ITACONIC ACID PRODUCTION FROM SORGHUM BRAN – A BIOREFINING APPROACH

AMINA AHMED EL-IMAM; C. DU; P. S. DYER

27TH AUGUST, 2015

OUTLINEIntroduction

a. Itaconic acid fermentation and potential mitigantsb. Sorghum branc. Biorefineries d. Motivation & Objectives

Methods & Findings a. Compositional analysesb. Hydrolysate preparationc. Determination of potential inhibitorsd. Hydrolysate enrichment and detoxificatione. Fermentation

Summary

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

INTRODUCTION

Sorghum grains

Sorghum bran

Pap/Ogi

Itaconic acid Fermentation

Figure 1. Reference: El-Imam, A. A., & Du, C. (2014). Fermentative Itaconic Acid Production. J Biodivers Biopros Dev, 1(119),

Pretreatment• Physical• Biological• Chemical

Uses/Applications

PROPOSED FLOWCHART FOR ITACONIC ACID PRODUCTION

MOTIVATION & OBJECTIVESMOTIVATION:

• Sorghum waste is relatively underutilised though it accumulates in large amounts

• Renewable Itaconic acid is beneficial to the environment

OBJECTIVES:A. Conduct compositional analyses of sorghum branB. Screen several isolates for desirable traitsC. Determine potential inhibitors of itaconic acid

fermentationD. Detoxification of the mediumE. Produce itaconic acid in economical amounts

A: COMPOSITIONAL ANALYSES

• a

Proximate composition of sorghum brans. Values are means ± SD of triplicate measurements

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:

OR

PROCESSFEEDSTOCK PRODUCTS APPLICATIONS

SORGHUM GRAINS

MILLING

DILUTE ACID HYDROLYSIS

SUGARS

STARCH

ENZYMATIC HYDROLYSIS

BRAN

A. terreus FERMENTATION

HYDROLYSISRESIDUE

S. cerevisiae/YEAST FERMENTATION

ETHANOL

VARIOUS INDUSTRIAL USES

FOOD

FEED

INDUSTRIAL PRODUCTS

HEAT

PYROLYSIS PRODUCTS

ITACONIC ACID

FUEL/ CHEMICAL

PROPOSED BIOREFINING ROUTE FOR THE PRODUCTION OF ITACONIC ACID AND HIGH VALUE CO-PRODUCTS

PHENOLICS

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

C: FERMENTATIONMICROORGANISMS• 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

Figure 2: Screening strains of Aspergillus terreus for itaconic acid production on glucose medium.

49-1

49-2

49-3

49-4

49-5

49-6

49-7

49-8

49-9

49-1

049

-11

49-1

249

-13

49-1

449

-15

49-1

649

-17

49-1

849

-19

49-2

049

-21

49-2

249

-23

49-2

449

-25

49-2

649

-27

49-2

849

-29

49-3

049

-31

49-3

249

-33

49-3

449

-35

49-3

649

-37

49-3

849

-39

49-4

049

-41

49-4

249

-43

49-4

449

-45

0

5

10

15

20

25

30

35

40

45

50

YIELDS ON GLUCOSE MEDIUM

ITACONIC ACID

Aspergillus terreus BDUN Strain ID

Itaco

nic

acid

yie

ld (g

/L)

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

INHIBITOR NAME

CONCENTRATION (mg/L)  

HMF FURFURALVANILLIC ACID

FERULIC ACID

ACETIC ACID SYR

Sorghum Hydrolysate 59.0 30.1 5.6 19.6 ND ND

COMMON FERMENTATION INHIBITORS

TIME COURSE EXPERIMENTSOrdinary Hydrolysate (55g/L) Supplemented Hydrolysate (120g/L)

2 3 4 5 7 90.00

1.00

2.00

3.00

4.00

5.00

6.00RBDAH

49-549-2249-45

TIME (DAYS)

ITA

CO

NIC

AC

ID Y

IELD

(g/L

)

2 3 4 5 7 90.00

5.00

10.00

15.00

20.00

25.00

30.00GLUCOSE MEDIUM

49-5

49-22

49-45

TIME (DAYS)

ITA

CO

NIC

AC

ID Y

IELD

(g/L

)

SUMMARYSUBSTRATE

• 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

ACKNOWLEDGEMENTSThe Federal Government of NigeriaBioenergy and Fungal Biology groups

THANK YOU FOR LISTENING