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Unlocking the full potentials of biomass: Higher value products from biorefining

Lene LangeProfessor, PhD et Dr.Scient.Center for Bioprocess EngineeringDTU, Technical University of Denmark

Developing the Bioeconomy(Background for the presentation)

• 20 years in Biotech Industry, Novo Nordisk & Novozymes• Last 10 years back in University

Advisory Roles*Scientific Committee for EU JU: Bio-Based Industry, 3.7bill Euro*Expert Group, reviewing EU Bioeconomy Strategy*Nordic Bioeconomy Panel *Danish Bioeconomy Panel *Danish Reference Group on Bioeconomy, Ministry of Research *International Advisory Boards: BIOTEC & NSTDA, Thailand

Global challenges: Bioeconomy can deliver!

• Feeding the world –Getting more out of land and harvest

• Mitigating Climate Change –Substitute fossils with renewables

• Bioeconomy -An important part of the Circular Economy

The Global Focus in Bioeconomy is changing

Before: • Biomass to Bioenergy; using only the energy content

New trend: • Use the full potentials of the biomass:

– Cascading use of biomass: Use its complexity– Smaller biorefineries; many types of biomass– Focus also on health and nutrition

• Proteins, Lipids (Omega-3); and Prebiotics

DTU Chemical Engineering, Technical University of Denmark

The Biomass Cascade Pyramide

DTU Chemical Engineering, Technical University of Denmark6 29 May 2017

The Circular Bioeconomy– so much more than a sugar platform and biofuel


Bioeconomy -many types of Biorefineries=> many new enzymes needed!

• The Yellow Biorefinery (straw, corn stover, wood)

• The Green Biorefinery (fresh green biomass)

• The Blue Biorefinery (fish by-catch & cut offs; sea weeds)

• The Red biorefinery (slaughterhouse waste)

• The White Biorefinery (agroindustry side streams)

• The Brown Biorefinery (sludge & household waste)

Products: Food & Feed ingredients; Health & Nutrition; Wound & Skin care; Bioplastics; Chemicals; New materials; Fuel; Fertilizer


New Enzyme Discovery Technology:Peptide Pattern Recognition, PPR

•A non-alignment based sequence analysis approach• Algorithm-based: identifies conserved peptide patterns;

create groupings, correlated to enzyme function• Can predict function of enzymes with 80 % - 97 % accuracy• Can be used to find more enzymes of same function; also

very distantly related

• Fast, easy, automatic => Suitable for fast track mining of (meta) genomes and (meta) transcriptomes

References:Busk & Lange 2012 Patent application IPC G06F 19/20Busk & Lange 2013 Applied and Environmental Microbiology. 79(11): 3380-91 Busk & Lange 2013 AMB Express 3, 47Busk, Pilgaard, Lange 2014 PLOS ONE 9(12)Busk & Lange 2015 BMC Genomics 16: 368


AALAALVAGAAAQQACSLTTETHPRLTWKRCTSGGNCSTVNGAVTIDANWRWTHTVSGSTNCYT

MRTAKFATLAALVASAAAQQACSLTTERHPSLSWKKCTAGGQCQTVQASITLDSNWRWTHQVSGSTNCYT

MVSAKFAALAALVASASAQQVCSLTPESHPPLTWQRCSAGGSCTNVAGSVTLDSNWRWTHTLQGSTNCYS

MMYKKF


M ALAALVAGAAAQQACSLTTETHPRLTWKRCTSGGNCSTVNGAVTIDANWRWTHTVSGSTNCYT



MYKKFA


MM LAALVAGAAAQQACSLTTETHPRLTWKRCTSGGNCSTVNGAVTIDANWRWTHTVSGSTNCYT



YKKFAA


Protein list Peptide list

Output: group of proteins; sharing a list of conserved peptides


Hotpep -for function targeted protein discovery

Finds all genes of interest in a genome, transcriptomeor metagenome/transcriptome (use the peptide patterns)Speed is 30 megabases per hour per 100 protein familiesOutput: Listing protein families found; converted into list

of functions (EC numbers) being present in the data base Synergy: Hotpep of genome plus MS on Secretome =>

document protein composition plus abundance!

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The Yellow Biorefinery -straw, stover, wood

Many Value Chains: From Cellulose• Sugar platform for biochemicals, biomaterials and biofuels

From Hemicellulose• Gut Health promoting Prebiotic animal feed and food

ingredients

From lignin• A broad spectrum of materials, binders and chemicals

NEW: Wooden biomass mixed with algae => Growfungi on it; use this FUNGAL protein for animal feed!

