Discovery of nickel hyperaccumulators from Kinabalu Park, Sabah (Malaysia) for potential utilization in phytomining Antony van der Ent*, David Mulligan & Peter Erskine Centre for Mined Land Rehabilitation, Sustainable Minerals Institute, The University of Queensland, Brisbane, PhD Research Scholar, +61 (7) 3346 4055,
[email protected] Centre for Mined Land Rehabilitation, Sustainable Minerals Institute, The University of Queensland, Brisbane, Director, +61 (7) 3346 4005,
[email protected] Centre for Mined Land Rehabilitation, Sustainable Minerals Institute, The University of Queensland, Brisbane, Senior Research Fellow, +61 (7) 3346 4065,
[email protected] ABSTRACT Ultramafic soils are naturally enriched in nickel and form major mining targets for this metal in tropical regions such as Indonesia and New Caledonia. These soils also host unique plants termed nickel hyperaccumulators that are able to sequester and accumulate nickel in excess of 0.1% in their leaves. Some of these remarkable plants can achieve up 3% in leaves and this observation has prompted the development of nickel phytomining technology, which aims at growing such plants at an agricultural scale to harvest a ‘nickel crop’. However, nickel hyperaccumulation is a rare phenomenon, and despite the knowledge of the plant diversity, records for nickel hyperaccumulators in Sabah (Malaysia), a tropical region with over 3500 km2 of ultramafic soils, were scant, with only six species previously known. Identification of nickel hyperaccumulators is necessary to facilitate their potential utilization in nickel phytomining. In this study, we screened for nickel hyperaccumulation in the flora of Kinabalu Park in Sabah, and discovered nine new nickel hyperaccumulator species. Some of these are among the strongest known globally and have high potential for utilization in future phytomining. The results also show that nickel hyperaccumulators occur on soils with circum-‐‑neutral pH and relatively high DTPA extractable nickel concentrations. *Corresponding author: Sustainable Minerals Institute, CMLR, PhD Research Scholar, The University of Queensland, St Lucia QLD 4072. Phone: +61 (7) 3346 4055. Email:
[email protected] Keywords: nickel hyperaccumulators, phytomining, Sabah. INTRODUCTION Ultramafic soils are derived from ultramafic bedrock and occur all around the world. These soils are naturally enriched in certain trace elements, mainly nickel, cobalt and chromium and are hence –1–
primary targets for nickel mining operations, particularly in tropical regions. These soils are considered adverse to many plants species because of the high concentrations of potentially toxic trace elements (mainly nickel), and because of cation imbalances and nutrient deficiencies (Brooks, 1987; Proctor, 2003). However, some plants restricted to ultramafic soils have evolved to accumulate nickel in their leaves (and other tissues), and are called nickel hyperaccumulators when having in excess of 0.1% nickel in their leaves (Reeves, 1992; Van der Ent et al., 2012). This phenomenon is rare, and known from around 450 plant species globally. Nickel hyperaccumulators are represented in many different plant families, although on a global scale the strongest represented are the Brassicaceae, Euphorbiaceae (inclusive of what is now the Phyllanthaceae), Asteraceae, Flacourtiaceae (now mostly Salicaceae), Buxaceae and Rubiaceae (Reeves, 2006). The growth forms of nickel hyperaccumulators in tropical regions are mainly (large) trees, shrubs, and occasionally climbers. Table 1 Nickel hyperaccumulators previously reported from the region. Hyperaccumulator
Ni % (leaves)
Occurrence
Source
Rinorea bengalensis
1.8
Throughout SE Asia
Brooks & Wither, 1977
Rinorea javanica
0.2
Throughout SE Asia
Brooks et al., 1977
Phyllanthus balgooyi
1.6
Dichapetalum gelonioides
2.7
Psychotria cf. gracilis
1.06
Sabah, Malaysia, Philippines Philippines and Sabah, Malaysia Sabah, Malaysia
Shorea tenuiramulosa
0.1
Sabah, Malaysia
Hoffman et al., 2003 Baker et al., 1992 Reeves, 2003 Proctor et al., 1989
