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1. Background

Reclaimed sites depend on artificial soil cover to restore soil function and vegetation (DePuit 1984;, Winter Sydnor and Redente 2002;, MacKenzie and Naeth 2007). Common practices for oil sands reclaimation like in Alberta are use LFH and peat mineral mix as cover soils (Singh 2007). LFH is are a forest floor layers, with major components of identifiable litter (L), fragmented and fermenting litter (F) and humus (H). Peat mineral mix (hereafter referred to as peat) is composed of 1 m of organic peat and 0.4 m of underlying mineral soil (Oil Sands Revegetation Reclamation Committee 1998, Singh 2007) (hereafter referred to as peat). (This background information mainly comes from Brown's thesis…….)

Previous research has been done to see whether coarse woody debris application facilitate s reclamation and it has been found that woody plants abundance was positively associated with woody debris cover. P revious study has found that there is p ositive relationship between woody plant a bundance and woody debris cover (Brown 2010). This research aims to further explore and testify the benefits of coarse woody debris application in reclamation from the aspect of soil Coarsenitrogen. Newly reclaimed sites are sensitive to nitrogen fertilizer application, indicating that nitrogen is often limited (Tan and Kang, 2009) Nitrogen (N) is a macroelement that is often the limiting factor in crop production (Weinhold 2007). So our hypothesis is that due to CWD application, soil nitrogen availability enhanced. As a result, woody plants abundance increased. This research measured nitrogen mineralization and nitrification rates to test this hypothesis. The rate of N mineralization and nitrification can vary widely depending on numerous environment factors as well as biotic factors (Rice et al., 1987; Schimel et al., 1989; Hatch et al., 1990). Researchers have concluded that the intact core with ion exchange resin provided more reasonable estimates of mineralization after comparing several in situ methods (Hanselman et al., 2004; Raison et al., 1987). Furthermore, to better understand and model gross N cycling processes, 15N isotopic pool dilution has been proved to be a valuable tool. (Murphy et al., 2003).

Coarse woody debris (CWD) is “sound and rotting logs and stumps that provide habitat for plants, animals and insects and a source of nutrients for soil development.” (Parminter 1995). While Although CWD's role in providing habitat has been well studied, another function of CWD, which is the its which is the role in nutrient cycling, is comparatively obscure. So far, researchers still don't have not reached a consensus on this issue. For example, Laiho and Prescott (1999) found that CWD didn't seem to make a significant contribution to nitrogen cycling in forests. By contrast, Creed et al. (2004) result demonstrated that CWD played a particularly important role in nitrogen dynamics in N saturated forests.

2. Objectives

This research has two components: one for theas a field work experiment and the other is for thea laboratory incubation experiment. Both researches will focus on aspen (Populus tremuloides Michx. ) CWD's effects on soil nitrogen cycling. We chose aspen because it is the most widely distributed deciduous tree in North America (Bartos, 2001). Hence, studying aspen debris has a wide application. The field work will use intact soil core incubation with ion exchange resin (Plant Root Stimulator or PRS probes) and 15N isotopic pool dilution to measure soil nitrogen cycling in reclaimed sites that received CWD treatment on LFH and peat soils. The in-situ research will provide soil nitrogen cycling changes in real time with various natural abiotic and biotic factors. The nitrogen cycling procedure, in this part, includes soil nitrogen mineralization rat and soil nitrification rates.

On the other hand, the lab incubation will focus on the effects of CWD extractives to measureon soil nitrogen cycling using 15N isotopic pool dilution. Like As mentioned in previous paragraphearlier, there's confounding effects in separating CWD's two functions. This lab research, to our best knowledge, is the first to solely study CWD's role in soil nitrogen cycling, excluding CWD's role in providing habitat. In addition, by optimizing the abiotic factors and using CWD extractives, we can simulate the natural CWD's effects on soil nitrogen cycling in an accelerated fashion.

