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

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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

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UMI Number: 3235459

3235459 2006

UMI Microform Copyright

ProQuest Information and Learning Company

300 North Zeeb Road P.O Box 1346 Ann Arbor, MI 48106-1346

by ProQuest Information and Learning Company

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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

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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

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Abstract

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 KH2PO4 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

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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

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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

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TABLE OF CONTENTS

Page

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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

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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

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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

untreated and treated dried distillers grains with solubles 48

with solubles 49

21 Citric acid production by A niger ATCC 26550 grown on

with solubles 54

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LIST OF FIGURES

Figure Page

with solubles 59

with solubles 64

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

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

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LIST OF FIGURES

Figure Page

37 Citric acid production by A niger ATCC 9029, ATCC 9142,

38 Biomass production by A niger ATCC 9029, ATCC 9142,

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,

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

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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

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

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

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CHAPTER ONE INTRODUCTION

Citric acid (C6H8O7, 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

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(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

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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

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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

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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 CO2) and the other one to oxaloacetate (by fixing this mole

of CO2 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

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

Glucose

Pyruvate Pyruvate

Oxaloacetate Acetyl-CoA

Citrate

Pyruvate Dehydrogenase

Pyruvate Carboxylase

Tricarboxylic acid (TCA) cycle

Citrate synthase

CO2 Glycolysis

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(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

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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

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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

28oC, 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 28oC for 168

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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 30oC, and a 4% methanol concentration (Roukas, 1998) The presence of methanol was also able to stimulate citric acid

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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 30oC 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

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fermentation techniques for their potential as substrates for citric acid production

(Vandenberghe et al., 2000) Solid-state fermentation is characterized by the

development of microorganisms in a low-water activity environment on a soluble material acting as both a nutrient source and physical support (Pandey, 1992) A cost reduction can be achieved by using less expensive substrates such

non-as pineapple wnon-aste, coffee husk, sugarcane pressmud, cnon-assava fibrous wnon-aste,

kiwifruit peel and wheat bran (Vandenberghe et al., 2004) Use of solid-state

fermentation has been claimed to eliminate the metal sensitivity problems

(Shankaranand and Lonsane, 1994a) Solid-state fermentation also offers

several other advantages including higher product concentration, lower

downstream processing costs, reduced risk of bacterial contamination because

of low moisture level, reduction in water usage and less wastewater management

(Abou-Zeid and Ashy, 1984; Shankaranand et al., 1993)

Previous studies have investigated citric acid production by A niger using

solid-state fermentation on various substrates Using autoclaved pineapple waste

as a substrate for solid-state fermentation, A niger ATCC 9142 produced a

higher citric acid level than A niger ATCC 12846 or ATCC 10577 (Tran et al.,

1998) Relative to citric acid yield, it was found that a yield of 0.65 g citric acid/g

sugar consumed was produced by A niger ATCC 9142 on the pineapple waste (Tran et al., 1998) The addition of methanol to the pineapple waste slightly

increased citric acid production by the strains using solid-state fermentation (Tran

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et al., 1998) Fungal citric acid yields were highest in flasks while lower yields

were obtained in tray and rotating drum bioreactors (Tran et al., 1998)

When apple pomace and grape pomace were screened as substrates for

solid-state fermentation by A niger ATCC 9142, ATCC 10577, ATCC 12846, the

highest citric acid yield was produced by ATCC 9142 after 120 hours of growth It was also reported that 3% (v/w) methanol addition stimulated citric acid

production from apple pomace or grape pomace (Hang and Woodams, 1984,

1985) The highest concentration of citric acid produced by A niger ATCC 12846

from grape pomace using solid-state fermentation was observed after it was grown in the presence of 3% (v/w) methanol (Hang and Woodams, 1985) Citric acid production varied considerably depending upon the initial moisture content

of apple pomace (Hang and Woodams, 1987) Using kiwifruit peel as a substrate

to produce citric acid by A niger ATCC 12846 in solid-state fermentation, the

maximum citric acid concentration was produced in the presence of 2% methanol

at 30oC in 96 hours (Hang and Woodams, 1987) When figs (moisture level of 75% and pH of 7.0) were utilized as a substrate for the solid-state fermentation of

citric acid, A.niger ATCC 10577 produced the highest citric acid concentration

after 360 hours of growth at 30oC (Roukas, 2000) Using carob pods (moisture level of 65%) as a substrate for solid-state fermentation at 30oC for 288 hours at

pH 6.5, A niger ATCC 9142 produced the highest citric acid concentration when

6% (w/w) methanol was supplemented into the substrate (Roukas, 1999)

