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 UMI Microform 3235459 Copyright 2006 by ProQuest Information and Learning Company All rights reserved This microform edition is protected against unauthorized copying under Title 17, United States Code ProQuest Information and Learning Company 300 North Zeeb Road P.O Box 1346 Ann Arbor, MI 48106-1346 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 Dissertation Advisor Date 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 (20022003) 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 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 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…………………………………………….…………… CHAPTER TWO Review of Literature 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 Aspergillus niger strains used in this study…………………………… 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 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 Effect of initial moisture on citric acid and biomass production by A niger ATCC 9142……………………………………………… 25 Effect of temperature on specific productivity and citric acid yields by A niger ATCC 9142………………………………………… 27 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 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 Scheme of carbon flow from glucose to citrate in A niger Corn dry-milling process overview 16 Protocol used for solid-state fermentation 22 Protocol used for surface fermentation .25 Citric acid production by A niger ATCC 9029 grown on untreated and treated dried distillers grains with solubles .31 Biomass production by A niger ATCC 9029 grown on untreated and treated dried distillers grains with solubles .33 Citric acid specific productivity by A niger ATCC 9029 grown on untreated and treated dried distillers grains with solubles 34 Citric acid yield (%) by A niger ATCC 9029 grown on untreated and treated dried distillers grains with solubles .35 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…… 42 120 effect of temperature and moisture on citric acid production by ATCC 9142 using solid-state fermentation was studied ATCC 9142 produced its highest citric acid level at 25oC on phosphate-supplemented dried distillers grains with solubles at 82% moisture following 10 days of growth When ATCC 9142 was grown at higher temperatures, more abundant mycelial growth occurred This increased mycelial growth could account for the reduced citric acid yield due to competition for the available fermentable sugars present An earlier study reported that the maximum yield of citric acid yield was obtained between and 12 days of incubation at a temperature range of 24oC to 28oC (Doelger and Prescott, 1934) This finding is in agreement with the results obtained in this study It was found that a moisture content of 70% was necessary for optimal biomass production by ATCC 9142 on dried distillers grains with solubles For either fungal citric acid or biomass production, a high moisture content of the dried distillers grains with solubles was necessary It is known that low moisture levels lead to poor microbial growth and poor accessibility to nutrients during solid-state fermentation (Pandey, 1992) Similarly, it was shown that low moisture reduced substrate availability to the fungus which caused low citric acid production (Kumar et al., 2003) When wet distillers grains was used as a substrate, ATCC 9142 produced the highest citric acid level among the seven strains tested when grown at 25oC for 240 hours (Table 6) This finding is consistent with the previous studies using dried distillers grains with solubles as a substrate where ATCC 9142 was the 121 most effective strain for citric acid production using solid-state fermentation Autoclaving the wet distillers grains resulted in a reduction of citric acid production by the majority of the strains tested It was not very clear why there was a difference in citric acid production by some of the strains when grown on autoclaved grains although thermal pretreatments have been found to lower the trace metal content of substrates (Aravantinos-Zafiris et al., 1994) The trace metal ions can stimulate fungal citric acid production depending upon their concentration (Aravantinos-Zafiris et al., 1994) In general, autoclaving the wet distillers grains resulted in higher fungal biomass production which was also observed during the studies using autoclaved dried distillers grains with solubles During the surface fermentation studies using thin stillage as substrate for the seven strains screened, ATCC 201122 produced the highest level of citric acid (Table 7) ATCC 26550 or ATCC 9029 produced slightly lower citric acid levels than ATCC 201122 did The most effective strain for biomass production on thin stillage was ATCC 9029 (Table 7) The strain that exhibited the highest specific productivity was ATCC 201122 while a slightly lower specific productivity was produced by ATCC 26550 (Table 7) The highest citric acid yields were produced by ATCC 201122, ATCC 26550 or ATCC 9029 (Table 7) Overall, ATCC 201122 was the most effective strain to use for the surface fermentation of the thin stillage while ATCC 26550 was slightly less effective Similar to the findings on thin stillage, ATCC 201122 was the most effective strain for citric acid production on condensed corn distillers solubles 122 Table 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 Treatment Parameter Substrate Citric acid production Thin stillage Autoclaved Biomass production Thin stillage Autoclaved Specific productivity Thin stillage Autoclaved Citric acid yield Thin stillage Autoclaved Citric acid production Biomass production Condensed corn distillers solubles Condensed corn distillers solubles Condensed corn distillers solubles Condensed corn distillers solubles Autoclaved Specific productivity Citric acid yield Most effective strains ATCC 201122, ATCC 26550 & ATCC 9029 ATCC 9029 ATCC 201122 & ATCC 26550 ATCC 201122, ATCC 26550 & ATCC 9029 ATCC 201122 Autoclaved ATCC 10577 & ATCC 9029 ATCC 201122 Autoclaved ATCC 201122 Autoclaved 123 (Table 7) ATCC 201122 also produced the highest citric acid specific productivity and citric acid yield on condensed distillers solubles (Table 7) On the other hand, ATCC 10577 or ATCC 9029 produced the highest biomass levels on condensed distillers solubles (Table 7) The data collected indicated that ATCC 201122 was the most effective strain to use for the surface fermentation of condensed distillers solubles Unfortunately, very few studies have utilized this strain for citric acid production using surface fermentation The strong inhibition of Isocitrate dehydrogenase by glycerol was reported to responsible for citric acid accumulation by ATCC 201122 (Gradisnik-Grupulin and Legisa, 1996) With respect to other studies utilizing alcohol processing by products, it has been shown that similar levels of citric acid were produced by Aspergillus niger NRRL 337 grown on spent grain liquor (Hang et al., 1977) 124 CHAPTER SIX CONCLUSIONS This study shows that: The coproducts resulting from ethanol fermentation of corn can be utilized as substrates to produce citric acid by selected strains of Aspergillus niger The effect of the different substrate treatments on fungal citric acid production depended on the strain used Aspergillus niger ATCC 9142 produced the highest level of citric acid on dried distillers grains with solubles among the seven strains screened Aspergillus niger ATCC 9142 also produced the highest level of citric acid on wet distillers grains of the seven strains studied Several selected Aspergillus niger strains were effective for producing citric acid on thin stillage Aspergillus niger ATCC 201122 was the most effective strain for citric acid production on condensed distillers solubles among the seven strains screened The solid-state fermentation studies analyzing citric acid production on dried distillers grains with solubles and wet distillers grains indicated that higher citric acid levels could be produced on wet distillers grains than dried distillers grains with solubles This finding has some economic significance for producing citric acid at an ethanol plant because it will not be necessary to dry the grains for use 125 as a substrate Obviously, it will be necessary to scale-up the process from the current laboratory-scale procedure reported here This could be accomplished using solid-state fermentation by A niger ATCC 9142 grown on the surface of a layer of wet distillers grains on large shallow trays The organism could be 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acid specific productivity by A niger strains. .. 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... 48106-1346 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