Chemistry manufacture and applications of natural rubber

However, natural rubber is obtained almost entirely from a tropical plant, Hevea brasiliensis [8–10].. Hevea brasiliensis is the botanical name of a commercially grown plant producing na

Chemistry, Manufacture and Applications of

Natural Rubber

Smart polymers and their applications

Chemistry,

Manufacture and Applications of Natural Rubber

Edited by Shinzo Kohjiya and Yuko Ikeda

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Contributor contact details xiii

S K ohjiya , Kyoto University, Japan and Y i Keda , Kyoto Institute

of Technology, Japan

K C ornish , The Ohio State University, USA

J E P usKas and K C hiang , University of Akron, USA and

B B arKaKaty , Oak Ridge National Laboratory, USA, formerly of

University of Akron, USA

vi Contents

P P hinyoCheeP , Mahidol University, Thailand

Y i Keda , Kyoto Institute of Technology, Japan

network structure of sulfur cross-linked

S t oKi , National Metal and Materials Technology Center, Thailand

5.2 Temperature-induced crystallization (TIC) and

improve the mechanical properties of natural

A t ohsan and Y i Keda , Kyoto Institute of Technology, Japan

A K ato , NISSAN ARC Ltd, Japan and Y K oKuBo , R t sushi and

Y i Keda , Kyoto Institute of Technology, Japan

viii Contents

T n aKao , The University of Tokyo, Japan, formerly of Sumitomo

Bakelite Co Ltd, Japan and S K ohjiya , Kyoto University, Japan

A B n air and R j osePh , Cochin University of Science and

R C R n unes , Universidade Federal do Rio de Janeiro, Brazil

10.2 NR/cellulose composites 285

Y h irata , H K ondo and Y o zawa , Bridgestone Corporation, Japan

A S h ashim and S K o ng , Universiti Kuala Lumpur, Malaysia

x Contents

Y F uKahori , Queen Mary University of London, UK

A I i sayev , University of Akron, USA

17 Recycling of sulfur cross-linked natural rubber (NR)

Y i Keda , Kyoto Institute of Technology, Japan

T P alosuo , National Institute for Health and Welfare, Finland

Department of Food, Agricultural

and Biological Engineering

The Ohio State University

Ohio Agricultural Research and

Akron Engineering Research Center (AERC)

264 Wolf Ledges, Rm# 209Akron, OH 44325-3906, USAE-mail: jpuskas@uakron.edu

K ChiangDepartment of Polymer ScienceUniversity of Akron

Akron, OH 44325, USA

B BarkakatyCenter for Nanophase Materials Sciences

Oak Ridge National LaboratoryP.O Box 2008

Oak Ridge, TN 37831-6496, USA(* = main contact)

A Tohsan and Y Ikeda*

Kyoto Institute of Technology

1 Natsushima-choYokosuka 237-0061, Japan

Y Kokubo, R Tsushi and Y Ikeda*

Kyoto Institute of TechnologyMatsugasaki, Sakyo

Kyoto, 606-8585, JapanE-mail: yuko@kit.ac.jp

Chapter 8

T NakaoInstitute for Solid State Physics, Neutron Science LaboratoryThe University of Tokyo 5-1-5 KashiwanohaKashiwa, Chiba, 277-8581, JapanProfessor Emeritus S Kohjiya* Kyoto University

7-506, Ohnawaba 6Umezu, Ukyo-kuKyoto, 615-0925, JapanE-mail: kohjiyas@marble.ocn.ne.jp

xvContributor contact details

Chapter 9

A B Nair and R Joseph*

Department of Polymer Science

and Rubber Technology

Professora Eloisa Mano

Universidade Federal do Rio de

Bridgestone Corporation3-1-1 Ogawahigashi-choKodaira-shi, Tokyo 187-8531, Japan

E-mail: hirat3-y@bridgestone.co.jp

Chapter 13

A S Hashim* and S K OngUniversiti Kuala Lumpur Malaysian Institute of Chemical and Bioengineering TechnologyLot 1988, Kawasan Perindustrian Bandar Vendor

London E1 4NS, UKE-mail: yoshi-fukahori@mrj.biglobe.ne.jp

Mannerheimintie 166Helsinki, FIN-00271, FinlandE-mail: timo.palosuo@thl.fi

Introduction to the unique qualities of natural rubber

Natural rubber is in widespread daily use It is unique among types of rubber, biopolymers and other materials in general use [1–10] Its unique qualities may be summarised as follows:

1 Among rubbers, it is the only biomass [3, 7] All other rubbers are chemically synthesised [5] Natural rubber is extracted from a tropical

plant in which the cis-1,4-polyisoprene molecule is bio-synthesised.

