treated potato starch film. Food chemistry 2012, 132(1),

524

344-350.

525 526

36. Hoover, R., The impact of heat-moisture treatment on molecular structures and properties of starches isolated from different botanical 25



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

527

sources. Critical reviews in food science and nutrition. 2010, 50(9),

528

835-847.

529

Page 26 of 50

37. Kumar, A. K.; Sharma, S., Recent updates on different methods of

530

pretreatment of lignocellulosic feedstocks: a review. Bioresources and

531

Bioprocessing. 2017, 4(1),7.

532

38. Yao, G.; Li, Z.; Gao, L.; Wu, R.; Kan, Q.; Liu, G.; Qu, Y.,

533

Redesigning the regulatory pathway to enhance cellulase production

534

in Penicillium oxalicum. Biotechnol Biofuels. 2015, 8(1), 71.

535

39. Panouillé, M.; Jeanfrançois Thibault, A.; Bonnin, E., Cellulase and

536

Protease Preparations Can Extract Pectins from Various Plant

537

Byproducts. Journal of Agricultural & Food Chemistry. 2006, 54(23),

538

8926-8935.

539

40. Zhang, M.; Su, R.; Qi, W.; He, Z., Enhanced enzymatic hydrolysis of

540

lignocellulose by optimizing enzyme complexes. Applied

541

biochemistry and biotechnology. 2010, 160(5),1407-1414.

542

41. Merfort, M., Herrmann, U., Ha, S. W., Elfari, M., Bringer‐Meyer, S.,

543

Görisch, H., Sahm, H. Modification of the membrane‐bound

544

glucose oxidation system in Gluconobacter oxydans significantly

545

increases gluconate and 5 - keto - D - gluconic acid accumulation.

546

Biotechnology Journal. 2006, 1(5): 556-563.

547 548

42. Deppenmeier U, Hoffmeister M, Prust C. Biochemistry and biotechnological applications of Gluconobacter strains. Applied 26


Page 27 of 50

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

549 550


Microbiology and Biotechnology. 2002, 60(3), 233-242. 43. Wei, L., Zhu, D., Zhou, J., Zhang, J., Zhu, K., Du, L., Hua, Q.

551

Revealing in vivo glucose utilization of Gluconobacter oxydans 621H

552

Δmgdh strain by mutagenesis. Microbiological research. 2014, 169(5),

553

469-475.

554

44. Vassilev, N. B., Vassileva, M. C., Spassova, D. I. (1993). Production

555

of gluconic acid by Aspergillus niger immobilized on polyurethane

556

foam. Applied microbiology and biotechnology. 1993, 39(3), 285-288

557

45. Sankpal N V, Kulkarni B D. Optimization of fermentation conditions

558

for gluconic acid production using Aspergillus niger immobilized on

559

cellulose microfibrils[J]. Process Biochemistry. 2002, 37(12),

560

1343-1350.

561

46. Klein, J., Rosenberg, M., Markoš, J., Dolgoš, O., Krošlák, M..

562

Biotransformation of glucose to gluconic acid by Aspergillus

563

niger—study of mass transfer in an airlift bioreactor. Biochemical

564

Engineering Journal. 2002, 10(3), 197-205.

565

47. Zhang, H.; Zhang, J.; Bao, J., High titer gluconic acid fermentation

566

by Aspergillus niger from dry dilute acid pretreated corn stover

567

without detoxification. Bioresource technology. 2016, 203, 211-9.

568

48. Ikeda, Y.; Park, E. Y.; Okuda, N., Bioconversion of waste office

569

paper to gluconic acid in a turbine blade reactor by the filamentous

570

fungus Aspergillus niger. Bioresource technology. 2006, 97(8), 27



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

571 572

Page 28 of 50

1030-1035. 49. Roukas, T., Citric and gluconic acid production from fig by

573

Aspergillus niger using solid-state fermentation. Journal of industrial

574

microbiology & biotechnology 2000, 25(6), 298-304.

575

50. Singh, O. V.; Singh, R. P., Bioconversion of grape must into

576

modulated gluconic acid production by Aspergillus niger ORS-4.410.

