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Alder-Frankia Symbionts Enhance the Remediation and Revegetation of Oil Sands Tailings Elisabeth Lefrançois1,2, Ali Quoreshi3, Damase Khasa4, Martin Fung5, Lyle G. Whyte2 Sébastien Roy6 and Charles W. Greer1 1
National Research Council of Canada; 2Department of Natural Resource Sciences, McGill University; 3Symbiotech Research Inc.; 4Centre de Recherche en Biologie Forestière, Université Laval; 5Syncrude Canada Ltd.; 6Département de Biologie, Université de Sherbrooke
ABSTRACT Extraction of petroleum from the oil sands creates large quantities of oil sands processaffected water (OSPW) and tailings sand. The tailings sand has a low fertility, a low organic matter content, it is highly alkaline, compactable, and contains residual hydrocarbons, making it a very inhospitable growth environment. As the exploitation of this resource intensifies, increasing quantities of OSPW and tailings sand are being produced. The petroleum industry is currently involved in efforts to revegetate and remediate the tailings sand, and one approach is to revegetate the tailings sand with Frankia–inoculated alders. Alders are primary successor trees that have the ability to grow in nutrient poor and waterlogged environments, in part because they form a symbiotic relationship with the nitrogen-fixing actinomycete, Frankia. The effect of Frankia-inoculated alders on soil quality was evaluated by monitoring the chemical, physical and microbiological characteristics of soil (organic matter, pH, bulk density, salts and nutrients concentrations, microbial population density, activity and diversity). The impact on the indigenous microbial community was also studied using hydrocarbon mineralization assays, molecular biology tools such as denaturing gradient gel electrophoresis (DGGE), and catabolic gene probing, using genes from bacterial pathways for the degradation of various hydrocarbons. Plant parameters were measured to evaluate the impact of Frankia on alder health and growth (plant biomass, nitrogen content, plant height). Preliminary greenhouse trials demonstrated the immense potential of this approach. In 2005, field trials were established at Syncrude Canada Ltd. After two growth seasons, samples of the bulk soil showed an increase in hydrocarbon (hexadecane, naphthalene and phenanthrene) mineralization where the tailings sand had been planted with Frankia-inoculated alders compared to both unplanted and planted with noninoculated alders. The rhizosphere samples all had comparable hydrocarbon mineralization rates. Soil tests showed that alders inoculated with Frankia decreased the soil pH, increased buffering capacity and decreased the percent saturation in three main soil cations (Ca, K, and Mg). The field results have confirmed that the alder-Frankia combination results in improved remediation capabilities and improves soil quality. These improvements in the quality of the tailings sand will allow the subsequent establishment of more sensitive species, leading ultimately to the reforestation of the site.
INTRODUCTION Canada has one of the largest oil sands reserves in the world, mainly located in central Canada. The largest deposit, in the Athabasca region, is a near-surface deposit that allows recovery through surface mining (Fung & Macyk, 2000). The oil extraction process generates several by-products, one of which is the tailings, a liquid slurry that can be consolidated into composite tailings (CT) through the addition of gypsum. This material has a low nutrient content, a high salinity, a high pH, low or no organic matter and residual hydrocarbon products, including toxic naphthenic acids. This is a highly challenging material to reclaim. Such harsh environments reduce the ability of more sensitive plant species to establish, and leaves the soil almost bare. Increased litter and plant cover reduces erosion and increases soil water retention (Cerdà, 1997), moreover, continuous addition of organic matter, through living plants can contribute to soil stability (Huang et al. 2005). Hines et al. (2006) reported that an increase in plant biomass led to an increase in herbivore numbers. This is a first step in the improvement of overall biodiversity and the re-establishment of a balanced ecosystem. Parrotta (1999) found that nitrogen-fixing trees can facilitate vegetation development through addition of nitrogen to the system. Previous greenhouse trials have shown that Frankia-inoculated alders could successfully grow in tailings sand or composite tailings, and have a positive impact on the diversity and activity of the indigenous soil microbial populations. Another study previously demonstrated that alders had a positive impact on soil fertility and on the physiological activity of the soil microbial population (Selmants et al. 2005). The objective of this work was to develop a greenhouse production procedure for Frankia–inoculated alders for subsequent outplanting to oil sands tailings material. Frankia are nitrogen-fixing bacteria that form nodules in the roots of certain plant species, allowing these species to establish in nutrient-poor, harsh environments (Roy et al. 2007). In an earlier study, Frankia-inoculated alders were evaluated in a greenhouse setting to determine if this symbiotic association would enhance successful revegetation of tailings sand. In the present study, the work was extended to a field assessment: inoculated alders were outplanted onto a site containing a mixture of overburden and tailings sand and the site was monitored for changes in soil quality and indigenous soil microbial population composition and activity. Preliminary results demonstrated that this approach to revegetation and remediation of tailings sand was a promising method towards the re-establishment of a balanced forest ecosystem.