2 It is the only polymeric hydrocarbon among biopolymers, i.e,

cis-1,4-polyisoprene is composed of carbon and hydrogen atoms alone All other biopolymers contain other covalently bonded elements (not as impurities) such as nitrogen, oxygen, sulphur, in addition to carbon and hydrogen

3 A biopolymer may be obtained from a variety of natural sources, i.e., plants, animals or fungi However, natural rubber is obtained almost

entirely from a tropical plant, Hevea brasiliensis [8–10] Its natural

habitat is the Amazon River valley, but at present, 99% of natural rubber

is obtained from domesticated Hevea trees in Asia Figure 0.1 shows a Hevea tree under cultivation By means of tapping (making a cut in the

trunk), latex (a milky liquid containing rubber molecules) is exuded and drops into a cup The latex is collected and used in its original form or coagulated to give a solid natural rubber

4 Chemical synthesis of natural rubber has not yet been established, although many industrially valuable biopolymers have been successfully synthesised by chemists [10]

5 As it is an agricultural product, natural rubber is renewable

6 It is carbon neutral, as are many plant products The initiating material for the bio-synthesis of natural rubber is carbon dioxide, thus making it carbon neutral It therefore does not contribute to global warming [10]

At the end of its life, it decomposes to carbon dioxide, so there is no net increase of the gas

7 Natural rubber will remain available despite the depletion of petroleum

Introduction

S KOHJIYA, Kyoto University, Japan and Y IKEDA,

Kyoto Institute of Technology, Japan

and is expected to contribute to sustainable development throughout the twenty-first century (see Chapter 15) This is of particular importance

in organic industrial materials

8 Natural rubber is scientifically unique because of its elasticity From the thermodynamics viewpoint, this is due to an entropy change resembling that of ideal gas It differs from energetic elasticity and standard organic, inorganic or metallic solid materials [4, 10, 11]

material for automobile tyres, and has historically contributed to a society characterised by high-density transportation networks [10]

Hevea brasiliensis is the botanical name of a commercially grown plant producing natural rubber [8–10] Hevea is the generic name and brasiliensis

is one of the 11 species of the genus Hevea, in accordance with Linnaean

nomenclature Other plants growing in the wild have been used for the

extraction of natural rubber These include Castilla elastica (commonly

known as Castilloa), which is grown in Central and South America, and

Manihot glaziovii (Ceara), grown in Brazil Ficus elastica is widespread

in tropical Asia The genera Landlphia (vine rubbers) and Funtumia are

0.1 Cultivated Hevea tree in a plantation under tapping operation

(Photo taken by S Kohjiya in 1975.)

common in mid-western African jungles These are known as typical rubber producing trees [8–10] More types of rubber yielding trees are described

in Chapter 15

American scientists continue to work on Parthenium argentatum (Guayule)

[10, 12–16] (see Chapter 1), a shrub found in Mexican deserts This was cultivated in the United States during the Second World War, when synthetic rubbers underwent rapid development due to the scarcity of natural rubber [16–18]

Russian scientists cultivated Taraxacum kok-saghyz (Russian dandelion

rubber) during the 1930s and 1940s Thomas Alva Edison (1847–1931), with the support of Henry Ford, investigated many types of Goldenrods

as possible sources of rubber in addition to Cryptostegia grandiflora [16]

(Goldenrods are a group of weeds widely found in the United States which are now abundant in other countries, including Japan, as non-native plants.)

In addition to Hevea, more than 2,000 plants are now known to yield rubber, though the quality and quantity are inferior The superiority of Hevea

was recognised as early as the middle of the nineteenth century, although the well-known Collins report [19] failed to state this clearly Natural rubber

from Hevea brasiliensis has historically been preferred (see Chapter 15)

Neither the reasons for, nor the significance of, so many plants being rubber-yielding has yet been fully elucidated When one of the present authors visited RRIC (Rubber Research Institute of Ceylon, now RRISL) in 1977,

he asked bio-related officers (including a physiologist), why plants produce rubber The reply was that there is as yet no evidence for the physiological function of rubber in plants Although rubber appears to be of no use to the plants themselves, they enable the bio-synthesis of highly stereo-regular

cis-1,4-polyisoprene, the perfect stereo-regularity of which has not yet been

achieved by chemical synthesis [10, 20] This unsolved puzzle as to why

cis-1,4-polyisoprene (a polymeric isoprenoid) is produced in plants or in

vegetables may be a unique quality associated with natural rubber

The history of natural rubber

Rubber was first used during the Olmec civilisation (circa 1300–300 BC),

and its use continued among the Mayans (mainly on the Yucatan peninsula

in Mexico, from circa 300 BC to AD 1500), the Incas (the Andes highlands around Peru, from circa AD 1100 to 1500), and the Aztecs (from the twelfth

century in central Mexico) until the Spanish destruction of the Central and South American civilisations The Olmec had been known to tap plants,

most probably Castilla elastica, and to have made rubber goods.‘Olmec’

may mean ‘rubber people’

One of the most notable usages of rubber was the manufacture of balls These were thought to have been used in a game [2, 10, 21] which was

considered an important religious and political event, in which victory or defeat was used to determine the outcome of wars Figure 0.2 shows an athletic field at Chichen Itza, a well-known site of the Mayan civilisation A stone ring attached to the wall at a height of about seven metres is assumed

to be a goal This game is thought to symbolise the harmonious nature of civilisations in South and Central America In the twentieth century, rubber became an important military material, but remained a symbol of peace for the people associated with its origin

The discovery by Columbus of the ‘New World’, which may have marked the end of the Middle Ages in Europe, was the beginning of a European invasion of that new continent by a military force, despite there being few counter-attacks due to the peaceful nature of the local Indians The rubber ball which Columbus observed during his second voyage may be assumed

to have been manufactured by Olmec craftsmen using rubber obtained from

Castilla elastica trees [10, 22] A Spanish priest, P Martyre d’Anghiera, attached to the invading army, first wrote about rubber in his book ‘De Orbo Novo’, which was published in 1530 Further literature was published, but

the useful application of rubber remained unknown among Europeans for nearly 200 years

A breakthrough on rubber came from two French scientists [23] F Fresneau (1703–1770) was an agricultural scientist working at the colonial office in French Guiana While travelling in Guiana and the Amazon in search of economically useful plants, he became interested in rubber-producing trees

on which he prepared a report The other scientist, C M de la Condamine

0.2 Athletic field in the Mayan ancient site of Chichen Itza A

ring-shaped goal can be seen on the wall to the left (Photo from K Aoyama with permission.)

