Accepted Manuscript

Fermentative production of high titer gluconic and xylonic acids from corn
stover feedstock by Gluconobacter oxydans and techno-economic analysis

Hongsen Zhang, Gang Liu, Jian Zhang, Jie Bao

PII: S0960-8524(16)31047-1
DOI: http://dx.doi.org/10.1016/j.biortech.2016.07.068
Reference: BITE 16830

To appear in: Bioresource Technology

Received Date: 8 June 2016

Revised Date: 15 July 2016
Accepted Date: 18 July 2016

Please cite this article as: Zhang, H., Liu, G., Zhang, J., Bao, J., Fermentative production of high titer gluconic and
xylonic acids from corn stover feedstock by Gluconobacter oxydans and techno-economic analysis, Bioresource
Technology (2016), doi: http://dx.doi.org/10.1016/j.biortech.2016.07.068

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Original Research Paper manuscript submitted to Bioresource Technology

Fermentative production of high titer gluconic and xylonic acids from corn stover

feedstock by Gluconobacter oxydans and techno-economic analysis

Hongsen Zhang#, Gang Liu#, Jian Zhang, Jie Bao*

State Key Laboratory of Bioreactor Engineering, East China University of Science and

Technology, 130 Meilong Road, Shanghai 200237, China

#
These authors are equally contributed to this work. HSZ conducted the fermentation

experiment and cement additive evaluation; GL conducted the Aspen Plus modeling and

TEA calculation.

*Corresponding author. Tel/fax: +86 21 64251799; Email: [email protected]

Abstract: High titer gluconic acid and xylonic acid were simultaneously fermented by

Gluconobacter oxydans DSM 2003 using corn stover feedstock after dry dilute sulfuric acid

pretreatment, biodetoxification and high solids content hydrolysis. Maximum sodium

gluconate and xylonate were produced at the titer of 132.46 g/L and 38.86 g/L with the overall

yield of 97.12% from glucose and 90.02% from xylose, respectively. The drawbacks of

filamentous fungus Aspergillus niger including weak inhibitor tolerance, large pellet

formation and no xylose utilization were solved by using the bacterium strain G. oxydans. The

obtained sodium gluconate/xylonate product was highly competitive as cement retarder

additive to the commercial product from corn feedstock. The techno-economic analysis (TEA)

based on the Aspen Plus modeling was performed and the minimum sodium

gluconate/xylonate product selling price (MGSP) was calculated as $0.404/kg. This study

provided a practical and economic competitive process of lignocellulose utilization for

production of value-added biobased chemicals.

Key Words: Lignocellulose; Gluconate; Xylonate; Gluconobacter oxydans DSM 2003;

Aspen Plus modeling

1. Introduction

Sodium gluconate is the major cement additive used for delaying the setting time

(“retarding”) of cement paste during cement casting in construction industry (Ma et al., 2015).

Sodium xylonate is also a cement additive used for better fluidity by deflocculating cement

granules (Chun et al., 2006). In 2014, the world cement production is estimated to be 4 billion

tons and 70-80% of the cement is used in the large building casting with the requirement of

retarder additive addition such as sodium gluconate (Li, 2011; Li et al., 2015). The

requirement of sodium gluconate as cement retarder is approximate 1 million tons even at the

minimum addition of 0.03% (w/w) (Ma et al., 2015). The future market expansion of sodium

gluconate in accordance to the booming infrastructure construction in developing countries

requires the alternative feedstock to substitute traditional starch and sucrose as fermentation

feedstock for sodium gluconate production.

Among many feedstock options, lignocellulose biomass is the most abundant

carbohydrate feedstock with positive impact on environment and climate change. In our

previous study, the filamentous fungus Aspergillus niger SIIM M276 was used as the

fermenting strain of gluconic acid from corn stover feedstock and the sodium gluconate

product was successfully applied as cement retarder additive for extending the setting time of

cement paste (Zhang et al., 2016). However, several disadvantages on using A. niger were

observed including sensitive to inhibitor compounds in lignocellulosic hydrolysates, tended to

the quick formation of larger fungus pellets thus oxygen transfer to the cell mycelia was

reduced, and unable to utilize xylose. These drawbacks lead to the low productivity,

difficulties in the multiple seed culture propagation in commercial scale application, and

considerable xylose loss.

To overcome the problems on using filamentous fungus strain, a gram-negative

bacterium strain Gluconobacter oxydans comes into the consideration for its rapid oxidation
of monosaccharide into acids and ketones (Adachi et al 1980; Matsushita et al., 1981;

Silberbach et al., 2003; Elfari et al., 2005). A membrane protein of G. oxydans, glucose

dehydrogenase, oxidizes glucose into gluconic acid and xylose into xylonic acid as well as

other hexose and pentose sugars into the corresponding acids. The complete cell membrane

transfer of oxygen, glucose, xylose and products across the cell membrane is partially

lessened because of the location of glucose dehydrogenase on the cell membrane, instead of

the intracellular location (Merfort et al., 2006; Deppenmeier et al., 2002; Toivari et al., 2012;

Wei et al., 2014). In this study, we report a comprehensive and simultaneous fermentation of

glucose and xylose into gluconic and xylonic acids by G. oxydans DSM 2003 using corn

stover feedstock after it was pretreated by dry dilute sulfuric acid method (Zhang et al., 2011;

He et al., 2014), biodetoxified (Zhang et al., 2010a; He et al, 2016), and enzymatically

hydrolyzed into fermentable sugars. The obtained sodium gluconate and xylonate products

were tested as cement retarder for extending setting time of cement paste and showed a better

performance than commercial sodium gluconate from corn starch feedstock. The detailed

techno-economic analysis (TEA) was performed based on the Aspen Plus modeling,

minimum sodium gluconate/xylonate product selling price (MGSP) in this case was obtained,

it will provide economic theoretical basis for practical industrial cellulosic sodium

gluconate/xylonate production.

2. Materials and Methods

2.1. Raw materials

Corn stover (CS) was harvested from Nanyang, Henan, China in fall, 2014. The collected

raw corn stover materials were chopped to small chippings coarsely and washed to remove

solids dirt, stones and metals, then dried in oven until constant weight. The dried corn stover

materials were milled by hammer crusher with 10 mm circle diameter mesh. The composition

of corn stover after pre-handling treatment contained 35.78% of cellulose, 19.36% of

hemicellulose, 28.36% of lignin, 3.56% of ash on dry weight base (w/w) determined by

Cellulose Analyzer (Ankom 220, Ankom Technology, Macedon, NY, USA).

2.2. Enzymes and reagents

Commercial cellulase enzyme Youtell #7 was purchased from Hunan Youtell

Biochemical Co., Yueyang, Hunan, China. The filter paper activity was 63.0 FPU/g cellulase

according to the National Renewable Energy Laboratory (NREL) protocol LAP-006 (Adney

and Baker, 1996), the cellobiase activity was 102.0 IU/g cellulase determined using the

method of Ghose (1987), and the protein concentration was 49.5 mg/g cellulase detected by

Bradford method using BSA as protein standard.

Chromatographic grade sodium gluconate was purchased as the standard reference

chemical from Sigma-Aldrich, St. Louis, MO, USA. The sodium gluconate used as cement

additive was from Shandong Xiwang Group, Zouping, Shandong, China. Calcium xylonate

hydrate was from Santa Cruz Biotech., Dallas, Texas, USA. Glucose, sorbitol,

4-hydroxybenzaldehyde (HBA) and syringaldehyde were from Sangon Biotech., Shanghai,

China. Furfural, 5-hydroxymethylfurfural (HMF) were purchased from J&K Scientific Co.,

Beijing, China. Xylose and acetic acid were from Sinopharm Chemical Reagent Co.,

Shanghai, China. Vanillin was purchased from Aladdin Reagents Co., Shanghai, China. The

standard cement was purchased from Shandong cement Co., Shandong, China.

