In: Handbook of Phytoremediation ISBN: 978-1-61728-753-4 Editors: Ivan A. Golubev ©2010 Nova Science Publishers, Inc.

Chapter 31

FIELD-SCALE RHYZOREMEDIATION OF A CONTAMINATED SOIL WITH

HEXACHLOROCYCLOHEXANE (HCH) ISOMERS: THE POTENTIAL OF POPLARS FOR ENVIRONMENTAL

RESTORATION AND ECONOMICAL SUSTAINABILITY

D Bianconia, MR De Paolisa, AC Agnelloa, D Lippia, F Pietrinia, M Zacchinia, C Polcarob, E Donatib,

P Parisa, S Spinac and A Massacci* a

aInstitute of Agro-environment and Forest Biology, Rome Research Area, Monterotondo Scalo (Rome)

bInstitute of Chemical Methodologies , Rome Research Area, Monterotondo Scalo (Rome)

cCommissariato Starordinario per l’emergenza nel bacino del Fiume Sacco, Regione Lazio (Rome)

ABSTRACT

Three pre-selected poplar clones and two soil HCH degrader micro-organisms have been experimentally applied in a contaminated agricultural soil in the basin of Fiume Sacco near to Rome for its reclamation. The aim was to successfully associate soil cleaning by rhizoremediation with an economically sustainable biomass for energy production of large poplar plantations. Plants and micro-organisms were selected for the best association with bacteria to obtain 1) the maximum HCH concentration reduction in soil, 2) the minimum plant contamination with HCH, and 3) the maximum biomass production. Results showed that an association between all these traits is possible in a specific poplar clone inoculated with a selected HCH degrader bacterium. The need for a pre-remediation phase in situ to select best candidate plants and bacteria with lowest

* Corresponding author: angelo.massacci@ibaf.cnr.it ,Institute of Agro-environment and Forest Biology, Rome

Research Area, Via Salaria km 29, 00015 Monterotondo Scalo (Rome)

D Bianconi, MR De Paolis, AC Agnello, et al. 2

HCH accumulation in its organs is emphasized. Rhyzoremediation associated with the safe thermo-convertible biomass production is confirmed as a sustainable recovery of soils interdicted to food-agricultural activities.

INTRODUCTION Rhizoremediation is a type of phytoremediation that takes place in the rhizosphere by a

complex interaction of roots, root exudates and microorganisms with contaminants (Kuiper et al., 2001; Walker et al., 2003). The rhizosphere is by definition the volume of soil influenced by roots that extends approximately 1–3 mm from their surface (Shimp, et al., 1993; Schnook, 1998). Besides being capable to metabolically degrade some organics and extract some heavy metals, roots provide a niche and key nutrients or other substances that stimulate microorganism growth and remediation activities (Cardon and Gage, 2006). These substances include relatively small molecules such as amino acids, organic acids, sugars, phenolics, and large molecules, such as mucilage (high molecular weight polysaccharides) and enzymatic proteins (Cardon and Gage, 2006). Some of these molecules affect the rhizosphere by modifying its pH or its redox status and by significantly contributing to an easy to use and essential carbon pool for microorganism growth (El Shatnawi and Makhadmeh, 2001). Nevertheless, other molecules can specifically induce or enhance the enzymatic breakdown of complex organic contaminants finally providing further components to the carbon pool (Walker et al., 2003). The proliferation of soil organisms in this soil volume can be 3 or 4 orders of magnitude greater than the population of soil microorganisms in non-vegetated soils (ITRC, 2001). The rhizosphere is thus a crowded and active chemical laboratory where breakdown of organic contaminants and the complexation of heavy metals are facilitated.

