Strain development and medium optimization for fumaric acid production

Biotechnology and Bioprocess Engineering 15: 761-769 (2010)

DOI 10.1007/s12257-010-0081-4

Strain Development and Medium Optimization for Fumaric Acid

Production

Seong Woo Kang, Hawon Lee, Daeheum Kim, Dohoon Lee, Sangyong Kim, Gie-Taek Chun, Jinwon Lee,

Seung Wook Kim, and Chulhwan Park

Received: 11 March 2010 / Accepted: 30 March 2010

© The Korean Society for Biotechnology and Bioengineering and Springer 2010

Abstract Rhizopus oryzae RUR709 mutant was isolated

based on halo size from selection medium via mutagenesis

with UV and γ-rays, and the production of fumaric acid in

the submerged fermentation was assessed. The maximum

concentration of fumaric acid was obtained using 0.5%

corn steep liquor (CSL) as the nitrogen source. Organic

nitrogen sources were shown to be more effective in fumaric

acid production than inorganic nitrogen sources. Using

optimum medium obtained by response surface methodology

(RSM), the maximum concentration of fumaric acid

achieved in flask culture was 26.2 g/L, which is fairly close

to the 27.4 g/L predicted by the model. The highest

concentration of fumaric acid in the stirred-tank reactor

generated by the R. oryzae RUR709 mutant was 32.1 g/L

and yield (0.45 g/g) and productivity (0.32 g/L/h) were

highest at 4 days.

Keywords: Rhizopus oryzae, fumaric acid, response surface

methodology (RSM), mutagenesis, optimization

1. Introduction

Fumaric acid is applicable to a broad range of procedures.

Due to its structure (a carbon-carbon double bond and two

carboxylic groups), fumaric acid is considered as an effec-

tive intermediate for chemical synthesis reactions, includ-

ing esterification and polymerization [1]. Since fumaric

acid is non-toxic, it is also utilized as an acidulant in fruit

drinks, other beverages, and certain pharmaceutical prepa-

rations [2]. Fumaric acid is currently manufactured via the

isomerization of maleic acid (or maleic anhydride), which

is generated by the catalytic oxidation of benzene [3]. As

benzene is a well-known carcinogen, there is a obvious

need for an alternative method of fumaric acid production.

Fungi are natural producers of a variety of valuable

chemicals. Although most academic and industrial entities

focus primarily on their capacity to generate secondary

metabolites, they also have the potential to be mass pro-

ducers of commodity chemicals, including fumaric acid

[4,5]. Fumaric acid-producing genera identified thus far

include the Rhizopus, Mucor, Cunninghamella, and Circinella

species. Among these strains, Rhizopus species (nigricans,

arrhizus, oryzae, and formosa) are considered the best

microorganisms for fumaric acid production [6-9]. Although

considerable effort has been made to improve fumaric acid

production via bioprocess optimization and immobilization

in reactors [10-14], no fungal strain has been until now

developed for the purpose of fumaric acid production.

Analysis of variance (ANOVA) and response surface

methodology (RSM) have been successfully used to evaluate

Seong Woo Kang, Seung Wook Kim*

Department of Chemical and Biological Engineering, Korea University,Seoul 136-701, KoreaTel: +82-2-3290-3300; Fax: +82-2-926-6102E-mail: [email protected]

Hawon Lee, Daeheum Kim, Chulhwan Park*

Department of Chemical Engineering, Kwangwoon University, Seoul139-701, KoreaTel: +82-2-940-5173; Fax: +82-2-912-5173E-mail: [email protected]

Dohoon Lee, Sangyong KimGreen Process R&D Department, Korea Institute of Industrial Technology(KITECH), Chonan 330-825, Korea

Gie-Taek ChunSchool of Bioscience and Biotechnology, Kangwon National University,Chuncheon 200-701, Korea

Jinwon LeeDepartment of Chemical and Biomolecular Engineering, Sogang University,Seoul 121-742, Korea

RESEARCH PAPER

762 Biotechnology and Bioprocess Engineering 15: 761-769 (2010)

the relationship between a set of controllable experimental

factors and the observed results from medium and bio-

process optimization [12,15-18]. These statistical methods

have been proven as powerful, useful tools. We employed

a central composite design (CCD) in order to determine the

optimal concentrations of medium components in the pro-

duction medium.