RESULTS In 2005, Frankia-inoculated and non-inoculated green alders (Alnus crispa) were planted in a mixture of overburden and tailings sand material at Syncrude Canada Ltd., Alberta. After one and half years of growth, the soil material around the plants and the alders themselves were harvested for analysis. Soil quality parameters, plant biomass and plant nitrogen content were determined as well as the activity and composition of the microbial community in the bulk soil, rhizosphere soil and endophytic community (microorganisms present in the plant’s root).
Plant and soil characterization of overburden-tailings mixture planted with alders and Frankia-inoculated alders. Plant biomass and nitrogen content were measured to evaluate plant performance (Table 1). Average plant biomass of the Frankia-inoculated alders (Frankia-alder) (154.1 g) increased compared to the non-inoculated alders (111.8 g). Nitrogen percent was similar between inoculated (3.1%) and non-inoculated plants (3.0%). However, as average plant biomass was greater in Frankia-alder, the average total nitrogen content was also greater, 4.8 g per plant compared to 3.5 g per plant.
Table 1. Average plant biomass and nitrogen (N) content of non-inoculated alders (Control-alders) and Frankia-inoculated alders (Frankia-alders) planted in a mixture of overburden and oil sands tailings after 1.5 years of growth. Treatment
Controlalders
Frankiaalders
Dry weight (g) Total % N Total N (g) /plant
111.8 3.0 3.5
154.1 3.1 4.8
An overall improvement of soil characteristics after the establishment of Frankiainoculated alders was observed (Table 2). Compared to unplanted soil and soil planted with control-alder, soil planted with Frankia-inoculated alder decreased the pH and buffer-pH from 7.5 to 6.6, and from more than 7.5 to 7.0, respectively. The decrease in buffer-pH indicates an increase in the soil buffering capacity. Soil sodium content decreased, as did the saturation in major cations (K, Mg, and Ca) with Frankia-alder (85.3%) compared to the two other treatments (100%). This is confirmed by an increase in the cation exchange capacity (CEC) from 23.0 meq/100 for unplanted soil, and 24.5 meq/100 for control-alder planted soil to 27.2 for Frankia-alder planted soil.
Microbial community characterization of overburden-tailings mixture planted with alders and Frankia-alders. The mineralization activity of the indigenous microbial population in the control- and Frankia-alder planted and the unplanted soils, as well as in the control- and Frankia-alder rhizosphere was evaluated using three representative hydrocarbon substrates: hexadecane, naphthalene, and phenanthrene (Figures 1 and 2). In the bulk soil, mineralization increased, for all three substrates tested, for Frankia-alder treatment as compared to both unplanted and control alder treatments. This indicates that the presence of Frankia-inoculated alders had a positive effect on the hydrocarbon degradation capacity of the soil indigenous microbial population. However, in the rhizosphere similar mineralization rates were found for the control and Frankia-inoculated alders, which were high for naphthalene and phenanthrene but lower for hexadecane.
Table 2. Soil analysis of overburden-oil sands tailings either unplanted, planted with noninoculated alders (Control-alders) or Frankia-inoculated alders (Frankia-alders) after 1.5 years of growth.