The high concentrations of nickel in hyperaccumulators prompted the development of phytomining technology, which aims at growing such plants on an agricultural scale to produce ‘bio-‐‑ore’ from harvested biomass (Chaney, 1983; 1998). As a part of phytomining, selected hyperaccumulators are cultivated on ultramafic soils, followed by harvesting of the biomass and incineration to generate ash (Brooks et al., 1998; Nicks & Chambers, 1998). Successful scientific phytomining trials have taken place in the US, Italy and South Africa (Robinson et al., 1997a and b). However tropical regions have the greatest potential for phytomining because these regions (for example in Indonesia and New Caledonia) have some of the world’s largest exposures of ultramafic soils and also form the habitat of native nickel hyperaccumulator plants (Van der Ent et al., 2013). The ideal ‘phytomining crop’ has high biomass, high growth rate and high levels of nickel hyperaccumulation (Angle et al., 2001; Chaney et al., 2007). As suggested by Reeves (2003), most tropical nickel hyperaccumulators are relatively fast-‐‑growing woody shrubs (1–5 m), and have therefore potential for rehabilitation or phytomining on ultramafic soils. This therefore presents an incentive for greater screening of native plants in their habitat in tropical regions on ultramafic soils aimed at finding potential candidate nickel hyperaccumulators for future phytomining operations. Sabah (Malaysia) on the island of Borneo has very extensive ultramafic soils totaling over 3,500 km2 (Proctor, et al. 1988) and this region is known for high species richness (Beaman & Beaman, 1990; Beaman et al., 2005). Therefore, the region could have a high potential for the discovery of new –2–
nickel hyperaccumulator species. Prior to this project, six nickel hyperaccumulators were known from Sabah (Table 1). The objective of this study was to screen the flora of ultramafic outcrops in Sabah, mainly Kinabalu Park, for the occurrence of nickel hyperaccumulators. METHODOLOGY Study area and field collection Plants were screened for nickel hyperaccumulation in Kinabalu Park in Sabah, Malaysia. In the field, the leaves were pressed against white test paper impregnated with the nickel-‐‑specific colorimetric-‐‑reagent dimethylglyoxime (‘DMG’). Approximately 5,000 plant samples have been tested using this method. All samples that tested visually positive were re-‐‑collected (full-‐‑grown sun leaves, at least 2 m above the soil) by hand. Fresh plant leaves were put in paper bags to prevent decomposition before transport to the field station. Leaves were thoroughly washed with demineralised water to remove soil contamination and then dried at 70°C for 5 days in a drying oven, packed for transport to Australia and gamma irradiated at Steritech Pty. Ltd. in Brisbane following Australian Quarantine Regulations. Soil samples (near the base of the plant in mineral soil) were also collected for analysis. Chemical analyses of plant tissue samples Foliar samples were crushed and ground, and a subsample digested using 4 mL concentrated nitric acid (70%) and 1 mL hydrogen peroxide (30%) in a microwave oven, and diluted to 45 mL with TDI water before analysis with ICP-‐‑AES (Varian Vista Pro II). Quality controls included NIST and internal standards. The analytical package consisted of Ni, Co, Cr, Cu, Zn, Mn, Fe, Mg, Ca, Na, K, S and P. Chemical analyses of soil samples The soil samples (300 mg) were digested using freshly prepared Aqua Regia (4 mL 70% nitric acid and 3 mL 37% hydrochloric acid per sample) in a digestion block for 2 hours and diluted to 45 mL before analysis to give ‘pseudo-‐‑total’ concentrations. The method followed Rayment & Higginson (1992) method 17B1. Soil pH and electrical conductivity (EC) was obtained in a 1:2.5 soil: water mixture. Phytoavailable nickel (and other elements) was extracted with Diethylene triamine pentaacetic acid (DTPA) according to Lindsay & Norvell (1969), but with modifications from Bequer et al. (1995) (excluding TEA, buffered at pH 5.3). Exchangeable cations were extracted with silver-‐‑thiorea (Dohrmann, 2006) over 16 hours. All soil extractions were undertaken in 50 mL polypropylene (PP) centrifuge tubes. Soil samples were weighted using a four-‐‑decimal balance and weights recorded for correction of precise weights in the mass balance calculations. Samples were agitated for method-‐‑specific times using an end-‐‑over-‐‑end shaker at 400 rpm and subsequently centrifuged (10 minutes at 4000 rpm) and the supernatant was collected in 10 mL PP tubes. All soil samples were analysed with ICP-‐‑AES (Varian Vista Pro II) for Ni, Co, Cu, Zn, Mn, Fe, Mg, Ca, Na, K, S and P. RESULTS AND DISCUSSION Discovery and confirmation of nickel hyperaccumulators –3–
Laboratory analysis with ICP-‐‑AES confirmed the tentative testing in the field with dimethylglyoxime (DMG). Field-‐‑testing with DMG-‐‑paper therefore remains a reliable quick method for nickel hyperaccumulator reconnaissance. The levels of nickel hyperaccumulation (up to 2.3 % in Cleistanthus sp. nov.) are amongst the highest globally (Table 2). The majority of these species are relatively fast-‐‑growing shrubs with a high potential for phytomining. Nickel hyperaccumulators in Sabah appear to prefer open, bare ultramafic soils that are difficult for most plants to colonize. In such habitats, colonizing plants are especially susceptible to herbivores. Therefore, as a result of extremely high foliar nickel concentrations (able to be isolated from critical plant metabolic functions but still presented as toxic at whole leaf level), the capacity to hyperaccumulate (in this case nickel) may have evolved as an adaptation to reduce insect herbivory (‘elemental herbivory defense’) (Martens & Boyd, 1994; Martens & Boyd, 2002). However, this hypothesis needs to be further tested experimentally. Table 2 Newly discovered and confirmed nickel hyperaccumulators in Sabah. Nickel Ni Accumulation Nickel % pH (pseudo-‐‑ (DTPA factor (foliar SPECIES (foliar) (soil) total soil soil nickel/soil mg/kg) mg/kg) nickel) Cleistanthus sp. nov. 1.1 7.0 2025 226 5.4 Flacourtia kinabaluensis
