Pleanry lecture presented at 8th International symposium on Advanced Technologies for the Pulp, Paper and \corrugated Board Industry

1. Wood bark as valuable raw
material for compounds
with biological activity
Valentin I.Popa
Gheorghe Asachi Technical University of Iasi
Faculty of Chemical Engineering and Environmental
Protection
Blvd. Mangeron No.71, Iasi, 700050, Romania
e-mail vipopa@tuiasi.ro; vipopa15dece@yahoo.com

2. Structure of the bark
• Bark is a highly
heterogeneous and
chemically complex
section of woody
biomass. It is usually
divided into the living
inner bark and dead
outer bark, representing
10-15 % of the total
weight of the tree.

4. Component Softwoods Hardwoods
Wood Bark Wood Bark
Lignin 25-30 40-55 18-25 40-50
Polysaccharides 66-72 30-48 74-80 32-45
Extractives 2-9 2.-25 2-5 5-10
Ash 0.2-0.6 Up to 20 0.2-0.6 Up to 20
Composition by mass of lignin, polysaccharide, extractive and ash
in woods and barks. The non-extractive components are based on
extractive-free material. Taken from (USDA, 1971)
• Bark contains useful products waiting for the right
economic conditions or the development of
satisfactory commercial processes

5. Solvent Typical substances removed in whole or
part
Petroleum ether
ether, benzene,
chloroform
Alcohol, acetone,
aqueous alcohol,
aqueous acetone
Hot or cold water
Aqueous alkali
Acid hydrolysis
Terpenes and their derivatives, fats, waxes, free and
wax acids and alcohols, sterols, resins.
Simple polyphenols and their glycosides, tannins,
mono- and disaccharides (sugars).
Disaccharides, starch, gums, pectins, tannins,
mucilages.
Phlobaphenes, phenolic acids, some bark lignin and
hemicelluloses, suberin fragments.
Simple sugars and uronic acids derived from
holocellulose, leaves residue of “lignin.”
Fractionation of bark

6. Chemical composition of bark
Chemical compounds,
%
Softwood
bark
Hardwood
bark
Alcohol-benzene extract
Cold water extract
Hot water extract
Extract with NaOH,1 %
Cellulose
Lignin
Pentosans
Tannin
4.45-7.50
3.20-5.10
6.00-7.70
31.40-50.00
30.60-35.00
32.70-39.70
11.30-12.70
1.90-3.00
2.70-5.50
6.30-7.40
16.00-16.50
28.40-29.00
31.60-41.70
19.00-30.00
21.00-24.500
-
• C.I.Simionescu, V.I.Popa et al., Holzforschung und
Holzverwertung,40(6), 136 (1988)

7. biochemical
degradation
burningadditives
compost fodder
lignocelluloses
SEPARATION
BARK
EXTRACTION OF
SECONDARY
COMPOUNDS
Rough mixture of
hemicelluloses +
polyphenols
Hcell-OH
PF-OH
Hcell-OH
softwoods
24-26%
hardwoods
15-20%
-furfural
- galacotse
- glucose
- arabinose
- xylose
PF-OH
phenol
substitute
(adhesives)
softwoods
10-12%
hardwoods
4-6%
- C6 phenols
- C6-C1 phenolic acids
- C6-C2 acetophenone
- C6-C3 coumarone
- C6-C1-C6 xanthone
- C6-C3-C6 stilbens
-(C6-C3)2 lignans
PF O CH2
CH
O
CH2
OH PF OH+
PF O CH2
CH
O
CH2
OH PF OH+
O CH2PF CH CH2 O
OH
PF OH prepolymer crosslinking

8. Obtaining phenolformaldehyde
resins with vegetal alkaline extracts
Compound, g R1 R2 R3 R4 R5
Phenol
Extract from:
hardwood bark
softwood bark
Formaldehyde, 37 %
Paraformaldehyde
Sodium hydroxide
solution, 40 %
150
55
200
9
17
150
60
180
8
17
150
40
181
6
17
150
65
207
12
16
150
70
215
10
17

