Taraxacum Kok-Saghys as a Strong Candidate Alternative Natural Rubber Crop in Temperate Regions in the Case of Emergency

Abstract

Natural rubber (NR, cis-1,4-polyisoprene) used in over 50,000 products, has unique properties, which cannot be matched by synthetic rubber. Hevea brasiliensis Muell. Arg. is currently the only NR commercial source that is not secure because of Hevea tree diseases, increasing demand, high labor costs, price instability, trade politics, competition for land with other crops, and a deforestation ban preventing new H. brasiliensis acreage. Hence, alternative rubber-producing crops are required for increasing the geographic and biological diversity of NR production. The mechanical properties and molecular composition of Taraxacum kok-saghyz NR are nearly identical to those of H. brasiliensis NR. However, developing T. kok-saghyz as an industrial crop is faced with some problems. This plant can become a commercially viable rubber-producing crop by improving agronomic fitness, rubber yield, and extraction process efficiency. An efficient process should extract NR at a high yield without damaging its physical and mechanical properties. This chapter focuses on the potential ways to improve rubber production and extraction processes from T. kok-saghyz.

1. Introduction

Natural rubber (NR, Cis-1,4-polyisoprene) applied in over 50,000 products [1, 2] has unique properties which cannot be matched by synthetic rubber including abrasion and impact resistance, efficient heat dispersion, elasticity, malleability at cold temperatures, and resilience [1, 2]. Trans-polyisoprene has poorer low-temperature thermoplasticity (Tm = 54.5°C) and less flexibility compared with NR [1]. Synthetic rubbers are polymers of alkenes or dienes derived from petroleum which is a non-renewable resource [3]. Trans-polyisoprene possesses different characteristics which include high rigidity, very low coefficient of thermal contraction/expansion, outstanding insulation, and resistance to acid and alkali situations [1]. Due to its unique properties, this polymer is suitable for use in insulated cables, sporting goods, molds, dental products, and medical and scientific instruments.

H. brasiliensis Muell. Arg. is the only commercial NR source [1]. The amazon basin as a native region of H. brasiliensis only produces 2% of NR world production [4] because of South American leaf blight [a fatal endemic disease] on this continent. Southeast Asia countries, especially Thailand (4.85 million metric tons in 2019) and Indonesia (3.30 million metric tons in 2019) are the main NR producers [1, 2]. Attempts to develop H. brasiliensis clones having stable resistance and accepted yields have not been successful [1]. H. brasiliensis tree may enable to escape the disease by planting in escape or sub-optimal areas because long dry seasons interrupt the development cycle of fungus but yield decreases. The new clones are needed in such marginal and an agroforestry system can be planted with other perennial crops such as coffee or cocoa to achieve profitability [5, 6]. Currently, Global NR sources are not secure because of Hevea tree diseases, increasing demand, high labor costs, price instability, business politics, competition for land with other crops, and deforestation ban [1, 2]. Hence, alternative NR-producing plants are needed.

Many plants were studied in terms of rubber production potential when supply problems arose due to NR price or accessibility, particularly during World War I (the 1910s), World War II (the 1940s), and oil embargo (the 1970s). One of the prominent efforts is the establishment of the Edison Botanical Research Company in 1927. This company evaluated more than 17,000 rubber-producing plants in terms of NR content and quality [1].

2. Alternative rubber-producing plants to H. brasiliensis

About 2500 plant species produce cis-polyisoprene NR which is not always in a form of tappable latex [7]. It is noteworthy that molecular weight strongly correlates with quality and the most significant indication of usable rubber is a molecular weight of 1000 kg/mol or more [1, 2]. NR with an average molecular weight of over 1000 kg/mol is produced by only a few plants including para rubber tree (H. brasiliensis), Parthenium argentatum (guayule), T. kok-saghyz (rubber dandelion), Taraxacum brevicorniculatum, Scorzonera tau-saghyz, Scorzonera uzbe-kistanica, prickly lettuce (Lactuca serriola), lettuce (Lactuca sativa), Ficus bengalensis, and Madagascar rubber vine (Cryptostegia grandiflora) [1, 2].

The potential of Prickly lettuce as an alternative rubber source is not certain because of its low rubber content. Scorzonera uzbekistanica, S. tau-saghyz, and T. kok-saghyz can accumulate significant rubber content [1], but this takes years to accumulate.

Most T. kok-saghyz plants accumulate below 10% NR in the first cultivation year, but rare plants may accumulate above 20% NR [8]. The mechanical properties and molecular composition of T. kok-saghyz NR are nearly identical to those of H. brasiliensis NR, simplifying development, while P. argentatum rubber compounds need alternative formulations to meet product characteristics [9]. Many rubber companies are currently involved in P. argentatum and T. kok-saghyz rubber. However, it is not possible to go straight to a commodity tire market, so the success key is solving scalability issues.

