Acta Agronómica

Introduction

Phytoextraction is a technology under development consisting of the utilization of metal-accumulating plants for metal extraction from polluted or metal-rich soils (Chaney et al., 2007). This technique can be an alternative for metal extraction in zones where soil metal concentration is not enough for them to be extracted throughout conventional methods. Phytoextraction is suggested as a possible alternative not only to recover gold, but also nickel (Ni) and thallium (Ti) (Robinson, Bañuelos, Conesa, Evangelou & Schulin, 2009; Wilson-Corral, Anderson, Rodríguez-López, Arenas-Vargas & López-Pérez, 2011).

A hyperaccumulator plant is defined as a plant that accumulates heavy metals in concentrations one hundred times higher than any other plant could accumulate in the same substrate (Anderson, Brooks, Stewart & Simcock, 1998). In order to obtain a considered plant as a gold hyperaccumulator, it must present a concentration of 1mg.kg^-1 (Brooks, Chambers, Nicks & Robinson, 1998; Harris, Naidoo, Nokes, Walker, & Orton, 2009).

The major interest in the usage of hyperaccumulator plants is the phytoremediation of contaminated areas by heavy metals. In addition to soil restoration, hyperaccumulation may represent an economic advantage in mining for some highly valuable metals, such as gold (Lamb, Anderson & Haverkamp, 2001). After accumulation, the plants are harvested and ashed to recover target metals. (Kulkarni et al., 2013; Anderson, Moreno & Meech, 2005).

In 1998, Brooks, Chambers, Nicks & Robinson, called phytomining, the technique of using plants with phytoextraction abilities for mining purposes. This technique, consisting in the extraction of precious metals throughout plants, which has been recently studied especially for gold extraction (Anderson, Moreno & Meech, 2005; Girling & Peterson, 1980).

Despite having several reports on hyperaccumulator species, reports on capable plants of accumulating naturally gold do not exist (Harris et al., 2009). According to Lamb et al., 2001, a possible explanation is due to gold is present in the soil in forms in which is impossible to absorb throughout plants. However, Girling & Peterson (1980), reported that plants could actually absorb naturally gold. For instance, they found concentrations of 21.1 ppb of gold in stem samples of Phacelia sericea (Graham) A. Gray, which naturally developed in a substrate and presented a concentration of 1.6 ppb in a mining region in southern British Columbia, Canada.

Gold hyperaccumulation can be promoted by using chemical amendments, which have allowed to be in a form in which the plant can absorb the metal (Wilson-Corral et al., 2011). Among the most used chemical compounds are cyanide (CN^-), ammonium thiocyanate (NH₄SCN), and ammonium thiosulfate (NH₄)₂S2O_3. These compounds can be metabolized by soil microorganisms and later used as source of carbon, nitrogen and sulfur, according to Ebbs et al., 2010.

Chaney et al. (2007), mention two significant approaches for the development of phytoextraction technology as follows: the domestication of plant hyperaccumulators materials, the cloning of all genes needed for hyperaccumulation and tolerance to insert them and express them in a transgenic hyperaccumulator crop with high-biomass production. Therefore, is necessary an identification of high-metal-accumulator genotypes with fast growth, large potential of biomass production and a great response to nutrients (Bhargava, Carmona, Bhargava & Srivastava, 2012).

In this sense, Harris et al. (2009), mention the current interest in phytomining, which is mainly due to three aspects: a social pressure correlated to the environmental impact of conventional mining, the inability of current mining techniques to recover metals from substrates with low metal concentration in an economically acceptable way and the high cost of these metals in the present tense.

It is known that there are essentially two techniques to recover accumulated metal in soil biomass. The first technique is pyrolysis or combustion, which is followed by ash smelting. This technique is very attractive since the energy released can be used to produce electrical energy. The second technique is the acid digestion of the metal contained in plant biomass throughout electrowinning or solvent extraction to recover the metal (Harris et al., 2009). Other authors as Piccinin et al. (2007), show that phytomining is an economically viable technology. Some aspects to be considered for a successful phytoextraction are mentioned by Wilson-Corral et al. (2011).

Soils affected by mining present high levels of pollution due to a structure loss caused by soil aggregate destruction or the use of pollutants, which additionally affects the soil biota. These soils can be biologically stabilized and recovered throughout plant establishment with dense and deep root systems (Tordoff, Baker & Willis, 2000).

The plant establishment have allowed a soil protection against erosion while retains the soil particles due to an intervention roots to form a "network" that benefits the soil aggregation. This process restores the soil functional and structural attributes to the initial conditions previously to mining activity (Bronick & Lal, 2005). Moreover, a root secretion of exudates, facilitates the presence of fungi and bacteria from the rhizosphere, all of which, produce linking material which benefits flocculation process and soil particles aggregation. In fact, these processes enhance soil physical, chemical and biological properties (Angers & Caron, 1998; Bronick & Lal, 2005).