577

Journal of applied microbiology. 2006, 100(5), 1114-1122.

578

51. Buzzini, P.; Gobbetti, M.; Rossi, J.; Ribaldi, M., Utilization of grape

579

must and concentrated rectified grape must to produce gluconic acid

580

by Aspergillus niger, in batch fermentations. Biotechnology Letters.

581

1993, 15(2), 151-156.

582

52. Purane, N. K.; Sharma, S. K.; Salunkhe, P. D.; Labade, D. S.;

583

Tondlikar, M. M., Gluconic Acid Production from Golden Syrup by

584

Aspergillus niger Strain Using Semiautomatic Stirred-Tank Fermenter.

585

Journal of Microbial & Biochemical Technology. 2012, 4, 92-95.

586

53. Singh, O. V.; Kapur, N.; Singh, R. P., Evaluation of agro-food

587

byproducts for gluconic acid production by Aspergillus niger

588

ORS-4.410. World J Microb Biot. 2005, 21(4), 519-524.

589 590 591

28


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592

Figure captions

593

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

594

from potato pulp.

595

Figure 2. Glucose released from potato pulp by cellulolytic enzyme from

596

T. reesei TX or P. oxalicum JUA10-1. The concentration of potato pulp in

597

the reaction system was 25 % (w/v). The potato pulp was not treated at

598

121 °C (a) or treated at 121°C (b) for 30 minutes, and cellulolytic enzyme

599

was added at 0, 2, 4, 8, 10, and 20 FPU/g dry potato pulp. Two

600

independent replicates were performed. Hollow bars represent T. reesei

601

TX, and black bars represent P. oxalicum JUA10-1.

602 603

Figure 3. Glucose released from potato pulp by commercial pectinase.

604

The concentration of potato pulp in the reaction system was 25 % (w/v).

605

The potato pulp was treated at 121 °C for 30 minutes, and commercial

606

pectinase was added at 0, 500, 1,000, 2,000, 4,000, and 6,000 PGU/g dry

607

potato pulp. Two independent replicates were performed.

608 609

Figure 4. Glucose released from potato pulp by an enzyme cocktail of

610

commercial pectinase (2,000 PGU/g dry potato pulp) mixed with

611

cellulolytic enzyme from T. reesei TX (hollow bars) or P. oxalicum

612

JUA10-1 (black bars). The concentration of potato pulp in the reaction

613

system was 25 % (w/v). The potato pulp was treated at 121 °C for 30 29



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

614

Page 30 of 50

minutes. Two independent replicates were performed.

615 616

Figure 5. Glucose released from the potato pulp by an enzyme cocktail of

617

cellulolytic enzyme (8 FPU/g dry potato pulp) from T. reesei TX (hollow

618

bars) or P. oxalicum JUA10-1 (black bars) mixed with commercial

619

pectinase. The concentration of potato pulp in the reaction system was 25 %

620

(w/v). The potato pulp was treated at 121 °C for 30 minutes. Two

621

independent replicates were performed.

622 623

Figure 6. Gluconic acid production by G. oxydans DSM 2003. The

624

hydrolysate was prepared with hydrothermal treatment using T. reesei TX

625

(a) or P. oxalicum JUA10-1 (b) mixed with commercial pectinase (2000

626

PGU/g dry potato pulp) and then was further fermented with G. oxydans

627

DSM 2003 in a 3 L fermenter. The circle symbols represent glucose, ()

628

and the triangle symbols represent gluconic acid.

629 630

Tables

631

Table 1. Comparison of different processes using different substrates to

632

produce gluconic acid.

