citric acid production by aspergillus niger strains grown on corn substrates from ethanol fermentation

CITRIC ACID PRODUCTION BY ASPERGILLUS NIGER STRAINS GROWN
ON CORN SUBSTRATES FROM ETHANOL FERMENTATION






BY
GANG XIE







A thesis submitted in partial fulfillment of the requirements for the
Doctor of Philosophy
Major in Chemistry
South Dakota State University
2006

UMI Number: 3235459
3235459
2006
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ii
CITRIC ACID PRODUCTION BY ASPERGILLUS NIGER STRAINS GROWN
ON CORN SUBSTRATES FROM ETHANOL FERMENTATION


This dissertation is approved as a creditable and independent
investigation by a candidate for the Doctor of Philosophy degree and acceptable
for meeting the dissertation requirements for this degree. Acceptance of this
dissertation does not imply that the conclusions reached by the candidate are
necessarily the conclusions of the major department.








Dr. Thomas P. West Date
Dissertation Advisor

Dr. James A. Rice Date
Head, Department of Chemistry
and Biochemistry








iii
ACKNOWLEDGMENTS
I would like to express my sincere gratitude to Dr. Thomas P. West for his
valuable guidance, support, time and encouragement throughout my studies. He
has been my advisor, mentor and an example of the highest caliber of a research
scientist. I would also like to thank Dr. James Rice, Dr. Duane Matthees, Dr. Igor
Sergeev, and Dr. James Doolittle for their willingness to serve on my advisory
committee. I would like to thank Dr. Rice for my teaching assistantship (2002-
2003) and research assistantship (2003-2006). I also acknowledge the South
Dakota Agricultural Experiment Station for funding this research project as well
as my research assistantship.

iv
Abstract

CITRIC ACID PRODUCTION BY ASPERGILLUS NIGER STRAINS GROWN
ON CORN SUBSTRATES FROM ETHANOL FERMENTATION

Gang Xie

2006


Citric acid is an important specialty chemical which can be synthesized
biologically. It has a number of commercial applications including its use in foods,

pharmaceuticals and other industries. In this study, the coproducts resulting from
ethanol fermentation of corn were tested for their suitability to be utilized as
substrates for citric acid production using solid-state fermentation or surface
fermentation. These coproducts include dried corn distillers grains with solubles,
wet corn distillers grains, thin stillage and condensed corn distillers solubles.
Seven citric acid-producing strains of the fungus Aspergillus niger were selected
and screened for their ability to produce citric acid from these corn-based
substrates. The treatments of the substrates include autoclaving and mild-acid
hydrolysis. In addition, the effects of 3% (v/v) methanol addition and 30 mM
KH
2
PO
4
supplementation were also studied. The concentration of citric acid was
analyzed by a coupled enzyme assay. It was found that A. niger ATCC 9142
produced the highest level of citric acid on solid substrates including dried
distillers grains with solubles and wet distillers grains. On the other hand, A. niger
ATCC 12846 and ATCC 26550 produced the highest biomass level on dried
distillers grains with solubles and wet distillers grains, respectively. The effects of

v
methanol and phosphate supplementation on citric acid and biomass production
were strain-dependent. It was also found that A. niger ATCC 201122 was the
most effective strain for citric acid production on liquid substrates including thin
stillage and condensed distillers solubles. A. niger ATCC 201122 also produced
the highest specific productivity and citric acid yield on the liquid substrates.
Moreover, A. niger ATCC 9029 and ATCC 10577 produced the highest biomass
level on thin stillage and condensed distillers solubles, respectively. It was
concluded that A. niger strains could use corn-based coproducts from ethanol
fermentation as substrates for citric acid production.

vi
TABLE OF CONTENTS

Page

Abstract iv
Table of Content vi
List of Tables
List of Figures……………………………………………………………… ix
CHAPTER ONE
Introduction…………………………………………….…………… 1
CHAPTER TWO
Review of Literature 4
CHAPTER THREE
Materials and Methods……… …………………………………… 19
Chemicals…………………………………………………………… 19
Microorganisms……………….….…………………………………. 19

