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O R I G I N A L A R T I C L E

Citric acid production by Aspergillus nigeron wet corndistillers grainsG. Xie1 and T.P. West2

1 Department of Chemistry and Biochemistry, South Dakota State University, Brookings, SD, USA

2 Department of Biology and Microbiology, South Dakota State University, Brookings, SD, USA

Introduction

Citric acid has a variety of commercial applications in

foods, beverages and pharmaceuticals (Tran et al. 1998).

Nearly 1 million tons of citric acid are produced globally

every year (Karaffa et al. 2001). Previous investigations

have studied the use of the fungus Aspergillus niger to

produce citric acid from brewery wastes such as brewers

spent grain liquor and from ground corn using surface

fermentation (Moyer 1953; Hang et al. 1975, 1977; Rou-

kas and Kotzekidou 1986). Using solid-state fermentation,

A. nigerstrains were able to produce citric acid from sub-

strates such as apple pomace (Hang and Woodams 1984),

grape pomace (Hang and Woodams 1985), kiwifruit peel

(Hang and Woodams 1987), pineapple waste (Tran et al.

1998), figs (Roukas 2000) or cassava bagasse (Van-

denberghe et al. 2004). Wet corn distillers grains are a

major co-product resulting from ethanol fermentation

and it has not been determined whether the grains could

be utilized as a substrate by A. niger strains to produce

citric acid. For each bushel (3524 l) of corn processed at

ethanol plants, c. 18 pounds (82 kg) of dry corn distillers

grains are produced and over a million tons

(907 000 metric tons) of dry distillers grains are produced

each year. The primary use of wet corn distillers grains is

as a protein supplement in animal feeds (Ham et al.

1994). In this study, seven citric acid-producing A. niger

strains were screened for their ability to synthesize citric

Keywords

Aspergillus niger, biomass, citric acid, wet

corn distillers grains, yield.

Correspondence

Thomas P. West, Department of Biology and

Microbiology, South Dakota State University,

Box 2140D, Brookings, SD 57007, USA.E-mail: thomas.west@sdstate.edu

2005/1500: received 19 December 2005,

revised 5 April 2006 and accepted 13 April

2006

doi:10.1111/j.1472-765X.2006.01958.x

Abstract

Aims: To determine which citric acid-producing strain ofAspergillus nigerutil-

ized wet corn distillers grains most effectively to produce citric acid.

Methods and Results:Citric acid and biomass production by the fungal strains

were analysed on the untreated grains or autoclaved grains using an enzyme

assay and a gravimetric method respectively. Fungal citric acid production on

the grains was found to occur on the untreated or autoclaved grains. The high-est citric acid level on the grains was produced by A. niger ATCC 9142. The

autoclaved grains supported less citric acid production by the majority of

strains screened. Biomass production by the fungal strains on the untreated or

autoclaved grains was quite similar. The highest citric acid yields for A. niger

ATCC 9142, ATCC 10577, ATCC 11414, ATCC 12846 and ATCC 26550 were

found on the untreated grains. Treatment of the grains had little effect on citric

acid yields based on reducing sugars consumed by A. niger ATCC 9029 and

ATCC 201122.

Conclusions:It is feasible for citric acid-producing strains ofA. niger to excrete

citric acid on wet corn distillers grains whether the grains are treated or

untreated. The most effective citric acid-producing strain ofA. nigerwas ATCC

9142.Significance and Impact of the Study: The study shows that the ethanol pro-

cessing co-product wet corn distillers grains could be utilized as a substrate for

the commercial production of citric acid by A. nigerwithout treatment of the

grains.

Letters in Applied Microbiology ISSN 0266-8254

2006 The Authors

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acid on untreated and autoclaved wet corn distillers

grains by solid-state fermentation.

Methods

Micro-organisms and inoculum

Seven citric acid-producing strains, namely A. niger

ATCC 9029, ATCC 9142, ATCC 10577, ATCC 11414,

ATCC 12846, ATCC 26550 and ATCC 201122, were used

in this study. The source of the wet distillers grains was

Dakota Ethanol LLC (Wentworth, SD, USA) and it con-

tained 54% total carbohydrates. When sterilized by auto-

claving, the wet corn distillers grains was subjected to

121C at 1172 kPa of pressure for 20 min. A loopful of

fungal mycelium of A. niger strain was inoculated into

potato dextrose broth (20 ml), and the culture was grown

for 72 h at 25C. This culture served as the inoculum.