9. Shear strength of plywood glued (N/mm2) with
modified phenolformaldehyde resins
Plywood
made of:
Fenoplac R1 R2 R3 R4 R5
3 veneer
minimum
maximum
5 veneer
minimum
maximum
1.5
-
2.1
-
1.8
2.6
2.7
3.0
1.8
2.4
2.2
2.8
1.6
2.6
-
-
2.4
2.8
2.7
3.0
2.4
2.6
2.5
2.8

10. Influence of addition of the alkaline extract
from beech bark on the properties of wood
fiber boards (Transversal, L-longitudinal)
Degree
of resin
substituti
on, %
Strength,
kg/cm2
Density,
Kg/cm3
Water
absorption, %
Swelling,
%
0
10
20
30
40
T 340
L 340
T 665
L 568
T 350
L424
T 430
L 386
T 505
L 446
1000
1150
1100
1090
1090
30.00
13.70
23.30
23.68
23.90
17.5
12.5
12.8
11.5
15.2

11. Physico-mechanical properties of wood fiber
board obtained at industrial level (compared
with standard level)
Characteristics First quality Second
quality
Experimental
values
Apparent density,kg/m3
Water absorption (after
24 h immersion), %
Thickness swelling
(after 24 h immersion),
%
Static bending strength,
daN/cm2
Internal transversal
cohesion, daN/cm2
1000
(+10%,-5 %)
30
18
400
8.38
1000
(+10%,-5 %)
40
25
300
8.38
991-1021
12.55-17.29
13.80-15.60
327-424
8.63-12.80

12. Bark
Extraction
(II)
Resins
Extraction
(I)
Polyphenols
Fractionation
(I) Hemicelluloses
Fractionation
(II) Cellulose
Composting
Bioremediation
Lignin
Acid hydrolysis/ enzymatic
Nano- and micro
cellulose
NaOH solution

13. Polyphenols
• Secondary metabolites (more than 8000
compounds)
Properties:
antioxidants; prooxidants; anticancer
agents; apoptosis-inducing;
antibacterial, antiparasite; anti-HIV
activities; amelioration of
cardiovascular diseases; improvement
of endothelial function; modulation of
gamma-glutamylcysteine synthase
expression; improvement of health and
survival on high –fat diet; colouring
agents; chelating agents

15. 15

16. Polyphenols were tested in:
• Seed germination
• Plant cultivation
• Bioremediation
• Plant grafting
• Tissue plant culture
• Microorganism cultivation (carotenes
pigments obtaining, mutagenesis)
• Modulation of sugars metabolism
(diabete and alcoholic fermentation)
O.C. Bujor, I. A. Talmaciu, I. Volf, and V. I. Popa
Biorefining to recover aromatic compounds with biological
properties,
Tappi J.,14 (3) 187-193 (2015)

17. 17
POLYPHENOLS ENCAPSULATION BY
ELECTROSPINNING TO OBTAIN SUBSTRATES
WITH BIOLOGICAL ACTIVITY
 Polyphenols (gallic, vanillic, syringic acids, catechine,
spruce bark extract) were encapsulated in nanofibrous
membranes, using biocompatible polymers: [poly (2-
hydroxyethyl methacrylate (pHEMA), poly [(lactic acid)-
co-(glycolic acid)] (PLGA)]
Roxana-Elena Ghitescu, Ana-Maria Popa, Valentin I. Popa,
Rene M. Rossi, Giuseppino Fortunato
Encapsulation of polyphenols into pHEMA e-spun fibers and
determination of their antioxidant activities
International Journal of Pharmaceutics, 494, 278–287(2015)

18. • The immobilized polyphenols were tested
with very good results to inhibit the
reactive oxygen species produced by
carbon nanotubes in the cells A549
originated from an explant culture of lung
carcinomatous tissue from a 58-year-old
Caucasian male.