In this study, we developed a strain, via mutagenesis, for

the high-level production of fumaric acid. The production

of fumaric acid by isolated mutant was evaluated at differ-

ent C/N ratios, inoculum sizes, and with different nitrogen

sources, and the production medium was also optimized

via RSM.

2. Materials and Methods

2.1. Microorganisms

Rhizopus oryzae KCTC 6946 was provided by the Korean

Collection for Type Cultures and the R. oryzae RUR709

mutant was selected via mutagenesis. Strains were culti-

vated for 7 days at 32oC in 250 mL Erlenmeyer flasks

containing YMS media (1% glucose, 0.3% yeast extract,

0.3% malt extract, 0.5% bacto-peptone, 0.5% NaCl, 0.2%

MgSO4·7H2O, and 1.7% agar, pH 7.0).

2.2. Mutagenesis and mutant selection

For the spore solution, 30 mL of 0.2% Tween 80 solution

was added to 250 mL Erlenmeyer flasks containing YMS

media. After shaking, the spore suspensions were trans-

ferred to a beaker and were stirred for 30 min, followed by

filtration with a syringe packed with glass wool. For UV

mutagenesis, the spore solutions (0.1 mL) were spread onto

plates (15 mm × 100 mm) containing selection media (1%

glucose, 0.3% yeast extract, 0.3% malt extract, 0.5% bacto-

peptone, 0.1% Triton X-100, 0.06% bromocresol purple,

and 1.7% agar). These plates were exposed to UV light at

306 nm, 0.3 W/m2 and were then incubated for 3 days at

32oC. For γ-ray mutagenesis, the spore solutions (5 mL)

were irradiated with γ-rays at a dosage of 1 ~ 10 KGy. The

treated spores were appropriately diluted and spread onto

plates containing selection media, then incubated for 3

days at 32oC. UV (5 min) and γ-ray (6 kGy) treatments

resulted in reduced cell viabilities in excess of 99.9%. The

mutants were selected based on the ratio of colony to halo

diameter and were tested for fumaric acid production in

basal medium via shake-flask culturing.

2.3. Production of fumaric acid in flask and STR

Two percent malt extract solution was inoculated with

spore solution (20 mL, 1 × 107 spore/mL) obtained by the

above methods, and inoculated seed media (100 mL) was

cultivated for 12 h at 32oC in a shaking incubator at 200

rpm. Seed cultures (2%) transferred into 250 mL Erlen-

meyer flasks containing 100 mL of basal medium and were

incubated at 35oC and 250 rpm. The basal medium con-

sisted of 10% glucose, 0.5% corn steep liquor (CSL),

0.1% KH2PO4, 0.05% MgSO4·7H2O, 0.002% ZnSO4·7H2O,

2.0% CaCO3, and an adjusted pH of 6.5. Glucose was

added separately to the basal medium. To determine the

concentration of CSL, the glucose concentration was fixed

at 10% and the CSL concentration was varied between 0.1

and 2.0%. In order to evaluate the effects of inoculum size

on fumaric acid production, 2 ~ 10% seed cultures were

inoculated into the medium. Additionally, various nitrogen

sources were supplemented into the basal medium in an

effort to estimate their effect on fumaric acid production. In

order to optimize the production medium via statistical

methods, the individual test media were prepared in accor-

dance with the experimental design. For the stirred tank

reactor (STR), seed culture (4%) was transferred into a 5 L

STR containing 3 L of optimized medium. The operating

conditions were 35oC, 400 rpm, and 1.0 vvm.

