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Biofuels and Biotechnology > Lecture 4 - Modification of plants and biomass crops to increase biomass potential for biorefiniing > Flashcards

Lecture 4 - Modification of plants and biomass crops to increase biomass potential for biorefiniing Flashcards

1
Q

What is a biorefinery?

A

A facility that integrates biomass conversion processes and equipment to produce fuels, power heat and value added chemicals (ethanol) from biomass.

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2
Q

Use US maize crop to ouline gains in agriculture

A

20% deline in the area planted to produce maize in the past 100 years, even though the number of people are increasing as are consuming levels

This is because maize production have increased 6 fold in the same period.

700% yield increase in 50 years due to the initiation of the green revolution:

  • application of agrochemicals, genetics, intensification
  • improvement in yeild, disease resistance but not for biomass quantity and quality
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3
Q

What are the economics of liquid biofuel production?

A
  • making liquid biofuels is a business with tight margins therefore the ability to improve is important
  • competes with fossil fuels that are simply pumped out of the grond and refined
  • a high volume, low value business (CAPEX investments only become worthwhile at scales around 1m Tonne of biomass per year)
  • Sugarcane ethanol was government supported for 30 years in Brazil before it became competitive with gasoline
  • Lignocellulose is a challenging substrate as only ~70% of substrate is polysaccharide and only ~40% is composed of hexose sugars that are readily fermented (except in conifers)
  • Biggest challenge in lignocellulose is to get the potential sugars out in a cost-effective manner
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4
Q

What is the bioethanol cost challenge?

A

EU mandates that by 2020 20% of liquid transportation fuel should be biofuel, but that only 5 of this 20% can come from food commodities (first gen.)

1st gen biofuels: sugar/molasses AND starch/grains

2nd gen biofuels: straw/lignocellulose

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5
Q

What are the comparative features of lignocellulose vs. starch/grain as bioethanol feedstocks?

A

Lignocellulose

  • cost much higher
  • energy inputs higher
  • cost mostly from pre-treatment

Starch/grains

  • easy to digest
  • not too expensive
  • cheap commodity
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6
Q

What are the bioethanol process requirements of sugar/molasses; starch/grain; straw/lignocellulose?

A

Sugar/molasses: fermentation followed by distillation

Starch/grain: enzymatic hydroylsis, fermentation followed by distillation

Straw/lignocellulose: Pre-treatment, enzymatic hydrolysis, fermentation and distillation (Pre-treatment and enzymatic hydrolysis a third of the cost)

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7
Q

What is the costing of bioethanol? How much wheat straw is needed to produce 1 tonne of ethanol?

A

Fermentation metabolism = 50% productive

2g sugar produces 1g of ethanol and 1g of CO2

Wheat flour (1st gen): 80% starch, 10% protein

Wheat straw (2nd gen): 40% cellulose, 25% arabidnoxylan

Therefor 5 tonnes of wheat straw neccessary to make 1 tonne ethanol

(5 tonnes wheat straw: 5 X 0.4 = 2 tonnes cellulose. Fermentation metabolism 50% effective. 2 tonnes cellulose = 1 tonne ethanol)

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8
Q

What are the commodity proces of sugar, molasses, feed wheat, wheat straw and ethanol as of 2013? (£/t)

A

Sugar (refined): 410

Molasses: 180

Feed wheat: 150-200

Wheat straw: 60

Ethanol: 380

Wheat straw: 5 X 60 = £300 for 1 tonne ethanol; production and expenses also high (pretreatment)

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9
Q

What is biorefining?

A

The sustainable processing of biomass into a spectrum of biologically based poducts (food/feed/chemicals/materials) and bioenergy (biofuels/power/heat)

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10
Q

How do you biorefine first generation biofuels? (starch/grain)

A
  1. Milking, steaming (sterilise), amylase to hydrolyse starch
  2. produces glucose for fermentation
  3. spent grain and solubles are dried and sold as animal feed, with remaining biomass used in distillation
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11
Q

What is the process of biorefining in 2nd generation biofuels? (straw/lignocellulose)

A
  1. Chooping, steam explosion or mild acid hydrolysis
  2. Use of complex cellulase cocktails and high solids loadings
  3. results in mixed sugars for fermentation (yeast only ferments hexose sugars)
  4. Either use distillation or produce lignin and xylans as waste products

Only ends up fermenting ~40% biomass

Can hydrolyse starch to uncrease price for biofeed as an increased availaible protein

60% waste burnt to provide heat and power

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12
Q

Why do pretreamtents of second gen biofuels present such a significant cost to biorefining?