Polycarboxylate QS8020 was from Qishuo Industry Co., Shanghai, China. The other reagents

were all from Lingfeng Chemical Reagent Co., Shanghai, China.

2.3. Strains and media

Gluconobacter oxydans DSM 2003 was purchased from German Collection of

Microorganisms and Cell Cultures (DSMZ), Braunschweig, Germany. The culture medium

used for G. oxydans DSM 2003 included:

(1) Activation medium, containing 40.0 g of sorbitol, 20.0 g of yeast extract, 0.5 g of
MgSO4·7H2O, 1.5 g of KH2PO4, 1.5 g of (NH4)2SO4, 20.0 g of agar in one liter of deionized

water.

(2) Seed medium, containing 80.0 g of sorbitol, 20.0 g of yeast extract, 0.5 g of

MgSO4·7H2O, 1.5 g of KH2PO4, 1.5 g of (NH4)2SO4 in one liter of deionized water.

(3) Synthetic medium for fermentation, containing 80.0 g of glucose, 20.0 g of yeast

extract, 0.5 g of MgSO4·7H2O, 1.5 g of KH2PO4, 1.5 g of (NH4)2SO4 in one liter of deionized

water.

Biodetoxification fungus Amorphotheca resinae ZN1 was isolated in our previous works

and stored in China General Microorganism Collection Center (CGMCC), Beijing, China

with the registration number 7452 (Zhang et al., 2010a). The fungus A. resinae ZN1 was

maintained on a potato dextrose agar medium (PDA) slant. The PDA medium was prepared

by boiling 200 g of peeled and sliced potatoes in one liter deionized water for 30 min.

2.4. Pretreatment, biodetoxification and hydrolysate preparation

Dry dilute acid pretreatment (DDAP) method was used for pretreating the corn stover

feedstock in this study (Zhang et al., 2011; He et al., 2014). Briefly, 1,200 g of dried corn

stove and 600 g of 5% (w/w) dilute sulfuric acid solution was co-currently fed into the 20 L

pretreatment reactor under the helical agitation of the single helical impeller in the reactor.

The sulfuric acid dosage was 2.5 gram per 100 grams of dry corn stover feedstock. The

pretreatment temperature was remained at 175 ± 1 oC for 5 minutes under helically agitation.

The pretreated corn stover contained approximately 50% (w/w) of dry solid matter (DM) and

no free wastewater stream was generated. The pretreated corn stover solids (excluding water)

contained 37.24% of cellulose, 8.21% of hemicellulose, 5.86% of ash determined by two-step

acid hydrolysis method described in NREL protocols (Sluiter et al., 2008; 2012). The

inhibitors per gram of dry pretreated corn stover (DM) include 5.64 mg of furfural, 3.54 mg

of HMF, 18.43 mg of acetic acid, 0.23 mg of vanillin, 0.67 mg of syringaldehyde and 0.18 mg
of HBA.

Biodetoxification of the pretreated corn stover materials was carried out in a 15 L

bioreactor at 28 oC and 1 vvm of aeration for 36 hours (He et al., 2016). Briefly, A. resinae

ZN1 seeds were cultured on the pretreated corn stover materials at 28 oC for 5 days by

inoculation of spores from PDA slant. Then the seed solids were inoculated at 10% (w/w)

inoculation ratio onto the freshly pretreated corn stover for biodetoxification. The corn stover

was briefly mixed for approximately 1 min every 6-8 h. The detoxified corn stover was disk

milled before use.

Hydrolysis of the pretreated corn stover was carried out in the bioreactor equipped with

helical ribbon impeller for mixing (Zhang et al., 2010b). Both the freshly pretreated corn

stover and the biodetoxified and pretreated corn stover were hydrolyzed at 50 oC, pH 4.8 for

48 h at the cellulase dosage of 15 FPU/g DM. The hydrolysate slurry was centrifuged to

remove the solids, then autoclaved and filtered by filter paper before use.

2.5. Gluconic and xylonic acids fermentation

G. oxydans DSM 2003 was maintained at -80 oC freezer in vials containing 30% (v/v)

glycerol solution. One vial (2 mL) was inoculated into 20 mL of seed medium in 100 mL

flask and cultured at 30 oC, 220 rpm for 24 hours. For flask fermentation, the seed broth was

inoculated at 10% volume ratio into 50 mL of fermentation medium in a 250 mL flask and

fermented at 30 oC, 220 rpm for 24-72 hours. For fermentor fermentation, the seed broth was

inoculated at 10% (v/v) inoculation ratio into 3 L fermentor containing 900 mL corn stover

hydrolysate and fermented at 30 oC, 500 rpm, 2.5 vvm of aeration for 24-72 hours, where 5M

NaOH and 2M H2SO4 were used for pH control. All the experiments were conducted in

duplicate.

Sodium xylonate used for cement additive assay was prepared by fermenting xylose in

the bottom liquid of the distillation from ethanol fermentation broth, in which only glucose
 was utilized and xylose was left in the broth (Zhang et al., 2010b). Xylose in the bottom

liquid was converted into xylonic acid by G. oxydans DSM 2003 and neutralized into sodium

xylonate. No sodium gluconate was detected in the sodium xylonate broth.

2.6. Analysis of sugars, acids and inhibitors

Samples were periodically taken, centrifuged and filtrated through 0.22 µm filters before

analysis. Gluconic acid, xylonic acid and keto-gluconic acid (KGA) were analyzed using

HPLC (LC-20AT, UV/VIS detector SPD-20A, Shimadzu, Kyoto, Japan) with an Aminex

HPX-87H column (Bio-rad, Hercules, CA, USA) at 55 oC using the mobile phase of 5 mM

H2SO4 at the flow rate of 0.4 mL/min. The detection wavelength was 210 nm.

Glucose was measured by a biosensor (SBA-40D, Shandong Academy of Agriculture,

Jinan, China). Xylose was measured by HPLC (LC-20 AD, refractive index detector RID-10A,

Shimadzu, Kyoto, Japan) with Aminex HPX-87H column at 65 oC using the mobile phase of

12 mM NaHCO3 at the flow rate of 0.6 mL/min.

Furfural, acetic acid and HMF were measured by HPLC (LC-20 AD, refractive index

detector RID-10A, Shimadzu, Kyoto, Japan) with Aminex HPX-87H column at 65 oC using

the mobile phase of 5 mM H2SO4 at the flow rate of 0.6 mL/min. Vanillin, syringaldehyde and

4-hydroxybenzaldehyde (HBA) were analyzed using HPLC (LC-20AT, UV/VIS detector

SPD-20A, Shimadzu, Kyoto, Japan) with YMC-Pack ODS-A column (YMC, Tokyo, Japan) at

35 oC and 270 nm at the flow rate of 1.0 mL/min. Two mobile phase solutions were 0.1% (v/v)

formic acid and acetonitrile. The acetonitrile concentration in the mobile phase was adjusted

in the following procedure: 10% in the first 4 min; increased from 10% to 35% from 4 to 5

min, and maintained at 35% from 6 to 15 min; reduced from 35% to 10% from 15 to 21min,

and maintained at 10% from 21 to 30 min (Khoddami et al., 2013).