Solid-state fermentation and substrate processing

One millilitre inoculum (5 103 conidia) was added to

5 g corn distillers grains (65% moisture) in a sterile

125 ml Erlenmeyer flask and the fungus was grown for

240 h at 25C. After cultivation, the citric acid present in

each solid-state fermentation culture was collected by the

following procedure. To each culture, sterile water

(25 ml) was added. After shaking each culture for 60 min

at 25C, the grains were filtered through a Whatman no.

1 filter. The fungal biomass in each culture was washed

with sterile water (10 ml) and also filtered through a

Whatman no. 1 filter. The fungal biomass was separatedfrom the grains during the course of washing the biomass

with water. The filtrates from each culture were com-

bined. To precipitate any protein present in each culture

filtrate, ice-cold 05 mmol l)1 HClO4 (05 ml) was added

and the filtrate was stirred. Any protein precipitate pre-

sent was removed. The filtrate was subsequently neutral-

ized to pH 70 with 1 mmol l)1 NaOH. The volume of

each culture filtrate was recorded.

Citric acid and biomass determinations

The neutralized filtrate was assayed for its citric acid con-

tent using a coupled enzyme spectrophotometric assay

(Moellering and Gruber 1966; Henniger and Mascaro

1985). The modified assay mix (1 ml) contained

01 mmol l)1 glycylglycine buffer pH 78, 02 mmol l)1

NADH, 06 mmol l)1 ZnCl2, 5 U citrate lyase, 6 U malate

dehydrogenase, 3 U lactate dehydrogenase and sample.

Citric acid standards were also assayed. The reaction was

monitored at 340 nm by following the decrease in

absorbance that is proportional to the concentration of

citric acid present in the sample. To measure biomass

production, wet fungal biomass collected after 240 h of

growth was placed in a preweighed beaker and dried at

105C to constant weight. The beaker containing the dry

fungal biomass was re-weighed to derive the weights for

each culture. The weight of the inoculum added to each

culture determined by collection on preweighed filtersand drying to constant weight at 105C was determined

after 240 h by subtracting from the biomass levels. To

measure the reducing sugar levels in the untreated or

autoclaved grains, a sample of the grains was brought to

100% moisture content, subjected to high-speed mixing

and an aliquot was removed for analysis. The reducing

sugar levels were assayed by a previously described

method using glucose as a standard (Dygert et al. 1965).

All values represent the mean of three independent deter-

minations involving three separate cultures. The Students

t-test was used during statistical analysis. All determina-

tions were performed in triplicate.

Results

Seven citric acid-producing strains of A. niger were

screened for their ability to utilize wet corn distillers grains

as a substrate for citric acid production. The strain found

to produce the highest level of citric acid after growth for

240 h on corn distillers grains was ATCC 9142 (Fig. 1).

There was a significant difference (P< 005) in citric acid

levels between ATCC 9142 and ATCC 11414 but not

between ATCC 9142 and ATCC 12846. Compared with

citric acid production by ATCC 9142, it was determined

that ATCC 9029 and ATCC 10577 produced about three-fold less citric acid after 240 h. Citric acid production by

ATCC 26550 and ATCC 201122 was noted to be twofold

lower than the level produced by ATCC 9142. It was also

of interest to learn whether autoclaving the wet corn dis-

tillers grains had an effect on citric acid production by the

sevenA. niger strains. A prior study reported that autocla-

ving pineapple waste improved fungal citric acid produc-

tion (Tran et al. 1998). Citric acid production by ATCC

201122 on the autoclaved grains was found to be highest

after 240 h and citric acid production on the treated

grains was higher than on the untreated grains. On the

autoclaved grains, citric acid production by ATCC 11414

or ATCC 12846 was 13-fold lower than ATCC 201122

while ATCC 26550, ATCC 9142, ATCC 10577 and ATCC

9029 were 14-, 15-, 19- and 21-fold lower respectively.

Citric acid production by ATCC 11414, ATCC 12846 and

ATCC 9142 on the autoclaved grains was significantly

lower (P< 001) than observed on the untreated grains.

