Biotechnology and Bioprocess Engineering 16: 739-745 (2011)

DOI 10.1007/s12257-010-0347-x

Acetone-butanol-ethanol Fermentation and Isoflavone Extraction

Using Kudzu Roots

Lan Wang and Hongzhang Chen

Received: 30 September 2010 / Revised: 11 March 2011 / Accepted: 15 March 2011

© The Korean Society for Biotechnology and Bioengineering and Springer 2011

Abstract The economics of Acetone-butanol-ethanol

(ABE) fermentation is greatly affected by raw materials,

and the use of readily available starchy materials from

marginal farming lands could be a viable option for reduc-

ing costs. Kudzu, a rapidly growing perennial leguminous

vine, has been planted on marginal farming land and wide-

ly distributed in Asia and America. This study investigated

ABE fermentation by C. acetobutylicum ATCC 824 using

kudzu roots and isoflavone extraction from kudzu fermen-

tation residue (KFR). The kudzu roots could be used as a

sole substrate for ABE fermentation without nutritional

supplements. Batch culture containing 140 g kudzu/L

produced 17.99 ± 1.08 g/L solvent (ABE), including 11.20

± 0.79 g/L butanol, 5.54 ± 0.20 g/L acetone, and 1.15 ±

0.09 g/L ethanol, with a productivity of 0.19 g/(L/h) and a

yield of 0.33 g solvent/g sugar after 96 h of fermentation.

Isoflavone yield extracted from KFR was 1.90/100 g KFR,

approximately 48% higher compared with that extracted

from raw kudzu. A kinetic analysis of the extraction

process showed that both the isoflavone yield and the

extraction rate obtained from KFR were higher than the

corresponding values obtained from raw kudzu. These

results indicate that kudzu may provide a new potential raw

material for ABE production and the process of ABE

fermentation integrated with isoflavone extraction may

provide a new way to reduce fermentable substrate costs.

Keywords: kudzu, acetone-butanol-ethanol fermentation,

isoflavone extraction

1. Introduction

In recent decades, environmental and global energy pro-

blems have stimulated increased efforts toward biofuel

production from renewable resources. Butanol is a fuel

superior to ethanol with respect to gasoline blending,

distribution, and refueling. Besides, it is also an excellent

chemical in the plastics industry and a food-grade ex-

tractant in the food and flavor industry [1]. Since acetone,

butanol, and ethanol can be produced by Clostridium

acetobutylicum, this fermentation is often called ABE

fermentation. Presently, the most important economic

restriction on the viability of ABE production is the cost of

the substrate (corn, molasses, etc.), which makes up about

60% of the overall cost [2]. Although inexpensively avail-

able agricultural residues (e.g. straw, corn stover) have

been investigated for their potential application [3,4], low

butanol production caused by fermentation inhibitors from

ligno-cellulosic biomasses remains a challenging problem

[5]. Thus, the use of readily available starchy materials

from marginal farming lands seems to be a more viable

option for reducing the ABE fermentation costs.

Kudzu (Pueraria lobata) is a rapid-growing and high-

climbing perennial leguminous vine [6]. It is native to

eastern Asia (mainly in China, Japan, and Korea), and is

now naturalized in the United States, Australia and parts of

South America and South Africa. Kudzu has long been

semi-domesticated in China and Japan where it is used as

a forage crop and harvested for its root starch, fiber and

medicinal qualities. Its main active compounds, namely

isoflavones, are reported to have many important physio-

logical activities [7]. The main uses of kudzu in the United

States have been for erosion control and livestock feed.

Because of its aggressive growth, kudzu has come to be

regarded as a dense monoculture that has destroyed the

Lan Wang, Hongzhang Chen*

Key Laboratory of Biochemical Engineering, Institute of Process Engi-neering, Chinese Academy of Sciences, Beijing 100-190, ChinaTel: +86-10-8262-7067, Fax: +86-10-8262-7071E-mail: hzchen@home.ipe.ac.cn

RESEARCH PAPER

740 Biotechnology and Bioprocess Engineering 16: 739-745 (2011)

habitats of many native plants and animals, thus earning a

notorious reputation in the southern USA [8].