The lab soil incubation experiment aims to explore the influence of extractives of aspen CWD on soil nitrogen cycling. The nitrogen cycling procedure, in this part, includes soil nitrogen mineralization rate (gross and net), soil nitrification rate (gross and net), and microbial nitrogen. The lab incubation aims to analyze the differences of nitrogen cycling with and without CWD treatments from the angles of soil respiration rate, microbial carbon, carbon to nitrogen ratio and microbial growth efficiency (Hart et al., 1994).

3. Experiment 1: Effects of coarse woody debris on soil nitrogen cycling

3.1 Material and Methods

3.1.1. Study sites

Research was conducted at Suncor Energy Inc., 24 km north of Fort McMurray, Alberta, Canada. Average annual temperature is 0.7°C. Average annual precipitation: 455.5 mm, with 342.2 mm as rain and 155.8 cm as snow (Environment Canada 2003). Topography of this area is mainly upland forests, wetlands and rolling plains. A detailed description of the research sites can be referred to Brown (2010).

3.1.22 Experiment design and Plot layout

Research plots were are located on the southeast dump at Suncor Energy Inc. and covered 70 x 300 m² of land on a slight east facing slope (referred to Brown 2010). Two rows of plots were arranged horizontally along the slope of the study site in a completely randomized block design. Each plot (experiment unit) was 10 m wide and 30 m long. Each row contained three replicates of each treatment for a total of 24 treatment plots. Each plot was separated by a 5 m buffer and the two rows were separated by a 10 m buffer.

This is a 2*2 factorial experiment to study two soil types and coarse woody debris effects on soil nitrogen cycling. Half the cover soil treatment plots received LFH and half received peat.

3.1.33 Field labelling of intact core

We used 15N dilution method to measure the mineralization rate and nitrification rate. Basically we followed what Davison et al. described in 1991 (Davison et al., 1991) and Koyama et al., described in 2010 (Koyama et al., 2010).

In this experiment, a set of soil cores was defined as two groups of soil cores and each group contains two soil cores. We labeled a group of two soil cores (0~10cm) with (15NH4+)2SO4 and the other group with K15NO3.One of each group was broken and extracted with 2M KCl immediately(equivalent to 10 g oven-dried weight fresh soil extracted with 100 mL 2M KCl). In each woody debris plot, three sets of soil cores were made under large woody debris and another three sets of soil cores were made under small woody debris. In control plots, three sets of soil cores were made away from woody debris.

For each soil core, plastic cylinders (? cm diameter x 12? cm deep) were driven into the soil. Then larger metal cylinders (? cm diameter x 12? cm deep) were driven around the plastic cylinders so that each pair formed concentric circles. The pair was removed and the soil between the two cylinders was placed in plastic bags, mixed, immediately subsampled for extraction with 2 M KCI (equivalent to 10 g oven-dried weight). The remaining soil were mixed, double bagged, placed directly on dry ice (-78 °C), transported on dry ice and stored in a freezer (-20 °C) until analysis for volumetric water content (drying at 105°C for 24 h), soil texture, total organic carbon, carbon to nitrogen ratio, pH, electrical conductivity and microbial carbon and nitrogen and lab incubation.

(In the final paper, I'll say the detailed injection procedures and labeling were basically the same of what Davison et al. described in 1991 (Davison et al., 1991) and I will delete the below two paragraphs.)

In each core for field incubation, a cap was placed on the top (surface), and the core was inverted. Solutions of either (15NH4+)2SO4 or K15NO3 (both are 99 atom % 15N), were injected from the bottom end of the core (which was now facing up). In each core, six 1 cm3 injections were made to ensure evenly spread. A metal wire was first inserted into the soil core to create space for needle, to avoid clogging. The metal wire was then removed and a filled 1 cm3 syringe attached. The plunger of the syringe was pushed down as the needle and syringe was lifted up, leaving a column of solution injected into the core. A second cap was then placed on the core and the core turned upright to allow any solution that had pooled at the opposite end (surface) to flow back down into the soil.

The time elapsed between injection and extraction was about 15 min. The other core for each label at each plot was reburied and extracted 24 h later. The amounts of 15N solution injected into cores were based on previously measured inorganic soil N concentrations (approximate 1? week before the experiments) at each plot. The final 15N concentration was approximate 2mg 15N/Kg dry soil.”

