Nanofibers Produced from Agro-Industrial Plant Waste Using Entirely

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Nanofibers produced from agro-industrial plant waste using entirely enzymatic pretreatments Claire Holland, Alixander Perzon, Pierre R.C. Cassland, John P Jensen, Birger Langebeck, Ole Bandsholm Sørensen, Eric Whale, David Hepworth, Robyn Plaice-Inglis, Øjvind Moestrup, Peter Ulvskov, and Bodil Jørgensen Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.8b01435 • Publication Date (Web): 10 Dec 2018 Downloaded from http://pubs.acs.org on December 16, 2018

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is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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Biomacromolecules

Nanofibers produced from agro-industrial plant waste using entirely enzymatic pretreatments Claire Hollanda, Alixander Perzona, Pierre RC Casslandb, John P Jensenc, Birger Langebeckc, Ole Bandsholm Sørensend, Eric Whalee, David

Hepworthe, Robyn Plaice-Inglise, Øjvind Moestrupf, Peter

Ulvskova and Bodil Jørgensena* a

Department of Plant and Environmental Sciences, Section for

Glycobiology, University of Copenhagen, Thorvaldsensvej 40, 1871 Frederiksberg C, Denmark b

c

Novozymes, Krogshoejvej 36, 2880 Bagsvaerd, Denmark

Nordzucker, Technology & Innovation, Falckvænget 1, 4900 Nakskov, Denmark d

e

KMC, Herningvej 60, 7330 Brande, Denmark

Cellucomp Ltd, Unit 3, West Dock, Harbour Place, Burntisland, Fife, KY3 9DW, UK

f

Department of Biology, University of Copenhagen, 2100 København Ø

ACS Paragon Plus Environment

1

Biomacromolecules 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 50

Key Words: Cellulose nanofibers (CNF), nanofibrilated cellulose (NFC),

agro-industrial

by-products,

cell

wall,

enzymatic

degradation

Abstract:

Cellulose fibers can be freed from the cell-wall skeleton via high-shear homogenization, to produce cellulose nanofibers (CNF) that

can

be

used,

for

example,

as

the

reinforcing

phase

in

composite materials. Nanofiber production from agro-industrial by-products normally involves harsh chemical-pretreatments and high temperatures to remove non-cellulosic polysaccharides (2070% of dry weight). However, this is expensive for large-scale processing pretreatment

and to

environmentally obtain

CNF

from

damaging.

An

agro-industrial

enzyme-only by-products

(potato and sugar beet) was developed, with targeted commercial enzyme

mixtures.

It

is

hypothesized

that

cellulose

can

be

isolated from the biomass, using enzymes only, due to the low lignin content, facilitating greater liberation of CNF via highshear homogenization. Comprehensive Microarray Polymer Profiling (CoMPP) measured remaining extractable polysaccharides, showing


2

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Biomacromolecules

that

the

enzyme-pretreatment

was

more

successful

at

removing

non-cellulosic polysaccharides than alkaline- or acid-hydrolysis alone.

Whilst

effective

alone,

the

effect

of

the

enzyme-

pretreatment was bolstered via combination with a mild high-pH pretreatment.

Dynamic

rheology

was

used

to

estimate

the

proportion of CNF in resultant suspensions. Enzyme-pretreated suspensions showed 4-fold and 10-fold increases in the storage modulus

for

potato

and

untreated samples.

sugar

beet

respectively,

compared

to

A greener yet facile method for producing

CNF from vegetable waste is presented here.

Introduction: Cellulose – the predominant plant cell wall polysaccharide and most

abundant

natural

biopolymer

–

is

a

long,

linear

homo-

polysaccharide comprised of thousands of unbranched -(1→4)-Dglucopyranose residues. Hydrogen-bond formation between adjacent glucose produces

residues densely

(approximately

3

on

several

packed nm

wide

neighboring

and

highly

and

several

cellulose

rigid

chains

microfibrils

micrometers

long)1,2.

Cellulose microfibrils are separate entities that bundle within


3


Page 4 of 50

the cell wall to form larger fibers that associate to form the main-load bearing scaffold of the plant cell wall, providing resistance to the internal osmotic pressure of the cell as a result

of

its

high

tensile

strength3,4.

This

microfibril

arrangement is combined with hemicelluloses, pectin, lignin, and proteoglycan, with proportions of each dependent on the plant species and type of cell wall. With respect to cellulose, the term “microfibril” is generally used

to

describe

fibrous

cellulose

structures,

formed

during

cellulose biosynthesis in higher plants5. Microfibrils can be separated

from

homogenizers6,7.

the The

cell

wall

complex

microfibrils,

compared

by to

high-pressure larger

fibers,

have superior reinforcing potential in composite materials. To facilitate delamination of large fibers and prevent clogging of high-pressure often such

homogenizers/micro

subjected as

to

chemical

TEMPO

fluidizers,

pulping

oxidation

or

the

oxidative

(oxidation

by

fibers

are

treatments, 2,2,6,6-

tetramethylpiperidinyloxyl)8. The resultant material is commonly referred to as “microfibrilated cellulose”, “nanofibrils”,

or

“nanofibers”9. The material typically contains high-aspect ratio (length-to-width)

cellulose

structures

with

at

least

one

dimension in the nanoscale. There are at least two other methods for

producing

extracted

nanoscale

low-aspect

cellulose,

ratio

one

crystalline

is

based

on

“nanocrystals”,

acidand


4

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Biomacromolecules

another is production of bacterial nanocellulose (high-aspect ratio). A recent review by Klemm el al8 provides an excellent overview of the various types of nanoscale celluloses, their production

methods

nanoscale

cellulose

and

applications.

is

superior

The

for

use

of

high

fabricating

aspect

composite

materials; it has been used to reinforce plastic and cement, to synthesize materials for packaging (coating, films, paper and filler reinforcement) and for filtration10,11,12. The market demand for

nanoscale

societies’

cellulose

increasing

sustainable

is

to

a

demands

materials.

large

for

extent

green,

Furthermore,

driven

non-toxic

producing

by and

nanoscale

cellulose from waste products is interesting from a bioeconomic point of view. Henceforth, isolated

this

from

the

paper cell

will wall

refer as

to

cellulose

cellulosic

fibers,

moieties and

the

nanoscale fibers produced following high-shear homogenization as cellulose nanofibers (CNF). Cellulose is endowed with numerous attractive and exploitable properties such as high strength and stiffness,

low

weight,

and

biodegradability13.

However,

its

utilization is not without difficulties. Exploitation of this resource has been hindered due to the inherent difficulty of gently liberating fermentable sugars and non-cellulosic polymers from the wall whilst leaving cellulose undamaged.


5


To

fully

exploit

the

reinforcing

Page 6 of 50

potential

of

cellulose

in

composite materials, CNFs need to be isolated from the cell wall skeleton. Within a composite, the nanoscale dimensions of the cellulosic fibers will maximize intra-fiber as well as fibermatrix interactions, increasing the strength and stiffness of the material. Cellulose nanofibers are composed of crystalline and amorphous regions, with only the former tolerating harsh chemical

treatments.

preserved

amorphous

It

is

parts

desirable to

improve

to

liberate

their

CNF

performance

with in

composite materials due to higher aspect ratios of the fibrils14. A high degree of conversion of cell wall material into CNF has been achieved by chemical pre-treatments and subsequent highshear homogenization. However, these treatments have high-energy requirements and can invoke irreversible changes in the fibers15. Biological

pretreatments

are

hypothesized

to

be

essentially

environmentally impact free; exploiting cell wall active enzymes or accessory proteins15. However, at present, to be effective, biological

treatments

need

to

be

coupled

with

a

chemical

pretreatment as, without this, enzyme activity can be limited due

to

the

encapsulation

lignin.