Growth medium………………… …………………………………. 19
Solid-state fermentation…………………… 21
Surface fermentation………………………………………………. 24
Citric acid assay…………………………………………………… 27
Biomass determinations………………………………………… 28
Reducing sugar assay……………………………………………… 28
Statistics……………………………………………………………… 29


vii
TABLE OF CONTENTS

Page

CHAPTER FOUR
RESULTS……………………………………………………………. 30
CHAPTER FIVE
DISCUSSION……………………………………….…………….… .110
CHAPTER SIX
CONCLUSIONS 124
BIBLIOGRAPHY…………………………………………………………… 126

viii
LIST OF TABLES
Table Page
1. Aspergillus niger strains used in this study…………………………… 5

2. Citric acid and biomass production by ATCC 9142 grown for
240 h on dried distillers grains with solubles supplemented with
30 mM phosphate at selected incubation temperatures………… .15

3. Citric acid specific productivity and yield by ATCC 9142 grown
for 240 h on dried distillers grains with solubles supplemented
with 30 mM phosphate at selected incubation temperatures……… 24

4. Effect of initial moisture on citric acid and biomass production
by A. niger ATCC 9142………………………………………………. 25

5. Effect of temperature on specific productivity and citric acid
yields by A. niger ATCC 9142………………………………………… 27

6. Most effective strains for citric acid production, biomass
production, specific productivity and citric acid yield on dried
distillers grains with solubles and wet distillers’ grains using
solid-state fermentation relative to treatment…………………… 118

7. Most effective strains for citric acid production, biomass

production, specific productivity and citric acid yield on thin
stillage and condensed corn distillers solubles using surface
fermentation relative to treatment………………………………… 122
















ix
LIST OF FIGURES

Figure Page
1. Scheme of carbon flow from glucose to citrate in A niger 4
2. Corn dry-milling process overview 16
3. Protocol used for solid-state fermentation 22
4. Protocol used for surface fermentation 25

5. Citric acid production by A. niger ATCC 9029 grown on
untreated and treated dried distillers grains with solubles 31

6. Biomass production by A. niger ATCC 9029 grown on
untreated and treated dried distillers grains with solubles 33

7. Citric acid specific productivity by A. niger ATCC 9029
grown on untreated and treated dried distillers grains
with solubles 34

8. Citric acid yield (%) by A. niger ATCC 9029 grown on
untreated and treated dried distillers grains with solubles 35

9. Citric acid production by A. niger ATCC 11414 grown on
untreated and treated dried distillers grains with solubles 37

10. Biomass production by A. niger ATCC 11414 grown on
untreated and treated dried distillers grains with solubles 38

11. Citric acid specific productivity by A. niger ATCC 11414
grown on untreated and treated dried distillers grains
with solubles……………………………… 39

12. Citric acid yield (%) by A. niger ATCC 11414 grown on
untreated and treated dried distillers grains with solubles 40

13. Citric acid production by A. niger ATCC 10577 grown on
untreated and treated dried distillers grains with solubles…… 42

x
LIST OF FIGURES

Figure Page
14. Biomass production by A. niger ATCC 10577 grown on
untreated and treated dried distillers grains with solubles… 43

15. Citric acid specific productivity by A. niger ATCC 10577
grown on untreated and treated dried distillers grains
with solubles………………………………………………… 44

16. Citric acid yield (%) by A. niger ATCC 10577 grown on
untreated and treated dried distillers grains with solubles… 45

17. Citric acid production by A. niger ATCC 12846 grown
on untreated and treated dried distillers grains with solubles 47