Citric acid production by ATCC 9029, ATCC 10577 and

ATCC 26550 on the autoclaved grains was also lower than

on the untreated grains.

Fungal citric acid production on grains G. Xie and T.P. West

270 Journal compilation 2006 The Society for Applied Microbiology, Letters in Applied Microbiology 43 (2006) 2692732006 The Authors

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Biomass production by the strains after growth on wet

corn distillers grains for 240 h was investigated. The cell

weights after 240 h growth on wet corn distillers grains

were similar for ATCC 10577, ATCC 9029, ATCC 11414,

ATCC 12846 and ATCC 201122 (Fig. 2). Only biomass

production by ATCC 9142 and ATCC 26550 was slightly

lower. The effect of autoclaving the grains on biomass

production by the strains was examined. Although bio-

mass production by the seven strains was similar, biomass

production by ATCC 26550 on the autoclaved grains was

the highest of the strains tested. Biomass production by

ATCC 26550 was significantly higher (P< 001) on the

autoclaved grains compared with the untreated grains.

The highest citric acid yields were observed for ATCC

9142, ATCC 11414, ATCC 12846 and ATCC 26550

(Fig. 3). Slightly lower citric acid yields were measured

for ATCC 26550, ATCC 10577 and ATCC 201122. The

lowest citric acid yield was observed for ATCC 9029. On

the autoclaved grains, the highest yield was observed for

ATCC 201122, which was significantly higher (P< 001)

than the yields calculated for ATCC 9029, ATCC 10577,

ATCC 11414, ATCC 12846 and ATCC 26550. The citric

acid yields on the autoclaved grains were significantly

lower (P< 001) for ATCC 9142, ATCC 11414, ATCC

12846 and ATCC 26550 compared with the yieldsobserved for these strains on the untreated grains. This

0

2

4

6

8

10

12

14

ATCC

9029

ATCC

9142

ATCC

10577

ATCC

11414

ATCC

12846

ATCC

26550

ATCC

201122

Strain

CitricAcid(gk

g1)

Figure 1 Citric acid production [g citric acid

(kg wet corn distillers grains))1] byAspergillus

nigerATCC 9029, ATCC 9142, ATCC 10577,

ATCC 11414, ATCC 12846 and ATCC 20112-

2 grown on untreated (() and autoclaved

( ) corn distillers grains for 240 h at 25C.

Data represent the mean of three separate

trials SD.

0

01

02

03

04

05

06

07

08

ATCC

9029

ATCC

9142

ATCC

10577

ATCC

11414

ATCC

12846

ATCC

26550

ATCC

201122

Strain

Biomass(gg1)

Figure 2 Biomass production [g biomass

(g wet corn distillers grains))1] byAspergillus

nigerATCC 9029, ATCC 9142, ATCC 10577,

ATCC 11414, ATCC 12846 and ATCC 20112-

2 grown on untreated (() and autoclaved

( ) corn distillers grains for 240 h at 25C.

Data represent the mean of three separate

trials SD.

G. Xie and T.P. West Fungal citric acid production on grains

2006 The Authors

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occurred despite the initial reducing sugar concentration

(mean of three trials SD) of the untreated wet distillers

grains being 2783 511 g reducing sugar (kg grains))1

and the initial reducing sugar concentration of the auto-

claved grains being 4935 454 g reducing sugar (kg

grains))1. The specific productivity (mean of three tri-

als SD) of ATCC 9142 on the untreated grains was

012 003 g citric acid (kg biomass))1 h)1 and it was

the highest specific productivity of the strains screened.

On the autoclaved grains, the highest specific productivity

(mean of three trials SD) was ATCC 201122 at

005 0

01 g citric acid (kg biomass)

)1

h)1

. The specificproductivity of ATCC 201122 was identical whether

untreated or autoclaved grains served as the substrate.

Discussion

Previous studies have investigated citric acid production

by the strains of A. niger utilized in this study using

solid-state fermentation on various substrates (Hang et al.

