Micro-organisms in latex and natural rubber coagula of Hevea brasiliensis and their impact on rubber composition, structure and properties

Authors


Summary

Natural rubber, produced by coagulation of the latex from the tree Hevea brasiliensis, is an important biopolymer used in many applications for its outstanding properties. Besides polyisoprene, latex is rich in many nonisoprene components such as carbohydrates, proteins and lipids and thereby constitutes a favourable medium for the development of micro-organisms. The fresh rubber coagula obtained by latex coagulation are not immediately processed, allowing the development of various microbial communities. The time period between tree tapping and coagula processing is called maturation, during which an evolution of the properties of the corresponding dry natural rubber occurs. This evolution is partly related to the activity of micro-organisms and to the modification of the biochemical composition. This review synthesizes the current knowledge on microbial populations in latex and natural rubber coagula of H. brasiliensis and the changes they induce on the biochemistry and technical properties of natural rubber during maturation.

Introduction

Natural rubber (NR) produced from the latex of the tree Hevea brasiliensis is a natural material of first importance for the tire industry and for many other applications such as the general rubber good industries (latex gloves, condoms, etc.) and vibration isolation. In 2012, 11 million tons of NR have been consumed (International Rubber Study Group statistics), which represents 42·2% of the world consumption of natural and synthetic rubbers. In 2010, 75% of NR consumption was dedicated to the tire industry (Vaysse et al. 2012). Indeed, NR is an indispensable material for applications like airplane or big truck tires. NR mainly differs from synthetic rubbers through, among other things, its ability to crystallize at high strain rates, which helps to strengthen mechanical properties with regard to rigidity, fatigue, tensile strength and limitation of crack propagation (Vaysse et al. 2012).

About 70% of the raw NR produced in the world is marketed as technically specified rubber grades TSR 10 and TSR 20 (ISO 2000:2003 guidelines). For these two grades, latex is collected from tapped trees into open cups, where it coagulates naturally after a few hours in the field. Rubber coagula are then subjected to a maturation step that corresponds to the phenomena occurring before their processing in factory (Intapun et al. 2010).

Maturation is of first importance as dry rubber properties are known to evolve during this step (Na-Ranong et al. 1995; Ehabe et al. 2002; Varghese et al. 2005; Cocard et al. 2006; Fri et al. 2007; Zhong et al. 2009; Intapun et al. 2010). Wallace initial plasticity (P0) and Plasticity Retention Index (PRI) have been especially studied in the maturation context. P0 provides information on the rheological flow behaviour of NR. P30 is the plasticity after ageing at 140°C during 30 min. PRI reflects the susceptibility of NR to thermo-oxidation, given by the ratio of P30 to P0 (in%). Indeed, the main evolution of P0 occurs during the first days of maturation and this parameter can increase or decrease. Maturation is generally also associated with a decrease of the PRI value.

NR can also be characterized by the molar mass distribution and the weight-averaged molar mass (Mw) of its polyisoprene chains and its gel content. Mw is a parameter allowing us to evaluate the average length of the polyisoprene chains (Ehabe et al. 2007). P0 partly depends on Mw (Bonfils et al. 1999; Ehabe et al. 2002, 2007; Vaysse et al. 2003). The gel content corresponds to the part of NR nonsoluble in an organic solvent (Tanaka and Tarachiwin 2009). Studies have shown a decrease of Mw during maturation by an average of 50–300 kg mol−1, whereas the quantity of gel increased all along maturation. This increase of the gel content is quite variable, from a negligible value in some cases (about 1%) to about 25% for others studies (Ehabe et al. 2002; Cocard et al. 2006; Intapun et al. 2010). The decrease of Mw has been proposed to be linked to a reduction of the length of the polyisoprene chains by scission mechanisms.

The specific properties of NR are linked not only to its polyisoprene chains but also to its nonisoprene components, presented in Table 1 (Boucher and Carlier 1964; Gregg and Macey 1973; Hasma 1990; Tuampoemsab and Sakdapipanich 2007). Indeed, lipids and proteins have been proposed to be involved in the structuration of the gel phase (Tanaka and Tarachiwin 2009). Some compounds among amino acids, phospholipids and some neutral lipids such as free tocotrienols and some phytosterols have been shown to have a strong antioxidant activity, thus increasing PRI (Boucher and Carlier 1964; Nadarajah et al. 1971; IRCA 1974; Hasma 1990; Na-Ranong et al. 1995; Liengprayoon 2008). To the contrary, some other compounds are strong pro-oxidants and have a negative effect on PRI, like free fatty acids (especially linoleic acid) and free metal ions such as copper and iron (Hasma 1990; Arnold and Evans 1991).