Kudzu contains approximately 30 ~ 50% (w/w, dry

mass) of starch and 1.77 ~ 12.0% (w/w, dry mass) of total

isoflavones, depending on its growing conditions [9]. With

respect to starch content and yield, kudzu is considered to

rival the carbohydrate production from maize and sugar

cane [8]. Several studies have examined the fermentation

of kudzu to ethanol [10,11], but there is no information

regarding kudzu fermentation for butanol production in the

literature. Because isoflavones mainly exist within the cells

of the plant material, it is difficult to extract isoflavones

from kudzu. To improve extraction efficiency, other assisted-

extraction techniques have been applied that facilitate

better penetration of the solvent into the raw plant materials

[9,12-14]. Currently, the most significant applications of

kudzu in China are isoflavone extraction and starch

processing. Because it lacked a combined process for the

production of isoflavones and kudzu starch, the waste of

raw materials was common problem in pharmaceutical and

food factories [15]. In this study, kudzu is identified as a

potential substrate for ABE production and kudzu fermen-

tation residue (KFR) is used for isoflavone production. The

integrated process of ABE fermentation and isoflavone

extraction was established for the feasibility of effective

utilization of kudzu.

2. Materials and Methods

2.1. Raw material pre-treatment

Kudzu was collected from Hunan Province, China in

November 2008. The roots of washed kudzu were cut into

30 ~ 40 mm pieces, dried in a well-aired area, and then

stored at room temperature. The moisture content of dried

roots was 3% (w/w). The composition of kudzu root is

shown in Table 1. Unless stated otherwise, dried kudzu

roots were used in the following experiments.

2.2. Microorganism and inoculum preparation

Clostridium acetobutylicum ATCC 824 was maintained as

a spore suspension in 6% (w/v) corn mash at 4°C. The pre-

culture medium contained the following components per

liter of distilled water: glucose 30 g, CH3COONH4 4.3 g,

KH2PO4 1.768 g, K2HPO4 2.938 g, p-aminobenzoic acid

10 mg, biotin 10 mg, and 1 mL of a mineral salts solution

[16]. These salts were of analytical grade and obtained

from Beijing Chemicals Factory, Beijing, China. The initial

pH of the medium was adjusted to 6.5 ± 0.2 with 1 M

NaOH or 1 M HCl. The medium was sterilized at 115°C

for 15 min. Cells were grown anaerobically at 37°C for 20

~ 36 h without agitation before being transferred into the

fermentation medium.

2.3. ABE fermentation

The kudzu fermentation medium contained 60 g/L of

kudzu, 4.3 g/L CH3COONH4, 1.768 g/L of KH2PO4, 2.938

g/L of K2HPO4, 10 mg/L of p-aminobenzoic acid, 10 mg/

L of biotin, and 1 mL/L of a mineral salts solution [16]. To

examine the effects of kudzu starch concentration on ABE

production, the concentrations of kudzu were varied from

60 to 180 g/L. To examine the direct fermentation of kudzu

to ABE, 140 g/L kudzu was used as the sole substrate

without any supplementary components.

All the batch fermentation studies were carried out in

250-mL serum bottles with a 100 mL working volume. The

pH of the medium was not adjusted before autoclaving at

121°C for 15 min. Upon cooling to room temperature, the

fermentation culture was inoculated with 10 mL of the

inoculum and then sparged with filtered oxygen-free nitro-

gen gas to maintain strict anaerobic conditions. Cultures

were incubated at 37°C for 96 h without agitation. Samples

were periodically withdrawn for ABE, acid, starch, pH,

and cell growth analysis.

All experiments were carried out at least twice to ensure

reproducibility.

2.4. Isoflavone extraction

KFR samples for isoflavone extraction were obtained

directly after the fermentation of kudzu to ABE described

in Sections 2.3. The residual solids, obtained by drying the

fermented culture in a 60°C oven for 3 ~ 4 day to constant

weight, were referred to as KFR. Prior to isoflavone ex-

traction, KFR and kudzu were ground using an electrical

mill (XJZF-1A-type plant grinder by Yinhe Instrument

Factory, JiangYan, China). Both the ground material and

un-ground material (particle size < 1 mm) were used for

isoflavone extraction.