CNF

has

of

previously

cellulose been

by

produced

hemicellulose from

and

wood-derived

kraft-pulp and bleached sulfite-pulp by subsequent endoglucanase treatment

and

high-shear

homogenization16,17.

However,

chemical

pulping of wood (i.e. kraft- or sulfite-processing) is still


6

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Biomacromolecules

required to reduce the high lignin content of the secondary cell walls

before

any

further

processing

can

be

carried

out.

Secondary cell walls are richer in cellulose than primary cell walls, making them a common source of CNF in both research and industry. Nevertheless, the presence of lignin – a hydrophobic phenolic polymer – makes secondary cell walls less flexible, more impermeable to water, and more resistant to enzymatic and microbial attack. Isolation of CNF from secondary cell walls is therefore only possible if the lignin barrier is overcome and water is “pushed” back into the structure. Furthermore, TEMPOcatalyzed

NaClO

oxidation,

which

introduces

negative

surface

charges onto cellulose, is an effective method for producing CNF from secondary cell walls18. Another

abundant

originating

from

source

of

industrial

cellulose production

is of

agricultural starch,

waste

sugar,

and

juices19,20. Depending on crop species, 30-80% of the vegetable pulp dry mass consists of cellulose21,22. In addition, due to a much lower lignin content in these cell walls compared to wood, disintegration

into

CNF

requires

less

energy

and

is

more

amenable to enzymes. Although conversion of vegetable pulp into CNF can be achieved via chemical pretreatment and subsequent high-shear

homogenization

–

cellulose

nanofibers

have

been

produced from sugar beet, potato, and carrot pulps by similar methods23,24,25

–

it

is

costly

for

large-scale

processes.

The


7


mildest

chemical

pretreatments

Page 8 of 50

typically

consist

of

soaking

pulps in 0.5 M NaOH at 80 °C and subsequent oxidation with sodium chlorite at 70 °C under acidic conditions, which requires specialist

equipment,

high-energy

input,

chemicals,

and

neutralization steps involving excessive washing. This work examines how an enzymatic pre-treatment, consisting of various commercial technical-grade enzymes that target noncellulosic polysaccharides, can be applied to produce CNF from potato

and

homogenization. pectin-,

and

sugar

beet

The

selected

starch

pulps,

activities

following

enzymes

have

as

as

well

high-shear

hemicellulose-,

limited

cellulase

activity (Table 1). The idea is to release intact CNF from the primary

cell

wall

matrix

by

avoiding

major

use

of

multi-

component cellulolytic enzymes which tends to degrade cellulose into

soluble

sugars.

Instead,

a

predominant

endoglucanase

(FiberCare, cellulase) is used, which has been shown to gently loosen

the cellulose microfibril network without compromising

the length of individual fibrils26. When cellulose is converted into

CNF

the

surface

area

increases

considerably,

and

this

results in the formation of a hydrogel at low concentrations (around 1% w/w). The storage modulus (elasticity of the sample) can

thus

be

used

to

estimate

the

resulting

increment

of

cellulose-cellulose interactions within the gel27.


8

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Biomacromolecules

The work presented here is essentially different from woodbased

work

previously

conducted

due

to

exploitation

of

the

primary rather than the secondary cell wall of plants sourced from agro-industrial by-products. Although the cellulose content of the primary cell wall is lower than that of the secondary cell

wall,

and

the

wall

itself

is

more

complex

due

to

the

presence of a more diverse set of matrix polysaccharides and proteoglycans, undamaged cellulosic fibers can theoretically be liberated

via

enzymatic

degradation

of

all

non-cellulosic

biopolymers. The inherent difficulty of achieving this is that some matrix polysaccharides feature

the same ß-1,4-glycosidic

linkages as cellulose whilst others bind very strongly to it. Although

breaking

chemical

or

ß-1,4-glycosidic

enzymatic

methods

is

linkages

through

simplistic,

mild

specifically

breaking hemicellulosic or cellulosic ß-1,4-glycosidic linkages, rather than all of those present, is more problematic. Here a greener method for production of CNF from agricultural waste through

the

optimization

demonstrated,

with

specifically

remove

and

of

enzymatic

without

non-cellulosic

chemical

pretreatments

is

assistance,

to

polysaccharides

whilst

emphasizing the preservation of cellulose.


9


Page 10 of 50

Materials and Methods: Plant Material Plant materials, in the form of ground sugar beet and potato (particle sizes around 10 mm) from agro-industrial by-products post-starch and -sugar extraction, were kindly provided by KMC and

Nordic

Sugar.

Both

materials

were

homogenized

to

pulps

(particle sizes around 1 mm) in a food processor and then heated to 100 °C. Pulps were diluted in water to a 2.5% w/v solution to form the starting material for all assays.

Low and high pH-preincubation Low (Ph=2, 4 or 5.6) and high (pH=9) pH-preincubations (as specified in Table 2 and 3) were established by the addition of HNO3 and NaOH respectively until the desired pH was achieved. The pH was monitored throughout the preincubation to ensure it was maintained.

The

pH

of

each

preincubation,

as

well

as

the

temperature and duration of this procedure, is outlined in Table 2 (potato samples) and Table 3 (sugar beet samples).

Enzyme Treatments Enzyme treatments were carried out for 24 h at 40 °C and pH 4 (0.1 M sodium acetate buffer) for both sugar beet and potato at pulp dry weight concentrations of 2.5 % w/v. The enzymes used for

potato

samples

comprised of

Viscozyme L

(15 µL/g pulp),


10

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Biomacromolecules

Pectinex Ultra Clear (15 µL/g pulp), and Aquazym 240 L (5 µL/g pulp) while Viscozym L (10 µL/g pulp), Pectinex Ultra Clear (10 µL/g pulp), Pulpzyme HC (10 µL/g pulp), and Aquazyme 240 L (10 µL/g pulp) were used for sugar beet samples. The main activities of

the

enzyme-products

are

listed

in

Table

1.

It

is

worth

emphasizing is that the enzymes used are multi-enzyme complexes that

contain

cellulase

and

hemicellulase

side

activities

as

well. The amount and composition of enzymes used for each crop is

the

best

composition

based

on

trial

and

error

(data

not

shown) to determine optimal removal of non-cellulosic moieties using CoMPP. Both enzyme cocktails were further supplemented by the addition of Fibercare R (5 µL/g pulp). After the reaction, the enzymes were inactivated by heating the solutions to 100 °C for

a

few

minutes.

pressed

(to

reduce

further

analysis.

The water

The

solutions content)

were and

post-treatments

finally stored were

filtered at

made

and

-20°C

for

within

the

process line of industrial preservation of starch- and sugarextracted pulp.

Table 1: Declared main enzyme-activities in Novozymes products incorporated into the potato and/ or sugar beet enzyme cocktails. (FBG = Fungal Beta-Glucanase Units, AXU = Endo Xylanase Units, ECU = endo cellulase units, PGNU = Polygalacturonase units, KNU = Kilo Novo Units alpha-amylase units all units as specified by Novozymes) Product

Main enzyme activity

Declared activity


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Viscozyme L

Page 12 of 50

Beta-glucanase (endo- 100 FBG/g 1,3(4)-)

Pectinex Ultra Polygalacturonase Clear

7900 PGNU/ml

Pulpzyme HC

Endo-xylanase 1,4-)

(endo- 1000 AXU/g

FiberCare R

Cellulase

4500 ECU/g

Aquazym 240 L

Alpha-amylase

240 KNU/g

Termamyl

Alpha-amylase stable)

(heat 120 KNU/g

Naming system for potato and sugar beet samples: Samples are labelled according to the following system, which is representative of the conditions used and relevant order of processes from start to finish:

Plant material: Mechanical treatment/ pH preincubation – time (hrs) – temperature (C)/ Enzyme treatment Plant Material: Potato (P) or Sugar beet (SB) Mechanical-pretreatments:

Dry-

(D)

or

wet-milling

(W).