Table 1. Typical composition of latex and natural rubber
ComponentLatex (% w/v of latex)Latex (% w/w dry rubber)Natural rubber (% w/w dry wt)Reference
  1. nd, no data; A, (Vaysse et al. 2012); B, (D'Auzac et al. 1989); C, (Subramaniam 1995); D, (Liengprayoon et al. 2013); E, (Liengprayoon et al. 2011).

  2. a

    Calculated for a dry rubber content of 40%. Lipid composition is given for clone RRIM600.

Water60A
Polyisoprene358794A
Proteins1·53·72·2A
Carbohydrates (total)1·53·70·4A
Quebrachitol12·5ndB
Sucrose0·41ndA
Glucose0·0040·01ndC
Lipids (total)1·36a3·42·3D
Neutral lipids (total)0·69a1·721·88D
TriacylglycerolsndndndB
Sterols0·49a1·230·75D
Tocotrienols0·07a0·180·18D
Free fatty acids0·02a0·060·55D
Glycolipids (total)0·35a0·880·28D
Digalactosyl diacylglycerols0·160·41ndE
Steryl glycosides0·120·30ndE
Esterified steryl glycosides0·040·11ndE
Monogalactosyl diacylglycerols0·030·07ndE
Phospholipids (total)0·36a0·900·09D
Phosphatidyl choline0·220·56ndD
Lysophosphatidyl choline0·070·17ndD
Phosphatidic acid0·040·10ndD
Phosphatidyl ethanolamine0·010·04ndD
Phosphatidyl inositol0·010·03ndD
Lysophosphatidyl inositol0·010·02ndD
Minerals0·51·25ndA
Organic acids0·41B
Amino acids0·20·5ndB

Latex and NR coagula have been early suspected to host a strong microbial activity during maturation, especially because of the richness of latex and fresh coagula in organic compounds (Table 1) but also by the presence of bubbles in maturated coagula as well as the strong odours present on the fields and around the rubber coagula piles. As not only polyisoprene but also nonisoprene components are involved in NR structure and properties, it can be expected that the modification of the composition of rubber coagula by microbial activities during maturation has an effect on NR characteristics. Micro-organisms in fresh latex of H. brasiliensis have been mainly studied from 1930 to 1975, and the activities of these micro-organisms during maturation were indeed rapidly linked with evolutions of the physical properties of NR (Spence and John 1939). However, very few studies have been published after this period of time until the recent works of Varghese et al. (2005) and Intapun et al. (2010). After a first part dedicated to microbial communities identified in latex and rubber coagula, their known impact on natural rubber composition, structure and properties will be presented.

Microbial communities in latex and natural rubber coagula

Microbial population in latex

To our knowledge, the oldest publication referring to the study of micro-organisms in the latex from H. brasiliensis is from Corbet (1930a), referring to an organism named Bacillus pandora. The latex from an untapped Hevea tree is sterile (Satchuthananthavale 1971a), but after the first tapping, the latex vessels in the vicinity of the cut are contaminated by micro-organisms (Taysum 1958, 1961b; John and Taysum 1963). For example, the level of contamination of a latex harvested in aseptic conditions from trees already in production, determined by plate counting of colony-forming units (CFU) on PCA medium by Intapun et al. (2010), was 105 CFU mL−1. The latex flowing into the cup is then further contaminated by the environment, and values up to 107 CFU mL−1 have been found in latex harvested without specific precautions in earlier studies (Taysum 1961b; Shum and Wren 1977). In the study of John and Taysum (1963), a hundred times more bacteria than yeasts were found in latex. The biochemical composition of latex also influences the level of its contamination. The richer the latex in carbohydrates and phosphorus, the more important its microbial population (Chat et al. 1961).