(1701–1774), was a geographer, and a member of the expedition to Quito (1735–1745) whose task was to measure longitude just below the equator While in Cayenne in French Guiana, he obtained the report authored by Fresneau, and later gave a lecture on rubber at the meeting of the French Academy of Science in Paris This was the first scientific report on rubber [10] (Historically, this achievement may be attributed to Fresneau [10, 23, 24].)

In 1765, an encyclopedia entitled ‘Encyclopedie, ou dictionaire raisonne des sciences, des arts et des métiers’ was published in France It included the term ‘caoutchouc’ – the French word for rubber It is probable that one

of the editors, Denis Diderot (1713–1784) drew on Condamine’s scientific paper In England, the chemist Joseph Priestley, who discovered oxygen, noticed in 1770 that pencil marks could be erased (rubbed out) by rubber [25] This means of erasure led Priestly to coin the English word ‘rubber’

In the nineteenth century two Englishmen, Charles Macintosh (1766–1843),

an entrepreneur and Thomas Hancock (1786–1865), an engineer, began the industrialisation of rubber products [25, 26] Macintosh applied rubber solution

to a cloth and found that it became highly water-resistant In cooperation with Hancock, he began to manufacture raincoats using the rubberised cloth [10, 26] London coachmen were the first to welcome this material which then grew in popularity due to its excellent water-proof performance

As the use of rubber products became more widespread, a significant defect was recognised: at low temperatures they became hard and lost elasticity, while at high temperatures they became too soft to retain their original shape This change of properties was found difficult to control, even though much work was devoted to the problem In 1839, Charles Goodyear developed the process of ‘vulcanisation’ [1, 10, 25] This consisted of a cross-linking reaction of rubber molecules with sulphur to give a three-dimensional and stable network structure As a result of this process, natural rubber became

an industrially important resource and a strategically indispensable material during times of war However, the mechanical details of the reaction have only recently been investigated and a full explanation of the process has not yet appeared [27]

It was necessary for natural rubber to show its potential for mass production

if the demands of modern industry were to be met Hevea brasiliensis was

introduced into Britain from the Amazon in the nineteenth century [8–10,

28–32] From Kew Gardens in London, Hevea was transplanted to Ceylon

where it was successfully cultivated, and later spread to the Malay Peninsula

[10, 30, 31] Figure 0.3 shows a Hevea tree transplanted from Britain to

Ceylon in 1876 This is one of the ‘Wickham trees’, the seeds of which were brought by H Wickham (1846–1928) from the Tapajos River Valley to the Royal Botanic Gardens at Kew [8–10, 28–33] These trees were successfully cultivated at the Henaratgoda Botanic Gardens in Ceylon [10, 34, 35] and

the seeds widely distributed in South and South-east Asia by H Ridley (1855–1956), Director of the Singapore Botanic Gardens [10, 28–33] The production of natural rubber from Asian estates was timely in the light of growing demands from the automobile industries, especially in the

United States The Ford Motor company attempted to establish a Hevea

plantation in the Amazon (which it named Fordlandia) to supply natural rubber for their automobile tyres [10, 36–38] Figure 0.4 shows Fordlandia seen from the Tapajos River Its symbol was the water tank which is still in use However, the venture failed and although Ref 38 describes it in detail, there is insufficient information to draw conclusions from an agricultural point of view [10]

During the Second World War, synthetic rubbers were developed in the United States [6, 17, 18, 39], Germany and Soviet Russia to supply tyres to the military [6] After the war, industrially manufactured synthetic rubbers became widespread in the international rubber market [39], and since that time, natural and synthetic rubbers have continued to co-exist [10, 40–42] However, natural rubber is still preferred for many applications, probably

0.3 Wickham tree transplanted in 1876 to the Henaratgoda Botanic

Gardens in Ceylon Its seed was collected in the Amazon by H Wickham, and transported via the Royal Botanic Gardens at Kew to

be cultivated in Ceylon It was 101 years old and a huge tree In the plantations, the trees were replaced every 30 years, and did not grow

as tall as the tree in this figure (Photo taken by S Kohjiya in 1977.)

because of its strain-induced crystallisation ability [10, 11, 43–47] This trend is likely to continue for the foreseeable future due to the superior characteristics of natural rubber, as described in this book

Types of rubber tree

A note on a familiar ‘rubber tree’ may be necessary for some readers It

differs from Hevea or the ‘Para’ rubber tree and should not be confused with the rubber tree described in this book This is the popular house plant Ficus elastica, which is a native of South and South-east Asia Among the genus Ficus, Ficus benjamina is also well known as the ‘Banyan’ or ‘umbrella’

tree, and is found in many urban areas of tropical Asia where it is planted

to provide shade

The Hevea trees described in this book are native to the Amazon and are

botanically different from so-called ‘rubber trees’, although both produce

natural rubber However, Ficus has not been widely used for the extraction

of natural rubber as both the quality and quantity are inferior to that of

Hevea trees [10] Almost all natural rubber for tyres and other rubber articles comes from Hevea brasiliensis Hevea rubber collected from wild trees in the Amazon Valley contributes less than 1% of current total natural rubber

consumption The concluding remarks of Professor R E Schultes on the

history of taxonomic studies in Hevea should be noted [48]:

0.4 Fordlandia on the right bank of the Tapajos River (Photo taken by

Y Ikeda in 2005.)