Besides breakdown activities, the microbiological consortium perform several other important functions in the rhizosphere such as promotion of plant growth, protection against plant pathogens, and the production of chelators for absorbing and translocating essential nutrients to the plant (Walker et al., 2003; El Shatnawi and Makhadmeh, 2001). To this latter process are known to strongly concur mycorrhizal fungi associated with roots of some plants (e.g. poplars). The mycorrhizal infection of roots is usually a mutualistic relationship, with the fungi receiving sugars from the host plant in exchange for tremendously increasing the plant’s absorption surface of essential mineral nutrients and eventually bioavailable contaminants (Quoreshi and Khasa, 2008). All of these rhizosphere activities involving the indigenous microbial population can be indicated as natural attenuation. Recent research has shown that by the use of specific amendments both growth and activities of these microorganisms can be stimulated to accelerate or enhance the natural attenuation. In alternative, microorganisms isolated from the soil and proved to degrade an organic contaminat can be re-injected into the rhizosphere as a concentrated culture (bioaugmentation). The application of either of these processes to cope with environmental contamination is still limited. In the few cases of “in situ” preliminary assessments the remediation results were promising despite some post-remediation problems that have still to be solved. It is evident that rhizoremediation bears most positive aspects out of all phytotechnologies. It is, for example, a relatively low cost, mostly energy sun-driven and eco-sustainable technology. It has no size restrictions for sites, it uses organic biodegradable materials, it has a discrete versatility to treat a diverse range of contaminants. Furthermore, it

Field-Scale Rhyzoremediation of a Contaminated Soil … 3

can potentially be employed in any geographical area that supports plant growth. On the other hand, the so far evidenced drawbacks of rhizoremediation are mainly related to factors expected from plant characteristics. It takes time that tree plants, as being the ones with most useful root apparatus, grow and develop a large rhizosphere to obtain acceptable effects. Many contaminants, especially the organic ones, diffuse little and, if outside of the rhizosphere, are scarcely bioavailable to degrader microorganisms. These microorganisms are, besides, aerobic and their growth is limited by the scarcity of oxygen deep below 0.3-0.4 m in the soil. In addition, stressors affecting rhizoremediation in the field are numerous and unforeseen (Wenzel, 2009). Nevertheless, substantial research efforts have improved and can continue to improve efficiency of the rhizosphere processes by eliminating or at least minimizing these drawbacks. For example, bioavailability of contaminants, one of the factors that most limit their degradation, can be improved by investigating the use of suitable surfactants, amphipathic molecules with both a hydrophobic and a hydrophilic part. These molecules are able to lower the surface tension, and can form micelles where substances that are generally insoluble in water, such as the chlorinated organics, for example, may be accumulated at interfaces and then solubilized. In turn, the solubilization renders these molecules more available for degradation by microorganisms (Lafrance and Lapointe, 2007). However, chemical surfactants are themselves a source of further and persistent pollution. By contrast, many microorganisms (Pseudomonas aeruginosa, Bacillus subtilis, Pichia pastoris, etc.) can produce diversified structurally surface-active agents, referred to as biosurfactants. Biosurfactants are much less harmful for natural ecosystems due to their low toxicity and biodegradable nature. A further help to improve bioavailability of some contaminants is to exploit the anastomosis ability of some plants. Poplars that have been selected for high density plantation (i.e. short rotation coppicing, SRC) with 7-8,000 plants ha-1 can root to a depth between 0.80 and 2.43 m (Jordahl et al., 1997; Trapp and Karlson 2001; Zacchini et al., 2008) and already at the end of the first growth season they form a complex root mat within rows (Coleman et al., 2004). This mat is useful as a diffusion pathway for exudates, microorganisms, nutrients, oxygen and contaminants that become more biovailable to rhizodegradation activities..

RHIZOREMEDIATION WITH POPLARS Poplar trees have specific characteristics that, beside their important rooting ability, make

them suitable for rhizoremediation. They are perennial, easily propagated, fast growing especially in the juvenile phase, large biomass producers and when they are planted at high density for short rotation coppicing (SRC) poplar trees can be used for producing heat and/or electric power. Furthermore, poplars show a high leaking capacity of organic carbon into the rhizosphere. According to Lynch (1987) plants release about 30% of photosynthates to the soil. A similar or even slightly higher percentage (35%) has been estimated for poplars. This amount of released carbon is one of the most likely reasons that make poplars a very good host of microorganisms in the rhizosphere and thus capable of higher rhizoremediation of organic xenobiotics as compared to other trees (Jordahl et al., 1997). Indeed, field and greenhouse studies showed that poplars have already themselves an important role in the uptake and degradation of particular chlorinated organics. They can degrade trichloroethylene

