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

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

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 $2.0-2.5 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

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),

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

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

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

B: HYDROLYSATE / MEDIA PREPARATION DILUTE ACID HYDROLYSATE + 10% Bran slurry 1% H2SO4 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

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.

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.

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

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

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

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