Mehlich-III
Estimated CEC
Saturation (%)
Treatment
Unplanted
Control-alders
Frankia-alders
pH buffer-pH K (kg/ha) Mg (kg/ha) Ca (kg/ha)
7.53 >7.5 211 1533 7633
7.53 >7.5 1697 8017
6.60 7.03 116 1637 7597
Na (kg/ha)
498
359
160
217
(meq/100)
23.0
24.5
27.2
K Mg
1.1 25.1
1.0 25.9
0.5 22.4
Ca
73.8
73.1
62.4
K+Mg+Ca
100
100
85.3
Total microbial community DNA was analyzed to evaluate the structure of the total microbial population without the bias associated with the use of culturing techniques. Total DNA was extracted from rhizosphere and alder roots (endophytic community), and a portion of the 16S rRNA gene, a universal bacterial target, was amplified using the polymerase chain reaction (PCR). The resulting fragment mixture was resolved using denaturing gradient gel electrophoresis (DGGE) (Figure 3). This method separates fragments of the same size on the basis of their nucleotide sequence differences. Theoretically, each band on the gel is due to a different microorganism, so the pattern of diversity can be compared between samples. Also, by determining nucleotide sequence of individual bands, it is possible to identify microorganisms responsible for the band of interest. For both rhizosphere and endophytes, the general band pattern does not show any differences between the control and Frankia-inoculated treatments. Some bands of interest were extracted, amplified, purified and sequenced (Table 3). Certain bands at the same positions in different lanes had similar nucleotide sequences (e.g. a, b, and c; d, and e; 1, 12, and 16; 3, 14, and 17;etc.) For the rhizosphere, when compared to databases, most of the bands sequences relate to soil or rhizosphere microorganisms, and are often associated with petroleum or other contaminants. Surprisingly, the sequence of two of the three dominant bands (a, b, c, and d, e, f) of each of the endophyte samples had high similarity with plant DNA, and not bacterial DNA. However, as expected, the bottom band (g, h, and i) had high similarity to the nucleotide sequence of Frankia sp. Total microbial community DNA from the different soils, rhizosphere and alder roots (endophytes) was also analyzed for the presence of the alkB gene, which encodes a key enzyme (alkane monooxygenase) in the degradation of aliphatic hydrocarbons, such as hexadecane. Total microbial DNA was also analyzed for the intergenic spacer region between the nifD-K genes (Hahn et al. 1999; Dai et al. 2004), genes involved in nitrogen fixation, specific to the sequence of Frankia F9, the strain that was used to inoculate the
80
Hexadecane
70 60 50 40 30 20 10 0
Naphthalene
Mineralization (%)
70 60 50 40 30 20 10 0
Phenanthrene
70
Sterile Unplanted Planted Frankia-inoculated
60 50 40 30 20 10 0 0
10
20
30
40
50
60
70
80
Time (days)
Figure 1. Mineralization of representative hydrocarbon substrates by overburden-oil sands tailings mixture either unplanted, planted with non-inoculated alders (Planted) or Frankia-inoculated alders after 1.5 years of growth.
60
Hexadecane 50 40 Sterile Planted Frankia-inoculated
30 20 10 0
Naphthalene
Mineralization (%)
70 60 50 40 30 20 10 0
Phenanthrene
70 60 50 40 30 20 10 0
0
10
20
30
40
50
60
70
80
Time (days)
Figure 2. Mineralization of representative hydrocarbon substrates in the rhizosphere of non-inoculated alders (Planted) or Frankia-inoculated alders after 1.5 years of growth in an overburden-oil sands tailings mixture.
Figure 3. Denaturing Gradient Gel Electrophoresis (DGGE) of 16S rRNA gene fragments PCR amplified from total DNA extracted from rhizosphere and roots of alders inoculated or not with Frankia strain F9 after 1.5 years growth in a mixture of overburden and oil sands tailings. Ca, Cc, and Fa, Fb identifies field replicates of noninoculated alders and Frankia-inoculated alders, respectively. F9 is the pure culture of Frankia strain F9, and the arrow indicates the position of its dominant band. Numbers (1-17) and letters (a-i) indicate bands that have been extracted for subsequent nucleotide sequencing (see Table 3).