2.4. Experimental design and statistical analysis

Response surface methodology (RSM) consists of a set of

experimental techniques designed to evaluate the relation-

ships between a cluster of controlled experimental vari-

ables and measured responses (y), according to one or

more selected criteria [19]. The central composite design

(CCD) is the most extensively utilized experimental design

for determining maximum or minimum response points.

CCD consists of a factorial portion (± 1 levels) that is

increased via the addition of axial or star points [α = (2n)1/4]

as well as several center points (0). The variables were

assigned in accordance with the following equation (1):

xi = (Xi − X0)/ΔXi i = 1, 2, 3 …, j (1)

where xi is the coded value of an independent variable, Xi

is the real value of an independent variable, X0 is the real

value of an independent variable at the center point, and

ΔXi is the step change value. The behavior of the system

was explained by the following second-order polynomial

equation (2):

y = β0 + Σβixi + Σβiixi2 + Σβijxixj (2)

where y is the predicted response, β0 (offset term), βi

(linear effect), βii (squared effect), and βij (interaction

effect) are constant coefficients, and x represents the coded

level of the independent variable. The SAS 9.1 package

program was employed for regression analysis of the

obtained experimental data, and also to estimate the coeffi-

cients of the regression equation. Maximum fumaric acid

production value was the response of the design experi-

Strain Development and Medium Optimization for Fumaric Acid Production 763

ments.

2.5. Analytical methods

In order to estimate dry cell weight (DCW), the culture

broth was diluted via the addition of distilled water and

1.0 N HCl to neutralize excessive CaCO3, followed by 6 h

of heating at 80oC. After cooling, pretreated culture broth

was filtered using a GF/C filter (47 mm Ø, Whatman)

and dried overnight at 80oC. The filtrates were acquired

for the analysis of glucose, fumaric acid, and ethanol using

an HPLC system equipped with a pump (HP 1100), a

refractive index detector (HP 1047), and an automatic

injector (HP 1050). The mobile phase was 0.006 N H2SO4,

and HPLC was conducted using an Aminex HPX-87H

column (300 × 7.8 mm; BioRad) at 45oC at a flow rate of

0.6 mL/min.

3. Results and Discussion

3.1. Selection of mutants for high-level fumaric acid

production

There have been no reports made concerning the develop-

ment of a Rhizopus strain via mutagenesis for the produc-

tion of fumaric acid. First, selection medium based on

color change was developed to detect mutants producing

abundant quantities of fumaric acid. Triton X-100 and

bromocresol purple in the selection medium were used to

restrict the growth of the mutant substrate mycelium, and

the medium changed from dark purple to yellow according

to the amount of acid produced by the mutants. In order to

select the proper R. oryzae mutant for abundant fumaric

acid production, the spore solution of wild type R. oryzae

KCTC 6946 was treated with UV and γ-rays followed by

spreading on selection medium plates. About 50 isolated

mutants had a lower than 0.2 ratio according to the

methods described above. The production of fumaric acid

was then evaluated in the submerged fermentation. The

concentrations of fumaric acid produced by 6 mutants were

measured in the range of 19.3 ~ 22.9 g/L at 4 days of

cultivation (Table 1). Among the mutants tested, the highest

fumaric acid concentration was produced by the R. oryzae

RUR709 mutant. Therefore, the R. oryzae RUR709 mutant

was ultimately selected for further study. The production of

fumaric acid by wild type R. oryzae KCTC 6946 and R.

oryzae RUR709 mutant was compared in basal medium

(Fig. 1). Maximum fumaric acid production (17 g/L in

KCTC 6946 and 21.4 g/L in RUR709) was noted at 4 days

of cultivation. During cultivation, ethanol was generated

and maintained at concentrations between 21 and 26 g/L

following 60 h of cultivation. The pH and DCW profiles of

R. oryzae RUR709 were similar to those of R. oryzae

KCTC 6946. The level of fumaric acid produced by R.

oryzae RUR709 was approximately 26% higher than that

of R. oryzae KCTC 6946, and the fumaric acid yield

(Yp/s) and productivity were 0.32 g/g and 0.22 g/L/h,

respectively.