A
  • High energy and specialised equipment- high temperature and pressure (enzymes in effective until lignocellulose broken up)
  • Or high ammonia concentrations and pressure, with associated ammonia recovery- expensive to construct
  • Or acid resistant equipment

Represents 30% of the cost of processing

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13
Q

Why are pre-treatments needed and how is this acheived?

A

Pre-treatments are needed to allow enzymic saccharification by:

  • opening up the structure of the biomass to give increased enzyme accessibility
  • breaking up the lignin network
  • opening up cellulose structure
  • remove/avoid inhibitors of saccharification and fermentation (high temperature generates inhibitors from the feedstock)
  • sterilize the feedstock for fermentation
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14
Q

What inhibitors of fermentation are produced during pre-treatment?

A
  • Hydroxy Methyl Furfural (HMF), Furfural (formed by dehydration of hexose and pentose sugars at high temperature and low pH)
  • Cinnamaldehyde, p-hydroxybenzaldehyde and syringaldehyde (from lignin breakdown)
  • Acetate, released from acetyl esters of hemicelluloses at high temperature

These inhibit the actions of microorganisms

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15
Q

What are the features of the enzymes used in biorefining?

A
  • Cellulase cocktails based on culture extracts from filamentous fungi (Trichoderma reesei)
  • Enzyme companies have invested heavily to improve activities
  • Even so, typically need more than 5% dry weight loadings to get effective digestion
  • Enzymes can account for 10-20% of overall process costs
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16
Q

What advantages could come with improving the digestibility of plant biomass?

A
  • Lower energy/chemicals for effective pretreatment
  • Lower enzyme loading
  • Decrease inhibitor production (generally a product pretreatment severity)
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17
Q

What are the roles of lignin?

A

Not in the primary cell walls

  • strong but extensible
  • polymer networks are highly hydrated
  • held together with non-covalent crosslinks between polymers

Are in the Secondary cell walls:

  • strength and rigidity
  • contain much less water
  • polymers are more tightly packed and permeated
  • sealed and crosslinked by the hydrophobic polyphenol lignin

Lignin increases wall rigidity and renders the network insoluble and resistant to degradation

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18
Q

Where and why has lignin modification been studied?

A

For the paper industry

  • lignin is essential for making pulp
  • requires a lot of chemicals and potentially toxic waste. which the industry has to dispose of

The forage industry

  • biomass digestibility limits the nutritional quality of agricultural residues and of forage grasses
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19
Q

What is the function of the monolignol biosyntheitc pathway?

A
  • production of H-, G-, and S- lignin
  • lots of genes involved
  • chemicals with very similar structures
20
Q

Outline the Chen and Dixon 2007 paper, ‘Lignin modification improves fermentable sugar yields for biofuel production’

A
  • resistance to sacchairifcation is a major limitation for the conversion of linocellulosic biomass to ethanol
  • Transgeic alfalfa lines were downregulated in 1 of 6 linin biosyntheitc enzymes
    • found that: resistance to acid pretreatment and enzyatic degradation was distinctly proportional to lignin content
    • some transgenics yield around two times more sugar from cell walls than the wildtype
  • HCT results in a doubling of yield of sugards
    • tested before and after treatment
  • whilst HCT plants were highly digestable they were also highly dwarfed
  • other genes C4H had a smaller effect on digestibility but not a huge effect on yield

Process: Measured sugar content with or without pre-treatment of digestibility mutants - found hct mutants - high sugar, dwarfed

  1. Biomass - dry, milled, extractive-free tissue (1.measured sugars with cellulase treatment) then treated with H2SO4<span> </span>(measured sugar levels) to produce lignocellulosic residue (measured sugars with cellulase treatment)
21
Q

Outline the Van Acker paper 2013, Selected arabidopsis K/O mutants for analysis of biomass digesatibility and lignin

A
  • arabidopsis not a crop
  • accumulated lots of variants and a comprehensive view of the lignin biosynthetic pathway
  • measured impacts of the gene K/O in monolignol and pheylpropanoid pathways on:
    • height
    • mass
    • cell wall residue (lignin content)
    • cellulase conversions
    • pre-treatment effect
  • Identified mutants that did not affect yield by did affect digestibility
  • some lines were incredibly digestible post hot water treatment and enzyme digestion
    • c4h-2
    • ccr1-3
    • ccr1-6
  • Identified a good correlation between lignin content and digestibility
    • although: as these were all pre-selected lignin mutants (not selected for digestibilty) this may not be the only influencing factor (bias)
22
Q

Outline the lignin and saccharification relationships in 22 wild accessions of brachypodium distachyon

A
  • took samples of brachypodium distachyon from around the world
  • showed good correlation with digestibility even though not specifically selected for anything

Reverse genetics approach to improving digestibility - hypothesis led, hypothesis testing -> went onto use forward genetic screening (needed a more empirical approach)