If

Termamyl was used instead of a mechanical pretreatment it is denoted by T. No mechanical treatment is shown by N. pH-preincubations: Low pH (A), high pH (B) or none (N). Enzyme-treatment: Enzyme (E) or no enzyme (N).


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Biomacromolecules

(h) refers to an extra homogenization step. Potato

underwent

the

following

enzyme-,

chemical-,

and

mechanical-treatments (Table 2) to produce three samples. The enzyme cocktail used is as described previously.

Table 2: Pretreatment and processing of potato samples ID

pH

Enzym e

Details

P: N/N/E

4

Yes

Enzyme treatment on potato pulp for 24 h at 40 °C

P: N/N/(h)E

4

Yes

Same as P: N/N/(h)E but were further homogenized with an IKA, Ultra Turrax T25 for 10 min

P: T/N/E

5.6 and 4

Yes

Potato pulp treated with Termamyl (0.5 KNU/g DM) for 90 min at pH 5.6 and 80 °C before the pH adjusted to pH 4 and enzyme treated for 24 h at 40 °C

The ten different sugar beet samples tested were produced under the following conditions (Table 3). Enzyme treatment refers to treatment

with

the

enzyme

cocktail

described

previously

for

sugar beet.

Table 3: Pretreatment and processing of sugar beet samples prior to microfibrilation ID SB: N/N/E

pH 4

Enzyme Details Yes

No pH preincubation. pH adjusted to pH 4


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Page 14 of 50

for enzyme treatment, 24 h at 40 °C SB: W/N/E

4

Yes

Wet-milling of pulp before treatment; pH 4, 24 h at 40 °C

enzyme

SB: U/N

N/B-1-

9

-

High pH preincubation for 1 h

SB: N/A-470/N

2

-

Low pH preincubation for 4 h at 70 °C

SB: U/E

N/B-1-

9

Yes

SB: N/A-470/E

2

Yes

Low pH preincubation for 4 h at 70 °C. pH adjusted to pH 4 for enzyme treatment, 24 h at 40 °C

SB: D/(h)B1-U(h)/E

9

Yes

Dry-milling, homogenized 10 min. 1 h high pH preincubation, homogenized 10 min, 24 h enzyme treatment at 40 °C

SB: D/(h)B1-U(h)/E(h)

9

Yes

Same as Batch homogenization separation

SB: D/(h)B2-U(h)/E

9

Yes

Dry-milling, homogenized 10 min. 2 h high pH preincubation, homogenized 10 min, 24 h enzyme treatment at 40 °C

SB: D/(h)B2-U(h)/E(h)

9

Yes

Same as SB: D/(h)B-2-U(h)/E plus homogenization for 10 min before separation

High pH preincubation for 1 h, and then adjusted to pH 4 for enzyme treatment, 24 h at 40 °C

SB: D/(h)B-1-U(h)/E plus for 10 min before

Comprehensive Microarray Polymer Profiling (CoMPP) Dried, finely-ground (particle sizes in the m range), aliquots of each sample (10 mg) were weighed out in triplicate and 300 μl of

50 mM

(CDTA)

trans-1,2-diaminocyclohexane-N,N,N′,N′-tetraacetic

pH

7.5

was

added

in

order

to

extract

acid

pectic

polysaccharides. The samples were gently shaken for 2 h at room


14

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Biomacromolecules

temperature (around 23 °C) before centrifugation at 2500 g for 10 min. The supernatant, which contains the solubilized pectin fraction, was removed and stored for later use. To the pellet, 300 μl of 4 M NaOH with 0.1% (v/v) NaBH4 was added in order to extract hemicellulosic polysaccharides. Again, the samples were shaken for 2 h, centrifuged for 10 min, and the supernatants containing the solubilized cell wall polymers were collected. The

supernatant

diluted

from

two-fold,

both

10-fold,

extractions

for

50-fold

250-fold

and

all

samples in

were

phosphate

buffered saline (PBS) buffer (140 mm NaCl, 2.7 mm KCl, 10 mm Na2HPO4, 1.7 mm KH2PO4, pH 7.5) and printed onto nitrocellulose membranes,

in

triplicate,

using

an

Arrayjet

Sprint

Inkjet

Microarrayer (Arrayjet, UK). Arrays were then cut-out, blocked for 1 h with 5% fat-free milk protein in PBS, and probed with primary

monoclonal

antibodies

(mAbs)

or

carbohydrate-binding

molecules (CBMs) for 2 h at room temperature, as described by Willats28 and McCartney29. After washing repeatedly in PBS, the arrays

were

probed

with

a

secondary

antibody

conjugated

to

alkaline phosphatase for a further 2 h. The arrays were washed again in PBS and once in dH2O prior to development with BCIP/NBT (5-bromo-4-chloro-3′-indolyphosphate/nitro-blue

tetrazolium

chloride). Once dry, the arrays were scanned into the computer using a flatbed scanner at 1200 dpi, and converted to negative image, 16-bit, grey-scale TIFFs, which were uploaded into the


15


Page 16 of 50

ImaGene 6.0 microarray analysis software. Processed and analyzed data were converted into a heatmap30.

Acid hydrolysis of polysaccharides Full-acid

hydrolysis

side-chains

to

their

was

used

to

constituent

hydrolyse

polysaccharides

monosaccharides.

This

was

conducted using 2 M trifluoro acectic acid (TFA) at 120 C for 1 h31.

Thin layer chromatography (TLC) Samples (10 g) were applied 20 mm from the bottom of a Merck silica-gel 60 TLC plate (VWR, Lutterworth, UK). Once dry, the TLC plate was run in ethyl acetate/ pyridine/ acetic acid/ dH2O (EPyAW, 6:3:1:1) for 4 h. Detection of digestion products were by thymol staining: 5% (v/v) sulfuric acid, 0.5% (w/v) thymol in ethanol. TLCs were air-dried prior to baking at 105°C for ~ 5 min32.

Transmission Electron Microscopy (TEM) Each

sample

was

microfibrilated placed

on

a

prepared

cellulose

carbon

coated

as

follows.

A

(MFC)-suspension copper

grid

volume (1.3

(150

%

mesh).

of w/w)

5

µL was

After

2


16

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Biomacromolecules

minutes, the excess of liquid was gently removed using filter paper

where

after

5

µL

of

Uranyless

(Electron

Microscopy

Sciences) was applied. After 5 minutes, the excess of liquid was gently removed using filter paper. The sample was dried at room temperature overnight. The images were acquired on a Jeol-1010 transmission electron microscope (Jeol, Tokyo, Japan), employing an accelerating voltage of 80 kV.

Light Microscopy Wet pulp (1.2% w/v) was loaded onto a glass slide and pressed under a cover slip. The autofluorescence of the samples were visualized using an emission filter (DAPI) and photographed with a

digital

molecules

camera. (phenolic

Autofluorescence compounds

in

happens

particular)

when are

certain

excited

by

wavelengths ranging from UV to visible light which causes them to emit light in the blue to green range. No staining agents were used. The images were acquired on an Olympus BX41 light microscope. To determine remaining starch 1 ml of 1.2% (w/v) pulp solution (potato and sugar beet) with 0.25 ml of a diluted (1:2, v/v) Lugol’s solution (2.6% KI + 26% I2) (Sigma-Aldrich).