Table 1. Fumaric acid production by isolated R. oryzae mutants in basal medium

Mutants

3 day 4 day

FinalpH

Used glucose(g/L)

Fumaric acid(g/L)

Ethanol(g/L)

FinalpH

Used glucose(g/L)

Fumaric acid(g/L)

Ethanol(g/L)

RUR501 4.39 ± 0.01 58.1 ± 0.8 17.4 ± 0.7 24.8 ± 0.1 4.13 ± 0.01 67.3 ± 0.3 19.5 ± 0.5 23.8 ± 0.4

RUR509 4.19 ± 0.05 73.8 ± 2.3 18.1 ± 1.8 30.7 ± 1.5 4.01 ± 0.03 82.1 ± 1.3 19.3 ± 0.1 30.0 ± 1.5

RUR516 4.34 ± 0.01 67.8 ± 0.1 18.5 ± 0.3 28.7 ± 0.1 4.15 ± 0.01 75.6 ± 1.4 20.1 ± 0.5 28.1 ± 0.3

RUR709 4.20 ± 0.01 67.7 ± 1.0 21.1 ± 0.1 23.6 ± 0.8 4.04 ± 0.01 76.1 ± 0.5 22.9 ± 0.1 27.1 ± 0.2

RUR710 4.31 ± 0.04 68.4 ± 1.3 19.4 ± 1.0 26.8 ± 0.7 4.11 ± 0.01 76.7 ± 0.4 20.5 ± 0.4 27.6 ± 2.3

RUR711 4.33 ± 0.01 64.4 ± 1.1 18.3 ± 0.1 25.3 ± 0.1 4.08 ± 0.01 74.1 ± 2.2 20.8 ± 0.6 26.1 ± 0.1

*Cultures were carried out at 35oC and 250 rpm.

Fig. 1. Cultivation of R. oryzae KCTC 6946 (closed symbol) andR. oryzae RUR709 mutant (open symbol) in basal medium.Cultures were carried out at 35oC and 250 rpm.


3.2. Effect of CSL concentration and inoculum size on

fumaric acid production

The carbon to nitrogen ratio (C/N ratio) is one of the most

important factors affecting the growth and metabolite

production of microorganisms. Some researchers have

reported that the most critical parameter in fumaric acid

production is the C/N ratio, which has been found in the

range of 120:1 ~ 200:1. In other word, high C/N ratios are

useful obtaining high yields of fumaric acid along with

control of cell growth [2,8,12,20]. The concentration of

glucose remained constant at 10% (w/v). To indirectly

evaluate C/N ratio, the effect of CSL concentration (0.1 ~

2.0%, v/v) on fumaric acid production was investigated

in basal medium (Fig. 2). The level of fumaric acid

production was enhanced until the CSL concentration

reached 0.5%, at this point it started to decline as the

amount of CSL was increased. Increases in CSL concent-

ration greatly increased the glucose consumption as well as

the levels of DCW and ethanol production. Cell growth was

not observed at 0.1% CSL, but ethanol production was

increased substantially upon increased nitrogen concent-

ration. The maximum concentration of fumaric acid was

achieved at 0.5% CSL. These results demonstrate that the

nitrogen source concentration was crucial for controlling

the dynamic between fumaric acid production and cell

growth. This finding is generally consistent with the

results reported by Zhou et al. [9] and Riscaldati et al.

[21] who cultured a fungal growth phase and acid pro-

duction phase in accordance with the amount of nitrogen

source. Further, regarding the effects of inoculum size on

fumaric acid production in basal medium, the most

remarkable feature was that fumaric acid production (22.2

~ 22.7 g/L) reached similar levels for all inoculum sizes,

except for 2%. Therefore, a 4% inoculum was employed

for further study.