23
Q

Suggest foward genetic screening approaches to improve digestibiility

A
  1. looking for correlations between disgestibility and composition across panels of natural diversity
  2. using assocaition screening and mutant screening
24
Q

What is the process of screening for mutants that affect digestibility

A
  1. Begin with a large population of chemically mutagenised plants
  2. screen plants for digestibility
  3. identify plants with high digestibility
  4. use future generations to map the mutation
25
Q

Outline Brachypodium as a model grass

A
  • •Small genome (300 Mbp)
  • •Fully sequenced
  • •Genetic tool available
  • •Diploid
  • •Small and easy to grow
  • •Self fertile
  • •Short life cycle
    *
26
Q

What are the differences between monocot vs. dicot secondary cell walls?

A

Monocot

  • main hemicellulose: glucuronoarabinoxylans
  • ferulates present and form cross links
  • mixed link glycans present
  • higher lignin content

Dicot

  • Glucuronoxylans main hemicellulose
  • mannans and glucomannans in higher quantities
27
Q

What was the process by which high digestibility mutants were identified

A
  1. Screened for saccharification potential and looked at the accessions at the extremes, particularly high (highest saccharification +72%) and particularly low (lowest saccharification -51%)
  2. Selected mutants that were passed down through generations (mendelian segregation - mutation in germ cell)
  • 37 high digestibility mutants selected
  • 12 mutant lines out of 3000 had heritable increase in digestibility
  1. Lignin content was measured (acetyl bromide method)
    * Only half mutants were lignin mitants therefore lignin not the only thing important in digestibility
  2. sac4 identified as a cadidate gene
28
Q

Outline sac4 as a candidate gene?

A

SNP in Bradi5g14720

  • Proline to leucine
  • preicted to affect stucture or function
  • confirmed mutation against WT and reference sequence
29
Q

Where does HCT act in linin monomer synthesis?

A

Acts to catalyse the reaction from p-coumaroyl CoA

to

Caffeoyl CoA

Conversion from making H monomer to G or S monomer

30
Q

What was the HCT activity in WT and mutant (sac4) stem extracts?

A

WT: high HCT activity

sac4: lower HCT activity, 50% reduction

Boiiled extract and extract only as negative controls

31
Q

What was the activity of the WT and sac4 HCT gene products produced in E.coli?

A

Measured activity of the mutated protein, found to be totally innactive (comparable to the empty vector)

As there are other mutants this protein has to be redundant

32
Q

What does HCT down-regulation cause in Arabidopsis, Medicago, Tobacco and Bracypodia? Why?

A

HCT down regulation causes stunting in Arabidopsis, Medicago and Tobacco

Doesn’t cause stunting in the brachypodium sac4 mutant.

K/O is lethal in arabidopsis, 1 copy

In brachypodia the gene family is increased (three functional HCT genes) has multiple copies so redundancy

sac4 is a K/O mutation in just one of the three functional HCT genes in brachypodium

33
Q

What is the sac1 mutant?

A

mutation in a 61 glycosyltransferases family

Impact of the study:

  • lignin is important
  • sac1 represents a biosynthetic gene for linking lignin and hemicelluloses in brachypodium
  • sac1 has a big impact on digestibility of plant materials, significant reduction in the concentration of ferulic acid
34
Q

What is the significance of ferulic acid linkages in sac1?

A
  • Hemicellulose to hemicellulose linkage
  • Hemicellulose to lignin linkage
  • Form a nucleation point
  • Sac1 mutant affects linkage between hemicellulose and lignin
35
Q

What is the importance of Xylan feruloylation?

A
  • sac1 mutants show the biggest impact on dingestibility in brachypodium
  • xax1 mutants in rice are highly digestible
  • may be more digestible than most lignin mutants
  • ferulic acid esters cross link oxidatively to form dimers and trimers crosslinking arabinoxylan polymers to one another
  • form the points of covelalent connection between arabinoxylans and lignin
36
Q

Describe the gux mutant

A
  • In dicots the major hemicellulose is not arabinoxylose
  • lignin may link to me-glucuronic acid residues
  • But gux mutant (lacking glucuronic side chains) does not have significantly reduced digestibility
  • Xylan more easy to extract from stems
37
Q

How have association genetics in barley made use of natural diversity?