Dynamic Rheology


17


Page 18 of 50

Dynamic rheology measurements were carried on a Discovery HR-3 Rheometer (TA instruments). An acrylic 50 mm plate with a gap of 500 µm was used for all samples. Before each measurement, the samples were allowed to rest for 5 minutes and the temperature was

fixed

determined sweeps

at by

were

25

°C.

strain measured

The

linear

sweeps

for

between

viscoelastic

all

suspensions.

strains

of

0.01-

region

was

Oscillation 100

%

at

a

frequency of 1 Hz. The chosen strain for the frequency sweep measurements was 0.5 % for both samples. The frequency sweeps were carried out in the range of 0.01 – 1000 Hz.

Preparation of nanofibers Wet pulps were diluted to 2 % w/v in 200 ml distilled water and subjected to homogenization (Kinematica Polytron PT 3100) for 20 seconds at 15000 rpm or until the average particle size was a few hundred micrometers. Each pulp was subsequently washed with 5 L of water on a 32 µm sieve to remove soluble compounds and remaining enzymes (see ‘Enzyme treatments’ section). The washed pulps were suspended in 200 ml distilled water to approximately 2% w/v (due to pulps soaking up different amounts of water after the washing). A volume of 20 ml of each sample was placed in an oven at 50 °C for 24 hours to determine the exact dry weight of the suspensions. After obtaining the dry weight of the samples, each

of

them

were

diluted

to

exactly

1.2%

w/v

in

distilled


18

Page 19 of 50 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

water.

Each

homogenizer

sample

(100

ml)

(Microfluidizer

was

circulated

materials

in

processor

a M

high-shear 110-P

with

orifices of 200 and 400 µm) under a pressure of 500 bars for 18 minutes to produce the nanofibers. The samples were stored at 4 °C for further characterization.


19


Page 20 of 50

Results and discussion: Comprehensive Microarray Polymer Profiling (CoMPP) is a highthroughput method30 that

uses monoclonal antibodies

(mAbs) to

detect the relative abundance of CDTA- and concentrated NaOHlabile

cell-wall

glycans

still

present

within

a

sample,

therefore allowing to determine the effectiveness of the various pH-,

enzymatic-,

and

combined-treatments

in

removing

non-

cellulosic moieties. It is important to understand that, during CoMPP,

the

remaining

polysaccharides

and

proteins

still

associated with the CNF are extracted, and not those, which have been degraded by the enzymes; the latter will be of insufficient size to contain the required mAb epitope or even bind to the nitrocellulose. greater

removal

As of

such, a

lower

detection

particular

pectic-

values or

indicate

a

hemicellulosic-

polysaccharide, or cell wall-associated protein, meaning a more effective method for nanofiber isolation. Samples,

with

respect

to

the

plant

material

and

treatments

used, are labelled according to the following template, which indicates the actual flow of processes from mechanical-treatment to pH-preincubation, and then to enzyme-treatment: Plant material: Mechanical treatment/ pH preincubation – time (hrs) – temperature (C)/ Enzyme treatment ACS Paragon Plus Environment

20

Page 21 of 50 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

Plant samples used, which were sourced from agro-industrial byproducts, were sugar beet (SB) and potato (P). The mechanical pretreatments used were dry- (D) or wet-milling (W), or none (N), and these were conducted prior to any pH-preincubations or enzyme-treatments.

The

use

of

a

thermostable

-amylase

(Termamyl), which breaks down non-resistant starch into small molecular weight products, was also included at this stage for one potato sample and is denoted by T. The pH-preincubations were either at low pH (A), high pH (B), or there was none (N), and further details as to treatment time and temperature are included; U if conducted at ambient temperature. If an enzymetreatment was used it is denoted by E but if it was absent then N is used instead. Extra homogenization steps are indicated by (h),

and

are indicative

of their position in the

processing

sequence of the sample. For example, a sugar beet sample that has undergone a low pH preincubation for 4 h at 70 C prior to enzyme treatment with no mechanical pretreatment is identified by the moniker SB: N/ A-4-70/E.

Isolation of purer cellulosic fibers from agro-industrial crops is possible using enzymatic methods


21


Potato

samples

consisting

of

were

incubated

commercially

Page 22 of 50

with

available

an

enzyme

enzymes.

cocktail

Potato

samples

were subjected to enzyme-treatment both alone and combined with mechanical- and chemical-treatments (Table 2). The resulting dry residue underwent CDTA- and NaOH-extraction, which was analyzed via

CoMPP,

to

determine

each

methods

efficacy

in

removing

pectin, hemicellulose, and cell wall proteins for the isolation of nanofibers. CDTA

(50

mM)

remaining

was

pectic

used

predominately

moieties

still

for

bound

the to

extraction the

of

nanofibers

following treatment. Enzyme-treatment dramatically reduced the level of detectable pectin and hemicellulose in the potato pulp, often to below the 5% cut-off specified, compared to the crude untreated potato control (Figure 1). Although small traces of arabinan were still detectable in the CDTA extract of enzymeonly

treated

homogenized this

was

potato

samples

around

samples, (P:

both

N/N/(h)E

10-fold

lower

and than

in P:

homogenized N/N/E

and

non-

respectively),

observed

for

the

crude

potato. When the potato sample was pretreated with Termamyl (P: T/N/E)

no

arabinan

was

detected

in

the

CDTA

fraction.

Interestingly, although enzyme-treatment reduced

the level

Arabinogalactan

extensins

less

proteins

(AGPs),

pronounced, and even

Termamyl

pretreatment.

the

effect

on

increased upon the

This

suggests

that

the

of was

inclusion of extensins

a

are


22

Page 23 of 50 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

strongly starch,

bound and

to

the

the

high

cellulose

in

temperatures

potato. (80

C)

The

removal

involved

in

of the

Termamyl treatment that may lead to swelling of the fibers, is hypothesized to expose extensin moieties that were previously inaccessible to the mAbs. NaOH (4 M + 0.1% NaBH4) is primarily used to remove remaining hemicelluloses from cellulose fibrils. While a similar pattern is observed in the NaOH fraction as in the CDTA fraction (i.e. a dramatic

decrease

in

hemicellulose

content

in

enzyme-treated

samples), higher levels of mannan and xylan are detected by mAbs BS-400-4 and LM11 respectively compared to the control. This is taken

to

be

the

result

of

a

demasking

of

tightly

bound

populations of xylan and mannan. Despite this, it is clear that the enzyme-treatment employed here is effective at removing most of the non-cellulosic polysaccharides.


23

HG Partially methylesterified


(1→4)-β-D-galactan

(1→5)-α-L-arabinan

Linearised (1→5)-α-L-arabinan

Feruloylate on any polymer

Xyloglucan

(1→4)-β-D-xylan

(1→4)-β-D-xylan/arabinoxylan

Extensin

AGP

LM5

LM6

LM13

LM12

BS-400-4 (1→4)-β-D-mannan

(1→4)-β-D-mannan

LM20

LM21

LM25

LM10

LM11

LM3

JIM13

Sample P: N/N/E P: N/N/(h)E P: T/N/E Potato Crude P: N/N/E P: N/N/(h)E P: T/N/E Potato Crude

Page 24 of 50

LM18

NaOH

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

CDTA

Biomacromolecules

0 0 8 0 0 7 0 0 0 0 100 75 0 0 0 0 0 0 0 0 9 0 18 36

0 0 0 7 0 0 0 0

0 0 0 0 0 0 0 0

0 0 0 0 43 27 33 16

0 0 0 0 0 0 0 0

0 0 0 47 0 0 0 0

0 0 0 0 0 0 0 0 0 18 0 0 15 0 35 7 0 38 22 22 12 26 0 9

15 0 24 0 37 0 33 23 0 0 0 0 0 0 0 0

Figure 1: Heatmap showing the relative abundance of cell-wall glycans as recognized by various probes in a range of potato samples following different treatments (enzyme-only, or combined with

mechanical-

or

chemical-treatments).