Few economic plants have more deeply affected civilisation than the Para

rubber tree, Hevea brasiliensis, the product of which has made possible

present-day transportation and much of modern industry and technology Furthermore, this tropical tree represents one of man’s most recently domesticated plants

Here, ‘Para rubber’ refers to wild natural rubber exported from the eastern Brazilian port of Para (now Belem) in Para state

north-Future trends

The introduction explains the characteristic features of natural rubber which make it indispensable to contemporary society It seems probable that natural rubber will contribute to sustainable development for the foreseeable future,

as described in Chapter 15

Currently, the main natural rubber-producing countries are Thailand, Indonesia, Malaysia, India, China, Sri Lanka and Vietnam As the demand for natural rubber grows, Cambodia, Laos, Bangladesh and some African countries may also become major producers

The application of techniques such as genome analysis is likely to become significant in the scientific study of natural rubber, particularly among biochemists and agriculturalists [10, 49, 50] A deeper understanding of the unique qualities of natural rubber is also an important area for scientific and academic study Discussions on the performance of natural rubber are expected to give rise to new applications

References

1 Goodyear, C.: ‘Gum-Elastic and Its Varieties, with a Detailed Account of Its Application and Uses and of the Discovery of Vulcanization’, published for the author, New Haven (1855) (Reprinted in 1939 by the Rubber Division, American Chemical Society.)

2 Davis, C.C., Blake, J.T., eds.: The Chemistry and Technology of Rubber, Reinhold

Publishing Co., New York (1937).

3 Bateman, L., ed.: The Chemistry and Physics of Rubber-Like Substances, Maclaren

& Sons, London (1963).

4 Treloar, L.R.G.: The Physics of Rubber Elasticity, 3rd edn, Clarendon Press, Oxford

9 Sethuraj, M.R & Mathew, N.M., eds.: Natural Rubber: Biology, Cultivation and

Technology, Elsevier, Amsterdam (1992).

10 Kohjiya, S.: ‘Ten-nen Gomu no Rekisi’, History of Natural Rubber, Kyoto University

Press (2013), (in Japanese).

11 Tosaka, M., Murakami, S., Poompradub, S., Kohjiya, S., Ikeda, Y., Toki, S., Sics,

I., Hsiao, B S.: Macromolecules, 37, 3299 (2004).

12 Johnson, J.D., Hinman, C.W.: Science, 208, 460 (1980).

13 Cornish, K., Backhaus, R.A.: Phytochemistry, 29, 3809 (1990).

14 Cornish, K., Siler, D.J.: Journal of Plant Physiology, 147, 301 (1995).

15 Cataldo, F.: Progress in Rubber and Plastic Technology, 16, no 1, 31 (2000).

16 Vanderbilt, B.M.: Thomas Edison, Chemist, American Chemical Society, Washington,

DC (1971).

17 Wilson, C.M.: Trees and Test Tube – The Story of Rubber, Henry Holt and Company,

New York (1943).

18 Morris, P.J.T.: The American Synthetic Rubber Research Program, University of

Pennsylvania Press, Philadelphia, PA (1989).

19 Collins, J.: ‘Report of the Caoutchouc of Commerce’, printed by the order of Her Majesty’s Secretary of State for India in Council, London (1872).

20 Tanaka, Y.: Rubber Chemistry and Technology, 74, 355 (2001)

21 Garrett, W.E.: National Geographic, August, 145 (1968).

22 Hosler, D., Burkett, S.L., Tarkanian, M.J.: Science, 284, 1988 (1999).

23 Trystram, F.: Le Proces des Etoiles, Editions Seghere, Paris (1979).

24 Loadman, J.: Tears of the Tree – The Story of Rubber, Oxford University Press,

Oxford (2005).

25 Singer, C., Holmyard, E.J., Hall, A.R., Williams, T.I.: A History of Technology, 8

vols, Oxford University Press, Oxford (1954–78).

26 Woodruff, W.: The Rise of the British Rubber Industry during the Nineteenth Century,

Liverpool University Press, Liverpool (1958).

27 Ikeda, Y., Higashitani, N., Hijikata, K., Kokubo, Y., Morita Y., Shibayama, M.,

Osaka, N., Suzuki, T., Endo, H., Kohjiya, S.: Macromolecules, 42, 2741 (2009).

28 Drabble, J.H.: Rubber in Malaya 1876–1922, Oxford University Press, Kuala Lumpur

(1973).

29 Schultes, R.E.: Endeavour, New Series, 1, No 3/4, 133 (1977).

30 Brockway, L.H.: Science and Colonial Expansion – The Role of the British Royal

Botanic Gardens, Academic Press, New York (1979).

31 Dean, W.: Brazil and the Struggle for Rubber: A Study in Environmental History,

Cambridge University Press, Cambridge (1987).

32 Jackson, J.: The Thief at the End of the World, Viking, New York (2008).

33 Desmond, R.: The History of the Royal Botanic Gardens Kew, 2nd edn, Kew

Publishing, London (2007).