Few studies are available on the identification of micro-organisms in latex from H. brasiliensis. Cultivable bacteria in latex have been studied in early works by Taysum (1957) and the Institut de Recherches sur le Caoutchouc (IRCA 1973a). Taysum has isolated 1000 strains of eubacteria from freshly collected latex and its derivatives (ammoniated latex and ammoniated latex concentrate). A total of 12 families and 100 species were identified. Many of the isolated micro-organisms were from soil and water and were often described as acid producers and carbohydrate fermenters. Table 2 presents the bacteria observed in H. brasiliensis fresh latex in several studies. The main genera found in latex were Bacillus, Lactococcus, Enterobacter, Serratia, Streptomyces and Micrococcus. To our knowledge, no study is available on the identification of yeasts in latex.

Table 2. List of bacteria described in H. brasiliensis latex according to different studies (Ref) with their respective abundance (Ab). A, (Taysum 1957); B, (IRCA 1973a); C, (John and O'Connell 1967); D, (Taysum 1956); E, (Corbet 1930b). Horizontal lines delimit the taxonomic orders. 1 (bold): common to very common; 2: fairly common; 3: rare, fairly rare to moderately rare; 4: outbreak according to (Taysum 1957)
Taxonomy with bacteria species updated Ab Ref
Firmicutes; Bacilli; Bacillales; Bacillaceae; Bacillus; Bacillus sp.3A
Firmicutes; Bacilli; Bacillales; Bacillaceae; Bacillus; Bacillus subtilis 1 A
Firmicutes; Bacilli; Bacillales; Bacillaceae; Bacillus; Bacillus megaterium2A
Firmicutes; Bacilli; Bacillales; Bacillaceae; Bacillus; Bacillus cereus var. mycoides 1 A
Firmicutes; Bacilli; Bacillales; Bacillaceae; Bacillus; Bacillus indicus (syn. Serratia indica)3A
Firmicutes; Bacilli; Bacillales; Bacillaceae; Bacillus; Bacillus circulans3A
Firmicutes; Bacilli; Bacillales; Paenibacillaceae; Paenibacillus; Paenibacillus polymyxa (syn. Bacillus pandora)3D; E
Firmicutes; Bacilli; Bacillales; Planococcaceae; Lysinibacillus; Lysinibacillus sphaericus (syn. bacillus sphaericus)3A
Firmicutes; Bacilli; Lactobacillales; Streptococcaceae; Lactococcus; Lactococcus lactis subsp lactis (syn. Streptococcus lactis) 1 A
Firmicutes; Bacilli; Lactobacillales; Streptococcaceae; Lactococcus; Lactococcus lactis subsp cremoris (syn. Streptococcus cremoris) 1 A
Firmicutes; Bacilli; Lactobacillales; Streptococcaceae; Streptococcus; Streptococcus pyogenes3A
Firmicutes; Bacilli; Lactobacillales; Streptococcaceae; Streptococcus; Streptococcus agalactiae3A
Firmicutes; Bacilli; Lactobacillales; Enterococcaceae; Enterococcus; Enterococcus faecalis (syn streptococcus faecalis)3A
Firmicutes; Bacilli; Lactobacillales; Lactobacillaceae; Lactobacillus; Lactobacillus delbrueckii subsp. Lactis (syn. Lactobacillus lactis)3A
Firmicutes; Bacilli; Lactobacillales; Lactobacillaceae; Lactobacillus; Lactobacillus casei3A
Firmicutes; Bacilli; Lactobacillales; Leuconostocaceae; Leuconostoc; Leuconostoc mesenteroides3A
Firmicutes; Clostridia; Clostridiales; Clostridiaceae; Clostridium; Clostridium perfringens (syn. Clostridium welchii)2A
Firmicutes; Clostridia; Clostridiales; Clostridiaceae; Clostridium; Clostridium tetani3A
Firmicutes; Clostridia; Clostridiales; Clostridiaceae; Sarcina; Sarcina flava (syn. ?)2A
Firmicutes; Clostridia; Clostridiales; Eubacteriaceae; Eubacterium; Eubacterium limosum (syn. butyribacterium rettgeri)4A
Proteobacteria; Gammaproteobacteria; Enterobacteriales; Enterobacteriaceae; Escherichia-Shigella; Escherichia coli2A
Proteobacteria; Gammaproteobacteria; Enterobacteriales; Enterobacteriaceae; Enterobacter; Enterobacter aerogenes (syn. Aerobacter aerogenes) 1 A, C