3.1.4 Soil core incubation with PRS probes

We used ion exchangeable membrane (IEM) plant root simulator (PRS) probes to measure in situ soil chemistry changes in a quick and relatively inexpensive fashion (Drohan et al., 2005).

(The number of probes needed is depended on the homogeneity of soil nitrogen status. )

We inserted PRS probes horizontally in pair within 1 m2 of the soil core incubation sites. In each pair, one probe was laid right beneath CWD layer and the other probe was inserted to the soil at the 10cm depth.

3.2 Soil background information analysis

Soil pH was measured at 1:5 ratio (equivalent to 10 g oven-dried weight :50 mL deionized water) by a DMP-2 mV/pH meter. Soil organic N and organic C concentrations were determined using a CN analyzer (NA Series 2, CE Instruments, Italy) (Cheng et al., 2011).

3.3 Analysis of 15N in KCl extracts

All KCl solutions were filtered through Whatman No. 1 filters that had been rinsed with KC1. The 15N abundance of insoluble organic N and NH4+And NO3-samples was determined by a stable isotope ratio mass spectrometer (Optima-EA; Micromass, Crewe, UK) linked to a CN analyzer (NA Series 2, CE Instruments, Italy) at the Lethbridge Research Centre of Agriculture and Agri-Food Canada.”

The PRS probes were sent to Western Ag. Innovation Company for analysis.

3.4 Calculation and statistical analysis

M0=initial 14+15N pool (μg N g-1 dry soil)

M1=post-incubation 14+15N pool (μg N g-1 dry soil)

H0=initial 15N pool (μg Ng-1 dry soil)

H1 =post-incubation 15N pool (μg N g-1 dry soil)

m=mineralization rate (μg N g-1 soil d-1)

c=consumption rate (μg N g-1 soil d-1)

t= time (1 d for the present study)

where m ≠c. Kirkham and Bartholomew (1954) provide another equation for the condition when m = c, which was ? not encountered in this study.

For samples that received 15NH4+, the NH4+pool was used for M and H. For samples that received 15NO3, the 15NO3pool was used for M and H and the symbol m was replaced by n, which is the nitrification rate (μg N g-1 soil d-1). The term 'consumption' refers to the sum of all consumptive processes of the labelled pool.

The data were tested for normality and homogeneity in order to proceed with two-way ANOVA. (if the data don't meet the assumptions of normality and homogeneity of variance, log-transformation or maybe square root, inverse sine or arcsin transformation will be performed to fill the assumptions. If the data are extremely skewed, then a non-parametric test will be performed.)

3.2 Results and Discussions (to be continued…)

3.3 Conclusions (to be continued…)

4. Experiment 2: Effects of coarse woody debris extractives on soil N cycling

4.1. Materials and methods

4.1.1 Experimental design

3.6 Lab incubation

This lab incubation aims to explore CWD extractives' effects on two types of soil—LFH and peat. We first grounded fresh aspen woody debris with little or no sign of decay into powders and extracted the powers with locally collected rainwater at 1:2 ratio (10 g CWD: 20ml). We'll do the chemical analysis of cellulose, hemicelluoses and lignin as well as the microbes communities using PLFH.

Soil cover














Basically we followed what Cheng et al. described in 2011(Cheng et al., 2011). We used soil between the two cylinders or we can use augur to sample soils (0-10cm) near where the soil cores were incubated. In order to achieve evenly distributed 15N application, the soil depth is less than 2 cm (Barraclough and Puri, 1995; Garcia-Montiel and Binkley, 1998; Scott et al., 1998; Willison et al., 1998; Murphy et al., 2003). (In Cheng's paper, he didn't mention it. He used 30g mineral soil and 10g fresh forest floor soil incubated in 250 ml flasks. I did some calculation based on the circumaviate diameter of the 250 ml flask. The soil depths in both cases are less than 2 cm. but since I will do microbial carbon and nitrogen measurements as well, I need more soil. So I will use 40 g mineral soil and 20g fresh forest floor soil. They qualifies as well.)