18. Biomass production by A. niger ATCC 12846 grown on
untreated and treated dried distillers grains with solubles 48

19. Citric acid specific productivity by A. niger ATCC 12846
grown on untreated and treated dried distillers grains
with solubles 49

20. Citric acid yield (%) by A. niger ATCC 12846 grown on
untreated and treated dried distillers grains with solubles 50

21. Citric acid production by A. niger ATCC 26550 grown on
untreated and treated dried distillers grains with solubles 52

22. Biomass production by A. niger ATCC 26550 grown on
untreated and treated dried distillers grains with solubles 53

23. Citric acid specific productivity by A. niger ATCC 26550
grown on untreated and treated dried distillers grains
with solubles 54

24. Citric acid yield (%) by A. niger ATCC 26550 grown on
untreated and treated dried distillers grains with solubles 55

25. Citric acid production by A. niger ATCC 201122 grown on
untreated and treated dried distillers grains with solubles 57



xi


LIST OF FIGURES

Figure Page
26. Biomass production by A. niger ATCC 201122 grown on
untreated and treated dried distillers grains with solubles 58

27. Citric acid specific productivity by A. niger ATCC 201122
grown on untreated and treated dried distillers grains
with solubles 59

28. Citric acid yield (%) by A. niger ATCC 201122 grown on

untreated and treated dried distillers grains with solubles 61

29. Citric acid production by A. niger ATCC 9142 grown on
untreated and treated dried distillers grains with solubles 62

30. Biomass production by A. niger ATCC 9142 grown on
untreated and treated dried distillers grains with solubles 63

31. Citric acid specific productivity by A. niger ATCC 9142
grown on untreated and treated dried distillers grains
with solubles 64

32. Citric acid yield (%) by A. niger ATCC 9142 grown on
untreated and treated dried distillers grains with solubles 66

33. Citric acid production by A. niger ATCC 11414, ATCC 26550
and ATCC 201122 grown on untreated and methanol-treated
dried distillers grains with solubles 67

34. Biomass production by A. niger ATCC 11414, ATCC 26550
and ATCC 201122 grown on untreated and methanol-treated
dried distillers grains with solubles 69

35. Citric acid specific productivity by A. niger ATCC 11414,
ATCC 26550 and ATCC 201122 grown on untreated and
methanol-treated dried distillers grains with solubles 70

36. Citric acid yields (%) by A. niger ATCC 11414, ATCC 26550
and ATCC 201122 grown on untreated and methanol-treated
dried distillers grains with solubles 71

xii


LIST OF FIGURES

Figure Page
37. Citric acid production by A. niger ATCC 9029, ATCC 9142,
ATCC 10577 and ATCC 12846 grown on untreated and
methanol-treated dried distillers grains with solubles 73

38. Biomass production by A. niger ATCC 9029, ATCC 9142,
ATCC 10577 and ATCC 12846 grown on untreated and
methanol-treated dried distillers grains with solubles 74

39. Citric acid specific productivity by A. niger ATCC 9029,
ATCC 9142, ATCC 10577 and ATCC 12846 grown on
untreated and methanol-treated dried distillers grains
with solubles 75

40. Citric acid yields (%) by A. niger ATCC 9029, ATCC 9142,
ATCC 10577 and ATCC 12846 grown on untreated and
methanol-treated dried distillers grains with solubles 76

41. Citric acid production by A. niger strains grown on untreated
and phosphate supplemented dried distillers grains
with solubles 78

42. Biomass production by A. niger strains grown on untreated
and phosphate supplemented dried distillers grains with

solubles 79

43. Citric acid specific productivity by A. niger strains grown on
untreated and phosphate supplemented dried distillers grains
with solubles 81

44. Citric acid yields (%) by A. niger strains grown on untreated
and phosphate supplemented dried distillers grains with
solubles 82

