Comparative evaluation of some brown midrib sorghum mutants for the production of food grain and 2,3-butanediol

Pacific Rim Summit on Industrial Biotechnology and Bioenergy

December 9, 2014

Yadhu N Guragain1, K.S. Vinutha2, G.S. Anil Kumar2, Reggeany Barrios1, P.V. VaraPrasad3, P. Srinivasa Rao2, Praveen V. Vadlani1

1Bioprocessing and Renewable Energy Laboratory, Grain Science and Industry, Kansas State University, USA2International Crops Research Institute for the Semi-Arid Tropics (ICRISAT), India3Department of Agronomy, Kansas State University (KSU), USA

Bioprocessing and Renewable Energy Laboratory, KSU Dr. Praveen Vadlani: -Director Two Post-doc Four Ph D students One Master Student Undergraduate students Research Scientists Summer REU students

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Research Areas

Biofuels – cellulosic ethanol, biobutanol

Biochemicals – 2,3-butanediol, lactic acid, succinic acid

Co-product utilization – animal feed, bioproducts

Specialty chemicals – flavor, pre and probiotics, fragrances, neutraceuticals

HPLC

GC-MSUV-VIS

5 L

fermenterParr

Reactor

GC

Pretreatment

Hydrolysis Monomer

sugars

Fermentation

Product recovery

Fuels and

Chemicals

Lignin

(Enzymes)

High Value aromatic

compounds and

macromolecules

Cellulose and

Hemicellulose

(Holocellulose)

Bioprocessing of Lignocellulosic Biomass

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Source: - http://crf.sandia.gov/index.php/

A lot of challenges in each steps

Major Challenges for lignocellulosic biorefineries

1. Pretreatment of biomass

Cost Production of inhibitors

• Phenolics, furan compounds, aliphatic acids

Environmental issues

2. Cost of enzymes

3. Valorization of lignin

4. Handling and storage of bulky biomass

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Source: - http://crf.sandia.gov/index.php/

5

37

22

29

12

Douglas Fir (Softwood)

40

22

21

17

Poplar (Hardwood)

Glucan XylanLignin Others

35

29

17

20

Switchgrass (Grass)

31

1916

34

Corn stover (Grass)

35

21

20

23

Sorghum stalk (Grass)

In general, alkali pretreatment efficiency ∞ lignin content;

however, this is not always true.

• Among five biomass here, douglas fir the most difficult and corn stover is the easiest for delignification

• Switchgrass is little more difficult than sorghum, and poplar is much more difficult than sorghum

Each biomass must be separately evaluated !

Sorghum is a model energy crop because of its high photosynthetic efficiency,abiotic stress tolerance, and wide applications as food, feed, and fuels.

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bmr mutation of sorghum leads to change in composition of its stover

bmr sorghum as a potential feedstock for bioenergy and biochemicals production needs investigation

Brown midrib White midrib

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Bio

pro

cess

ing

and

Re

new

able

En

erg

y La

bo

rato

ry, K

SU

Bacillus licheniformis

Both bmr mutations had longer flowering time and lower yields than their background, except stover yield of Kansas Collier

8

0

10

20

30

40

50

60

70

80

90

0

1

2

3

4

5

6

7

Early Hegari(EH)

bmr6 (EH) bmr12 (EH) Atlas (AT) bmr6 (AT) bmr12 (AT) KansasCollier (KC)

bmr6 (KC) bmr12 (KC)

50

% F

low

erin

g ti

me

(day

s)

Yie

ld (

t/h

a)

Genotype

Agronomy dataStover yield

Grain yield

50% Flowering time

bmr mutation led to decrease in lignin (10 to 25%) and carbohydrate polymers (2 to 9%), and increase in extractives (6 to 43% )for Atlas and Kansas collier

This was not true for bmr 12 of Early Hegari.

9

0

10

20

30

40

50

Cellulose + Hemicellulose Lignin Extractives

Co

mp

osi

tio

n (

%, d

ry w

eigh

t b

asis

)

Biomass Component

Composition of biomassEarly Hegari (EH) bmr 6 (EH) bmr 12 (EH)

Atlas (AT) bmr 6 (AT) bmr 12 (AT)

Kansas Collier (KC) bmr 6 (KC) bmr 12 (KC)

Significant proportion of extractives in soluble sugars (sucrose, glucose and fructose)

Sugar proportion significantly higher in bmr mutants in all wild types, except,bmr12 of Kansas Collier (consistent with composition data)

10

0

10

20

30

40

50

60

70

Early Hegari(EH)

bmr 6 (EH) bmr 12 (EH) Atlas (AT) bmr 6 (AT) bmr 12 (AT( KansasCollier (KC)

bmr 6 (KC) bmr 12 (KC)

Am

ou

nt

(%, W

/W))

Genotype

Total extractives Vs sugarsTotal extractives

Sugars in water extraction

Sugar proportion

Both bmr mutats had higher sugar yield than wild type in all biomass, except bmr12 of Kansas Collier

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0

10

20

30

40

50

60

70

80

90

0 10 20 30 40 50

Suga

r yi

eld

(%

, g/g

pre

tre

ate

d b

iom

ass)

Hydrolysis time (h)

Hydrolysis yield for monomer sugars from pretreated biomass

Early Hegari (EH) bmr 6 (EH) bmr 12 (EH)

Atlas (AT) bmr 6 (AT) bmr 12 (AT)

Kansas Collier (KC) bmr 6 (KC) bmr 12 (KC)

2,3-butanediol yield was around 0.3 g/g sugars or lower (theoretical maximum 0.5)

Significant byproduct formation: glycerol, acetic acid, ethanol

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0.27 0.26 0.27 0.26 0.27 0.260.28

0.330.31

0.27

0.0

0.1

0.2

0.3

EarlyHageri (EH)

bmr6 (EH) bmr12 (EH) Atlas (AT) bmr6 (AT) bmr12 (AT) KansasCollier (KC)

bmr6 (KC) bmr12 (KC) Control2,3

-bu

tan

ed

iol y

ield

(g/

g su

gar

con

sum

ed

)

Axis Title

Shake flask fermentation using Bacillus licheniformis

Overall mass balance: Stover to fermentable sugars

Hydrolysis

Pretreatment

Pretreated biomass

Liquid stream

Released sugars

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Ground Biomass

100100100

100100100

100100100

KCATEH

172826

112121

15193

KCATEH

Wild→bmr 6→

bmr 12→

44394

454345

454555

KCATEH

293134

313035

343741

KCATEH

445959

415157

505542

KCATEH8

1716

71410

17153

KCATEH

Sugars

333

333

434

KCATEH

Phenolics

Sugar extraction (in water)

% increment in sugar yieldEH AT KC

bmr 6 10 25 33

bmr 12 -16 38 33

% increment in sugar yieldEH AT KC

bmr 6 6 -1 5

bmr 12 19 16 14

Grain and stover yield significantly vary among bmrsorghum mutants

1.25% (W/V) NaOH is optimum for pretreatment of bmrsorghum

bmr mutation alters the biomass composition, andimproves hydrolysis efficiency of stalk. However, theeffects significantly vary among sorghum varietiesindicating background effect.

bmr mutation leads to increase in non-structural sugarcontent in sorghum, which must be extracted beforepretreatment

Biomass derived sugar can be used for 2,3-butanediolproduction using Bacillus licheniformis.

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Conclusion:

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Acknowledgement:

ICRISAT-KSU bmr sorghum project from USAID-CGIAR-US varsity linkage program

The Small Business Innovation Research (SBIR),Navy, and Department of Defense, USA

Thank you for

your attention

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