D Bianconi, MR De Paolis, AC Agnello, et al. 4

(TCE) to trichloroethanol, di and trichloroacetic acid, or complete mineralization to CO2 (Gordon et al., 1998). In addition, they can uptake, hydrolyze, and dealkylate atrazine to less toxic metabolites (Burken and Schnoor 1996, 1997). It has also been shown that they can take up and transpire dioxane in both hydroponic and soil experiments (Aitchison et al., 2000). Further, they can take up and translocate from root to shoots lesser-chlorinated polychlorinated biphenyls (PCBs), being the translocation of PCBs to stems inversely related to PCB hydrophobicity (Liu and Schnoor, 2008). Moreover, in vivo metabolism of 3,3′,4,4′-tetrachlorobiphenyl through hidroxilation was observed (Liu et al., 2009). It is noteworthy to report that poplars are among the few tree genera that can associate in a mutualistic symbiosis with both types of mycorrhizal fungi (ectomycorrhizae and vesicular arbuscular mycorrhizae), which have a fundamental role in increasing the root surface up to 800 fold. However, it is not clear if the fungi can directly degrade the contaminant or if they stimulate bacterial degradation by enhancing plant growth (McCutcheon and Schnoor, 2003; Karlinski et al., 2009; Ma et al., 2009). Lastly, poplars have a high intraspecific genetic polymorphism and genetic diversity. As a consequence, a number of different genotypes could be available with a high degree of adaptability to a given climate and able to tolerate and uptake/degrade a given contaminant. Exploring the poplar diversity can provide specific material that can be applied in most environmental conditions and for rhizodegrading a variety of organic contaminants.

THE CASE STUDY OF POPLAR RHIZOREMEDIATION IN THE VALLE DEL SACCO AREA NEAR TO ROME

A land of about 8 Kmq in the Rome and Frosinone provinces of South Lazio Region of

Italy is contaminated by hexachlorocyclohexane (α, β, γ and δ HCH) isomers with concentrations that are spotty above law soil limits fixed for industrial or green areas (figure 1). The area is 100 m wide along the both sides of the Sacco river and was part of agricultural lands used for growing forage. Sediments of the river were polluted by nearby industrial landfill percolations, probably in the eighties, containing co-products of the lindane (the γ-HCH) synthesis. The forage crop irrigation with the river water and river floodings that occurred few times have widespread the contaminated sediments in that stretch of land. In the year 2005 a high concentration of the β-HCH was detected in the milk produced in local farms and further analysis in the surrounding soils confirmed the presence of this and other isomers. These results immediately led to the interdiction of the above-indicated large area. Such a decision aiming to reduce health risk to the population caused an important economic loss to farmers. Health risks and socio-economic problems thereby induced environmental authorities to include the area of Valle del Sacco in the list of national interest contaminated sites. The HCH isomer contamination has become the target of many activities, including the removal of active sources and the cleaning of agricultural soils.

It is important to consider that HCH isomers show marked differences in some physicochemical properties and their relative diffusion pathway in the environment, their relative persistence, and toxicity are different. They are produced by the photochemical chlorination of benzene during the manufacturing of the technical lindane (t-HCH). The production of one ton of lindane is accompanied with the production of 6-10 tons of other

Field-Scale Rhyzoremediation of a Contaminated Soil … 5

isomers (IHPA, 2006) characterized by different chlorine atom orientations in the cyclohexane molecule (axial or equatorial position). This structural difference seems to significantly affect the individual chemical-physical and biological properties of some isomers. For example, it has been shown that isomers with the chlorine atoms in axial position are less persistent than β-HCH having this latter all its six chlorine atoms in the equatorial orientation (Bachmann et al. 1988b; Beurskens et al. 1991). It can be argued that this can be the reason because the β-HCH comprises above 80% of HCH residues of technical lindane in a ten year contaminated field surrounding an industrial landfill in Germany (ATSDR, 2005). It has to be considered also that biotic and abiotic factors may lead to the isomerization process that enables the interconversion between HCH chemical isomers (Malaiyandi et al. 1982; Huhnerfuss et al. 1992).

Figure 1. The contaminated surface (red color) in the Valle del Sacco area.