34 Ferguson, J.: All about Rubber: All Varieties in all Countries, with Harvesting and

Preparation, 3rd edn, A.M & J Ferguson, Colombo (1899).

35 Wright, H.: Hevea brasiliensis or Para Rubber – Its Botany, Cultivation, Chemistry

and Diseases, 3rd edn, A.M & J Ferguson, Colombo (1908).

36 Galey, J.: Journal of Interamerican Studies and World Affairs, 21, 261 (1979).

37 Dempsey, M.A.: Michigan History Magazine, July/August, 24 (1994).

38 Grandin, G.: Fordlandia: The Rise and Fall of Henry Ford’s Forgotten Jungle City,

Metropolitan Books, New York (2009).

39 Whitby, G.S., Davis, C.C., Dunnbrook, R.F., eds.: Synthetic Rubber, John Wiley &

Sons, New York (1954).

40 Brydson, J.A.: Rubbery Materials and Their Compounds, Elsevier Applied Science,

London (1988).

41 Hofmann, W.: Rubber Technology Handbook, Hanser Publishers, Munich (1989).

42 Morton, M., ed.: Rubber Technology, 3rd edn, Chapman & Hall, London (1995).

43 Murakami, S., Senoo, K., Toki, S., Kohjiya, S.: Polymer, 43, 2117 (2002).

44 Toki, S., Sics, I., Ran, S., Liu, L., Hsiao, B., Murakami, S., Senoo, K., Kohjiya, S.:

Macromolecules, 35, 6578 (2002).

45 Trabelsi, S., Albouy, P.A., Rault, J.: Macromolecules, 35, 10054 (2002).

46 Kohjiya, S., Tosaka, M., Furutani, M., Ikeda, Y., Toki, S., Hsiao, B S.: Polymer,

48, 3801 (2007).

47 Ikeda, Y., Yasuda, Y., Hijikata, K., Tosaka, M., Kohjiya, S.: Macromolecules, 41,

5876 (2008).

48 Schultes, R.E.: Botanical Review, 36, 197 (1970).

49 Liyanage, K.K.: Bulletin of the Rubber Research Institute of Sri Lanka, 48, 16

(2007).

50 Okumura, A., Hayashi, Y., Kato, N.: Nippon Gomu Kyokaishi, 82, 424 (2009), (in

Japanese).

Part I

Properties and processing of

natural rubber

© 2014 Woodhead Publishing Limited

Abstract: rubber biosynthesis in plants underpins the production of this

strategically vital polymer A fundamental understanding of the regulation of rate and polymer quality is essential to the development of alternate rubber- producing crops and new rubber materials with novel properties Alternate rubber crops are needed to meet the projected shortfalls in global rubber production caused by the burgeoning economies of China and india

Key words: biochemistry, initiator, monomer, natural rubber, polyisoprene,

polymer.

natural rubber is the fourth most important natural resource of the modern earth, after air, water, and petroleum (history Channel, June 9, 2004) however, due to its pervasive utility, it is also one of our most underrated, taken-for-granted natural products There are at least 40,000 different products made with natural rubber and over 400 medical devices (Mooibroek and Cornish, 2000) The large amount of irreplaceable natural rubber needed in the military, industrial, transportation, medical and consumer sectors have led to natural rubber being repeatedly defined as a strategic raw material over the last 70 years Although many synthetic rubber (derived from petroleum) applications can be met with natural rubber, the converse is not true For example, although all tires contain a significant proportion of natural rubber, the higher the performance required, the greater the amount of the natural rubber component: truck tires are 90–100%, airplane tires are 100%, and navy jet tires on aircraft carriers are single-use 100% natural rubber tires Almost all commercial natural rubber is tapped from a single species,

Hevea brasiliensis, the para rubber tree Production predominately occurs in

plantations and small holdings in south-east Asia, a region which produces about 90% of global natural rubber Africa produces around 10% and south America less than 1% because of the endemic south American leaf blight, a

fatal fungal disease caused by Microcyclus ulei infection (Furtado et al., 2008; Lieberei, 2007; rocha et al., 2011) The genetic diversity of cultivated H brasiliensis is extremely low, advanced lines are grown as clonal scions on

seedling root stocks, and most, if not all, modern lines are M ulei sensitive Thus, H brasiliensis is at constant risk of crop failure.

The expanding economies of China and india have already eroded the small gains achieved in the rubber supply during the economic downturn

of 2008–11, and shortages are burgeoning (Fig 1.1) The independent international rubber study Group (irsG, singapore) has predicted a 1.5–3 million metric ton global shortfall between supply and demand by 2020 – the United states imports 1.2 million MT/yr Like many countries, the United states is currently totally dependent upon the import of natural rubber (nr);

in its case, for more than 1.2 million metric tons per year from grown sources The economic importance of maintaining a steady supply of

tropically-nr is highlighted by the fact that the United states rubber products trade in

2011 was worth over $18 billion

The use of a single species to generate the global supply of a strategy commodity is not necessary, and has happened for rubber more as a matter

of chance than anything else in contrast, many crops are used to supply starch to humans even though the composition and quality of the different crops is dissimilar (e.g., potatoes, wheat, and rice) similarly, many different

plants (Mooibroek and Cornish, 2000), and some Lactarius sp fungi (Mekkriengkrai et al., 2004; ohya et al., 1997, 1998) make rubber and a

few of these, as wild plants, have been used over past centuries and still

1.1 Annual global production and consumption of natural rubber

from 1995 to 2012 with projections to 2020 The economic downturn reduced the rate of consumption from 2007 to 2009, allowing earlier projected shortfalls to be halved (data from the International Rubber Study Group) It should be noted that some of the shortfall could

be made up by increased tapping of existing trees (Dock No, IRSG, personal communication), labor permitting.

