The authors have declared that no competing interests exist.
Conceived and designed the experiments: JHK SXC MAN. Performed the experiments: JHK. Analyzed the data: JHK. Contributed reagents/materials/analysis tools: SXC MAN. Wrote the paper: JHK. Reviewed and revised the paper: SXC MAN WS. Study plot establishment and management: MAN. Discussed the results and contributed in this manuscript: JHK SXC MAN WS.
Forest floor mineral soil mix (FMM) and peat mineral soil mix (PMM) are cover soils commonly used for upland reclamation post open-pit oil sands mining in northern Alberta, Canada. Coarse woody debris (CWD) can be used to regulate soil temperature and water content, to increase organic matter content, and to create microsites for the establishment of microorganisms and vegetation in upland reclamation. We studied the effects of CWD on soil microbial community level physiological profile (CLPP) and soil enzyme activities in FMM and PMM in a reclaimed landscape in the oil sands. This experiment was conducted with a 2 (FMM vs PMM) × 2 (near CWD vs away from CWD) factorial design with 6 replications. The study plots were established with
The Athabasca oil sands region in northern Alberta, Canada, is the largest single oil sands deposit in the world with an estimated 1.6 trillion barrels of bitumen, a low quality crude oil mixed with sands and water [
A common reclamation practice in this region is returning disturbed land to upland boreal forests. Substrates, such as overburden materials or tailing sands, are not suitable for plant growth due to lack of nutrients, high salinity, and high concentrations of toxic materials including naphthenic acids, polycyclic aromatic hydrocarbons, phenolic compounds and trace metals, and therefore, approximately 30 cm of cover soils are applied over a substrate to support plant growth to supply nutrients and to improve soil properties [
Soil microbial community and enzyme activities are affected by substrate quality and quantity [
Forest floor (litter, fragmented litter, and humus) mineral soil mix (hereafter FMM) and peat mineral soil mix (hereafter PMM) are cover soils commonly used for oil sands reclamation in northern Alberta. Peat mineral soil mix is readily available in northern Alberta and has been used for oil sands reclamation, while the availability of FMM is limited. Applying FMM for oil sands reclamation has recently been used and FMM has several advantages over PMM when used for soil sands reclamation. Properties of cover soils, such as FMM and PMM, used for oil sands reclamation in northern Alberta have been compared [
Coarse woody debris (CWD), including large branches, logs, standing dead trees, and dead coarse roots, plays important ecological roles in forest ecosystems [
Many studies assessed the effect of CWD on microbial community and enzyme activities in natural forest ecosystems. Coarse woody debris increased fungal to bacterial ratio, soil respiration, and microbial biomass in temperate coniferous forests [
This study was conducted to determine if applying CWD on reclaimed oil sands soils amended with FMM or PMM will affect microbial community functional diversity and soil enzyme activities thereby improving soil fertility and accelerating upland reclamation. We hypothesized that 1) soil microbial CLPP would be different between the two cover soils due to their contrasting properties, and microbial biomass would be greater in FMM than in PMM, 2) enzyme activities would be greater in FMM than in PMM regardless of CWD application due to greater microbial biomass and vegetation cover in FMM, 3) CWD would change microbial CLPP due to increased labile C content coming from CWD leachate, and 4) CWD would enhance microbial biomass, average well color development in Biolog Ecoplates, and enzyme activities due to increased availability of microsites. To test these hypotheses, we conducted field experiments 6 and 7 years after land reclamation in an open pit oil sands mining area in the Athabasca oil sands region in northern Alberta.