19. 19
DCF 60 minutes
0
100
200
300
400
500
600
700
800
20µg/ml
MWCNT
100 50 25 12.5 6.25 3.12 1.5 0.78
Concentration of catechin (%)
Fluorescence
485nm[MW-
blanks]
no catechin 0.25% catechin in PLGA fibers 0.5% catechin in PLGA fibers
DCF 120 minutes
0
100
200
300
400
500
600
700
800
20µg/ml
MWCNT
100 50 25 12.5 6.25 3.12 1.5 0.78
Concentration of catechin (%)
Fluorescence485
nm[MW-blanks]
no catechin 0.25% catechin in PLGA fibers 0.5% catechin in PLGA fibers

20. Hemicelluloses based products

21. 21
• Valentin I.Popa
-Hemicelluloses in
pharmacy and
medicine in
Polysaccharides in
medicinal and
pharmaceutical
application, Edited
by Valentin I.Popa,
Smithers, Rapra,
2011

22. Filtrate
discarded
sodium chlorite oxidation
Residue (Holocellulose)
Extractive-free wood bark
Filtrate
(Pectic
substances
and water-soluble
polysaccharides)
* Yield – 12. 5 % Filtrate
(Mixture of pectic
substances and
acidic xylan)
Residue
Filtrate (Xylan, “glucan”
and water-soluble galacto-
glucomann)
Residue
Residue
Filtrate
(Mainly glucomannan)
Yield – 2.5%
Residue (pure cellulose)
Yield – 30.3%
Three successive preparations with aqueous
barium hydroxide
Pure glucomannan (alkali-soluble)
Yield – 2.0%; Percent composition:
Galactose -4; Glucose – 25; Mannose – 71.
*All yields reported in this
scheme are based on dry
extractive- free bark
Hot ammonium oxalate solution
10% (w/w) sodium
carbonate solution
24% (w/w)
Potassium hydroxide
Yield – 4.3%
Yield – 8.5%
17% (w/w)
sodium hydroxide
+ 4% boric acid
Fig.1 Fractionation scheme for Extraction of Hemicelluloses from Bark of Engelmann Spruce

23. 24% Potassium hydroxide Extract
Uronic acid – 7.3; Galactose – 8.8; Glucose – 29.4;
Mannose – 3.3; Arabinose – 8.8; Xylose – 42.5
Composition:
(relative percent)
Precipitate
Yield – 2.2 %
Filtrate
(see fig. 3)
Uronic acid – 3.3; Galactose – 11.4; Glucose – 6.64;
Mannose – 10.8; Arabinose – Trace; Xylose – 27.9
Composition:
(relative percent)
Insoluble Portion
Yield – 0.3%
Soluble Portion
Yield – 1.8%
Uronic acid – 2; Galactose – 10; Glucose –38;
Mannose – 40; Arabinose – Trace; Xylose – 10
Composition:
(relative percent)
Uronic acid – 2; Galactose – 11; Glucose – 57;
Mannose – 2; Arabinose – Trace; Xylose – 28
Composition:
(relative percent)
Three successive
Precipitations with
Fehling’s solution
Aqueous
Barium hydroxide (5%)
(This fraction consist mainly of the water-soluble galactoglucomannan ) (This fraction consist mainly of the heteropolymeric “glucan”)
Fig.2 Fractionation scheme for Extraction of Hemicelluloses from Bark of Engelmann Spruce: Resolution of 24%
Potassium hydroxide Extract