45. Citric acid and biomass production by A. niger ATCC 9142 as
a function of fermentation time 90

46. Citric acid specific productivity and yield by A. niger ATCC 9142
as a function of fermentation time 92

xiii


LIST OF FIGURES

Figure Page
47. Citric acid production by A. niger strains grown on untreated
and autoclaved wet corn distillers grains 94

48. Biomass production by A. niger strains grown on untreated
and autoclaved wet distillers grains 96

49. Citric acid specific productivity by A. niger strains grown
on untreated and autoclaved wet distillers grains 97

50. Citric acid yields (%) by A. niger strains grow on untreated
and autoclaved wet distillers grains 98

51. Citric acid production by A. niger strains grown on thin stillage 100

52. Biomass production by A. niger strains grown on thin stillage 101

53. Citric acid specific productivity by A. niger strains grown on
thin stillage 103

54. Citric acid yields (%) by A. niger strains grown on thin stillage 104

55. Citric acid production by A. niger strains grown on
condensed corn distillers solubles 105

56. Biomass production by A. niger strains grown on condensed
corn distillers solubles 107

57. Citric acid specific productivity by A. niger strains grown on
condensed corn distillers solubles 108

58. Citric acid yields (%) by A. niger strains grown on condensed
corn distillers solubles 109

59. Formation of furfural and hydroxymethyl furfural in acid
hydrolysate of hemicellulose and cellulose 113

1

CHAPTER ONE
INTRODUCTION

Citric acid (C
6
H
8
O
7
, 2-hydroxy-propane-1,2,3-tricarboxylic acid) is a
commercially important specialty chemical with a number of applications
including its use in food (70%), pharmaceuticals (12%) and other industries
(18%) (Vandenberghe et al., 2004). Citric acid combines a pleasant sour taste
with low toxicity and high solubility which has made it a common food additive. It
is utilized to regulate the acidic flavor of soft drinks, fruit and vegetable drinks,
wines, ciders, jams, jellies, preserves and pie fillings (Karaffa et al., 2001). Citric
acid is also able to chelate metal ions and is therefore applied in the stabilization
of oils and fats during ion-catalyzed oxidation reactions (Karaffa and Kubicek,
2003). Among the organic acids industrially produced, citric acid is the most
important in quantitative terms with an estimated annual global production of over
9,000,000 tons, and almost the entire production is carried out by fermentation
(Karaffa and Kubicek, 2003). There is a constant increase (3.5-4%) each year in
its consumption which indicates the need of finding new substrate alternatives for
its manufacture (Vandenberghe et al., 1999).
New value-added approaches to produce the specialty chemical citric acid
are needed. One such value-added approach could involve the use of
coproducts from ethanol production. In recent years, ethanol is increasingly being
used as a renewable fuel because ethanol contains oxygen, which improves fuel

2
combustion and reduces emissions. For this reason, the ethanol industry in the
United States has grown rapidly from a production volume of 10 million gallons in
1979 to 2.81 billion gallons in 2003 (Bothast and Schlicher, 2005). The increased
ethanol demand primarily comes from the dry-milling of corn. Once the ethanol is
removed by distillation, a number of coproducts result from the dry-milling of corn
for ethanol production. The coproducts from ethanol production include dried
distillers grains with solubles, wet distillers grains, thin stillage and condensed
distillers solubles. During the final stages of corn dry-milling process, the water
and all solids are collected and centrifuged to separate the coarse solids
(referred to as wet distillers grains) from the liquid (referred to as thin stillage).
Thin stillage can be concentrated in the evaporator to become condensed
distillers solubles. Wet distillers grains and condensed distillers solubles are then
combined and dried in a rotary dryer to form dried distillers grains with solubles.
About 18 pounds of 90% dried distillers’ grains with solubles are produced from
each bushel of corn processed at ethanol plants. Currently, dry-milling ethanol
plants produce over 3.8 million tons of dried distillers grains with solubles
annually. Large amounts of the other coproducts also remain. Currently, these
coproducts are used as protein supplements in animal feeds (Ham et al., 1994).
In addition to having a high nitrogen content, these coproducts also contain
fermentable sugars and starch that could be utilized further by microorganisms
as a source of carbon. Considering the fact that a million tons of corn-based
coproducts from ethanol production are available, it was of interest to learn