This study was conducted on an oil sands mine site (56° 58’N, 111° 22’W), approximately 24 km north of Fort McMurray, Alberta, Canada. The site was cleared in 1999 for open pit oil sands mining, and thereafter, was used as a saline-sodic overburden waste dump until 2004. The owner of the land (Suncor Energy Inc.) gave permission to conduct the study on this site. A detailed description of the research site and experimental plots is provided in
For the study area, average annual temperature from 1981 to 2010 was 1.0°C and average annual precipitation was 418.6 mm, with 316.3 mm as rainfall and 133.8 cm as snowfall [
A factorial experiment consisted of 2 cover soil types (FMM vs PMM) and 2 sampling distances from CWD (near vs away from CWD) with 6 replications was designed for this research. Study plots 10 × 30 m in size were established between November 2007 and February 2008. Six plots were covered with FMM and six plots were covered with PMM. The FMM was applied at a depth of 20 cm on top of 30 cm of B and C horizon mixed subsoil layer over 100 cm of clean overburden. The PMM was applied at a depth of 30 cm over 100 cm of clean overburden [
Plots were covered by naturally established forbs, grasses, shrubs, and mosses [
Soil was sampled in the 0 to 10 cm layer using an auger on July 26, August 26, and September 28 in 2013 and on July 8, August 8, and September 4 in 2014. Three soil samples, approximately 30 g each, were collected from each treatment, three within 5 cm from CWD and three more than 100 cm from CWD, in each plot and bulked to form a composite sample of each treatment; a total of 24 composite soil samples were collected [4 treatments (2 cover soils × 2 sampling distance from CWD) × 6 replications] in each sampling. Soil samples were transported to the laboratory on ice packs in a cooler. Fresh soil samples were passed through a 2-mm sieve, and stored in a refrigerator at 4°C until analysis. All analyses were completed within 4 days after sampling. A sub-sample of each soil sample was used to determine available N, microbial biomass C (MBC) and N (MBN), dissolved organic C (DOC) and N (DON), soil microbial community level physiological profile, and extracellular enzyme activities. The remainder of each sample was air dried at room temperature and used to determine pH and electrical conductivity (EC). A portion of the air dried sample was ground into fine powder using a ball mill (MM200, REtsch GmbH, Haan, Germany) and used for total C and total N analyses.
Soil pH was determined using a pH meter (Orion, Thermo Fisher Scientific Inc., Beverly, MA, USA) and EC using an AP75 portable waterproof conductivity/TDS meter (Thermo Fisher Scientific Inc., Waltham, MA, USA) at a 1:5 soil weight to deionized water volume ratio. Available N including ammonium (NH4+) and nitrate (NO3−) concentrations were determined via steam distillation [
Soil microbial CLPP was determined using a Biolog Ecoplate (Biolog Inc., Hayward, CA, USA), which contains 31 C substrates and one control with 3 replications in a 96 well microplate. One gram of each fresh soil sample was put into a sterile flask with a 100 mL of 0.87% sterilized sodium chloride solution, shaken for 30 min and diluted 1,000 times with 0.87% sterilized sodium chloride solution. A 150 μL aliquot of each soil suspension was inoculated into each well of the Ecoplate. Ecoplates were incubated for 168 hours at 25°C in the dark. The optical density of each well was read at 590 nm every 24 hours using a microplate reader (Emax, Biolog Microstation, CA, USA).
To describe the soil microbial community functional diversity (average well color development in Biolog Ecoplates) and area under the curve (
The area under the curve
Extracellular enzyme activities involved in C and N cycling, including β-1,4-N-acetylglucosaminidase (NAGase, enzyme classification (EC) number 3.2.1.14), β-1,4-glucosidase (EC 3.2.1.21), cellobiohydrolase (EC 3.2.1.91), and peroxidase (EC 1.11.1.x) were measured according to
For NAGase, β-1,4-glucosidase, and cellobiohydrolase activities, 200 μL of soil suspension and 50 μL of 200 μmol L−1 of each substrate were pipetted into black 96 well plates. Reference standards and quench controls were added to each reference and quench well in each plate. The plates were incubated at 20°C in the dark for 3, 3, and 7 hours for NAGase, β-1,4-glucosidase, and cellobiohydrolase, respectively. After the incubation, a 20 μL of 0.5 mol L−1 sodium hydroxide solution was added to each well to stop the enzyme reaction. Fluorescence was measured at 360 nm excitation and 460 nm emissions using a multi-detection microplate reader (Synergy HT, Bio-Tek Instruments, Winooski, VT, USA).
For peroxidase activity, 200 μL of soil suspension and 50 μL of 200 μmol L−1 of substrate (3,4-dihydroxy-L-phenylalanine) were pipetted onto clean transparent 96 well plates. A 10 μL 0.3% hydrogen peroxide solution was added to each well after the substrate. The plates were incubated at 20°C in the dark for 5 hours. After incubation, absorbance was measured at 460 nm using the multi-detection microplate reader.
