1

Biorefining of Protein Containing

Biomass

Wim Mulder

Biofuels Summer School

2018

22 June 2018

Wageningen UR;

university and 9 applied research institutes

Introduction

Wageningen UR Food & Biobased Research

Food & Biobased Research

● Fresh, Food & Chains

● Biobased Products

Biobased Products

● Biobased Chemicals

● Biobased Materials

● Biorefinery & Bioenergy

Content

Biobased Economy / circulair economy

Use of biomass

Proteins

● Structure

● Use/application

● Biorefinery of protein-rich biomass

5

Fossil resources

6

Biobased Economy

7

Biobased economy

Severn Suzuki, Rio Summit 1992

● http://www.youtube.com/watch?v=oJJGuIZVfLM

8

Biobased Economy

9

Circular

bioeconomy:

co-production

of variety of

products

In closed loop

biorefinery processes

production of food, feed,

chemical, materials,

biofuels

Definitions

Sustainability

Brundtland Report

Cradle to cradle

People, Planet, Profit (triple P) philosophy

Life cycle analysis (LCA)

Biobased Economy

http://www.youtube.com/watch?v=3ibJsOQdeoc

12

https://www.youtube.com/watch?v=XX6911pS0Kg

From biomass to biomaterials

13

From biomass to biomaterials

Value biomass products

● Pharma and cosmetics ~10 euro/kg

● Chemistry 1-10 euro/kg

● Minerals and energy < 1 euro/kg

Biomass can be divided into:

● 20% high value molecules

● 40% transport fuel

● 40% energy (burning)

Biorefinery: a tool for value increase

Pretreatment&

Extraction

New biomass

Byproducts

CropsLignocellulose

Fresh biomass

Aquatic biomass

Residues

Feed & Food

Materials

Chemicals

Energy & Fuels

Biomass chain aspects: availability, quality, sustainability, logistics

Oils & fats

Sugars

Fibres

Proteins

(Bio

)chem

ical

Convers

ion

Biomass chain design and policy advise

Protein content < 5 % 5 % 15 % 35 % 50 %

ExamplesWheat

straw

Corncobs

Sugarcaneleaf

Rapestraw

Beet leaf

Rapemeal

Soy meal

Cost (€/ton) 50-80 50-110 100-140 150-180 300-350

Protein groups

0

100

200

300

400

500

600

700

800

0

10

20

30

40

50

€/

ton

Biorefinery of rapemeal increases

value components

Animalfeed

Protein

Aminoacids

Ferment.substrates

Ligno-cellulose

Fibres

Phosphorus

Rest

Need for a sustainable protein supply

World

population

continues to

grow

Need durable

protein supply

Need efficient

protein

extraction

methods

Question??

Do we have enough land to feed 9 billion people in 2050?

Do we have enough protein?

7 billion now and 9 billion people in 2050

0.8 g protein/day.kg body weight

About 500 million tons protein produced via mais, wheat, etc

19

Question: do we have enough protein?

With average body weight about 50 g protein per day

With 7 billion people 122 million ton protein needed (500 million tons produced)

No problem?

Food waste (30-40% to the bin)

For 1 kilo meat we need:

● Chicken 2.5 kg wheat

● Cow 7 kg wheat

More reasons

20

21

Primary Protein structure

22

Question: what is the structure and the name of the simplest amino acid?

Protein secondary structure

23

CNO

CH

C

R

H

C

N

C

C

N

O

CH

H

O

H

R

C N

O

C

H

C

C N

O

C

C

H

C N

O

C

C

H

Trans orientation of the chain around the peptide bond

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CHN

CCH

NC

CHN

CCH

NC

CH

O O

CHC

NCH

CN

CHC

NCH

CN

CH

H H

O

R O

H

R

O RR

O

H

R

R

H R

H

O

R

H R

H

O

R

the antiparallel -sheet structure

Secondary structures

Alpha helix

Tertiary structure

25

Orange: random coil

Yellow: β sheet

Green: α helix

Quaternary structure

26

Keratin fibre

Current ‘classes’ of protein

Whey proteins:

Soluble over pH rangeHeat denaturation

Soy proteins:

Insoluble pH 4- 6Heat denaturation

Egg proteins:

Heat denaturation

Caseins:

Insoluble pH 4- 6(random coil)

Gelatin:

Reversible with heating(random coil)

Novel protein sources:

Size, structure, solubility, charge,

hydrophobicity

How are proteins isolated?

What are the molecular differences between proteins?

How to study modifications / unfolding?