3.3. Effect of nitrogen source on fumaric acid produc-

tion

The effects of various nitrogen sources on the production

of fumaric acid were assessed in this study. First, when

nitrogen sources (0.5%, w/v) were added to the medium,

the concentrations of fumaric acid (4.7 ~ 9.6 g/L) were

measured using polypeptone, polypeptone-S, beef extract,

NZ-amine A, and tryptone. Fumaric acid was not detected

in any other cases (data not shown). In the presence of high

concentrations of organic nitrogen, cell growth was high

and the level of fumaric acid was low, similar to that of a

high CSL concentration. Therefore, various nitrogen sources

(0.1%, w/v) were added to basal medium containing 10%

(w/v) glucose. Organic nitrogen sources were more effec-

tive in fumaric acid production than inorganic nitrogen

sources. When organic nitrogen sources (with the exception

of beef extract) were utilized, the fumaric acid concent-

rations were detected in a range of 18.2 ~ 25.3 g/L, with

NZ-amine A showing the highest level of fumaric acid

production. For beef extract, although the protein content

(over 80%) was greater than other organic nitrogen sources,

glucose consumption was low due to the presence of

unusable high molecular weight proteins. These results

indicate that enzymatic hydrolysates of casein, such as NZ-

amine A and tryptone, are the most appropriate organic

nitrogen sources (Table 2). When inorganic nitrogen sources

were employed, the level of fumaric acid production was

very low. In particular, the highest concentration of ethanol,

47.2 g/L, was produced when using urea. Some researchers

have also utilized ammonium sulfate to produce fumaric

acid [12,13,20]. Although ammonium sulfate is the most

frequently used nitrogen source for the production of

fumaric acid, ammonium sulfate in this study yielded a low

concentration of fumaric acid (5.7 g/L). These findings

indicate that the proper selection of a nitrogen source is

essential for effective fumaric acid production. Carta et al.

[8] reported that KNO3 was an appropriate nitrogen source

for the production of fumaric acid by R. formosa MUCL

28422. In this study, the growth of R. oryzae RUR709

proved unsatisfactory, and no fumaric acid was detected

when KNO3 was used alone as a nitrogen source. On the

other hand, the production of fumaric acid was stable and

increased by approximately 10% when basal medium

containing 0.5% CSL was supplemented with KNO3 (data

not shown). Based on this result, we suggest that KNO3

may have a physiological effect when combined with

another organic nitrogen source, although our strain did not

assimilate KNO3 when it was used as the sole nitrogen

source.

Fig. 2. Effect of CSL concentration on fumaric acid production byR. oryzae RUR709 mutant in basal medium. Cultures were carriedout at 35oC and 250 rpm for 4 days.


3.4. Optimization of production medium through RSM

RSM was conducted to determine the optimal concent-

rations of medium components affecting fumaric acid pro-

duction. These components were selected based on pre-

liminary experiments in shake-flask cultures. The indepen-

dent variables and their levels are provided and the experi-

ment was conducted using four independent variables,

glucose (X1), NZ-amine A (X2), KNO3 (X3), and CaCO3

(X4), using a 24 full factorial design experiment with eight

star points (α = ± 2) and four replicates at the center point

(Table 3). Regression analysis was conducted to fit the

response function with the experimental data, and the results

are provided in Table 4. The value of the determination

coefficient (R2 = 88.3), a measure of the goodness of fit of

the model, indicates that 88.3% of the variability in the

response could be explained by the model. The coefficient

of variation (CV) indicates the degree of precision with

which the treatments are compared. Generally, a higher CV

value indicates that the reliability of the experiment is low.

According to this result, a lower CV value (16.83%) is

reflective of highly reliable experimental factors. Addition-

ally, the F-value and P-value were 7.03 and 0.0006, respec-

tively. The tested model is statistically significant at a

significance level of 1%. This indicates that the response

equation provided a suitable model for the response surface

of the fumaric acid production experiment. The response

equation obtained via multiple regression analysis is as

follows:

y = 24.85 + 1.479x1 + 2.429x2 + 0.288x3 − 3.554x4

− 0.893x1x1 − 4.105x2x2 − 0.48x3x3 − 1.805 x4x4

+ 0.569x1x2 + 0.256x1x3 + 0.019x1x4 − 0.006x2x3

+ 1.256x2x4 + 0.244x3x4 (3)

where x1 = coded value of glucose, x2 = coded value of

NZ-amine A, x3 = coded value of KNO3, x4 = coded value

of CaCO3.