Proteobacteria; Gammaproteobacteria; Enterobacteriales; Enterobacteriaceae; Enterobacter; Enterobacter cloacae (syn. Aerobacter cloacae)3A
Proteobacteria; Gammaproteobacteria; Enterobacteriales; Enterobacteriaceae; Enterobacter; Enterobacter nimipressuralis (syn. Erwinia nimipressuralis)3A
Proteobacteria; Gammaproteobacteria; Enterobacteriales; Enterobacteriaceae; Proteus; Proteus vulgaris2A; B
Proteobacteria; Gammaproteobacteria; Enterobacteriales; Enterobacteriaceae; Proteus; Proteus mirabilis3A
Proteobacteria; Gammaproteobacteria; Enterobacteriales; Enterobacteriaceae; Serratia; Serratia marcescens 1 A
Proteobacteria; Gammaproteobacteria; Enterobacteriales; Enterobacteriaceae; Salmonella; Salmonella enterica (syn. Salmonella arizona)B
Proteobacteria; Gammaproteobacteria; Enterobacteriales; Enterobacteriaceae; Citrobacter; Citrobacter freundii (syn. Escherichia freundii)2A; B
Proteobacteria; Gammaproteobacteria; Enterobacteriales; Enterobacteriaceae; Klebsiella; Klebsiella pneumoniaeB
Proteobacteria; Gammaproteobacteria; Pseudomonadales; Pseudomonadaceae; Azotobacter; Azotobacter chroococcum2A
Proteobacteria; Gammaproteobacteria; Pseudomonadales; Pseudomonadaceae; Pseudomonas; Pseudomonas sp.2A
Proteobacteria; Gammaproteobacteria; Pseudomonadales; Pseudomonadaceae; Pseudomonas; Pseudomonas aeruginosaB
Proteobacteria; Gammaproteobacteria; Pseudomonadales; Pseudomonadaceae; Pseudomonas; Pseudomonas fluorescens2A
Proteobacteria; Gammaproteobacteria; Vibrionales; Vibrionaceae; Vibrio; Vibrio sp.3A
Proteobacteria; Deltaproteobacteria; Desulfovibrionales; Desulfovibrionaceae; Desulfovibrio; Desulfovibrio desulfuricans3A
Proteobacteria; Betaproteobacteria; Burkholderiales; Alcaligenaceae; Achromobacter; Alcaligenes sp.3A
Proteobacteria; Betaproteobacteria; Nitrosomonadales; Nitrosomonadaceae; Nitrosomonas; Nitrosomonas europaea (syn.Nitrosomonas monocella)3A
Proteobacteria; Betaproteobacteria; Neisseriales; Neisseriaceae; Aquaspirillum; Aquaspirillum serpens (syn. Spirillum serpens, Vibrio serpens)3A
Proteobacteria; Alphaproteobacteria; Rhodospirillales; Acetobacteraceae; Acetobacter; Acetobacter aceti3A
Proteobacteria; Alphaproteobacteria; Rhodospirillales; Acetobacteraceae; Gluconobacter; Gluconobacter oxydans (syn. Acetobacter suboxydans)3A
Actinobacteria; Actinobacteria; Streptomycetales; Streptomycetaceae; Streptomyces; Streptomyces albus 1 A
Actinobacteria; Actinobacteria; Streptomycetales; Streptomycetaceae; Streptomyces; Streptomyces coelicolor 1 A
Actinobacteria; Actinobacteria; Micrococcales; Micrococcaceae; Micrococcus; Micrococcus luteus (syn. Sarcina lutea)2A, C
Actinobacteria; Actinobacteria; Micrococcales; Micrococcaceae; Micrococcus; Micrococcus candidus (syn. ?) 1 A
Actinobacteria; Actinobacteria; Micrococcales; Micrococcaceae; Micrococcus; Micrococcus aurantiacus (syn. ?) 1 A
Actinobacteria; Actinobacteria; Micrococcales; Microbacteriaceae; Microbacterium; Microbacterium lacticum3A
Actinobacteria; Actinobacteria; Propionibacteriales; Propionibacteriaceae; Propionibacterium; Propionibacterium jensenii (syn. Propionibacterium peterssonii, Propionibacterium zeae)4A
Actinobacteria; Actinobacteria; Propionibacteriales; Propionibacteriaceae; Propionibacterium; Propionibacterium pentosaceum (syn. ?)4A
Actinobacteria; Actinobacteria; Propionibacteriales; Propionibacteriaceae; Propionibacterium; Propionibacterium freudenreichii3A
Actinobacteria; Actinobacteria; Propionibacteriales; Propionibacteriaceae; Propionibacterium; Propionibacterium acidipropionici (pentosaceum)4A
Actinobacteria; Actinobacteria; Corynebacteriales; Mycobacteriaceae; Mycobacterium; Mycobacterium phlei3A
Bacterium fulvum (syn. ?) 1 A
Bacterium zenkiri (syn. ?) 1 A
Bacterium mycoides (syn.?)3A