5Biosynthesis of NR in different rubber-producing species

could be developed as crops Hevea brasiliensis is actually quite a recent

crop because, until 120 years ago, we did not know how to compound rubber and generate desired product performance (Finlay, 2013) Wild rubber was used but had few applications since then, the enormous investment in all aspects of commercial production has led to the remarkable expansion of the crop that we have seen, particularly in south-east Asia, and until 2005, consumption of rubber has closely matched production (Fig 1.2)

of the many plants capable of natural rubber production, two temperate

species stand out as commercial candidates, Parthenium argentatum (guayule) and Taraxacum kok-saghyz (Kazak dandelion, also known as

russian dandelion and Buckeye Gold) These alternate rubber species are under development at a number of universities and companies on several

continents At the present time, P argentatum is ahead of T kok-saghyz, commercially however, there is a large pilot plant for T kok-saghyz processing

in Wooster, ohio, UsA Also, rubber and latex from T kok-saghyz is similar

to that from H brasiliensis in composition and performance (Cornish et al.,

2012) This similarity includes latex and rubber-particle bound proteins that

cross react with Type i latex allergy, and so this rubber is a supplement to

H brasiliensis rubber, but not a circumallergenic rubber or latex, like that from P argentatum (Cornish, 2012).

Future improvements in rubber yield per area of H brasiliensis and the

development of alternate natural rubber crops require an understanding of

Consumption Production Global natural rubber

1.2 Annual global production and consumption of natural rubber

from 1900 to 2008 The impact of World War II is clear and led to the Emergency Rubber Project However, the world needs ten times more rubber than it needed in the 1940s, and demand will at least double by 2030 (data from the International Rubber Study Group).

how the rubber is made, and how these mechanisms relate to both yield and quality This understanding can direct both genetic engineering approaches, and plant breeding Without such knowledge, such efforts rely heavily on serendipitous discoveries, and research over many years is required in this chapter, i attempt to describe some of the commonalities and complexities

of rubber biosynthesis and what we know, at this time, about the regulation

of rubber biosynthetic rate, chain transfer and final molecular weight in evolutionarily-divergent rubber-producing species

rubber biosynthesis requires two distinct pyrophosphate substrates and

a divalent cation activator, usually magnesium ions in the living plant system (Archer and Audley, 1967, 1987; Cornish, 2001a, 2001b; Cornish

(PP) substrates into the active site The first substrate, which initiates the polymerization reaction, is an allylic pyrophosphate (APP), which appears

single initiator, the rest of the rubber polymer is made from the non-allylic-PP, isopentenyl pyrophosphate (iPP) (Fig 1.3a) iPP is isomerized to DMAPP

(Fig 1.3b) and then two condensation reactions, catalyzed by the trans prenyl

transferase farnesyl pyrophosphate synthase, sequentially add two iPPs to

the DMAPP to make FPP Thus, the final rubber polymer is actually trans trans-[cis] n -polyisoprene, when n is an indeterminate number that is around

30,000 for most high performance rubbers The head group of the elongating rubber molecule functions as the APP during polymerization The reaction is

an alkylation by prenyl transfer from the non-allylic pyrophosphate monomer iPP (nucleophile) to the initiator APP (electrophile) (Walsh, 1979)

1.3 (a) Structure of IPP; (b) structure of APP, where APP is

dimethyl allylic pyrophosphate (DMAPP) if R = H, APP is geranyl pyrophosphate (GPP) if R = C5H9, APP is farnesyl pyrophosphate (FPP) if R = C10H17; (c) structure of FPP In solution, the

pyrophosphate groups will be ionized to varying degrees.

7Biosynthesis of NR in different rubber-producing species

Each rubber polymerase can produce many rubber molecules sequentially, and multiple enzymes are present on each enzymatically-active rubber particle (Castillón and Cornish, 1999) however, the mechanism by which chain termination occurs is poorly understood and may not be the same in different

species In vitro, chain length, and therefore termination, is closely tied to

the rate of the chain transfer reaction (the displacement of the elongating rubber molecule by a new APP initiator) which, in turn, is governed by the concentration, APP identity, and APP:iPP ratio, the magnesium cofactor

these compounds (Castillón and Cornish, 1999; Cornish, 1993, Cornish and

Backhaus, 1990; Cornish et al., 2000; Cornish and scott, 2005; Cornish and siler, 1995, 1996; da Costa et al., 2005, 2006; Espy et al., 2006; scott et al.,

2003; siler and Cornish, 1995) however, if this were the only mechanism, the concentration of substrates and activators in rubber-producing tissues must be very tightly regulated, at least in species making rubber in laticifers,

because the rubber polydispersity is quite narrow (Cornish et al., 1993) Low

polymer polydispersity means that the same molecular weight is made all the time in a specific species, even though different species make rubber of different chain lengths We have only found broad-based rubber transferase-

regulated polymer chain length in Parthenium argentatum (guayule), a species

that makes high molecular weight rubber in generalized bark parenchyma cells Tight control of cytoplasmic APP and iPP concentrations may be possible in a latificer, because this organ is partially separated from the tissues and functions essential to life however, these isoprenoid substrates are used by many other enzymatic processes in plants, and their abundance varies with season, stage of plant growth and development This suggests that other endogenous factors may be involved in the maintenance of low polydispersity