1977; Hang and Woodams 1984, 1985; Tran et al. 1998;

Roukas 2000; Vandenberghe et al. 2004). Apple pomace

or grape pomace were utilized as substrates for solid-state

fermentation by A. niger ATCC 9142, ATCC 11414 and

ATCC 12846. The highest citric acid yield on untreated

apple pomace (30%) was produced by ATCC 9142 after

96 h of growth (Hang and Woodams 1984). On the

untreated grape pomace, the highest citric acid yield

(48%) was produced by ATCC 9142 after 120 h of

growth (Hang and Woodams 1985). Citric acid produc-

tion by A. niger ATCC 9142, ATCC 11414 and ATCC

12846 on kiwifruit peel was also studied and it was found

that ATCC 12846 produced 70 g citric acid (kg kiwifruit

peel))1 which was the highest level observed after 120 h

of growth (Hang and Woodams 1987). With autoclaved

pineapple waste as a substrate for solid-state fermenta-

tion, A. niger ATCC 9142 and ATCC 12846 were able to

produce citric acid after 168 h but ATCC 9142 produced

the highest citric acid yield (Tran et al. 1998). A yield of

065 g citric acid (g sugar consumed))1 was produced by

ATCC 9142 on the pineapple waste (Tran et al. 1998).

When figs served as a substrate for solid-state fermenta-

tion, A. niger ATCC 10577 produced 64 g citric acid (kg

figs))1 after 360 h with a citric acid yield of 8% (Roukas

2000). Citric acid was produced at a level of 78 g citricacid (kg dry bagasse))1 byA. niger ATCC 9142 following

the solid-state fermentation of cassava bagasse after 144 h

(Vandenberghe et al. 2004). In this study, the citric acid

level produced by ATCC 9142 on the wet corn distillers

grains after 240 h was the highest level observed for the

seven strains tested. Although the concentration of citric

acid produced by ATCC 9142 on wet corn distillers grains

was several-fold lower than the concentration produced

by ATCC 12846 on kiwifruit peel or by ATCC 10577 on

figs, its concentration was found to be about 13-fold

higher than when ATCC 9142 was grown on cassava

bagasse (Vandenberghe et al. 2004). The citric acid yield

produced by ATCC 9142 on the grains was comparable

with the citric acid yield produced by ATCC 9142 on

apple pomace but was lower than the yield on grape

pomace (Hang and Woodams 1984, 1985). In addition,

the citric acid yield produced by ATCC 9142 on the

grains was higher than the citric acid yield of ATCC

10577 on figs (Roukas 2000). It was not clear why there

was a difference in citric acid production by some of the

strains when grown on the autoclaved grains. It may be

0

5

10

15

20

25

30

ATCC

9029

ATCC

9142

ATCC

10577

ATCC

11414

ATCC

12846

ATCC

26550

ATCC

201122

Strain

Yield(gg1

100%)

Figure 3 Citric acid yields [g citric acid

(g reducing sugar consumed))1 100%] by

Aspergillus nigerATCC 9029, ATCC 9142,

ATCC 10577, ATCC 11414, ATCC 12846 and

ATCC 201122 grown on untreated (() and

autoclaved ( ) corn distillers grains for 240 h

at 25C. Data represent the mean of three

separate trials SD.

Fungal citric acid production on grains G. Xie and T.P. West

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that autoclaving the grains resulted in less available sub-

strates for citric acid production by some of the strains or

in fermentation inhibitors being formed. Despite the cit-

ric acid level produced by ATCC 9142 on the grains being

low compared with a high sugar substrate such as figs,

the wet corn distillers grains still has the potential as a

substrate for fungal citric acid production considering thequantity of grains being produced each year. The solid-

state fermentation process may be further optimized for

possible commercial use by adjusting the moisture con-

tent of the grains or by adding supplements such as

methanol to the grains (Roukas 2000).

Overall, citric acid was produced by the seven strains

of A. niger used in this study whether they were grown

on untreated or autoclaved corn distillers grains. The

concentration of citric acid and the citric acid yields were

found to be highest for A. niger ATCC 9142 on the

untreated corn distillers grains.

Acknowledgements

Published as paper 3539, Journal Series, South Dakota

Agricultural Experiment Station. This work was supported

by the South Dakota AES and the South Dakota Corn

Utilization Council. This paper reports results of research

only and the mention of brand or firm names does not

constitute an endorsement by the South Dakota AES over

those mentioned but of a similar nature.

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