Evolution of microbial population during maturation

The initial population of micro-organisms in latex is evolving in terms of quantity and diversity during maturation. Latex, before and just after its coagulation, has been more studied than the later coagulum stage. It has been shown by Taysum (1958) that the time between tapping and latex coagulation varies depending on the initial content of micro-organisms. A latex harvested on field usually coagulates after 8–48 h, with a very fast coagulation called precoagulation (D'Auzac et al. 1960), being observed when the initial content of micro-organisms is high. According to Taysum (1958), microbial growth displays a lag phase with no detectable growth of micro-organisms after tapping, followed by a logarithmic growth phase. The lag phase lasts between 1 and 4 h depending on the initial concentration of micro-organisms. In the study of Taysum (1958), after 8 h, latex contained 109 CFU ml−1 and had begun to coagulate. Comparatively, D'Auzac et al. (1960) observed that coagulation started when the microbial population reached 1011–1012 CFU ml−1. The link between micro-organisms and latex coagulation has also been shown by Satchuthananthavale (1971a), who observed that a sterile latex did not coagulate during 5-day experiments. This was confirmed by Intapun (2009), who showed that a latex stabilized with sodium azide did not coagulate during 2 weeks.

However, the dynamics of the quantity and diversity of micro-organisms during and after latex coagulation is not yet well known. According to Taysum (1961b), the coagulation may be induced by acid-producing bacteria such as Streptococcus and Lactobacillus, and then, the microbial population would diversify and the nonisoprene components be degraded by anaerobic bacteria such as Clostridium. On the contrary, another study proposed that aerobic bacteria are responsible for the degradation of nonisoprene components (Lowe 1959). The only recently published study on the micro-organisms present in the serum of fresh coagula is the one by Intapun et al. (2010). In this study, latex was inoculated with micro-organisms from the serum of field coagula obtained 2 days after tapping. The inoculum contained 10CFU ml−1. Amounts of yeasts, Gram-positive bacteria, Gram-negative bacteria and lactic acid bacteria were 4 × 108, 3 × 108, 4 × 107 and 9 × 107 CFU mL−1, respectively. Twelve different species of bacteria were isolated on plates from this inoculum (Table 3), with mainly enterobacteria. The four families found included Lactobacteriaceae, Enterobacteriaceae, Pseudomonadaceae and Bacillaceae (Intapun 2009).

Table 3. Genera and species of bacteria isolated from inoculum, according to Intapun (2009)
NoGram typeGenus and speciesGeneral characters
1+Bacillus sp.Lactose+, acid+, gas+
2 Klebsiella pneumoniae Lactose+, acid+, gas+
3Acinetobacter sp.Strictly aerobic, nonfermentating
4 Enterobacter cloacae Lactose+, acid+, gas+
5 Klebsiella oxytoca Lactose+, acid+, gas+
6 Citrobacter freundii Lactose+, acid+, H2S+
7+γ-Streptococcus sp.Production of lactic acid
8 Pseudomonas alcaligenes Lactose+, acid+, H2S+
9 Escherichia coli Lactose+, acid+, H2S+
10+ Staphylococcus Glucose+, acid+, gas+
11+EnterococciProduction of lactic acid
12+Sphingobacterium spp. 

Besides, some studies have described micro-organisms that were isolated from the rubber-processing environment. One study described the characterization of bacteria and fungi present in the wastewater of a processing unit (Atagana et al. 1999a,b), which reflects the microbial communities in serum from matured coagula. Arthrobacter, Bacillus, Lactobacillus, Pseudomonas and Streptococcus were the main genera identified in factory serum effluents (Atagana et al. 1999b), with Arthrobacter sp. as the dominant species. The growth of Arthrobacter in both effluent and natural rubber waste serum (NRWS) was related to pH, with the highest growth rates recorded at pH 8·5 and 7·5 for effluent and NRWS, respectively. Besides, the fungi identified were Mucor racemous, Mucor sp. and Aspergillus niger.