T. kok-saghyz can be planted as an annual crop in temperate regions. Development of T. kok-saghyz as an industrial crop is faced with some problems including self-incompatibility, high heterozygosity, very variable rubber concentration, considerable rubber yield only quantifiable at maturity, its need for continual moisture during germination, low rate of growth, and poor competition with weeds [10, 11]. Hence, conventional and molecular breeding is required for improving agronomic fitness and NR yield to convert this species into a commercially reasonable rubber crop. Also, converting T. kok-saghyz into an industrial rubber-producing crop requires a cost-effective and environmentally friendly rubber extraction process proven at a commercial scale. This chapter focuses on the potential ways to improve rubber production and different latex and solid rubber extraction processes from T. kok-saghyz.

3. NR biosynthesis pathway

NR is composed of isopentenyl monomers derived from isopentenyl pyrophosphate, synthesized primarily from the cytosolic mevalonate pathway and likely also from the plastidic 2-C-methyl-D-erythritol-4-phosphate pathway (Figure 1) [1]. Geranyl pyro-phosphate, farnesyl pyrophosphate (FPP), and geranylgeranyl pyrophosphate can serve as rubber molecule initiators [12, 13]. FPP is most likely the leading in vivo initiator.

Figure 1.

The pathway of natural rubber (cis-1,4-poly isoprene), 2-C-methyl-D-erythritol-4-phosphate (MEP), mevalonate (MVA), and the oligomeric allylic pyrophosphate biosynthesis [1]. DXS: 1-deoxy-D-xylulose-5-phosphate synthase; DXR: 1-deoxy-D-xylulose-5-phosphate reductoisomerase; MCT: 2-C-methyl-D-erythritol-4-phosphate cytidyl transferase; CMK: 4-(cytidine-5′-diphospho)-2-C-methyl-D-erythritol kinase; MCS: 2-C-methyl-D-erythritol-2,4-cyclodiphosphate synthase; HDS: 4-hydroxy-3methylbut-2-enyl diphosphate synthase; HDR: 4-hydroxy-3-methylbut-2-enyl diphosphate reductase; ACAT: acetyl coenzyme A acetyltransferase; HMGS: hydroxymethylglutaryl coenzyme A synthase; HMGR: hydroxymethylglutaryl coenzyme A reductase; MVK: mevalonate kinase; PMK: phosphomevalonate kinase; PMD: diphosphomevalonate decarboxylase.

Rubber biosynthesis (Figure 1) is catalyzed by RT-ase (EC 2.5.1.20) at the rubber particle surface [14]. RT-ase is the only cis-prenyltransferase (CPT) that can biosynthesize high molecular weight cis-polyisoprene (>1000 kg/mol). Even CPTs that are not part of RT-ase, can affect rubber biosynthesis because CPTs are associated with sterol biosynthesis, and they are possibly required for forming rubber particle membranes. Also, most CPTs produce short-chain cis-allylic pyrophosphates which can function as rubber polymer initiators [15]. It seems that small rubber particle protein and rubber elongation factor are involved in NR biosynthesis [16, 17, 18] and they are effective targets for knockout or overexpression. A substantial mechanism regulating the secondary metabolite pathway is the transcriptional co-regulation of its pathway genes [19, 20]. Hence, this may demonstrate a main research scope in the future. Also, CPT-Like/CPT-binding proteins seem to be involved in NR biosynthesis [21, 22, 23] and may supply structural scaffolds [22].

4. Potential ways to improve NR yield

Theoretically, the reconstitution of rubber particles, a rubber synthetic machinery has not yet been achieved [1]. A eukaryotic organism with an endomembrane is required for ectopic rubber biosynthesis because eukaryotic post-translation modification may modify the RT-ase and also, the biogenesis of rubber particles likely occurs in Golgi or endoplasmic reticulum [24]. The overexpression of hydroxymethylglutaryl coenzyme A reductase [25] and allylic pyrophosphates [26] may increase rubber production, but it may harm rubber quality especially molecular weight [26]. It was noteworthy that the result of isoprenoid overexpression studies depends on the genetic background [1]. There is a negative correlation between rubber and inulin contents in T. kok-saghyz [14, 27, 28]. The target secondary metabolite content may be increased by blocking the branch pathways [29]. The content of short-chain polyisoprene was increased (two folds) in T. kok-saghyz and T. brevicorniculatum roots by over-expressing fructan 1-exohydrolase, which catalyzes the degradation of inulin to fructose and sucrose [30]. In brief, high molecular weight rubber biosynthesis in plants, yeasts, and bacteria has not been achieved by molecular genetic studies, and only isoprenoid production is slightly increased [14, 22, 30, 31, 32, 33, 34]. This indicates that our knowledge about feedback mechanisms and other rate-limiting enzymes is incomplete.