The isoflavone was extracted according to previously

described procedures [7,17]. Briefly, 4.0 g of sample and

200 mL of 70% ethanol were added into a 500-mL tri-neck

Table 1. Composition of kudzu root

Component g/100 g (dry weight)

Starcha 51.2 ± 4.0

Total nitrogenb 7.2 ± 1.5

Crude fiberc 22.5 ± 5.0

Total isoflavonesd 2.8 ± 0.5

Ashf 8.2 ± 2.0

aStarch content was determined by the methods of Madihah et al. [20].bTotal nitrogen was determined by the Kjeldahl method.cCrude fiber was determinded by the method of GB/T 5009.10-2003(Deterimination of crude fiber in vegetable foods).

dTotal isoflavones were determined by Xu and Hu methods [7,17] .fAsh was determined by the method of GB 5009.4-85 (Determinationof ash in foods).

Acetone-butanol-ethanol Fermentation and Isoflavone Extraction Using Kudzu Roots 741

flask partially immersed in an 80°C water bath. Reflux

extraction was performed for 90 min, during which 0.5 mL

of the ethanol-extract was periodically obtained for analysis.

Prior to obtaining extracts, the flask was shaken gently for

2 min.

2.5. Analytical procedures

2.5.1. Determination of kudzu starch content and utili-

zation

The starch content of the sample was determined using a

modified method of Holm et al. [18]. A mixture containing

1.0 g of the sample and 0.05 g α-amylase with an activity

of 2 × 104 IU/g (supplied by Beijing Donghuaqiangsheng,

Biotechnology Co. Ltd., Beijing, China) was suspended in

25 mL distilled water added a 50-mL flask. The flask was

incubated at 60°C for 8 h and shaken for 2 min every 2 h.

The suspension was filtered, and then transferred to a 50-

mL volumetric flask filled with water to the mark. The

glucose concentration of this suspension was determined

by the phenol-sulphuric acid method of Dubois et al. [19].

The starch content of the sample was calculated from Eq.

(1):

Starch (%) = (1)

where a is the dilution factor and b is the correction factor

for glucose to starch.

Starch utilization was calculated from Eq. (2):

Starch utilization (%) = (2)

where mr is the final dry weight of KFR (g), cr is the final

starch content of KFR (g/g), mi is the initial dry weight of

kudzu (g), and ci is the initial starch content of kudzu (g/g).

2.5.2. Determination of dry cell weight

The cell concentration was determined using a modified

method of Madihah et al. [20]. A portion of 0.2 mL α-

amylase with an activity of 200 IU/mL (supplied by Beij-

ing Donghuaqiangsheng, Biotechnology Co. Ltd., Beijing,

China) was added to 4 mL kudzu fermentation medium

and then incubated at 60°C for 2 h to hydrolyze the starch

in the medium to soluble sugars. Samples were centrifuged

at 3,000 rev/min for 10 min and the supernatant was

decanted. The remaining solid was re-suspended in 4 mL

distilled water and re-centrifuged. After discharging the

supernatant, the cells were dried at 105°C for 4 h to deter-

mine the dry cell weight.

2.5.3. Determination of ABE and organic acids

ABE and acid (acetic and butyric) were measured using a

gas chromatograph (4890D, Agilent, USA) equipped with

a flame ionization detector (FID) and a 30 m × 0.25 mm

capillary column (Innowax, 19095N-123, Agilent Techno-

logies, Beijing, China). The oven temperature was main-

tained at 85°C for 4.5 min, programmed at increments of

20°C per min to 170°C. The final temperature was held for

2.5 min. The injector and detector temperatures were set at

250°C. Nitrogen was used as the carrier gas and isobutanol

was used as the internal standard. The productivity was

calculated as the total solvent concentration achieved at the

end of fermentation divided by the fermentation time and

is expressed as g/(L/h). The yield was calculated as the

total amount of solvents produced divided by the amount

of kudzu starch utilized and is expressed as g/g.

2.5.4. Determination of isoflavones yield

Since several isoflavones exist in kudzu root, the term

“total isoflavones” was adopted to describe the characteri-

stics of isoflavones. Unless stated otherwise, the use of the

word isoflavone refers to total isoflavones in this paper.

The extracted isoflavones were analyzed at 250 nm using

an Ultraviolet spectrophotometer (UVmini-1240, Shimadzu,

Japan). Puerarin (supplied by the National Institute for the

Control of Pharmaceutical and Biological Products, Beijing,

China), a main isoflavone compound in kudzu, was used as

the standard to determine the isoflavone concentration. The

standard curve of absorbance versus puerarin concentration

was obtained according to the method of Hu et al. [17].

