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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
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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
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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
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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
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DTU Chemical Engineering, Technical University of Denmark
The Biomass Cascade Pyramide
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DTU Chemical Engineering, Technical University of Denmark6 29 May 2017
The Circular Bioeconomy– so much more than a sugar platform and biofuel
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DTU Chemical Engineering, Technical University of Denmark
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
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DTU Chemical Engineering, Technical University of Denmark
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
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DTU Chemical Engineering, Technical University of Denmark
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DTU Chemical Engineering, Technical University of Denmark
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DTU Chemical Engineering, Technical University of Denmark
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DTU Chemical Engineering, Technical University of Denmark
Protein list Peptide list
Output: group of proteins; sharing a list of conserved peptides
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DTU Chemical Engineering, Technical University of Denmark
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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DTU Chemical Engineering, Technical University of Denmark
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
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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
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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)
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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!
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Hemicellulose polymers: enzyme discovery => the best C5-oligos, prebiotic feed ingredients (=> improved gut health; less use of antibiotics)
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DTU Chemical Engineering, Technical University of Denmark
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
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DTU Chemical Engineering, Technical University of Denmark
Blue Biorefinery: upgrade of marine biomass
•Seaweeds
•Fish, discard and innards
•Fish, by-catch
•Mussels as biomass
•Invertebrates (sea cucumber)
20 29 May 2017
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DTU Chemical Engineering, Technical University of Denmark
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
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Macroalgae/seaweeds Value chains
Products:
• Food and Feed ingredients• Health promoting compounds (prebiotics etc)• Skin care & Cosmetics• Wound healing compounds
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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)
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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
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DTU Chemical Engineering, Technical University of Denmark
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
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DTU Chemical Engineering, Technical University of Denmark
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
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DTU Chemical Engineering, Technical University of Denmark27 29 May 2017
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)
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DTU Chemical Engineering, Technical University of Denmark
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
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DTU Chemical Engineering, Technical University of Denmark29 29 May 2017
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)
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DTU Chemical Engineering, Technical University of Denmark
Cluster analysis of the 11 AA11expanded PPR groups Busk & Lange 2015 BMC Genomics 16:368 doi:10.1186/s12864-015-1601-6
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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)
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DTU Chemical Engineering, Technical University of Denmark
b-chitin_LPMO#5767
32 29 May 2017
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DTU Chemical Engineering, Technical University of Denmark
PASC-LPMO No activity found on cellulose!
33 29 May 2017
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DTU Chemical Engineering, Technical University of Denmark
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)
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New Focus, early lineage fungi:Rhizophydium keratinophilum, a zoosporic Chytridiomycota
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Genome sequencing and Hotpep mining of consortiumof R.keratinophilum and associated microbial flora
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Heat map of PPR found Proteases from the keratin degrading microbiome, Chytrid/Bacteria/Protozoes
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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
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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
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BasidiomycotaAscomycotaZygomycota
Zoosporic fungiAnimalsBacteriaArchaea
Neocalli
Chytrid
Rumen bac
Chytrid
Possible horizontal transfer events
GH5endo-glucanaseEC 3.2.1.4
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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
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DTU Chemical Engineering, Technical University of Denmark
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
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The importance of Bioeconomy for meeting the UN Sustainable Development Goals
byLene Lange, Professor, PhD et Dr. scient.
Center for Bioprocess Engineering