Two-way analysis of variance (ANOVA) was conducted to determine the differences in soil properties and each enzyme activity using the SAS software (SAS Institute Inc., NC, USA). Before performing the ANOVA, the normality of distribution and homogeneity of variance were checked with Kolmogorov-Smirnov and Levene’s tests. A repeated measures ANOVA was conducted to assess cover soil type and distance from CWD effects over time on MBC, MBN, each enzyme activity, and average well color development using the PROC MIXED model using the SAS 9.3 software. Distance from CWD was used as a split-plot factor and the month of each sampling was considered a repeated measures variable for determining seasonal variations in 2013 and 2014. In this analysis, the output statistics passed tests for compound symmetry. Tukey’s HSD test was used to determine the significant differences between cover soil type, distance from CWD, month of sampling and their interactions. Pearson correlation and multiple regression analyses were used to determine which soil parameters have strong relationships with MBC, MBN, enzyme activities, and average well color development in Biolog Ecoplates. Slope of changes of average well color development over time was compared using an analysis of covariance.
Color development in each Ecoplate well followed a sigmoidal curve with time and the response of each substrate was different with time; some had short response times while others had longer lags [
The
Soil pH, total C, and total N were significantly affected by cover soil type but not by distance from CWD or their interactions, and they were higher in PMM than in FMM (
Treatment | pH | EC |
Total C | Total N | C:N |
|
---|---|---|---|---|---|---|
Cover soil | Distance from CWD | (dS m−1) | (g kg−1) | (g kg−1) | ||
FMM | Near | 5.90 (0.12) | 0.18 (0.01) | 39.7 (4.9) | 1.4 (0.2) | 30.1 (3.7) |
Away | 6.05 (0.10) | 0.21 (0.02) | 40.8 (5.6) | 1.4 (0.3) | 32.8 (2.9) | |
PMM | Near | 7.06 (0.03) | 0.37 (0.02) | 68.4 (8.2) | 2.4 (0.3) | 28.6 (1.6) |
Away | 7.18 (0.04) | 0.45 (0.03) | 72.7 (10.3) | 2.4 (0.4) | 31.3 (1.3) | |
Cover soil (S) | ns | |||||
Distance from CWD (D) | ns | ns | ns | ns | ||
S x D | ns | ns | ns | ns | ns |
Abbreviations:
aEC = electrical conductivity and
bC:N = carbon to nitrogen ratio
Values are means with SE (n = 6);
c* = p < 0.05;
*** = p < 0.001;
ns = not significant;
Soil DOC concentrations in 2013 were not affected by cover soil type, distance from CWD, or their interactions, except for September 2013 (