0.3
7.3
1216
157
2.4
Glochidion mindorense
0.1
7.4
322
146
3.1
Kibara coriacea
0.4
5.8
1510
196
2.6
Mischocarpus sundaicus
0.3
6.9
2135
69
1.4
Phyllanthus balgooyi
0.8
6.2
1455
29
5.5
Phyllanthus cf. securinegoides
2.3
5.6
1520
110
15.1
Psychotria sarmentosa
0.7
6.3
2481
90
2.8
Rinorea bengalensis
0.5
6.8
3401
442
1.4
Rinorea javanica
0.6
6.8
2004
122
2.9
Walsura pinnata
0.4
6.9
1015
166
3.9
Xylosma luzoniensis
0.1
6.7
2863
169
0.3
Nickel hyperaccumulator soils chemistry Foliar nickel hyperaccumulation is generally in excess of (pseudo)-‐‑total soil nickel concentrations (Table 2). Compared with nickel concentrations in ultramafic soils elsewhere in the region (Proctor, 2003; Van der Ent et al., 2013), pseudo-‐‑total nickel concentrations in Sabah are relatively low. However, potential plant-‐‑available nickel (as DTPA-‐‑extractable) concentrations are high. The soil pH is circum-‐‑neutral (5.6 -‐‑ 7.4 with mean of 6.6). CONCLUSIONS AND RECOMMENDATIONS Given that only approximately 10% of the total flora has been screened, it is expected that more species with such characteristics will be recorded in the near future. However, the fact that nickel –4–
hyperaccumulators are confined to ultramafic soils, which are principal mining targets, indicates that these species are potentially under threat (Baker et al., 2010). The nickel mining industry could capitalize on hyperaccumulator species that might occur on mine leases by developing phytomining trials. As such, this could offer opportunities to reduce the legacy of strip-‐‑mining and gain income from progressive rehabilitation. Unfortunately, as of yet, phytomining has not been developed or widely trialed in tropical regions. Indonesia, in particular, has a high potential for nickel phytomining because of the concomitant situation of large swaths of ultramafic soils, rich biodiversity and large-‐‑scale strip-‐‑mining creating land in need of rehabilitation (Van der Ent et al., 2013). ACKNOWLEDGEMENTS We wish to thank Rimi Repin, Rositti Karim, Sukaibin Sumail (Sabah Parks) and John Sugau and Postar Miun (Sabah Forestry Department) for their support. We would like to express our gratitude to Sabah Parks for their support and thank the SaBC for granting permission for conducting research in Sabah. The University of Queensland is gratefully acknowledged for financial support that made this project possible. Antony van der Ent is the recipient of IPRS and UQRS scholarships in Australia. REFERENCES Angle, J., Chaney, R., Baker, A., Li, Y., Reeves, R., Volk, V., Roseberg, R., Brewer, E., Burke, S., Nelkin, J. (2001) Developing commercial phytoextraction technologies: practical considerations. South African Journal Of Science, 97(11-‐‑12), 619–623. Baker A.J.M., Proctor J., Van Balgooy M.M.J., Reeves R.D. (1992) Hyperaccumulation of nickel by the flora of the ultramafics of Palawan, Republic of the Philippines. In: ‘The vegetation of ultramafic (serpentine) soils’. (Eds AJM Baker, J Proctor, RD Reeves), (Intercept: Andover, UK), 291–304. Baker, A.J.M., Ernst, W.H.O., Van der Ent, A., Malaisse, F., Ginocchio, R. (2010) Metallophytes: the unique biological resource, its ecology and conservational status in Europe, central Africa and Latin America. In: ‘Ecology of industrial pollution’. Cambridge University Press, Cambridge, 7–40. Becquer, T., Bourdon, E., Pétard, J. (1995) Disponibilité du nickel le long d'ʹune toposéquence de sols développés sur roches ultramafiques de Nouvelle-‐‑Calédonie. Comptes rendus de l'ʹAcadémie des sciences. Série 2. Sciences de la terre et des planètes, 321(7), 585–592. Beaman J.H. (2005) Mount Kinabalu: Hotspot of plant diversity in Borneo. Biologiske Skrifter 55, 103-‐‑127. Beaman J.H., Beaman R.S. (1990) Diversity and distribution patterns in the flora of Mount Kinabalu. In: Baas, P, Kalkman, K, Geesink, R. (eds.) ‘The plant diversity of Malesia’. Kluwer Academic Publishers, 147-‐‑160. Brooks, R.R. 1987. Serpentine and its vegetation: A multidisciplinary approach. Dioscorides Press, Portland, Oregon.
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