Isoflavone yield was calculated as the mass of the extracted

isoflavones divided by the mass of the original dry sample

and is expressed as g/100 g.

2.5.5. Isoflavone extraction kinetics model

The mechanism of isoflavone extraction is described using

the exponential Eq. (3) [21,22]. Based on Fick’s law:

(3)

where Yt is the isoflavone yield in the ethanol-extract at

time t (% dry weight), Y∞ is the maximum isoflavone yield

when the time approaches infinity (% dry weight), and K

is the specific rate of the isoflavone yield (/min).

All the data presented here represent the average of

triplicate experiments.

3. Results and Discussion

3.1. Evaluation of ABE fermentation using kudzu root

In order to evaluate the fermentation characteristics of

kudzu starch to ABE by C. acetobutylicum ATCC 824, an

initial trial was performed with 60 g/L kudzu in the

fermentation culture using CH3COONH4 as the nitrogen

mg of glucose 10a× 0.9

b×sample weight 1.00 g( )

------------------------------------------------------------ 100×

1m

rcr

×m

ici

×----------------–⎝ ⎠

⎛ ⎞ 100×

Yt = Y

∞1 exp Kt–( )–[ ]×

742 Biotechnology and Bioprocess Engineering 16: 739-745 (2011)

source. This medium contained approximately 30 g/L kudzu

starch due to the 51.2% starch content of raw kudzu. For

comparison, a control culture using 30 g/L glucose as

carbon source was set up, with the assumption that all the

starch in the system is converted to glucose. After 96 h of

fermentation, the kudzu fermentation medium achieved a

solvent production of 8.04 g/L (Fig. 1), in comparison with

6.60 g/L achieved by the control culture. Meanwhile, the

solvent yield and starch utilization of kudzu were 0.30 and

96% (Fig. 1), respectively. These results reveal that kudzu

starch could be comparable to glucose for ABE fermentation

by C. acetobutylicum ATCC 824.

3.2. Optimization of kudzu root to ABE production

In order to enhance ABE production, the effect of variation

in kudzu starch concentration on ABE fermentation was

first evaluated. As shown in Fig. 1, the total solvent pro-

duction (ethanol, acetone, and butanol) increased dramati-

cally when the kudzu concentration was increased from 60

to 140 g/L, whereas further increase in kudzu concentration

from 140 to 180 g/L lead to no significant differences in

total solvent. The highest total solvent production (18.25 ±

0.42 g/L) was obtained when 140 g/L kudzu was used. The

total acid production (acetic and butyric) fluctuated within

a small range of 5.2 ~ 6.6 g/L in the different kudzu

fermentation media. More than 90% of kudzu starch was

utilized when the kudzu concentration was less than 100 g/

L (corresponding to 50 g/L starch). However, the starch

utilization decreased with increased kudzu concentration,

and was 77% in the culture containing 180 g/L kudzu

(corresponding to 90 g/L starch).

It has been proved that a sugar concentration of more

than 60 g/L is undesirable for starch utilization due to

butanol inhibition [23]. Besides, mass transfer could be

limited when more than 140 g/L (corresponding to 70 g/L

starch) of kudzu was used in fermentation culture without

agitation. Taking above factors into consideration, a kudzu

concentration of 140 g/L was selected as the optimum

substrate concentration for subsequent fermentation experi-

ments.

Previous work reported that kudzu could provide a

vitamin-enriched source of starch for ethanol and yeast

fermentation [11]. The protein content of kudzu used in

this work was approximately 7.2 ± 1.5% (Table 1). In order

to examine whether or not the kudzu provides sufficient

nutritional requirements for cell growth, kudzu was used as

the sole substrate for AEB fermentation. Fig. 2 shows the

typical time course of ABE fermentation by C. acetobut-

ylicum ATCC 824 using 140 g/L kudzu. During the first 36

h of fermentation, the pH of the fermentation broth de-

creased from 6.59 to 4.68 (Fig. 2A). Subsequently, the pH

increased and remained at 5.27 until the end of fermen-

tation. Maximum concentration of cell density of 2.41 ±

0.24 g/L was reached after 24 h of fermentation (Fig. 2A).

Starch was utilized during the fermentation, with 75% of

the available starch being degraded after 96 h (Fig. 2A).