24. Filtrate remaining after addition of Fehling’s Solution to Potassium hydroxide extract (See fig. 2)
Yield – 6.0%
Uronic acid – 7.9; Galactose – 9.1; Glucose – 25.1; Mannose – Nil;
Arabinose – 14.3; Xylose – 43.6
Composition:
(relative percent)
Insoluble Portion
Yield – 0.5%
Soluble Portion
Yield – 4.8%
Uronic acid – 10; Galactose – 12;
Glucose – 15; Mannose – Nil;
Arabinose – 33; Xylose – 30
Composition:
(relative percent)
Uronic acid – 7; Galactose – 20;
Glucose – 25.1; Mannose – Nil;
Arabinose – 10; Xylose -56
Composition:
(relative percent)
Soluble Portion
Yield – 4.5%
Small precipitate
(Discarded)
Uronic acid – 7; Galactose –6;
Glucose – 12; Mannose – Nil;
Arabinose – 10; Xylose – 65
Composition:
(relative percent)
Aqueous barium hydroxide
CTA-OH + aqueous
Sodium hydroxide
(This fraction consist mainly of the acidic arabinoxylan)
Fig.3 Fractionation scheme for Extraction of Hemicelluloses from Bark of Engelmann Spruce: Resolution of 24%
Potassium hydroxide Extract (continued)

25. Reactions:
• Acid hydrolysis
• Enzymatic hydrolysis
• Esterification
• Etherification
• Enzymatic modification (treatment
with laccase of hemicelluloses
from annual plants allow obtaining
gels)

26. Directions to use hemicelluloses:
• ethanol-fermentation C6
• polyols-2,3 butylene glycol-aerobic
fermentation
• lactic, acetic, butyric acids-fermentation
• fodder yeast (50 % proteins; 2-7 % fats-
vitamins) Candida utilis
• xylitol- xylose reducing- sweetener
• furfural-furfurilic alcohol,furan resins,
poly(amide) -4,6

27. Derivatives of de xylan:
-acetates; -butyrates;
-benzoates- extrusion agents for fatty acids
-carboxymethylxylan- (surfactants, flocculants,
adhesives for paper coating);-eating packages;
-xylan sulfate- (antiHIV, antitumor, antioxidant,
anticoagulant, antimicrobial, decrease of
cholesterol);
-biofilms (xyloglucan/chitosan) –immobilisation of
streptomicyn, antioxidants, antifungal and,
antimicrobials agents, dyes, nutrients,
packagings).

28. -arabinoxylans- emulsifying agents; thickening,
food stabilisers, immunotherapy agents;
-4-O-methylglucuronoxylan-antitumor.
Advantages to use hemicelluloses in
pharmacy, cosmetics and medicine:
- are accessible
-are not toxic
-can be chemically and enzymatically modified
-are biodegradable
-are biocompatible

29. Health benefic effects:
-they improve lipids and minerals
metabolism;
-they improve the function of colon and
assure protection against cancer;
-they reduce the risk of heart diseases
Examples:
-regeneration of tissues
-support for controlled delivery of drugs
--gels for cells immobilisation

30. Cellulose based products

31. Nanocellulose-supermaterial
• Eco-friendly
• Lightweight
• Ductile
• Stronger than steel and Kevlar
• The super-material is theoretically derived from plant
matter that has been reduced to small bit and pieces,
and then purified by a homogenizer to remove non-
cellulose components like lignin. The remaining
cellulose fibers are finally separated and processed into
a thick substrate that boasts of long polymers or
crystallized structures. This ultimately results in what is
termed as nanocrystalline cellulose or nanocellulose
‘paste’, an incredible material with flexibility,
malleability, super-strength as well as low-impact
credentials.

32. Uses of nanocellulose
• Composites
• Paper and boards
• Food
• Hygiene and absorbent products
• Emulsion and dispersion
• Oil recovery
• Medical , cosmetic and phramaceutical

33. • V.I.Popa
Nanotechnology and nanocellulose
Celuloză şi Hârtie, 63 (4), 14-23 (2014)
• V.I.Popa
Obtaining of nanocellulose (I)
Celuloză şi Hârtie, 64 (1), 3-10 (2015)
• V.I.Popa
Obtaining nanocellulose (II)
Celuloză şi Hârtie, in press

34. Nanofibrile

35. Schematic representation of (a) the
homogenizer and (b) the
microfluidizer

36. Procedure for individualizing cellulose
nanofibers by ultrasonication

37. Nanocellulose

38. 1) High-strength yet lightweight
body armor -

39. 2) Low-impact (fuel efficient) yet
super-durable vehicles

40. 3) Medical usages

41. 4) Bendable battery systems

42. 5) Flexible electronic displays
• -

43. 6) Bio-fuel can be a by-product
when ‘growing’ nanocellulose
• Sugars resulted in the hydrolysis
pretreatments colud be used by
fermentation to obtain biofuels or
other valuable bioproducts, thus
contributing to the efficiency of the
porocess of nanocellulose fabrication.