Treatment | DOC (mg kg−1) | DON (mg kg−1) | SWC (%) | MBC (mg kg−1) | MBN (mg kg−1) | |||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Cover soil | Distance from CWD | July | Aug. | Sept. | July | Aug. | Sept. | July | Aug. | Sept. | July | Aug. | Sept. | July | Aug. | Sept. |
FMM | Near | 243.5 (39.8) | 302.3 (81.7) | 259.8 (23.9) | 9.3 (2.0) | 8.7 (2.5) | 5.4 (1.9) | 29.9 (6.9) | 25.7 (4.1) | 20.6 (4.5) | 400.3 (96.0) | 382.7 (92.2) | 299.7 (65.2) | 87.0 (22.9) | 65.4 (15.7) | 57.8 (11.9) |
Away | 220.8 (27.8) | 227.4 (21.7) | 298.1 (25.4) | 7.1 (1.0) | 5.7 (0.9) | 5.0 (1.5) | 26.5 (6.6) | 25.0 (5.1) | 20.9 (6.7) | 267.3 (38.9) | 286.6 (39.8) | 248.4 (74.0) | 55.1 (8.3) | 45.7 (10.8) | 40.7 (10.1) | |
PMM | Near | 305.8 (26.4) | 319.7 (36.0) | 323.4 (27.3) | 5.6 (1.0) | 6.8 (1.1) | 2.4 (0.7) | 43.2 (6.0) | 46.4 (7.2) | 32.0 (3.3) | 212.1 (44.2) | 220.6 (45.5) | 279.9 (50.7) | 39.9 (7.2) | 35.5 (8.2) | 37.4 (4.6) |
Away | 286.9 (19.1) | 332.6 (52.4) | 383.7 (38.1) | 6.0 (0.9) | 7.8 (1.3) | 5.1 (1.2) | 41.5 (4.6) | 47.5 (8.3) | 28.2 (4.7) | 196.4 (24.6) | 173.7 (51.1) | 176.4 (35.5) | 37.2 (7.0) | 33.3 (8.1) | 26.1 (6.0) | |
Cover soil (S) | ns | ns | ns | ns | ns | ns | ns | ns | ns | ns | ||||||
Distance from CWD (D) | ns | ns | ns | ns | ns | ns | ns | ns | ns | ns | ||||||
S x D | ns | ns | ns | ns | ns | ns | ns | ns | ns | ns | ns | ns | ns | ns | ns | |
FMM | Near | 158.7 (25.7) | 181.4 (19.3) | 150.2 (15.0) | 8.6 (0.8) | 11.3 (1.7) | 8.8 (0.6) | 26.1 (4.9) | 11.8 (2.6) | 20.3 (4.3) | 251.8 (52.1) | 279.3 (54.6) | 347.6 (50.5) | 58.2 (11.3) | 42.6 (8.3) | 59.6 (9.9) |
Away | 132.0 (22.6) | 146.3 (10.5) | 118.0 (7.5) | 8.1 (1.8) | 10.9 (0.1) | 6.8 (0.7) | 20.5 (4.7) | 10.7 (2.0) | 22.0 (2.7) | 200.8 (21.1) | 250.8 (38.5) | 309.9 (54.1) | 44.9 (5.8) | 34.8 (5.0) | 53.2 (8.8) | |
PMM | Near | 192.0 (26.0) | 220.4 (21.6) | 184.1 (22.6) | 7.1 (0.8) | 10.2 (1.8) | 6.5 (1.0) | 38.0 (6.2) | 19.6 (2.8) | 28.3 (4.6) | 144.7 (38.2) | 300.3 (57.9) | 276.9 (46.3) | 35.4 (7.6) | 31.7 (6.1) | 39.8 (6.9) |
Away | 216.1 (23.6) | 246.1 (30.1) | 176.5 (19.4) | 9.7 (1.4) | 9.7 (1.1) | 6.6 (0.7) | 37.9 (5.9) | 18.7 (2.2) | 32.5 (4.4) | 91.5 (28.4) | 197.6 (35.1) | 237.2 (43.4) | 28.3 (6.5) | 23.8 (3.5) | 33.1 (6.2) | |
Cover soil (S) | ns | ns | ns | ns | ns | ns | ns | ns | ||||||||
Distance from CWD (D) | ns | ns | ns | ns | ns | ns | ns | ns | ns | ns | ||||||
S x D | ns | ns | ns | ns | ns | ns | ns | ns | ns | ns | ns | ns | ns | ns | ns |
Values are mean with SE (n = 18);
a* = p < 0.05;
** = p < 0.01;
ns = not significant.