Glycans

were

sequentially extracted using CDTA and NaOH and probed with a range

of

indicated.

mAbs

or

Color

intensity

signal.

The

assigned

a

accordingly.

cellulose

highest value

of

mean 100%

is

binding

molecules

proportional

spot

signal

in

all

other

and

A signal minimum of

to the

(CBMs)

the data

signals

mean set

as spot was

adjusted

5% was imposed, and

values

below this are represented as zero. N means none, (h) means


24

Page 25 of 50 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

extra

homogenization,

T

means

Termamyl

treated,

and

E

means

enzymatic digestion as specified in the Materials and Methods.

Following the successful development of an enzyme-only method for the isolation of purer cellulosic fibrils from potato, the aim

was

to

determine

whether

this

could

be

applied

across

species or whether optimization would be required on a speciesby-species basis. Given the importance and prevalence of sugar beet

from

genetic

an

and

agro-industrial

physiological

perspective,

differences

to

and

its

potato,

distinct

whether

an

enzyme-only approach was sufficient to isolate nanofibers from this source too was tested. Sugar beet pulp is a more recalcitrant material than potato pulp. As described in the Materials and Methods, a different enzyme

cocktail

composition

was

used

for

the

optimal

CNF-

isolation from sugar beet compared to potato. As such, it is pertinent to plant

note

samples,

conducted

on

recalcitrant

a

that,

enzyme

for effective cocktail

optimization

species-by-species

nature

of

sugar

isolation of

beet,

basis. we

Due

also

CNF from

needs to

to

the

introduced

be more pH-

preincubations at either low pH (pH 2, indicated with an A in the sample identifiers) or high-pH (pH 9, indicated with a B), for the purpose of controls.


25


High

and

walls,

low

pH-treatments

disrupting

cause

hydrogen-bonding

Page 26 of 50

swelling and

of

isolated

unmasking

cell

internally-

associated hemicelluloses, pectins, and cell wall proteins. If this holds true then extraction of polysaccharides during CoMPP, especially

by

detection.

These

homogalacturonan pectic

arabinan

involving

CDTA,

will

be

phenomena

in

the

in

CDTA

the

arabinofuranosyl

NaOH

promoted are

clearly

extract,

are

to

higher

illustrated

and

extract.

residues

leading

by

tightly

Glycosidic acid

by

bound

linkages

labile.

Low

pH-

treatment effectively reduces the LM13-epitope (unbranched) but not

the

LM6-epitiope

(branched

arabinan)

anticipated that, when combined with pH-pretreatments

will

facilitate

(Figure

2a).

It

is

enzyme-treatments, these

enzyme

access

to

their

substrates.


26

(1→4)-β-D-xylan

(1→4)-β-D-xylan/arabinoxylan

Extensin

AGP

LM10

LM11

LM3

JIM13

Feruloylate on any polymer LM12

Xyloglucan

Linearised (1→5)-α-L-arabinan LM13

LM25

(1→5)-α-L-arabinan LM6

(1→4)-β-D-mannan

(1→4)-β-D-galactan LM5

BS-400-4 (1→4)-β-D-mannan


5 83 100 6 0 0 0 0 12 9 41 19 37 61 18 12 7 0 0 12 0 13

LM20

32 18 61 43 19 36 48 35 58 36 23 0 0 0 0 0 0 0 0 0 0 0


0 0 0 53 0 7 0 11 0 0 0 54 18 64 32 34 61 0 50 19 0 0 45 65 78 75 6 64 0 40 24 0 0 0 0 0 57 6 33 0 19 6 0 0 0 0 0 35 0 11 0 17 0 0 0 0 0 0 58 0 19 0 20 0 0 0 0 0 0 43 0 0 0 16 0 0 0 0 0 0 59 29 28 0 25 0 0 0 0 0 0 66 18 12 0 20 0 0 0 0 0 0 45 14 9 0 18 0 0 0 0 14 13 36 0 23 0 14 0 0 0 0 0 0 69 16 0 67 15 31 35 17 0 0 16 74 38 0 18 7 26 43 36 0 0 16 74 0 0 43 22 41 48 72 0 0 0 45 0 0 50 0 20 60 6 0 0 0 26 0 0 40 7 26 47 48 0 0 0 38 6 0 41 6 35 48 38 0 0 0 39 0 0 24 0 37 17 7 0 0 0 12 0 0 17 0 9 0 9 0 0 0 26 0 0 31 0 32 7 37 0 0 0 14 0 0 12 0 8 0 22 0 0 0 35 10 0 25 0 19 0 7

LM18

Sample SB: N/N/E SB: N/B-1-U/N SB: N/A-4-70/N SB: N/B-1-U/E SB: N/A-4-70/E SB: W/N/E SB: D/B-1-U(h)/E SB: D/(h)B-1-U(h)/E SB: D/(h)B-2-U/E SB: D/(h)B-2-U(h)/E Sugarbeet Crude SB: N/N/E SB: N/B-1-U/N SB: N/A-4-70/N SB: N/B-1-U/E SB: N/A-4-70/E SB: W/N/E SB: D/B-1-U(h)/E SB: D/(h)B-1-U(h)/E SB: D/(h)B-2-U/E SB: D/(h)B-2-U(h)/E Sugarbeet Crude

LM21

NaOH

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

CDTA

Page 27 of 50

2.a. Enzyme/ pH only 2.b. Enzyme-pH combined 2.c. Mechanical + combined enzyme-pH treatment 2.a-c. Control 2.a. Enzyme/ pH only 2.b. Enzyme-pH combined 2.c. Mechanical + combined enzyme-pH treatment 2.a-c. Control

Figure 2(a-c): Heat map showing the relative abundance of cellwall

glycans,

undergone

in

various

treatments,

as

a

range

of

mechanical-,

recognized

by

sugar pH-,

beet

samples

enzyme-,

various

or

probes.

that

have

combinationGlycans

were


27


Page 28 of 50

sequentially extracted using CDTA and NaOH and probed with a range of mAbs or CBMs as indicated in the top row.

Simultaneous

facilitation

polysaccharides

and

of

of

enzymatic

extraction

removal

of

the

of

residual

polysaccharides after enzyme digestion is so balanced that some treatments appear to raise the levels of some polysaccharides about that of the control (Figure 2a-c). A dot-blot assay was conducted on solid sugar beet samples from untreated, enzymetreated only, high pH-treated only, and combined enzyme and pHtreated samples. This was

then

(Figure

access

3a).

The

promoted

probed for

for HG content (LM18) enzymes

obviously

also

holds true for mAbs, thus enhancing the detection. As expected, the crude sugar beet sample showed a higher HG content than the enzyme-treated higher

levels

sample, of

while

detectable

the

pH-treated

pectin,

sample

even

though

had

much

internal

epitopes could not be reached by the mAbs in the control sample. Combining enzyme- and high pH-treatments (pH=9) was shown to remove

a

greater

amount

of

HG

and

other

non-cellulosic

polysaccharides than when using enzymes alone, presumably due to the increased accessibility afforded by the high-pH treatment (Figure 2b).