2.7. Yield calculation of gluconic and xylonic acids

The gluconic acid yield is defined as the ratio of the glucose converted into the gluconic
acid according to the stoichiometric equation:

[GA] × V − [GA]0 × V 0
Yield (%) = × 100%
[Glu ]0 × V 0 × 1.089

where [Glu]0, the initial glucose concentration (g/L); [GA]0 and [GA], the initial and final

gluconic acid concentrations (g/L); 1.089 is the conversion factor for glucose to equivalent

gluconic acid; V and V0, the initial and final volumes of fermentation broth (L).

The xylonic acid yield is defined as the ratio of the xylose converted into the xylonic

acid according to the stoichiometric equation:

[ XA] × V − [ XA]0 × V 0
Yield (%) = × 100%
[ Xyl ]0 × V 0 × 1.107

where [Xyl]0, the initial xylose concentration (g/L); [XA]0 and [XA], the initial and final

xylonic acid concentrations (g/L); 1.107 is the conversion factor for xylose to equivalent

xylonic acid; V and V0, the initial and final volumes of fermentation broth (L).

2.8. Assay of setting time, fluidity and strength of cement paste

The fermentation broth of gluconic and xylonic acids was purified by filtration and

decoloration to remove the solids and dark colors. The obtained solution contained 132.46 g/L

of sodium gluconate and 15.90 g/L sodium xylonate. The dosage of cellulosic sodium

gluconate addition was based on the weight of the actual sodium gluconate and xylonate in

the solution. Xylonic acid fermentation broth was prepared by fermenting xylose in the

bottom liquid of the distillation from ethanol fermentation broth, was also purified by

filtration and decoloration to remove the solids and dark colors. The obtained solution

contained 80.50 g/L sodium xylonate. The dosage of cellulosic sodium xylonate addition was

based on the weight of the actual sodium xylonate in the solution. The purity of the

commercial sodium gluconate was 98% (w/w) and the dosage of commercial sodium

gluconate addition was based on the weight.

The consistency of the cement paste was measured by Vicat apparatus (Luda
Construction Co., Shanghai, China) and adjusted by water addition into the standard range.

The setting time of the cement paste was also determined by Vicat apparatus according to

Chinese Standard Protocol GB/T 1346-2011. Briefly, 500 g of the standard cement was mixed

by required amount of water into cement paste mixer (NJ-160, Wuxi Construction Co.,

Jiangsu, China). The cement paste was mixed by the rotation at 140 rpm and the revolution at

62 rpm for 120 seconds, then stop for 15 seconds; again mixed at the rotation at 285 rpm and

the revolution at 125 rpm for 120 seconds.

The fluidity of cement paste was determined according to the Chinese Standard Protocol

GB/T 2419-2005. Briefly, 300 g of standard cement was mixed with 87 mL of water and 0.18

g polycarboxylate as water reducer into cement paste mixer (NJ-160, Wuxi construction Co.,

Jiangsu, China) and mixed at the rotation at 140 rpm and the revolution at 62 rpm for 180

seconds. The cement paste was fully filled into a standard copper conical cylinder (60 mm in

height, 50 mm in top circle diameter, and 75 mm in bottom circle diameter), then the cylinder

was lifted up promptly to let the cement paste flowing on the glass plate after 30 seconds. The

circle diameter of the cement paste slurry on the glass plate was measured as the indicator of

the fluidity of cement paste (mm).

The flexural and compressive strength of the cement mortars were measured according

to Chinese Standards Protocol GB/T17671-1999. Briefly, the cement paste was prepared with

standard sand according to Chinese Standard GB/T17671-1999 for preparing the standard

specimens (40 × 40 × 160 mm) with the weight ratio of cement, sand and water at 2:6:1. The

flexural strength was carried out on the long surface of the cement mortar specimen using a

cement bending tester (300KN, Jinan Kaine Testing Mechanics Co., Shandong, China) and

three specimens were tested for each sample. The compressive tests were carried out on the

cement pressure tester (DKZ-5000, Jinan Kaine Testing Mechanics Co., Shandong, China)

and six specimens were tested for one sample to get the average value.
2.9. Process model on Aspen Plus platform and economic analysis method

The process model was developed using Aspen Plus software (AspenTech Co.,

Cambridge, MA, USA). The basic model was applied the NREL design report (Humbird et al.,

2011) with the changes in four areas: (1) The pretreatment area was changed from the

conventional dilute acid pretreatment in the NREL report into the dry dilute acid pretreatment

(DDAP) (Zhang et al., 2011); (2) the detoxification area was changed from ammonia

overliming into the biodetoxification (He et al., 2016); (3) the saccharification and

fermentation area was changed from 20% (w/w) solids loading into 30% (w/w) solids loading;

(4) the product recovery area was changed from ethanol into sodium gluconate and xylonate.

The plant size is 900 tons processing capacity of corn stover each day (300,000 tons annually)

with an annual operation of 8,000 hours.

The detailed Aspen plus flowsheet contains ten process areas (Supplemental Materials

Fig. S1): feedstock handing, pretreatment, biodetoxification, enzymatic hydrolysis and

fermentation, cellulase production, product recovery, wastewater treatment, residue

combustion, storage, and utilities system. Area 200 is the dry dilute acid pretreatment (DDAP)

unit, in which the feedstock is treated by dilute acid at the corn stover solids of 66.7% (w/w).

Area 300 is the biodetoxification using A. resinae ZN1 to remove most of the inhibitors in 36

hours. Area 400 is the enzymatic hydrolysis and fermentation, in which the pretreated and

biodetoxified corn stover is hydrolyzed and fermented to sodium gluconate and xylonate by

Gluconobacter oxydans DSM 2003 at 30% (w/w) solids loading. Area 500 is the product

recovery of sodium gluconate and xylonate to meet the technical grade of 98% (w/w) by

solid/liquid separation, decoloration and multiple evaporation steps. Others Areas in the

Aspen plus flowsheet are maintained the same with the NREL model.

The material and energy balance data from Aspen plus modeling are used to design the

equipment and determine the chemical usage. The year of 2013 is used as the reference year.
The exchange rate from US dollar ($) to Chinese Yuan (CNY) is 1: 6.2. The general purpose

equipment of pumps, conveyors and evaporators are quoted from the NREL price (Humbird et

al., 2011). The specific equipment of the pretreatment reactors, fermentors and helical

agitators, chemicals, and staff wages are modified according to actual situation in China. The

raw material composition, main equipment and chemical used in the process and their prices

are shown in Supplemental Materials Tables S1, S2 and S3. Note that the price of net

electricity to the grid is 0.75 CNY/kWh based on the government regulation for renewable

electricity pricing (“Regulations on Electricity Prices on Renewable Energy Use”, the

National Development and Reform Commission of China,

http://www.gov.cn/ztzl/2006-01/20/content_165910.htm).

A discounted cash flow rate of return to determine the minimum sodium

gluconate/xylonate product selling price (MGSP, $/kg) requires a net present value of zero for

8% internal rate of return after taxes. Table S4 shows the assumptive parameters in the

discounted cash flow analysis.

3. Results and Discussion

3.1. Gluconic and xylonic acids fermentation by G. oxydans cells using corn stover feedstock

Gluconic acid fermentability of G. oxydans DSM 2003 was evaluated in both synthetic

medium and corn stover hydrolysate (without inhibitor removal) in flasks (Fig. 1). Gluconic

acid productivity and the cell growth of G. oxydans DSM 2003 in the inhibitor containing

corn stover hydrolysate were almost same to that in synthetic medium, even without any

nutrients or inorganic salts addition to the hydrolysate (Fig. 1a). G. oxydans cells showed

stronger inhibitor degradation capacity than A. niger SIIM M276 (Zhang et al., 2016), and

well adapted to the hydrolysate environment by maintaining stable cell morphology (Fig. 1b).