Response surface plots provide a method by which

responses for different test values of variables can be

predicted, and the contours of the plots help to identify the

type of interactions between test variables. Two-dimen-

sional (2D) contour plots represented an infinite number of

combinations of the two independent variables, with the

other variables maintained at their zero levels. The effect of

each variable can be observed by analysis of the 2D-con-

tour plots. In Fig. 3, most of the contour plots are elliptical.

Therefore, according to our analysis of 2D-contour plots,

NZ-amine A and CaCO3 showed more effective compari-

son to other factors when considering the production of

fumaric acid. In particular, alteration in the NZ-amine A

concentration resulted in a rapid change in the level of

fumaric acid production (Figs. 3A, 3D, and 3E). However,

for all other components, more gradual changes were

observed (Figs. 3B and 3C). Thus, NZ-amine A was

identified as the factor most affecting the production of

fumaric acid. The optimum points of each variable that

yield maximal production of fumaric acid are 7.47%

glucose (x1 = 0.469), 0.105% NZ-amine A (x2 = 0.113),

0.107% KNO3 (x3 = 0.163), and 2.56% CaCO3 (x4 =

−0.440), respectively, and the maximum value of fumaric

Table 2. Effect of nitrogen source on the production of fumaric acid by R. oryzae RUR709 mutant in basal medium

Nitrogen source(0.1%, w/v)

Final pHUsed glucose

(g/L)Fumaric acid

(g/L)Yp/s

Ethanol(g/L)

Control 4.21 ± 0.01 68.4 ± 0.4 22.3 ± 0.2 0.326 ± 0.001 21.3 ± 0.5

Bacto-peptone 4.57 ± 0.04 56.9 ± 0.8 20.4 ± 0.4 0.359 ± 0.002 20.5 ± 1.8

Polypeptone 4.03 ± 0.02 78.1 ± 0.5 22.1 ± 0.1 0.283 ± 0.001 27.6 ± 0.3

Polypeptone-S 4.30 ± 0.02 54.3 ± 0.3 22.8 ± 0.5 0.420 ± 0.001 16.2 ± 0.3

Proteose peptone 4.64 ± 0.03 73.9 ± 1.7 18.2 ± 0.6 0.247 ± 0.002 27.7 ± 0.7

Beef extract 5.74 ± 0.05 26.2 ± 0.8 7.4 ± 0.3 0.283 ± 0.002 7.9 ± 0.3

Yeast extract 4.42 ± 0.02 68.3 ± 0.3 20.4 ± 0.4 0.299 ± 0.004 22.8 ± 0.2

NZ-amine A 4.13 ± 0.01 71.7 ± 0.3 25.3 ± 0.1 0.353 ± 0.000 24.0 ± 0.4

Tryptone 4.10 ± 0.01 77.8 ± 0.6 24.1 ± 0.3 0.310 ± 0.001 26.4 ± 0.3

Casamino acid 4.64 ± 0.05 69.1 ± 1.1 18.3 ± 0.7 0.265 ± 0.006 25.4 ± 1.2

Urea 4.80 ± 0.04 96.4 ± 0.6 7.6 ± 0.2 0.079 ± 0.001 47.2 ± 0.5

(NH4) 2SO4 5.46 ± 0.02 87.6 ± 1.4 5.7 ± 0.5 0.065 ± 0.005 38.4 ± 0.8

NH4H2PO4 5.17 ± 0.01 75.2 ± 0.4 12.6 ± 0.1 0.168 ± 0.000 31.3 ± 1.0

NH4NO3 5.09 ± 0.02 82.3 ± 1.3 9.8 ± 0.6 0.119 ± 0.005 35.4 ± 0.6

NaNO3 7.72 ± 0.01 0 0 0 0

KNO3 7.71 ± 0.01 0 0 0 0

*The concentration of nitrogen source was 0.1% (w/v) and that of control was 0.5% CSL (v/v). Cultures were carried out at 35oC and 250 rpm for4 days.


acid predicted from the model is 27.4 g/L.