Soil MBC was greater in FMM than in PMM and CWD increased MBC in cover soils; however, effects of cover soils and CWD were variable among sampling months (
Variable |
DOC | DON | SWC | MBC | MBN | NAGase | GLU | CEL | PER |
---|---|---|---|---|---|---|---|---|---|
MBC | 0.68 |
0.58 |
0.78 |
||||||
MBN | 0.52 |
0.60 |
0.77 |
0.92 |
|||||
NAGase | 0.52 |
-0.08 | 0.28 | 0.47 |
0.26 | ||||
GLU | 0.37 | 0.23 | 0.66 |
0.51 |
0.46 |
0.53 |
|||
CEL | 0.42 |
0.29 | 0.57 |
0.58 |
0.58 |
0.40 | 0.43 |
||
PER | -0.11 | -0.09 | -0.01 | 0.18 | 0.15 | -0.13 | -0.42 | -0.05 | |
MBC | 0.47 |
0.26 | 0.65 |
||||||
MBN | 0.36 |
0.50 |
0.79 |
0.79 |
|||||
NAGase | 0.65 |
0.26 | 0.35 |
0.63 |
0.48 |
||||
GLU | 0.45 |
0.50 |
0.72 |
0.77 |
0.79 |
0.76 |
|||
CEL | 0.38 |
0.31 | 0.29 | 0.49 |
0.54 |
0.40 | 0.54 |
||
PER | 0.01 | 0.17 | 0.14 | 0.09 | 0.11 | 0.03 | 0.22 | 0.10 | |
MBC | 0.68 |
0.46 |
0.54 |
||||||
MBN | 0.57 |
0.26 | 0.78 |
0.88 |
|||||
NAGase | 0.84 |
0.75 |
0.39 |
0.64 |
0.53 |
||||
GLU | 0.69 |
0.61 |
0.38 |
0.48 |
0.42 |
0.82 |
|||
CEL | 0.76 |
0.64 |
0.60 |
0.57 |
0.59 |
0.84 |
0.90 |
||
PER | 0.11 | 0.07 | 0.14 | -0.12 | 0.11 | 0.04 | 0.24 | 0.31 | |
AWCD | 0.33 |
0.16 | 0.65 |
0.33 |
0.51 |
0.42 |
0.46 |
0.54 |
0.11 |
MBC | 0.31 |
0.27 | 0.28 | ||||||
MBN | 0.38 |
0.27 | 0.72 |
0.76 |
|||||
NAGase | 0.71 |
0.72 |
0.29 | 0.47 |
0.49 |
||||
GLU | 0.63 |
0.64 |
0.44 |
0.44 |
0.62 |
0.94 |
|||
CEL | 0.65 |
0.70 |
0.30 | 0.55 |
0.56 |
0.91 |
0.91 |
||
PER | 0.05 | 0.10 | 0.26 | -0.35 | 0.09 | 0.20 | 0.29 | 0.13 | |
AWCD | 0.30 |
0.27 | 0.75 |
0.11 | 0.64 |
0.45 |
0.60 |
0.49 |
0.53 |
aVariables: DOC = dissolved organic C, DON = dissolved organic N, MBC = microbial biomass C, MBN = microbial biomass N, SWC = gravimetric soil water content, NAGase = β-1,4-N-acetylglucosaminidase, GLU = β-1,4-glucosidase, CEL = cellobiohydrolase, PER = Peroxidase, and AWCD = average well color development measured after 168 hours of incubation;
Values are Pearson correlation coefficient;
* = p < 0.05;
** = p < 0.01.
Soil MBN showed a similar pattern to that of MBC and was significantly increased by CWD in most samplings, except for September 2014 (
Average well color development in Biolog Ecoplates was significantly affected by cover soil type, distance from CWD, and sampling time (
Soil properties |
Cover soils (S) | Distance from CWD (D) | Sampling time (T) | S × D | S × T | D × T | S × D × T | |||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
F value | F value | F value | F value | F value | F value | F value | ||||||||
MBC | 3.13 | 0.111 | 7.20 | 0.025 | 0.15 | 0.857 | 0.14 | 0.716 | 1.30 | 0.286 | 0.62 | 0.534 | 4.00 | 0.028 |
MBN | 3.67 | 0.088 | 13.84 | 0.005 | 9.15 | 0.001 | 6.44 | 0.031 | 1.69 | 0.199 | 0.40 | 0.675 | 0.95 | 0.397 |
NAGase | 2.45 | 0.152 | 1.41 | 0.264 | 77.80 | <0.001 | 0.09 | 0.768 | 8.58 | 0.001 | 3.06 | 0.060 | 0.88 | 0.424 |
GLU | 9.20 | 0.014 | 0.40 | 0.541 | 5.24 | 0.011 | 0.72 | 0.419 | 1.36 | 0.271 | 0.00 | 0.999 | 0.30 | 0.745 |
CEL | 10.43 | 0.010 | 0.76 | 0.406 | 12.25 | <0.001 | 0.05 | 0.836 | 2.74 | 0.078 | 0.78 | 0.467 | 0.72 | 0.492 |
PER | 7.76 | 0.013 | 0.84 | 0.371 | 2.09 | 0.142 | 2.17 | 0.159 | 0.08 | 0.927 | 1.94 | 0.161 | 0.90 | 0.418 |
MBC | 1.44 | 0.258 | 18.35 | 0.002 | 31.32 | <0.001 | 1.47 | 0.253 | 5.16 | 0.010 | 0.39 | 0.681 | 0.75 | 0.480 |
MBN | 3.15 | 0.106 | 15.03 | 0.003 | 16.87 | <0.001 | 0.20 | 0.667 | 2.42 | 0.103 | 0.36 | 0.701 | 0.37 | 0.691 |
NAGase | 3.16 | 0.106 | 4.59 | 0.056 | 32.66 | <0.001 | 1.62 | 0.230 | 0.64 | 0.531 | 0.66 | 0.522 | 0.59 | 0.559 |