3
whether these low-value coproducts could be further fermented to produce value-
added specialty chemicals such as citric acid. The fermentation of ethanol
processing coproducts to the specialty chemical citric acid seemed feasible
considering that almost all the citric acid produced commercially is obtained

through sugar or starch fermentation using the filamentous fungus Aspergillus
niger (Vandenberghe et al., 2000).
Since the worldwide demand for citric acid far exceeds its production
(Tran et al., 1998), a need exists to find alternative cheaper substrates of
considerable availability to produce citric acid. The objective of this project was to
investigate the suitability of the coproducts from ethanol production as substrates
for citric acid production.














4
CHAPTER TWO

REVIEW OF THE LITERATURE

Citric acid, originally described as a constituent from citrus plants and
known as an intermediate of the tricarboxylic acid cycle for 69 years, is widely
used in food and pharmaceutical industries. Citric acid was first isolated from
lemon juice and crystallized from lemon juice and crystallized as calcium citrate

by Scheele in 1784 (Tsay and To, 1987). This acid is widespread in many fruits
such as citrus fruits, pineapples, pears and figs. Since citric acid is present in
almost every life form, it is easily metabolized and eliminated from the body. It is
viewed as a “natural” substance because it is fully biodegradable in the
environment. Citric acid has a pleasant acid taste, high solubility and ready
assimilability. It also has GRAS status (generally regarded as safe by the United
States Food and Drug Administration) because of its very low toxicity. These
properties lead to its main applications in food and beverage industry.
Pharmaceutical applications of citrates include their uses in blood transfusions
while the free acid is used in effervescent products (Abou-Zeid and Ashy, 1984).
Another very important property of citric acid is its ability to chelate heavy metal
ions such as iron and copper. Therefore, citric acid is used as an antioxidant and
a preservative in foods. For example, citric acid can stabilize oils and fats during
ion-catalyzed oxidation reactions (Karaffa and Kubicek, 2003). The ability of
citric acid to chelate metal ions also leads to its use in soaps and laundry
detergents. By chelating metal ions in hard water, it lets these cleaners produce

5
foam and work better without the need for water softening. In the cosmetic
industry, citric acid is useful as a buffering agent over a broad range of pH values
(2 to 7) because of its three acid groups with different pKa values (Karaffa et al.,
2001). Other applications include its use as a metal cleaning and sequestering
agent in industrial processes. A recent application for citric acid use in
environmental remediation has been devised where citric acid can be used as a
scrubber to remove sulfur dioxide from pollutant gases (Tsao et al., 1999).
Considering the economic significance of citric acid, many studies have
investigated the biochemistry of citric acid production in Aspergillus niger (Tsao
et al., 1999). Previous studies have focused on A. niger because numerous
strains of this fungus have been isolated that produce high levels of citric acid
with minimal formation of undesired side products while other species produced

citric acid with low yields (Hockenhull, 1960). Also, A.niger is generally regarded
as a safe organism because no toxic byproducts were secreted by this fungus
(Schuster et al., 2002). Prior studies demonstrated that citric acid biosynthesis
involves glycolytic catabolism of glucose to two moles of pyruvate (Figure 1)
(Karaffa and Kubicek, 2003), of which one is converted to acetyl-CoA (by
releasing one mole of CO
2
) and the other one to oxaloacetate (by fixing this mole
of CO
2
onto the second pyruvate). These two precursors are subsequently
condensed to citric acid (Figure 1). The final condensation reaction is catalyzed
by the enzyme citrate synthase, which is located exclusively in the mitochondria.
The product is then transported out of the mitochondria and finally out of the cell