14 29 May 2017

The Green Biorefinery

• Decentralized & many value chains– Small scale– On farm and in local community

• Feedstock: fresh green leaves, grass, clover etc• Simple processing: screw press => pulp and juice

• Low investment• Mobile equipment is an option

Green Biorefinery: Change in agricultural practice=> more product, less pollution

Cereal (barley/wheat) stop photosynthesis 3rd week of July =>No photosynthesis in August, September & October!

Doubling BIOMASS per hectar: less use of fertilizerFull use of sunlight if changing to perennial grass and clover ⇒ Twice us much biomass!

POLLUTION –cut down with 50%:Grass root systems grow year around => Less negative impact on environment as less surplus of nutrient run-off to freshwater (rivers and lakes) and sea (marine)

Green biorefinery:many new products!-need for new enzymes

• Protein rich animal feed –substituting for imported soy protein– Soluble feed protein recovered by precipitation– Additional protein extracted by protease treatment of pulp (Rubisco

protein; 40% more for Food (ref: Dotsenko & Lange 2016)

• Prebiotic feed ingredients from hemicellulose – For non-ruminants, pigs, chicken and fish

• Minerals used as fertilizer: circulated back to the soil!

Hemicellulose polymers: enzyme discovery => the best C5-oligos, prebiotic feed ingredients (=> improved gut health; less use of antibiotics)


Discovery from nature´s own biomass conversion!-prototypes of yellow and green biorefinery found in nature -cow rumen is the anaerobic version of a biorefinery

•Termite larvae (first studies of gut-channel, cDNA, NZ 2000)

• Termite fungal garden

•Cow rumen, rumen fungi

•Leaf cutter ant, fungal garden

•Ectomycorrhizal fungi-are also cellulose degraders


Blue Biorefinery: upgrade of marine biomass

•Seaweeds

•Fish, discard and innards

•Fish, by-catch

•Mussels as biomass

•Invertebrates (sea cucumber)

20 29 May 2017


Blue Biorefinery-the value cascade from macroalgae

Seaweeds components:– Alginate– Laminarin– Fucoidans– Proteins– Antioxidants

•New EU JU BBI: ”MacroCascade”; Value added products from algae

– new enzymes needed!

21 29 May 2017

Macroalgae/seaweeds Value chains

Products:

• Food and Feed ingredients• Health promoting compounds (prebiotics etc)• Skin care & Cosmetics• Wound healing compounds

Upgrade of organic household waste

• Central sorting and separation now possible:– REnescience -steaming & liquefaction by enzyme treatment

• Processing– Cascading use of all organic components

• Products– Organic acids, Materials, Fertilizer, (Animal feed?)

• Challenges –steaming of organic waste => chemical residues from plastic bags! (one more reason for bioplastic)

A modern slaughterhouse is a Red Biorefinery : -a new resource for upgrade to higher value products

• Blood-based, Iron-rich food supplement and drugs• Protein-dense products for elderly and convalescence• In Denmark: Danish Crown Ingredients, DCI


New target: Produce Protein-rich animal feed from chicken featherand pig bristles, using a blend of enzymesResearch: enzyme degradation, recalcitrant protein

25 29 May 2017


Expression of all three keratin degradingproteases of O.corvina in Pichia!

Series of Experiments:PPR, comparative genomics, Fractionation, MS, activity etc

• Three types of proteases needed, S8, M28 and M3 –All successfully expressed in Pichia, (bioreactor)

• Protein could be recovered from all three (/HIS-tag)

• All three proteases were active


The proteinaceous structure of keratin can be decomposed by a synergistic effect of three proteases

(Huang, Busk & Lange, 2015 doi.org/10.1016/j.enzmictec.2015.03.001)(Lange, Huang & Busk, 2016, DOI 10.1007/s00253-015-7262-1)


Mapping of PPR determined conserved peptides in AA9, AA10, & AA11 on 3D proteins structures

Busk & Lange 2015, BMC Genomics. 16:368 doi:10.1186/s12864-015-1601-6


LPMO-AA11 found by PPR (on expanded families) to beoverrepresented in keratin degrading fungi:The dermatophytic ascomycetes had on average more than three times as many LPMOs as the non-dermatophytic