At the same time, the solvent concentration reached a

Fig. 1. Comparison of total solvent, total acids, and starchutilization in cultures with different concentration of kudzu after96 h of fermentation.

Fig. 2. Time course of ABE fermentation using 140 g/L kudzu asa sole substrate by C. acetobutylicum ATCC 824. (A) pH, cellgrowth, and starch utilization; (B) ABE and acids.

Acetone-butanol-ethanol Fermentation and Isoflavone Extraction Using Kudzu Roots 743

maximum of 17.99 ± 1.08 g/L (Fig. 2B), including 11.29

± 0.79 g/L butanol, 5.54 ± 0.20 g/L acetone, and 1.15 ±

0.09 g/L ethanol. The final concentrations of acetic acid

and butyric acid were 3.07 ± 0.14 g/L and 0.76 ± 0.09 g/

L, respectively. A solvent productivity of 0.19 g/(L/h) and

a yield of 0.33 was achieved, indicating that kudzu could

be used for ABE production as a sole substrate.

Table 2 shows the comparisons of ABE production

between kudzu and other starch-based materials [3,24-26].

Similar to corn and potatoes, kudzu was fermented into

solvent without an extra nitrogen source. Kudzu was also

comparable to cassava starch and starch-based packing

peanuts in terms of starch utilization and solvent produc-

tivity. The slightly lower yield in kudzu fermentation may

be attributed to the bacterial strain used in the experiment.

The strain used in our experiments was the common C.

acetobutlycium, rather than the hyper-solvent producing

strains such as C. saccharoperbutylacetonicum N1-4, C.

acetobutylicum EA 2018, or C. beijerinckii BA101. Noting

that kudzu root is high in isoflavones, which have many

important biological and medicinal activities and consider-

ing that KFR can be used for isoflavone production, kudzu

could be a competitive raw material for ABE production.

Isoflavones Extraction from Kudzu Fermentation Residues

In order to investigate the effect of ABE fermentation on

isoflavone extraction, the kinetics of isoflavone extraction

from kudzu and KFR were analyzed. As shown in Fig. 3,

isoflavone yields in all experiments increased significantly

during the initial 30 min and leveled off thereafter. Both

ground and un-ground KFR have higher extraction yield

than the raw kudzu. The highest isoflavone yield of 1.90/

100 g was obtained from ground KFR. This yield is 48%

higher than that produced from ground raw kudzu (1.29/

100 g). Continuous curves in Fig. 3 represent the kinetic

model obtained by fitting the experimental data to the Eq.

(3). The curves fit the data well, as shown by the high

value of the coefficients of determination (R2 > 0.99) given

in Table 3. Both the Y

and K values of KFR are higher than

that of raw kudzu. No significant difference was found in

K value (0.1018 vs. 0.1037, Table 3) between KFR and

ground KFR, suggesting that additional grinding has no

effect on extraction rate of KFR except for the increased

yield.

The higher isoflavone yield extracted from KFR could

be attributed to the increased concentration of isoflavones

after most of the kudzu starch had been consumed by C.

acetobutylicum ATCC 824 during ABE fermentation. The

higher extraction rate of KFR was likely due to cell disrup-

tion after the fermentation, allowing the ethanol-extract to

easily penetrate into the cell. This effect has also been

confirmed by several reports of an enhanced extraction yield

when smaller milled berries and Rosa mosqueta particles

were subjected to extraction procedures [27,28]. These

Table 2. Comparison of ABE production using different starch-based materials in batch fermentation

Substrate Strain Nitrogensource

Starch concentration

(g/L)

Starch utilization

Produc-tivity

(g/(L/h))

Yield(g/g)

Fermen-tation time

(h)

Confer-ence

Kudzu C. acetobutylicum ATCC 824 − 72 75% 0.19 0.33a 96 this work

Cassava starchC. saccharoperbutylacetonicum N1-4

CH3COONH4, 3 g/L 60 78.2% 0.44 0.41a 48 [24]

Corn C. acetobutylicum EA 2018 − 60 Un 0.42 Un 48 [25]

Starch-based packing peanuts

C. beijerinckii BA101CH3COONH4, 2.2 g/L; yeast extract, 1 g/L

69.6 84% 0.20 0.37a 110 [3]

Sago starchC. saccharoperbutylacetonicum N1-4

CH3COONH4, 3 g/L 60 69.1% 0.27 0.43b 72 [24]

Potatoes C. beijerinckii NRRL B592 − 42 Un 0.42 Un 45 [26]

−means no supplements.aThe yield based on starch consumed.bThe yield based on glucose consumed.Un = Unpublished.