alders. The alkB gene was detected in all of the Frankia-inoculated soil replicates, in 2 of the 3 replicates of the control-alder soil and in 2 of the 3 unplanted soil replicates (Figure 4). It was also detected in all of the rhizosphere samples, but it was not detected in any of the endophyte samples (Figure 5). These results indicated that the alkB gene appeared to be more abundant in soil that had been planted with alders, whether they had been inoculated or not. Frankia F9 was detected in all of the control and Frankia-inoculated endophyte samples (Figure 6). However, from root and nodule observation it was noted that the number and size of the nodules was greater in inoculated samples than in noninoculated ones. The detection of Frankia F9 in the non-inoculated alders suggested that the plants had been exposed to the strain under greenhouse conditions, since analysis of indigenous alders adjacent to the field sites showed the presence of nodules, but they were not positive for Frankia F9. Frankia F9 was not detected in any of the rhizosphere samples or in the bulk soil (data not shown).
Table 3. Nearest microorganisms of sequenced DGGE bands seen in Figure 3.
Closest microbial match
% Similarity BLAST
% Similarity FASTA
Characteristics
EF018110
Uncultured bacterium
97
97.994
trembling aspen rhizosphere
AM403320
Uncultured alpha proteobacterium
97
97.167
uranium mining site
EF540402
Uncultured soil bacterium
96
96.884
semi-coke
U59505
Hyphomicrobium sp.
94
93.768
Rhizobiale
3
AY177365
Rhizobium sp.
97
97.734
phenanthrene degrader
4
EF018177
Uncultured proteobacterium
94
94.595
trembling aspen rhizosphere
5
EF617349
Arthrobacter globiformis
100
NP
salt affected soil
DQ129877
Arthrobactersp.
NP
100
hypersaline lake
EF393416
Uncultured bacterium
95
95.652
river sediment
DQ297984
Uncultured soil bacterium
94
94.783
Hydrocarbon contaminated soil
EF393416
Uncultured bacterium
97
97.796
river sediment
DQ297984
Uncultured soil bacterium
96
96.970
Hydrocarbon contaminated soil
EF507099
Uncultured bacterium
98
98.125
Bi-phenyl utilizing
AB258386
Kaistobacter terrae
97
97.812
AM403320
Uncultured alpha proteobacterium
98
98.154
uranium mining site
EF540402
Uncultured soil bacterium
97
97.846
semi-coke
AF370880
Nordella oligomobilis
94
94.462
rhizobiale
AM176541
Arthrobacter sp.
NP
100
PAH-degrading
DQ129877
Arthrobacter sp.
100
100
hypersaline lake
12
EF018110
Uncultured proteobacterium
95
95.402
trembling aspen rhizosphere
13
AM403320
Uncultured alpha proteobacterium
93
93.438
uranium mining site
EF540402
Uncultured soil bacterium
93
93.125
semi-coke
14
AY177365
Rhizobium sp.
94
94.044
phenanthrene degrader
15
EF018177
Uncultured proteobacterium
93
93.373
trembling aspen rhizosphere
16
EF018110
Uncultured proteobacterium
99
99.703
trembling aspen rhizosphere
17
EU072713
95
NP
soil
AM236080
Rhizobium sp. Rhizobium bv.viciae
NP
95.077
EF665114
Uncultured alpha proteobacterium
95
94.884
Forest soil
EF019618
Uncultured proteobacterium
94
94.884
trembling aspen rhizosphere
EF135033
Uncultured actinobacterium
97.802
agricultural soil
EF540541
Uncultured soil bacterium
97
97.527
semi-coke
Y12848
Frankia sp.
96
96.703
host: Alnus glutinosa
Plant DNA
99
CT573213 Frankia alni str. ACN14A NP: Not present in the database
99
DGGE Band
Accession number
1 2
6 7 8 9
11
18 19
a-f g, h, i
leguminosarum
Various plant species 99.437
Figure 4. PCR amplification of the alkB gene from total DNA extracted from bulk soil of unplanted, planted with non-inoculated alders (control planted) or Frankia-inoculated alders. The alkB gene was amplified using the consensus primers, alkH1F and alkH3R. The arrow indicates the position of the amplified 575 bp fragment, The 100 bp ladder (M) was electrophoresed along the samples. Letters (A-C) identify field triplicates for each treatment; (+) and (-) indicate the positive and negative controls, respectively.