3.5. Production of fumaric acid using optimized medium

in flask and STR

To verify the optimal conditions, experimental rechecking

was conducted under the optimal conditions as detailed

above. Rhizopus species tend to grow on the walls, baffles,

and stirrer of the reactor. Therefore, fermentation can suffer

from oxygen limitation due to the formation of clumps. To

solve this, enhanced fumaric acid production was reported

as the agitation speed and dissolved oxygen level were

increased [10,22]. These results are based on the increased

mass transfer of oxygen by high agitation speed, resulting

in a sufficient supply of dissolved oxygen as an electron

acceptor into the TCA cycle. In this study, fumaric acid

production in a STR was carried out at operating condi-

tions of 400 rpm and 1.0 vvm. Fig. 4 shows the time course

profiles of fumaric acid production by the R. oryzae

RUR709 mutant using optimized medium in the flask and

STR. The high correlation between the experimental and

statistical results confirms the validity of the response model

as well as the existence of an optimal point. The fumaric

Table 3. Real and coded values of the factors and experimental design and results for response surface methodology (RSM)

RunGlucose(x1, g/L)

NZ-amine A(x2, g/L)

KNO3

(x3, g/L)CaCO3

(x4, g/L)

Result

Final pH

Used glucose(g/L)

Fumaric acid(g/L)

Ethanol(g/L)