28

Page 29 of 50 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

Hydrolysis itself)

by

of

matrix

polysaccharides

trifluoro

acetic

acid

(but

(TFA)

not

does

of

not

cellulose depend

on

accessibility. The monosaccharide profiles of the TFA-released material

were

analyzed

by

thin

layer

chromatography

(TLC)

(Figure 3b). The profiles indicate in all cases that the tightly bound material is largely HG+RG-I with a smaller contribution of glucose

and

xylose

indicative

of

xyloglucan,

although

some

xylose may originate from xylan. Meanwhile, the serial dilutions illustrate that the enzyme treatment, when combined with a pHpreincubation, has some ability to access, and degrade, these populations

of

tightly-bound

matrix

polysaccharides

as

the

overall monosaccharide content here is reduced compared to the other three samples. Although the enzyme-only treatment used here was able to remove non-cellulosic polysaccharides and proteins from the sugar beet pulp,

the

high

proportion

of

untouched

internal

moieties

uncovered by the pH-treatment suggests that this approach is insufficient to effectively isolate nanofibers. However, it was found that pH-preincubation of sugar beet can bolster the effect of

enzyme-treatments,

fibers.

As

such,

therefore,

both

mechanical-

producing and

purer

cellulosic

pH-pretreatments

were

combined with the enzyme-treatment in an attempt to optimize the removal of non-cellulosic polysaccharides. This study focused on high-pH

rather

than

low-pH

preincubations

for

the

following


29


Page 30 of 50

samples as high-pH incubations correlated generally with a more effective removal of non-cellulosic polysaccharides. Also,

as

high-pH incubation is effective at room temperature and with short

incubation

periods,

it

is

a

more

industrially

viable

process, requires less external energy input, and is essentially a greener process.


30

Page 31 of 50 a) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Sample Dilution # HG (LM18)

Biomacromolecules SB Crude 1 2 3 4 5 6 36 33 41 43 30 17

7 0

1 6

High pH + Enzyme 2 3 4 5 6 8 12 10 13 11

High pH Only 7 1 2 3 4 5 6 5 100 90 69 38 13 0

7 0

Enzyme Only 1 2 3 4 5 6 16 23 12 13 14 9

7 8

b)

1 2 3 4 Enzyme Only

Figure 3: Heatmap showing the relative abundance of HG (LM18) in sugar beet samples representing an untreated control, alkalinepretreatment

and

subsequent

enzymatic-treatment

combined,

alkaline-pretreatment only, and enzyme-treatment only. Detection was

via

dot-blot

on

an

intact

solid

sample

at

a

range

of

dilutions (1-7; 4x stepwise dilution from 1 to 7). SB crude, high pH-only and enzyme only samples show detectable levels of monosaccharide in dilutions 1-3, while monosaccharides in the


31


high

pH

+

dilution

enzyme

2.

This

sample

are

only

indicates

a

Page 32 of 50

clearly

lower

detectable

overall

up

to

monosaccharide

content in the high pH + enzyme sample and, therefore, a greater efficacy of non-cellulosic polysaccharide removal prior to TFAhydrolysis. To determine whether the efficacy of pH- and enzyme-treatments could

be

further

enhanced

through

the

use

of

mechanical

treatments, the effect of including dry- and wet-milling as well as

additional

Combining

mid-flow

wet-milling

homogenization with

steps

was

enzyme-treatment

tested.

significantly

reduced the amount of all hemicelluloses present, presumably due to increased surface area for enzyme activity. However, this was unlikely to resolve the issue of the internal hemicelluloses that

can

only

be

accessed

when

in

combination

with

a

pH-

preincubation. As such, combining enzyme-, mechanical- and pHtreatments

was

removal

and,

Although

it

treatment

(2

tested,

which

therefore, was h)

determined provided

led

optimal that some

to

greater

isolation a

slightly

reduction

hemicellulose

of

nanofibers.

longer in

high

pH-

non-cellulosic

moieties compared with a shorter incubation period (1 h), it was the extra homogenization steps that had the most dramatic effect (Figure 2c).


32

Page 33 of 50 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

Although

enzymatic-pretreatments

are

effective

at

isolating

cellulosic fibers from agro-industrial crops, it is clear that significantly purer cellulosic fibers can be obtained from more recalcitrant crops, such as sugar beet, when mechanical-, pHand enzyme-treatments are combined. We used the purest cellulose samples from potato (P: N/N/E) and sugar beet (SB: D/(h)B-U2(h)/E)

to

produce

CNF

via

micro

fluidization

(high-sheer

homogenization). The physical properties of the resultant CNF for each sample were then tested to determine the effect of the enzyme treatments with respect to improving CNF liberation.

Enzymatic-pretreatments

significantly

improve

CNF

liberation

from agro-industrial crops To determine the efficacy of CNF liberation from the enzymepretreated samples, rheology was used to estimate CNF release based

on

the

sample’s

gel

properties

post

high-shear

homogenization (micro fluidization). When nanofibers interact, a network higher

structure storage

network,

which

arises

that

modulus becomes

in

has

the

viscoelastic

sample

particularly

properties.

indicates

apparent

with

a

A

stronger increasing

concentration of CNF12. The storage modulus (G′) represents the deformation-energy

stored

by

a

sample

in

an

elastic

manner,

while the loss modulus (G′′) measures the dissipated energy. In


33


Page 34 of 50

an ideal gel the storage modulus will be independent of the frequency and G′ >> G′′, while in a classical viscous fluid the storage- and loss-modulus will have a characteristic frequency dependency and G′ >

enzyme-pretreated

G′′).

Moreover,

samples

an

indicates

a

stronger network formation compared to the untreated sample. As the presence of

non-cellulosic polysaccharides is drastically

reduced in the enzyme-treated samples, the higher G′ observed is likely due to an increase in exposed cellulose surface area. Staining of the enzyme treated pulps with Lugol’s solution was used

to

identify

any

remaining

starch

in

the

samples

(see

Supplemental Figure S1). Nevertheless, as only a few areas were stained in the potato sample, and none in the sugar beet sample, we rule out the possibility that bulk rheological properties would be influenced by starch. With

respect

to

the

non-treated

samples,

which

contain

relatively large amounts of pectin and hemicelluloses, the G′ may represent other interactions as well, in addition to the nanofiber-nanofiber interactions. Therefore, the 4-fold and 10fold increase in G′ displayed by the enzyme-pretreated samples compared

to

the

untreated

samples

indicates

a

pronounced

increase in CNF release. The values of the storage moduli at 1 Hz

(6.28

rads/s)

in

the

enzyme-treated

samples;

510

Pa

for


34

Page 35 of 50 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

potato, and 273 Pa for sugar beet are of the same magnitude as wood-extracted

nanofibers12.

Nevertheless,

additional

measurements of the samples 24 hours after micro fluidization (storage at 23 °C) revealed a drop in G′ (Figure 4), likely to occur due to local aggregation of fibrils, which disrupt the homogenous

network

of

fibrils

within

the

gels.

The

unstable

dispersions of CNF may need to be overcome by functionalization of the fibers to introduce negative charges, depending on the end use of the CNF.


35


Page 36 of 50

Figure 4: The storage modulus (G′) and the loss modulus (G′′) as a function of frequency for crude potato-CNF (P), crude sugar beet-CNF enzyme

(SB),

enzyme

pre-treated

Measurements

to

pre-treated

sugar the

potato-CNF

beet-CNF

left

were

(SB:

(P:

T/N/E),

and

D/(h)B-2-U(h)/E(h))).

carried

out

after

micro

fluidization (high-shear homogenization), while measurements to the

right

were

carried

out

24

h

after

micro

fluidization

(storage at 23 °C). Geometry used: acrylic plate. Temperature was set to 25 °C.


36

Page 37 of 50 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

To visualize the nanoscale elements, electron microscopy images of potato and sugar beet CNF-suspensions are shown in Figure 5a)-b) and Figure 5c)-d), respectively, where Figure 5b) and d) show

suspensions

that

were

subjected

to

enzyme-treatments.