6


Figure 1. Scheme of carbon flow from glucose to citrate in A. niger





Glucose
Pyruvate Pyruvate
Oxaloacetate Acetyl-CoA
Citrate
Pyruvate
Dehydrogenase

Pyruvate
Carboxylase
Tricarbox
y
lic acid
(
TCA
)
c
y
cle
Citrate synthase
CO
2

Glycolysis

7
(Karaffa and Kubicek, 2003).
There is general agreement that only selected strains of A. niger are
useful citric acid producers (Hockenhull, 1960). For example, there are a total of
168 mutant strains of A. niger available from The American Type Culture
Collection (ATCC), but only 27 strains of them have been found to be capable of
producing high concentrations of citric acid on a variety of substrates. The
selection of a suitable strain is the most important step from a process viewpoint
because once a strain is selected it dominates the development and potential
success of the process (Hockenhull, 1960). Seven citric acid-producing strains,
namely A. niger ATCC 9029 (Somkuti and Bencivengo, 1981), ATCC 9142 (Kiel
et al., 1981), ATCC 10577 (Roukas, 2000), ATCC 11414 (Hang and Woodams,
1984), ATCC 12846 (Hang et al., 1987), ATCC 26550 (Wold et al., 1973) and

ATCC 201122 (Gradisnik-Grapulin and Legisa, 1996), have previously been
shown to excrete high levels of citric acid. Therefore, these seven citric acid-
producing strains of A. niger were selected for use in this study.
The accumulation of citric acid by selected mutants of A. niger has
attracted the interests of many researchers. For example, the biochemical
mechanism responsible for citric acid accumulation by A. niger ATCC 201122
was studied (Gradisnik-Grupulin and Legisa, 1996). It was found that isocitrate
dehydrogenase, which catalyzes the oxidation of isocitrate to ketoglutarate in
tricarboxylic acid (TCA) cycle, was much more strongly inhibited by glycerol in
the citric acid-accumulating strain A. niger ATCC 201122 than in other non-

8
producing strains including A. niger A 116, A. niger A 113, A. foetidus A 117 and
A. wentii A 6. Inhibition of isocitrate dehydrogenase caused a diminished
metabolic flux through the TCA cycle and consequently intracellular accumulation
of citric acid (Gradisnik-Grupulin and Legisa, 1996). From a biochemical point of
view, it is apparent that the regulation of glycolysis with respect to the
accumulated citrate is the major factor governing the rate of citric acid
accumulation (Kubicek, 1987). Hexokinase and phosphofructokinase are key
enzymes for glycolysis. In the A. niger B60 mutant strain which produced high
yields of citric acid, activities of these two enzymes were found to be 2-fold
higher than their activities in the parent strain (Schreferl et al., 1986).
Fermentation is the predominant way of producing citric acid and accounts
for more than 90 percent of the world production. In fact, citric acid is one of the
world’s largest tonnage fermentation products. The reasons for this are two-fold.
First, extraction of citric acid from fruit is not economical and is rarely used due to
the low yield and the high cost of production. Second, the chemical synthesis of
citric acid is possible but not economically feasible due to the expense of the raw
materials required for a complicated low-yielding reaction process (Karaffa et al.,
2001). There are many microorganisms, including fungi, yeasts, and bacteria,

that can ferment citric acid from sugar-containing substrates. However, the
majority of these microorganisms are not as efficient as certain strains of the
filamentous fungus A. niger. It has been demonstrated that citric acid can be
produced in high productivity and high yields by the fermentation of simple