633

30


Page 31 of 50

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Figure 1

31



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Page 32 of 50

Figure 2

32


Page 33 of 50

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Figure 3

33



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

Page 34 of 50

Figure 4

34


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Figure 5

35



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Figure 6

36


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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47


Graphic for table of contents Table 1 Comparision of gluconic acid production with different processes using different substrates Materials

Potato pulp

Pretreatment Hydrolysi Strains

Gluconic

Fermentatio Productivit Glucose

Gluconi

method

acid

n time

to

c acid

concentratio

gluconic

Yield

n

acid

( g/g,

( g/L )

conversio dry

s method

Hydrotherm

Enzymati

G. oxydans 81.40±1.06

al treatment

c

DSM 2003

20 hours

y ( g/L/h )

4.07

n(%)

weight )

94.90 %

0.57

This stud

hydrolysi

y

s Corn stover

Dilute acid

Enzymati

Aspergillus 76.67

88 hours

0.87

94.83 %

-

47

37



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47

pretreatment c hydrolysi

Page 38 of 50

niger SIIM M276

s Waste

Cut without

Enzymati

Aspergillus 80-100

office

further

c

niger

paper

pretreatment hydrolysi

72 hours

1.11-1.39

60 %

-

48

63 %

0.685

49

95.8 %

0.804

50

IAM2094

s Semidried

Chopped

figs

without

niger

further

ATCC

pretreatment

10577

Rectified grape must

-

-

-

Aspergillus -

Aspergillus 80-85

15 days

10 days

niger

0.033-0.03 5

38


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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47


ORS-4.410 Concentrate -

-

Aspergillus 67.43

d rectified

niger

grape must

ATCC6036

72 hours

0.937

96 %

-

51

44 hours

1.936

86.97 %

-

52

144 hours

0.481

72.4 %

-

53

3 Golden

-

-

syrup

Aspergillus 85.2 niger NCIM 530

Banana-mu

Thermal

st

treatment

-

Aspergillus 69.3 niger ORS-4.410

39



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Page 40 of 50

For Abstract Graphic Use Only Gluconic acid production from potato waste by Gluconobacter oxydans using sequential hydrolysis and fermentation Yi Jiang1§, Kuimei Liu1, 3§, Hongsen Zhang2, Yingli Wang4, Quanquan Yuan1, Ning Su1, Jie Bao2, Xu Fang1,*

Synopsis: An environmentally friendly method for production of gluconic acid from sustainable agriculture waste via enzymatic hydrolysis was established.

40


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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42


Gluconic acid production from potato waste by Gluconobacter oxydans using sequential hydrolysis and fermentation Yi Jiang1§, Kuimei Liu1, 3§, Hongsen Zhang2, Yingli Wang4,

Quanquan Yuan1, Ning Su1, Jie Bao2, Xu Fang1,*



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42

1. Yi Jiang, Shandong University, State Key Laboratory of Microbial Technology, School of Life Sciences, [email protected]

2. Kuimei Liu, Shandong University, State Key Laboratory of Microbial Technology, School of Life Sciences; Harbin University of Science and Technology, Rongcheng Campus, Weihai, China, [email protected] 3. Hongsen Zhang, East China University of Science and Technology, State Key Laboratory of Bioreactor Engineering, Shanghai, China, [email protected] 4. Yingli Wang, Weihai Aquatic school, Weihai, China, [email protected] 5. Quanquan Yuan, Shandong University, State Key Laboratory of Microbial Technology, School of Life Sciences, [email protected]

6. Ning Su, Shandong University, State Key Laboratory of Microbial Technology, School of Life Sciences, [email protected] 7. Jie Bao, East China University of Science and Technology,130 Meilong Road, Shanghai, [email protected] 8. Xu Fang (Corresponding Author), Shandong University, State Key Laboratory of Microbial Technology, School of Life Sciences, [email protected], Tel: +86-531-88364004; Fax: +86-531-88364363 ACS Paragon Plus Environment

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42


Starch extraction

Potato Pulp Hydrothermal Pretreatment Enzymatic hydrolysis Gluconobacter oxydans DSM 2003 Fermentation

Gluconic acid

ACS Paragon Plus Environment Figure 1. A scheme of over-process of gluconic acid production from potato pulp.