1 −1 (60) −1 (0.6) −1 (0.6) −1 (20) 4.14 46.4 22.9 11.7

2 +1 (80) −1 (0.6) −1 (0.6) −1 (20) 4.22 46.3 23.3 10.8

3 −1 (60) +1 (1.4) −1 (0.6) −1 (20) 4.37 59.4 20.7 17.2

4 +1 (80) +1 (1.4) −1 (0.6) −1 (20) 4.04 77.3 25.9 23.9

5 −1 (60) −1 (0.6) +1 (1.4) −1 (20) 4.60 40.2 20.6 8.9

6 +1 (80) −1 (0.6) +1 (1.4) −1 (20) 4.27 47.1 22.1 11.4

7 −1 (60) +1 (1.4) +1 (1.4) −1 (20) 4.49 59.5 20.3 17.0

8 +1 (80) +1 (1.4) +1 (1.4) −1 (20) 4.05 77.4 26.0 22.7

9 −1 (60) −1 (0.6) −1 (0.6) +1 (40) 5.58 24.8 8.0 5.5

10 +1 (80) −1 (0.6) −1 (0.6) +1 (40) 5.49 32.0 11.5 7.6

11 −1 (60) +1 (1.4) −1 (0.6) +1 (40) 5.28 59.5 15.7 16.7

12 +1 (80) +1 (1.4) −1 (0.6) +1 (40) 5.10 71.5 17.5 23.0

13 −1 (60) −1 (0.6) +1 (1.4) +1 (40) 5.59 29.6 9.1 7.0

14 +1 (80) −1 (0.6) +1 (1.4) +1 (40) 5.44 35.0 12.1 7.8

15 −1 (60) +1 (1.4) +1 (1.4) +1 (40) 5.48 59.5 13.4 17.5

16 +1 (80) +1 (1.4) +1 (1.4) +1 (40) 5.14 72.7 18.2 23.3

17 −2 (50) 0 (1.0) 0 (1.0) 0 (30) 5.17 49.5 18.1 11.0

18 +2 (80) 0 (1.0) 0 (1.0) 0 (30) 4.77 68.5 22.9 16.9

19 0 (70) −2 (0.2) 0 (1.0) 0 (30) 6.53 3.5 0.1 0

20 0 (70) +2 (1.8) 0 (1.0) 0 (30) 4.84 69.1 15.2 21.6

21 0 (70) 0 (1.0) −2 (0.2) 0 (30) 4.86 61.9 19.5 15.5

22 0 (70) 0 (1.0) +2 (1.8) 0 (30) 5.11 65.7 24.8 16.1

23 0 (70) 0 (1.0) 0 (1.0) −2 (10) 3.48 66.3 19.1 20.3

24 0 (70) 0 (1.0) 0 (1.0) +2 (50) 5.40 57.5 14.6 13.9

25 0 (70) 0 (1.0) 0 (1.0) 0 (30) 4.80 64.0 25.3 17.1

26 0 (70) 0 (1.0) 0 (1.0) 0 (30) 4.67 65.8 23.9 15.5

27 0 (70) 0 (1.0) 0 (1.0) 0 (30) 4.74 64.7 23.8 15.6

28 0 (70) 0 (1.0) 0 (1.0) 0 (30) 4.61 66.5 26.4 15.8

Table 4. Analysis of variance (ANOVA) for the production of fumaric acid by R. oryzae RUR709 mutant

Source Sum of squares Degrees of freedom Mean square F-value P > F

Model 965.668 14 68.976 7.03 0.0006

Error 127.511 13 9.809

Corrected total 1093.179 27

Coefficient of variation (CV) =16.83%, coefficient of determination (R2) = 0.883.


acid concentration was increased for 5 days until glucose

was exhausted. The concentrations of fumaric acid obtained

at 4 days of cultivation in the flask and STR were 26.2 and

30.2 g/L, respectively. In the flask culture, R. oryzae

RUR709 produced fumaric acid at a concentration of 26.9

g/L at 5 days, which is 58% higher than that of R. oryzae

Fig. 3. Contour plots showing the effect of medium components on fumaric acid production. A, glucose and NZ-amine A; B, glucose andKNO3; C, glucose and CaCO3; D, NZ-amine A and KNO3; E, NZ-amine A and CaCO3; F, KNO3 and CaCO3.


KCTC 6946. For the STR, the highest concentration of

fumaric acid (32.1 g/L at 5 days) obtained using R. oryzae

RUR709 mutant was approximately 89% higher than that

of R. oryzae KCTC 6946. Additionally, the yield and pro-

ductivity obtained at 4 days of cultivation in the STR were

approximately 0.45 g/g and 0.32 g/L/h, respectively, while

ethanol production (7 g/L) was reduced by 70% compared

to that of R. oryzae KCTC 6946. Compared to the flask

culture, a sufficient supply of oxygen in the STR resulted

in decreased ethanol production as well as increased

fumaric acid production [11,23].

4. Conclusion

Rhizopus species represent a potential biological source of

fumaric acid for use in industrial applications. Although

considerable effort has been made to improve the produc-

tion of fumaric acid by bioprocess optimization in a reactor,

no fungal strain has yet been developed for the production

of fumaric acid. In this study, we developed a strain via

mutagenesis for the high-level production of fumaric acid

by examining the effects of culture conditions and nitrogen

source. The results of this study show that the C/N ratio

and organic nitrogen source are the most significant factors

affecting the production of fumaric acid. This study also

used a statistical method for optimization of the production

media. In optimal medium, the maximum concentration of

fumaric acid was 26.2 g/L, which is similar to the 27.4 g/L

predicted by the model. The highest fumaric acid concent-

ration obtained from the R. oryzae RUR709 mutant was

32.1 g/L, which was approximately 1.9-fold higher than

that from R. oryzae KCTC 6946.

Acknowledgements

The authors gratefully acknowledge the financial support

provided by the Korea Energy Management Cooperation

(KEMCO). This research was also supported by a Research

Grant from Kwangwoon University in 2008.

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Strain development and medium optimization for fumaric acid production

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