9
sugars such as glucose or sucrose as well as a variety of cheap raw materials by
A. niger (Tsao et al., 1999).
Currently, there are three different types of fermentation processes in use
for citric acid production. These are surface fermentation, submerged
fermentation and solid-state fermentation. Among them, surface fermentation
and submerged fermentation processes involve liquid fermentation and are used
more extensively than solid-state fermentation.
Microbial production of citric acid is a highly aerobic process. Surface
fermentation involves allowing a mat of fungal hyphae to grow on the surface of
the medium (Drysdale and McKay, 1995). Surface fermentation does not require
the energy input for agitation and aeration used in submerged fermentation and
avoids the associated problem of foaming (Drysdale and McKay, 1995). For this
reason, surface fermentation accounts for a substantial part of citric acid
fermentation capacity worldwide.
Previous investigations have used a variety of substrates for
biosynthesis of citric acid by surface fermentation. Using beet molasses as a
substrate for citric acid production by A. niger ATCC 10577 for 240 hours at
28
o
C, citric acid yields were highest when the initial culture medium pH was 6.0
(Clement, 1952). Citric acid was also produced from beet molasses by
immobilized A. niger ATCC 9142 cells in shake flask cultures where the
maximum citric acid concentration was observed after 28 days (Roukas, 1991).
In another study, cane molasses supported citric acid production at 28

o
C for 168

10
hours by A. niger T55 using surface fermentation. These studies found that citric
acid biosynthesis is greatly impaired by organic and inorganic inhibitors. It was
later shown that treatment of molasses was needed to remove the inhibitors in
order to increase citric acid production (Kundu et al., 1984). Acid-hydrolyzed
cotton waste was also examined for its potential to support citric acid production
by A. niger ATCC 9142. The hydrolyzed cotton waste failed to support citric acid
production but did support biomass production (Kiel et al., 1981). On the brewery
waste spent grain liquor, A. niger ATCC 9142 produced more citric acid by
surface fermentation than did A. niger ATCC 10577 (Roukas and Kotzekidou,
1986). Lager tank sediment also supported citric acid production by the fungus
(Roukas and Kotzekidou, 1986).
During the surface or submerged fermentation of citric acid by A. niger, it
was observed that methanol addition stimulated citric acid production (Moyer,
1953a). Higher levels of zinc, iron, and manganese can be tolerated in either
surface or submerged culture for citric acid fermentation if a slightly toxic
concentration of methanol is present (Moyer, 1953a). It is not known why
methanol stimulates citric acid production by the fungus although its effect may
be related to increased membrane permeability. Using carob pod extract as a
substrate for the surface fermentation of citric acid by A. niger ATCC 9142, the
highest citric acid concentration was achieved at an initial sugar concentration of
200 g/L, pH of 6.5, a temperature of 30
o
C, and a 4% methanol concentration
(Roukas, 1998). The presence of methanol was also able to stimulate citric acid

11

production by a strain of A. niger using sugar cane bagasse as a substrate
(Lakshminarayana et al., 1975). In this study, the average yield of citric acid
under surface culture conditions by the strain was higher than that under
submerged culture conditions and the yield of citric acid is increased in the
presence of methanol (Lakshminarayana et al., 1975). A methanol concentration
between 2-4% added to acid-hydrolyzed whey permeate was suitable for the
production of citric acid by a strain of A. niger using shake flask cultures. The
optimal production of citric acid occurred in the shake flask cultures after 8-12
days at 30
o
C following methanol supplementation (Somkuti and Bencivengo,
1981).
In addition to methanol, the supplementation of phosphate to the medium
has been reported to stimulate citric acid production by the fungus. In an earlier
study, it was noted the addition of phosphate to the medium stimulated citric acid
production by A. niger ATCC (Shu and Johnson, 1948). Another study reported
that phosphate addition to extracts prepared from dates promoted citric acid
production likely due to the ability of phosphate to chelate high levels of inhibitory
metal ions like Mn, Fe, and Zn present in the extract (Roukos and Kotzekidou,
1997).
In recent years, considerable interest has been shown in using
agricultural products and their residues as alternative carbon sources for citric
acid production by A. niger (Vandenberghe et al., 2004). A variety of agro-
industrial residues and byproducts have been investigated with solid-state