Without an enzyme-pretreatment, nanofibers were less prevalent in both samples and difficult to locate. In contrast, there was an abundance of nanofibers in the enzyme-pretreated samples. The width of a nanofiber is approximately 5 nm (Figure 5(1)), with an occurrence of larger bundles with widths up to 130 nm (Figure 5(2)) Nanofibers were easier to locate in the enzyme-pretreated potato

sample

than

indicating they are

in

corresponding

more

prevalent in

sugar

beet

sample,

the former sample.

In

addition, it should be noted that larger fragments of cellulose were

easily

washed

off

during

sample

preparation,

which

may

explain the absence of any fibrous structures in the untreated samples (Figures 5a and c).


37


Figure 5:

Page 38 of 50

TEM pictures of a) micro fluidized crude potato pulp,

b) enzyme-treated and micro fluidized potato pulp (P: T/N/E), c) micro fluidized crude sugar beet pulp, d) enzyme-treated and micro fluidized sugar beet pulp (SB: D/(h)B-2-U(h)/E(h)). (1) shows a single cellulose nanofibril (5 nm wide) while (2) shows a bundle of cellulose nanofibrils (130 nm wide).

Apart

from

nano-scale

elements,

light

microscopy

pictures

reveal the presence of micron-sized particles in both potato (Figure broken

6)

and

down

in

sugar the

beet

(Figure

7)

samples

that

microfluidizer.

In

Figures

6

were

and

7,

not (a)


38

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Biomacromolecules

denotes

untreated

sample

and

(b)

denotes

enzyme

pre-treated

sample. The autofluorescence of these particles reveal that the samples

contain

suberin-rich digestion.

phenolic

fragments In

compounds, that

comparison,

are chemical

indicating resistant

ligninto

pre-treatments

and

enzymatic usually

contain an oxidation step to degrade these fragments. As they would interfere in the construction of nanostructured composites we propose that these fragments may be removed by sieving.


39


Page 40 of 50

Figure 6: Light-microscopy pictures and auto-fluorescence of (a) Potato-CNF

suspension

without

pre-treatment

(crude),

and

(b)

Potato-CNF suspension with enzymatic pre-treatment (P: T/N/E).

Figure 7: Light-microscopy pictures and auto-fluorescence of a) Sugar beet-CNF suspension without pre-treatment (crude), and b) Sugar

beet-CNF

suspension

with

enzymatic

pre-treatment

(SB:

D/(h)B-2-U(h)/E(h)).


40

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Biomacromolecules

Concluding remarks It has been shown here that isolation of cellulose nanofibers is possible using entirely enzymatic treatments. By comparing two species with significantly different pectin structures it is revealed that there is no “one-size fits all” enzyme digestion protocol. Instead, enzyme cocktail optimization is required on a species-by-species basis and needs to be carried out for each new

raw

materials,

material. such

as

In

the

sugar

case

beet,

it

of

more

has

been

recalcitrant shown

that

raw the

efficacy of enzyme-treatments can be further bolstered through the combined use of enzyme-, mechanical-, and pH-treatments. It is proposed that the increased storage moduli observed in the optimally prepared materials from both species, when compared with the untreated controls, are directly due to more effective CNF liberation during micro fluidization. The process for cellulose fiber isolation outlined in this work presents a greener and more industrially viable process than currently used as it does not require high temperature or large volumes of harmful chemicals (and therefore water). However, the unstable dispersions of CNF remain a challenge; the observed decline in the storage modulus of both crops with time suggests that the gel-like networks are subject to rearrangements by which CNFs increasingly self-associate and eventually flocculate. The prevalence of suberized and lignified particles in sugar beet is also a hindrance, e.g. for fabrication of nanomaterials, but this work indicates that appropriate mechanical separation will bring an environmentally friendly process within reach.


41


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FUNDING SOURCES The work presented here was partly supported by the Innovation foundation Denmark with the project 5152-00001B; ASSEMBLY: Reverse engineering the cell wall for high performance Bio composites.

ACKNOWLEDGMENTS We would like to thank the Innovation Foundation Denmark, and previously The European FP7 Framework program, for funding. Potato pulp and sugar beet pulp were kindly donated by KMC and Nordic Sugar respectively. Enzymes were donated by Novozymes.

ABBREVIATIONS AGP, arabinogalactan protein; AXU, Endo Xylanase Units; CBM, cellulose binding molecule; CNF, cellulose nanofibril; CoMPP, Comprehensive Microarray Polymer Profiling; ECU, endo cellulase units; FBG, Fungal Beta-Glucanase Units; HG, homogalacturonan; mAbs monoclonal antibodies; KNU, Kilo Novo Units alpha-amylase units; MFC, microfibrilated cellulose; PGNU, Polygalacturonase units; RG-I, rhamnogalacturonan-1; TFA, trifluoroacetic acid; TLC, thin layer chromatography;

REFERENCES (1) Somerville, C.; Bauer, S.; Brininstool, G.; Facette, M.; Hamann, T.; Milne, J.; Osborne, E.; Paredez, A.; Persson, S.; Raab, T. Toward a systems approach to understanding plant


42

Page 43 of 50 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

cell

walls.

Science

2004,

306(5705)

2206-2211,

DOI:

10.1126/science.1102765 (2) McNamara,

J.T.;

description

of

Morgan,

cellulose

J.L.;

Zimmer,

biosynthesis.

J.

A

Annual

molecular review

of

biochemistry 2015, 84, 895-921, DOI: 10.1146/annurev-biochem060614-033930 (3) O’Sullivan, A.C. Cellulose: the structure slowly unravels. Cellulose 1997, 4(3) 173-207. (4) Gorshkova,

T.A.;

Mikshina,

P.V.;

Gurjanov,

O.P.;

Chemikosova, S.B. Formation of plant cell wall supramolecular structure. Biochemistry 2010, 75(2) 159-172. (5) Krässig, H.A. In: Cellulose: structure, accessibility and reactivity; Krässig, H.A. Eds.; Gordon and Breach Science Publishers: Yverdon, Switzerland, 1993; p 307–314. (6) Herrick, F.W.; Casebier, K.R.

Microfibrillated

accessibility.

J.

Appl.

(United States) 1983,

R.L.; Hamilton, J.K.; Sandberg, Cellulose: Polym.

Sci.

morphology Appl.

Polym.

and Symp.

37(9) 797–813,

(7) Turbak, A.F.; Snyder, F.W.; Sandberg, K.R. Microfibrillated cellulose, a new cellulose product: properties, uses, and commercial potential. J. Appl. Polym. Sci. Appl. Polym. Symp. (United States) 1983. 37(9) 815–827


43


Page 44 of 50

(8) Klemm, D.; Cranston, E.D.; Fischer, D.; Gama, M.; Kedzior, S.A.; Kralisch, D.; Kramer, F.; Kondo, T.; Lindström, T.; Nietzche, S.; Petzold-Welcke, K.: Rauchfuß, F. Nanocellulose as

a

natural

source

for

groundbreaking

applications

in

materials science: Today’s state. Materials Today 2018, 21(7) 720-748, DOI: (9) Klemm,

D.;

10.1016/j.mattod.2018.02.001 Kramer,

F.;

Moritz,

S.;

Lindström,

T.;

Ankerfors, M.; Gray, D.; Dorris, A. Nanocelluloses: a new family

of

International

nature-based Edition

materials. 2011,

Angewandte

50(24):

Chemie

5438-5466,

DOI:

10.1002/anie.201001273 (10)

Shatkin, J.A.; Wegner, T.H.; Bilek, E.M.; Cowie, J.

Market projections of cellulose nanomaterial-enabled products – Part 1: Applications. Tappi Journal 2014, 13(5) 9-16 (11)

Cowie, J.; Bilek, E.M., Wegner, T.H.; Shatkin, J.A.