Further, it is to underline that the β-HCH transportation through the environment doesn’t much follow the diffusion water path. Its octanol-water partition coefficient (log KOW) is, in fact, close to 4. Remarkably, its vapour pressure at 25°C is 6.3 10-5 mmHg. This means that in a moist soil surface at this temperature a significant amount of β-HCH can be released to the atmosphere and can probably move for long distances carried by wind bound to small dust particles (Wania et al., 1999). Luckily, this isomer can be degraded in the atmosphere by reacting with photochemically produced hydroxyl radicals. An important target of HCH isomer phytoremediation is then the degradation of the recalcitrant β-isomer. Degradation of HCHs has been observed under both oxic and anoxic conditions (Phillips et al., 2005; Nagata et al., 2007). However, the mineralization (complete degradation of the pollutant to CO2) generally occurs in oxic systems. Over time, several species of microorganisms (bacteria and fungi) have demonstrated the ability to degrade specific HCH isomers in the two mentioned

D Bianconi, MR De Paolis, AC Agnello, et al. 6

conditions (summarized by Phillips et al., 2005). It has also been reported that the aerobic bacteria Arthrobacter citreus can degrade γ-HCH and use it as a sole source of carbon (Datta et al., 2000). In certain cases, one single strain might not have all the enzymes involved in the complete catabolic pathway of a pollutant. A consortium of bacteria, each with different parts of the degradation route has been reported to be very efficient (Rahman et al., 2002).

THE EXPERIMENTAL APPLICATION The HCH remediation in the Valle del Sacco area has been designed considering present

knowledge in the use of poplars in association with microorganisms to reduce chlorinated organic concentration from the soil and to produce biomass for energy. Preliminary studies have been performed to select candidate bacteria and hybrid poplars that combine the most efficient contaminant reduction in the soil, the highest biomass production and the uncontamination of harvestable biomass. Bacteria isolated from contaminated soil samples of Valle del Sacco and those isolated and characterized for previous studies on organic xenobiotic metabolism have been tested and selected to obtain an efficient β-HCH degradation. Figure 2 reports the growth curves of two Arthrobacter strains in culture with the β-HCH isomer as the sole carbon source. This persistent isomer was degraded by about 50% (N:P:K) in 48 h (De Paolis et al., 2009) and given its highest persistence in the environment the two strains were chosen for association studies with six hybrid poplar clones. These clones were among those more successful in terms of biomass production in the Valle del Sacco area outside the contaminated soils. These studies were performed in the glasshouse and three candidate clones were finally selected for an experimental “in situ” application of 0.4 Ha in the contaminated area. I-214, AF2 and Monviso were those showing growth characteristics and response to contaminants that have been considered useful for a rhizodegradation application. I-214 showed itself good β-HCH degradation ability without bacterial inoculation, while AF-2 showed the highest uncontaminated biomass production, and Monviso both the highest HCH degradation with inoculated bacteria and a high biomass production. It is remarkable that bacteria inoculated in the potted contaminated soil without poplar cutting degraded 50% less HCH isomers than in association with Monviso. This indicates that this hybrid can provide rhizosphere conditions stimulating the HCH degradation of the two agrobacters.

Cuttings of these three hybrid poplar clones were mechanical planted in 2008 in a site of about 0.5 ha. Plant density in 5 replicated parcels of 10x9 m for each treatment was about 8000 ha-1. Treatments were: a) poplar clone alone, b) poplar clone inoculated with bacteria, c) poplar clone on the soil amended with commercial compost and with oxygen release compound (ORC, IXPER® 75C Calcium Peroxide, Solvay, S.A). The addition of the compost (produced on plant wastes) was due to assess whether its high microbic count could be alternative to the inoculation, which is technologically complicated and a bit more expensive than composting. The simultaneous presence of ORC served to enhance the degrading microbiological activity by the continuum oxygen supply of this compound in presence of water and by its own degradation ability of chlorinated organics (Arienzo, 2000; Cassidy and Irvine, 1999). A drip irrigation system was placed to provide evapotranspired water restitution twice a week. Before plantation the soil physico-chemical and biological