Role of micro-organisms in the evolution of rubber properties during maturation

The understanding of the main factors driving the evolution of properties during maturation is of first importance to ensure more consistency in NR technological properties and, therefore, in its processability. Many factors were reported to influence NR properties, such as season, clone, age of the tree, frequency of tapping, maturation time and process. The evolution of NR properties during maturation has also been linked with the activity of micro-organisms (Cocard et al. 2006; Ehabe et al. 2007).

Impact of micro-organisms on rubber properties during maturation

Early studies have reported the improvement of properties, especially PRI, with the destruction or inhibition of micro-organisms during maturation (IRCA 1973b). According to Lévêque (1975), the use of fungicides had no impact on the improvement of PRI comparing with bactericides. The most efficient antibiotics reducing the decrease of PRI during maturation were identified as those acting against Gram-negative bacteria (IRCA 1973b). In contrast to PRI, the impact of antibiotics on P0 was not found to be as straightforward (IRCA 1973b; Intapun et al. 2010). More recently, Varghese et al. (2005) have also reported the improvement of properties (P0 and PRI) by soaking the coagulum in a bactericide (formalin) solution.

The study of Intapun et al. (2010) has clearly shown the link between NR properties and microbial activity by controlling the initial quantity of micro-organisms in latex and by inducing coagulation with acid. The evolution of P0 was clearly dependent on the amount of inoculum, with a constant value in the absence of micro-organisms and a significant variation (up to 38% increase and 20% decrease) during time with various inoculum concentrations. It is interesting to note that micro-organisms have been proposed to promote both cross-linking and scission of polyisoprene macromolecules (Varghese et al. 2005), leading, respectively, to an increase or a decrease of P0. Likewise, the evolution of PRI over maturation time was found to be clearly dependent on the initial quantity of micro-organisms in latex. Indeed, the decreasing rate of the PRI value was proportional to the initial micro-organism concentration. Concerning the mesostructure of NR, only a slight decrease of Mw with maturation time was observed for the samples stabilized with sodium azide, while Mw values dropped from 1500 down to 1150 kg mol−1 after 6 days of maturation in the presence of active micro-organisms (Intapun et al. 2010). The drop rate of Mw increased with the quantity of micro-organisms, and it was shown that micro-organisms led to chain scission. For all treatments, gel quantity increased with maturation time and reached a plateau after 2 days of maturation. Yet, the value associated with this plateau increased with the initial quantity of micro-organisms, from 45% for biocide-treated samples to 55% for the treatment with the highest initial micro-organism content.

Intapun (2009) also performed 45 days of controlled (temperature, humidity, oxygen, inoculation) maturations with different coagulation modes. This study showed that the impact of micro-organisms on NR properties during maturation was lower under anaerobic conditions or when performing acid coagulation. Indeed, P0 and PRI parameters were higher in anaerobic than in aerobic conditions, which suggests that the decrease of NR property values might be due to oxidative microbial mechanisms.

The recent comprehensive review by Yikmis and Steinbüchel (2012) has shown that many studies have been carried out on the degradation by micro-organisms of both pure rubber and vulcanized rubber products. Some mechanisms might be similar during maturation. The oldest study reporting NR polyisoprene degradation is from Spence and Niel (1936). Sterile dialysed latex was inoculated with strains of actinomycetes, and after 4 weeks of incubation at 30°C in the dark, a decrease of 70% of the dry weight of polyisoprene was observed. The same result was observed by inoculating dialysed latex with contaminated particles of soil, with a 20% decrease of the dry weight of rubber after 35 days of incubation. The gel content also decreased with the incubation time, and it was supposed that actinomycetes could degrade rubber hydrocarbons and reduce the gel content of NR (Spence and Niel 1936). According to recent studies, the most potent rubber-degrading bacteria currently identified are actinobacteria (Yikmis and Steinbüchel 2012), where two groups could be distinguished: the CNM (Corynebacterium, Nocardia, Mycobacterium) group and ‘clear zones forming bacteria’. Bacteria of the CNM group required direct contact with the rubber substrate and did not produce translucent halos on NR latex agar plates. The other group of rubber-degrading bacteria formed clear zones and generally belonged to actinomycetes (Actinoplanes, Streptomyces and Micromonospora). The degradation mechanism of the polyisoprene chain has been described as an oxidative cleavage of the double bond of the carbon chain (Yikmis and Steinbüchel 2012), the main functional groups produced being aldehydes and ketones. Two key enzymes have been identified in this mechanism: RoxA (rubber oxygenase A) (65 Kda) isolated from Xanthomonas sp. strain 35Y and LcpK30 isolated from Streptomyces sp. K30 (Yikmis and Steinbüchel 2012). It has been also demonstrated that radical-generating enzymes required a substrate acting as a radical precursor (e.g. lipoxygenase–linoleic acid) to degrade polyisoprene.

