1. ITACONIC ACID PRODUCTION FROM SORGHUM BRAN – A BIOREFINING APPROACH
AMINA AHMED EL-IMAM; C. DU; P. S. DYER
27TH AUGUST, 2015

2. OUTLINE
Introduction
Itaconic acid fermentation and potential mitigants
Sorghum bran
Biorefineries
Motivation & Objectives
Methods & Findings
Compositional analyses
Hydrolysate preparation
Determination of potential inhibitors
Hydrolysate enrichment and detoxification
Fermentation
Summary
Itaconic acid and its uses

3. INTRODUCTION ITACONIC ACID (IA):
A versatile drop-in organic acid monomer
Produced by fermentation of sugars by Aspergillus terreus
High cost limits application – need for cheaper substrates
SORGHUM BRAN (SB):
Sorghum (Sorghum bicolor) is an important cereal
Used in making various foods, feed, ethanol etc
Grain processing generates bran as waste
BIOREFINERY
Facility that integrates biomass conversion with fuels, power and chemical production
Most promising route to creation of biobased industries
For commercial IA production, the yield based on the amount of glucose consumed is a very important economic consideration because the cost of the carbon source is reported to be more than 25% of the total production cost (Kobayashi and Nakamura 1964;
Costs about $ per kg; slightly more expensive than acrylic acid etc
expansion of the IA market is only possible by further reduction of production costs (Willke & Vorlop, 2001).
Notably in Nigeria with >6 million MT in 2014
Sorghum parts all useful

4. INTRODUCTION PROPOSED FLOWCHART FOR ITACONIC ACID PRODUCTION
Pretreatment
Physical
Biological
Chemical
Sorghum bran
Sorghum grains
Pap/Ogi
In an ideal world, this is a simplified version of how the fermentation process will proceed
Fermentation
Uses/Applications
Itaconic acid
Figure 1. Reference: El-Imam, A. A., & Du, C. (2014). Fermentative Itaconic Acid Production. J Biodivers Biopros Dev, 1(119),

5. MOTIVATION & OBJECTIVES
Sorghum waste is relatively underutilised though it accumulates in large amounts
Renewable Itaconic acid is beneficial to the environment
OBJECTIVES:
Conduct compositional analyses of sorghum bran
Screen several isolates for desirable traits
Determine potential inhibitors of itaconic acid fermentation
Detoxification of the medium
Produce itaconic acid in economical amounts
MENTION NIGERIA/MY COUNTRY

6. A: COMPOSITIONAL ANALYSES
COMPONENT
WHITE BRAN
RED BRAN
MOISTURE
11.6 ± 0.3
6.3 ± 0.1
PROTEIN
19.2 ± 0.6
21.4 ± 0.1
STARCH
52.9 ± 0.1
57.0 ± 0.2
LIPID
3.2 ± 0.8
3.01 ± 0.7
LIGNIN
6.7 ± 0.01
10.5 ± 0.04
ASH
1.8± 0.04
1.4± 0.05
SUM (%)
95.4
99.6
High Starch, protein and lipid contents
Low ash content: a OR
Proximate composition of sorghum brans. Values are means ± SD of triplicate measurements

7. PROPOSED BIOREFINING ROUTE FOR THE PRODUCTION OF ITACONIC ACID AND HIGH VALUE CO-PRODUCTS
PROCESS
PRODUCTS
APPLICATIONS
FEEDSTOCK
MILLING
STARCH
FOOD
FEED
BRAN
PHENOLICS
INDUSTRIAL PRODUCTS
DILUTE ACID HYDROLYSIS
HEAT
HYDROLYSISRESIDUE
PYROLYSIS PRODUCTS
SORGHUM GRAINS
ENZYMATIC HYDROLYSIS
SUGARS
VARIOUS INDUSTRIAL USES
ITACONIC ACID
A. terreus FERMENTATION
FUEL/ CHEMICAL
S. cerevisiae/YEAST FERMENTATION
ETHANOL

8. B: HYDROLYSATE / MEDIA PREPARATION
DILUTE ACID HYDROLYSATE +
10% Bran slurry % H2SO Bioreactor
DEFINED MEDIA/SOLUTIONS
Fermentation medium and all solutions prepared according to standard methods
Glucose conc. in hydrolysate (g/L)
22.08 ± .011
The hydrolysates were then adjusted to the desired pH, sterile filtered and stored at 4oC pending use

9. C: FERMENTATION MICROORGANISMS
44 strains of Aspergillus terreus - BDUN collection
1 strain - DSMZ
SUBMERGED FERMENTATION
1 x 106 spores/ml
Incubated at 37oC and 200 rpm for 7 days
ITACONIC ACID ESTIMATION
Jasco® HPLC with a Hi-Plex column and R.I. detector
HYDROLYSATE INHIBITOR ANALYSES
Determined using a Waters® Techsphere ODS-2 column and a Photodiode Array detector.

10. C: FERMENTATION
Introduction:
The various samples generated: sugars, OAs. And inhibitors were estimated by HPLC
The samples were run by a gradient method using a mixture of neat methanol and a 1:1 methanol : water mixture at a flow rate of 0.5 ml/minute. The data was acquired with a …. software.
Figure 2: Screening strains of Aspergillus terreus for itaconic acid production on glucose medium.

11. D: DETERMINATION OF POTENTIAL INHIBITORS
Tannins:-
Phosphorus:- Determined by ICPMS-CCT, along with other elements
Sulphates:- The effect of neutralization salt Na2SO4 on defined medium was investigated
Common fermentation Inhibitors:- estimated by HPLC
SAMPLE
C.E. (g/100 g)
RED BRAN
0.18 ± 12.7
COMMON FERMENTATION INHIBITORS
INHIBITOR NAME
CONCENTRATION (mg/L)
HMF
FURFURAL
VANILLIC ACID
FERULIC ACID
ACETIC ACID
SYR
Sorghum Hydrolysate
59.0
30.1
5.6
19.6 ND
Say: The amounts of the main potential inhibitors in the hydrolysates: tannins, sulphates and phosphates were investigated
method of Hagerman and Butler (1978) thus:
Into screw-top test tubes, 200 mg of SB was measured and 5 mL of ethyl ether added.
The tubes were capped tightly and rotated for 15 minutes to de-fat it.
Mixture was centrifuged at 4000 rpm and the ether extract was discarded.
The residue was extracted for 15 min with 5 mL of methanol centrifuged and the extract assayed for tannin within 8 h of the extraction.
Since the PO4 molecule is three times as heavy as the P atom, results reported as PO4 are three times the concentration of those reported as P. For example, if you measure 0.06 mg/L as PO4, that's equivalent to 0.02 mg/L as P. To convert PO4 to P, divide by 3. To convert P to PO4, multiply by 3.
Na2SO4 was produced in large amounts because of the buffering ability of the sorghum bran hydrolysate

12. TIME COURSE EXPERIMENTS
Ordinary Hydrolysate (55g/L)
Supplemented Hydrolysate (120g/L)

13. SUMMARY
SUBSTRATE
Sorghum bran is nutritionally suitable for bioconversions
Inhibitors: tannins and phosphates present
SCREENING
Three strains show promise on the hydrolysate
Glucose augmentation improved yields by ~87%
FERMENTATION
About a quarter of the yield of the glucose medium obtained by 49-22
Peak IA yield was obtained on Day 4
FUTURE WORK
More detoxification attempts are currently being run and the results appear promising with up to 200% increase in yield

14. THANK YOU FOR LISTENING
ACKNOWLEDGEMENTS
The Federal Government of Nigeria
Bioenergy and Fungal Biology groups
THANK YOU FOR LISTENING