Fig. 3. Comparison of kinetics of isoflavone extraction fromkudzu fermentation residues (KFR) and kudzu. Extraction solvent,70% ethanol; solid/liquid ratio (g/mL), 1:50; temperature, 80°C.

744 Biotechnology and Bioprocess Engineering 16: 739-745 (2011)

results indicate that the process of ABE fermentation

integrated with isoflavone extraction appears feasible for

the effective utilization of kudzu.

4. Conclusion

Kudzu was demonstrated to be a possible sole substrate for

C. acetobutylicum ATCC 824 growth and was comparable

to commercially available starch-based resource in terms of

ABE yield. The isoflavone yield extracted from KFR

increased by 48% compared with that from raw kudzu. The

process of ABE fermentation integrated with isoflavone

extraction is feasible for comprehensive kudzu utilization

and reduction of substrate cost.

Acknowledgements

This work was financially supported by the Important

National Basic Research Program of China (No.

2011CB707400) and the Knowledge Innovation Program

of the Chinese Academy of Sciences (No. 2011CB707401).

References

1. Dürre, P. (2007) Biobutanol: An attractive biofuel. Biotechnol. J.2: 1525-1534.

2. Jones, D. and D. Woods (1986) Acetone-butanol fermentationrevisited. Microbiol. Rev. 50: 484-524.

3. Jesse, T. W., T. C. Ezeji, N. Qureshi, and H. P. Blaschek (2002)Production of butanol from starch-based waste packing peanutsand agricultural waste. J. Ind. Microbiol. Biotechnol. 29: 117-123.

4. Qureshi, N., T. C. Ezeji, J. Ebener, B. S. Dien, M. A. Cotta, andH. P. Blaschek (2008) Butanol production by Clostridium beijer-inckii. Part I: Use of acid and enzyme hydrolyzed corn fiber.Bioresour. Technol. 99: 5915-5922.

5. Qureshi, N., B. C. Saha, and M. A. Cotta (2007) Butanol pro-duction from wheat straw hydrolysate using Clostridium beijer-inckii. Bioproc. Biosyst. Eng. 30: 419-427.

6. Yang, L. and T. Tan (2008) Enhancement of the isolation selec-tivity of isoflavonoid puerarin using oligo-β-cyclodextrin cou-pled polystyrene-based media. Biochem. Eng. J. 40: 189-198.

7. Xu, H. N. and C. H. He (2007) Extraction of isoflavones fromstem of Pueraria lobata (Willd.) Ohwi using n-butanol/water

two-phase solvent system and separation of daidzein. Sep. Purif.Technol. 56: 85-89.

8. Sage, R. F., H. A. Coiner, D. A. Way, G. Brett Runion, S. A. Prior,H. Allen Torbert, R. Sicher, and L. Ziska (2009) Kudzu [Puer-aria montana (Lour.) Merr. Variety lobata]: A new source of car-bohydrate for bioethanol production. Biomass Bioenerg. 33: 57-61.

9. Kwun, K. H., G. J. Kim, and H. J. Shin (2009) Ultrasonicationassistance increases the efficiency of isoflavones extraction fromkudzu (Pueraria lobata Ohwi) roots waste. Biotechnol. Bioproc.Eng. 14: 345-348.

10. Fu, X. G., H. Z. Chen, and W. D. Wang (2008) Production of eth-anol and isoflavones from steam pretreated Radix Puerariae bysolid state fermentation (Chinese). Chin. J. Biotechnol. 24: 957-961.

11. Tanner, R. D. and S. S. Hussain (1979) Kudzu (Pueraria lobata)root starch as a substrate for the lysine-enriched baker's yeast andethanol fermentation process. J. Agric. Food Chem. 27: 22-27.

12. Lee, M. H. and C. C. Lin (2007) Comparison of techniques forextraction of isoflavones from the root of Radix Puerariae: Ultra-sonic and pressurized solvent extractions. Food Chem. 105: 223-228.