Maximal citric acid production from cassava bagasse by A niger strain LPB-21

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using solid-state fermentation was reached at an initial moisture of 62% at 26oC

for 120 hours (Vandenberghe et al., 2000) Sugarcane-pressmud, a by-product of

cane-sugar manufacture, and coffee husk also supported citric acid synthesis by

A niger strain CFTRI 30 using solid-state fermentation at 30oC after 120 h at pH 5.5 to 5.7 (Shankaranand and Lonsane, 1993, 1994b) Other novel agricultural substrates, such as kumara (a starch-containing root crop grown extensively in New Zealand) and taro, were tested for their ability to support citric acid

production by at A niger using solid-state fermentation (Lu et al., 1995) It was found that kumara and taro supported excellent citric acid production (Lu et al.,

1995) Another substrate tested for its ability to support fungal citric acid

production was the potato but it was found to be a poor substrate In contrast, the

potato did support excellent fungal biomass production (Lu et al., 1995)

A clear need exists to find additional alternative substrates of low

economic value and of considerable availability to produce citric acid because

the world-wide demand for citric acid far greatly exceeds its production (Tran et

al., 1998), Coproducts resulting from ethanol production could meet this need since they are highly available substrates with a relatively low economic value Over recent years, ethanol has increasingly been used as a renewable fuel

because ethanol burns cleanly and increases the octane level of gasoline as a fuel component Fuel ethanol is an important gasoline oxygenate, required to meet the U.S Clear Air Act Standards for vehicle emissions For this reason, the ethanol industry in the United States is rapidly expanding Today, most fuel

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ethanol in the United States is produced from corn by either the dry-milling

process (67%) or the wet-milling process (33%) The corn dry-milling process is illustrated in Figure 2 (Bothast and Schlicher, 2005) The first two steps involve cleaning and breaking down the kernel into fine particles using a hammer mill Subsequently, a slurry is formed by mixing the ground kernels with water The enzyme alpha amylase is added to the slurry to facilitate the hydrolysis of corn starch to dextrin (long chain sugars) This step is referred to as liquefaction Following complete liquefaction, glucoamylase is added This enzyme converts the dextrin into the simple sugar dextrose An inoculum of a species of yeast,

usually Saccharomyces cerevisae, is then added to metabolically convert the

dextrose to ethanol and CO2 The entire fermentation process is completed

within 40-60 h at 32oC Upon completion, the fermentation mash is transferred into a distillation area to strip away the ethanol Once the ethanol is removed, the water and all solids collected from the distillation base are referred to as whole stillage From the whole stillage, a number of coproducts from ethanol production can be obtained The whole stillage is centrifuged to separate the coarse solids (defined as wet corn distillers grains) from the liquid (defined as thin stillage) To recover additional water, thin stillage can be concentrated in an evaporator to form what is known as condensed corn distillers solubles Wet corn distillers grains and the condensed corn distillers solubles can be combined and dried in a rotary dryer to form dried corn distillers grains with solubles (Bothast and

Schlicher, 2005) With the expanding ethanol industry, these coproducts are

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Figure 2 Corn dry-milling process overview

Corn Corn cleaning

Cooker

Fermentation

D i s t i l l a t i o n

Centrifuge

Rotary Drier

Ethanol

Thin Stillage

Condensed Corn Distillers Solubles

Alpha Amylase

Yeast and Glucoamylase Whole Stillage

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becoming increasingly available in large amounts For example, more than 3.8 million tons of wet corn distillers grains and dried corn distillers grains with

solubles are produced from ethanol production per year (Weil et al., 2002) The

primary uses for the coproducts resulting from ethanol production are as animal feed supplements Dried corn distillers grains with solubles has been reported to

be an excellent protein supplement in animal rations (Ham et al 1994) Although

yeast fermentation of the corn mash to ethanol has reduced the concentration of fermentable sugars, dried corn distillers grains with solubles still contains

fermentable sugars and starch that could be utilized as a carbon source by

microorganisms (Moyer, 1953b; Nguyen et al., 1992) It has been shown that ground corn or corn starch was able to support citric acid production by A niger ATCC 12846 (Moyer, 1953b; Nguyen et al., 1992) The low-value coproducts

from ethanol production would seem to be an excellent source of substrates that can be used to produce a specialty chemical such as citric acid