Figure 5. PCR amplification of the alkB gene from total DNA extracted from rhizosphere and root (endophyte) samples. The alkB gene was amplified using the consensus primers, alkH1F and alkH3R. The arrow indicates the position of the amplified 575 bp fragment. The 100 bp ladder (M) was electrophoresed along both sides of the gel. Field replicates of non-inoculated alders (Ca, Cb) and Frankia-inoculated (Fa, Fb) alders are identified; (+) and (-) indicate the positive and negative controls, respectively.
Figure 6. PCR amplification of the intergenic nifD-K spacer region in Frankia strain F9 using total DNA extracted from different rhizosphere (rhizo) and root (endo) samples. The fragment was amplified using the primers, Fr-IGS-F, and Fr-IGS-R. The arrow indicates the position of the amplified 269 bp fragment. The 100 bp ladder (M) was electrophoresed along both sides of the gel. Ca, Cb, Cc and Fa, Fb identifies Field replicates of non-inoculated (Ca, Cb, Cc) and Frankia-inoculated (Fa, Fb) alders, are identified; (+) and (-) indicate the positive and negative controls, respectively.
CONCLUSIONS • Characterization of plant and soil under the different treatments demonstrated that greenhouse Frankia-inoculated alders have a positive impact on plant establishment, and resulted in an overall improvement in soil quality parameters following growth in the field over a period of 1.5 years. • Characterization of the indigenous soil microbial population indicated an increase in the hydrocarbon degradation capacity when alders were inoculated with Frankia, even though the alkB gene was detected in all of the treatments. • The detection of Frankia F9 in the non-inoculated alders was the result of exposure to the strain under greenhouse conditions, since this strain was not detected in nodules from indigenous alders growing around the field sites. Further work will be conducted to determine if these indigenous Frankia could serve as alder-inoculants, since they are clearly adapted to the site conditions. • Overall, the results demonstrated that Frankia-inoculated alders are a promising biotechnological approach for the remediation and revegetation of the oil sands tailings.
ACKNOWLEDGMENTS
The authors acknowledge financial support from the NRCan PERD program, and to EL from the Silverhill Institute of Environmental Research and Conservation. REFERENCES Cerdà, A. 1997. The effect of patchy distribution of Stipa tenacissima L. on runoff and erosion. Journal of Arid Environment 36: 37-51. Dai, Y.M., He, X.Y., Zhang, C.G., Zhang, Z.Z. 2004. Characterization of genetic diversity of Frankia strains in nodules of Alnus nepalensis (D. Don) from the Hengduan Mountains on the basis of PCR-RFLP analysis if the nifD-nifK IGS. Plant and Soil 267: 207-212. Fung, M.Y.P., and Macyk, T.M. 2000. Reclamation of Oil Sands Mining Areas. pp.755774. In: Barnhisel, R.I., Darmody, R.G., Daniels, W.L. (eds.) Reclamation of Drastically Disturbed Lands, Agronomy Monograph no.41. 1082p. Hahn, D., Nickel, A., Dawson, J. 1999. Assesing Frankia populations in plants and soil using molecular methods. FEMS Microbiology Ecology 29: 215-227. Hines, J., Megonigal, J.P., Denno, R.F. 2006. Nutrient subsidies to belowground microbes impact aboveground food web interactions. Ecology 87(6): 1542-1555. Huang, P.-M., Wang, M.-K., Chiu, C.-C. 2005. Soil mineral-organic matter-microbe interactions : Impact on biogeochemical processes and biodiversity in soils. Pedobiologia 49: 609-635. Parrotta, J.A. 1999. Productivity, nutrient cycling, and succession in single- and mixedspecies plantations of Casuarine equisetifolia, Eucalyptus robusta, and Leucanea leucocephala in Puerto Rico. Forest Ecology and Management 124: 45-77. Roy, S., Khasa, D.P., Greer, C.W. 2007. Combining alders, frankiae, and mycorrhizae for soil remediation and revegetation. Canadian Journal of Botany 85: 237-251 Selmants, P.C., Hart, S.C., Boyle, S.I., Stark, J.M. 2005. Red alder (Alnus rubra) alters community-level soil microbial function in conifer forests of the Pacific Northwest, USA. Soil Biology & Biochemistry 37: 1860-1868.