Then the fermentation was conducted in fermentors with accurate control of pH,

temperature, dissolved oxygen level (Fig. 2). In the hydrolysate prepared from 15% (w/w) of
the freshly pretreated corn stover, gluconic acid productivity in fermentors increased

significantly to 2.82 g/(L·h) from 1.90 g/(L·h) in flasks (Fig. 2a) and 0.28 g/(L·h) in flasks by

A. niger (Zhang et al., 2016). When the sugars increased by increasing solids content of

freshly pretreated corns stover hydrolysate preparation to 20% (w/w), the cell growth rate

decreased with the inhibitors increased but gluconic acid productivity was not inhibited

obviously comparing with the one in 15% (w/w) freshly pretreated corn stover hydrolysate

(Fig. 2b). Xylose started to convert to xylonic acid (Fig. 2c) after the complete consumption

of glucose, but also gluconic acid started to convert to keto-gluconic acid (KGA). The

existence of xylonic acid in the fermentation products is a positive factor because sodium

xylonate is an excellent water reducer of cement (Chun et al., 2006), but the function of

keto-gluconic acid is not clear as cement additive. Furfural and HMF inhibitors were quickly

degraded, and acetic acid was almost constant or slightly increased but no obvious inhibition

on gluconic acid productivity was observed (Fig. 2d). However, when the sugar concentration

increased by increasing solids content in hydrolysis step to 25% (w/w), G. oxydans DSM 2003

was not able to grow.

To obtain the maximum yield and titer of gluconic and xylonic acids, the inhibitors in the

pretreated corn stover were removed as completely as possible by applying the

biodetoxification on the solid pretreated corn stover (Fig. 3). The residue furfural and HMF

were completely degraded in the first 16 hours of the fermentation (Fig. 3d). G. oxydans cells

growth in the detoxified corn stover hydrolysates increased 2-3 folds than that in the

hydrolysate without inhibitor removal, even similar to that in synthetic medium (Fig. 3a).

Gluconic acid productivity maintained considerably high with the maximum titer of 102.10

g/L and yield of 91.13 % (Fig. 3b), and xylose was converted to xylonic acid with the

maximum titer of 34.31 g/L and yield of 90.02% (Fig. 3c). The maximum gluconic acid and

xylonic acid could be obtained by avoiding unnecessary conversion of gluconic acid to

keto-gluconic acid at specific time point.

The fermentation was further scaled up in a 50 L bioreactor (35 L of working volume)

from the 3 L fermentors (1 L of working volume) using the corn stover hydrolysate prepared

at 30% (w/w) solids loading of the pretreated and biodetoxified corn stover feedstock (Fig. 4).

119.10 g/L of gluconic acid (equivalent to 132.46 g/L of sodium gluconate) with 4.14 g/(L·h)

of productivity and 97.12 % of yield within 32 h were received. The productivity was slightly

low because of the reduced inoculum size (10% in 3 L, but 1% in 50 L) and oxygen transfer

(2.5 vvm of aeration and 500 rpm of agitation rate in 3L, but 1.5 vvm and 300 rpm in 50L) for

considerations of practical commercial scale operation. The fermentation was stopped at 32 h

to prevent the possible conversion of gluconic acid to keto-gluconic acid, thus xylose

conversion to xylonic acid was not complete (14.04 g/L of xylonic acid, equivalent to 15.90

g/L of sodium xylonate).

The robust inhibitor tolerance, easy cell growth, and co-production of high titer sodium

gluconate and xylonate by G. oxydans DSM 2003 successfully overcome the drawbacks of

low productivity, difficult seed propagation, and xylose loss by A. niger SIIM M276.

3.2. Sodium gluconate and xylonate product from corn stover as cement retarder additives

Sodium gluconate broth produced using corn stover feedstock in the 50L fermentor was

directly used as the cement retarder additive without any concentrating operation. The cement

additive assay was conducted by testing the addition of sodium gluconate broth on the setting

time, fluidity and strength of cement paste and compared with the commercial sodium

gluconate from corn starch feedstock (Table 1). The initial setting time indicates the start of

cement paste setting, hardening, and plasticity loss; and the final setting time indicates the

complete loss of plasticity and behaving structural strength. The fluidity indicates the

uniformity and stability of cement paste. The corn stover derived sodium gluconate/xylonate

showed the obvious cement retarding properties of longer setting time and greater fluidity
than that of the commercial sodium gluconate in the range of sodium gluconate dosage of

0.01%-0.03% (w/w) addition. When the sodium gluconate dosage exceeded 0.03% (w/w), the

plasticity of the cement paste was too high and the setting time was too long (more than 390

min). The addition of sodium xylonate only also showed considerable retarding property for

extending setting time in the range of 0.01%-0.06% (w/w) addition.

Strength test indicates the ability to withstand external force to cement mortar. The

sodium gluconate/xylonate showed similar flexural strength and the compressive strength

reached almost the same with the commercial sodium gluconate when the sodium gluconate

dosage was 0.03% (w/w).

The results indicate that the crude sodium gluconate/xylonate broth from corn stover

feedstock by G. oxydans DSM 2003 was a satisfactory cement retarder additive, similar to the

commercial product from corn starch.

3.3. Techno-economic analysis (TEA) of cellulosic sodium gluconate product

The Aspen Plus model was established for sodium gluconate and xylonate production as

described in the Method section based on the experiments results and the relevant TEA study

on L-lactic acid product from lignocellulose (Liu et al., 2015). The main process input data

are listed in Table 2. The sodium gluconate and sodium xylonate in fermentation broth are 132

g/L and 39 g/L in the process simulation, respectively, according to the maximum

experimental results. The recovery yield of sodium gluconate and xylonate in the product

recovery area is assumed to be the same with the sodium lactate recovery from corn stover

(92%) (Liu et al., 2015). The overall processing capacity is 300,000 tons of corn stover

annually and the total product output is 151,688 tons annually (75.65% of sodium gluconate,

22.35% of sodium xylonate, and 2.00% of water, w/w).

The detailed material balance of the overall process is shown in Fig. 5a at the time scale

of hour. The rigorous calculation shows that 506 kg of sodium gluconate product is obtained
from one ton of dry corn stover. For fresh water usage, 4.92 tons of fresh water is consumed

for producing one ton of product, but only 1.20 tons of fresh water is required if the water

cycling is taken into account in the overall process. The pretreatment area and the enzyme

hydrolysis area consume 19.16% and 57.12% of the total fresh water, respectively. For waste

water generation, 1.88 tons of waste water is generated for producing one ton of product. No

free waste water stream is released from dry dilute acid pretreatment, and equivalently 1.58

tons of waste water per ton of product is released in product recovery area to the waste water

area.

The detailed energy balance of the overall process is shown in Fig. 5b at the time scale of

hour. For heating steam usage, 6,349 MJ of heating value is required for producing one ton of

product, which is equivalent to 2.18 tons of hot steam (273 oC, 1.3 MPa). The pretreatment

steam and the triple-effect evaporation in the recovery area account for 18.05% and 73.65%

of heating steam usage, respectively, while the hydrolysis step takes 8.3 % of heating steam.