The substrates for rubber biosynthesis, iPP (the monomer), and its APP catabolites (the initiators), are synthesized from carbohydrates via acetyl-coenzyme A, 3-hydroxy-3-methylglutaryl-coenzyme A reductase and mevalonate The plastid-localized deoxy-xylulose/methyl-erythritol phosphate

pathway also produces iPP (Lichtenthaler et al., 1997; rohmer et al., 1993)

Plastidic iPP can move from the chloroplast to the cytosol, where it would

be available for rubber biosynthesis, but it is not known how much

cross-talk between the two compartments actually occurs (Kumar et al., 2012).

Rubber transferase is an unusual enzyme in that the specific binding of

molecule and does not directly involve the entire substrate (Cornish, 2001a,

APP initiator, with binding affinity increasing with initiator length until the interior of the rubber particle is reached Length, in this case, refers to the

minimized chemical structure (much shorter in an all cis-APP than an all

trans one) and not to the number of carbons in the polymer chain (Table 1.1) Many different APPs have been shown, in in vitro assays, to effectively

initiate rubber biosynthesis ((Archer and Audley, 1987; Castillón and Cornish, 1999; Cornish, 2001a, 2001b; Cornish and scott, 2005) (Tables 1.2–1.4) The use of labeled and derivatized APPs to study rubber biosynthesis is limited

Table 1.1 Intramolecular lengths of different substrates in rubber biosynthesis

Molecules measured are minimized structures

Substrate Entire molecule Entire hydro- Linear region Linear region

(terminal P to carbon region (from (from C

terminal C) (first to last C) terminal P) adjacent to OPP)

Note: All measurements are based on straight lines between the centers of the two

atoms designated and are expressed in nm.

Table 1.2 Binding constants (Km ) and maximum reaction velocity (V max ) of the

Parthenium argentatum rubber transferase

Substrate Size Stereoisomer Apparent K m V max

9Biosynthesis of NR in different rubber-producing species

Table 1.3 Binding constants (Km ) and maximum reaction velocity (V max ) of the

Hevea brasiliensis rubber transferase

Substrate Size Stereoisomer Apparent K m V max

geranyl pyrophosphate; GGPP, geranylgeranyl pyrophosphate; HPP, hexaprenyl pyrophosphate; NPP, neryl pyrophosphate; OPP octaprenyl pyrophosphate; SPP, solanesyl pyrophosphate

Table 1.4 Binding constants (Km ) and maximum reaction velocity (V max ) of the

Ficus elastica rubber transferase

Substrate Size Stereoisomer Apparent K m V max

geranyl pyrophosphate; GGPP, geranylgeranyl pyrophosphate; HPP, hexaprenyl pyrophospahe; NPP, neryl pyrophosphate; OPP octaprenyl pyrophosphate; SPP, solanesyl pyrophosphate

only by the solubility (Lichtenthaler et al., 1997; rohmer et al., 1993) of the

APP, and the solubility of large or complex APP molecules can be enhanced

by the use of noninhibitory concentrations of detergents This ability has proven a powerful tool in biochemical studies and has led to biochemically-based models of the active site (Cornish, 2001a, 2001b) Derivatized APPs

of various types (Xie et al., 2008; DeGraw et al., 2007; henry et al.,

2009)

In contrast, the IPP binding site is highly specific and no IPP analogs,

to date, have been incorporated into the rubber polymer in in vitro assays

There is some very interesting work, from the Puskas group, however, indicating isoprene can be incorporated under specific circumstances, but the mechanisms for this are not fully understood, and it may not be relevant

in planta (Chiang et al., 2009, 2011; Kostjuk et al., 2011; Forestier et al., 2009; Lindsay et al., 2008; Puskas et al., 2006; Puskas, 1994; Puskas and

Wilds, 1994)

it has also been shown that APP competitively inhibits iPP binding at the iPP binding site, but that iPP enhances APP binding at the APP binding

site (Castillón and Cornish, 1999; scott et al., 2003) Thus, it appears that

both nonallylic and allylic pyrophosphates are behaving as substrate analogs capable of allylic pyrophosphate substrate activation

1.3 Rubber particles and rubber biosynthesis

rubber particles from different species have highly species-specific complements of lipids and proteins, and these can change with rubber particle age one of the proteins, or protein complexes, is responsible for rubber biosynthesis This biological catalyst, rubber transferase or rubber polymerase (EC 2.5.1.20), is embedded, probably as a complex, in the

monolayer membrane of cytosolic rubber particles (Cornish et al., 1999; Wood and Cornish, 2000; siler et al., 1997; Backhaus and Walsh, 1983,

Cornish and Backhaus, 1990) A combination of structural and kinetic studies indicates that the substrates for rubber biosynthesis enter the rubber particle at the surface and the rubber polymer is elongated to the interior of the rubber particle on the far side of the monolayer biomembrane (Cornish,

2001a, 2001b; Cornish et al., 1999; Wood and Cornish, 2000) Extension

of the elongating rubber polymer into the hydrophobic rubber interior of the particle is probably essential to the continued polymerization reaction The aqueous-organic interface provided by the rubber particle monolayer biomembrane is probably required and accounts for the general lack of success

in identification of solubilized rubber transferase activity Only one report

has been published, for the P argentatum rubber transferase (Benedict et al., 2009), but this has not yet been reproduced.