Due to loss of structure, mining-degraded soils present problems for the establishment of plant populations. This affects the soil porosity and infiltration, which limits the plant growth and root system development (Bengough et al., 2006). When occurs a loss in soil structure, one of the first effects that appears is soil compaction, which inhibits root growth and present a decreasing in water and nutrient uptake ability, which can lead to a poor plant development (Bronick & Lal, 2005).

Conversely, induced changes in soil structure are important due to an improvement in the water storage and its availability to retain soil water (Angers & Caron, 1998). Soil aggregation, promoted by plant establishment, is greater when a high root density and a long root length is present. Therefore, the use of fibrous and extensive roots had achieved a major impact on soil aggregation (Bronick & Lal, 2005).

An inappropriate mining extraction causes a negative impact in the environment and human health due to the use of pollutants in the beneficiation process. Some of those pollutants are mercury (Hg) and cyanide (CN^-). In the case of Hg, it accumulates in animals, and provokes air and soil pollution. In fact, mercury is harmful to humans in concentrations in the air as low as 1µg.m^-3 (Veiga, 2006).

In Colombia, the usage of mercury reaches amounts up to 150 tons.year^-1 (Veiga, 2008), which makes necessary the quest for other alternatives for gold extraction as phytoextraction, since is less risky, and also contributes to soil reclamation.

The aim of this research was to determine if Brachiaria decumbens Stapf and Helianthus annuus L. have the potential to improve soil physical properties while had achieved a gold extraction.

Materials and methods

The research was carried out at Universidad Nacional de Colombia, Medellin campus (UNALMED), in Antioquia, Colombia (6°15'47.07''N-75°34'40.02''O), with an altitude of approximately 1524 m.a.s.l., with an average temperature of 23ºC, an average annual rainfall of 1397 mm and a relative humidity between 66% and 80%.

The substrate used corresponds to a soil sample from two mining zones in the department of Antioquia, Colombia as follows: The municipalities of Gómez Plata and Santa Fe de Antioquia, Colombia. These two soil samples were homogenized and chemically characterized in the Soil Analysis Laboratory of UNALMED, Medellin-Antioquia, Colombia.

The species used were sunflower (H. annuus) and Brachiaria (B. ducumbens), which were sown in containers with 2 kg of substrate, and they were fertilized with 30 g of vermicomposting.

Gold content determination in plants and substrate

Substrate

The soil sample was analyzed in the Laboratory of Mineralogy Institute CIMEX of UNALMED, Medellin-Antioquia, Colombia. Gold concentration was determined throughout atomic absorption spectrophotometry (device: ICE 3000 SERIES AA, Espectromole Thermoscientific). Gold concentration in soil sample was 1.2 ppm (Table 1).

Table 1: Soil physicochemical characterization used for phytoextraction evaluation

Plants

Ten days after the chemical amendment (NH₄SCN) was applied, the twenty-four experimental units were harvested, washed and finally dried in an oven at 105°C until constant weight was reached. The resulting dried material was grinded for subsequent analysis by atomic absorption spectrophotometry, which have allowed the determination of gold concentration in each experimental unity. A general diagram of the procedure carried out for phytoextraction is shown in Figure 1.

Figure 1: Procedure carried out for gold phytoextraction. Substrate homogenization (A). Substrate disposition (B). Application of vermicomposting (C). Phytoextractor plants (D). Grinder (E). Atomic absorption spectrophotometer (F).

Soil physical properties

A changes evaluation of soil physical properties in the substrate due to the presence of B. ducumbens and H. annus, was performed 45 days after sunflower and brachiaria were sown, respectively. The soil indicators used were as follows: bulk density, measured with a bulk density ring (IGAC, 2004), true density throughout an automatic density analyzer (PENTAPYC 5200e, Quantachrome Instruments, Florida USA), and soil aggregates, which were weighted and the average diameter were measured after wet screening (WADAWS) according to De Lehner & De Boot method, modified by IGAC (2006).

Results

Figure 2: Evidence of toxicity in leaf blade caused by ammonium thiocyanate (NH4SCN). With toxicity (A). Without toxicity (B).

B. decumbens and H. annuus showed the effect of plants in soil structure. In both species, bulk density decreased in 1 g.cm^-3, and weighted average diameter after wet screening (WADAWS) increased in an average of 250 µm (Figure 3).