GLU | 27.66 | <0.001 | 28.17 | 0.001 | 49.51 | <0.001 | 6.12 | 0.035 | 6.06 | 0.005 | 0.45 | 0.644 | 0.20 | 0.817 |
CEL | 17.16 | 0.002 | 3.47 | 0.091 | 25.62 | <0.001 | 0.61 | 0.452 | 2.25 | 0.119 | 0.53 | 0.592 | 2.53 | 0.093 |
PER | 46.63 | <0.001 | 0.10 | 0.765 | 50.06 | <0.001 | 0.07 | 0.796 | 2.29 | 0.116 | 3.00 | 0.062 | 0.40 | 0.675 |
AWCD | 6.51 | 0.029 | 8.18 | 0.016 | 19.49 | <0.001 | 0.01 | 0.905 | 3.28 | 0.049 | 2.57 | 0.089 | 0.34 | 0.717 |
aSoil properties: MBC = microbial biomass C, MBN = microbial biomass N, NAGase = β-1,4-N-acetylglucosaminidase, GLU = β-1,4-glucosidase, CEL = cellobiohydrolase, PER = Peroxidase, and AWCD = average well color development measured after 168 hours of incubation.
Error bars indicate standard errors (n = 6).
Soil microbial CLPP was different between FMM and PMM (
Principal component analysis (PCA) of the community level physiological profile in 2014 based on the area under the curve (
Cover soil type and sampling time significantly affected enzyme activities, including β-1,4-glucosidase, cellobiohydrolase and peroxidase in both 2013 and 2014; however, NAGase activities were not affected by cover soil type (
Error bars indicate standard errors (n = 6).
Soil microbial CLPP in the studied cover soils were significantly different (
Both above- and belowground litter and root exudates are primary energy and nutrient sources for soil microorganisms [
Applying CWD for land reclamation changed microbial CLPP in FMM but not in PMM (
Average well color development in Biolog Ecoplates was strongly affected by soil water content (
The greater soil enzyme activities in FMM than in PMM (
We had expected that CWD application would increase soil enzyme activities because of the effect of CWD on vegetation cover and microsites [
The two cover soils (FMM and PMM) commonly used for oil sands reclamation had different microbial CLPP, microbial biomass, and enzyme activities associated with contrasting soil properties and vegetation cover originated from different ecosystems. The FMM is a more favorable cover soil for land reclamation due to its greater microbial biomass and enzyme activities relative to PMM, therefore, organic matter decomposition and nutrient supply rates would be greater in FMM than in PMM. The CWD changed soil microbial CLPP in FMM and increased microbial biomass in most samplings and microbial community functional diversity (average well color development in Biolog Ecoplates) in both FMM and PMM. The CWD did not affect enzyme activities, suggesting that enzyme activities were resilient to CWD additions as an additional source of organic matter. Effects of CWD on microbial CLPP and microbial biomass were dependent on cover soil type and sampling time; CWD had greater effects when applied on FMM than on PMM. Increased microbial community functional diversity and microbial biomass by CWD in the studied cover soils suggest that applying CWD for land reclamation would benefit early ecosystem development by increasing organic matter decomposition and nutrient cycling. Applying CWD for land reclamation is recommended for accelerating upland reclamation and for recycling natural resources.
Funding for this work was provided by the Helmholtz-Alberta Initiative through a grant from the Alberta government. We thank Dr. Kangho Jung, Dr. Min Duan and Mr. Ghulam Murtaza Jamro for their help in the field and laboratory. We especially thank Suncor Energy Inc. for providing access to the study site.