Breeding of T. kok-saghyz is essential in terms of agronomic properties, vigor traits, and rubber yield to convert it to a commercial crop. Hybridization [Taraxacum officinale (common dandelion) × T. kok-saghyz], selection, hybrid breeding, and polyploidy breeding have been assessed to achieve this aim. Quantitative traits can be selected by the family selection in the best way. For example, population selection and half-sib family selection increased the germination rate from 5.8% in cycle 0 to 40.8% and 47.8% in cycle 3, respectively, under in vitro water stress [35]. Also, half-sib family, recurrent selection increased rubber yield from 0.15 to 0.22 g/plant after four selection cycles [36]. Interbreeding of diploid dandelion species failed to improve vigor and rubber concentration in single Taraxacum genotypes in early attempts [37]. However, recent European attempts have been successful to introgress vigor-related genetic elements of T. officinale into a rubber-producing genetic background of T. kok-saghyz [38]. The polyploidy breeding approach can enhance rubber concentration and/or root size and potentially end in high rubber yield. The self-incompatibility of T. kok-saghyz results in heterozygous and complex genetic backgrounds of its seeds, which may confound the chromosome doubling effects. Rare vigorous T. kok-saghyz tetraploids obtained in polyploid research [39, 40] propose that tetraploid breeding stands as a hopeful method for increasing T. kok-saghyz rubber yield. Developing the homozygous or inbred T. kok-saghyz lines are challenging because the self-incompatibility of diploid T. kok-saghyz prevents its self-fertilization. Development of T. kok-saghyz hybrids is possible using self-compatibility (SC) and cytoplasmic male sterility (CMS) traits [1]. CS and CMS have been detected [11] and identified [41] in T. kok-saghyz. Unfortunately, SC and CMS phenotypes are temperature-dependent [41].

Either hydroponic production or genetically modified in vitro cultures can be more rapidly scaled up than field crops and could provide essential, more expensive, NR in the case of emergency. Several strategies can enhance secondary metabolite production in in vitro cultures, for example, cell metabolism modification applying elicitors can result in increased secondary metabolite production in plant cells [42, 43, 44, 45].

5. Rubber extraction processes

Rubber exists as latex (a rubber particle aqueous emulsion) and solid rubber threads in T. kok-saghyz living roots [2]. For extracting the rubber particles in latex form, T. kok-saghyz roots must be mechanically homogenized [46, 47]. The latex and solid rubber extraction processes date back to the 1930s. Soviet research focused on latex extraction [48, 49, 50] and US Researchers simulated mastication using wet-milling to extract solid rubber in water [51, 52, 53, 54]. If the latex is not needed, it is better to recover all the NR as solid rubber by drying the roots [2].

For economic viability, all T. kok-saghyz components must be used [2]. Rubber, sugar syrups, soluble fiber, food, food and beverage ingredients, and biofuels can be generated from T. kok-saghyz. The leaves also can be applied in salads, teas, and tisanes [47]. Polar organic fractions contain useful compounds such as lubricants, cosmetic ingredients, insect pheromones, sealants, adhesives, surfactants, and emulsifiers [55].

5.1 Latex rubber extraction

Soviet researchers extracted latex from T. kok-saghyz roots using the flow method, in which thin circular cut roots are put in the extraction medium to extract the latex into a stabilizing buffer, then it is centrifuged to recover the latex [48, 49, 50]. USDA scientists developed the blender method to extract latex from P. argentatum at lab and pilot scales [56, 57]. This process can be applied to extract latex from T. kok-saghyz roots [2].

The latex colloidal stability can be assessed by zeta-potential measure [58]. The latex is likely coagulated when the emulsions go acidic [58]. The latex colloidal stabilization is ordinarily kept by adding hydroxides (most commonly, ammonium hydroxide [59], or ethanolamine (ETA) [58]. Some bases (like KOH or ammonia) and ETA possess enough bactericidal activity to maintain latex for several months [58]. ETA is a better stabilizer and more “green” compared with ammonia or KOH [2].

5.2 Solid rubber extraction

Drying the roots recover all the rubber as solid rubber. Several processes have been patented to extract rubber from dried T. kok-saghyz roots [47, 52, 53, 54, 55, 60, 61, 62] and some processes have pending applications. They include wet milling, enzyme digestion, solvent extraction, and dry milling processes.