1- Subunits built from an acidic (A) and basic (B) polypeptide chain

2- A and B chains linked by disulfide bridges

3- Association of subunits into larger structures (Trimer /Hexamer)

Acidic Basic

pH ~7‘11 S’

hexamer

Soy protein (glycinin)

ΔpH, T, I

Glycinin 7STrimer

Glycinin 3SMonomer

https://www.youtube.com/watch?v=wvTv8TqWC48

https://www.youtube.com/watch?v=qBRFIMcxZNM

30

Protein sources in compound feed

Bottle necks

Production (million tons) protein-rich crops

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

Europe

Wheat (195)

Potato (117)

Mais (94)

Barley (79)

Sunflower seed (24)

Rapeseed (22)

Oats (13)

Triticale (13)

Rye (12)

Soy bean (6)

Rice (4)

Peas (3)

Sorghum (0.8)

Millet (0.6)

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

World

Maize (875)

Rice (718)

Wheat (675)

Potato (368)

Soy bean (253)

Barley (132)

Rapeseed (65)

Sorghum (58)

Sunflower seed (37)

Millet (25)

Oats (21)

Rye (15)

Triticale (13)

Pea (10)

Commercial production of protein isolates

CerealsOil

cropsLegumes

Plant

tuberMeatBlood Milk Egg

Plant origin Animal origin

Wheat

protein

Soy

protein

Pea &

Lupine

protein

Potato

protein

plasma,

haemo-

globine

Casein

Whey

White

Yolk

Broad

range

Food Protein prices [€/kg]: high functionality

Vegetable proteins Animal derived proteins

Soy meal (40% protein) 0.8 Gelatine (low quality) 2

Soy concentrate 2 Gelatine (high quality) 4-6

Soy isolate 3 Collagen 4-6

Pea concentrate 2 Egg white powder 6-8

Pea isolate 3 Egg yolk powder 4-6

Lupine concentrate 2-4 Plasma powder 3-5

Potato protein (feed) 0.8 Haemoglobin powder 1

Potato protein (food) >3 Milk powder 2.2

Wheat gluten 1.2 Whey concentrate (30% protein) 1.6

Maize gluten feed 0.12 Whey concentrate (80% protein) 5.5

Zein 25 casein/caseinate 6.5

Prices (2015)

Product Price [€/kg]

Soybean 0.46

Soybean meal 0.40

Rapeseed meal 0.23

Sunflower seed meal 0.25

Peas 0.25

Lucerne 0.19

Wheat 0.17

Maize 0.17

Barley 0.19

Functional properties

Solubility

Gel formingEmulsification

Foaming

Protein concentrates and isolates

in food

Functionality related to structure

Protein functionality

You’re so hot, you

denature my proteins.

Protein functionality

Active (functional)

protein

Denatured protein

Peptides and free

amino acids

Newly launced products with plant proteins

in 2014 (Innova)

Main outlet food

and FeedRestriction new

proteins in food

due to Novel Food.

Potential in bio-

based

https://www.google.nl/url?sa=i&rct=j&q=&esrc=s&source=images&cd=&cad=rja&uact=8&ved=0ahUKEwiWj--54brSAhWB1RoKHeApDb4QjRwIBw&url=https://www.beautytopia.nl/review-brazilian-keratin-therapy-30-day-smoothing-treatment/&bvm=bv.148747831,d.d2s&psig=AFQjCNF2N20zdhtUasU5UsWW0Pf07pnVng&ust=1488645013461750

https://www.google.nl/url?sa=i&rct=j&q=&esrc=s&source=images&cd=&cad=rja&uact=8&ved=0ahUKEwiWj--54brSAhWB1RoKHeApDb4QjRwIBw&url=https://www.beautytopia.nl/review-brazilian-keratin-therapy-30-day-smoothing-treatment/&bvm=bv.148747831,d.d2s&psig=AFQjCNF2N20zdhtUasU5UsWW0Pf07pnVng&ust=1488645013461750

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Routekaart Biobased Economy

http://www.biobasedeconomy.nl/routekaart/

Question?

What are the most important oil crops in Europe?

What is PPO?

Which oils are used for biodiesel production. How much was produced in 2009?

What is DDGS? How it is formed and for what it is used?

Biorefinery of fresh biomass

Grass 5-15 % Duckweed 25- 40 %

Fountain herbWater pest Nettle Water nap

Beet leave 23 %

Green Biorefinery research

Fresh Biomass (leaves/grass)

Silage

Separation

Smart Separation

Pre-treated material

Juice

Cake

Coagulation

Smart Separation

Protein Cake

Amino acids

Organic acids

Minerals

Upgrading fractionisation

Pulping

Dissolving cellulose

Chemical pulp

FIller

Feed

Sugars

Bioethanol

Lactic acid

Fibres

Mechanical pulp

Isolation material

Composites

Chlorophyll

Mechanical pretreatment

Other components

Pellets

Rubisco

Sugar beet leaves: new protein products

Pilot scale white protein production

Reaction Centrifugation Microfiltration

Side product Proteins obtained by various processes

Duckweed

Fastest growing plant worldwide (doubles every 2 - 3 days)

yield 20-30 ton/ha (dry solids)