44. Lignina

45. Lignin based products

47. Biological properties of lignin
1. Lignins as antibacterials
2. Lignins as antioxidants and
photoprotectors
3. Lignins in reduction of
carcinogenesis
4. Anti-HIV properties of lignins
5. Lignin as spermicide

48. 48
• Valentin I.Popa,
Lignin in
biological
systems
in Polymeric
Biomaterials, 2
vol, Founding
Editor: Severian
Dumitriu, Editor:
Valentin I.Popa,
2013, CRC Press

49. Conversion of native lignin into lignophenol
derivatives and control of their functionality

50. Lignins as antibacterials/Escherichia coli

51. Influence of different lignin samples on pathogenic
bacteria sorption (Curan-commercial kraft lignin-
Borregaard Ltd)

52. The influence of lignin on phytopatogenic
microorganisms

53. Lignins as antioxidants and photoprotectors
• Inhibitory effect of
different lignin solutions
on haemolysis induced
by AAPH. [2,2’-azobis
(2-amidopropane)
dihydrochloride] a
peroxyl radical initiator.
LG-lignosulfonates, BG –
lignin from bagasse, SE
lignin from steam
explosion and CU- Curan
a commercial lignin.

54. Haemolysis and photohaemolysis of CPZ
(chlorpromazine a photohaemolytic compound) in the
presence and absence of different lignins

55. Relative ABTS-radical scavenging activity of
lignin samples
• The ABTS+* [ABTS - 2,2’-azino-
bis(3-ethylbenzo-thiazoline-6-
sulphonate)] cation radicals
were generated by an enzymatic
system consisting of
peroxidase and hydrogen
peroxide.
• He-hemp, Si-sisal, Ab-abaca, Ju-
jute, Fl-1-flax, SW-Ls-1-
lignosulfonate from softwood
(Boresperse 3A),SW-Kr-1- kraft
from softwood (Indulin AT), SW-
Ls-2- lignosulfonate from softwood
(Wafex P), Fl-2- soda flax
(Bioplast), Fl-ox-soda flax
oxidised, SW-Kr-2- kraft from
softwood (Curan 100), SW-SF-
1( soda from softwood
(precipitated at high pH), SW-Kr-
3-kraft from softwood, HW-
organosolv (Alcell) from mixed
hardwoods, SW-SF-2- soda
softwood (precipitated at low pH),
SW-Kr-4-kraft (Curan 2711P

56. Relative chain-breaking antioxidant
effect of lignin in lipid peroxidation

57. Precipitated lignin 3mg/mL; melatonin 1µM; quercetin
1µM; commercial lignin 3 mg/mL.
Sample
Superoxide aninon Hydroxyl radical
Precipitated lignin 51.44 ±1.29 33.68±0.91
Commercial lignin 47.15±2.04 27.81±1.30
Melatonin 79.06±0.32 53.89±1.07
Quercetin 71.46±0.85 53.07±1.13
Inhibition percentages of superoxide anion and hydroxyl radical generation

58. Antimutagenic activity of modified kraft
spruce lignin against 4-nitroquinoline-N-
oxide

59. LIGNIN
The are specialized phagocytic cells that attack foreign substances, infectious
microbes and cancer cells through destruction and ingestion

60. CONCLUSION
• Wood bark contains useful products
waiting for the right economic
conditions or the development of
satisfactory commercial processes.
• By applying the biorefining concept
wood bark could be used to obtain
compounds of high interest in the
biological field.

61. Thank you for your
attention!

62. Questions