Microbial activity and biochemical changes during maturation

Several studies have tried to explain the biochemical mechanisms occurring during maturation and their relation with the degradation or the production of the nonisoprene components partly responsible for NR properties. Initially, fresh latex of H. brasiliensis is composed of poly-(cis-1, 4-isoprene) and also proteins, carbohydrates and cyclitols, lipids, inorganic compounds, organic solutes and water. Quebrachitol that belongs to the cyclitol family is especially abundant in Hevea latex (1).

The pH of latex decreases during the first 24 h after tapping from 6·5 to around 5·2, leading to natural coagulation. Then, pH increases more or less fast up to pH 8 or 9 (Hanower et al. 1980; Intapun et al. 2010). Natural coagulation and pH drop are triggered by the activity of micro-organisms. The evolution of pH may be the result of a balance between an acidification by carbohydrate fermentation (Philpott and Sekar 1953) and an alkalization by organic acid consumption and release of ammonia by protein degradation (Chat et al. 1961; Satchuthananthavale 1971b; Intapun et al. 2010). In the study of Loyen and de Livonnière (1975), a low pH of coagula serum in the first 24 h after tapping seemed to be correlated with a high PRI.

According to Compagnon (1986), the major acids produced during the acidification phase are formic, acetic, lactic, butyric and propionic. Most of these acids are volatile fatty acids (VFA), and their quantity is directly linked to the quantity and activity of micro-organisms, especially lactic acid bacteria (Lactobacillales), except the genus Lactobacillus (Taysum 1957, 1961a; Lowe 1959). It is commonly assumed that VFA originate from sugar degradation, and VFA quantification is indeed one of the most important tests of quality of commercial concentrated natural latex. (Lowe 1960) showed that VFA production is also triggered by the addition of amino acids, suggesting that one of the substrates for VFA formation could be a glucose–amino complex. The contribution of quebrachitol, a major cyclitol in latex, to VFA formation during maturation is controversial. Indeed, during 11 days of storage of ammonia-preserved natural latex concentrate, quebrachitol, myo-inositol and L-inositol were not degraded in the study by Lowe (1959), and it was supposed that quebrachitol was not a substrate for VFA formation (Lowe 1960). On the other hand, John (1966) tested the capacity of 12 strains present in latex to metabolize carbohydrates and quebrachitol. Among them, Enterobacter aerogenes (syn. Aerobacter aerogenes) and Achromobacter delicatulus were able to metabolize quebrachitol via an oxidative metabolic pathway into acids, which implied that it was a possible substrate for acid production.

In several studies, the degradation of NR proteins during maturation was supposed to be the cause of the increase of pH by then release of ammonia. An experiment by Taysum (1961a) evidenced protein degradation during maturation that became significant 4 days after tapping, 8 days being then necessary to obtain 50% of deproteinization. More recently, Zhong et al. (2009) observed a decrease of the nitrogen content in TSR-grade rubber during maturation from 0·5% after natural coagulation to 0·2% after 30 days of maturation. The major reason for low PRI of field coagulum during storage was claimed to be related to the bacterial decomposition of proteins and other nonisoprene constituents, leading to the release of free copper, which is an active pro-oxidant (Varghese et al. 2005). Indeed, it was observed by Watson (1969) that the amount of copper in dry rubber increased during maturation and that soaking rubber coagula into an oxalic or phosphoric acid solution, to chelate copper, increased PRI.

The IRCA institute observed the evolution of the colour of crepes during 5 weeks of maturation of coagula from latex from two different H. brasiliensis clones, with or without micro-organisms (IRCA 1972). The colour of NR crepes did not evolve when micro-organisms were destructed by latex sterilization for 1 h at 100°C in an autoclave, whereas with living micro-organisms, the colour changed and becomes browner. Satchuthananthavale (1971a) also demonstrated that a latex inoculated with different bacterial strains allowed the production of dry rubber with different colours.