Field-Scale Rhyzoremediation of a Contaminated Soil … 7

characteristics were analysed on samples collected according to regulated sampling procedure for contaminated soils. Data are reported in the table 1. The soil appears equilibrated regarding the C/N ratio required by optimal plant and bacteria growth. A high carbon content would in fact disadvantage the bacterial use of contaminant carbon skeleton by competition with more easily metabolized organic compounds and consequently this would reduce degradation efficiency. A typical fertilization adopted for poplar plantation in SRC in the Valle del Sacco soils with ammonium nitrate at a rate of 150 Kg ha-1 was applied during soil mechanical preparation. Bacteria concentrated solution (106 CFU per plant) was inoculated at root level in early June after 60 days from plantation. At the end of the growing season (figure 3) in October soil and plants were sampled for HCH isomers analysis. Table 2 shows results of HCH degradation under the various treatments and evidences that the field application of rhizoremediation significantly reduced HCH content in the soil planted with Monviso and inoculated with the two agrobacter strains. With respect to the glasshouse studies the field degradation was lower in percentage (25:40) probably reflecting the different glasshouse and field conditions. Furthermore, in pots the interaction between degrading bacteria and contaminants is certainly facilitated. Interestingly, the compost and ORC treatment also reduced β-HCH content by a significant extent. However, the HCH chromatogramme showed that the disappearance of HCH isomers was compensated by the detection of a new, not yet identified, peak at high molecular weight. Further studies are in course to identify the chemical characteristic and toxicity of this compound.

Time (h)

0 6 12 18 24

CFU

x 1

06 ml-1

0

50

100

150

200

β − HCHα − HCH

Strain A2

Growth on α-HCH μmax = 0.84 ± 0.02 Lag = 0.72 ± 0.12 td = 0.82 ± 0.02

Growth on β-HCH μmax = 0.73 ± 0.02 Lag = 3.03 ± 0.11 td = 0.95 ± 0.02

Growth on α-HCH μmax = 0.86 ± 0.07 Lag = 6.38 ± 0.15 td = 0.80 ± 0.06

Growth on β-HCH μmax = 1.13 ± 0.05 Lag = 9.73 ± 0.07 td = 0.61 ± 0.03

Time (h)

0 6 12 18 24

CFU

x 1

06 ml-1

0

10

20

30

40

β - HCHα - HCH

Strain A3

Figure 2. Growth curves and parameters of Arthrobacter strains on α- and β-HCH. Two Arthrobacter strains, A2 e A3, were grown in mineral medium B7

+ containing α-HCH (10 mg/l) or β-HCH (5 mg/l) as sole carbon source. 3, 7 and 14 days old-cultures were analyzed for capacity to utilize HCH. Moreover bacterial growth curves on α- e β-HCH were recorded and their growth parameters were calculated by means of software MicroFit (ver. 1.0), following the mathematic model of Baranyi.

It can be expected that in the second growth season in the field, when the root system will be more extended and deep enough to completely explore the soil volume down to 0.8 m, the interaction of bacteria with contaminants will increase using the more complex and extended root as diffusion pathway.

D Bianconi, MR De Paolis, AC Agnello, et al. 8

Table 1. Some physico-chemical and biological characteristics of soil from experimental site.

Sand (%) 75.25 Silt + Clay (%) 24.75 pH (H2O) 7.72 Total N (g / Kg) 0.16 Organic C (g / Kg) 12.24 Organic matter (g / Kg) 21.11 Electrical conductivity (mS / cm) 462.0 Bacteria (CFU x 107 g-1) 58.80 Actinomycetes (CFU x 107 g-1) 13.10 Fungi (CFU x 105 g-1) 18.00 CFU: colony-forming unit.

Figure 3. The experimental plantations with inter-row distances of 200 cm and a spacing of 50 cm between cuttings within the row.

Another important result obtained in this experimental application was that the biomass production of the three clones (figure 4) was as good as in the uncontaminated areas of the Valle del Sacco, with a minimum of 5 and a maximum of 10 tons d.m. ha-1 year-1 with I-214 and AF-2 showing the minimum and maximum yield, respectively. Besides it is remarkable that the harvestable plant parts were only slightly contaminated. Only 10% of samples were contaminated by a 0.03 mg Kg-1 of the β-HCH isomer (data not shown).