Unfortunately, few studies have examined using coproducts resulting from alcohol fermentation as a feedstock for citric acid fermentation by citric acid-

producing strains of A niger Previous studies have focused on utilizing brewery

wastes as substrates for fungal fermentation of citric acid A major brewery

waste is spent grain liquor Citric acid production by A niger NRRL 337 was

maximal on spent grain liquor after 72 h of growth at 30oC (Hang et al., 1975) It

was also found that spent grain liquor supported elevated biomass production by

A niger NRRL 337 (Hang et al., 1975) Another investigation found that yields of

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citric acid varied from 3.5 to 12.3 g/L of liquor fermented after 96 h of A niger NRRL 337 growth on spent grain liquor (Hang et al., 1977) The yields ranged

widely because the initial sugar concentration in the spent liquor was variable

The addition of 2-4% methanol enhanced citric acid production by A niger NRRL

337 but diminished mycelial growth (Hang et al., 1977)

In this project, the overall objective is to learn whether the coproducts from ethanol production can be utilized as substrates for citric acid production With the coproducts such as dried corn distillers grains with solubles and wet corn distillers grains being in solid form, it will be necessary to employ solid-state

fermentation of these substrates using known citric acid-producing strains of A niger Surface fermentation by the citric acid-producing strains of the liquid

coproducts thin stillage and condensed corn distillers solubles will be used

Some of the selected strains, such as A niger ATCC 9142 and ATCC 10577,

were previously shown to produce citric acid following growth on brewery waste which is similar to the coproducts from ethanol production (Roukas and

Kotzekidou, 1986) Additional treatments of the solid coproducts such as

sterilization by autoclaving and mild acid-hydrolysis were studied in an effort to learn whether such treatments released fermentable sugars for subsequent

fungal citric acid production

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CHAPTER THREE MATERIALS AND METHODS

Chemicals

Glycylglycine, glycine, neocuproine, CuSO4.5H2O, lactate dehydrogenase, ZnCl2, KH2PO4 and NADH were obtained from the Sigma Chemical Company, St Louis, Missouri, USA Na2CO3 was obtained from Eastman Kodak Company, Rochester, New York, USA Malate dehydrogenase and citrate lyase were

obtained from Roche Diagnostic Corporation, Indianapolis, Indiana, USA

Methanol was purchased from Fisher Scientific Company, New Jersey, USA The source of potato dextrose broth was Difco Laboratories, Detroit, Michigan, USA The filters were obtained from Whatman International Ltd., Maidstone, England, Great Britain The corn distillers grains with solubles, thin stillage, wet corn

distillers grains and condensed corn distillers solubles were provided by the Dakota Ethanol, LLC Plant in Wentworth, South Dakota, USA All other

chemicals were of analytical grade

Microorganisms

Seven known citric acid-producing strains of Aspergillus niger were

utilized in this study and are listed in Table 1 The strains were purchased from the American Type culture Collection (ATCC), Manassas, Virginia, USA

Growth medium

The strains were maintained on potato dextrose agar plates, and the

plates were stored at 4oC The plates contained 10 g/L potato dextrose broth

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Table 1 Aspergillus niger strains utilized in this study

Strain Origin

Aspergillus niger ATCC 9029 Somkuti and Bencivengo (1981)

Aspergillus niger ATCC 9142 Kiel et al (1981)

Aspergillus niger ATCC 10577 Roukas (2000)

Aspergillus niger ATCC 11414 Hang and Woodams (1984)

Aspergillus niger ATCC 12846 Hang et al (1987)

Aspergillus niger ATCC 26550 Wold et al (1973)

Aspergillus niger ATCC 201122 Gradisnik-Grapulin and Legisa (1996)

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and 20 g/L agar To prepare the inoculum of each strain used for the solid-state

and surface fermentation experiments, the strains were grown in potato dextrose

broth that contained 10 g/L potato dextrose broth

Solid-state fermentation

Solid-state fermentation was used for citric acid production from the solid

substrates including dried corn distillers grains with solubles and wet corn

distillers grains An outline of the protocol used for solid-state fermentation is

illustrated in Figure 3 Initially, the dried corn distillers grains with solubles (5 g)