Few studies have described the evolution of lipids during maturation. Phakagrong (2010) compared the lipid composition of NR from unsmoked sheet (USS, obtained by diluted latex acid coagulation, lamination and outdoor air-drying; P0 38; PRI 88) and NR from ‘maturated USS’ (obtained by diluted latex natural coagulation, 5–7 days of maturation of coagulum in nondrilled container, lamination and outdoor air-drying; P0 45; PRI 28). No significant difference concerning the total lipid content was observed. However, among neutral lipids, triglycerides were not detected anymore in maturated rubber. The free fatty acid content was lower in maturated USS rubber (9% of total lipids, vs 22% for nonmaturated USS rubber). The proportion of unsaturated fatty acids (linoleic acid, linoleic acid and furanic acid) in total fatty acids was found to decrease with maturation. The amount of α-tocotrienol was also reported to be lower, while stigmasterol content was higher in maturated rubber (Phakagrong 2010). It has also been shown that polar lipids are less present in unsmoked sheet than in latex. Indeed, these lipids could be lost during the washing steps of the dry rubber process or rapidly hydrolysed, thus liberating the significant amount of free fatty acids detected in dry rubber (Liengprayoon et al. 2013).

These biochemical modifications could be linked to microbial activities or to native enzymes present in latex. To our knowledge, no data have been published on the enzymatic activities (either endogenous or microbial) which could be responsible for the observed changes in nonisoprene composition during maturation.

Conclusions

The existence of a strong link between the development of micro-organisms in latex and rubber coagula during maturation and the evolution of rubber structure and properties has been evidenced for a long time. The current knowledge on the impact of micro-organisms on natural rubber properties is summarized in Table 4. Both yeasts and bacteria are naturally present in freshly collected H. brasiliensis latex, with total amounts of 10–107 CFU ml−1 observed in several studies. Mainly Lactobacillales, Enterobacteriales, Actinobacteriales and Bacillales are present in fresh latex. Micro-organism concentration increases during the first hours of maturation up to 109 CFU ml−1, and their activity induces latex coagulation by acidification. Indeed, a pH change occurs during maturation with a first phase of acidification, followed by alkalization. During acidification, many volatile fatty acids and other organic acids are produced by the activity of micro-organisms in part by degradation of sugars. The alkalization phase might be linked to both the consumption of organic acids by the successive microbial communities and by the release of ammonia during protein degradation.

Table 4. Summary of known impacts of micro-organisms on dry rubber properties during maturation of natural rubber coagula
ParametersCorrelation with the quantity of micro-organisms
PRINegative correlation: microbial activity leads to a natural rubber more sensitive to thermo-oxidation
P0Complex: micro-organisms seem to promote both cross-linking (positive correlation) and scissions (negative correlation) of the rubber chain
MwNegative correlation: microbial activity leads to scission of rubber chain directly by their own activity or indirectly during the drying of natural rubber
Gel contentPositive correlation

The degradation of nonisoprene compounds by micro-organisms during maturation leads to modifications of rubber quality indicators. The decrease of PRI during maturation is supposed to be partly linked to the release of metal ions in natural rubber, possibly when metallo-proteins are degraded by micro-organisms, whereas the evolution of P0 might be linked to the length of the rubber chain (MW) and to the gel content. The microbial mechanisms involved in the evolution of P0 are less straightforward. Some micro-organisms belonging to Actinomycetales are also able to degrade pure polyisoprene, and this mechanism, although slow, could be present during maturation of coagula and modify the length of the polyisoprene chain.

Although a number of studies have described the evolution of natural rubber properties during maturation, only a few have dealt with biochemical and microbial population evolutions in parallel, especially after latex coagulation. The correlations between the evolution of rubber properties and the development of micro-organisms are not straightforward and thus need more investigations. The use of modern approaches such as high-throughput DNA sequencing and metagenomics appears as a necessity to further explore the microbial communities in latex and NR coagula and describe their evolution during maturation. It would be very useful to identify the key micro-organisms and enzymes involved in the degradation of nonisoprene components leading to modifications of rubber properties. These investigations could help to improve the handling of field coagula and, through a suitable control of the development of micro-organisms, to favour a higher quality, with better consistency, of technically specified natural rubber.

Conflict of interest

The authors declare no conflict of interest.

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