contains 25-40 % high quality protein

protein yield per ha 5 – 7 x higher than soybean

also source for fibre, antioxidant, minerals, biogas

used for feed worldwide

used for food (vegetable) in SE-Asia

Integrated Lemna Farming Systems in

Indonesia

BiodigesterMethane for

cookingDigestate

Lemna pond

Feed replacement

Manure

Cows, pigs, chickens,

ducks, fish

5-20% of commercial feed replaced by LemnaSavings up to 10% of annual net income

Microalgae

Fish feed

Food supplement

Biodiesel

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Harvesting

Flocculation and centrifugation

60

Flocculation Centrifugation

Cell disruption

61

Bead millingHigh pressure homogenisation

Before cell disruption After cell disruption

Extraction

Lipid pigment extraction by organic solvents

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Filtration

63

Starch

Proteins

Pigments

Aqueous phase

Flow

FiltrateRetentate

Cell disruption

N.

ole

oa

bu

nd

an

s

Centrifugation

N.

ole

oa

bu

nd

an

s

Pellet Pellet

Aq

ue

ou

s p

has

e

Pellet Pellet Pellet

30

0 k

Da

Products

64

Products

65

Oil

Pigments

1, 2, 3 are pigments

Recovery from fresh biomass

0

20

40

60

80

100

0 20 40 60 80 100

Pro

tein

pu

rity

(w

/w %

)

Yield (%)

SBL

SBL

Alfalfa

Series3

Algae2

Duckweed

Coliflower

Soy

Lupine

Rapeseed

Pulses

Thesis Angelica Tamayo Tenorio

Question

What is the reason why protein extraction from green biomass is more complex than from the traditional crops (soy, wheat etc)

67

68

Green biomass: • Only rubisco – large pool• Heterogeneous – membrane

proteins

Green biomass vs. protein crops

Protein crops: • Large pool, similar properties

(albumins, globulins, prolamins)

Insoluble SolubleSoluble

RubiscoMembrane

proteins

Photosystem I (Dekker & Boekema, 2005)

Albumin16%

Globulin70%

Glutelin8%

Prolamin2% 4%

Leaf proteins Soy proteins

(Siwela & Amonsou, 2016)

69

Green biomass: • Only rubisco – large pool• Heterogeneous – membrane

proteins• No storage protein / Enzymatic role• Small length scale arrangement

Green biomass vs. protein crops

Protein crops: • Large pool, similar properties

(albumins, globulins, prolamins)• Storage protein• Larger arrangement: Protein

bodies

0.3 µm

Thylakoids membranes Photosystem I

10 nm

Fragment of legumes

200 µm

Starch

Protein bodies< 3 µm

70

Green biomass: • Only rubisco – large pool• Heterogeneous – membrane

proteins• No storage protein / Enzymatic role• Small length scale arrangement• Moisture content: 85-95%

Green biomass vs. protein crops

Protein crops: • Large pool, similar properties

(albumins, globulins, prolamins)• Storage protein• Larger arrangement: Protein

bodies• Moisture content: 10-15%

High perishability

Quick stabilisation/processing

Low bulk density - transportation

Process development

• Decentralised configurations

• Process right after harvest

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Green biomass: • No storage protein / Enzymatic role• Small length scale arrangement• Only rubisco – large pool• Heterogeneous – membrane

proteins• Moisture content: 85-95%• Low yield

Green biomass vs. protein crops

Protein crops: • Storage protein• Larger arrangement: Protein

bodies• Large pool, similar properties

(albumins, globulins, prolamins)• Moisture content: 10-15%• High yields, high purity

Biorefining in energy sector

Energy sector deals with big amounts

Feed uses large amounts of protein containing biomass

Balance between amounts AND functionality AND price

Interesting biorefinery concepts:

● Bioethanol production (brewers’ spent grain,DDGS)

● Biogas production

● Oil seeds refinery (e.g palm kernel meal,rapeseed)

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Pelletizing

PREPARATION

EXTRACTION

Dehulling

Crude oil

Seeds

Cooking

Flaking

Flakes

Cleaning

Cracking

Pressing oil

Cake

Pressing

Pellets

Pelletizing

Extraction

oil

Distillation

Meal

Désolventization

Marc Miscella

Hexane

Extraction

Sunflower

Rapeseed

75

seeds

flaking

cooking, pre-pressing

dehulling methane

cake

cold pressing

Crude oil

refining

biodiesel

GAME, CO2

alcohol extraction

oil meal

water extraction

oil quality

proteins

Etc.

GAME, CO2

quality

proteins

Etc.

oil

cakes biopolymers

Biorefinery options

Optimise use of protein containing sources

- Which sources for which end applications?

More knowledge necessary on interaction matrix components in

relation to extractability (biology, biorefinery, physica etc)

How much biorefinery (purity) and separation

stages is needed for which end markets?

- Use biomass as such

- Enriched fractions

- High purity, high value