were weighed into sterile 125 mL Erlenmeyer flasks The grains were brought to

82% moisture with water, 0.5% H2SO4, 1.0% H2SO4, 1.5% H2SO4, 2.0% H2SO4 or

2.5% H2SO4 The grains were subjected to sterilization by autoclaving at 17

pounds/square inch of pressure for 20 min at 121oC Flasks containing 5 g of

grains brought to 82% moisture without sterilization served as the control

cultures Following sterilization, the pH of the acid-hydrolyzed grains was

adjusted to 6.0 Next, the wet corn distillers grains was used as a substrate for

solid-state fermentation The wet corn distillers grains (5 g) containing 68% initial

moisture were weighed into sterile 125 mL Erlenmeyer flasks One set of flasks

containing the wet corn distillers grains was sterilized by autoclaving at 17

pounds/square inch of pressure for 20 min at 121oC while a control set of flasks

containing the wet grains was not

When determining the effect of methanol supplementation on citric acid

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Assay for citric acid

Figure 3 Protocol used for solid-state fermentation.

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production, 3% (v/v) methanol was added to each culture When the effect of

methanol addition upon citric acid production by ATCC 11414, ATCC 26550 or ATCC 201122 was utilized, the strain was grown for a period of 240 h Due to significant growth inhibition of ATCC 9029, ATCC 9142, ATCC 10577 and ATCC

201122 following methanol addition, an incubation period of 480 h at 25oC was used When testing the effect of phosphate addition on citric acid production by the strains, 30 mM KH2PO4 was added to the culture and the fungal cultures were grown for a period of 240 h at 25oC To initiate solid-state fermentation of the

grains, each flask was inoculated with approximately 2000 conidia/mL of each A niger strain The flasks were incubated at 25oC for 240 or 480 h

When the effect of temperature on citric acid production by A niger ATCC

9142 was investigated using corn distillers grains with solubles as a substrate, the fungus was grown on the grains (82% initial moisture) with 30 mM KH2PO4 supplementation at 25oC, 28oC and 30oC for 240 h When the effect of initial moisture content of the grains with solubles on citric acid production by ATCC

9142 was explored, the fungus was grown with initial moisture of 55%, 70%, 78% and 82% Each culture was incubated at 25oC for 240 h The initial moisture of the grains was determined by placing the grains in a preweighed beaker and weighing the grains After drying the grains in an oven at 105oC to constant

weight, the dry weight of the grains was measured The difference in weight was used to derive the initial moisture of the grains In the time course study of citric

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To precipitate any protein present in each culture filtrate, ice cold 0.5 N HClO4 (0.5 mL) was added and the filtrate was stirred Any protein precipitate present was removed The filtrate was subsequently neutralized to pH 7.0 with 1 N NaOH The volume of each culture filtrate was recorded The neutralized filtrate was assayed for its citric acid content Citric acid production and biomass production are

expressed as g citric acid produced per kg grains and kg biomass per kg grains, respectively Specific citric acid productivity is given as g citric acid/kg biomass/h

Surface fermentation

Surface fermentation of the corn processing substrates thin stillage and

condensed corn distillers solubles by strains of A niger were tested for their

ability to produce citric acid An outline of the protocol is shown in Figure 4 The thin stillage was prepared as a substrate by initially adjusting its pH to 6.0 and filtering it through a Whatman No 1 filter to remove insoluble material The

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Assay for citric acid

Figure 4 Protocol used for surface fermentation.

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clarified stillage was sterilized at 17 pounds/square inch of pressure for 20 min at

121oC The condensed corn distillers solubles was prepared as a substate by suspending the solubles (200 g/L) in water The pH of the suspended solubles was adjusted to pH 6.0 and the mixture was filtered through a Whatman No 1 filter to remove insoluble matter The solubles were autoclaved for 20 min at 17 pounds/square inch of pressure at 121oC A volume of the processed thin

stillage and condensed corn distillers solubles were added to sterile 250 mL Erlenmeyer flasks Each flask was inoculated with approximately 2000

conidia/mL of each A niger strain to initiate the surface fermentation of the

substrates The cultures were placed in a rotary shaker and shaken at 200

revolutions/min at 25oC for a period of 144 h Following the incubation period of

144 h, the cultures were processed to allow the assaying of citric acid and

biomass levels Each culture was 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 removed and utilized during the subsequent biomass determination To precipitate any protein present in each culture filtrate, ice cold 0.5 N HClO4 (0.5 mL) was added and the filtrate was stirred After any protein precipitate present was removed, the filtrate was subsequently neutralized to pH 7.0 with 1 N NaOH The volume of each

culture filtrate was recorded The neutralized filtrate was assayed for its citric acid content Citric acid and biomass levels are expressed as g citric acid produced

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