A
  • Looking for peaks of inheritance of QTL for digestibility
  • High density single nucleotide polymorphism markers defined in 850 elite barley cultivars from around the world
  • Phenotype screening of these cultivars alonside SNP association mapping can define quantitative trait loci QTL at high resolution
  • Quantitative traits (e.g. digestibility) are determined by many genes, QTL analysis allows the identification of major genetic contributors to a trait
  • Identifies the trait among elite crop cultivars
38
Q

Describe the Association genetics experiment 2010

A

(Intro)

  • 640 elite 2-row spring barley genotypes
  • grown in polytunnel in 2010, 5 reps
  • various phenotypes measured (including straw biomass)
  • samples collected and SNP genotypes
  • 3240 samples of 2nd internode base collected and powered
  • evaluated for sacharrification

(results)

  • 640 genotypes of elite spring barley ranked by yield of sugar released in a saccharification assay (datapoints are means of 5 replicate plants per genotype - 3 technical assay replicates per plant)
  • GWAS identify severel QTL for digestibility in barley straw. Results indicate:
    1. Other factors besides lignin content affect digestibility
    2. Can increase digestibility without affecting stem strength
39
Q

Why do genome studies in Rice?

A
  • Wheat genome: massive, hexaploid, not fully sequenced
  • Barley genome: Large, has not been fully assembled, diploid, can serve as a model for wheat
  • Rice genome: Smallest genome of any cereal, 2 X arabidopsis, second largest crop produced worlwide
40
Q

Why would Rice be used as a model and feedstock for developing a cereal straw biorefinery?

A
  • Vietnam produces more than 60m tonnes of rice straw
  • Most is burned (around 700m tonnes year/globally)
  • Could be used for bioenergy or biofuels
  • Air pollution by burning leads to premature deaths, shading of crops and reduced crop yeild, generation of trophospheric ozone, global warming from black carbon
  • Bad quality for animal feed (high silica content - up to 10% dry weight), needs to be removed so can have next crop (flooded rice field production)
  • High levels of silica make it unpractical for combustion to produce bioelectricity
41
Q

Outline the experimental process for developing a cereal straw biorefinery using rice as a model and feedstock

A

Rice diversity panel for GWAS studies

  1. assembled diversity of 180 rice accessions with the vietnam acadamy of agricultural sciences
  2. mapped SNPs using genotype by sequencing methods
  3. measured digestibility on our robotic platform
  4. silica content mmeasured using X-ray fluoresence
  5. Looked at digestibility over two seasons
  6. 5 QTL identified reproducibly over 2 seasons (environmentally robust QTL), one identified as a QTL for lignin content
  7. Identified a weak correlation between lignin and digestibility
  8. Saw a stronger correlation between digestibility and silicon content
  9. Identified a particularly digestible line with low lignin and low silica - commercial variety, grown as has good lodging resistance and lots of biomass (wider stem - geometric stability)
  10. Did a GWAS for Si content - identified four strong QTL, one as the same as identified for high digestibility. Looked at the connections between silica and hemicellulose in rice.
42
Q

Outline the impacts of improving lignocellulose digestibility

A
  • Can be done without impacting field performance
  • Can help decrease the need for high-energy pretreatment
  • Lignin is a major determinant - innovation that led to the success of land plants
  • Xylans are imporant (ferulic acid linkages)
  • There are good enzyme systems for digesting the inherantly indigestible cellulose
43
Q

Outline silicon in grasses and rice

A
  • Grasses differ from other angiosperms in accumulating significant quantites of silica
  • Si appears to remove stress and pathogen resistance in grasses
  • High Si grasses are unpalatable to herbivores
  • Rice accumulated high Si (up to 10% dry weight) and requirews Si for growth
44
Q

Outline gymnosperms as an alternative source of lignocellulose

A
  • Major forestry crop
  • High lignin and low digestibility
  • Major hemicellulose is galactoglucomannan
    • all hexose sugars which most fermentation organisms prefer, in contrast to angiosperms such as hardwoods or grasses where it is xylans (mostly pentose sugars)
  • Roughly 70% of conifer wood can yeild fermentable hexose sugars compared to 30-40% angiosperms
45
Q

Outline the role of grasses in biofuels and biorefineries

A
  • Major staple crops are cereals - abundant residues produced in parallel with food, cereal straw ro sugarcane bagasse
  • C4 grasses are some of the highest yielding crops under low water conditions
    • sorghum, maize, energy cane, switch grass, some miscanthus
  • Unique features of grass biomass - complex hemicellulose (arabinoxylans), lower lignin, role of silicon (emerging)
46
Q

What are the important factors in biomass digestibility?

A
  • Limiting factor for producing fermentation feedstock from plant biomass and for cost effective biofuel production
  • Lignin is a major factor limiting digestibility especially in broad leaf plants
  • connections between hemicelluloses and lignin are very important in determining digestibilty
  • cellulose crystallinity makes it hard to diest, but there are no good demonstrations that this can be manipulated to a significant extent
  • grasses typically have less lignin tha other plants and in rice Si content is an important factor
47
Q
A

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