(a)

(b)

Glucan conversion (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42

Page 44 of 50

Loading amount of enzymes (Unit/g)

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 OC (a) or treated at 121OC (b), and cellulolytic enzyme was added at 0, 2, 4, 8, 10, and 20 FPU/g. Two independent replicates were performed. Hollow bars represent T. reesei TX, and black bars represent P. oxalicum JUA10-1(a and b). ACS Paragon Plus Environment

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42



Page 45 of 50

Loading amount of enzymes (Unit/g)

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 minutes, and commercial pectinase was added at 0, 500, 1,000, 2,000, 4,000, and 6,000 PGU/g. Two independent replicates were performed. ACS Paragon Plus Environment

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42

Page 46 of 50



Loading amount of cellulytic enzyme (FPU/g)

Figure4 Glucose released from potato pulp by cocktail enzyme of 2000PGU/g commercial pectinase mixed with cellulolytic enzyme from T. reesei TX , P. oxalicum JUA10-1. The concentration of potato pulp in the reaction system was 25 % (w/v). The potato pulp was treated at 121 °C for 30 minutes, Hollow bars represents 2000 PGU/g commercial pectinase combined with different loading amount of cellulolytic enzyme from cellulolytic enzyme from T. reesei TX and black bars represents that of P. oxalicum JUA10-1. Three independent replicates were performed. ACS Paragon Plus Environment

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42



Page 47 of 50

Loading amount of commercial pectinase (PGU/g)

Figure5. Glucose released from potato pulp by cocktail enzyme of 8FPU/g cellulolytic enzyme from T. reesei TX or P. oxalicum JUA10-1 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 minutes, Hollow bars represents 8FPU/g cellulolytic enzyme combined with different loading amount of commercial pectinase and black bars represents that of P. oxalicum JUA10-1. Three independent replicates were performed. ACS Paragon Plus Environment

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42

Glucose and gluconic acid (g/L)


100

(a)

Page 48 of 50

(b)

80 60 40 20 0 0

4

8

12

16

20

24

28

32

0

Time ( h )

4

8

12

16

20

24

28

32

Time ( h )

Figure 6 Gluconic acid fermentation by Gluconobacter oxydans DSM 2003. The hydrolysate from T. reesei TX (a) or P. oxalicum JUA10-1 (b) was further fermented with Gluconobacter oxydans DSM 2003 in 3L fermentor, converting glucose (circle symbol) into gluconic acid (triangle symbol).


Page 49 of 50

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42

Table 1 Comparision of different processes using different substrates to produce gluconic acid

Materials

Pretreatment

ACS Sustainable Chemistry & Engineering Hydrolysis Strains Gluconic acid Fermentation time Productivity (

Glucose to

Gluconic acid

method

method

gluconic acid

Yield ( g/g, dry

conversion ( % )

weight )

94.90 %

0.57

concentration

g/L/h )

( g/L ) Potato pulp

Hydrothermal

Enzymatic

G. oxydans DSM

treatment

hydrolysis

2003

Dilute acid

Enzymatic

Aspergillus niger

pretreatment

hydrolysis

SIIM M276

Waste office

Cut without

Enzymatic

Aspergillus niger

paper

further

hydrolysis

IAM2094

-

Aspergillus niger

Corn stover

81.40±1.06

20 hours

4.07

This

study 76.67

88 hours

0.87

94.83 %

-

47

80-100

72 hours

1.11-1.39

60 %

-

48

-

15 days

63 %

0.685

49

80-85

10 days

0.033-0.035

95.8 %

0.804

50

67.43

72 hours

0.937

96 %

-

51

85.2

44 hours

1.936

86.97 %

-

52

144 hours

0.481

72.4 %

-

53

pretreatment Semidried figs

Chopped without further

ATCC 10577

pretreatment Rectified grape

-

-

must Concentrated

Aspergillus niger ORS-4.410

-

-

rectified grape

Aspergillus niger ATCC60363

must Golden syrup

-

-

Aspergillus niger

530 Plus Environment ACSNCIM Paragon Banana-must

Thermal treatment -

Aspergillus niger

69.3

StarchACS Sustainable Chemistry & Engineering 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42

Page 50 of 50

For Abstract Graphic Use Only Fungus

Starch extraction Hydrolysates

Gluconobacter oxydans DSM 2003

Enzymatic hydrolysis Potato pulp

Submerged fermentation

Synopsis: An environmentally friendly method for production of gluconic acid from sustainable agriculture waste via enzymatic hydrolysis was established. ACS Paragon Plus Environment Gluconic acid

Gluconic Acid Production from Potato Waste by Gluconobacter

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