13. Guo, Z., Q. Jin, G. Fan, Y. Duan, C. Qin, and M. Wen (2001)Microwave-assisted extraction of effective constituents from aChinese herbal medicine Radix Puerariae. Anal. Chim. Acta 436:41-47.

14. Xu, H., Y. Zhang, and C. He (2007) Ultrasonically assistedextraction of isoflavones from stem of Pueraria lobata (Willd.)Ohwi and its mathematical model. Chin. J. Chem. Eng. 15: 861-867.

15. Chen, H. Z. and L. Wang (2009) Method of direct fermentation ofRadix Puerariae to acetone-butanol-ethanol combined withisoflavones extraction. China Patent 200910090098.3.

16. George, H. A., J. L. Johnson, W. E. C. Moore, L. V. Holdeman,and J. S. Chen (1983) Acetone, isopropanol, and butanol produc-tion by Clostridium beijerinckii (syn. Clostridium butylicum) andClostridium aurantibutyricum. Appl. Environ. Microbiol. 45:1160-1163.

17. Hu, Y., T. Wang, S. F. Han, P. Y. Wan, and M. H. Fan (2008)Extraction of isoflavonoids from Pueraria by combining ultra-sound with microwave vacuum. Chem. Eng. Proc. 47: 2256-2261.

18. Holm, J., I. Björck, A. Drews, and N. G. Asp (1986) A rapidmethod for the analysis of starch. Starch/Stärke 38: 224-226.

19. Dubois, M., K. A. Gilles, J. K. Hamilton, P. A. Rebers, and F.Smith (1956) Colorimetric method for determination of sugarsand related substances. Anal. Chem. 28: 350-356.

20. Madihah, M. S., A. B. Ariff, K. M. Sahaid, A. A. Suraini, and M.I. A. Karim (2001) Direct fermentation of gelatinized sago starchto acetone-butanol-ethanol by Clostridium acetobutylicum.World J. Microbiol. Biotechnol. 17: 567-576.

21. Wardhani, D. H., J. A. Vázquez, and S. S. Pandiella (2010) Opti-misation of antioxidants extraction from soybeans fermented byAspergillus oryzae. Food Chem. 118: 731-739.

22. Xu, H. N., W. N. Huang, and C. H. He (2008) Modeling forextraction of isoflavones from stem of Pueraria lobata (Willd.)Ohwi using n-butanol/water two-phase solvent system. Sep.Purif. Technol. 62: 590-595.

23. Ezeji, T. C., N. Qureshi, and H. P. Blaschek (2004) Butanol fer-mentation research: upstream and downstream manipulations.Chem. Rec. 4: 305-314.

24. Thang, V. H., K. Kanda, and G. Kobayashi (2010) Production ofAcetone-Butanol-Ethanol (ABE) in direct fermentation of cas-sava by Clostridium saccharoperbutylacetonicum N1-4(pub-lished online). Appl. Biochem. Biotechnol. 161: 157-170.

25. Gu, Y., S. Y. Hu, J. Chen, L. J. Shao, H. Q. He, Y. L. Yang, S.

Table 3. Values resulting from kinetic data of isoflavone extractionto Eq. (3): Yt = Y

∞ × [1−exp(−Kt)]

Sample Y∞

K R2

KFR 1.5137 0.1018 0.994

Kudzu 0.9965 0.0699 0.992

Gound KFR 1.9066 0.1037 0.992

Gound kudzu 1.2885 0.1070 0.996

Acetone-butanol-ethanol Fermentation and Isoflavone Extraction Using Kudzu Roots 745

Yang, and W. H. Jiang (2009) Ammonium acetate enhances sol-vent production by Clostridium acetobutylicum EA 2018 usingcassava as a fermentation medium. J. Ind. Microbiol. Biotechnol.36: 1225-1232.

26. Nimcevic, D., M. Schuster, and J. R. Gapes (1998) Solvent pro-duction by Clostridium beijerinckii NRRL B592 growing on dif-ferent potato media. Appl. Microbiol. Biotechnol. 50: 426-428.

27. Cacace, J. E. and G. Mazza (2003) Mass transfer process duringextraction of phenolic compounds from milled berries. J. FoodEng. 59: 379-389.

28. Franco, D., M. Pinelo, J. Sineiro, and M. J. Núñez (2007) Pro-cessing of Rosa rubiginosa: Extraction of oil and antioxidantsubstances. Bioresour. Technol. 98: 3506-3512.