I will use (15NH4+)2SO4 and K15NO3 for the lab incubation. The other parts are the same as Cheng.

Microbial Nitrogen and Carbon will be analyzed using chloroform (CHCl3) fumigation extraction (Brookes et al., 1985; Vance et al., 1987; Davidson et al., 1989) at 1:5 ratio (10g soil: 50ml 0.5mol/L K2SO4 ).

(the below part needs adaptation to avoid plagiarism.)

“The 15N labeled solution were added by pipetting solutions evenly over the soil surface, and the ¬nal soil moisture contentwas adjusted to 60% water holding capacity (WHC) with deionized water. Subsequently, the ¬‚asks were sealed with silicone rubber stoppers and then incubated in the dark at 25 ℃. The samples were aerated by removing the stoppers for 1 h after 23 h of incubation. Half of the (15NH4+)2SO4 and K15NO3 labeled ¬‚asks were extracted in a 2 mol/L KCl solution after 1 h of incubation and the remaining ¬‚asks were extracted after 48 h of incubation. The soil samples were extracted at a soil to KCl solution ratio of 1:5 (mass:volume). After the samples were shaking at 250 rpm on a mechanical shaker for 1 h at 25℃, they were ¬ltered through Whatman No. 42 ¬lter papers. A portion of the KCl extract was steam-distilled with MgO to determine NH4+concentrations on a steam distillation system (Vapodest 20, C. Gerhardt, Königswinter, Germany), thereafter the sample in the ¬‚ask was distilled again after addition of Devar da's alloy to determine NO3 concentrations (Lu, 2000). The liberated NH3 was collected in H3BO3 solutions. To prevent isotopic cross-contamination between samples, 25 mL of reagent-grade ethanol was added to distillation ¬‚asks and steam-distilled for 3 min between sample distillations (Hauck, 1982). After titration to determine the NH4+ and NO3-concentrations, the distillates were acidi¬ed and dried in an oven at 60℃ and sent for 15N analysis as described below. The residual soil was subsequently washed with distilled water, oven-dried at 60℃ till constant weight, and ground to pass a 0.15 mm sieve for 15N analysis of insoluble organic N as described below.”

4.2 Results and Discussions (to be continued…)

4.3 Conclusions (to be continued…)

45. Future work

The main chemical components of broadleafaves trees are cellulose 40-50%, lignin 17-35%, 15-35% hemicelluoses depending on tree species and growth conditions. So I'd like to measure soil enzymes activities.

Cellulose: EG¼ŒEndo-β-Glucanase (including EGⅠ、 EGⅡ、 EG â…¢ andâ…¤) ¼ŒCBHâ… &â…¡ CBH (Cx, C1 and β-Glucosidase). The products are carbohydrates.

Hemicelluoses Hemicelluloses: hemicellulase. The products are carbohydrates.

Lignin: phenol oxidase and phenol peroxidase. The products are polyphenol and phenol.

Another aspect to explore may be a more detailed analysis of CWD. We first separate bark as a layer (hereafter referred as bark layer) and sapwood and heartwood together as the other layer (hereafter referred as wood layer) from the CWD samples. These samples were dried at 80 ° C to constant weight and milled to 0.5 or 1 mm. extract 10 g ? CWD powder with 50 ml ? water at 80° C water bath. Shake for 1 hr on a reciprocating shaker. Lay the bottle on its side in order to have good extraction. apply the extracting solution on soils. Same amount of water (or acid) was applied on soil (both on LFH and peat) as control. Then we incubated the soil for four months starting at ? , 2011 and ends at ?2012).

In all samples, the concentrations of cellulose, acid-insoluble lignin and macronutrients N, P and K were determined.

65 Acknowledgements

We would like to thank the province of Alberta? for funding through the Helmholtz-Alberta Initiative. Thank you to the following people for their support, friendship, assistance and hard work.

Dr. Scott Chang, as my supervisor, provides kind and in-time feedback through the whole project. He is always ready to offer to help me. Without him, I couldn't have finished the thesis proposal.

Chou Pak Chow provided technical support.

Some ideas appeared after discussions with Ms. Shanghua Sun.

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