For electricity consumption, the electricity consumption is approximately 504 kWh for

producing one ton of product. The disk milling of pretreated corn stover, drying and grinding

of fresh corn stover and the hydrolysis mixing of highly viscous material account for 26.51%,

21.36%, and 11.73%, respectively. On the other hand, the combustion of lignin residue in

boiler area generates sufficient heat and electricity to meet all the needs of the overall

processing with 251 kWh of excessive electricity to the grid as by-product credit for

producing one ton of product.

Fig. 5c illustrates the detailed contribution of capital, operations and fixed costs to the

overall cost. The dry dilute acid pretreatment and biodetoxification cost is significantly

reduced to $0.030 per kg product compared to other pretreatment technologies (Humbird et

al., 2011). The cost of corn stover and sodium hydroxide is $0.122 and $0.090 per kg product,

respectively, accounting for 78% of the cost of saccharification and fermentation area, as well
as 52% of the total cost. The cost of combustion step is only $0.024 per kg product due to

electricity credit. The total capital investment is about $166.9 MM for this biorefinery plant

with 300,000 tons of corn stover feedstock annually. The minimum sodium

gluconate/xylonate product selling price (MGSP) is calculated to be $0.404 per kg product, in

which the feedstock, enzyme production and non-enzyme conversion cost were $0.122,

$0.095 and $0.187 per kg, respectively. Compared with a typical commercial sodium

gluconate product using corn feedstock on Chinese market ($0.476/kg, Xiwang Group Co. on

Alibaba Enterpriser website https://www.1688.com), the cellulosic sodium gluconate/xylonate

product show a certain competitive.

Dry milling biorefining process (DMBP) played the key role on realizing a competitive

production of sodium gluconate/xylonate to the corn based product by its low capital

investment, low steam and water usage, low waste water generation, and high fermentation

performance (Zhang et al., 2010a; Zhang et al., 2011; He et al., 2014; He et al, 2016). Note

that no extra nutrients were required for fermentation and the crude fermentation broth only

was an excellent cement retarder additive product, which provides an important alternative for

traditional sodium gluconate from corn. The minimum sodium gluconate/xylonate selling

price (MGSP) based on the Aspen plus modeling is only $0.404 per kg, which was obviously

competitive than ethanol from corn stover (MESP at $2.15 per gallon, equivalent to $0.720

per kg, Humbird et al., 2011) using the same calculation principle and similar processing

flowsheet), and competitive to the commercial sodium gluconate ($0.476 per kg) from starch

in China market. The biorefining technology practice on value added biochemical production

provides the practical and competitive economic benefits on the future utilization of

lignocellulose biomass.

4. Conclusion

Gluconobacter oxydans DSM 2003 showed an excellent gluconic and xylonic acids
fermentability using the dry dilute acid pretreated and biodetoxified corn stover feedstock.

Maximum titer of sodium gluconate at 132.46 g/L and sodium xylonate at 38.86 g/L were

obtained and the fermentation was scaled up. Sodium gluconate/xylonate product from corn

stover showed competitive performance as cement retarder than the commercial sodium

gluconate. The techno-economic analysis based on Aspen plus modeling demonstrated high

economic and technical competitiveness to the corn starch based commercial technology. This

study provided a potential of lignocellulose biomass commercially applicable for sodium

gluconate and xylonate industry production.

Acknowledgement

This research was supported by the National High-Tech Program of China

(2014AA021901), the Natural Science Foundation of China (21306048), and the Fundamental

Research Funds for the Central Universities of China (WF1514325).

References

1. Adachi, O., Matsushita, K., Shinagawa, E., Ameyama M., 1980. Crystallization and

characterization of NADP-dependent D-Glucose dehydrogenase from Gluconobacter

suboxydans. Agric. Biol. Chem. 44 (2), 301-308.

2. Adney, B., Baker, J., 1996. Measurement of cellulase activities. LAP-006. NREL.

Analytical Procedure. National Renewable Energy Laboratory, Golden, CO.

3. Chun, B., Dair, B., Macuch, P. Wiebe, D., Porteneuve, C., Jeknavorian, A., 2006. The

development of cement and concrete additive: based on xylonic acid derived via

bioconversion of xylose. Appl. Biochem. Biotechnol. 131, 645-658.

4. Deppenmeier, U., Hoffmeister, M., Prust, C., 2002. Biochemistry and biotechnological

applications of Gluconobacter strains. Appl. Microbiol. Biotechnol. 60, 233-242.

5. Elfari, M., Ha, S., Bremus, C., Merfort, M., Khodaverdi, V., Herrmann, U., Sahm, H.,

Görisch, H., 2005. A Gluconobacter oxydans mutant converting glucose almost

 quantitatively to 5-keto-gluconic acid. Appl. Microbiol. Biotechnol. 66, 668-674.

6. Ghose, T.K., 1987. Measurement of cellulase activities. Pure Appl. Chem. 59, 257-268.

7. He, Y., Zhang, J., Bao, J., 2014. Dry dilute acid pretreatment by co-currently feeding of

corn stover feedstock and dilute acid solution without impregnation. Bioresour. Technol.

158, 360-364.

8. He, Y., Zhang, J., Bao, J., 2016. Acceleration of biodetoxification on dilute acid

pretreated lignocellulose feedstock by aeration and the consequent ethanol fermentation

evaluation. Biotechnol. Biofuels 9, 19.

9. Humbird, D., Davis, R., Tao, L., Kinchin, C., Hsu, D., Aden, A., Schoen, P., Lukas, J.,

Olthof, B., Worley, M., Sexton, D., Dudgeon, D., 2011. Process Design and Economics

for Biochemical Conversion of Lignocellulosic Biomass to Ethanol.

NREL/TP-5100-47764. National Renewable Energy Laboratory, Golden, CO.

10. Khoddami, A., Wilkes, M., Roberts, T., 2013. Techniques for analysis of plant phenolic

compounds. Molecules 18, 2328-2375.

11. Li, D., 2011.The formulation and preparation of cement concrete additive, first ed. China

Textile Press, Beijing.

12. Li, N., Ma, D., Chen, W., 2015. Projection of cement demand and analysis of the impacts

of carbon tax on cement industry in China. Energy Procedia. 75, 1766-1771.

13. Liu, G., Sun, J., Zhang, J., Tu, Y., Bao, J., 2015. High titer L-lactic acid production from

corn stover with minimum wastewater generation and techno-economic evaluation based

on Aspen plus modeling. Bioresour. Technol. 198, 803-810.

14. Ma, S., Li, W., Zhang, S., Ge, D., Yu, J., Shen, X., 2015. Influence of sodium gluconate

on the performance and hydration of Portland cement. Constr. Build. Mater. 91, 138-144.

15. Matsushita, K., Shinagawa, E., Adachi, Q., Ameyama M., 1989. Reactivity with

ubiquinone of quinoprotein D-glucose dehydrogenase from Gluconobacter suboxydans. J.

 Biochem. 105, 633-637.

16. Merfort, M., Herrmann, U., Ha, S., Elfari, M., Bringer-Meyer, S., Görisch, H., Sahm, H.,

2006. Modification of the membrane-bound glucose oxidation system in Gluconobacter

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

Biotechnol. J. 1, 556-563.

17. Silberbach, M., Maier, B., Zimmermann, M., Büchs, J., 2003. Glucose oxidation by

Gluconobacter oxydans: characterization in shaking-flasks, scale-up and optimization of

the pH profile. Appl. Microbiol. Biotechnol. 62, 92-98.

18. Sluiter, A., Hames, B., Ruiz, R., Scarlata, C., Sluiter, J., Templeton, D., 2008.

Determination of sugars, byproducts, and degradation products in liquid fraction process

samples. NREL/TP-510-42623. National Renewable Energy Laboratory, Golden, CO.