11Biosynthesis of NR in different rubber-producing species

The organic aqueous interface between the aqueous cytosol and the organic rubber interior is essential to the synthesis of high molecular weight hydrocarbon rubber chains This is because the rubber is synthesized from hydrophilic pyrophosphates in the cytoplasm, and then the hydrocarbon rubber polymer must be elongated into the hydrophobic interior of the rubber particles

A combination of structural and kinetic studies indicates that the polymer passes through a hydrophobic column as it traverses the membrane (Cornish,

2001a, 2001b; Cornish et al., 1999) The physical length of this column was

determined by kinetic characterization, using different APP initiators, and electroparamagnetic spin probe analysis These contrasting methods yielded the same conclusions, with the distance from the APP specific binding site

to the rubber polymer particle interior being equivalent to the size of an all

trans GGPP for H brasiliensis and F.elastica and an all trans FPP for P argentatum (Table 1.4)

Extension of the elongating rubber polymer into the hydrophobic rubber interior of the particle is probably essential to the continued polymerization reaction Without the hydrophobic compartment drawing the polymer from the enzyme, the polymer would rapidly block the active site This type of blockage has been shown in GGPP synthase when site-directed mutagenesis opened the floor of the enzyme’s binding pocket In this case, a significantly

longer trans-polyisoprene molecule was synthesized, but once this polymer

obstructed the channel through hydrophobic interactions coiling up the

polymer against the protein, synthesis halted (Tarshis et al., 1996)

The physical interaction between the hydrophobic rubber particle interior and the elongating rubber molecule may increase the physical ‘tug’ on the chain and eventually encourage the chain transfer reaction, by increasing the vibration or strain on the head group and pulling the APP terminus away from the binding site at the moment of prenyl transfer and transient substrate release It is also possible that the fluidity of the membrane enhances the

chain release from the active site Electron para magenetic spin probe analysis demonstrated that the H brasiliensis and P argentatum rubber particle membranes are fluid, whereas those of Euphorbia lactiflua (high in protein) and F elastica are stiff (Cornish et al., 1999) Furthermore, the fatty acid

analysis (Table 1.5) suggests that particle size may be related to fatty acid

size (siler et al., 1997) The smallest rubber particles (E lactiflua, mean

particle diameter 200 nm) also have the shortest fatty acids in the particle

membrane, whereas the largest particles (F elastica, mean particle diameter

1.5) scanning electron microscopy also clearly shows the stiff membrane

of the F elastica particle, and proves that the inner rubber core is fluid (Fig

1.4) (Wood and Cornish, 2000)

1.4 Kinetic analyses of rubber transferase

rubber biosynthesis is dependent upon the concentrations of APP (initiator), iPP (monomer), and magnesium ions (activator) Kinetic constants are best determined for each by varying the concentration of one at a time, while the other two are present at non-limiting, but non-inhibitory, concentrations These constants can vary over several orders of magnitude in different rubber-producing species for the initiator, and over at least one order of magnitude for the magnesium ion activator, and other activators can be different again Thus, several species-specific experiments may be required

to find the appropriate concentration ranges for good kinetic data Depending upon which aspect of rubber biosynthesis is under investigation, the initiation reaction, the polymerization reaction, or both simultaneously (as is most common), different kinetic analyses are appropriate (segel, 1993) We have

found that the Michaelis–Menton plot of 1/v versus 1/[S] generally results

The Eadie–hosftee plot of v/[S] versus [S] generates a linear plot over

most concentrations for the iPP polymerization reaction, in the presence

of non-limiting initiator concentrations, but very low iPP concentrations and non-limiting iPP concentrations should be deleted The gradient of

Table 1.5 Sum of neutral, phospho and glycol lipids in rubber particle membranes

from four species

13Biosynthesis of NR in different rubber-producing species

initiator used However, due to the non-specific hydrophobic binding region

in the rubber transferase active site, short initiators also generate curved v/ [S] versus [S] plots in these circumstances, we suggest using the hill plot

value where y = 1.

Similarly, cofactor investigations can be problematical It is difficult to

kinetics, because of its tight affinity with the active site, without taking so much time that appreciable enzyme activity is lost during the purification This problem can be solved by the addition of EDTA to chelate the essential magnesium cation activator and bring the enzyme activity to baseline Titrating back magnesium will indicate the true origin as activity rises above the baseline value That particular magnesium concentration becomes the true

origin and is subtracted to adjust the x-axis values to the origin informative

kinetic plots can then be constructed and rate constants determined These analyses underpin the next sections of the chapter A recent publication can

be consulted to obtain details of rubber transferase assays and related topics (Cornish and Xie, 2012)

1.5 Regulation of biosynthetic rate

1.5.1 Allylic and non-allylic pyrophosphates

Enzymological investigations ideally are performed using a soluble enzyme system where

such reactions follow Michaelis–Menton kinetics however, this equation does not adequately describe rubber biosynthesis, which involves a membrane-bound enzyme, two substrates, a cofactor and a polymeric product that is not fully released from the active site at each substrate addition This system can be described as:

(rubber) Membrane-bound enzyme reactions can be investigated intact