Figure 3: Change in physical variables (Bd = bulk density, WADAWS = weighted average diameter after wet screening) evaluated in the substrate with sunflower (Helianthus annuus L.) and the substrate with Brachiaria grass (Brachiaria decumbens). DAS: days after sowing.

It should be taken into account that this indicates an improvement in soil physical properties due to the action of system root in the soil. In fact, an improvement in the substrate was also evident since the initial substrate had not structure (fine grain) (Figure 1A), compared to the final substrate which presented united particles, which formed a structure (Figure 4).

Figure 4: Evidence of phytostructute in soil generated by the action of sunflower (Helianthus annuus) (A) and (B) Brachiaria grass (Brachiaria decumbens)

Given these concerns, the treatment which presented greatest gold accumulation in dry material was Brachiaria 2.48, which had achieved a concentration of 0.05 mg.kg^-1. In the other hand, the treatment with lowest gold accumulation was Sunflower 0, with 0 mg.kg^-1. The maximum absorption rate was of 0.09 mg.kg^-1 in B2.48, followed by B1.24 with 0.05 mg.kg^-1 (Table 2).

Table 2: Gold concentration in B. decumbens and sunflower H. annus, under different concentrations of NH4SCN.

Treatments with different concentrations of NH₄SCN: Brachiaria 0; 0 g.kg^-1; Brachiaria 1.24: 1.24 g.kg^-1; Brachiaria 2.48; 2.48 g.kg^-1; Sunflower 0;0 g.kg^-1; Sunflower 1.24, 1.24 g.kg^-1; Sunflower 2.48, 2.48 g.kg^-1 of soil. Where dm stands for dry material accordingly to Kruskal-Wallis test (α = 0.05).

Table 3, shows significant differences among treatments. In fact, throughout Wilcoxon test was found that B1.24, was significantly different from treatments with sunflower and B0 g.kg^-1, but was not significantly different from B2.48. Despite having a higher mean B2.48 than B1.24, B2.48 was not significantly different from all treatments. This could be due to high dispersions on B2.48 data. A possible explanation for this is based on a plant toxicity, which translates into a quickly death plant (Table 2, Figure 5).

Table 3: Kruskal-Wallis test (α = 0.05).

Same letters: indicates there are not significant differences. Therefore, different letters indicates significant differences. 0, 1.24, 2.48 grams of ammonium thiocyanate per kg of soil.

Discussion

Compared to gold concentration values of 1.64-55.6 mg.kg^-1 in sunflower Helianthus annuus L., and 5-76 mg.kg^-1 in mustard, Brassica juncea (L.) Czern., reported by Wilson-Corral et al. (2011), and Lamb et al. (2001), gold concentration values obtained in this research were very low, since the highest concentration in this experiment was of 0.09 mg.kg^-1 in B. decumbens. This could be due to a strong toxicity in plants treated with NH₄SCN, which generated necrosis in the leaf blade (Figure 2).

Nonetheless, it is worth noting that the substrates used in this research, presented a higher gold concentration (2.35 mg.kg^-1 and 5 mg.kg^-1, respectively), which is according to previously reported by Wilson-Corral et al. (2011), where is considered as one of the factors to be taken into account for a successful gold extraction.

The application form of NH₄SCN could have disfavored the physiological mechanisms which have allowed the plant to absorb gold. Conversely, dose applications at different timing or in lower doses, which confirmed the reported data and obtained by Anderson et al. (1998), could increase the plant ability to absorb gold from soil in a more effective way and during a longer period of time due to a lower toxicity, which could lead to a greater metal absorption.

This drawback can be overcome by potential of B. decumbes acting as gold extractor, perhaps related to a genetic condition and due this occurs in high density planting. This type of grass is constituted with a large transpiratory surface compared to sunflower, which have allowed a great movement of gold released on the soil solution throughout xylematic continuum.

Conclusion

Brachiaria decumbens has potential for gold phytoextraction, presenting a maximum extraction value of 0.09 mg.kg^-1 of dm, which is higher than 0.008 mg.kg^-1 found on sunflower Helianthus annuus L., a species with reported potential for gold phytoextraction. The application of ammonium thiocyanate are thought to contribute and modules the gold absorption process since produces a plant toxicity, which is more evident in the leaf blade death in both species. Alternatively, is believed to be an outcome of hampering transpiration and gold uptake from the substrate. Both species, Brachiaria decumbens Stapf. and Helianthus annuus L., had achieved a soil physical properties improvement with an increasing soil aggregation and soil weighted average diameter after wet screening (WADAWS) with an average of 250 µm. Therefore, both species also contributes to a decreasing soil bulk density (Bd) with an average of 1 g.cm^-3.