Field-Scale Rhyzoremediation of a Contaminated Soil … 9

Table 2. Mean of individual HCH isomers and total HCH (� + � + � + �) concentrations (± S.E.) in soil and rhizosphere samples (ppm) under three treatments. Means (n = 3)

with different letters indicate significant differences at p < 0.05 level (Anova). S.E.: standard error.

SAMPLE α-HCH β-HCH γ-HCH δ-HCH Total HCH Soil before poplar cuttings planting 0.044 ±0.031 ab 0.058 ±0.052 a 0.017 ±0.002 abc 0.006 ±0.001 a 0.125 ±0.008 a

I-214 0.044 ±0.074 ab 0.046 ±0.015 abcd 0.011 ±0.001 cd 0.002 ±0.001 c 0.103 ±0.009 abc I-214 + bacteria 0.042 ±0.015 ab 0.047 ±0.058 abcd 0.016 ±0.001 abc 0.004 ±0.003 abc 0.108 ±0.007 ab I-214 + ORC + compost 0.047 ±0.035 a 0.054 ±0.055 abc 0.020 ±0.003 a 0.006 ±0.027 ab 0.126 ±0.006 a

AF-2 0.044 ±0.061 ab 0.039 ±0.019 bcd 0.014 ±0.001 abcd 0.004 ±0.006 abc 0.102 ±0.008 abc AF2 + bacteria 0.045 ±0.035 ab 0.053 ±0.044 abc 0.018 ±0.001 ab 0.005 ±0.001 abc 0.120 ±0.008 ab AF2 + ORC + compost 0.039 ±0.054 ab 0.038 ±0.063 cd 0.012 ±0.002 bcd 0.003 ±0.001 abc 0.093 ±0.010 bcd

Monviso 0.038 ±0.062 abc 0.057 ±0.015 ab 0.013 ±0.001 bcd 0.004 ±0.001 abc 0.112 ±0.008 ab Monviso + bacteria 0.025 ±0.007 c 0.032 ±0.099 d 0.010 ±0.001 cd 0.002 ±0.001 bc 0.070 ±0.011 d Monviso + ORC +compost 0.032 ±0.012 bc 0.030 ±0.020 d 0.013 ±0.001 bcd 0.003 ±0.001 abc 0.079 ±0.002 cd

Tota

l bio

mas

s (K

g pl

ant-1

)

0.0

0.1

0.2

0.3

0.4

0.5

0.6

I-214I-214 + bacteriaI-214 + compost + ORCAF2AF2 + bacteriaAF2 + compost + ORCMonvisoMonviso + bacteriaMonviso + compost + ORC

a

bc

ab

cd

de

efef

de

f

Figure 4. Dry biomass (Kg plant-1) measured in plants of three poplar clones at the end of the first year from plantation. Means values with a same letter were not significantly different (P < 0.05, ANOVA; LSD mean comparisons test). Values are the mean of five replicates. Error bars indicate standard error.

CONCLUSION

Some important aspects of the poplar rhizoremediation and biomass production in the

Valle del Sacco contaminated area have still to be dealt with. It is, for example, important to

D Bianconi, MR De Paolis, AC Agnello, et al. 10

test a large scale bacteria injection into the rhizosphere. Adaptation of commercial agricultural machines is under evaluation as well as the distribution of inoculums via the buried drip irrigation system. The choice will be affected by the ongoing studies on the agrobacters persistence in the soil after the inoculation. It is evident that the improvement of the inoculation techniques providing a higher distribution of degrading bacteria in the contaminated soil will be an important goal of experimental activities that will increase the HCH bioavailability and bacterial degradation capacity. Results achieved are positive indications that the adopted strategy can be effective in reducing soil HCH contamination, offering local farmers to produce bioenergy as an important payback of incomes lost by the interdiction of the agricultural activities. It is evident that the thermoconvertible biomass production adds an important economic value. So far, the problem of remedying HCH isomer contamination in the Valle del Sacco seems to be more of answering the question: does the biomass produced in a contaminated site have to be considered a waste or is it thermoconvertible as good biomass?

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