19. Sluiter, A., Hames, B., Ruiz, R., Scarlata, C., Sluiter, J., Templeton, D., Crocker, D.,

2012. Determination of structural carbohydrates and lignin in biomass.

NREL/TP-510-42618. National Renewable Energy Laboratory, Golden, CO.

20. Toivari, M., Nygård, Y., Penttilä, M., Ruohonen, L., Wiebe, M., 2012. Microbial

D-xylonate production. Appl. Microbiol. Biotechnol. 96, 1-8.

21. Wei, L., Zhu, D., Zhou J, Zhang, J., Zhu, K., Du, L., Hua, Q., 2014. Revealing in vivo

glucose utilization of Gluconobacter oxydans 621H △mgdh strain by mutagenesis.

Microbiol. Res. 169, 469-475.

22. Zhang, H., Zhang, J., Bao, J., 2016. High titer gluconic acid fermentation by Aspergillus

niger from dry dilute acid pretreated corn stover without detoxification. Bioresour.

Technol. 203, 211-219.

23. Zhang, J., Chu, D., Huang, J., Yu, Z., Dai, G., Bao, J., 2010b. Simultaneous

saccharification and ethanol fermentation at high corn stover solids loading in a helical

stirring bioreactor. Biotechnol. Bioeng. 105, 718-728.

24. Zhang, J., Wang, X., Chu, D., He, Y., Bao, J., 2011. Dry pretreatment of lignocellulose

with extremely low steam and water usage for bioethanol production. Bioresour. Technol.

102, 4480-4488.

25. Zhang, J., Zhu, Z., Wang, X., Wang, N., Wang, W., Bao, J., 2010a. Biodetoxification of

toxins generated from lignocellulose pretreatment using a newly isolated fungus

Amorphotheca resinae ZN1 and the consequent ethanol fermentation. Biotechnol.

Biofuels 3, 26.
Figure captions
Figure 1 Gluconic acid fermentation of G. oxydans DSM 2003 in flasks using synthetic
medium and inhibitors containing corn stover hydrolysate. (a) Conversions; (b) Inhibitors
degradation. Hydrolysate was prepared from freshly pretreated corn stover without
detoxification. The fermentation was carried out at 30 oC, pH 5.5, 220 rpm and the inoculum
size 10%.
Figure 2 Gluconic and xylonic acids fermentation of G. oxydans DSM 2003 in fermentors
using inhibitor containing corn stover hydrolysates. (a) Cell growth; (b) Gluconic acid
generation; (c) Xylonic acid generation; (d) Inhibitors degradation. The fermentation was
carried out at 30 oC, pH 5.5, 500 rpm, 2.5 vvm, inoculum size 10%. Corn stover hydrolysate
contained: (1) 15 % solids loading, 54.49 g/L of glucose, 23.53 g/L of xylose, 3.24 g/L of
acetic acid, 0.20 g/L of HMF, 0.31 g/L of furfural; (2) 20 % solids loading, 72.20 g/L of
glucose, 37.60 g/L of xylose, 4.28 g/L of acetic acid, 0.48 g/L of HMF, 0.51 g/L of furfural.
Figure 3 Gluconic and xylonic acids fermentation of G. oxydans DSM 2003 in fermentors
using biodetoxified corn stover hydrolysates. (a) Cell growth; (b) Gluconic acid generation; (c)
Xylonic acid generation; (d) Inhibitors degradation. Conditions: 30 oC, pH 5.5, 500 rpm, 2.5
vvm, the inoculum size 10%, 1 L liquid in 3 L fermentor. Corn stover hydrolysates: (1) 15 %
solids, 62.72 g/L of glucose, 23.88 g/L of xylose, 1.09 g/L of acetic acid, 0.02 g/L of HMF,
0.07 g/L of furfural; (2) 20 % solids, 88.47 g/L of glucose, 27.13 g/L of xylose, 1.03 g/L of
acetic acid, 0.06 g/L of HMF, 0.07 g/L of furfural; (3) 25 % solids, 109.78 g/L of glucose,
31.46 g/L of xylose, 1.13 g/L of acetic acid, 0.06 g/L of HMF, 0.08 g/L of furfural; (4) 30 %
solids, 124.80 g/L of glucose, 40.03 g/L of xylose, 1.46 g/L of acetic acid, 0.12 g/L of HMF,
0.10 g/L of furfural.
Figure 4 Large scale sodium gluconate fermentation in 50 L fermentor in corn stover
hydrolysate from 30% (w/w) pretreated and biodetoxified corn stover. The fermentation was
carried out at 30 oC, pH 5.5, 300 rpm, 1.5 vvm, inoculum size 1%, liquid volume 35 L in 50 L
fermentor. Corn stover hydrolysate contained 124.80 g/L of glucose, 40.03 g/L of xylose, 1.46
g/L of acetic acid, 0.12 g/L of HMF, 0.10 g/L of furfural. 119.10 g/L gluconic acid and 14.04
g/L xylonic acid were obtained at 32 h, equivalent to 132.46 g/L of sodium gluconate and
15.90 g/L of sodium xylonate, respectively.
Figure 5 Materials, energy and cost balance of cellulosic sodium gluconate production from
corn stover feedstock on the hour basis (tons per hour). (a) Materials balance; (b) Energy
balance; (c) Cost contribution details from each process area (per kg sodium
gluconate/xylonate product). Abbreviations: CS, Corn stover; DDAP, Dry dilute acid
pretreatment; PCS, Pretreated corn stover; BPCS: Pretreated biodetoxification corn stover;
CSL, Corn steep liquor; CSH, Corn stover hydrolysate; DAP: Ammonium phosphate. NaGA,
Sodium gluconate; NaXA, Sodium xylonate.
Table 1 Assay of sodium gluconate and xylonate as cement retarder additives

Properties Addition Commercial Cellulosic Cellulosic

(%, w/w) sodium sodium sodium

gluconate gluconate xylonate

163±4 163±4
Setting Time Initial 0 163±4
185±0 186±6
(min) 0.01 165±3
223±4 239±5
0.02 170±0
308±4 325±7
0.03 230±4
>390 >390
0.06 225±4
>390 >390
0.1 ND
220±0 220±0
Final 0 220±0
248±4 253±4
0.01 215±4
288±4 303±4
0.02 225±4
355±7 380±7
0.03 280±7
>390 >390
0.06 275±4
>390 >390
0.1 ND
243±1 243±1
Fluidity 0 243±1
271±2 276±4
(mm) 0.01 246±3
281±1 282±4 244±4
0.02
289±1 286±1 246±1
0.03
253±4 240±6 240±7
0.06
198±4 153±4 ND
0.1
 5.4±0.3 5.3±0.3 ND
Flexural 3 days 0.01
6.1±0.4 5.6±0.4
strength 0.02 ND
5.5±0.5 5.7±0.3
(MPa) 0.03 ND