5.2.1 Wet-milling process

Wet milling simulates mastication using pebble milling to extract rubber in water [9, 51, 52, 53, 54, 63]. In the Eskew process, at first, inulin and other water-soluble components are extracted from dried and chopped T. kok-saghyz roots by mixing the roots with hot water, then rubber is separated from root biomass using pebble milling, and in the end, the rubber is retrieved by flotation [51, 52, 53, 54]. No enzymes or chemicals are needed in the Eskew process (wet milling) [52], so it is a low-cost process [2]. The rubber purity reduces in this method because root biomass is trapped in the rubber phase [2]. Eskew process can retrieve nearly 75% of the rubber in T. kok-saghyz roots with a dirt content of 12–15% w/w dry NR [53], which hurts rubber mechanical characteristics and restricts its commercial application. Two processes were patented for improving the yield and purity of rubber extracted by the Eskew process. One of them reported that the rubber yield was increased to 90% by milling whole roots instead of chopped roots [52], but the rubber impurity was still 10–15% [53]. In another patent, the crude rubber extracted by Eskew process was scrubbed in NaOH solution, and then it was neutralized with stearic acid to improve rubber purity (1.47% impurities) [53].

5.2.2 Enzyme digestion process

About 77% (w/w) of rubber impurities extracted using the Eskew process include cellulose, hemicellulose, lignin, and pectin [64]. So, in the enzymatic digestion process, after carbohydrate extraction from dried and crushed roots (less than 1 mm), rubber is further purified by a mixture of industrial cellulose, hemicellulose, and xylanase enzymes [47, 61, 64].

The PENRA III, an enzyme-based aqueous process was developed by the team of PENRA (Program for Excellence in Natural Rubber Alternatives). This process consists of extracting with hot water, alkaline pre-treatment, treating with enzyme, centrifuging, pebble milling, floating, and filtrating [63]. The rubber yield and purity were 80% (of the theoretical T. kok-saghyz NR yield) [64] and 99.5% (at lab-scale) [65], respectively in this process. The enzymes secreted from Thermomyces lanuginosus fungus and enzymes contained in transgenic maize flour may be used as low-cost enzymes in the commercial extraction process [2].

5.2.3 Solvent extraction

In solvent extraction, inulin is preliminary extracted from dried and ground T. kok-saghyz roots using hot water, then polar and non-polar solvents are sequentially or simultaneously used [66] for extracting and purifying rubber. The purity requirements of 99.8% are met in solvent extraction [67] but may lead to safety and environmental concerns. Also, it is faced with scale-up challenges and is expensive. Also, this process may result in lower rubber yield because T. kok-saghyz rubber has naturally crosslinked rubber gel which difficulty dissolves in solvents [68].

5.2.4 Dry milling process

T. kok-saghyz roots dried to 7.5% moisture were ground by a gristmill, then rubber threads were separated from root tissue applying a mesh screen (mesh size: 2 mm). The rubber threads were shaken in warm water, settled, floating rubber threads, skimmed off, and re-stirred in warm water [60]. This process can recover 97.5% of the extractable rubber with a purity of 99.8% after 5 repetitions.

5.3 Natural rubber quality

On the molecular level, the determinants of rubber quality consist of polymer molecular weight, macromolecular structure (branching), gel content, and content and composition of non-rubber components like lipids and proteins [1, 2]. Hence, the extracted rubber should be assessed in terms of rubber purity, gel content of the solid rubber, resin content, molecular characterization, and NR composition. An impurity content of less than 0.2% is required for preventing unallowable tear initiation and propagation according to ASTM D1278-91a [69].

6. Conclusions and perspectives

Trying to establish H. brasiliensis clones having acceptable yield and durable resistance has not been successful. T. kok-saghyz as a strong candidate alternative rubber crop needs optimized agronomic practices and extraction processes. New T. kok-saghyz plants can be developed using gene editing and breeding research. None of the studied processes for T. kok-saghyz rubber extraction are fully satisfactory and each of them has intrinsic advantages and disadvantages. It is necessary to optimize the extraction process that can produce rubber with high purity and high quality, is cost-effective, and does not cause environmental concerns.

Conflict of interest

The authors declare no conflict of interest.

Abbreviations

NR	natural rubber
FPP	Farnesyl pyrophosphate
CPT	cis-prenyltransferase
SC	self-compatibility
CMS	cytoplasmic male sterility
ETA	Ethanolamine
PENRA	program for excellence in natural rubber alternatives