(Busk, Lange, Pilgaard & Lange, 2014)


Cluster analysis of the 11 AA11expanded PPR groups Busk & Lange 2015 BMC Genomics 16:368 doi:10.1186/s12864-015-1601-6

DTU Chemical Engineering, Technical University of Denmark31

Hypothesis: LPMOs (AA11 #5767) break the β-1,4-bonds between N-acetylglucosamine moieties in the glycosylation of serine and threonine in the non-coiled head structure of the keratin filaments

(Lange, Huang & Busk, 2016; DOI 10.1007/s00253-015-7262-1)


b-chitin_LPMO#5767

32 29 May 2017


PASC-LPMO No activity found on cellulose!

33 29 May 2017


3 hypotheses for LPMO activity on Keratin-a highly recalcitrant proteinaceous polymer

• LPMO acts upon keratin by de-glycosylation of proteins by oxidizing its GlcNAc-moities (same moity as in β-chitin)

• LPMO acts on keratin by oxidative cleavage of the peptidebonds

• LPMO acts on keratin by a combination both of the above(the four AA11 LPMOs in Onygena corvina may have different modes of action)

New Focus, early lineage fungi:Rhizophydium keratinophilum, a zoosporic Chytridiomycota

Genome sequencing and Hotpep mining of consortiumof R.keratinophilum and associated microbial flora

Heat map of PPR found Proteases from the keratin degrading microbiome, Chytrid/Bacteria/Protozoes

Enzyme discovery from the Chytrid model species: Rhizophlyctis rosea

• Isolate: leg. et det. Peter Letcher

• Collaboration: F.H. Gleason

Ref: Gleason, F.H., Letcher, P.M., and McGee, P.A. (2004) Mycol. Res. 108, 583–589

GH-enzyme activity profile of R.rosea culturebroth

Semi quantitative AZCL-plate assay from Megazyme International Ireland. Units in mm238

PPR & Hotpep Enzyme DiscoveryR.rosea GH enzyme functions as compared to GH families/functionsof other aerobic chytrids and the pathogen B. dendrobatidis

Chytridiomycota

EC Function Rhizophlyctis rosea Spizellomycespunctatus

Homoloaphlyctispolyrhiza

Batrachochytriumdendrobatidis

Total EC/GH 40 13 10 83.2.1.4 endo-1,4-β-D-glucanase 13 GH45 GH5 GH6 GH7 GH93.2.1.91 1,4-β-cellobiosidase (non-reduc) 5 GH63.2.1.176 1,4-β-cellobiosidase (reduc ) 3 GH73.2.1.8 endo-1,4-β-xylanase 19 GH10 GH11 GH303.2.1.78 endo-1,4-β-mannosidase 5 GH26 GH53.2.1.55 α-N-arabinofuranosidase 1 GH433.2.1.37 1,4-β-xylosidase 4 GH3 GH43 GH53.2.1.15 polygalacturonase 3 GH283.2.1.132 Chitosanase 1 GH463.2.1.17 lysozyme 1 GH243.2.1.14 Chitinase 1 GH18 GH18 GH18 GH183.2.1.20 α-glucosidase 2 GH13 GH31 GH31 GH31 GH313.2.1.28 α-trehalase 2 GH37 GH37 GH37 GH373.2.1.3 1,4-α-glucosidase 2 GH15 GH15 GH15 GH153.2.1.45 glucosylceramidase 3 GH5 GH5 GH5 GH5

39

BasidiomycotaAscomycotaZygomycota

Zoosporic fungiAnimalsBacteriaArchaea

Neocalli

Chytrid

Rumen bac

Chytrid

Possible horizontal transfer events

GH5endo-glucanaseEC 3.2.1.4

Gene Structure of five GH5 in the chytrid R.rosea-one of them acquired by horizontal gene transfer?

In clade with bacteria in the GH5 tree

Grouped with otherfungi in GH5 tree


Take home messages:•Many types of biomass=> need for new enzymes

–Much more to learn from Nature

•Sense of Urgency:–Climate change challenged agriculture–Need for improved use of bio-resources

•International Collaboration–sharing best practice in R&D, Agriculture, regularory and incentives/policy

•Think Global –act Local Lene42 29 May 2017

The importance of Bioeconomy for meeting the UN Sustainable Development Goals

byLene Lange, Professor, PhD et Dr. scient.

Center for Bioprocess Engineering

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