28 days 0.01 9.1±0.4 8.8±0.2 ND

0.02 8.6±0.3 8.6±0.3 ND

0.03 8.8±0.2 8.7±0.2 ND

Compressive 3 days 0.01 28.5±0.8 26.4±0.6 ND

strength 0.02 28.3±0.8 26.1±0.6 ND

(MPa) 0.03 26.6±0.6 26.9±0.9 ND

28 days 0.01 53.8±1.1 50.3±0.8 ND

0.02 52.4±0.8 51.0±0.6 ND

0.03 50.2±1.0 50.9±0.5 ND

Fluidity and setting time assays were conducted according to Chinese Standard Protocol GB/T
1346-2011 and GB/T 2419-2005, respectively. Strength assay was conducted according to
Chinese Standard Protocol GB/T 17671-1999. Cellulosic sodium gluconate fermentation
broth contained 132.46 g/L of sodium gluconate and 15.90 g/L of sodium xylonate. Cellulosic
sodium xylonate fermentation broth contained 80.50 g/L sodium xylonate. Polycarboxylate
was supplemented into the cement paste at the dosage of 0.06% (w/w) when the fluidity was
tested as water reducer. The final setting time over 390 min was not counted according to
Chinese Standard Protocol GB/T 2419-2005. ND demonstrated not detected.
Table 2 Main process input data for the established Aspen plus simulation model

Features Values

Pretreatment

Sulfuric acid dosage (%) 2.5

Residence time (min) 5

Temperature (oC) 175

Pressure (MPa) 0.89

Solids after pretreatment (%) 50

Glucose yield from glucan (%) 4

Hemicellulose sugar yields (%) 40

Furfural yield from xylan (%) 3.3

Acetic acid hydrolysis ratio (%) 60

Biodetoxification

Temperature (oC) 28

Residence time (hour) 36

Furfural conversion (%) 100

Acetic acid conversion (%) 70

Glucose consumed for cell growth (%) 5

Xylose consumed for cell growth (%) 90

H2SO4 neutralized (%) 100

Saccharification and Fermentation

Temperature for hydrolysis (oC) 50

 Temperature for fermentation (oC) 33

Residence time for hydrolysis (hour) 48

Residence time for fermentation (hour) 24

Solids loading (%) 30

Cellulase dosage (mg protein/g cellulose) 28

Glucan conversion to glucose (%) 87

Xylan conversion to xylose (%) 82

Sodium gluconate yield from glucose (%) 97

Sodium xylonate yield from xylose (%) 90

Glycerol yield from glucose (%) 1

Glucose consumed for cell growth (%) 2

Sodium gluconate concentration (g/L) 132

Sodium xylonate concentration (g/L) 39

Product recovery

Purity (sodium gluconate and sodium xylonate) 98% (w/w)

Water content 2% (w/w)

 (a) Gluconic acid production in pure medium and hydrolysate
Glucose Synthetic Medium Hydrolysate
Gluconic acid Synthetic Medium Hydrolysate
70 OD600 3.5
Glucose and gluconic acid (g/L) Synthetic Medium Hydrolysate

60 3.0

50 2.5

40 2.0

OD600
30 1.5

20 1.0

10 0.5

0 0.0
0 4 8 12 16 20 24 28 32
Time (h)
(b) Inhibitor degradation in corn stover hydrolysate
Furfural HMF
0.5 Vanillin Syringaldehyde 5.0
4-Hydroxybenzaldehyde Acetic acid
Aldehyde inhibitors (g/L)

0.4 4.0

Acetic acid (g/L)

0.3 3.0

0.2 2.0

0.1 1.0

0.0 0.0
0 4 8 12 16 20 24 28 32
Time (h)
Figure 1 Gluconic acid fermentation of G. oxydans DSM 2003 in flasks using synthetic
medium and inhibitors containing corn stover hydrolysate. (a) Conversions; (b) Inhibitors
degradation. Hydrolysate was prepared from freshly pretreated corn stover without
detoxification. The fermentation was carried out at 30 oC, pH 5.5, 220 rpm and the inoculum
size 10%.
 (a) Cell growth
OD600 15% 20%
1.8
1.6
1.4
1.2
OD600

1.0
0.8
0.6
0.4
0.2
0.0
0 4 8 12 16 20 24 28 32 36 40 44 48 52 56
Time (h)
(b) Gluconic acid production
90 Glucose 15% 20%
Glucose, gluconic acid, KGA (g/L)

Gluconic acid 15% 20%

80 Keto-gluconic acid 15% 20%
70
60
50
40
30
20
10
0
0 4 8 12 16 20 24 28 32 36 40 44 48 52 56
Time (h)
 (c) Xylonic acid production
40 Xylose 15% 20%
Xylose and xylonic acid (g/L) Xylonic acid 15% 20%
35

30

25

20

15

10

0
0 4 8 12 16 20 24 28 32 36 40 44 48 52 56
Time (h)
(d) Inhibitors degradation
0.4 Furfural 15% 20% 8
HMF 15% 20%
Acetic acid 15% 20% 7
Furfural and HMF (g/L)

0.3 6

Acetic acid (g/L)

5

0.2 4

0.1 2

0 0
0 4 8 12 16 20 24 28 32 36 40 44 48 52 56
Time (h)
Figure 2 Gluconic and xylonic acids fermentation of G. oxydans DSM 2003 in fermentors
using inhibitor containing corn stover hydrolysates. (a) Cell growth; (b) Gluconic acid
generation; (c) Xylonic acid generation; (d) Inhibitors degradation. The fermentation was
carried out at 30 oC, pH 5.5, 500 rpm, 2.5 vvm, inoculum size 10%. Corn stover hydrolysate
contained: (1) 15 % solids loading, 54.49 g/L of glucose, 23.53 g/L of xylose, 3.24 g/L of
acetic acid, 0.20 g/L of HMF, 0.31 g/L of furfural; (2) 20 % solids loading, 72.20 g/L of
glucose, 37.60 g/L of xylose, 4.28 g/L of acetic acid, 0.48 g/L of HMF, 0.51 g/L of furfural.
 (a) Cell growth
3.5 OD600 15% 20% 25% 30%

3.0

2.5

2.0
OD600

1.5

1.0

0.5

0.0
0 4 8 12 16 20 24 28 32
Time (h)
(b) Gluconic acid production
140 Glucose 15% 20% 25% 30%
Glucose, gluconic acid, KGA (g/L)

Gluconic acid 15% 20% 25% 30%

120 Keto-gluconic acid 15% 20% 25% 30%

100

80

60

40

20

0
0 4 8 12 16 20 24 28 32
Time (h)
 (c) Xylonic acid production
50 Xylose 15% 20% 25% 30%
Xylose and xylonic acid (g/L) Xylonic acid 15% 20% 25% 30%

40

30

20

10

0
0 4 8 12 16 20 24 28 32
Time (h)
(d) Inhibitors degradation
0.20 Furfural 15% 20% 25% 30% 2.0
HMF 15% 20% 25% 30%
Acetic acid 15% 20% 25% 30%
0.16 1.6
Furfural, HMF (g/L)

Acetic acid (g/L)

0.12 1.2

0.08 0.8

0.04 0.4

0.00 0.0
0 16 4 20 8
24 12 28 32
Time (h)
Figure 3 Gluconic and xylonic acids fermentation of G. oxydans DSM 2003 in fermentors
using biodetoxified corn stover hydrolysates. (a) Cell growth; (b) Gluconic acid generation; (c)
Xylonic acid generation; (d) Inhibitors degradation. Conditions: 30 oC, pH 5.5, 500 rpm, 2.5
vvm, the inoculum size 10%, 1 L liquid in 3 L fermentor. Corn stover hydrolysates: (1) 15 %
solids, 62.72 g/L of glucose, 23.88 g/L of xylose, 1.09 g/L of acetic acid, 0.02 g/L of HMF,
0.07 g/L of furfural; (2) 20 % solids, 88.47 g/L of glucose, 27.13 g/L of xylose, 1.03 g/L of
acetic acid, 0.06 g/L of HMF, 0.07 g/L of furfural; (3) 25 % solids, 109.78 g/L of glucose,
31.46 g/L of xylose, 1.13 g/L of acetic acid, 0.06 g/L of HMF, 0.08 g/L of furfural; (4) 30 %
solids, 124.80 g/L of glucose, 40.03 g/L of xylose, 1.46 g/L of acetic acid, 0.12 g/L of HMF,
0.10 g/L of furfural.
 Glucose Gluconic acid
140 Xylose Xylonic acid 50
Glucose and gluconic acid (g/L)