Market projections of cellulose nanomaterial-enable products – Part 2: Volume estimates. Tappi Journal 2014, 13(6) 57-69 (12)

Kargarzadeh,

H.;

Mariano,

M.;

Huang,

J.;

Lin,

N.;

Ahmad, I.; Dufresne, A.; Thomas; S. Recent developments on nanocellulose reinforced polymer nanocomposites: A review. Polymer

2007,

132,

368-393,

DOI:

10.1016/j.polymer.2017.09.043


44

Page 45 of 50 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

(13)

Moon, R. J.; Martini, A.; Nairn, J.; Simonsen, J.;

Youngblood,

J.

Cellulose

nanomaterials

review:

structure,

properties and nanocomposites, Chemical Society Reviews 2011, 40(7) 3941-3994, DOI: 10.1039/c0cs00108b (14)

Saïd

Dufresne,

Azizi A.

Samir,

Tangling

M.A.; effect

Alloin, in

F.;

Paillet,

fibrillated

M.;

cellulose

reinforced nanocomposites. Macromolecules 2004, 37(11) 43134316, DOI: 10.1021/ma035939u (15) M.E.;

Procentese, Rehmann,

pretreatment

A.; L.;

with

Raganati,

F.;

Marzocchella,

deep

eutectic

Olivieri, A.

G.;

Low-energy

solvents

for

Russo, biomass

bio-butanol

production. Bioresource Technology 2017, 243(1) 464-473, DOI: 10.1016/j.biortech.2017.06.143. (16)

Liu,

expansins

X.;

Ma,

and

lignocellulose

Y.;

Zhang.

expansion-like degradation.

M.

Research

proteins

Biotechnology

advances

in

involved Letters

in 2015,

37(8) 1541-1551, DOI: 10.1007/s10529-015-1842-0 (17)

Henriksson,

M.;

Henriksson,

G.;

Berglund,

L.;

Lindström, T. An environmentally friendly method for enzymeassisted

preparation

of

microfibrillated

cellulose

(MFC)

nanofibers. European Polymer Journal 2007, 43(8) 3434-3441.


45


(18)

Page 46 of 50

Pääkkö, M.; Ankerfors, M.; Kosonen, H.; Nykänan, A.;

Ahola,

S.;

Osterberg,

M.;

Ruokolainen,

J.;

Laine,

J.;

Larsson, P.T.; Ikkala, O.; Lindström, T. Enzymatic hydrolysis combined

with

homogenization gels.

mechanical for

nanoscale

Biomacromolecules

shearing

and

cellulose

fibrils

2007,

8(6)

high-pressure and

strong

1934-1941,

DOI:

10.1021/bm061215p (19)

Saito,

T.;

Kimura,

S.;

Nishiyama,

Y.;

Isogai,

A.

Cellulose nanofibers prepared by TEMPO-mediated oxidation of native

cellulose.

Biomacromolecules

2007,

8(8)

2485-2491,

DOI: 10.1021/bm0703970 (20)

Procentese,

A.;

Raganati,

F.;

Olivieri,

G.;

Russo,

M.E.; Rehmann, L.; Marzocchella, A. Deep Eutectic Solvents pretreatment of agro-industrial food waste. Biotechnology for Biofuels 2018, 11:37, DOI: 10.1186/s13068-018-1034-y. (21)

Stenmarck,

Estimates

of

A.;

Jensen,

European

Environmental

food

Research

C.;

Quested,

waste

T.;

levels.

Institute

Moates,

G.

IVL

Swedish

2016,

DOI:

10.13140/RG.2.1.4658.4721 (22)

Rani,

B.;

Kawatra,

A.

Fibre

constituents

of

some

foods. Plant foods for human nutrition 1994, 45(4) 343-347.


46

Page 47 of 50 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

(23)

Vincken,

J.-P.;

Borkhardt,

B.;

Bush,

M.;

Doeswijk-

Voragen, C.; Dopico, B.; Labrador, E.; Lange, L.; McCann, M.; Morvan, C.; Muñoz, F.; Oomen, R.; Peugnet, I.; Rudolph, B.; Schols, H.; Sørensen, S.; Ulvskov, P.; Voragen, A.; Visser, R. Remodelling pectin structure in potato. In: Phytosphere ’99 – Highlights in European biotechnology; de Vries, G.E., Metzlaff, K. Eds.; Elsevier Academic Press: New York, NY, 2000; p 245–256. (24)

Dinand, E.; Chanzy, H.; Vignon, M.R. Parenchymal cell

cellulose from sugar beet pulp: preparation and properties. Cellulose 1996, 3(1) 183-188, DOI: 10.1007/BF02228800 (25)

Dufresne,

microfibrils

A.;

from

Dupeyre, potato

D.;

Vignon,

tuber

cells:

M.R.

Cellulose

processing

and

characterization of starch-cellulose microfibril composites. J. of Applied Polymer Science 2000, 76(14) 2080-2092. (26) A.P.

Siqueira,

G.;

Oksman,

K.;

Tadokoro,

S.K.;

Matthew,

Re-dispersible carrot nanofibers with high mechanical

properties

and

reinforcing

capacity

for

use

in

composite

materials. Composites Science and Technology 2016, 123, 4956, DOI: 10.1016/j.compscitech.2015.12.001 (27)

Henriksson,

M.;

Henriksson,

G.;

Berglund,

L.A.;

Lindström, T. An environmentally friendly method for enzyme-


47


assisted

preparation

of

Page 48 of 50

microfibrillated

cellulose

(MFC)

nanofibers. European Polymer Journal 2007, 43(8) 3434-3441, DOI: 10.1016/j.eurpolymj.2007.05.038 (28) a

Willats, W. G.; Marcus, S.E.; Knox, J.P. Generation of monoclonal

antibody

specific

to

(15)-alpha-L-arabinan.

Carbohydrate Research 1998, 308(1-2) 149-152 (29)

McCartney,

antibodies

to

L.;

plant

Marcus, cell

S.E.;

wall

Knox,

xylans

J.P.

and

Monoclonal

arabinoxylans.

Journal of Histochemistry & Cytochemistry 2005, 53(4) 543546, DOI: 10.1369/jhc.4B6578.2005 (30)

Moller, I.; Sørensen, I.; Bernal, A.J.; Blaukopf, C.;

Lee, K.; Obro, J.; Pettolino, F.; Roberts, A.; Mikkelsen, J.D.; Knox, J.P.; Bacic, A.; Willats, W.G. High-throughput mapping of cell-wall polymers within and between plants using novel microarrays. Plant Journal 2007, 50(6) 1118-1128, DOI: 10.1111/j.1365-313X.2007.03114. (31) xylans

Kerr,

E.M.;

increase

Fry,

in

S.C.

molecular

Pre-formed weight

xyloglucans

in

three

and

distinct

compartments of a maize cell-suspension culture, Planta 2003, 217(2) 327–339, DOI: 10.1007/S00425-003-1027-2 (32)

Jork, H.; Funk, W.; Fischer, W.; Wimmer, H. In: Thin-

Layer Chromatography Reagents and Detection Methods; physical


48

Page 49 of 50

and chemical detection methods: fundamentals, reagents, Vol 1a; VCH; Verlags gesellschart: Weinheim, Cambridge, New York, 1990

Agro-industrial waste: - Renewable cellulose source

Traditional Chemical Methods 1. High energy requirements 2. Harsh chemicals 3. Industrially expensive

New Enzymatic Pretreatment 1. Ambient temperatures 2. Gentler CNF isolation 3. Industrially viable

Can invoke irreversible changes (e.g. additional charge) that can impair functionality downstream

Facilitates greater liberation of intact CNF following high-sheer homogenization

Cellulose Nanofibers

TOC graphic

Cellulose Source

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules


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Supplemental


50

Nanofibers Produced from Agro-Industrial Plant Waste Using Entirely

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