Xylose and xylonic acid (g/L)

120
40
100

30
80

60
20

40
10
20

0 0
0 4 8 12 16 20 24 28 32
Time (h)

Figure 4 Large scale sodium gluconate fermentation in 50 L fermentor in corn stover

hydrolysate from 30% (w/w) pretreated and biodetoxified corn stover. The fermentation was

carried out at 30 oC, pH 5.5, 300 rpm, 1.5 vvm, inoculum size 1%, liquid volume 35 L in 50 L

fermentor. Corn stover hydrolysate contained 124.80 g/L of glucose, 40.03 g/L of xylose, 1.46

g/L of acetic acid, 0.12 g/L of HMF, 0.10 g/L of furfural. 119.10 g/L gluconic acid and 14.04

g/L xylonic acid were obtained at 32 h, equivalent to 132.46 g/L of sodium gluconate and

15.90 g/L of sodium xylonate, respectively.

 DAP: 0.026 t
Enzymes: 9.245 t
(a) Acid solution: 18.821 t Lime slurry: 3.592 t CSL: 0.063 t Sterile air: 11.820 t
Protein: 0.365 t
H2SO4: 0.938 t Ca(OH)2: 0.720 t H2O: 8.696 t NaOH: 18.298 t O2: 2.700 t
H2O: 17.883 t H2O: 2.918 t Activated carbon: 0.500 t
Others: 0.184 t NaOH: 3.660 t N2: 8.967 t
Steam: 7.543 t Oxygen: 0.700 t H2O: 14.638 t H2O: 0.154 t Water: 5.120 t
Water: 53.312 t

Corn Stover
(CS) Product
Dry Dilute Acid High-Solids
Biological Gluconic Acid
Pretreatment Loading Recovery
Detoxification Fermentation
(DDAP) Hydrolysis
CS: 44.118 t PCS: 70.481 t BPCS: 73.815 t CSH: 136.372 t Broth: 154.507 t Product: 18.961 t
Glucan:13.429 t Glucan: 13.094 t Glucan: 13.094 t NaGA: 14.496 t
Glucose: 12.997 t NaGA: 15.714 t
Xylan: 7.324 t Xylan: 4.153 t Xylan: 4.153 t NaXA: 4.288 t
Xylose: 4.152 t NaXA: 4.648 t
Lignin: 5.910 t Lignin: 5.910 t Lignin: 5.910 t Glucan: 1.702 t H2O: 0.383 t
Lignin: 5.910 t
Ash: 1.849 t Glucose: 0.358 t Glucose: 0.339 t Xylan: 0.768 t H2O: 110.175 t
H2O: 6.618 t Xylose: 3.329 t Xylose: 0.306 t Lignin: 5.910 t Others: 18.060 t
Others: 8.988 t Furfural: 0.189 t Acetic acid: H2O: 95.916 t
HMF: 0.069 0.102 t Others: 14.927 t
Acetic acid: 0.367 t H2O: 35.656 t
H2O: 31.534 t CaSO4: 1.301 t
Others: 11.478 t Others: 12.954 t

CO2: 0.958 t Waste Gas: Lignin Residue: 25.717 t

12.072 t Lignin: 5.880 t
Glucan: 1.694 t
CaSO4: 1.295 t
H2O: 8.438 t
Others: 8.410 t
Waste Water: 30.141 t
NaGA: 0.763 t
NaXA: 0.226 t
H2O: 21.371 t
Others: 7.781 t
Condensate Water: 85.102 t
(b) ：1020 kW
Electricity： Electricity: 800 kW ：1266 kW
Electricity： Electricity: 560 kW Electricity: 793 kW Electricity: 337 kW

Dry Dilute Acid High-Solids

Feed Handing Biological Gluconic Acid Recovery
Pretreatment Loading
(DDAP) Detoxification Fermentation
Hydrolysis

Heat duty: 21725 MJ Heat duty: 9993 MJ Heat duty: 88913 MJ

 (c) Capital Recovery Charge Raw Materials & Waste Process Electricity
Grid Electricity Total Plant Electricity Fixed Costs

Feedstock + Handling: 12.2¢

Pretreatment : 1.1¢
Detoxification: 1.9¢
Enzyme Production: 9.5¢
Hydrolysis and Fermentation: 11.6¢
Product Recovery: 3.2¢
Boilier/Turbogengerator: -2.4¢
Storage: 0.4¢
Wastewater Treatment: 1.7¢
Utilities: 1.0¢

-$0.150 -$0.100 -$0.050 $0.000 $0.050 $0.100 $0.150

Summary Minimum Sodium Gluconate Selling Price (MGSP) $0.404 per kg product
Feedstock contribution $0.122 per kg product
Enzyme contribution $0.095 per kg product
Non-enzyme conversion contribution $0.187 per kg product

Figure 5 Materials, energy and cost balance of cellulosic sodium gluconate production from corn stover feedstock on the hour basis (tons per
hour). (a) Materials balance; (b) Energy balance; (c) Cost contribution details from each process area (per kg sodium gluconate/xylonate product).
Abbreviations: CS, Corn stover; DDAP, Dry dilute acid pretreatment; PCS, Pretreated corn stover; BPCS: Pretreated biodetoxification corn
stover; CSL, Corn steep liquor; CSH, Corn stover hydrolysate; DAP: Ammonium phosphate. NaGA, Sodium gluconate; NaXA, Sodium
xylonate.
 Corn stover feedstock

Dry dilute acid pretreatment

Biodetoxification

Gluconobacter oxydans Enzymatic hydrolysis

DSMZ 2003
Fermentation
Zero extra nutrients addition

QC-A300
QC H-A300
WA300

Aspen plus
W KB400
QC -B400

Cement paste
QC H -B400

Fe ed Ha ndli ng Pre tre at men t De to x ifi cat io n Enz yme

A200 Pro du ctio n Bo il e r
A100 A300
CS 102 209 B400 QT405 A600

HIERARCHY HI ERARCHY HI ERARCHY HIE RARCHY

HI ERARC HY

retarder test modeling Feme nta t ion

A400
323

432
QH-A500

514
804
814
666

WTOTAL
QH405
440
HIE RARCHY Wate r treatme nt QM611
A800 WKA600
QH 801
Re co very HI ERARC HY
Sto ra ge A500 513
A700

HIERAR CHY
H IERARCHY S-GA WKA800

944
WKA500 Utillit es
QC -A400 A900
QC -A200
W A400
W KA200
W KA100
W KA700
HIERARCHY
903

Sodium gluconate: 132 g/L Application as TEA analysis

Sodium xylonate: 39 g/L cement additive

Graphical abstract
Highlights (85 characters)

Dilute acid pretreated and biodetoxified corn stover is used for this fermentation.

Glucose and xylose are simultaneously converted by Gluconobacter oxydans DSM 2003.

Highest 132.46 g/L of sodium gluconate and 38.86 g/L sodium xylonate were obtained.

Cellulosic sodium gluconate and sodium xylonate are both used as cement additive.

TEA based on Aspen plus is performed for the sodium gluconate/xylonate product.